JP7716729B2 - Golgi apparatus-localized compounds and uses thereof - Google Patents

Description

The present invention relates to compounds that localize to the Golgi apparatus and uses thereof. More specifically, the present invention relates to novel compounds, Golgi apparatus labeling agents, drug delivery systems that target the Golgi apparatus, agents for localizing tagged proteins to the Golgi apparatus, methods for controlling the localization of target proteins in cells, and kits for controlling the localization of tagged proteins in cells.

The Golgi apparatus is an organelle present in eukaryotic cells. Most secretory and membrane proteins undergo modifications such as glycosylation and cleavage in the Golgi apparatus before being sorted and delivered to organelles in the secretory pathway, the cell membrane, or outside the cell. Dysfunction in the structure or function of the Golgi apparatus causes abnormalities in the structure, function, and delivery of these proteins, and is increasingly being linked to neurodegenerative diseases, glycosylation disorders, carcinogenesis, and other conditions.

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

The inventors have previously synthesized a compound (mgcTMP) that combines trimethoprim (TMP), a ligand for Escherichia coli-derived dihydrofolate reductase (eDHFR), with a motif for localization to the cell membrane or 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 Golgi apparatus. The chemical formula of trimethoprim is shown below in formula (A1).

The chemical formula for mgcTMP is shown below in formula (A2). The portion of mgcTMP consisting of a myristoyl group, a glycine residue, and a cysteine residue functions as a motif for localization to the cell membrane or Golgi apparatus.

Lipsky N. G. and Pagano R. E., A vital stain for the Golgi apparatus, Science, 228 (4700): 745-747, 1985. Toth B., et al., Phosphatidylinositol 4-Kinase III beta Regulates the Transport of Ceramide between the Endoplasmic Reticulum and Golgi, J. Biol. Chem., 281 (47), 36369-36377, 2006. Yadav R. B., et al., mTOR direct interactions with Rheb-GTPase and raptor: sub-cellular localization using fluorescence lifetime imaging, BMC Cell Biology, 14:3, 2013. Ishida M., et al., Synthetic self-localizing ligands that control the spatial location of proteins in living cells., J Am Chem Soc., 135(34), 12684-12689, 2013.

However, conventional ceramide derivatives have room for improvement in terms of their specificity for the Golgi apparatus. Furthermore, mgcTMP is a compound that also localizes in the cell membrane, and is not a compound that specifically localizes only in the Golgi apparatus. Therefore, an object of the present invention is to provide a compound that specifically localizes in the Golgi apparatus.

The present invention includes the following aspects.
[1] A compound represented by the following formula (1):
[In formula (1), Z represents a group represented by formula (2) or (3) below; L represents a divalent hydrocarbon group having 1 to 50 carbon atoms which may be interrupted by substitution of -CH ₂ - with -O-, -CO-, -NH-, or a group represented by formula (4) below; R ¹ represents a group derived from a labeling substance or a group derived from a drug, a group which functions as a ligand for a tagged protein, or a group in which a photodegradable protecting group is further bonded to the group which functions as a ligand for the tagged protein; in formulas (2) and (3) below, R ² represents a saturated or unsaturated linear hydrocarbon group having 9 or more carbon atoms; R ³ each independently represents a hydrogen atom or a methyl group; and * indicates an asymmetric carbon atom.]
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[2] The compound according to [1], wherein the combination of the tag protein and the group that functions as a ligand for the tag protein is a combination of Escherichia coli dihydrofolate reductase (eDHFR) protein and a group represented by the following formula (5), a combination of FK506 binding protein and a group represented by the following formula (6), a combination of carbonic anhydrase I protein or carbonic anhydrase II protein and a group represented by the following formula (7), a combination of SNAP-tag (registered trademark) protein and a group represented by the following formula (8) or (9), a combination of HaloTag (registered trademark) protein and a group represented by the following formula (10), or a combination of photoactive yellow protein and a group represented by the following formula (11).
[3] The compound according to [1] or [2], wherein the photodegradable protecting group is a group represented by any one of the following formulas (12) to (20):
[4] A Golgi apparatus labeling agent comprising the compound according to [1], wherein R ¹ is a group derived from a labeling substance.
[5] A drug delivery system targeting the Golgi apparatus, comprising the compound according to [1], wherein R ¹ is a group derived from a drug.
[6] An agent for localizing a tagged protein to a Golgi apparatus, comprising the compound according to any one of [1] to [3], wherein R ¹ is a group that functions as a ligand for the tagged protein.
[7] A method for controlling the localization of a target protein in a cell, the method comprising the step (a) of introducing into the cell the localization agent according to [6], wherein ^R1 is a group that functions as a ligand for the tagged protein, so that the group that functions as a ligand of the localization agent binds to the tagged protein and the tagged protein is localized in the Golgi apparatus together with the target protein, and the cell is a cell that expresses a fusion protein of the target protein and the tagged protein.
[8] The method according to [7], further comprising, after step (a), step (b) of introducing a ligand of the tagged protein into the cell, wherein in step (b), the group functioning as the ligand competes with the ligand, and the ligand replaces the group functioning as the ligand in at least a portion of the tagged protein localized in the Golgi apparatus, resulting in the target protein being translocated from the Golgi apparatus.
[9] A method for controlling the localization of a target protein in a cell, the method comprising: a step (a1) of introducing into the cell the localization agent according to [6], wherein ^R1 is a group formed by further binding a photolabile protecting group to a group that functions as a ligand of the tagged protein; and a step (a2) of irradiating the cell with light after the step (a1), such that the photolabile protecting group is detached from the localization agent, the group that functions as a ligand of the localization agent binds to the tagged protein, and the tagged protein is localized together with the target protein in a Golgi apparatus, wherein the cell is a cell that expresses a fusion protein of the target protein and the tagged protein.
[10] The method according to [9], further comprising, after step (a2), step (b) of introducing a ligand of the tagged protein into the cell, wherein in step (b), the group functioning as a ligand competes with the ligand, and the ligand replaces the group functioning as a ligand in at least a portion of the tagged protein localized in the Golgi apparatus, resulting in the target protein being translocated 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 tagged protein in a cell, comprising the localization agent according to [6].
[13] The kit described in [12], further comprising a cell expressing a fusion protein of the target protein and the tag protein, an expression vector for 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.
[14] The kit according to [12] or [13], further comprising a ligand for the tagged protein.

The present invention provides compounds that specifically localize in the Golgi apparatus.

1(a) is a confocal laser microscope photograph showing the results of Experimental Example 2. 1(b) is a graph showing the numerical value of the ratio (F/F ₀ ) of the fluorescence intensity F in the cytoplasm after the addition of a compound to the fluorescence intensity F ₀ in the cytoplasm before the addition of a compound over time in Experimental Example 2. (a), (b), (d), and (e) are confocal laser microscope photographs showing the results of Experimental Example 2. (c) is a graph plotting fluorescence intensity along the straight line shown in (b). (f) is a graph plotting fluorescence intensity along the straight line shown in (e). (a) is a fluorescence micrograph showing the results of Experimental Example 4. (b) is a composite image of a fluorescence micrograph and a differential interference contrast micrograph in the same field of view as (a). (c) is a graph plotting fluorescence intensity along the straight line shown in (b). (a) is a fluorescence microscope photograph showing the results of Experimental Example 4. (b) is a fluorescence microscope photograph of mCherry fluorescence observed in the same field of view as (a). (c) is a composite image of (a) and (b). (a) is a fluorescence micrograph showing the results of Experimental Example 4. (b) is a composite image of a fluorescence micrograph and a differential interference contrast micrograph in the same field of view as (a). (c) is a graph plotting fluorescence intensity along the straight line shown in (b). (a) is a fluorescence microscope photograph showing the results of Experimental Example 4. (b) is a fluorescence microscope photograph of mCherry fluorescence observed in the same field of view as (a). (c) is a composite image of (a) and (b). (a) is a fluorescence micrograph showing the results of Experimental Example 4. (b) is a composite image of a fluorescence micrograph and a differential interference contrast micrograph in the same field of view as (a). (c) is a graph plotting fluorescence intensity along the straight line shown in (b). (a) is a fluorescence microscope photograph showing the results of Experimental Example 4. (b) is a fluorescence microscope photograph of mCherry fluorescence observed in the same field of view as (a). (c) is a composite image of (a) and (b). 10(a) to 10(d) are confocal laser microscope photographs showing the results of Experimental Example 5. 10(a) to 10(h) are confocal laser microscope photographs showing the results of Experimental Example 6. 10(a) to 10(d) are confocal laser microscope photographs showing the results of Experimental Example 7. FIG. 10 is a schematic diagram illustrating Experimental Example 8. 10( a ) and 10 ( b ) are confocal laser microscope photographs showing the results of Experimental Example 8. 10( a ) and 10 ( b ) are graphs showing the results of Experimental Example 8. FIG. 10 is a schematic diagram illustrating Experimental Example 9. 10( a ) and 10 ( b ) are confocal laser microscope photographs showing the results of Experimental Example 9. 10(a) to 10(d) are graphs showing the results of Experimental Example 9. (a) is a schematic diagram showing the structure of the fusion protein expressed in Experimental Example 11. (b) to (d) are confocal laser microscope photographs showing the results of Experimental Example 11. (e) is a graph showing the results of Experimental Example 11. 10 is a graph showing the results of measuring the absorption spectrum of m ^D c ^NVOC TMP in Experimental Example 12. 10 is a graph showing the results of Experimental Example 12. 10(a) to 10(h) are confocal laser microscope photographs showing the results of Experimental Example 13. 10(i) is a graph showing the results of Experimental Example 13. (a) to (d) are confocal laser microscope photographs showing the results of Experimental Example 14. (e) is an image showing areas where the cell area increased or decreased in Experimental Example 14. (a) to (d) are confocal laser microscope photographs showing the results of Experimental Example 15. (e) is an image showing areas where the cell area increased or decreased in Experimental Example 17. 10(a) to 10(d) are images showing areas where the cell area increased or decreased in Experimental Example 16. Graph (a) shows the increase in area of the protruding region (region where the area increased) of the cell in Experimental Example 16. Graph (b) shows the decrease in area of the retracting region (region where the area decreased) of the cell in Experimental Example 16. 10 is a graph showing the results of measuring the absorption spectra of m ^D c ^NVOC TMP and m ^D c ^DEACM TMP in Experimental Example 17. 10(a) and 10(b) are confocal laser microscope photographs showing the results of Experimental Example 18. 10(c) and 10(d) are graphs showing the results of Experimental Example 18.

