Proximal Dependency Labeling Method

Abstract

This method enables highly efficient proximal-dependent labeling using biotin. [Solution] A method for proximal-dependent labeling of a biotin ligase fusion protein using the first biotin derivative, comprising the following steps (A) and (B): (A) introducing a first biotin derivative having a photodegradable protecting group into a cell containing a biotin ligase fusion protein; and (B) irradiating the cell with light to carry out a deprotection reaction of the first biotin derivative to obtain biotin that binds to the biotin ligase fusion protein from the first biotin derivative.

Description

Technical Field

[0001] The present invention relates to a method for proximally dependent labeling of a neighboring protein of a biotin ligase fusion protein using a biotin derivative having a photocleavable protecting group.

Background Art

[0002] Basic research has been conducted to understand at the molecular level as much as possible how cells control the intracellular transport of proteins. In recent years, a proximally dependent labeling method has been developed and has come to be used as a means for analyzing the intracellular transport of proteins. Proximally dependent labeling is a technique that enables labeling of proteins in the vicinity of a target protein, and a method of labeling proteins by biotinylation is known as an example. For example, Non-Patent Document 1 discloses the biotinylation of proteins using TurboID as a biotin ligase.

[0003] In addition, as a method enabling the time-course observation of the intracellular transport of proteins, the retention using selective hooks method (RUSH method) is known (Non-Patent Documents 2 and 3). The RUSH method is a method that enables the time-course observation of the intracellular transport of a target protein by the following steps (i) and (ii). (i) Retain the target protein in the endoplasmic reticulum by a hook. (ii) Then, by adding biotin into the endoplasmic reticulum, release the target protein from the hook and initiate the transport of the target protein.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

[0005] When performing proximity-dependent labeling using biotin, although biotin is present in principle in all living cells, its amount is extremely small, so biotin may be supplemented from outside the cells. However, the cell membrane permeability of biotin is low. Therefore, the rate at which biotin enters the cells is slow. Thus, there was room for improvement from the viewpoint of efficiency in the conventional proximity-dependent labeling method using biotin.

[0006] One aspect of the present invention aims to realize a proximity-dependent labeling method using biotin with excellent efficiency. [Means for Solving the Problems]

[0007] The inventors diligently conducted research to solve the above problems. As a result, they found that esterifying the carboxyl group of biotin improves cell membrane permeability compared to biotin. In fact, they confirmed that when biotin is esterified with methanol to form biotin methyl ester, cell membrane permeability is improved compared to biotin.

[0008] On the other hand, when interacting with biotin ligase within cells, it is preferable that biotin not be esterified, from the viewpoint of controlling the timing of the interaction. Therefore, after further investigation by the inventors, we found that both (i) and (ii) below can be achieved by esterifying biotin with a photodegradable protecting group. (i) Improve cell membrane permeability. (ii) Since esterified biotin can be deesterified by light irradiation at any desired timing, the timing of interaction with biotin ligase can be controlled.

[0009] The present invention includes the following embodiments.

[0010] <1> The following steps (A) and (B) (A) A step of introducing a first biotin derivative having a photodegradable protecting group into cells containing a biotin ligase fusion protein. (B) A step of irradiating the cells with light to carry out a deprotection reaction of the first biotin derivative, thereby obtaining biotin from the first biotin derivative that binds to the biotin ligase fusion protein. A method for proximal-dependent labeling of a biotin ligase fusion protein using the first biotin derivative comprising the first biotin derivative.

[0011] <2> The aforementioned cells further contain endoplasmic reticulum-localized proteins having a biotin-binding site, The biotin ligase fusion protein has a binding sequence with the biotin binding site, The process further includes introducing into the cell a second biotin derivative having a protecting group different from the photodegradable protecting group of the first biotin derivative and exhibiting the ability to bind to the biotin binding site of the endoplasmic reticulum-localized protein, prior to step (B) above. <1> Methods used.

[0012] <3> The aforementioned photodegradable protecting group is a group derived from a compound represented by any one of the following structural formulas 1 to 3. <1> or <2> Methods used.

[0013] [ka]

[0014] <4> In step (B) above, the wavelength of the irradiated light is 300 nm to 400 nm. <1> ~ <3> The method described in any one of the following ways. [Effects of the Invention]

[0015] According to one aspect of the present invention, a biotin-based proximal-dependent labeling method that is highly efficient is provided. [Brief explanation of the drawing]

[0016] [Figure 1] This shows the experimental setup for the deprotection group reaction in Example 1 of this application. [Figure 2] The results of tracking the deprotection group reaction in Example 1 of this application using 1H NMR spectroscopy are shown. [Figure 3] In the synchronized transport initiation experiment using the RUSH method in Reference Example 1 of this application, the time at which cargo proteins accumulate most in the Golgi apparatus in each cell is shown. [Figure 4] In the synchronized transport initiation experiment using the RUSH method described in Reference Example 1 of this application, the localization of the cargo protein after a specific time has elapsed since the initiation of transport is shown. [Figure 5] The results of detecting protein biotinylation and TurboID fusion proteins in Reference Example 2 of this application are shown. [Figure 6] Reference Example 2 of this application shows the results of measuring the amount of biotin introduced per TurboID fusion protein. [Figure 7] Reference Example 3 of this application shows the results of detecting TurboID fusion proteins and biotinylated proteins using fluorescent proteins and fluorescently labeled streptavidin. [Figure 8] Reference Example 3 of this application shows the results of measuring the amount of biotin introduced per TurboID fusion protein. [Figure 9] The results of detecting protein biotinylation and TurboID fusion proteins in Reference Example 4 of this application are shown. [Figure 10] Reference Example 4 of this application shows the results of measuring the amount of biotin introduced per TurboID fusion protein. [Figure 11] In the synchronized transport initiation experiment using the RUSH method in Reference Example 5 of this application, the percentage of cells in which the cargo protein began to move after a specific time period following the addition of biotin, biotin methyl ester, or biotin tert-butyl ester is shown. [Figure 12] In the synchronized transport initiation experiment using the RUSH method described in Reference Example 6 of this application, the localization of the cargo protein before the administration of desthiobiotin methyl ester and 4 minutes after administration is shown. [Figure 13] Reference Example 6 of this application shows the results of detecting protein biotinylation and TurboID fusion proteins. [Figure 14] Reference Example 6 of this application shows the results of measuring the amount of biotin introduced per TurboID fusion protein. [Figure 15] In Example 2 of this application, we observed whether biotinylation of proximal proteins by TurboID was induced in a light-irradiation-dependent manner when a derivative having a photodegradable protecting group was used. The results are shown below. [Figure 16]In Example 3 of this application, the localization of the cargo protein after a specific time has elapsed since the start of transport is shown in a synchronized transport initiation experiment using the RUSH method with a derivative having a photodegradable protecting group. [Modes for carrying out the invention]

[0017] The following describes in detail some examples of embodiments of the present invention. However, the present invention is not limited to the embodiments described below, and various modifications may be made within the scope of the claims. Embodiments that combine the technical means described in different embodiments are also included in the technical scope of the present invention. Unless otherwise specified in this specification, "A to B" representing a numerical range means "greater than or equal to A and less than or equal to B".

[0018] [Proximal Dependence Labeling Method] A method for proximal-dependent labeling of neighboring proteins of a biotin ligase fusion protein according to one aspect of the present invention (also referred to herein simply as the "proximal-dependent labeling method") comprises the following steps (A) and (B) (A) A step of introducing a first biotin derivative having a photodegradable protecting group into cells containing a biotin ligase fusion protein. (B) A step of irradiating the cells with light to carry out a deprotection reaction of the first biotin derivative, thereby obtaining biotin from the first biotin derivative that binds to the biotin ligase fusion protein. The method comprises the first biotin derivative and involves proximal-dependent labeling of neighboring proteins of the biotin ligase fusion protein.

