WO2019168125A1 - Development of novel technique based on chemical crosslinking for visualization of in-vivo receptors - Google Patents

Abstract

The present disclosure provides a labeling method for biological samples, the method comprising: (a) a step in which an immobilizing agent is applied to a cell-containing biological sample; and (b) a step in which a labeling reagent is applied to the cell-containing biological sample (either one among step (a) or step (b) can be performed first, or step (a) and step (b) can be performed simultaneously). The labeling reagent contains: a ligand for a cell surface receptor; a labeling substance; and at least one reactive group that reacts with the immobilizing agent. Some of the cells contained in the cell-containing biological sample include said cell surface receptor. The cell surface receptor comprises a ligand binding site and at least one reactive group that reacts with the immobilizing agent. The immobilizing agent reacts with the reactive group of the labeling reagent and the reactive group of the cell surface receptor to form a covalent bond, whereby the labeling reagent and the cell surface receptor are connected to each other.

Description

Development of new in vivo receptor visualization technology based on chemical cross-linking method

The present specification discloses a labeling method for a biological sample, a labeled cell-containing biological sample, and a labeling agent.

Cell surface receptors are important for cell recognition, signal transduction, mass transport into and out of cells, and the action point of drugs, but they are hydrophobic proteins embedded in cell membranes and are difficult to separate and purify. Labeling with antibodies to surface receptors has been performed.

In the case of labeling cells exposed on the surface of a biological sample, it is possible to label cells using antibodies, but when the cell layer is thick, large molecules such as antibodies are difficult to penetrate deeply, so The sign was difficult.

Patent Document 1 discloses a technique for labeling a neurotransmitter receptor with a compound having a ligand and a labeling group for the neurotransmitter receptor, and the labeling using the compound described above is the stability of the compound, There was room for improvement in terms of overall animal labeling.

Table 2015/125851

Although the prior art has the problems exemplified above, it is possible to label the deep part of the tissue or the whole animal, and to provide a stable in vivo receptor visualization technique even when stored for a long time according to the present disclosure. This is one of the problems that can be solved.

As one aspect of the present disclosure, the following labeling method for a biological sample, labeled cell-containing biological sample, and labeling reagent are provided.
Item 1. A method for labeling a biological sample, the method comprising:
(A) applying a fixing agent to the cell-containing biological sample, and (b) applying a labeling agent to the cell-containing biological sample,
(Step (a) and step (b) may be performed first, step (a) and step (b) may be performed simultaneously),
here,
The labeling agent has at least one reactive group that reacts with a ligand for a cell surface receptor, a labeling substance, and an immobilizing agent;
Some cells contained in the cell-containing biological sample include the cell surface receptor,
The cell surface receptor has at least one reactive group that reacts with the immobilizing agent and a ligand binding site,
The immobilizing agent reacts with a reactive group of the labeling agent and a reactive group of the cell surface receptor to form a covalent bond, thereby linking the labeling agent and the cell surface receptor; A method for labeling biological samples.
Item 2. Item 2. The method for labeling a biological sample according to Item 1, wherein the number of reactive groups of the labeling agent involved in the covalent bond with the immobilizing agent is the same as the number of reactive groups of the cell surface receptor.
Item 3. The reactive group of the cell surface receptor is selected from the group consisting of NH ₂ , OH and SH, and the reactive group of the labeling agent is selected from the group consisting of NH ₂ , NHNH ₂ , CONHNH ₂ , OH and SH. Item 3. The method for labeling a biological sample according to any one of Items 1 to 2, which is selected.
Item 4. Item 4. The method for labeling a biological sample according to any one of Items 1 to 3, wherein the step (a) is performed simultaneously with the step (b).
Item 5. Either by providing a cell-containing biological sample pre-fixed with the immobilizing agent, or by further coexisting the immobilizing agent with a cell-containing biological sample pre-fixed with the immobilizing agent. (a) is achieved,
Item 5. The method for labeling a biological sample according to any one of Items 1 to 4, wherein in the step (b), the biological sample immobilized with the immobilizing agent reacts with the labeling agent.
Item 6. Item 6. The method for labeling a biological sample according to any one of Items 1 to 5, wherein the reaction between the reactive group of the cell surface receptor, the reactive group of the labeling agent, and the immobilizing agent is reversible. .
Item 7. Item 7. The method for labeling a biological sample according to any one of Items 1 to 6, wherein the cell surface receptor is a receptor for a neurotransmitter.
Item 8. Item 8. The biological sample label according to any one of Items 1 to 7, wherein the immobilizing agent is at least one selected from the group consisting of formaldehyde, paraformaldehyde, glutaraldehyde, glyoxal, methylglyoxal, and dimethyl suberimidate. Method.
Item 9. The cell-containing biological sample is brain, heart, liver, gallbladder, kidney, adrenal gland, pancreas, lung, thyroid, esophagus, stomach, duodenum, small intestine, large intestine, ureter, bladder, prostate, uterus, breast, bronchi, blood vessel Item 9. The method for labeling a biological sample according to any one of Items 1 to 8, comprising at least one selected from the group consisting of lymphatic vessels, skin, bones, joints, muscles, oral mucosa, and nasal mucosa.
Item 10. A labeled cell-containing biological sample labeled with a labeling agent, wherein the labeling agent is covalently linked by a cell surface receptor of the biological sample and a fixing agent. .
Item 11. Having at least one reactive group that reacts with a ligand for a cell surface receptor, a labeling substance, and an immobilizing agent, and is used for linking to the cell surface receptor of a cell-containing biological sample by the immobilizing agent. Labeling agent.

Receptors that receive ligands such as neurotransmitters are important drug targets.

According to one aspect of the present disclosure, a deep part of a living body or an entire animal can be labeled. For example, cell surface receptors such as neurotransmitter receptors can be comprehensively labeled and visualized. it can.

According to another aspect of the present disclosure, since a biological sample fixed with an immobilizing agent can be labeled, for example, an enormous amount of immobilized biological sample stock including an immobilized disease site is used. Leveraging and labeling in this embodiment can contribute to clarifying the relationship between disease and cell surface receptors.

Further, according to still another aspect of the present disclosure, a plurality of cell surface receptors can be labeled simultaneously, which can contribute to clarifying the mutual relationship between them. According to the method of the present disclosure, it is possible to visualize samples such as cultured nerve cells, brain slices, whole brains, whole animals, and calcium channels.

Furthermore, the labeling method of the present disclosure can be used in combination with a normal immunostaining method.

The labeling of a biological sample according to an embodiment can contribute to elucidation of cancer formation / metastasis mechanism, memory mechanism (brain connectome research), and the like.