[New compound]
In one embodiment, the present invention provides a compound represented by the following formula (1):
[In formula (1), Z represents a group represented by formula (2) or (3) below; L represents a divalent hydrocarbon group having 1 to 50 carbon atoms which may be interrupted by substitution of -CH ₂ - with -O-, -CO-, -NH-, or a group represented by formula (4) below; R ¹ represents a group derived from a labeling substance or a group derived from a drug, a group which functions as a ligand for a tagged protein, or a group in which a photodegradable protecting group is further bonded to the group which functions as a ligand for the tagged protein; in formulas (2) and (3) below, R ² represents a saturated or unsaturated linear hydrocarbon group having 9 or more carbon atoms; R ³ each independently represents a hydrogen atom or a methyl group; and * indicates an asymmetric carbon atom.]
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In the above formulas (2) and (3), at least one ^R3 is preferably a methyl group. As will be described later in the Examples, the compound of this embodiment is localized in the Golgi apparatus. More specifically, the compound of this embodiment is localized on the cytoplasmic side of the membrane of the Golgi apparatus. Furthermore, by using the compound of this embodiment, a tagged protein can be localized in the Golgi apparatus. Furthermore, by expressing the tagged protein in a cell as a fusion protein with a target protein, any target protein can be localized in the Golgi apparatus together with the tagged protein.

Furthermore, as will be described later in the Examples, the structure of the group represented by formula (2) or (3) above is particularly important for the compound of this embodiment to localize to the Golgi apparatus. The group represented by formula (2) or (3) above has a structure in which the portion consisting of the myristoyl group, glycine residue, and cysteine residue in the mgcTMP described above has been modified.

Furthermore, as will be described later in the Examples, the inventors have clarified that the Golgi apparatus can be localized when the group represented by ^R2 has 9 or more carbon atoms. The group represented by ^R2 is preferably a saturated linear hydrocarbon group having 9 or more carbon atoms.

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

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), the groups represented by R ² and R ³ are the same as those in the above formula (2).

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

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), the groups represented by R ² and R ³ are the same as those in the above formula (3).

The SH group in the above formula (3-1) can also be said to be the side chain of an L-cysteine residue. Also, the SH group in the above formula (3-2) can also be said to be the side chain of a D-cysteine residue.

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

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

Specific examples of combinations of tag proteins and groups that function as ligands for the tag proteins include a combination of Escherichia coli dihydrofolate reductase (eDHFR) protein and a group represented by formula (5) below, a combination of FK506 binding protein (FKBP) and a group represented by formula (6) below, a combination of carbonic anhydrase I protein or carbonic anhydrase II protein and a group represented by formula (7) below, a combination of SNAP-tag (registered trademark) protein and a group represented by formula (8) or (9) below, a combination of HaloTag (registered trademark) protein and a group represented by formula (10) below, or a combination of photoactive yellow protein and a group represented by formula (11) below.

The tagged protein and the group that functions as a ligand for the tagged protein may be modified, as long as they can bind to each other. For example, the tagged protein may have an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to the amino acid sequence of the tagged protein described above. Here, "one or several" may mean, for example, 1 to 10, 1 to 5, or 1 to 3. Furthermore, the group that functions as a ligand for the tagged protein may have a substituent in the group described above.

The compound of this embodiment may further have a photolabile protecting group bound to the group that functions as a ligand for the tagged protein. Such compounds are called caged compounds.

As will be described later in the Examples, in the compound of this embodiment, when a photolabile protecting group is further bound to a group that functions as a ligand for a tagged protein, it is believed that this function can be controlled by light irradiation, etc.

When a photolabile protecting group is further bound to the group that functions as a ligand of the tagged protein, the tagged protein and the group that functions as a ligand of the tagged protein do not bind. However, when the photolabile protecting group is deprotected, for example by irradiating it with light of a specific wavelength, the tagged protein and the group that functions as a ligand of the tagged protein can bind.

There are no particular limitations on the photodegradable protecting group, and any type can be used. Specific photodegradable protecting groups 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), and a (7-diethylaminothiocoumarin-4-yl)methyloxycarbonyl (DEACM) group represented by the following formula (16). Examples of such groups include an oxycarbonyl (DEATCOC) group, 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), and a 3-nitrodibenzofuran-2-ylmethyl (NDBF) group represented by the following formula (20).

[Golgi apparatus labeling agent]
In one embodiment, the present invention provides a Golgi apparatus labeling agent comprising a compound represented by formula (1) above, wherein R ¹ is a group derived from a labeling substance.

Examples of labeling substances include fluorescent substances, Raman tags, peptide tags, biotin, etc.

Fluorescent substances include low molecular weight compounds such as fluorescein, coumarin, rhodamine, cyanine dyes, and BODIPY, as well as their derivatives.

Raman tags include alkyne compounds and nitrile compounds.

Examples of peptide tags include HA tags, myc tags, and His tags.

Examples of groups derived from labeling substances include monovalent groups formed by removing a hydrogen atom, a hydroxyl group, etc. from the above-mentioned labeling substances.

As described below in the Examples, the Golgi apparatus labeling agent of this embodiment can specifically label the Golgi apparatus.

[Drug delivery system targeting the Golgi apparatus]
In one embodiment, the present invention provides a drug delivery system targeted to the Golgi apparatus, which comprises a compound represented by the above formula (1), wherein R ¹ is a group derived from a drug.

Drugs are not particularly limited, and examples include anticancer drugs and vesicle transport inhibitors. Anticancer drugs include compounds that exert anticancer activity by being delivered to the Golgi apparatus. Vesicle transport inhibitors include (+)-brefeldin A and its derivatives.

Examples of groups derived from drugs include monovalent groups formed by removing a hydrogen atom, hydroxyl group, etc. from the above-mentioned drugs.

The drug delivery system of this embodiment can deliver drugs specifically to the Golgi apparatus.

[Agent for localizing tagged proteins to the Golgi apparatus]
In one embodiment, the present invention provides an agent for localizing a tagged protein to the Golgi apparatus, which comprises a compound represented by the above formula (1), wherein R ¹ is a group that functions as a ligand for the tagged protein.

The tagged protein and the group that functions as a ligand for the tagged protein are the same as those described above. As will be described later in the Examples, the localization agent of this embodiment can be used to localize the tagged protein to the Golgi apparatus.

[Method for controlling the localization of a target protein in a cell]
(First embodiment)
A first embodiment of the present invention relates to a method for controlling the localization of a target protein in a cell, the method comprising the step (a) of introducing into the cell the above-described localization agent, wherein ^R1 is a group that functions as a ligand for the tagged protein, so that the group that functions as a ligand of the localization agent binds to the tagged protein and the tagged protein is localized in the Golgi apparatus together with the target protein, wherein the cell is a cell that has expressed a fusion protein of the target protein and the tagged protein. The first embodiment of the present invention relates to a method for controlling the localization of a target protein in a cell using a localization agent that does not have a photolabile protecting group.

In the method of the first embodiment, cells that express a fusion protein of a target protein and a tag protein are used. The cells are preferably eukaryotic cells. Examples of eukaryotic cells include animal cells, insect cells, plant cells, and yeast cells.

The fusion protein may have one or more target proteins. Furthermore, the fusion protein may have one or more tag proteins. Here, when there are multiple target proteins, those target proteins may be one type or two or more types. Furthermore, when there are multiple tag proteins, those tag proteins may be one type or two or more types.

Furthermore, in the fusion protein, the order in which the target protein and tag protein are arranged is not particularly limited, and either may be located at the N-terminus. For example, the target protein and tag protein may be arranged in that order from the N-terminus, or the tag protein and target protein may be arranged in that order from the N-terminus. Furthermore, when there are multiple target proteins and multiple tag proteins, the order in which they are arranged in the fusion protein is not particularly limited.