[0019] The first biotin derivative has a photodegradable protecting group. "Having a photodegradable protecting group" means that the carboxyl group of biotin is esterified with a photodegradable protecting group. A photodegradable protecting group is a substituent that can undergo a deprotection reaction of the photodegradable protecting group by irradiating the first biotin derivative with light of a specific wavelength.

[0020] The photodegradable protecting group is not particularly limited, but examples include a group derived from a compound represented by any one of the following structural formulas 1 to 5. From the viewpoint of thermal stability and irradiation wavelength characteristics, the photodegradable protecting group is preferably a group derived from a compound represented by any one of the following structural formulas 1 to 3, and more preferably a group derived from a compound represented by the following structural formula 3.

[0021] [ka]

[0022] The first biotin derivative may be a commercially available product or may be prepared by synthesis. For specific synthesis methods, please refer to the production examples in this invention.

[0023] A biotin ligase fusion protein is a fusion protein of a biotin ligase and any other protein. The biotin ligase is not particularly limited, but examples include TurboID, miniTurbo, BioID, BioID2, and AirID. From the viewpoint of rapidly biotinylating proteins, TurboID is preferred as the biotin ligase.

[0024] According to a proximal-dependent labeling method according to one embodiment of the present invention, neighboring proteins located several nanometers away from a biotin ligase fusion protein can be biotinylated. That is, neighboring proteins of any protein to be fused with biotin ligase can be comprehensively identified within the cell. The biotin ligase fusion protein may be labeled with a fluorescent substance or the like.

[0025] The biotin ligase fusion protein may be a commercially available product, or it may be expressed in cells using, for example, an expression vector. The method for expressing the expression vector in cells is not particularly limited, and conventional methods for introducing known vectors into cells can be employed. The method for introducing the vector into cells is not particularly limited, and any known genetic engineering technique can be used. For example, lipofection, microinjection, electroporation, etc., can be used.

[0026] The cell type into which the first biotin derivative is introduced is not particularly limited, but may be established cell lines such as HeLa cells, HEK293, and PRE-1, or it may be unestablished cells such as primary cultured cells. The proximal-dependent labeling method according to one embodiment of the present invention may further include a step of introducing an expression vector for a biotin ligase fusion protein into these cells. Furthermore, the proximal-dependent labeling method may be carried out using a cell line that has already expressed a biotin ligase fusion protein.

[0027] The method for introducing the first biotin derivative into cells containing a biotin ligase fusion protein is not particularly limited. For example, the first biotin derivative can be introduced into the culture medium of cells expressing a biotin ligase fusion protein, and the cells and the first biotin derivative can be brought into contact. Alternatively, the cells expressing the biotin ligase fusion protein and the first biotin derivative may be left to stand or mixed by inversion for a predetermined time. The predetermined time is not particularly limited, but may be, for example, 1 minute or more, and preferably 5 minutes or more.

[0028] In step (B), the "deprotection reaction" refers to a reaction in which the photodegradable protecting group is removed from the first biotin derivative by irradiating it with light of a specific wavelength. The wavelength of the light irradiated when carrying out the deprotection reaction of the first biotin derivative is not particularly limited and may be set appropriately depending on the type of photodegradable protecting group contained in the first biotin derivative. For example, the lower limit of the wavelength of the irradiated light may be 300 nm or more, preferably 320 nm or more, and more preferably 340 nm or more. The upper limit of the wavelength of the irradiated light may be 400 nm or less, preferably 390 nm or less, and more preferably 380 nm or less.

[0029] In step (B), there are no particular limitations on the lower limit of the duration of light irradiation, but it may be, for example, 1 second or more, preferably 5 seconds or more, and more preferably 10 seconds or more. The upper limit of the duration of light irradiation may be 600 seconds or less, preferably 100 seconds or less, and more preferably 50 seconds or less.

[0030] According to a proximal-dependent labeling method according to one embodiment of the present invention, a first biotin derivative having a photodegradable protecting group is used in step (A). The inventors have found that biotin derivatives obtained by esterifying the carboxyl group of biotin and attaching a protecting group have superior membrane permeability compared to biotin. Therefore, because the first biotin derivative has superior membrane permeability compared to biotin, the first biotin derivative can be rapidly introduced into cells.

[0031] Furthermore, in step (B), the timing of obtaining biotin can be controlled by initiating the deprotection reaction of the first biotin derivative through light irradiation at an arbitrary timing. As a result, the timing of the interaction between biotin and the biotin ligase fusion protein can be controlled, enabling efficient proximal-dependent labeling of proteins near the biotin ligase fusion protein.

[0032] [Combination of proximal-dependent labeling method and RUSH method] In a proximal-dependent labeling method according to one embodiment of the present invention, a retention method using selective hooks (also referred to as the "RUSH method" in this specification) may be performed before proximal-dependent labeling, for example, before step (B). The RUSH method is a method that enables the observation of intracellular transport of the target protein over time.

[0033] When performing the RUSH method, cells containing biotin ligase fusion proteins may further contain endoplasmic reticulum-localized proteins that have a biotin-binding site. Furthermore, the biotin ligase fusion protein may also have a binding sequence for the biotin-binding site. Here, "endoplasmic reticulum-localized protein" refers to a protein localized in the endoplasmic reticulum. The endoplasmic reticulum-localized protein exists bound to the biotin ligase fusion protein within the endoplasmic reticulum via the binding of its biotin-binding site to a binding sequence for the biotin-binding site.

[0034] The biotin binding site may be a protein that binds to biotin, such as avidin, streptavidin, and neutraavidin, or modified proteins thereof. The biotin ligase fusion protein may be a fusion protein containing at least a portion of such avidin, streptavidin, and neutraavidin. The binding sequence to the biotin binding site is a peptide having an amino acid sequence that recognizes such a binding site to the biotin binding site, such as SBP (streptavidin-binding peptide).

[0035] Furthermore, the proximal-dependent labeling method according to one embodiment of the present invention may further include a step of introducing a second biotin derivative that exhibits the ability to bind to the biotin binding site of the endoplasmic reticulum-localized protein into the cell before step (B). The second biotin derivative is a biotin derivative that has the ability to bind to the biotin binding site but does not have the ability to bind to the biotin ligase fusion protein. Thus, the second biotin derivative has the function of initiating transport by the RUSH method, but does not cause proximal-dependent labeling in the derivative state.

[0036] The second biotin derivative may be the same biotin derivative as the first biotin derivative, having the same photodegradable protecting group as the first biotin derivative, as long as the above conditions are met, and may have a protecting group different from the photodegradable protecting group of the first biotin derivative.

[0037] The protecting group of the second biotin derivative may be any of the photodegradable protecting groups described above in the description of the first biotin derivative, or it may be any other protecting group. Other protecting groups include, for example, linear or branched alkyl groups having two or more carbon atoms. An example of such alkyl groups is a tert-butyl group. A second biotin derivative having such other protecting groups has the ability to bind to a biotin binding site, but not to a biotin ligase fusion protein.

[0038] Furthermore, examples of secondary biotin derivatives include compounds in which a protecting group is added to desthiobiotin. Desthiobiotin is a compound obtained by removing a sulfur atom from a biotin molecule and is a type of biotin derivative. An example of desthiobiotin with a protecting group is desthiobiotin methyl ester (DBME), which has a methyl group as the protecting group. Such secondary biotin derivatives also have the ability to bind to biotin binding sites, but do not have the ability to bind to biotin ligase fusion proteins.