FIG. 1 schematically shows Ligand-directed chemistry disclosed in Patent Document 1. FIG. 2 shows a schematic diagram of labeling in one exemplary embodiment of the present disclosure. The lower column (a) and (b) in FIG. 2 schematically shows the following in relation to the reaction equation in the upper column: (a) The labeling agent does not decompose → applicable to deep tissue, ( b) Labeling when immobilizing tissues and individuals. FIG. 3 shows an example of labeling of AMPAR (AMPA glutamate receptor) in living cells. The upper column of FIG. 3 schematically shows the process in Test Example 1. The column in FIG. 3 shows the chemical structure of the labeling agent used in Test Example 1. A labeling substance contained in the labeling agent is indicated by a circle. The lower column of FIG. 3 shows the result of confocal microscope observation for each of the labeling agents used in Test Example 1. Specifically, the lower column (a) of FIG. 3 shows an image obtained when Compound 1 (Fluorescein) was used as a labeling agent. The juxtaposed image illustrates the result of visualizing the presence of AMPAR by displaying AMPAR in green and the inside of the cell in red. On the other hand, the lower column (b) of FIG. 3 shows an image obtained when Compound 1 (Alexa 647) was used as a labeling agent. The juxtaposed image illustrates the result of visualizing the presence of AMPAR by displaying AMPAR in red and displaying the inside of the cell in green. FIG. 4 illustrates (a) labeling of live cells with mGlu1 (metabotropic glutamate receptor) and (b) labeling with GABA _A R (GABA _A receptor). FIG. 4 (a) shows the chemical structure of the used labeling agent on the left side and an image obtained by visualization on the right side. In the image, mGlu1 visualized in red (the cell surface is colored) and mGlu1 expressing cells visualized in green (the whole cell is colored) are grasped. On the other hand, FIG. 4 (b) also shows the chemical structure of the used labeling agent on the left side and an image obtained by visualization on the right side. In the image, GABA _A R visualized in red (cell surface is colored) and GABA _A R expressing cells visualized in green (the whole cell is colored) are grasped. FIG. 5 is a schematic diagram illustrating an example of the timing of using a labeling agent (Labeling® Reagent) and a fixing agent. This figure illustrates protocols 1 and 2 and illustrates the flexibility of the procedure of the technology of the present disclosure. FIG. 6 shows an example of visualization of mGlu1 in a cerebellar slice using the technique of the present disclosure. FIG. 6 (a) shows the procedure for visualizing the cerebellar slice (thickness 300 μm) and the chemical structure of the labeling agent used. FIG. 6 (b) shows an example of the visualized result in the cerebellar slice, FIG. 6 (c) shows the cerebellar slice schematically shown, and FIG. 6 (d) shows that mGlu1 is a molecule in the cerebellar slice. It indicates that the layer is localized (the arrow indicates the molecular layer). FIG. 7 shows a schematic diagram of a cell recombination agent and a labeled recombination reaction according to one embodiment of the present disclosure. FIG. 8 shows the protocol and results of the immobilization drive labeling method of the present disclosure in hippocampal neurons. Red indicates spines with Alexa647 bound, and green indicates MAP2 protein present in immunostained dendrites. FIG. 9 shows an example of simultaneous staining of multiple receptors in a cerebellar slice. The upper column of FIG. 9 schematically shows the procedure of this test. The column in FIG. 9 shows the chemical structure of the labeling agent for mGlu1 and the labeling agent for AMPAR used in this test. In the lower column of FIG. 9, an image (mGlu1 (FL), AMPAR (Alexa647), superposition of these (Merge) showing the staining result of the receptor together with a schematic diagram showing the functional position of the receptor described above. ) And an image (+ FITM, + NBQX, superposition of these (Merge)) when an inhibitor is used in combination with the labeling agent. FIG. 10 shows an example of labeling for a voltage-dependent calcium channel (VDCC: Voltage-dependent Ca2 + channel) in a cerebellar slice. The upper column of FIG. 10 shows the three-dimensional structure and primary sequence of a VDCC peptide labeling agent (peptide-based labeling reagent for VDCC) as well as the subunit structure of VDCC. The lower column of FIG. 10 shows the result of imaging in the presence of a competitive inhibitor in the same system as the result of visualization of the channel in the cerebellar slice (confocal microscope when using a labeling reagent). Show FIG. 11 shows the protocol and results of labeling to mGlu1 in the whole cerebellum. The upper column of FIG. 11 shows a photograph of the whole mouse cerebellum taken out after the treatment together with the protocol. The lower column of FIG. 11 shows the relationship between the distance (unit: mm) from the injection point (injection point) of the labeling agent and the visualization result (image) of mGlu1 in the sliced tissue section.

The fixing agent used in one embodiment of the present disclosure can be the one used for tissue and organ fixation. Specific examples include suberic acid dialkyl esters such as formaldehyde, paraformaldehyde, glutaraldehyde, glyoxal, methylglyoxal, and dimethyl suberimidate, and these may be used alone or in combination of two or more. Can do. Known conditions can be applied as specific conditions (concentration of fixing agent, temperature, time, etc.) for immobilizing an organ, tissue, or whole animal (whole animal).

The cell surface receptor to be labeled is typically one in which part or all of the cell surface receptor is exposed on the outer surface of the cell membrane and can interact with substances outside the cell membrane. Does not contain receptors. These cell surface receptors can function as cell recognition, signal transduction, mass transport into and out of cells, and a site of action of drugs. As used herein, the term cell surface receptor includes ion channels. As ion channels, voltage-dependent calcium channels (VDCC: Voltage-dependent Ca2 + channel), voltage-dependent potassium channels, voltage-dependent sodium channels, calcium-activated potassium channels, HCN (Hyperpolarization-activated cyclic nucleotide-gated) channels , CNG (cyclic nucleotide-gated) channel, voltage-gated proton channel, two-pore channel, inward rectifier potassium channel, acid-sensitive ion channel (Acid-sensing channel; ASIC), epithelial sodium channel (epithelial Na + channel; ENaC), chloride channel, ligand-dependent channel (Cys-loop channel, glutamate receptor, P2X receptor) and the like.

In one embodiment of the present disclosure, the cell surface receptor to be fluorescently labeled is any receptor, ion channel to be labeled, and is not particularly limited. For example, a neurotransmitter receptor, G protein-coupled receptor Body (GPCR), tyrosine kinase receptor, voltage-dependent Ca2 + channel (VDCC) and the like, specifically, but not limited to the following receptors:
GABA receptor, NMDAR1 receptor, NGF receptor, folate receptor, glycine receptor, selectin receptor, erythropoietin receptor, glucagon receptor, muscarinic acetylcholine receptor (M1, M2, M3, M4, M5), nicotine Sex acetylcholine receptor, adenosine receptor (A1, A2A, A2B, A3), adrenergic receptor (α1A, α1B, α1D, α2A, α2B, α2C, β1, β2, β3), angiotensin receptor (AT1, AT2), Bombesin receptors (BB1, BB2, BB3), bradykinin receptors (B1, B2), calcitonin / iniline / CGRP and adrenomedrin receptors, cannabinoid receptors (CB1, CB2), chemokine receptors (CCR1, CCR2, CCR3, CCR4) , CCR5, CCR6, CR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CX3CR1, XCR1), keniotactic peptide receptors (C3a, C5a, fMLP), cholecystokinin and gastrin receptors (CCK1, CCK2), Corticotropin releasing factor receptor (CRF1, CRF2), dopamine receptor (D1, D2, D3, D4, D5), endothelin receptor (TA, ETB), galanin receptor (GAL1, GAL2, GAL3), glutamate receptor ( mGlu1, mGlu2, mGlu3, mGlu4, mGlu5, mGlu6, mGlu7, mGlu8), glycoprotein hormone receptors (FSH, LSH, TSH), histamine receptors (H1, H2, H3, H4), 5-HT ( Rotonin) receptor (5-HT1A, 5-HT1B, 5-HT1D, 5-ht1B, 5-ht1F, 5-HT2A, 5-HT2F, 5-HT2C, 5-HT3, 5-HT4, 5-ht5A, 5 -Ht5B, 5-HT6, 5-HT7), leukotriene receptors (BLT, CysLT1, CysLT2), lysophospholipid receptors (edg1, edg2, edg3, edg4), melanocortin receptors (MC1, MC2, MC3, MC4) MC5), melatonin receptors (MT1, MT2, MT3), neuropeptide Y receptors: Y1, Y2, Y4, Y5, Y6), neurotensin receptors (NTS1, NTS2), opioid receptors (DOP, KOP, MOP, NOP), P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y) 1, P2Y12), prostanoid receptor (DP, FP, IP, TP, EP1, EP2, EP3, EP4), protease activated receptor (PAR1, PAR2, PAR3, PAR4), somatostatin receptor (sst1, sst2, sst3, sst4, sst5), tahikinin receptors (NK1, NK2, NK3), thyrotropin releasing hormone receptors (TRH1, TRH2), urotensin-II receptors, vasopressin and oxytocin receptors (V1a, V1b, V2, OT), Tyrosine kinase receptor, insulin receptor, voltage-dependent calcium channel (VDCC) and EGF receptor.