For example, as described below in the Examples, a fusion protein may have one tag protein and two target proteins. The target proteins are not particularly limited and can be fluorescent proteins, signaling proteins, enzymes, etc.

In the method of the first embodiment, in step (a), the above-mentioned localization agent is introduced into cells expressing a fusion protein of a target protein and a tag protein. Introduction of the localization agent can be performed, for example, by adding the localization agent to the cell culture medium.

As described below in the Examples, when a localization agent is introduced into a cell, a group that functions as a ligand for the localization agent binds to the tagged protein, causing the tagged protein to specifically localize to the Golgi apparatus along with the target protein.

The method of the first embodiment may further include, after step (a), step (b) of introducing a ligand for the tagged protein into the cell. In this case, in step (b), the group functioning as a ligand competes with the ligand, and the group functioning as a ligand is replaced by the ligand in at least a portion of the tagged protein localized in the Golgi apparatus, resulting in the target protein being translocated from the Golgi apparatus. In other words, the target protein is delocalized. Step (b) allows the localization of the localized target substance to be controlled and delocalized.

Second Embodiment
A method of a second embodiment is a method for controlling the localization of a target protein in a cell, the method comprising: a step (a1) of introducing into the cell the above-mentioned localization agent, wherein ^R1 is a group formed by further binding a photolabile protecting group to a group that functions as a ligand for the tagged protein; and a step (a2) of irradiating the cell with light after the step (a1), such that the photolabile protecting group is cleaved from the localization agent, the group that functions as a ligand of the localization agent binds to the tagged protein, and the tagged protein localizes to the Golgi apparatus together with the target protein, wherein the cell is a cell that has expressed a fusion protein of the target protein and the tagged protein. The method of the second embodiment differs from the method of the first embodiment mainly in that a localization agent that is a caged compound is used.

In the method of the second embodiment, in step (a1), a localization agent, which is a caged compound, is introduced into cells expressing a fusion protein of a target protein and a tag protein. In the method of the second embodiment, the method for introducing the cells and the localization agent into the cells is the same as in the method of the first embodiment.

Next, after step (a1), step (a2) is carried out, in which the cells are irradiated with light. The light irradiated in step (a2) may be light that satisfies conditions that allow the photolabile protecting group of the caged compound to be deprotected, and may be adjusted appropriately depending on the photolabile protecting group used. For example, as described below in the Examples, when an NVOC group is used as the photolabile protecting group, irradiation with light having a wavelength of 405 nm may be used. Furthermore, when a DEACM group is used as the photolabile protecting group, irradiation with light having a wavelength of 405 nm, light having a wavelength of 445 nm, or the like may be used.

Upon irradiation with light, the photolabile protecting group is released from the localization agent. Subsequently, the group that functions as a ligand on the localization agent binds to the tagged protein, and the tagged protein is localized in the Golgi apparatus along with the target protein.

As described below in the Examples, the method of the second embodiment makes it possible to control the localization of tagged proteins at the single-cell level or in specific regions on a single cell.

The method of the second embodiment may further include, after step (a2), step (b) of introducing a ligand for the tagged protein into the cells. Step (b) in the method of the second embodiment is the same as step (b) in the method of the first embodiment.

In this case, in step (b), the group functioning as a ligand competes with the ligand, and the ligand is substituted for the group functioning as a ligand in at least a portion of the tagged protein localized in the Golgi apparatus, resulting in the target protein being translocated from the Golgi apparatus. In other words, the target protein is delocalized. Step (b) allows the localization of the localized target substance to be controlled and delocalized.

[kit]
In one embodiment, the present invention provides a kit for controlling the localization of a tagged protein in a cell, comprising the localization agent described above. By using the kit of this embodiment, the localization of the tagged protein in the cell can be controlled.

In the kit of this embodiment, the localization agent may or may not have a photolabile protecting group.

The kit of this embodiment may further include cells expressing a fusion protein of a target protein and a tag protein, an expression vector for the fusion protein of a target protein and a tag protein, or a vector for producing the fusion protein of a target protein and a tag protein.

In the kit of this embodiment, the same cells as those described above can be used. When the kit of this embodiment includes cells expressing a fusion protein of a target protein and a tag protein, the localization of the tag protein within the cells can be controlled by introducing the localization agent described above into the cells.

When the kit of this embodiment includes an expression vector for a fusion protein of a target protein and a tag protein, cells expressing the fusion protein of the target protein and the tag protein can be prepared by introducing the expression vector into desired cells and expressing the vector. By introducing the above-mentioned localization agent into these cells, the localization of the tag protein within the cells can be controlled.

An example of a vector for producing a fusion protein of a target protein and a tag protein is a vector that includes a gene encoding the tag protein and a multi-cloning site downstream of a promoter. Either the gene encoding the tag protein or the multi-cloning site may be located on the promoter side. An expression vector for a fusion protein of a target protein and a tag protein can be prepared by cloning a gene encoding the desired target protein into the multi-cloning site of a vector for producing a fusion protein.

Then, by introducing the expression vector into desired cells and expressing it, cells expressing a fusion protein of the target protein and the tagged protein can be prepared. By introducing the above-mentioned localization agent into these cells, the localization of the tagged protein within the cells can be controlled.

The kit of this embodiment may further include a ligand for the tagged protein. As described below in the Examples, after localizing the tagged protein, the localization of the tagged protein can be eliminated by introducing the ligand for the tagged protein into cells.

The present invention will now be described in more detail using examples, but the present invention is not limited to the following examples.

[Experimental Example 1]
(Synthesis of Compounds)
mgc(1Me)TMP represented by the chemical formula (21) below, mgc(2Me)TMP represented by the chemical formula (22) below, mgc(3Me)TMP represented by the chemical formula (23) below, and mc(2Me)TMP represented by the chemical formula (24) below were each synthesized.

Synthesis of mgc(1Me)TMP
mgc(1Me)TMP was synthesized according to the following scheme (1).

Specifically, mgc(1Me)TMP was synthesized on Rink amide resin using standard solid-phase peptide synthesis utilizing the 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group and N-methylation protocols.

Fmoc deprotection was carried out using 20% piperidine in N,N-dimethylformamide (DMF) for 15 minutes at room temperature. Amino acid coupling reactions for standard (unmethylated) amino acids were carried out 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 at room temperature. Additionally, amino acid coupling reactions for N-methylated amino acids were carried out at room temperature using a mixture of Fmoc-protected amino acid (3.1 equivalents), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[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 the Kaiser test and chloranil test. All washing procedures were performed using DMF or NMP.

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, followed by washing with DMF.

Next, using Fmoc-Adox-OH as the building block, the Fmoc deprotection and coupling reaction was repeated three times.

The Fmoc-Adox terminus on the resin was deprotected with Fmoc, followed by N-methylation of the terminal amino group. N-methylation was first achieved by (1) treating the resin with 2-nitrobenzenesulfonyl chloride (4.0 equivalents) and 2,4,6-collidine (10.0 equivalents) in NMP at room temperature for 15 minutes to achieve 2-nitrobenzenesulfonylation. (2) After washing with NMP, the resin was treated with DBU (3.0 equivalents) in NMP for 3 minutes. (3) Dimethyl sulfate (10 equivalents) was then added and treated for an additional 2 minutes. N-methylation was achieved by repeating steps (2) and (3) once more. Next, the resin was treated with 2-mercaptoethanol (10.0 equivalents) and DBU (5.0 equivalents) in NMP for 5 minutes. The 2-nitrobenzenesulfonyl group was deprotected by repeating this procedure once more.

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

The N-terminus was myristoylated using a mixture of myristic acid (4.1 equiv.), HBTU (4.0 equiv.), HOBt (4.0 equiv.) and DIPEA (8.0 equiv.) in DMF/CH ₂ Cl ₂ (1:1).

The resin was then treated with Pd(PPh ₃ ) ₄ (3.0 equivalents), acetic acid (5%), and 4-methylmorpholine (2.5%) in chloroform to selectively deprotect the Aloc group. The resin was washed with DMF containing DIPEA (0.5%) and then with DMF containing sodium diethyldithiocarbamate (0.5%).

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.

Trt deprotection and cleavage from the resin were carried out using trifluoroacetic acid (TFA) containing 2.5% triisopropylsilane (TIPS). The crude product was then 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 give mgc(1Me)TMP as a white solid.

^1H NMR (400MHz, _CD3 OD): δ7.21 (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).

Synthesis of mgc(2Me)TMP
mgc(2Me)TMP was synthesized according to the following scheme (2).

In scheme (2), mgc(2Me)TMP was synthesized by N-methylating Cys in addition to the N-methylation of Adox in scheme (1).

^1H NMR (400MHz, _CD3 OD): δ7.21 (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).

Synthesis of mgc(3Me)TMP
mgc(3Me)TMP was synthesized according to the following scheme (3).