[0039] The method for introducing the second biotin derivative into cells is not particularly limited, and the method described above as an example for introducing the first biotin derivative into cells can be used as appropriate. Furthermore, if the first biotin derivative and the second biotin derivative are the same biotin derivative, the step of introducing the second biotin derivative can be considered to be performed simultaneously with the step of introducing the first biotin derivative into cells.

[0040] An example of the RUSH method procedure is described below. (1) Express a hook containing an endoplasmic reticulum localization signal and a biotin-binding site, along with a cargo protein having a binding sequence for the biotin-binding site, within the cell. Specifically, introduce expression vectors for the hook and the cargo protein into the cell, respectively, to express the hook and the cargo protein within the cell. At this time, marker proteins such as trans-Golgi markers and Golgi markers may also be expressed within the cell. (2) Next, biotin is introduced into the cells. This causes the biotin to be taken up into the cells, and the competitive binding of the biotin to the biotin binding site causes the hook and the cargo protein to dissociate.

[0041] As described in (1) and (2) above, it becomes possible to track the transport of cargo proteins from the endoplasmic reticulum to the Golgi apparatus over time, starting from the introduction of the second biotin derivative into cells.

[0042] Next, an example of the procedure for implementing the RUSH method in the proximal-dependent labeling method according to one embodiment of the present invention is described below. (1) Express an endoplasmic reticulum-localized protein having a biotin-binding site in a cell containing a biotin-ligase fusion protein having a binding sequence for a biotin-binding site. One method for expressing the endoplasmic reticulum-localized protein and the biotin-ligase fusion protein in a cell is to introduce an expression vector into the cell. Alternatively, cells that have already expressed at least one of the endoplasmic reticulum-localized protein and the biotin-ligase fusion protein may be used. (2) Next, the second biotin derivative is introduced into the cell. The second biotin derivative competitively binds to the biotin binding site, causing the endoplasmic reticulum-localized protein and the biotin ligase fusion protein to dissociate. This initiates the transport of the biotin ligase fusion protein. The method for introducing the second biotin derivative into the cell can be the method exemplified above as a method for contacting the cell with the first biotin derivative.

[0043] In other words, in a typical RUSH method, the "hook" and "cargo protein" may be, in one embodiment of the present invention, a "endoplasmic reticulum-localized protein" and a "biotin ligase fusion protein," respectively.

[0044] The RUSH method may be performed before step (A) or after step (A), i.e., between step (A) and step (B). After the RUSH method is performed, the biotin obtained by light irradiation in step (B) interacts with the biotin ligase fusion protein transported by the RUSH method, enabling proximal-dependent labeling. [Examples]

[0045] The following describes examples of the preparation of the compounds used in the embodiments and reference examples of this application. In the descriptions of the preparation examples, compound names may be abbreviated. Abbreviations: MeOH = methanol, Hex = hexane, siRNA = ethyl acetate, THF = tetrahydrofuran, EDC = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, DMAP = 4-dimethylaminopyridine

[0046] In addition, 1 1H NMR spectrum and 13¹³C NMR spectra were measured using a Varian 400MR. For mass spectrometry, a high-performance hybrid mass spectrometer, Thermo Fisher Scientific·LTQ Orbitrap XL, was used for electrospray ionization (ESI). For gas chromatography (GC) measurements, a time-of-flight mass spectrometer, JEOL JMS-T100 GCV, was used. Infrared absorption spectra (FTIR) were measured using a JASCO FT / IR-6300. Unless otherwise specified, materials and reaction reagents were purchased from Tokyo Chemical Industry Co., Ltd. and used as is. When using solvents in the reaction, dichloromethane was distilled using calcium chloride as a drying agent immediately before the reaction, and low-moisture grade tetrahydrofuran (THF) from Nacalai Tesque Co., Ltd. was used. For silica gel column chromatography, a FL100D from Fuji Silysia Chemical Co., Ltd. was used. For light irradiation experiments, a LIGHTNINGCURE LC-L1V5 from Hamamatsu Photonics K.K. was used at a distance of 1 cm from the NMR tube to observe the decomposition reaction. 1 The results were tracked using 1H NMR spectroscopy.

[0047] [Manufacturing Example 1] Synthesis of biotin methyl ester (BME, compound 2)

[0048] [ka]

[0049] 51.2 mg (0.2095 mmol) of biotin (compound 1) was suspended in 1.2 mL of MeOH under a nitrogen stream, and 45 mL (0.6241 mmol) of thionyl chloride was added to initiate the reaction. The mixture was stirred overnight at room temperature, and then the solvent and excess thionyl chloride were removed under reduced pressure. The resulting reaction mixture was purified by silica gel column chromatography (Â1 / MeOH = 6:1) to obtain 51.1 mg (0.1978 mmol) of compound 2 (94% yield). 1H NMR (400MHz, CDCl3): δ 5.55(1H,d,J=5.7Hz),5.15(1H,d,J=4.0Hz),4.53-4.50(1H,m),4.33-4.30(1H,m),3.67(3H,s),3.18-3.14(1H,m) ,2.92(1H,dd,J=12.8,5.0Hz),2.74(1H,d,J=12.8Hz),2.34(2H,t,J=7.5Hz),1.75-1.62(4H,m),1.49-1.42(2H,m). 13 C NMR (100MHz, CDCl3): δ 174.2, 107.6, 62.1, 60.4, 55.5, 51.8, 40.8, 33.8, 28.5, 28.4, 24.9. HRMS(ESI): m / z[M+Na] + calcd for [C 11 H 18 [N2O3NaS] + 281.09303; found 281.09290. FTIR(NaCl):γ=3272 broad,3195 broad,3114 broad,3061,2945,2923,2881,2850,1745,1710cm -1 .

[0050] [Manufacturing example 2] Synthesis of ビオチンtert-BtBE, compound 3

[0051]

change

[0052] 38.0 mg (0.1555 mmol) of biotin (compound 1) was mixed with 5 mL of thionyl chloride under a nitrogen stream and stirred for 1 hour while cooling on ice, then stirred for another 2 hours at room temperature. After removing excess thionyl chloride under reduced pressure, 93.3 mg (1.1106 mmol) of sodium bicarbonate and 5 mL of 2-methyl-2-propanol were added, and the mixture was stirred overnight at 30°C under a nitrogen stream. The resulting insoluble matter was removed by filtration, and the solvent was removed under reduced pressure to obtain 46.8 mg of crude product. The obtained crude product was purified by silica gel column chromatography (Â10:1~6:1) to obtain 17.1 mg (0.0569 mmol) of compound 3 (yield 37%) as a white solid. 1 H NMR(400MHz,CD3OD):δ 4.48(1H,dd,J=7.9,4.9Hz),4.30(1H,dd,J=7.9,4.5Hz),3.22-3.17(1H,m),2.93(1H,dd,J=12. 7,4.9Hz),2.70(1H,d,J=12.7Hz),2.23(2H,t,J=7.3Hz),1.74-1.55(4H,m),1.47-1.41(11H,m). 13 C NMR(100MHz,CD3OD):δ 175.0,166.1,81.5,63.4,61.6,57.0,41.0,36.17,29.7,29.5,28.4,28.3,26.1. HRMS(ESI):m / z[M+Na] + calcd for C 14 H 24 N2O3NaS 323.13998;found 323.13992. FTIR (NaCl):γ=3224 broad,2932 broad,2862 broad,2399 broad,1730,1668,1522,1463,1365,1318,1259cm -1 .