Examples of the ligand include a ligand that binds to the ligand binding site of the cell surface receptor. The ligand includes not only a full agonist (eg, acetylcholine with respect to the acetylcholine receptor) that is originally bound to the receptor, but also other agonists, partial agonists, antagonists and the like as long as it binds to the ligand binding site. Full agonists include, but are not limited to:
・ Muscarinic acetylcholine receptor: acetylcholine, muscarinic / adenosine receptor: adenosine, caffeine / adrenergic receptor: adrenaline, noradrenaline / GABA receptor: GABA
Angiotensin receptor: Angiotensin cannabinoid receptor: Cannabis component and anandamide Cholecystokinin receptor: Cholecystokinin dopamine receptor: Dopamine glucagon receptor: Glucagon histamine receptor: Histamine opioid receptor: Cocaine Opium components such as morphine and heroin and endogenous peptide ligands (enkephalins, endorphins, etc.)
・ Secretin receptor: Secretin ・ Serotonin receptor: Serotonin ・ Somatostatin receptor: Somatostatin ・ Gastrin receptor: Gastrin ・ Erythropoietin receptor: Erythropoietin ・ Insulin receptor ・ Nicotinic acetylcholine receptor: Acetylcholine, Nicotine glycine receptor: Nerve Glycine as a transmitter, strykinin / folate receptor (FR): folate / EGF receptor (EGFR): epidermal growth factor (EGF)

A cell-containing biological sample (hereinafter sometimes abbreviated as “biological sample”) is an organism (single cell organism, multicellular organism) itself, or a unit constituting the organism (ie, a multicellular organism). Units that formally distinguish from the surroundings and bear the function of the unit as a whole, such as tissues (structural units composed of multiple cells), organs (multiple tissues) Is a structural unit composed of Examples of multicellular organisms include, but are not limited to, animals (generally multicellular organisms with motor skills and sensations) in one embodiment. Examples of animals include, but are not limited to, mammals. Such cell-containing biological samples derived from such animals include, but are not limited to, animals themselves, animal tissues, organs, and the like. Examples of mammals include, but are not limited to, humans, mice, rats, rabbits, hamsters, goats, cows, horses, pigs, dogs, cats, monkeys, chimpanzees and the like. In certain embodiments, examples of such mammals include humans, mice, and rats, and humans may be more preferable. In one aspect, when the labeling agent is administered systemically to a living mammal, systemic cell surface receptors can be labeled and when the labeling agent is administered locally to the living mammal. Can label around the local administration site.

In one embodiment of the present disclosure, when two or more labeling agents are applied to a biological sample, a plurality of cell surface receptors can be visualized simultaneously. In this case, the labeling substances of the two or more labeling agents preferably have different wavelengths of fluorescence, and the difference in fluorescence wavelength between the two or more labeling substances is preferably 30 nm or more, more preferably 40 nm or more.

Examples of animal organs and tissues include parts of the central nervous system (cerebellum, cerebral cortex, amygdala, hippocampus, striatum, substantia nigra, thalamus, hypothalamus, midbrain, bridge, medulla, spinal cord, etc.) , Spinal cord, pituitary, stomach, pancreas, kidney, liver, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, gastrointestinal tract, blood vessel, heart, thymus, spleen, submandibular gland, prostate, testis, testis, ovary , Placenta, uterus, breast, teeth, bones, joints, skeletal muscle, etc., or a portion thereof. Tissues and organs include thin samples such as sliced slices, but even a thick biological sample may be labeled to a deeper depth using the method of the present disclosure.

Examples of the labeling substance (Fl) include, but are not limited to, fluorescent substances. Further specific examples of labeling substances include, but are not limited to, biotin, 5-carboxyfluorescein or 6-carboxyfluorescein (FAM), VIC, NED, IRD-700 / 800, Alexa-350 , Alexa-405, Alexa-430, Alexa-488, Alexa-500, Alexa-514, Alexa-532, Alexa-546, Alexa-555, Alexa-568, Alexa-594, Alexa-610, Alexa-633, Alexa -647, Alexa-660, Alexa-680, Alexa-700, Alexa-750, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, BODIPY 505/515, Atto390, Atto425, Atto465, Atto488, Atto495, Atto520, Atto532, Atto550, Atto565, Atto590, Atto594, Atto620, Atto633, Atto 647N, Atto655RhoG6, Atto Rho11, Atto Rho12, Atto Rho101, BMN-5, BMN-6, CEQ8000 D2, CEQ8000 D3, CEQ8000D DY-480XL, DY-485XL, DY-495, DY-505, DY-510XL, DY-521XL, DY-521XL, DY-530, DY-547, DY-550, DY-555, DY-610, DY- 615, DY-630, DY-631, DY-633, DY-635, DY-647, DY-651, DY-675 DY-676, DY-680, DY-681, DY-700, DY-701, DY-730, DY-731, DY-732, DY-750, DY-751, DY-776, DY-780, DY- 781, DY-782, ethidium bromide, acridinium dye, carbazole dye, phenoxazine dye, porphyrin dye, polymethine dye, PET, fluorescein, fluorescein inthiocyanate (FITC), xanthene, 6-carboxy-2 ', 4', 7 ', 4,7-hexachlorofluorescein (HEX), 6-carboxy-1,4-dichloro-2', 7'-dichlorofluorescein (TET), 6-carboxy-4 ', 5'-dichloro-2', 7'-dimethoxyfluorescein (JOE), N, N, N ', N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 5-carboxyrhodamine-6G (R6G5) , 6-carboxyrhodamine-6G (RG6), rhodamine, rhodamine green, rhodamine red, rhodamine 110, Rhodamin 6G, Oregon green, U Berylferon, eosin isothiocyanate, PE, ATTO655, CypHer5E, Rhodamine B, BODIPY 580/605, Texas Red, California Red, Yakima Yellow, APC, TET, CAL FluorOrange560, CAL FluorGRed590, CAL Fluor Red610CAL , IRDye700Dx, IRDye800CW, Marina Blue, Pacific Blue, LC Red610, LC Red640, LC Red670, LC Red705, indocyanine green, lanthanoid complexes such as europium and samarium. In certain embodiments, the labeling substance may be preferably Alexa-488, Oregon green, Alexa-546, Alexa-568, ATTO655, CypHer5E.

The at least one reactive group of the labeling agent which reacts with the immobilized _{agent, OH, SH, NH 2,} NHNH 2, and CO-NHNH ₂ include, but are not limited to. In one embodiment, the reactive group is NH ₂ or CO—NHNH ₂ , and in another embodiment, the reactive group is NH ₂ .

The reactive groups of the cell surface receptor that reacts with the immobilized agent, OH, SH, and NH ₂ and the like.

The labeling agent has at least one reactive group (●) that reacts with a ligand (Lg; ligand) for the cell surface receptor, a labeling substance (Fl), and an immobilizing agent. The labeling agent usually has one ligand.

In an exemplary embodiment of the labeling agent of the present disclosure, the ligand (Lg) and the labeling substance (Fl) are bound via a divalent linking group (Z), and a reactive group (●) is bound to the linking group. Further combined.

The number (n) of reactive groups (“●” in FIG. 2) of the cell surface receptor that reacts with the immobilizing agent may be 1 or more (for example, 2, 3, or 4). There may be. The number of reactive groups (●) on the cell surface receptor and the number (n) of reactive groups (●) on the labeling agent are usually the same. This is because the immobilizing agent has a function of linking the reactive group (●) of the cell surface receptor and the reactive group (●) of the labeling agent in a one-to-one relationship (FIG. 2).

In an exemplary embodiment of the present disclosure, the labeling agent may be represented by, for example, the following formula (I).

(In the formula, Lg represents a ligand, Fl represents a labeling substance, ● represents a reactive group, Z represents a divalent linking group, n represents an integer of 1 to 4, Ya and Yb represent (The same or different, CONH or NHCO is indicated.)
The divalent linking group represented by Z is not particularly limited, and any divalent linking group can be used.

Examples of the divalent linking group represented by Z include, for example, a linear or branched alkylene group having 2 to 20 carbon atoms, an arylene group, an aralkylene group, an ether group, a group derived from an amino acid or an oligopeptide, or a group thereof. Combinations are mentioned, and these groups may be intervened by one or two or more divalent hetero groups (CONH, NHCO, O, S, CO, NH). Specific examples of the divalent linking group represented by Z are shown below.

(In the formula, m1 represents an integer of 1 to 20, and m2 represents an integer of 1 to 10. R ¹ , R ² and R ³ are the same or different and are on the side of 20 natural amino acids constituting a protein. Indicates a chain.)
Twenty natural amino acids constituting the protein include Glu, Asp, Gln, Asn, Gly, Ala, Ser, Thr, Cys, Met, Leu, Ile, Val, Phe, Tyr, Trp, His, Arg, Lys, Pro.

The arylene group and aralkylene group may have a substituent. As substituents, alkyl, cycloalkyl, alkoxy, halogen atoms (F, Cl, Br, I), OH, CN, NO ₂ , COOH, NH ₂ , phenyl, benzyl, acetylamino, acetyl, acetyloxy, methoxycarbonyl , Ethoxycarbonyl, butoxycarbonyl, trifluoromethyl, and the like.

Alkyl includes linear or branched C _1-18 alkyl such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, hexyl and the like. .

Cycloalkyl includes C _3-10 cycloalkyl such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. A part of the ring may be substituted with a hetero element or may have a substituent.