In scheme (3), mgc(3Me)TMP was synthesized by N-methylating Cys and Gly in addition to the N-methylation of Adox in scheme (1). Furthermore, Siber amide resin was used instead of Rink amide resin in scheme (1), Fmoc-Cys(Mmt)-OH was used instead of Fmoc-Cys(Trt)-OH, and NMP was used instead of DMF as the condensation solvent for myristic acid. Mmt deprotection and cleavage from the resin were carried out using CH ₂ Cl ₂ containing 2.5% triisopropylsilane (TIPS) and 5% TFA.

^1H NMR (400MHz, _CD3 OD): δ7.21 (1H, s), 6.56 (2H, s), 5.66-5.44 (1H, m), 4.41 (1H, m), 4.03 (2H, s), 3.9 9 (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).

Synthesis of mc(2Me)TMP
mc(2Me)TMP was synthesized according to the following scheme (4).

In scheme (4), mc(2Me)TMP was synthesized by removing the Gly coupling in scheme (3).

^1H NMR (400MHz, _CD3 OD): δ7.22 (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).

[Experimental Example 2]
(Consideration of localization)
The localization of a target protein in cells was investigated using mgcTMP, mgc(1Me)TMP, mgc(2Me)TMP, mgc(3Me)TMP, and mc(2Me)TMP. Specifically, a fusion protein of a target protein and a tag protein was first 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 positioned at the N-terminus of the fusion protein, and EGFP at the C-terminus. Hereinafter, this fusion protein may be referred to as "DG." The amino acid sequence of DG is shown in SEQ ID NO: 1.

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

Figure 1(a) is a confocal laser microscope photograph. In Figure 1(a), "mgc" indicates the result after the addition of mgcTMP, "mgc(1Me)" indicates the result after the addition of mgc(1Me)TMP, "mgc(2Me)" indicates the result after the addition of mgc(2Me)TMP, "mgc(3Me)" indicates the result after the addition of mgc(3Me)TMP, "mc(2Me)" indicates the result after the addition of 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.

Figure 1(b) is a graph showing the ratio (F/F ₀ ) of the fluorescence intensity in the cytoplasm after the addition _of a compound to the fluorescence intensity in the cytoplasm before the addition of a compound, as quantified over time. When EGFP is localized to the Golgi apparatus, the fluorescence intensity in the cytoplasm decreases. In Figure 1(b), the vertical axis represents the normalized fluorescence intensity in the cytoplasm (relative value), and the horizontal axis represents the time (minutes) after the addition of the compound.

The results showed that when mgcTMP was added, EGFP localized to the cell membrane and Golgi apparatus 5 minutes after addition. Furthermore, it was revealed that EGFP localization spontaneously disappeared 40 minutes after the addition of mgcTMP.

Furthermore, when mgc(1Me)TMP was added, it was found that EGFP localized to the Golgi apparatus 5 minutes after addition. Furthermore, it was found that EGFP localization spontaneously disappeared 60 minutes after the addition of mgc(1Me)TMP.

Furthermore, when mgc(2Me)TMP, mgc(3Me)TMP, or mc(2Me)TMP was added, EGFP localized to the Golgi apparatus 5 minutes after addition. Furthermore, when these compounds were added, EGFP localization to the Golgi apparatus was maintained even 60 minutes later. In particular, mgc(3Me)TMP was found to be highly specific to the Golgi apparatus and tended to maintain its localization to the Golgi apparatus for long periods of time.

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

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

As a result, it was confirmed that the addition of these compounds could localize the fusion protein to the Golgi apparatus.

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

Synthesis of mgc(3Me)FDA
mgc(3Me)FDA was synthesized according to the following scheme (5).

Specifically, mgc(Trt)(3Me)K was first synthesized on Sieber amide resin following a protocol similar to that used to synthesize mgc(3Me)TMP.

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

After Fmoc deprotection of the Fmoc-Adox terminus on the resin, N-methylation was performed using a method similar to that in Scheme (3). Subsequently, coupling reactions, Fmoc deprotection, and N-methylation were repeated using Fmoc-Cys(Trt)-OH and Fmoc-Gly-OH as building blocks. Then, myristoylation of the N-terminus was performed.

Subsequently, the Aloc group was selectively deprotected using a method similar to that used in Scheme (1).

The resin was then treated with _CH2Cl2 containing 5% TFA for 5 minutes, and _the solution was collected, this procedure repeated five times. Toluene was added to the collected solution, and the mixture was concentrated to give mgc(Trt)(3Me)K. The crude product was then purified by reverse-phase HPLC on a semi-preparative C4 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA to give mgc(Trt)(3Me)K as a white solid.

Subsequently, 5-carboxyfluorescein diacetate (FDA) was introduced into the side chain of the lysine residue by reacting mgc(Trt)(3Me)K with 5-carboxyfluorescein diacetate (1.0 equiv.), HBTU (1.0 equiv.), and DIPEA (3.0 equiv.) in DMF. After evaporation of the solvent, Trt deprotection was carried out with CH ₂ Cl ₂ containing 25% TFA and 2.5% TIPS. The crude product was purified by reverse-phase HPLC on a semi-preparative C4 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA to give mgc(Trt)(3Me)FDA as a white solid.

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

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.

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

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

Golgi apparatus staining with mgc(3Me)FDA 1
Mgc(3Me)FDA was added to the HeLa cell culture medium 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 bovine serum albumin (BSA) and observed under a microscope.

Figure 3(a) is a fluorescence micrograph. Excitation light with a wavelength of 488 nm was irradiated, and fluorescence with wavelengths of 500 to 540 nm was observed. The scale bar is 20 μm. Figure 3(b) is a composite image of a fluorescence micrograph and a differential interference contrast micrograph taken in the same field of view as Figure 3(a). Figure 3(c) is a graph plotting fluorescence intensity along the straight line shown in Figure 3(b).

The results demonstrated that mgc(3Me)FDA can specifically stain the Golgi apparatus.

Golgi Apparatus Staining with mgc(3Me)FDA 2
HeLa cells expressing mCherry-Giantin, a fusion protein of the fluorescent protein mCherry and the Golgi apparatus 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 cell culture medium to a final concentration of 1 μM, and the cells were incubated at room temperature for 5 minutes. The cells were then washed with medium containing 3 mg/mL BSA and observed under a microscope.

Figure 4(a) is a fluorescence microscope photograph. Excitation light with a wavelength of 488 nm was irradiated, and fluorescence with wavelengths of 500 to 540 nm was observed. The scale bar is 20 μm. Figure 4(b) is a fluorescence microscope photograph of mCherry fluorescence observed in the same field of view as Figure 4(a). Figure 4(c) is a composite image of Figures 4(a) and 4(b).

The results showed that the fluorescence of mgc(3Me)FDA coincided with that of mCherry. This result further supports the idea that mgc(3Me)FDA can specifically stain the Golgi apparatus.

Golgi staining with mgc(3Me)DEAC 1
Mgc(3Me)DEAC was added to the HeLa cell culture medium 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.

Figure 5(a) is a fluorescence micrograph. Excitation light with a wavelength of 405 nm was irradiated, and fluorescence with wavelengths of 430 to 470 nm was observed. The scale bar is 20 μm. Figure 5(b) is a composite image of a fluorescence micrograph and a differential interference contrast micrograph taken in the same field of view as Figure 5(a). Figure 5(c) is a graph plotting fluorescence intensity along the straight line shown in Figure 5(b).

The results demonstrated that mgc(3Me)DEAC can specifically stain the Golgi apparatus.

<Golgi Apparatus Staining with mgc(3Me)DEAC 2>
HeLa cells expressing mCherry-Giantin, a fusion protein of the fluorescent protein mCherry and the Golgi apparatus marker protein Giantin, were prepared. mgc(3Me)DEAC was added to the cell culture medium to a final concentration of 1 μM and incubated at room temperature for 5 minutes. The cells were then washed with medium containing 3 mg/mL BSA and observed under a microscope.

Figure 6(a) is a fluorescence microscope photograph. Excitation light with a wavelength of 405 nm was irradiated, and fluorescence with wavelengths of 430 to 470 nm was observed. The scale bar is 20 μm. Figure 6(b) is a fluorescence microscope photograph of mCherry fluorescence observed in the same field of view as Figure 6(a). Figure 6(c) is a composite image of Figures 6(a) and 6(b).

The results showed that the fluorescence of mgc(3Me)DEAC coincided with that of mCherry. This result further supports the idea that mgc(3Me)DEAC can specifically stain the Golgi apparatus.

<Golgi Apparatus Staining with BODIPY FL C5-Ceramide 1>
A complex of BODIPY FL C5-Ceramide (product number "D3521", Thermo Fisher Scientific) and BSA was added to the HeLa cell culture medium to a final concentration of 5 μM, and the cells were incubated for 30 minutes at 4° C. Subsequently, the cells were washed with buffer, further incubated for 30 minutes at 37° C., and then observed under a microscope.

Figure 7(a) is a fluorescence micrograph. Excitation light with a wavelength of 488 nm was irradiated, and fluorescence with wavelengths of 500 to 540 nm was observed. The scale bar is 20 μm. Figure 7(b) is a composite image of a fluorescence micrograph and a differential interference contrast micrograph taken in the same field of view as Figure 7(a). Figure 7(c) is a graph plotting fluorescence intensity along the straight line shown in Figure 7(b).