[0053] [Manufacturing Example 3] Synthesis of Compound 5

[0054] [ka]

[0055] Under a nitrogen atmosphere, 1001 mg (5.55 mmol) of compound 4, dissolved in 3 mL of acetic anhydride, was slowly added to 6 mL of 69% nitric acid cooled to 0°C, and the mixture was stirred for 2 hours. The reaction solution was poured into 30 mL of cold water, and the precipitated yellow crystals were filtered by suction. The resulting crude product was recrystallized from an ethanol / water = 1:1 solution to obtain 521.9 mg of yellow crystals. These yellow crystals were purified by silica gel column chromatography (Hex / Â=3:1) to obtain 422.4 mg (1.87 mmol) of nitrate 5 (yield 34%). 1 H NMR (400MHz, CDCl3): δ 7.60(1H,s),6.75(1H,s),3.97(6H,s),2.49(3H,s). 13 C NMR(100MHz,CDCl3):δ 199.7,154.0,149.7,138.7,132.8,108.8,107.1,56.7,56.6,30.2. HRMS(ESI):m / z[M+Na] + calcd for [C 10 H 11 [NO5Na] + 248.05294; found 248.05281. FTIR(NaCl):γ=3584,3065,2972,2918,2843,1699,1618,1576,1515cm -1 .

[0056] [Manufacturing Example 4] Synthesis of Compound 6

[0057] [ka]

[0058] Compound 5 (233.7 mg, 1.038 mmol) was dissolved in 8 mL of anhydrous THF under a nitrogen stream. 8 mL of MeOH was added, and the mixture was cooled to 0°C. Then, 85.1 mg (2.250 mmol) of sodium borohydride was added, and the mixture was stirred for 2 hours. The ice bath was removed, and the mixture was stirred overnight at room temperature. Water was then gradually added to stop the reaction. The solvent was removed using a rotary evaporator. The reaction mixture was extracted three times with ethyl acetate. The collected ethyl acetate layer was dried over sodium sulfate, and the solvent was removed to obtain 228.7 mg of crude product. The crude product was purified by silica gel column chromatography (Hex / Â=2:1~1:1) to obtain 208.9 mg (0.919 mmol) of compound 6 (89% yield). 1 H NMR (400MHz, CDCl3): δ 7.57(1H,s),7.31(1H,s),5.58(1H,q,J=6.2Hz),4.00(3H,s),3.94(3H,s),2.28(1H,brs),1.56(3H,d,J=6.2Hz). 13 C NMR (100MHz, CDCl3): δ 153.9,147.9,136.7,108.7,107.9,65.8,56.4,24.3. HRMS(ESI):m / z[M+Na] + calcd for [C 10 H 13 [NO5Na] + 250.06859; found 250.06844. FTIR(NaCl):γ=3583,3515 broad,3369 broad,3102,2974,2935,2848,1615,1580,1519cm -1 .

[0059] [Manufacturing Example 5] Synthesis of Compound 7

[0060] [ka]

[0061] 123.1 mg (0.5039 mmol) of biotin (compound 1), 112.0 mg (0.493 mmol) of compound 6, 1.4105 g (7.358 mmol) of EDC, and 63.2 mg (0.5173 mmol) of DMAP were mixed with 15 mL of dichloromethane and stirred overnight at room temperature under a nitrogen stream. The reaction solution was extracted three times with dichloromethane, and the resulting dichloromethane layers were combined and dried over sodium sulfate. The solvent was then removed to obtain 230.5 mg of crude product. The crude product was purified by silica gel column chromatography (Â1 / MeOH = 6:1) to obtain 104.2 mg (0.229 mmol) of compound 7 (yield 47%). 1 H NMR(400MHz,CDCl3):δ7.52(1H,s),6.97(1H,s),6.40(1H,q,J=6.4Hz),6.23(1H,brs),5.77(1H,brs),4.43(1H,brs),4.23(1H,brs),3.94(3H,s),3 .89(3H,s),3.09-3.06(1H,m),2.84-2.81(1H,m),2.65(1H,d,J=12.8Hz), 2.39-2.26(2H,m),1.63(4H,m),1.57(3H,d,J=6.4Hz),1.40-1.36(2H,m). 13 C NMR(100MHz,CDCl3):δ 172.2,163.8,153.6,148.0,139.9,132.9,108.2,107.7,68.0,61.9,60.0,56.3,56.2,55.3,40.3,33.9,28.3,28.2,24.7,21.8. HRMS(ESI):m / z[M+Na] + calcd for [C 20 H 27 N3O7NaS] + 476.14619; found 476.14633. FTIR(NaCl):γ=3398 broad,3243 broad,3013 broad,2935,2857,1703,1616,1582,1521cm -1 .

[0062] [Manufacturing Example 6] Synthesis of compound 9

[0063] [ka]

[0064] Compound 8 (1050 mg, 6.318 mmol) was dissolved in 3 mL of acetic anhydride, then added to 7.0 mL of 69% HNO3 (115.7 mmol, 18.32 eq) cooled to 0°C, and stirred under a nitrogen stream for 2 hours. The reaction solution was poured into cold water, and the precipitated yellow crystals were filtered by suction. The resulting solid was recrystallized using 120 mL of EtOH / H2O = 1:1 to obtain 716.9 mg (3.397 mmol) of compound 9 (yield 54%) as yellow crystals. 1 H NMR (400MHz, CDCl3): δ 10.45(1H,s),7.62(1H,s),7.42(1H,s),3.97(3H,s),3.96(3H,s). 13 C NMR (100MHz, CDCl3): δ 187.9,153.5,152.5,129.0,125.7,109.9,107.3,56.95,56.89. HRMS(ESI):m / z[MH] - calcd for [C9H8O5N] - 210.4080; found 210.4097. FTIR(NaCl):γ=2980,2941,2897,1685,1604,1572,1520,1458,1438,1396cm -1 .

[0065] [Manufacturing Example 7] Synthesis of compound 10

[0066] [ka]

[0067] Compound 9 (716.9 mg, 3.397 mmol) and NaBH 4 (260.87 mg, 6.896 mmol, 2.030 eq) were mixed under a nitrogen stream with 20 mL of methanol and 18 mL of THF, and stirred for 2 hours while cooling on ice. The mixture was then stirred overnight at room temperature, and the reaction was stopped by carefully adding water. The reaction solution was extracted three times with ethyl acetate, and the collected ethyl acetate layer was dried with sodium sulfate. The solvent was then removed under reduced pressure to obtain the crude product (569.1 mg). The crude product was purified by silica gel column chromatography (Hex / Â=1:1) to obtain 327.7 mg (1.537 mmol) of compound 10 (yield 45%) as a yellow solid. 1 H NMR (400MHz, CDCl3): δ 7.71(1H,s),7.18(1H,s),4.96(2H,d,J=6.5Hz),4.01(3H,s),3.96(3H,s),2.62(1H,d,J=6.5Hz). 13 C NMR (100MHz, CDCl3): δ 154.0,148.1,139.9,132.4,111.2,108.3,62.98,56.63,56.55. HRMS(ESI):m / z[M+Na] + calcd for [C9H 11 O5NNa] + 236.05294; found 236.05280. FTIR(NaCl):γ=3496,3242 broad,3103,3040,3013,2944,2924,2847,1614,1580cm -1 .