Alkoxy includes linear or branched methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, n-pentyloxy, isopentyloxy, hexyloxy, polyethylene glycol derivatives, etc. C _1-18 Alkoshi.

The labeling agent of the present disclosure can be synthesized according to the following Scheme A-Scheme F.

(In the formula, Lg, Fl, Z, Ya, Yb, ●, and n are as defined above. Z1 and Z2 are the same or different and represent a divalent linking group. N1 is an integer of 0 or more. N2 represents an integer of 0 or more, and n1 + n2 = n, Pro represents a protecting group, R represents OH, Cl, Br, methoxy, ethoxy, propoxy, butoxy, phenoxy, benzyloxy or cyanomethyloxy N3 is 1 when ● is NH ₂ , NHNH ₂ , CO—NHNH ₂ , and is 0 when ● is OH or SH.)
Protecting groups represented by Pro include Boc (tert-butoxycarbonyl), Cbz (benzyloxycarbonyl), Fmoc (9-fluorenylmethyloxycarbonyl), Alloc (allyloxycarbonyl), Troc (2,2, 2-trichloroethoxycarbonyl), TMS-ethylcarbonyl, cyanoethylcarbonyl and the like.

The first reactions of Schemes A to F are all amide bond (CONH) formation reactions. In this reaction, about 0.8 to 1.2 mol of the compound (2, 4, 6, 8, 10, or 12) is used per 1 mol of the compound (1, 3, 5, 7, 9, or 11). The compound of the formula (Ia), (Ib) or (Id) proceeds advantageously by reacting in a solvent in the presence of a condensing agent, if necessary, from room temperature to the boiling temperature of the solvent for about 1 to 24 hours. Get. Solvents include dimethylformamide, tetrahydrofuran, dioxane, diethyl ether, diisopropyl ether and other ethers, dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane and other halogenated hydrocarbons, benzene, toluene, xylene and other aromatics Examples include hydrocarbons, aliphatic hydrocarbons such as hexane, acetonitrile, methanol, ethanol, and the like. Examples of the condensing agent include DCC, water-soluble carbodiimide (WSC), DIC, carbonyldiimidazole (CDI), DMT-MM, etc. In combination with 1-hydroxybenzotriazole (HOBt), hydroxysuccinimide (HOSu), etc. Also good. What is necessary is just to use a condensing agent 1 mol-excess amount with respect to 1 mol of compounds (1,3,5,7,9 or 11).

The target compound of formula (I) can be obtained by deprotecting the compound of formula (Ia) obtained in Schemes B to D in the same manner as in scheme 1.

The deprotection reaction in Scheme A, E, F can be deprotected according to the well-known deprotection conditions of each protecting group. For example, the Boc group can be deprotected under strongly acidic conditions such as trifluoroacetic acid, HCl-ethyl acetate solution, HCl-dioxane solution, Cbz can be deprotected by hydrogenation reaction with Pd / C, Birch reduction, etc. Fmoc It can be deprotected with a secondary amine such as piperidine, Troc can be deprotected with zinc powder-acetic acid, etc., and Alloc can be deprotected with the addition of an amine in the presence of a palladium catalyst. Protecting groups other than these can also be deprotected according to conventional methods.

-Labeling method of biological sample with labeling agent A biological sample can be labeled by allowing a labeling agent and an immobilizing agent to act on a biological sample containing cells expressing a receptor.

The biological sample labeling method includes the following steps (a) and (b).
(A) applying a fixing agent to the cell-containing biological sample, and (b) applying a labeling agent to the cell-containing biological sample,
Here, step (b) may be performed after step (a), step (a) may be performed after step (b), and step (a) and step (b) are performed simultaneously. Also good.

The amount of the immobilizing agent used for 100 g of the biological sample is about 1 to 300 mg.

The amount of labeling agent used for 100 g of biological samples is about 1 to 10 mg.

An excessive amount of the fixing agent and the labeling agent may be used with respect to the biological sample.

Step (a) may be performed with an unfixed biological sample or may be performed with an immobilized biological sample.

When the cell-containing biological sample used in step (a) is not fixed, a fixing agent is applied to the biological sample. Application of the immobilizing agent to the biological sample can be performed according to a conventional method. For example, the step (a) can be performed by immersing the biological sample in the immobilizing agent solution. The application temperature of the fixing agent is about 0 ° C. to 37 ° C., and the application time of the fixing agent is about 1 to 48 hours.

When using a biological sample that has already been immobilized, step (a) provides a cell-containing biological sample that has been immobilized in advance with an immobilizing agent, or the immobilization together with the biological sample that has been immobilized in advance with the immobilizing agent. It can be carried out either by further coexisting the agent.

Step (b) can be performed by dissolving the labeling agent in a solvent and applying it to the biological sample. When performing step (b) on a biological sample that has not been immobilized, the biological sample is immersed in a solution containing a labeling agent and reacted at a temperature of about 0 ° C. to 37 ° C. for about 1 to 48 hours to label the biological sample. The ligand of the agent can be bound to the ligand binding site of the cell surface receptor. Excess labeling agent is removed from the biological sample by washing. When step (b) is performed before step (a), since there is no immobilizing agent, the labeling agent is not covalently bound to the cell surface receptor in step (b), but after step (b) By performing the step (a) of applying the immobilizing agent, the labeling agent can be covalently bound to the cell surface receptor.

Steps (a) and (b) are performed by immersing an unfixed biological sample in a solution containing both the immobilizing agent and the labeling agent and reacting at a temperature of about 0 ° C. to 37 ° C. for about 1 to 48 hours. ) Can be performed simultaneously.

Since immobilization of a biological sample with an immobilizing agent is a reversible equilibrium reaction, when a labeling agent is allowed to act on an immobilized biological sample, a part of the immobilizing agent bound to the reactive group of the receptor. Is separated from the biological sample, and the newly exposed receptor reactive group and the reactive group of the labeling agent are covalently bonded to the immobilizing agent, whereby the receptor can be labeled with the labeling agent. As shown in FIG. 7, the labeling agent is strongly bound to the receptor (“Protein” in FIG. 7) by the ligand. This substitution or recombination reaction takes place (Figure 7). As a solution containing a labeling agent and, if necessary, a fixing agent, a buffer solution having a pH of about 4 to 7 can be used. The labeling agent is preferably used in excess relative to the receptor. The labeling agent labels the receptor when the receptor and the ligand (Lg) are bound, but non-specific labeling is suppressed. After the labeling reaction is completed, excess labeling agent can be removed by immersing the labeled biological sample in a solution containing no labeling agent. This washing operation can be repeated until the labeling agent is removed.

Numerous literatures have been published regarding the method of making a biological sample transparent, and for example, the following literature can be referred to.
Title: Tainaka K et al, Cell. 2014; 159: 911-24, Chung K et al, Nature. 2013; 497: 332-7, Hama H et al, Nat Neurosci. 2011; 14: 1481-8, Renier N et al, Cell. 2014; 159: 896-910, Hama H et al, Nat Neurosci. 2015; 18: 1518-29.
By making the biological sample transparent, it becomes easier to understand the labeling situation. It is also possible to label a plurality of receptors with labeling substances that can be recognized separately by wavelength or the like. The biological sample may be clarified before labeling or after labeling.

Solvents are solvents in which water or compound (I) can be dissolved, for example, lower alcohols such as methanol, ethanol, propanol, acetone, tetrahydrofuran (THF), dioxane, N-methylpyrrolidone, dimethylformamide (DMF), acetonitrile, dimethyl Water miscible solvents such as acetamide and dimethyl sulfoxide (DMSO) can be mentioned, but are not limited thereto. Examples of the buffer include, but are not limited to, HEPES buffer, phosphate buffer, Tris buffer, and the like.