As a result, it became clear that it was difficult to specifically stain only the Golgi apparatus using BODIPY FL C5-Ceramide.

<Golgi Apparatus Staining with BODIPY FL C5-Ceramide 2>
HeLa cells expressing mCherry-Giantin, a fusion protein of the fluorescent protein mCherry and the Golgi apparatus marker protein Giantin, were prepared. A complex of BODIPY FL C5-Ceramide (product number "D3521", Thermo Fisher Scientific) and BSA was added to the cell culture medium to a final concentration of 5 μM, and the cells were incubated at 4°C for 30 minutes. The cells were then washed with buffer, further incubated at 37°C for 30 minutes, and then observed under a microscope.

Figure 8(a) is a fluorescence microscope photograph. Excitation light with a wavelength of 488 nm was irradiated, and fluorescence with wavelengths of 500 to 540 nm was observed. The scale bar is 20 μm. Figure 8(b) is a fluorescence microscope photograph of mCherry fluorescence observed in the same field of view as Figure 8(a). Figure 8(c) is a composite image of Figures 8(a) and 8(b).

The results revealed that the fluorescence of BODIPY FL C5-Ceramide did not match that of mCherry. This result further supports the finding that it is difficult to specifically stain the Golgi apparatus using BODIPY FL C5-Ceramide.

[Experimental Example 5]
(Examination of localization using mg ^D c(3Me)TMP)
A localization agent in which the cysteine residue of mgc(3Me)TMP was substituted with the D-isomer (hereinafter, sometimes referred to as " ^mgDc (3Me)TMP") was synthesized, and its ability to localize tagged proteins was examined.

Synthesis of mg ^D c(3Me)TMP
First, mg ^D c(3Me)TMP was synthesized according to the following scheme (7).

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

^1H NMR (400MHz, _CD3 OD): δ7.22 (1H, s), 6.56 (2H, s), 5.61-5.39 (1H, m), 4.41 (1H, m), 4.03 (2H, s), 3.9 9 (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).

<<Study on localization using ^mgDc (3Me)TMP>>
mg ^Dc (3Me)TMP was added to the medium of DG-expressing cells to a final concentration of 10 μM, and EGFP fluorescence was observed using a confocal laser microscope.

Figures 9(a) to (d) are confocal laser microscope photographs. 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. Figure 9(b) is a photograph of DG-expressing cells taken 5 minutes after the addition of mg ^{D c} (3Me)TMP. Figure 9(c) is a photograph of DG-expressing cells taken 30 minutes after the addition of mg D c(3Me)TMP. Figure 9(d) is a photograph of DG-expressing cells taken 60 minutes after the addition of mg ^D c(3Me)TMP.

The results showed that EGFP localized to the Golgi apparatus 5 minutes after the addition of mg ^D c(3Me)TMP, and that this localization was maintained even 60 minutes after the addition of mg ^D c(3Me)TMP.

[Experimental Example 6]
(Study on the structure of the myristoyl group moiety of mgc(3Me)TMP)
The number of carbon atoms in the myristoyl group moiety of mgc(3Me)TMP was modified to examine the localization ability of tagged proteins. First, a compound represented by the following formula (27) in which the group represented by R ² is the octyl group shown in the following formula (28) (hereinafter, this may be referred to as "cap-gc(3Me)TMP"), and a compound represented by the following formula (27) in which the group represented by R ² is the nonyl group shown in the following formula (29) (hereinafter, this may be referred to as "pel-gc(3Me)TMP") were synthesized.

[In formula (27), ^R2 represents a saturated or unsaturated linear hydrocarbon group having 9 or more carbon atoms.]

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) of Experimental Example 1 was carried out, except that capric acid was used instead of myristic acid. Subsequently, the product was purified by reverse-phase HPLC to obtain cap-gc(3Me)TMP as a white solid.

^1H NMR (400MHz, _CD3 OD): δ7.22 (1H, s), 6.56 (2H, s), 5.61-5.39 (1H, m), 4.41 (1H, m), 4.03 (2H, s), 4.0 0 (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).

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

^1H NMR (400MHz, _CD3 OD): δ7.22 (1H, s), 6.56 (2H, s), 5.61-5.39 (1H, m), 4.41 (1H, m), 4.03 (2H, s), 4.0 0 (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).

<<Study on localization using cap-gc(3Me)TMP and pel-gc(3Me)TMP>>
Cap-gc(3Me)TMP was added to the culture medium of DG-expressing cells to a final concentration of 30 μM, and EGFP fluorescence was observed using a confocal laser microscope. Also, pel-gc(3Me)TMP was added to the culture medium of DG-expressing cells to a final concentration of 60 μM, and EGFP fluorescence was observed using a confocal laser microscope.

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

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 was revealed that localization of EGFP to the Golgi apparatus was significantly reduced. From these results, it was considered that in order to localize the compound represented by formula (25) above to the Golgi apparatus, the group represented by R2 must have ⁹ or more carbon atoms.

[Experimental Example 7]
(Examination of localization using mgc(3Me)SLF ^* )
A fusion protein of a target protein and a tag protein was expressed in HeLa cells. EGFP was used as the target protein, and FKBP (F36V) was used as the tag protein. FKBP (F36V) was positioned at the N-terminus of the fusion protein, and EGFP was positioned at the C-terminus. 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.

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 a compound represented by compound X2 (SLF ^* -COOH) was used instead of compound X1. The product was then purified by reverse-phase HPLC to obtain mgc(3Me)SLF ^* as a white solid.

^1H NMR (400MHz, _CD3OD ): δ7.19 (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).

<<Study on localization using mgc(3Me)SLF ^* >>
Mgc(3Me)SLF ^* was added to the culture 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, EGFP fluorescence was observed using a confocal laser microscope.

Figures 11(a) to (d) are confocal laser scanning 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 ^* . Figure 11(b) is a photograph of FKBP(F36V)-EGFP-expressing cells taken 5 minutes after the addition of mgc(3Me)SLF ^* . Figure 11(c) is a photograph of FKBP(F36V)-EGFP-expressing cells taken 30 minutes after the addition of mgc(3Me)SLF ^* . Figure 11(d) is a photograph of FKBP(F36V)-EGFP-expressing cells taken 60 minutes after the addition of mgc(3Me)SLF ^* .

The results showed that EGFP was localized to the Golgi apparatus 5 minutes after the addition of mgc(3Me)SLF ^* , and that the Golgi localization of EGFP was maintained even 60 minutes after the addition of mgc(3Me)SLF ^* .

These results demonstrate that tagged proteins can be localized even when the combination of tagged protein and group functioning as a ligand is other than the combination of eDHFR and TMP.

[Experimental Example 8]
(Control of intracellular signaling using mgc(3Me)TMP 1)
Intracellular signaling was controlled using mgc(3Me)TMP. First, an expression vector for a fusion protein of a target protein and a tag protein was prepared. Red fluorescent protein (RFP) and RasGEF were used as the target proteins. eDHFR was used as the tag protein. RFP, eDHFR, and RasGEF were arranged in this order from the N-terminus 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.

For comparison, an expression vector for a fusion protein in which RFP and eDHFR were arranged in that order from the N-terminus 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.

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 also prepared. The amino acid sequence of BFP-RBD is shown in SEQ ID NO: 6.

An expression vector for the EGFP and NRas fusion protein (hereinafter sometimes referred to as "EGFP-NRas") was also prepared. The amino acid sequence of EGFP-NRas is shown in SEQ ID NO: 7.

Next, HeLa cells expressing RD-RasGEF, EGFP-NRas, and BFP-RBD were generated. For comparison, HeLa cells expressing RD, EGFP-NRas, and BFP-RBD were also generated.

Next, mgc(3Me)TMP was added to the culture medium of each cell to a final concentration of 2.5 μM. The RFP, BFP, and EGFP fluorescence were then observed using a confocal laser microscope.

Figure 12 is a schematic diagram illustrating this experimental example. As shown in Figure 12, NRas is localized to the cell membrane and Golgi apparatus. BFP-RBD is localized to the nucleus and cytoplasm. Adding mgc(3Me)TMP to the culture medium localizes RasGEF to the Golgi apparatus, activating EGFP-NRas localized to the Golgi apparatus. As a result, BFP-RBD binds to the activated NRas and translocates to the Golgi apparatus.

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

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 that the photograph was taken before the addition of mgc(3Me)TMP, and "+mgcTMP_3Me" indicates that the photograph was taken 30 minutes after the addition of mgc(3Me)TMP. In Figures 13(a) and (b), the scale bars indicate 20 μm.

Figure 14(a) is a graph showing the results of measuring the localization of RD-RasGEF to the Golgi apparatus and the percentage of BFP-RBD localized to the Golgi apparatus or nucleus over time in HeLa cells expressing RD-RasGEF, EGFP-NRas, and BFP-RBD (n=5). In Figure 14(a), the values on the graph represent the mean ± standard deviation. The vertical axis on the left represents the percentage of localization to the Golgi apparatus or nucleus, with higher values indicating a higher percentage of localization to the Golgi apparatus. The vertical axis on the right represents the percentage of localization to the Golgi apparatus, with higher values indicating specific localization to the Golgi apparatus. The horizontal axis represents time (minutes).