[0068] [Manufacturing Example 8] Condensation of compound 10 with biotin (synthesis of compound 11)

[0069] [ka]

[0070] Compound 10 (309.4 mg, 1.451 mmol), biotin (compound 1) (357.8 mg, 1.464 mmol), EDC (2085 mg, 13.43 mmol), and DMAP (554.6 mg, 4.540 mmol) were added to 50 mL of dichloromethane and stirred overnight under a nitrogen stream. The reaction solution was extracted three times with dichloromethane, and the resulting dichloromethane layers were combined and dried over sodium sulfate. The solvent was then removed under reduced pressure to obtain the crude product (896.1 mg). The crude product was purified by silica gel column chromatography (CH2Cl2 / Â / MeOH = 7:3:1) to obtain 366.7 mg (0.8443 mmol) of compound 11 (yield 58%) as a pale yellow solid. 1 H NMR(400MHz,CDCl3):δ 7.71(1H,s),6.99(1H,s),5.48(2H,s),5.36(1H,d,J=7.0Hz),4.93(1H,brd ,J=7.4Hz),4.51(1H,dd,J=7.0,5.1Hz),4.32(1H,dd,J=7.4,4.6Hz),3.99(3 H,s),3.96(3H,s),3.15(1H,dt,J=11.9,4.6Hz),2.92(1H,dd,J=12.8,5.1H z),2.26(1H,d,J=12.8Hz),2.44(2H,t,J=7.3Hz),1.71(4H,m),1.46(2H,m). 13 C NMR(100MHz,CDCl3):δ 174.2,164.0,154.2,149.6,140.6,126.9,111.2,106.6,63.24,61.98,60.17,55.55,51.56,40.57,39.03,33.71,28.40,28.25,24.79. HRMS(ESI):m / z[M+Na] + calcd for [C 19 H 25 [O7N3NaS] + 462.13054; found 462.13025. FTIR(NaCl):γ=3267,3204,3115,3008,2923,2880,2851,1738,1712,1604cm -1 .

[0071] [Manufacturing Example 9] Condensation of compound 12 with biotin (synthesis of compound 13)

[0072] [ka]

[0073] A 20 mL dichloromethane solution containing 47.8 mg (0.312 mmol) of compound 12, 82.0 mg (0.336 mmol, 1.07 eq) of biotin (compound 1), 763.4 mg (4.917 mmol, 15.7 eq) of EDC, and 100.10 mg (0.8194 mmol, 2.62 eq) of DMAP was stirred overnight at room temperature under a nitrogen stream. The reaction solution was extracted three times with dichloromethane, and the resulting dichloromethane layers were combined and dried over sodium sulfate. The solvent was then removed under reduced pressure to obtain the crude product (135.7 mg). The crude product was purified by silica gel column chromatography (Â / MeOH = 6:1) to obtain 65.67 mg (0.1730 mmol) of compound 13 (yield 55%) as a white solid. 1 H NMR(400MHz,CDCl3):δ 8.09(1H,dd,J=8.2,0.8Hz),7.66(1H,ddd,J=7.1,6.8,0.8Hz),7.57(1H,dd,J=7.1,1.0Hz),7 .50(1H,ddd,J=8.2,6.8,1.0Hz),5.50(2H,s),5.10(1H,d,J=7.6Hz),4.75(1H,brd,J=6.4Hz), 4.52(1H,dd,J=7.6,5.1Hz),4.33(1H,dd,J=6.4,4.6Hz),3.17(1H,dt,J=8.3,4.6Hz),2.93(1H ,dd,J=12.8,5.1Hz),2.73(1H,d,J=12.8Hz),2.44(2H,t,J=7.3Hz),1.71(4H,m),1.47(2H,m). 13C NMR(100MHz,CDCl3):δ 173.1,164.0,147.1,133.9,132.1,129.4,129.0,125.1,63.02,62.03,60.22,55.61, 40.65,33.88,28.44,28.30,24.83. HRMS (ESI): m / z[M+Na] + calcd for [C 17 H 21 [O5N3NaS] + 402.10941; found 402.10910. FTIR(NaCl):γ 3218,3119,3074,2927,2858,1737,1703,1613,1597,1525cm -1 .

[0074] [Manufacturing Example 10] Synthesis of desthiobiotin methyl ester (DBME, compound 15)

[0075] [ka]

[0076] To 2 mL of a MeOH solution containing 20.3 mg (0.0948 mmol) of desthiobiotin (compound 14), 90 μL (1.24 mmol) of thionyl chloride was added under a nitrogen stream and the mixture was stirred overnight at room temperature. The solvent and excess thionyl chloride were removed under reduced pressure to obtain 20.6 mg (0.0944 mmol) of compound 15 (99% yield). 1 H NMR(500MHz,CD3OD):δ 3.86(1H,dq,J=7.4,6.5Hz),3.73(1H,dt,J=7.4,6.5Hz),3.65(3H,s),2.33(2H,t ,J=7.4Hz),1.64(2H,tt,J=7.4,7.4Hz),1.50-1.30(6H,m),1.12(3H,d,J=6.5Hz). 1313C NMR (125 MHz, CD3OD): δ 175.9, 165.6, 58.1, 53.6, 52.0, 34.6, 30.4, 30.1, 27.0, 25.81, 15.4. HRMS (ESI, m / z): [M+Na] + calcd. for C 11 H 20 N2O3, 251.13661; found, 251.13666. FTIR (NaCl): ν = 3227 broad, 2934, 2863, 1734, 1686 cm -1 .

[0077] [Example 1: Photodegradation Reaction of Compound 7 (Deprotection Reaction)] A CDCl3 solution of Compound 7 (2.5 mg / 0.5 mL) was placed in a 5-mm-diameter sample tube (Shigemi EC-57), and irradiated with 365-nm light from a distance of 1 cm from the sample tube using LIGHTNINGCURE LC-L1V5 manufactured by Hamamatsu Photonics K.K. The state of the deprotection reaction experiment is shown in Fig. 1. The deprotection reactions by irradiation for 1 minute, 3 minutes, 9 minutes, and 24 minutes were respectively 1 monitored by 1H NMR spectrum. The results are shown in Fig. 2. As shown in Fig. 2, while the characteristic signals of Compound 7 attenuated due to the deprotection reaction, new signals increased. The signal of biotin generated by the deprotection reaction could not be confirmed, but insoluble matter formed in the sample tube as the deprotection reaction proceeded. This insoluble matter was collected after the reaction and analyzed by mass spectrometry, and it was confirmed to be biotin. There has been no report of isolating and assigning the compound showing the newly generated signal so far, and the structure shown in Fig. 2 is a deduced structure. From this example, it was confirmed that by irradiating light on biotin esterified with a photodegradable protecting group (biotin derivative), a deprotection reaction occurs and biotin can be obtained.

[0078] [Reference Example] [Culture of HeLa Cells] The HeLa cells used in Reference Examples 1-6 and Example 3 of this application were cultured under the following conditions. The HeLa cells were cultured at 37°C in a medium consisting of D-MEM (High Glucose, Fujifilm Wako: 045-30285) supplemented with penicillin, streptomycin, and L-glutamine solution (Fujifilm Wako: 161-23501), pyruvate (final concentration 1 mM, Fujifilm Wako: 190-14881), HEPES (final concentration 25 mM, pH 7.3, Nakarai), and 10% inactivated fetal bovine serum, under a 5% CO2 environment.