Hereinafter, the items disclosed in the present specification will be described in more detail using examples.
Example 1

Scheme 1. Synthesis of Compound 1 (Fluorescein)

Compound 7
Compound 6 (30 mg, 67 μmol) (ref. 1) was dissolved in 1.5 ml of dry DMF. Under nitrogen atmosphere, WSC-HCl (166 mg, 870 μmol), HOBt-H ₂ O (120 mg, 870 μmol), 4- (tert-Butoxycarbonyl) aminobutyric acid 17.7 mg (87 μmol), DIEA 221 μl (1.27 mmol) was added and stirred at room temperature for 5 hours. After the solvent was distilled off, purification was performed by column chromatography (silica, CHCl ₃ / MeOH = 15/1) to obtain 3.4 mg (5.7 μmol) of Compound 7. Nuclear proton magnetic resonance spectroscopy ( ¹ H NMR spectroscopy) (400 MHz, CD ₃ OD) δ 7.61 (s, 1H), 7.30 (s, 1H), 6.81-6.79 (m, 2H), 6.24-6.23 (m, 1H), 5.04 (s, 2H), 4,26-4.21 (m, 4H), 3.08-3.05 (m, 2H), 2.22 (t, J = 7.6 Hz, 2H), 1.76 (t, J = 7.2 Hz , 2H), 1.42 (s, 9H), 1.27 (t, J = 7.2 Hz, 3H).

Compound 8
Compound 7 (3.4 mg, 5.7 μmol) was suspended in 1.2 ml of dichloromethane. 0.24 ml of TFA was added and stirred at room temperature for 1 hour. After distilling off the solvent, TFA was carefully removed by azeotropy with toluene to obtain Compound 8 (4.3 mg, 7.1 μmol). ¹ H NMR spectroscopy (400 MHz, CD ₃ OD) δ 7.61 (s, 1H), 7.27 (s, 1H), 6.81-6.80 (m, 2H), 6.24-6.23 (m, 1H), 5.04 (s, 2H ), 4,27-4.21 (m, 4H), 2.99-2.96 (m, 2H), 2.39-2.36 (m, 2H), 1.95-1.91 (m, 2H), 1.27 (t, J = 7.2 Hz, 3H ).

Compound 9
5-Carboxyfluorescein diacetate N-succinimidyl ester (50 mg, 90 μmol) (ref. 2) was dissolved in 2.7 ml of dichloromethane. Under a nitrogen atmosphere, N ^6- (tert-Butoxycarbonyl) -L-lysine (26.4 mg, 107 μmol) and TEA 37.8 μl (269 μmol) were added, and the mixture was stirred at room temperature for 4 hours. Next, N ^6- (tert-Butoxycarbonyl) -L-lysine (6.6 mg, 27 mmol) was added to this solution, and the mixture was stirred at room temperature for 2 hours under a nitrogen atmosphere. Thereafter, dichloromethane (30 mL) was added, and the organic layer was washed with 0.01 M HCl and 10 mL saturated brine, dried over Na ₂ SO ₄ , and evaporated under reduced pressure to obtain 48.3 mg of a crude compound. The obtained product was directly used for the next reaction.

The crude compound (48.3 mg) was dissolved in 1.4 ml of THF, 0.42 ml (210 μmol) of 0.5 M lithium hydroxide aqueous solution was added, and the mixture was stirred at room temperature for 3 hours. Next, 0.42 ml (210 μmol) of 0.5 M lithium hydroxide aqueous solution was added to this solution, and the mixture was stirred at room temperature for 5 hours. After neutralizing with 1N hydrochloric acid, the mixture was dried under reduced pressure. The obtained solid was purified by HPLC (acetonitrile (0.1% TFA) / water (0.1% TFA)) and dried to obtain Compound 9 (9.8 mg, 16.2 μmol). ¹ H NMR spectroscopy (400 MHz, CD ₃ OD) δ 8.55 (s, 1H), 8.27-8.24 (m, 1H), 7.36 (d, J = 8.0 Hz, 1H), 6.80 (s, 2H), 6.75 ( d, J = 8.8 Hz, 2H), 6.75 (d, J = 8.8 Hz, 2H), 6.65 (d, J = 9.2 Hz, 2H), 4.64 (t, J = 4.8 Hz, 1H), 3.07-3.05 ( m, 2H), 1.79-1.75 (m, 2H), 1.57-1.53 (m, 4H), 1.40 (s, 9H).

Compound 1 (Fluorescein)
Compound 8 (4.3 mg, 7.1 μmol) was dissolved in 0.5 ml of dry DMF. HBTU 3.96 mg (10.6 μmol), Compound 9 (6.4 mg, 10.6 μmol) and DIEA 4.98 μl (28.4 μmol) were added, and the mixture was stirred at room temperature for 10 hours. The solvent was distilled off to obtain 19.5 mg of a crude compound.

The obtained crude compound (19.5 mg) was dissolved in 0.14 ml of THF, and 0.14 ml (71 μmol) of 0.5 M aqueous solution of lithium hydroxide was added and stirred at room temperature for 8.5 hours. The mixture was neutralized with 1N hydrochloric acid and then dried under reduced pressure to obtain 27.7 mg of crude compound.

The crude compound (27.7 mg) was suspended in 1.5 ml of dichloromethane. 0.3 ml of TFA was added and stirred at room temperature for 1 hour. After the solvent was distilled off, TFA was carefully removed by azeotropy with toluene. The obtained crude compound was purified by HPLC (acetonitrile (0.1% TFA) / water (0.1% TFA)) and dried to obtain Compound 1 (Fluorescein) (1.29 mg, 1.21 μmol). ¹ H NMR spectroscopy (400 MHz, CD ₃ OD) δ 8.52 (d, J = 0.8 Hz, 1H), 8.26-8.23 (m, 1H), 7.59 (m, 1H), 7.31-7.29 (m, 1H), 7.23 (m, 1H), 6.70-6.69 (m, 2H), 6.59-6.56 (m, 2H), 6.52-6.50 (m, 2H), 6.21-6.20 (m, 1H), 5.01 (s, 1H), 4.57-4.53 (m, 1H), 4.22 (s, 2H), 2.96-2.94 (m, 2H), 2.86-2.85 (m, 2H), 2.28 (t, J = 7.6 Hz, 2H), 1.93 (t, J = 7.6 Hz, 2H), 1.89-1.82 (m, 2H), 1.74-1.72 (m, 2H), 1.59-1.51 (m, 2H).

Scheme 2. Synthesis of Compound 1 (Alexa647)

Compound 10
N-α-Benzyloxycarbonyl-N-ε-tert-butoxycarbonyl-L-lysine (0.5 g, 1.31 μmol) was dissolved in 13 ml of dry DMF. Add WSC-HCl 330 mg (1.71 μmol), HOBt-H ₂ O 260 mg (1.71 μmol), 4-Aminobutyric acid methyl ester hydrochloride 240 mg (1.58 μmol), DIEA 183 μl (5.26 μmol), and add 10 at room temperature. Stir for hours. After the solvent was distilled off, AcOEt (60 mL) was added to the crude compound. The organic layer was washed with 0.01 M HCl (20 ml x 2), saturated aqueous NaHCO ₃ solution (20 mL x 2) and saturated brine (20 ml), dried over Na ₂ SO ₄ and compound 10 (546 mg, 1.14 μmol) was added. Obtained as a colorless oil. ¹ H NMR spectroscopy (400 MHz, CD ₃ OD) δ 7.38-7.29 (m, 5H), 5.08 (s, 2H), 4.02-4.00 (m, 1H), 3.65 (s, 3H), 3.32-3.19 (m , 2H), 3.03-2.99 (m, 2H), 2.37-2.33 (m, 2H), 1.80-1.72 (m, 2H), 1.63-1.60 (m, 2H), 1.42 (s, 9H), 1.37-1.32 (m, 4H).

Compound 11
Compound 10 (393 mg, 0.82 mmol) was dissolved in THF 8.2 ml, 1 M aqueous sodium hydroxide solution 0.82 ml (0.82 mmol) was added, and the mixture was stirred at room temperature for 1 hour. Next, 0.82 ml (0.82 mmol) of 1 M aqueous sodium hydroxide solution was added to this solution and stirred at room temperature for 1 hour. Next, 0.82 ml (0.82 mmol) of 1 M aqueous sodium hydroxide solution was added to this solution and stirred at room temperature for 1 hour. Next, 0.82 ml (0.82 mmol) of 1 M aqueous sodium hydroxide solution was added to this solution and stirred at room temperature for 1 hour. After neutralizing with 1N hydrochloric acid, the mixture was dried under reduced pressure. Chloroform (60 ml) was added to the obtained solid, and the product was extracted into the organic layer. The organic layer was washed with 0.1 M HCl (20 ml × 2) and saturated brine (20 mL), and dried over Na ₂ SO ₄ . The obtained crude compound was purified by column chromatography (silica, CHCl ₃ / MeOH = 30/1 + 1% AcOH) to obtain Compound 11 (64 mg, 0.13 mmol). ¹ H NMR spectroscopy (400 MHz, CD ₃ OD) δ 7.38-7.27 (m, 5H), 5.08 (s, 2H), 4.04-4.01 (m, 1H), 3.24-3.21 (m, 2H), 3.03-2.98 (m, 2H), 2.34-2.30 (m, 2H), 1.82-1.71 (m, 3H), 1.65-1.62 (m, 1H), 1.42 (s, 9H), 1.37-1.32 (m, 4H).