Figure 14(b) is a graph showing the results of measuring the localization of RD to the Golgi apparatus and the percentage of BFP-RBD localized to the Golgi apparatus or nucleus over time in HeLa cells expressing RD, EGFP-NRas, and BFP-RBD (n=5). In Figure 14(b), the values on the graph represent the mean ± standard deviation. The vertical axis on the left represents the percentage of localization to the Golgi apparatus or nucleus, with higher values indicating a higher percentage of localization to the Golgi apparatus. The vertical axis on the right represents the percentage of localization to the Golgi apparatus, with higher values indicating specific localization to the Golgi apparatus. The horizontal axis represents time (minutes).

The results showed that in HeLa cells expressing RD-RasGEF, EGFP-NRas, and BFP-RBD, RD-RasGEF localized to the Golgi apparatus and BFP-RBD localized to the Golgi apparatus after the addition of mgc(3Me)TMP.

On the other hand, in HeLa cells expressing RD, EGFP-NRas, and BFP-RBD, RD localized to the Golgi apparatus after the addition of mgc(3Me)TMP, but BFP-RBD did not localize to the Golgi apparatus.

These results demonstrate that mgc(3Me)TMP can be used to control intracellular signaling.

[Experimental Example 9]
(Control of intracellular signaling using mgc(3Me)TMP 2)
Intracellular signaling was 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 this order from the N-terminus 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.

For comparison, a fusion protein was also prepared containing, from the N-terminus, EGFP, eDHFR, and Sac1 lacking phosphatase activity (hereinafter sometimes referred to as "Sac1-dead"). Hereinafter, this fusion protein will be referred to as "GD-Sac1-dead." The amino acid sequence of GD-Sac1-dead is shown in SEQ ID NO: 9.

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

Next, GD-Sac1 and RFP-P4M-SidM were expressed in COS-7 cells, which are derived from African green monkey kidneys. For comparison, COS-7 cells expressing GD-Sac1-dead and RFP-P4M-SidM were also generated.

Next, mgc(3Me)TMP was added to the culture medium of each cell to a final concentration of 10 μM. EGFP fluorescence and RFP fluorescence were then observed using a confocal laser microscope.

Figure 15 is a schematic diagram illustrating this experimental example. As shown in Figure 15, SidM binds to phosphatidylinositol-4-monophosphate (PI4P) and is localized to the Golgi apparatus. When mgc(3Me)TMP is added to the culture medium to localize Sac1 to the Golgi apparatus, Sac1 dephosphorylates PI4P, resulting in the translocation of SidM from the Golgi apparatus to the cytoplasm.

By observing EGFP fluorescence, the localization of GD-Sac1 can be detected. Furthermore, by observing RFP fluorescence, the localization of RFP-P4M-SidM can be detected.

Figure 16(a) is a fluorescence micrograph of COS-7 cells expressing GD-Sac1 and RFP-P4M-SidM. 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 that the photograph was taken before the addition of mgc(3Me)TMP, and "+mgcTMP_3Me" indicates that the photograph was taken 30 minutes after the addition of mgc(3Me)TMP.

Figure 17(a) is a graph showing the results of measuring the percentage of GD-Sac1 localized 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 percentage of RFP-P4M-SidM localized 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 on the graphs represent the mean ± standard deviation. The vertical axis indicates the degree of localization to the Golgi apparatus, with higher values indicating more specific localization to the Golgi apparatus. The horizontal axis indicates time (minutes).

Figure 17(c) is a graph showing the results of measuring the percentage of GD-Sac1 localized 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 percentage of RFP-P4M-SidM localized 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 shown on the graphs represent the mean ± standard deviation. The vertical axis indicates the degree of localization to the Golgi apparatus, with higher values indicating more specific localization to the Golgi apparatus. The horizontal axis indicates time (minutes).

The results showed that in COS-7 cells expressing GD-Sac1 and RFP-P4M-SidM, GD-Sac1 localized to the Golgi apparatus after the addition of mgc(3Me)TMP, and RFP-P4M-SidM translocated to the cytoplasm.

In contrast, in COS-7 cells expressing GD-Sac1-dead and RFP-P4M-SidM, GD-Sac1-dead localized to the Golgi apparatus after the addition of mgc(3Me)TMP, but RFP-P4M-SidM did not translocate to the cytoplasm.

These results further support the idea that mgc(3Me)TMP can be used to control intracellular signaling.

[Experimental Example 10]
(Synthesis of ^{mDcNVOC TMP} )
A caged localization agent was synthesized. Specifically, ^mDcNVOCTMP was synthesized, which is a compound in which a photolabile protecting group is further bonded to the trimethoprim group of ^mDcTMP . The ^NVOC group represented by the above formula (12) was used as the photolabile protecting group. The ^NVOC group can be deprotected by irradiation with light having a wavelength of 405 nm, for example. ^mDcNVOCTMP was synthesized according to the following scheme (11).

¹ H NMR (400MHz, 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).

[Experimental Example 11]
(Localization induction by light irradiation 1)
A fusion protein of a target protein and a tag protein was expressed in HeLa cells. The target protein was the fluorescent protein mScarlet I, and the tag protein was eDHFR. A K6 tag (a peptide tag consisting of six linked lysine residues) was placed at the N-terminus of eDHFR.

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.

Next, ^mDcNVOC TMP 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. Five minutes after the addition of ^mDcNVOC TMP, the cells were washed. For comparison, ^K6 -DS-expressing cells to which ^{mDcNVOC TMP} had not been added were also prepared. The cells were then irradiated with light at a wavelength of 405 nm for 2.6 ^seconds , and the fluorescence of mScarlet I was observed using a confocal laser microscope.

Figures 18(b) to (d) are confocal laser microscope photographs of K6-DS-expressing cells with the addition ^{of mDcNVOC} TMP. Figure 18(b) is a photograph of K6-DS-expressing cells before light irradiation. Figure 18(c) is a photograph of K6-DS-expressing cells 10 seconds after the start of the experiment. Figure 18(d) is a photograph of K6-DS-expressing cells 60 seconds after the start of the experiment. Figure 18(e) is a graph quantifying the experimental results. In Figure 18(e), the horizontal axis represents time (seconds) and the vertical axis represents the ^cytoplasmic fluorescence intensity ratio. "+ ^mDcNVOC TMP" indicates the results for ^K6 -DS-expressing cells ^with the addition of ^mDcNVOC TMP, and " ^-mDcNVOC TMP" indicates the results for K6-DS-expressing cells without the addition ^{of mDcNVOC} TMP.

As a result, it was revealed that in K6-DS expressing cells ^to which ^mDcNVOC TMP had been added, mScarlet I was localized in the cell membrane immediately after light irradiation.

These results demonstrate that by using a compound with a photolabile protecting group, it is possible to control the localization of tagged proteins by light irradiation.

[Experimental Example 12]
(Localization induction by light irradiation 2)
19 is a graph showing the results of measuring the absorption spectrum ^{of mDcNVOC} TMP. As shown in Fig. 19, it was revealed that ^mDcNVOC TMP absorbs light with a wavelength of ⁴⁰⁵ nm, but hardly absorbs light with a wavelength of 445 nm.

Next, K6-DS-expressing cells were exposed to various combinations ^{of mDcNVOC} TMP, 405 nm light irradiation, 445 nm light irradiation, and free trimethoprim (hereinafter sometimes referred to as "TMP"), and the fluorescence of mScarlet I was observed using a confocal laser microscope. Subsequently, the localization efficiency of the tagged protein was measured based on the confocal laser microscope images. ^mDcNVOC TMP was added to a final concentration of 10 μM, and cells were irradiated with 405 nm light for 2.6 seconds ^and with 445 nm light for 2.6 seconds. TMP was added to a final concentration of 100 μM.

20 is a graph showing ^the results of measuring the localization efficiency of tagged proteins. In FIG. 20, "-" indicates that ^mDcNVOC TMP or TMP was not added, or that irradiation with light at a wavelength of 405 nm or 445 nm was not performed, and "+" indicates that ^mDcNVOC TMP ^or TMP was added, or that irradiation with light at a wavelength of 405 nm or 445 nm was performed.

The vertical axis of the graph represents the localization efficiency calculated by the following formula (F1).
Localization efficiency = 1 - (fluorescence intensity in the cytoplasm 5 minutes after the start of the experiment / fluorescence intensity in the cytoplasm at the start of the experiment (0 minutes)) ... (F1)

The results showed that mScarlet I was localized on the cell membrane when ^mDcNVOC TMP was added and irradiated with light at a wavelength of ⁴⁰⁵ nm, and that the localization of mScarlet I on the cell membrane was suppressed in the presence of TMP.