[0079] <Plasmid vector> (Reference example 1) ·Str_KDEL_SBP-EGFP-GPI Addgene's Plasmid #65294 (Reference example 5) ·CT7-Rush-2NG-GPI Based on CT7-Rush-2NG-VSVG, which was created in the experiment described in the paper below, the VSVG gene was replaced with the GPI gene in Str_KDEL_SBP-EGFP-GPI to create this gene. Tago et al. RudLOV is an optically synchronized cargo transport method revealing unexpected effects of dynasore. EMBO Rep (2024) (Reference examples 2~4, 6) ·CMV-SP-SBP-TurboID-LL-KDEL The TurboID gene was excised from the plasmid V5-TurboID-NES_pCDNA3 (Plasmid #107169, manufactured by Addgene), which contained the TurboID gene and was donated by Dr. Alice Ting. The fusion protein expressed by this plasmid under the CMV promoter has the following at the N-terminus of TurboID. • IL-2 derived signal peptide (SEQ ID NO: 1) • Streptavidin binding sequence (SEQ ID NO: 2) • Linker (Sequence ID 3) • Two copies of the mClover3 gene • Linker (Sequence ID 4) Furthermore, TurboID has a KDEL sequence and a stop codon at its C-terminus via a 74-amino acid linker (SEQ ID NO: 5). The molecular weight of this fusion protein, excluding the signal peptide, is 101 kDa. (Example 3) ·CT7-Rush-2NG-GPI

[0080] <Transfection of plasmid vectors> In Reference Examples 1-6 and Example 3 of this application, cells were seeded in one of the following devices for live imaging, antibody staining, and cell lysate preparation, and transfection was performed the following day. • 8-well cell culture chamber (μ-Slide 8 Well ibiTreat, ibidi / Nippon Genetics) • 18-well cell culture chamber (μ-Slide 18 Well ibiTreat, ibidi / Nippon Genetics) • 6-well cell culture plate (Cell Culture plate True line, TR5000) • 12-well cell culture plate (Cell Culture plate True line,TR5001) • 24-well cell culture plate (Cell Culture plate True (line,TR5002) • 100mm cell culture dish (Nunclon™ Delta Surface, Themo Scientific: 150464)

[0081] JetOptimus (Polyplus-Transfection) was used for plasmid vector transfection. In the standard protocol, 800 ng of plasmid DNA was suspended in 50 μL of JetOptimus buffer, 1.2 μL of JetOptimus reagent was added, and the mixture was vigorously mixed with a vortex mixer for 20 seconds. After standing at room temperature for at least 10 minutes, the mixture was diluted in 2 mL of standard medium and layered onto cells from which the old medium had been removed. The medium was replaced with fresh medium after 6–18 hours.

[0082] <Cell fixation and observation using indirect immunofluorescence in Reference Example 3> The culture medium was removed from the well, and the cells were fixed by placing 4% paraformaldehyde fixative on top and letting it stand at room temperature for 5 minutes. After removing the fixative, the cells were washed three times with 1×PBS and then stored with 1×PBS on top to prevent drying. After removing the 1×PBS from the wells on which the cells were attached and fixed, the cells were permeabilized with 0.1% Triton-100 in 1×PBS for 2 minutes at room temperature to allow antibody to permeate into the cells. After washing the cells three times with 1×PBS, they were incubated with 10% FBS in 1×PBS for 10 minutes at room temperature. Then, they were incubated with Streptavidin-Alexa Fluor 568 (1 / 1,000 in 10% FBS) for 1 hour at room temperature.

[0083] Before observation, cells were incubated in 40% sRIMS at room temperature for 5 minutes, then mounted in 80% sRIMS containing 0.5% N-propyl-gallate and observed using a confocal laser microscope (FV3000, Olympus). Images containing multiple Z-stacks were processed using deconvolution with cellSens. 40% sRIMS: 40% (wt / vol) sorbitol, 0.02 M phosphate buffer pH 7.5, and 0.01% (wt / vol) azide 80% RIMS: 80% (wt / vol) sorbitol, 0.02 M phosphate buffer pH 7.5, and 0.01% (wt / vol) azide 0.5% N-propyl-gallate: Fujifilm Wako 102747

[0084] <Live imaging of HeLa cells using FV3000 in Reference Examples 1 and 6> After transfection of the cells inoculated into an 8-well cell culture chamber, the cells were observed under conditions of 37°C and 5% CO2 using a confocal laser microscope FV3000 with a CO2 incubator attached.

[0085] <Synchronized transport initiation experiment using the RUSH method in Reference Examples 1, 5, 6 and Example 3> HeLa cells constitutively expressing GalT::iRFP were transfected with Golgi markers and cargo proteins. After 6–10 hours, 10–25 μM biliverdin was added. 18–20 hours after transfection, biotin, biotin methyl ester, biotin tert-butyl ester, desthiobiotin methyl ester, or compound 7 was administered to initiate cargo protein transport.

[0086] <Preparation of cell lysates in reference examples 2, 4, and 6> TurboID fusion protein GBF1 BFAres Cells were expressed, biotinylated by administering a biotin derivative, and immediately cooled on ice and washed with PBS. The cells were then lysed using Lysis Buffer to obtain cell lysates. The lysates were centrifuged at 16,000 G at 4°C for 5 minutes to ensure no precipitate formed. 4× sample buffer was added until the final volume was 1× sample buffer, and the mixture was heated at 95°C for 5 minutes before being stored at -25°C.

[0087] <Blotting in Reference Examples 2, 4, and 6> After thawing, the prepared lysate was centrifuged at 16,000G at 4°C for 5 minutes to ensure no precipitate formed, and then 5 μL was applied to an SDS-PAGE gel. PrecisionPlus Protein Dual Color Standards (BioRad) were used as the marker, and 5 μL was applied. Electrophoresis was performed at a constant current of 30 mA per gel for 55 minutes.

[0088] Protein transfer from the gel to the PVDF membrane was performed using a Mini Trans-Blot (BioRad) at a constant voltage of 100V for 60 minutes. The membrane with the transferred proteins was rinsed three times with 1×TTBS, and then blocked for 30-40 minutes with Blockingsoln (1×TTBS containing 3% bovine serum and 0.02% azide) or Blocking One. After blocking, the membrane was rinsed three times with 1×TTBS, and the PVDF membrane was placed in the primary antibody and rotated and shaken overnight at 4°C. Three washes of 5 minutes each were performed with 1×TTBS, and the membrane was placed in a secondary antibody solution conjugated with HRP (Reference Examples 2 and 4) or a fluorescently labeled streptavidin solution (Reference Example 6) and shaken at room temperature for 1-2 hours. This membrane was then washed three times with 1×TTBS for 5 minutes each, and further washed for 1 minute with 1×TBS (prepared by diluting 10×TBS containing 12.1% Tris and 9.0% NaCl). After allowing the PVDF membrane to stand in a Clarity ECL substrate mixture for 5 minutes, the bands were detected using the Chemidoc MP system.

[0089] Primary antibody: Anti-GFP (Rabbit) 1 / 7500 (Life Technologies A6455) was added to 1×TTBS containing 0.15% bovine serum and 0.04% azide at the concentrations listed below. Secondary antibody conjugated with HRP: The following concentrations were added to 1×TTBS: Rb-HRP 1 / 20,000, Streptavidin-HRP 1 / 20,000. Fluorescently labeled streptavidin; Streptavidin-Alexa Fluor 568 (1 / 1,000 in 10% FBS).

[0090] [Reference Example 1: Improvement of the RUSH method using biotin methyl ester] Biotin at concentrations of 4 μM, 10 μM, and 40 μM, and biotin methyl ester at concentrations of 0.1 μM, 0.4 μM, 1 μM, 4 μM, 10 μM, and 40 μM were used. Synchronized transport initiation experiments of cargo proteins using the RUSH method described above were performed with these concentrations of biotin and biotin methyl ester. GalT::iRFP713 was used as the Golgi marker, and GPI-AP was used as the cargo protein.

[0091] The structures of biotin and biotin methyl ester (which may be referred to as "BME" in this specification) are as follows:

[0092] [ka]

[0093] Figure 3 shows a graph plotting the time it takes for GPI-AP (cargo protein) to accumulate most abundantly in the Golgi apparatus in each cell, using the RUSH method. Each dot in the graph represents one cell. PBS is typically used as the solution for dissolving biotin. However, biotin methyl ester does not dissolve in PBS and requires dissolution using dimethyl sulfoxide (DMSO). Therefore, in this reference example, a solution of biotin dissolved in DMSO was also prepared and used in the experiment. As shown in Figure 3, it was confirmed that when biotin was used, there was a large variation in the time it took for GPI-AP to accumulate most abundantly in the Golgi apparatus between cells, but when biotin methyl ester was used, the variation in this time was small.