Compound 12
Compound 6 (30 mg, 67 μmol) (ref. 1) was dissolved in 2.0 ml of dry DMF. Under a nitrogen atmosphere, add WSC-HCl 16.7 mg (87 μmol), HOBt-H ₂ O 13.4 mg (87 μmol), Compound 11 (42.5 mg, 87 μmol), DIEA 94 μl (269 μmol), and Stir for 8 hours. Compound 11 (6.75 mg, 13.4 μmol) was then added to this solution and stirred at room temperature for 2 hours. After the solvent was distilled off, chloroform (60 mL) was added to the crude compound. The organic layer was washed with 0.01 M HCl (20 ml x2), saturated aqueous NaHCO ₃ solution (20 mL x2) and saturated brine (20 ml), dried over Na ₂ SO ₄ and column chromatography (silica, CHCl ₃ / MeOH = 12/1, 10/1) to obtain 28.6 mg (33 μmol) of Compound 12. ¹ H NMR spectroscopy (400 MHz, CD ₃ OD) δ 7.59 (s, 1H), 7.33-7.26 (m, 6H), 6.81-6.78 (m, 2H), 5.05-5.04 (m, 2H), 4.26 (s , 2H), 4.25-4.20 (m, 2H), 4.01-3.98 (m, 1H), 3.21-3.19 (m, 2H), 3.03-2.99 (m, 2H), 2.23-2.20 (m, 2H), 1.83 -1.72 (m, 3H), 1.66-1.57 (m, 1H), 1.45 (s, 1H), 1.37-1.32 (m, 4H), 1.24 (t, J = 7.2 Hz, 3H).

Compound 13
Compound 12 (25.3 mg, 29.4 μmol) was dissolved in 1.0 ml of dry methanol. Under a nitrogen atmosphere, 10 mg of 10% Pd / C was added, and the mixture was stirred at room temperature for 4 hours under a hydrogen atmosphere. After removing palladium carbon by filtration, the solvent was distilled off to obtain 28.3 mg of Compound 12. ¹ H NMR spectroscopy (400 MHz, CD ₃ OD) δ 7.61 (s, 1H), 7.24 (s, 1H), 6.80-6.78 (m, 2H), 6.23-6.22 (m, 1H), 5.04 (s, 2H ), 4.26-4.20 (m, 4H), 4.14-4.06 (m, 1H), 3.23-3.19 (m, 2H), 3.04-3.00 (m, 2H), 2.27-2.22 (m, 2H), 1.82-1.79 (m, 2H), 1.67-1.54 (m, 1H), 1.51-1.47 (m, 2H), 1.45 (s, 9H), 1.38-1.34 (m, 2H), 1.24 (t, J = 7.2 Hz, 3H ).

Compound 14
Compound 13 (28.3 mg, 39 μmol) was dissolved in 1.1 ml of THF, 0.32 ml (117 μmol) of 0.5 M lithium hydroxide aqueous solution was added, and the mixture was stirred at room temperature for 3.5 hours. After neutralizing with 1N hydrochloric acid, the mixture was dried under reduced pressure. The obtained solid was dissolved in CHCl ₃ / MeOH = 2/1, filtered and the solvent was distilled off to obtain 32.8 mg of compound 14. ¹ H NMR spectroscopy (400 MHz, CD ₃ OD) δ 7.62 (s, 1H), 7.06 (s, 1H), 6.81-6.77 (m, 2H), 6.19-6.18 (m, 1H), 4.76 (s, 2H ), 3.28-3.27 (m, 1H), 3.25-3.21 (m, 2H), 3.04-3.00 (m, 2H), 2.28-2.22 (m, 2H), 1.84-1.81 (m, 2H), 1.64-1.49 (m, 2H), 1.45 (s, 9H), 1.37-1.28 (m, 4H).

Compound 1 (Alexa647)
Compound 14 (1.0 mg, 1.46 μmol) was dissolved in 0.1 ml of dry DMF. Under a nitrogen atmosphere, Alexa647-NHS-ester (1 mg) and DIEA 1.09 μl (3.13 μmol) were added, and the mixture was stirred at room temperature for 4 hours. DIEA 1.09 μl (3.13 μmol) was then added to the solution and stirred at room temperature for 4.5 hours. The solvent was distilled off to obtain 3.0 mg of a crude compound. The obtained product was directly used for the next reaction.

3.0 mg of crude compound was suspended in 1.0 ml of dichloromethane. 0.2 ml TFA was added and stirred at room temperature for 1 hour. After the solvent was distilled off, TFA was carefully removed by azeotropy with toluene. The obtained product was purified by HPLC (acetonitrile / 10 mM ammonium acetate aqueous solution) and lyophilized to obtain the target compound 1 (Alexa647). ESI-Mass ([M-2H] = 716.6962 (obs.), 716.6977 (calc.))

Scheme 3. Synthesis of Compound 2 (Fluorescein)

Compound 15
3- (tert-Butoxycarbonylamino) propionic acid (0.5 g, 4.8 mmol) was dissolved in 48.4 mL of dry DMF. Under nitrogen atmosphere, add WSC-HCl 1.2 g (6.3 mmol), HOBt-H ₂ O 0.87 g (6.3 mmol), β-Alanine methyl ester (0.6 g, 5.8 mmol), DIEA 4.2 ml (24 mmol) And stirred at room temperature for 5 hours. After the solvent was distilled off, AcOEt (100 mL) was added to the crude compound. The organic layer was washed with 10% citric acid (30 ml x2), saturated aqueous NaHCO ₃ solution (20 mL x2) and saturated brine (30 ml), dried over Na ₂ SO ₄ and compound 15 (0.43 g, 1.57 mmol). ) Was obtained as a white solid. ¹ H NMR spectroscopy (400 MHz, CD ₃ OD) δ 6.14 (s, 1H), 5.15 (s, 1H), 3.71 (s, 3H), 3.51 (q, J = 6.0 Hz, 2H), 3.39 (q, J = 6.0 Hz, 2H), 2.55 (t, J = 6.0 Hz, 2H), 2.37 (t, J = 6.0 Hz, 2H), 1.43 (s, 9H).

Compound 16
Compound 15 (0.35 g, 1.3 mmol) was suspended in 10 ml of dichloromethane. TFA 3.0 ml was added and it stirred at room temperature for 1 hour. After the solvent was distilled off, TFA was carefully removed by azeotropy with toluene to obtain 493 mg of a crude compound (including 1.3 mmol of compound 16). The obtained compound was directly used in the next reaction. ¹ H NMR spectroscopy (400 MHz, CD ₃ OD) δ 11.3 (s, 1H), 7.95-7.79 (m. 2H), 4.10 (s, 1H), 3.55 (t, J = 6.0 Hz, 2H), 3.35- 3.26 (m, 2H), 2.67 (t, J = 5.6 Hz), 2.55 (t, J = 6.0 Hz, 2H).

Compound 17
473 mg of the crude compound was dissolved in 20 mL of dry DMF. Under nitrogen atmosphere, WSC-HCl 27 mg (0.14 mmol), HOBt-H ₂ O 20 mg (0.14 mmol), N ⁴ -[(Benzyloxy) carbonyl] -N ² -[(tert-butyloxy) -carbonyl]- L-2,4-diaminobutyric acid (0.6 g, 5.8 mmol) and DIEA 189 μl (5.4 mmol) were added, and the mixture was stirred at room temperature for 30 minutes. Next, under a nitrogen atmosphere, WSC-HCl 27 mg (0.14 mmol) and HOBt-H ₂ O 20 mg (0.14 mmol) were added to this solution, and the mixture was stirred at room temperature for 13 hours. After evaporation of the solvent, AcOEt (90 mL) was added to the crude compound. The organic layer was washed with 10% citric acid (30 ml), saturated aqueous NaHCO ₃ solution (30 mL) and saturated brine (30 ml), dried over Na ₂ SO ₄ and compound 17 (195 mg, 0.39 mmol) was obtained. Obtained as a white solid. ¹ H NMR spectroscopy (400 MHz, CD ₃ OD) δ 7.35-7.28 (m. 5H), 5.07 (s, 1H), 4.01 (s, 1H), 3.67 (s, 3H), 3.44-3.38 (m, 4H ), 3.24-3.14 (m, 2H), 2.53 (t, J = 6.8 Hz), 2.36 (t, J = 6.4 Hz, 2H), 1.94-1.69 (m, 2H), 1.44 (s, 9H).