[Experimental Example 13]
(Localization induction by light irradiation 3)
We investigated the induction of localization by light irradiation at the single-cell level. First, ^mDcNVOC TMP was added to the culture medium of K6- ^DS -expressing cells at a final concentration of 10 μM, and the cells were observed using a confocal laser microscope. Figure 21(a) shows a confocal laser microscope image at the start of the experiment. Next, using the confocal laser microscope function, only the cell labeled "Cell ¹ " in Figure 21(a) was irradiated with light at a wavelength of 405 nm for 2.6 seconds. Figure 21(a) shows a confocal laser microscope image 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 labeled "Cell ^1. "

Figure 21(c) is a confocal laser microscope image taken 600 seconds after the start of the experiment. Next, using the confocal laser microscope function, only the cell marked "Cell ² " in Figure 21(c) was irradiated with light of 405 nm wavelength for 2.6 seconds. Figure 21(d) is a confocal laser microscope image 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 cell marked "Cell ^2. "

Figure 21(e) is a confocal laser microscope image taken 1200 seconds after the start of the experiment. Next, using the confocal laser microscope function, only the cell labeled "Cell ³ " in Figure 21(e) was irradiated with 405 nm light for 2.6 seconds. Figure 21(f) is a confocal laser microscope image 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 cell labeled "Cell ^3. " Figure 21(f) also shows the position of the cell labeled "Cell ^4. "

Figure 21(g) shows a confocal laser scanning microscope image 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) shows a confocal laser scanning microscope image 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 eliminated in the cells indicated as "Cell ¹ ,""Cell ² ," and "Cell ^3. "

Figure 21(i) is a graph showing the numerical values of the changes in the cytoplasmic fluorescence intensity ratio for the cells indicated as "Cell ¹ ,""Cell ² ,""Cell ³ ," and "Cell ⁴ " in Figures 21(a) to 21(h). The horizontal axis indicates the time (seconds) from the start of the experiment.

[Experimental Example 14]
(Localization induction by light irradiation 4)
We investigated the control of cell motility signals by light irradiation. 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 this order from the N-terminus of the fusion protein. In addition, a K6 tag was further arranged on the N-terminus 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.

We also prepared a fusion protein of Lifeact and mNeonGreen (hereinafter sometimes referred to as "Lifeact-mNeonGreen"). The amino acid sequence of Lifeact-mNeonGreen is shown in SEQ ID NO: 13. Lifeact is a peptide that specifically binds to F-actin.

Next, K6-DS-Tiam1 and Lifeact-mNeonGreen were expressed in NIH3T3 cells, which are mouse embryonic fibroblasts. Subsequently, mDcNVOC TMP was added to the medium of the NIH3T3 cells expressing K6-DS-Tiam1 and Lifeact-mNeonGreen (hereinafter, sometimes referred to as "K6-DS-Tiam1 and Lifeact- ^mNeonGreen -expressing cells") to a final concentration of 10 μM. Subsequently, ⁵ minutes after the addition of ^mDcNVOC TMP ^, the cells were washed.

Then, the fluorescence of mScarlet I and mNeonGreen was observed using a confocal laser microscope. Figures 22(a) and (c) are confocal laser microscope images taken at the start of the experiment. Next, using the confocal laser microscope's functions, the area enclosed by the square in Figure 22(a), i.e., the entire cell, was irradiated with light with a wavelength of 405 nm for 2.6 seconds.

Figures 22(b) and (d) are confocal laser microscope photographs taken 300 seconds after the start of the experiment. Figure 22(e) is an image showing areas of increased or decreased area, comparing K6-DS-Tiam1 and Lifeact-mNeonGreen expressing cells at the start of the experiment with those at 300 seconds after the start of the experiment. In Figure 22(e), "increased" indicates areas that have increased in area compared to the start of the experiment, and "decreased" indicates areas that have decreased in area compared to the start of the experiment.

The results showed that irradiation with 405 nm light changed the area of K6-DS-Tiam1 and Lifeact-mNeonGreen-expressing cells. These results demonstrate that the use of compounds with photolabile protecting groups makes it possible to control cell motility signals through light irradiation.

[Experimental Example 15]
(Localization induction by light irradiation 5)
^mDcNVOC TMP was added to the culture medium of K6-DS-Tiam1 and ^Lifeact -mNeonGreen expressing cells to a final concentration of 10 μM.

Then, the fluorescence of mScarlet I and mNeonGreen was observed using a confocal laser microscope. Figures 23(a) and (c) are confocal laser microscope images taken at the start of the experiment. Next, using the confocal laser microscope's functions, the circled area in Figure 23(a), i.e., a portion of the cell, was locally irradiated with light at a wavelength of 405 nm for 2.6 seconds.

Figures 23(b) and (d) are confocal laser microscope photographs taken 300 seconds after the start of the experiment. Figure 23(e) is an image showing areas of increased or decreased area, comparing K6-DS-Tiam1 and Lifeact-mNeonGreen expressing cells at the start of the experiment with those at 300 seconds after the start of the experiment. In Figure 23(e), "increased" indicates areas that have increased in area compared to the start of the experiment, and "decreased" indicates areas that have decreased in area compared to the start of the experiment.

The results showed that irradiation with 405 nm light changed the area of K6-DS-Tiam1 and Lifeact-mNeonGreen-expressing cells. These results further support the idea that light irradiation can be used to control cell motility signals by using compounds with photolabile protecting groups.

[Experimental Example 16]
(Localization induction by light irradiation 6)
^mDcNVOC TMP was added to the medium of NIH3T3 cells expressing K6-DS-Tiam1 (hereinafter sometimes referred to as "K6-DS-Tiam1-expressing cells") to a final concentration of 10 μM. For comparison, ^mDcNVOC TMP was also added to the medium of NIH3T3 cells expressing K6- ^DS (hereinafter sometimes referred to as " ^K6 -DS-expressing cells") to a final concentration of 10 μM.

Then, the mScarlet I fluorescence was observed using a confocal laser microscope. Next, using the confocal laser microscope's functions, the entire cell or a portion of the cell was locally irradiated with 405 nm light for 2.6 seconds. The changes in cell morphology were then observed using the confocal laser microscope.

Figures 24(a) to (d) are images showing areas that have increased or decreased in area, comparing K6-DS-Tiam1-expressing cells or K6-DS-expressing cells at the start of the experiment with those at 300 seconds after the start of the experiment. In Figures 24(a) to (d), "increased" indicates areas that have increased in area compared to the start of the experiment, and "decreased" indicates areas that have decreased in area compared to the start of the experiment.

Figure 24(a) shows typical results obtained by irradiating the entire cell with 405 nm light, while Figures 24(b) to (d) show typical results obtained by irradiating a portion of the cell with 405 nm light. In Figures 24(a) to (d), "+m ^D c ^NVOC TMP" indicates the addition of m ^D c ^NVOC TMP, "-m ^D c ^NVOC TMP" indicates the absence of m ^D c ^NVOC TMP, "+K6-DS-Tiam1" indicates the results for K6-DS-Tiam1-expressing cells, and "+K6-DS" indicates the results for K6-DS-expressing cells. Figures 24(c) and (d) show the results for the negative control. Furthermore, the circled areas in Figures 24(b) to (d) indicate the areas irradiated with 405 nm light.

25(a) is a graph showing the increase in the area of the protruding region of the cell (region with increased area), and FIG. 25(b) is a graph showing the decrease in the area of the retracted region of the cell (region with decreased area). In FIGS. 25(a) and 25(b), "whole" indicates the result of irradiating the whole cell with light at a wavelength of 405 nm, "local" indicates the result of irradiating a part of the cell locally with light at a wavelength of ⁴⁰⁵ nm, "+" indicates the result of adding ^mDcNVOCTMP , the result of K6-DS-Tiam1-expressing cells, or the result of K6-DS-expressing cells, and "-" indicates the result ^of not adding ^mDcNVOCTMP , the result of not using K6-DS-Tiam1-expressing cells, or the result of not using K6-DS-expressing cells.

The results showed that irradiation with 405 nm light changed the area of K6-DS-Tiam1-expressing cells. These results further support the idea that the use of compounds with photolabile protecting groups makes it possible to control cell motility signals through light irradiation.

[Experimental Example 17]
⁽ Synthesis of ^mDcDEACM TMP)
A caged localization agent was synthesized that has a different wavelength for deprotecting the ^photolabile protecting group from that of ^mDcNVOCTMP . Specifically, ^mDcDEACMTMP , a compound in which a photolabile protecting group is further bonded to the trimethoprim group of ^mDcTMP , was synthesized according to the following scheme (12). The ^DEACM group represented by the above formula (15) was used as the photolabile protecting group. The DEACM group can be deprotected by irradiation with light at a wavelength of 445 nm, for example.

¹ H NMR (400MHz, d-DMSO): δ0.85 (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).

26 is a graph showing the results of measuring the absorption spectra of ^mDcNVOC TMP and ^{mDcDEACM TMP} . As shown in Fig. 26, it was revealed ^{that mDcNVOC} TMP does not ^absorb light with a wavelength of 445 nm, but ^mDcDEACM TMP can absorb light with a wavelength ^of 445 nm.