[0094] Next, the upper panel of Figure 4 shows the localization of GPI-AP at 0 (pre), 10, 20, 30, 40, 50, 60, and 70 minutes after initiating GPI-AP transport from the endoplasmic reticulum using the RUSH method with 40 μM (in PBS) biotin. In Figure 4, the arrows indicate cells in which GPI-AP transport has begun. The lower panel of Figure 4 shows the localization of GPI-AP at 0 (pre), 10, 20, and 30 minutes after initiating GPI-AP transport from the endoplasmic reticulum using the RUSH method with 40 μM (in DMSO) biotin methyl ester. At 10 minutes after the start of transport, it was confirmed that GPI-AP transport had begun in all cells.

[0095] (Conclusion) The conclusions drawn from this experiment are as follows. (1) When the RUSH method is performed using biotin to initiate the transport of GPI-AP from the endoplasmic reticulum to the Golgi apparatus, the time from biotin addition until the start of transport varies depending on the cell. On the other hand, when biotin methyl ester is used to initiate the transport, transport starts immediately in all cells. (2) Rapid transport initiation by biotin methyl ester can be triggered even at a low concentration of 1 μM of biotin methyl ester.

[0096] [Reference Example 2: Improvement of TurboID biotinylation by biotin methyl ester 1 (Blot)] The amount of autobiotylation by TurboID was investigated using 1 μM, 5 μM, 10 μM, 50 μM, 100 μM, 200 μM, and 400 μM biotin and biotin methyl ester. HeLa cells were expressed with a TurboID fusion protein (Sp::2CV::TurboID::LL:KDEL) localized in the endoplasmic reticulum lumen, and biotin or biotin methyl ester was administered to the HeLa cells. After incubation at 37°C for 10 minutes, the HeLa cells were solubilized to prepare samples for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0097] After performing SDS-PAGE, blotting was carried out using the method described above. After blotting the samples, biotinylation of the proteins was detected using HRP (horseradish peroxidase)-labeled streptavidin. TurboID fusion proteins were also detected using an anti-GFP antibody. Figure 5 shows the results of detecting protein biotinylation and TurboID fusion proteins. Figure 6 shows the results of measuring and plotting the amount of biotin introduced per TurboID fusion protein from the blotting results.

[0098] (Conclusion) The conclusions drawn from this experiment are as follows. (1) Compared to biotin, biotin methyl ester was found to biotinate proteins at more than twice the rate at all concentrations. (2) It was found that, especially at low concentrations, biotinylation of proteins by biotin methyl ester occurs with more than five times the efficiency.

[0099] [Reference Example 3: Improvement of TurboID biotinylation by biotin methyl ester (fluorescence observation)] The amount of autobiotylation by TurboID was investigated using 1 μM, 5 μM, 10 μM, 50 μM, 100 μM, 200 μM, and 400 μM biotin and biotin methyl ester. HeLa cells were expressed with a TurboID fusion protein (Sp::2CV::TurboID::LL:KDEL) localized in the endoplasmic reticulum lumen, and biotin or biotin methyl ester was administered to the HeLa cells. After incubation at 37°C for 10 minutes, the cells were fixed.

[0100] After subjecting the fixed cells to permeabilization, they were stained with Streptavidin-Alexa Fluor 568, and fluorescence was observed using a confocal laser microscope FV3000. For details on cell fixation and fluorescence observation methods, please refer to the section "Cell Fixation and Observation by Indirect Immunofluorescence Method" above. Furthermore, the TurboID fusion protein was detected by the fluorescence of the fluorescent protein mClover3. The results are shown in Figure 7. In addition, the amount of biotin introduced per unit of TurboID fusion protein was measured and plotted from the above observation results, and the results are shown in Figure 8.

[0101] (Conclusion) The conclusions drawn from this experiment are as follows. (1) Compared to biotin, biotin methyl ester showed more than twice the biotinylation of proteins at all concentrations. (2) It is suggested that biotinylation of proteins by biotin methyl ester occurs more efficiently, especially at low concentrations.

[0102] [Reference Example 4: Improvement of TurboID biotinylation by biotin methyl ester 2 (Blot)] The amount of autobiotylation by TurboID was investigated using biotin and biotin methyl ester at various reaction times. HeLa cells were expressed with a TurboID fusion protein (Sp::2CV::TurboID::LL:KDEL) localized in the lumen of the endoplasmic reticulum, and 50 μM biotin or biotin methyl ester was administered to the HeLa cells. After incubation at 37°C for 1, 2, 5, 10, 30, or 60 minutes, the cells were soluble to prepare samples for SDS-PAGE.

[0103] After performing SDS-PAGE, blotting was carried out. Next, biotinylation of the protein was detected using HRP (horseradish peroxidase)-labeled streptavidin. Figure 9 shows the results of detecting protein biotinylation and TurboID fusion proteins. Figure 10 shows the results of measuring and plotting the amount of biotin introduced per TurboID fusion protein from the blotting results.

[0104] (Conclusion) The conclusions drawn from this experiment are as follows. (1) When biotin methyl ester was used, more than twice the biotinylation was observed up to 30 minutes after administration compared with when biotin was used. (2) In particular, it was found that biotinylation with biotin methyl ester occurs with more than five times the efficiency compared to when biotin is used, especially in short periods of time.

[0105] [Reference Example 5: Further improvement of the RUSH method using biotin tert-butyl ester] Synchronized transport initiation experiments of cargo proteins by the RUSH method were performed using 10 μM or 50 μM biotin, biotin methyl ester, or biotin tert-butyl ester (sometimes referred to as "BtBE" in this specification). GalT::iRFP713 was used as the Golgi marker, and GPI-AP was used as the cargo protein. The structure of biotin tert-butyl ester is as follows.

[0106] [ka]

[0107] Figure 11 shows the percentage of cells to which the cargo protein migrated at 2, 4, 6, 8, and 10 minutes after administration of each concentration of biotin, biotin methyl ester, or biotin tert-butyl ester.

[0108] When biotin was administered, cargo proteins had not moved 10 minutes after administration, regardless of whether the concentration was 10 μM or 50 μM.

[0109] Upon administration of biotin methyl ester, at a concentration of 10 μM, cargo protein migration began in 30% of cells at 6 minutes after administration and in 60% of cells at 8 minutes after administration. At a concentration of 50 μM, cargo protein migration began in 70% of cells at 6 minutes after administration.

[0110] Upon administration of biotin tert-butyl ester, at a concentration of 10 μM, cargo protein migration began in 10% of cells at 2 minutes after administration and in 60% of cells at 4 minutes after administration. At a concentration of 50 μM, cargo protein migration began in 10% of cells at 2 minutes and in 90% of cells at 4 minutes.

[0111] (Conclusion) The conclusions drawn from this experiment are as follows. (1) Both biotin tert-butyl ester and biotin methyl ester were found to induce the initiation of cargo protein transport in all cells more rapidly after administration than biotin. (2) Biotin tert-butyl ester was found to induce a more rapid initiation of cargo protein transport compared to biotin methyl ester.

[0112] [Reference Example 6: Further Improvement of the RUSH Method with Desthiobiotin Ester] Using desthiobiotin methyl ester (sometimes referred to as "DBME" in this specification), we performed synchronized transport initiation experiments of cargo proteins using the RUSH method and detected the amount of autobiotination using TurboID. The structure of desthiobiotin methyl ester is as shown as compound 15 above.