Compound 18
Compound 17 (51 mg, 0.1 mmol) was dissolved in 1.0 ml of THF, 0.5 ml of 1 M aqueous sodium hydroxide solution was added, and the mixture was stirred at room temperature for 5 hours. Chloroform 5.0 ml, pure water 5.0 ml, and 1 M HCl 0.5 ml were added to the reaction solution. Next, 10 ml of chloroform, 10 ml of pure water and 5.0 ml of saturated saline were added to separate the organic layer and the aqueous layer. The aqueous layer was extracted with chloroform (15 ml × 2), and the organic layer was recovered. Drying with Na ₂ SO ₄ gave compound 18 (44.9 mg, 0.09 mmol). ¹ H NMR spectroscopy (400 MHz, CD ₃ OD) δ 7.35-7.30 (m. 5H), 5.07 (s, 1H), 4.03-3.99 (m, 1H), 3.45-3.39 (m, 4H), 3.23-3.14 (m, 2H), 2.50 (t, J = 6.8 Hz), 2.37 (t, J = 6.4 Hz, 2H), 1.92-1.69 (m, 2H), 1.44 (s, 9H).

Compound 19
Compound 18 (40 mg, 72.6 μmol) was dissolved in 0.5 ml of dry DMF. Under a nitrogen atmosphere, add WSC-HCl 13.9 mg (72.6 μmol), HOBt-H ₂ O 11.1 mg (72.6 μmol), compound 6 (20 mg, 48.4 μmol), DIEA 12.6 μl (0.36 mmol), and Stir for 3.5 hours. After the solvent was distilled off, the residue was purified by column chromatography (silica, CHCl ₃ / MeOH = 8/1) to give 20.3 mg (23 μmol) of compound 19. ¹ H NMR spectroscopy (400 MHz, CD ₃ OD) δ 7.60 (s, 1H), 7.27-6.86 (m. 6H), 6.84-6.78 (m, 2H), 6.24-6.23 (m, 1H), 5.04 (s , 4H), 4.24 (q, J = 7.2 Hz, 4H), 4.02 (s, 1H), 3.44-3.41 (m, 4H), 3.21-3.13 (m, 2H), 2.40 (t, J = 6.8 Hz, 2H), 2.34 (t, J = 6.4 Hz, 2H), 1.92-1.70 (m, 2H), 1.43 (s, 9H), 1.26 (t, J = 6.8 Hz, 3H).

Compound 20
Compound 19 (18.7 mg, 21 μmol) was dissolved in 0.5 ml of methanol. Under a nitrogen atmosphere, 10 mg of 10% Pd / C was added, and the mixture was stirred at room temperature for 4.5 hours under a hydrogen atmosphere. Subsequently, 1.5 ml of methanol was added, 10% Pd / C 10 mg was added under a nitrogen atmosphere, and the mixture was stirred at room temperature for 6.5 hours under a hydrogen atmosphere. After removing palladium carbon by filtration, the solvent was distilled off to obtain 18.9 mg of a crude product. 18.9 mg of the crude product was dissolved in 2.0 ml of dry methanol. Under a nitrogen atmosphere, 10 mg of 10% Pd / C was added, and the mixture was stirred at room temperature for 10 hours under a hydrogen atmosphere. After removing palladium carbon by filtration, the solvent was distilled off to obtain Compound 20 (15.4 mg, 20.4 μmol). ¹ H NMR spectroscopy (400 MHz, CD ₃ OD) δ 7.61 (s, 1H), 7.29 (s. 1H), 6.81 (s, 2H), 6.24 (s, 1H), 5.05 (s, 2H), 4.28- 4.21 (m, 4H), 4.13 (s, 1H), 3.48-3.42 (m, 4H), 3.14-3.13 (m, 2H), 2.44-2.35 (m, 4H), 2.08-1.92 (m, 2H), 1.49-1.45 (m, 9H), 1.27 (t, J = 7.2 Hz, 3H).

Compound 2 (Fluorescein)
Compound 20 (15.4 mg, 20.4 μmol) was dissolved in 0.6 ml of dry DMF. Under a nitrogen atmosphere, 5-Carboxyfluorescein diacetate N-succinimidyl ester (12.5 mg, 22.4 μmol) (ref. 2) and TEA (8.6 μL, 61 μmol) were added, and the mixture was stirred at room temperature for 5 hours. Subsequently, 5-Carboxyfluorescein diacetate N-succinimidyl ester (2.27 mg, 4.1 μmol) (ref. 2) was added under a nitrogen atmosphere, and the mixture was stirred at room temperature for 5 hours. The solvent was distilled off to obtain 35.3 mg of a crude product. The obtained compound was directly used in the next reaction.

35.3 mg of the crude product was dissolved in 0.5 ml of methanol, 0.4 ml of 0.5 M lithium hydroxide aqueous solution was added and stirred at room temperature for 8.5 hours. The mixture was neutralized with 1M hydrochloric acid and then dried under reduced pressure to obtain 37.5 mg of a crude product. The obtained compound was directly used in the next reaction.

37.5 mg of the crude compound was suspended in 4.0 ml of dichloromethane. 0.8 ml of TFA was added and stirred at room temperature for 3 hours. After the solvent was distilled off, TFA was carefully removed by azeotropy with toluene. The obtained crude compound was purified by HPLC (acetonitrile (0.1% TFA) / water (0.1% TFA)) and dried to obtain Compound 2 (Fluorescein) (1.37 mg, 1.25 μmol). ¹ H NMR spectroscopy (400 MHz, CD ₃ OD) δ 8.48 (s, 1H), 8.22-8.21 (m, 2H), 7.58 (s, 1H), 7.19 (s, 1H), 6.77-6.75 (m, 2H ), 6.59-6.49 (m, 6H), 6.19-6.16 (m, 1H), 4.92 (s, 2H), 4.22 (s, 2H), 3.89 (t, J = 6.8 Hz, 1H), 3.49-3.45 ( m, 4H), 3.14-3.12 (m, 2H), 2.44-2.40 (m, 4H), 2.30-2.13 (m, 2H).

Scheme 4. Synthesis of Compound 3 (Fluorescein)

Compound 21
Methyl N-benzyloxycarbonyl-L-glutamate (0.5 g, 1.7 mmol) was dissolved in 17 ml of dry DMF. Under a nitrogen atmosphere, add WSC-HCl 0.42 g (2.2 mmol), HOBt-H ₂ O 0.34 g (2.2 mmol), Boc-hydrazine (0.25 g, 2.0 mmol), DIEA 236 μl (6.77 mmol) at room temperature. For 5 hours. After the solvent was distilled off, AcOEt (60 mL) was added to the crude compound. The organic layer was washed with 0.01 M HCl (20 ml x 2), saturated aqueous NaHCO ₃ solution (20 mL x 2) and saturated brine (20 ml), dried over Na ₂ SO ₄ and compound 21 (635 mg, 1.55 mmol) Was obtained as a white powder. ¹ H NMR spectroscopy (400 MHz, CD ₃ OD) δ 7.36-7.29 (m, 5H), 5.09 (s, 2H), 4.22-4.20 (m, 1H), 3.72 (s, 3H), 2.32-2.30 (m , 2H), 2.19-2.16 (m, 1H), 1.98-1.92 (m, 1H), 1.46 (s, 9H).

Compound 22
Compound 21 (635 mg, 1.55 mmol) was dissolved in 15.5 ml of THF, 7.7 ml of 1 M aqueous sodium hydroxide solution was added, and the mixture was stirred at room temperature for 45 minutes. After neutralizing with 1 M hydrochloric acid, the mixture was dried under reduced pressure. The obtained crude product was dissolved in chloroform (60 ml). The organic layer was washed with 0.1 M HCl (20 ml, x2) and saturated brine (20 ml) and dried over Na ₂ SO ₄ to obtain compound 22 (492 mg, 1.18 mmol) as a white powder. ¹ H NMR spectroscopy (400 MHz, CD ₃ OD) δ 7.38-7.27 (m, 5H), 5.09 (s, 2H), 4.22-4.18 (m, 1H), 2.36-2.34 (m, 2H), 2.24-2.19 (m, 1H), 1.99-1.94 (m, 1H), 1.46 (s, 9H).