[Experimental Example 18]
(Light-induced localization 7)
^mDcDEACM TMP was added to the culture medium of HeLa cells expressing K6-DS (hereinafter sometimes referred to as " ^{K6-DS-expressing cells") to a final concentration of 10 μM. Five minutes after the addition of mDcDEACM TMP ,} the cells were washed. The cells were then irradiated with light at a wavelength of 445 nm for 2.6 seconds, and the fluorescence of mScarlet I was observed using a confocal laser microscope.

27(a) and (b) are confocal laser microscope photographs of K6-DS expressing cells ^to which ^mDcDEACM 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.

As a result, it was revealed that in K6-DS expressing cells ^to which ^mDcDEACM TMP had been added, localization of the tagged protein could be induced by irradiation with light having a wavelength of 445 nm.

FIG. 27(c ^{) is a graph showing the results of measuring the localization efficiency of tagged proteins after irradiating K6-DS expressing cells containing mDcNVOC TMP} with light at a wavelength of 405 nm or 445 nm.

FIG. 27(d) is a graph showing the results of measuring the localization efficiency of tagged proteins after irradiating K6-DS expressing cells containing ^mDcDEACM TMP with light at a wavelength ^of 405 nm or 445 nm.

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

As a result, it was revealed ^that in K6-DS expressing cells supplemented with ^mDcNVOC TMP, the tagged protein localized to the cell membrane upon irradiation with 405 nm light, but that the localization of the tagged protein remained almost unchanged upon irradiation with 445 nm light.On the other hand, it was revealed that in K6-DS expressing cells supplemented with ^mDcDEACM TMP, the tagged protein localized to the cell membrane upon irradiation with both 405 nm and 445 ^nm light.

These results demonstrate that it is possible to control the induction of localization of tagged proteins by using compounds with photolabile protecting groups that have different absorption wavelengths and controlling the wavelength of the irradiated light.

The present invention provides compounds that specifically localize in the Golgi apparatus.

Claims (25)

A compound represented by the following formula (1):
[In formula (1), Z represents a group represented by the following formula (2) or (3); L represents a divalent hydrocarbon group having 1 to 50 carbon atoms which may be interrupted by substitution of —CH ₂ — with —O—, —CO—, —NH— or a group represented by the following formula (4)]; R ¹ represents a group derived from a labeling substance or a group derived from a drug, a group which functions as a ligand for a tagged protein, or a group in which a photodegradable protecting group is further bonded to a group which functions as a ligand for the tagged protein; the labeling substance is a fluorescent substance, Raman tag, peptide tag, or biotin; the drug is an anticancer drug or a vesicle transport inhibitor; in the following formulas (2) and (3), R ² represents a saturated or unsaturated linear hydrocarbon group having 9 or more carbon atoms; R ³ each independently represents a hydrogen atom or a methyl group; at least one of the multiple R ^3s is a methyl group; and * indicates an asymmetric carbon atom.]
］ The compound according to claim 1, wherein the combination of the tag protein and the group that functions as a ligand for the tag protein is a combination of Escherichia coli dihydrofolate reductase (eDHFR) protein and a group represented by the following formula (5), a combination of FK506 binding protein and a group represented by the following formula (6), a combination of carbonic anhydrase I protein or carbonic anhydrase II protein and a group represented by the following formula (7), a combination of SNAP-tag (registered trademark) protein and a group represented by the following formula (8) or (9), a combination of HaloTag (registered trademark) protein and a group represented by the following formula (10), or a combination of photoactive yellow protein and a group represented by the following formula (11).
The compound according to claim 1 or 2, wherein the photodegradable protecting group is a group represented by any one of the following formulas (12) to (20):
A Golgi apparatus labeling agent comprising the compound according to claim 1, wherein R ¹ is a group derived from a labeling substance. A drug delivery system targeted to the Golgi apparatus, comprising the compound of claim 1, wherein R ¹ is a group derived from a drug. An agent for localizing a tagged protein to the Golgi apparatus, comprising the compound according to any one of claims 1 to 3, wherein R ¹ is a group that functions as a ligand for the tagged protein. 1. A method for controlling the localization of a protein of interest in a cell in vitro, comprising :
a step (a) of introducing into the cell the localization agent according to claim 6, wherein ^R1 is a group that functions as a ligand for the tagged protein, so that the group that functions as a ligand of the localization agent binds to the tagged protein, and the tagged protein is localized to the Golgi apparatus together with the target protein;
The method, wherein the cell is a cell that expresses a fusion protein of the target protein and the tag protein. The method further comprises, after the step (a), a step (b) of introducing a ligand for the tagged protein into the cell,
The method of claim 7, wherein in step (b), the group functioning as a ligand competes with the ligand, and the ligand displaces the group functioning as a ligand in at least a portion of the tagged protein localized in the Golgi apparatus, resulting in the target protein being translocated from the Golgi apparatus. 1. A method for controlling the localization of a protein of interest in a cell in vitro, comprising :
a step (a1) of introducing into the cell the localization agent according to claim 6, wherein ^R1 is a group in which a photolabile protecting group is further bound to a group that functions as a ligand for the tagged protein;
and step (a2) of irradiating the cell with light after step (a1), thereby causing the photolabile protecting group to be cleaved from the localization agent, causing the group of the localization agent that functions as a ligand to bind to the tagged protein, and causing the tagged protein to localize in the Golgi apparatus together with the target protein,
The method, wherein the cell is a cell that expresses a fusion protein of the target protein and the tag protein. The method further comprises, after the step (a2), a step (b) of introducing a ligand for the tagged protein into the cell,
10. The method of claim 9, wherein in step (b), the group functioning as a ligand competes with the ligand, and the ligand displaces the group functioning as a ligand in at least a portion of the tagged protein localized in the Golgi apparatus, resulting in the target protein being translocated from the Golgi apparatus. The method of any one of claims 7 to 10, wherein the target protein is a signal transduction protein. A kit for controlling the localization of a tagged protein in a cell, comprising the localization agent of claim 6. a cell expressing a fusion protein of the target protein and the tag protein;
an expression vector for a fusion protein of the target protein and the tag protein, or
The kit according to claim 12 , further comprising a vector for producing a fusion protein between the target protein and the tag protein. The kit described in claim 12 or 13, further comprising a ligand for the tagged protein. A compound represented by the following formula (1):
[In formula (1), Z represents a group represented by formula (2) or (3) below; L represents a divalent hydrocarbon group having 1 to 50 carbon atoms which may be interrupted by substitution of —CH ₂ — with —O—, —CO—, —NH— or a group represented by formula (4) below; R 1 represents a group which functions as a ligand for a protein or a group in which a photodegradable protecting group is further bonded to the group which functions as a ligand for a protein; in formulas (2) and (3) below, R ² represents a saturated or unsaturated linear hydrocarbon group having 9 or more carbon atoms; R ^{3 each} independently represents a hydrogen atom or a methyl group, at least one of the multiple R ^3s is a methyl group; and * indicates an asymmetric carbon atom.]
］ The compound according to claim 15, wherein the combination of the protein and the group that functions as a ligand for the protein is a combination of Escherichia coli dihydrofolate reductase (eDHFR) protein and a group represented by the following formula (5), a combination of FK506 binding protein and a group represented by the following formula (6), a combination of carbonic anhydrase I protein or carbonic anhydrase II protein and a group represented by the following formula (7), a combination of SNAP-tag (registered trademark) protein and a group represented by the following formula (8) or (9), a combination of HaloTag (registered trademark) protein and a group represented by the following formula (10), or a combination of photoactive yellow protein and a group represented by the following formula (11).
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):
An agent for localizing a protein to the Golgi apparatus, comprising the compound described in any one of claims 15 to 17. 1. A method for controlling protein localization in a cell in vitro, comprising:
A method comprising the step (a) of introducing into the cell the localization agent described in claim 18, wherein ^R1 is a group that functions as a ligand for the protein, so that the group that functions as a ligand of the localization agent binds to the protein and the protein is localized to the Golgi apparatus. The method further comprises, after the step (a), a step (b) of introducing a ligand for the protein into the cell,
20. The method of claim 19, wherein in step (b), the group that functions as a ligand competes with the ligand, and the ligand displaces the group that functions as a ligand in at least a portion of the protein localized in the Golgi apparatus, resulting in the protein being translocated from the Golgi apparatus. 1. A method for controlling protein localization in a cell in vitro, comprising:
(a1) introducing into the cell the localization agent according to claim 18, wherein ^R1 is a group in which a photolabile protecting group is further bonded to a group that functions as a ligand for the protein;
and step (a2) of irradiating the cell with light after step (a1), thereby causing the photolabile protecting group to be cleaved from the localization agent, causing the group of the localization agent that functions as a ligand to bind to the protein, and localizing the protein to the Golgi apparatus. The method further comprises, after the step (a2), a step (b) of introducing a ligand for the protein into the cell,
22. The method of claim 21, wherein in step (b), the group that functions as a ligand competes with the ligand, displacing the group that functions as a ligand in at least a portion of the protein localized in the Golgi apparatus, resulting in the protein being translocated from the Golgi apparatus. The method of any one of claims 19 to 22, wherein the protein is a signal transduction protein. A kit for controlling protein localization in cells, comprising the localization agent of claim 18. The kit of claim 24, further comprising a ligand for the protein.