[0113] The RUSH method was used to initiate synchronized transport of cargo proteins, and the experiment was conducted in the same manner as in Reference Example 1 described above, except for the following two points. • Using 1 μM, 10 μM, and 100 μM desthiobiotin methyl ester • Detection of GPI-AP localization before administration and 4 minutes after administration.

[0114] Figure 12 shows the results of detecting the localization of GPI-AP under each concentration condition. As shown in Figure 12, when desthiobiotin methyl ester was administered, cargo protein transport began within a short time of 4 minutes after administration, regardless of the concentration (1 μM, 10 μM, or 100 μM).

[0115] The detection of autobiotinization levels using TurboID was performed using the same method as in Reference Example 2 described above, except for the following three points. The administration conditions were as follows: (A) control with no biotin derivative administration, (B) 50 μM biotin methyl ester, (C) 1 μM, 10 μM, and 50 μM desthiobiotin methyl ester, and (D) 1 μM, 10 μM, and 50 μM desthiobiotin methyl ester + 50 μM biotin methyl ester. • The incubation time after administration was 10 minutes for (B) above, 1 hour for (C) above, and for (D) above, 1 hour after administration of desthiobiotin methyl ester, followed by an additional 10 minutes of biotin methyl ester administration. • Biotinylated proteins were detected using Streptavidin-Alexa Fluor 568.

[0116] Figure 13 shows the results of detecting biotinylation of proteins and the detection of TurboID fusion proteins. Figure 14 shows the results of measuring the amount of biotin introduced per TurboID fusion protein from the blotting results.

[0117] (Conclusion) The conclusions drawn from this experiment are as follows. (1) Desthiobiotin methyl ester was found to rapidly induce the initiation of cargo protein transport in all cells after administration. (2) When desthiobiotin methyl ester was used, it was found that autobiotination by TurboID hardly occurred.

[0118] [Example 2: Induction of photo-induced biotinylation using compound 7] For compound 7 synthesized in Production Example 5, we investigated by cell observation whether it underwent light-dependent cleavage upon light irradiation and whether proximal protein biotinylation by TurboID was induced. GBF1 contains TurboID (TB), which is fused with the endoplasmic reticulum localization signal KDEL and the green fluorescent protein Clover (Cv). BFAres Cells expressing Cv::TB::KDEL were administered 10 μM of compound 7. Cells that were irradiated with light for 2 to 5 minutes after administration, and cells that were not irradiated with light, were fixed simultaneously (Figure 15, upper panel: Ctrl, lower panel: +UV). Light irradiation was performed using a confocal laser microscope (FV3000) with a UV filter (U-FUNA) without attenuating the epifluorescence. The wavelength of the light was 360-370 nm. After cell fixation, biotin was detected using Streptavidin-Alexa Fluor 568 and fluorescence was detected using FV3000. The results are shown in Figure 15. It was found that biotinylation of proximal proteins occurred in cells administered with compound 7 in a light irradiation-dependent manner.

[0119] [Example 3: Synchronized transport initiation experiment using compound 7 via the RUSH method] Synchronized transport initiation experiments of cargo proteins using the RUSH method were conducted with compound 7 synthesized in 3 μM, 10 μM, and 100 μM concentrations according to Production Example 5. GalT::iRFP713 was used as the Golgi marker, and GPI-AP was used as the cargo protein.

[0120] Figure 16 shows the localization of the cargo protein before administration of compound 7 and after a specific time has elapsed following administration of compound 7 at each concentration. In Figure 16, the top panel A shows the experimental results with a compound 7 (PhB) concentration of 3 μM, the middle panel B shows the experimental results with a compound 7 (PhB) concentration of 10 μM, and the bottom panel C shows the experimental results with a compound 7 (PhB) concentration of 100 μM.

[0121] Regardless of the concentration of the compound administered, GPI-AP transport was confirmed to begin at 3 or 4 minutes after administration, and GPI-AP was found to be localized in the Golgi apparatus.

[0122] 〔summary〕 Examples 1 and 2 confirmed that a deprotection reaction occurs when a biotin derivative having a photodegradable protecting group is irradiated with light, yielding biotin. Furthermore, Example 2 confirmed that the biotin derivative having a photodegradable protecting group interacts with TurboID in a light-irradiation-dependent manner, leading to biotinylation of nearby proteins, i.e., proximal-dependent labeling.

[0123] Furthermore, from Reference Examples 2-4 of this application, it was confirmed that even when biotin methyl ester is used, biotination can be performed more efficiently than when biotin is used, although the protecting group is different from a photodegradable protecting group. This is because deesterification occurs with biotin methyl ester without any stimulus, so biotin is produced, interacts with TurboID, and nearby proteins are biotinized. However, this deesterification cannot be controlled. Therefore, when biotin methyl ester is used, it is not possible to control the series of steps in proximal-dependent labeling. In the proximal-dependent labeling method according to one embodiment of the present invention, since the protecting group is a photodegradable protecting group, it is possible to control the timing of initiating proximal-dependent labeling.

[0124] Furthermore, based on the measurement results of the amount of biotin and biotin methyl ester introduced per TurboID fusion protein, as seen in Reference Example 2, biotin methyl ester is thought to have higher cell membrane permeability and be rapidly deesterified compared to biotin. This suggests that even when using a biotin derivative with a photodegradable protecting group instead of biotin methyl ester, cell membrane permeability is higher compared to biotin.

[0125] Furthermore, from Reference Examples 1 and 5, it was confirmed that the RUSH method was performed more efficiently when using biotin methyl ester or biotin tert-butyl ester, even though the protecting group was different from the photodegradable protecting group, compared to when using biotin. Also, from Example 3, it was confirmed that cargo protein transport was initiated even when the RUSH method was performed using a biotin derivative having a photodegradable protecting group. Thus, based on the results of Reference Examples 1 and 5, it is suggested that cargo protein transport is initiated more rapidly when using a biotin derivative having a photodegradable protecting group compared to when using biotin.

[0126] Furthermore, from Reference Example 6, it was confirmed that desthiobiotin methyl ester can initiate the transport of cargo proteins by the RUSH method, but does not induce autobiotinization by TurboID. In other words, it is suggested that desthiobiotin methyl ester is a biotin derivative that has the ability to bind to biotin binding sites but not to biotin ligase fusion proteins. It was confirmed that such desthiobiotin methyl ester can be suitably used as a second biotin derivative in one embodiment of the present invention. [Industrial applicability]

[0127] The present invention can be used, for example, in research using proximal-dependent labeling or in screening of drug candidates.

Claims

1. The following steps (A) and (B) (A) A step of introducing a first biotin derivative having a photodegradable protecting group into cells containing a biotin ligase fusion protein. (B) A step of irradiating the cells with light to carry out a deprotection reaction of the first biotin derivative, thereby obtaining biotin from the first biotin derivative that binds to the biotin ligase fusion protein. A method for proximal-dependent labeling of a biotin ligase fusion protein using the first biotin derivative comprising the first biotin derivative.

2. The aforementioned cells further contain endoplasmic reticulum-localized proteins having a biotin-binding site, The biotin ligase fusion protein has a binding sequence with the biotin binding site, The method according to claim 1, further comprising the step of introducing into the cell a second biotin derivative having a protecting group different from the photodegradable protecting group of the first biotin derivative and exhibiting the ability to bind to the biotin binding site of the endoplasmic reticulum localized protein, prior to step (B) above.

3. The method according to claim 1, wherein the photodegradable protecting group is a group derived from a compound represented by any one of the following structural formulas 1 to 3. 【Chemistry 1】

4. The method according to claim 1, wherein in step (B), the wavelength of the irradiated light is 300 nm to 400 nm.

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