Compound 23
Compound 22 (492 mg, 1.18 mmol) was dissolved in 12 ml of dry DMF. Under nitrogen atmosphere, WSC-HCl 293 mg (1.53 mmol), HOBt-H ₂ O 234 mg (1.53 mmol), 4-Aminobutyric acid methyl ester hydrochloride (217 mg, 1.41 mmol), DIEA 165 μl (4.72 mmol) In addition, the mixture was stirred at room temperature for 6 hours. After the solvent was distilled off, AcOEt (60 mL) was added to the crude compound. The organic layer was washed with 0.01 M HCl (20 ml x 2), saturated aqueous NaHCO ₃ solution (20 mL x 2) and saturated brine (20 ml), dried over Na ₂ SO ₄ and compound 23 (550 mg, 1.11 mmol) Was obtained as white crystals. ¹ H NMR spectroscopy (400 MHz, CD ₃ OD) δ 7.36-7.29 (m, 5H), 5.09 (s, 2H), 4.11-4.06 (m, 1H), 3.65 (s, 3H), 3.25-3.21 (m , 2H), 2.38-2.35 (m, 2H), 2.32-2.28 (m, 2H), 2.06-1.93 (m, 2H), 1.82-1.78 (m, 2H), 1.46 (s, 9H).

Compound 24
Compound 23 (550 mg, 1.11 mmol) was dissolved in 11 ml of THF, 1.1 ml of 1 M aqueous sodium hydroxide solution was added, and the mixture was stirred at room temperature for 2 hours. Next, 1.1 ml of 1 M aqueous sodium hydroxide solution was added, and the mixture was stirred at room temperature for 1.5 hours. After neutralizing with 1 M hydrochloric acid, the mixture was dried under reduced pressure. The obtained crude product was dissolved in chloroform (60 ml). The organic layer was washed with 0.01 M HCl (20 ml, x2) and saturated brine (20 ml) and dried over Na ₂ SO ₄ . The resulting crude compound was subjected to column chromatography (silica, CHCl ₃ / MeOH = 10/1 + 1% AcOH → CHCl ₃ / MeOH = 8/1 + 1% AcOH → CHCl ₃ / MeOH = 5/1 + 1% AcOH ) To give 366 mg (0.73 mmol) of compound 24. ¹ H NMR spectroscopy (400 MHz, CD ₃ OD) δ 7.35-7.28 (m, 5H), 5.08 (s, 2H), 4.13-4.11 (m, 1H), 3.25-3.22 (m, 2H), 2.35-2.30 (m, 4H), 2.15-2.00 (m, 1H), 1.95-1.94 (m, 1H), 1.83-1.77 (m, 2H), 1.45 (s, 9H).

Compound 25
Compound 24 (43.7 mg, 87 μmol) was dissolved in 2.0 ml of dry DMF. Under a nitrogen atmosphere, add WSC-HCl 16.7 mg (87 μmol), HOBt-H ₂ O 13.3 mg (87 μmol), Compound 6 (30 mg, 67 μmol), DIEA 94 μl (269 μmol), and Stir for 5 hours. After the solvent was distilled off, chloroform (60 mL) was added to the crude compound. The organic layer was washed with 0.01 M HCl (20 ml x2), saturated aqueous NaHCO ₃ solution (20 mL x2) and saturated brine (20 ml), dried over Na ₂ SO ₄ and column chromatography (silica, CHCl ₃ / MeOH = 10/1) to obtain 16.1 mg (18.4 μmol) of compound 25. ¹ H NMR spectroscopy (400 MHz, CD ₃ OD) δ 7.60 (s, 1H), 7.36-7.27 (m, 6H), 6.81-6.77 (m, 2H), 6.24-6.23 (m, 1H), 5.04-5.02 (m, 4H), 4.27-4.20 (m, 4H), 4.08-4.05 (m, 1H), 2.32-2.21 (m, 4H), 1.83-1.80 (m, 2H), 1.45 (s, 9H), 1.23 (t, J = 7.2 Hz, 3H).

Compound 26
Compound 25 (16.1 mg, 18.4 μmol) was dissolved in 1.0 ml of dry methanol. Under a nitrogen atmosphere, 10 mg of 10% Pd / C was added, and the mixture was stirred at room temperature for 13 hours under a hydrogen atmosphere. After removing palladium carbon by filtration, the solvent was distilled off to obtain 12.7 mg of compound 26 (17.2 μmol). ¹ H NMR spectroscopy (400 MHz, CD ₃ OD) δ 7.60 (s, 1H), 7.11 (s, 1H), 6.79-6.77 (m, 2H), 6.21 (s, 1H), 5.05 (s, 2H), 4.27-4.18 (m, 4H), 4.00-3.98 (m, 1H), 3.27-3.22 (m, 2H), 2.26-2.23 (m, 4H), 1.83-1.82 (m, 2H), 1.46 (s, 9H ), 1.24 (t, J = 7.2 Hz, 3H).

Compound 3 (Fluorescein)
Compound 26 (12.7 mg, 17.2 μmol) was dissolved in 1.0 ml of dry DMF. Under a nitrogen atmosphere, 5-Carboxyfluorescein N-succinimidyl ester (10.6 mg, 22.3 μmol) and DIEA (23.9 μL, 68.8 μmol) were added, and the mixture was stirred at room temperature for 19 hours. The solvent was distilled off to obtain 29.6 mg of a crude product. The obtained compound was directly used in the next reaction.

29.6 mg of the crude product was dissolved in 1.0 ml of THF, and 235 ml of 0.5 M lithium hydroxide aqueous solution was added and stirred at room temperature for 6 hours. The mixture was neutralized by adding 1M hydrochloric acid and then dried under reduced pressure. The obtained crude product was dissolved again in 1.0 ml of THF, 235 l of 0.5 M lithium hydroxide aqueous solution was added, and the mixture was stirred at room temperature for 4.5 hours. The mixture was neutralized with 1M hydrochloric acid and then dried under reduced pressure to obtain 41mg of crude compound. The obtained compound was directly used in the next reaction.

41 mg of crude compound was suspended in 2.0 ml of dichloromethane. 0.4 ml of TFA was added and stirred at room temperature for 40 minutes. After the solvent was distilled off, TFA was carefully removed by azeotropy with toluene. The obtained crude compound was purified by HPLC (acetonitrile (0.1% TFA) / water (0.1% TFA)) and dried to obtain Compound 3 (Fluorescein) (1.91 mg, 1.76 mol). ESI-MS ([M + H] = 969.2640 (obs.), 969.2661 (calc.)).

Scheme 5. Synthesis of Compound 4 (Fluorescein)

Compound 27
N ^ε -[(tert-Butoxy) carbonyl] -N ^α -[(benzyloxy) carbonyl] -L-lysine (12.6 mg, 33.6 μmol) was dissolved in 1.0 ml of dry DMF. Under a nitrogen atmosphere, HBTU (16.3 mg, 43 μmol), compound 6 (15 mg, 33.6 μmol) and DIEA 23.5 μl (134 μmol) were added, and the mixture was stirred at room temperature for 2 hours. After the solvent was distilled off, the residue was purified by column chromatography (silica, CHCl ₃ / MeOH = 25/1) to obtain 18.4 mg (24 μmol) of Compound 27. ¹ H NMR spectroscopy (400 MHz, CD ₃ OD) δ 7.60 (s, 1H), 7.38-7.25 (m, 6H), 6.80-6.78 (m, 2H), 6.22 (s, 1H), 5.09 (s, 2H ), 5.02 (s, 2H), 4.85-4.25 (s, 2H), 4.22 (t, J = 7.2 Hz, 2H), 4.08-4.05 (m, 1H), 3.03-2.99 (m, 2H), 1.76- 1.62 (m, 2H), 1.49-1.45 (m, 4H), 1.41 (s, 9H), 1.25 (t, J = 7.2 Hz, 3H).

Compound 28
Compound 25 (16.6 mg, 20 μmol) was dissolved in 0.5 ml of methanol. Under a nitrogen atmosphere, 10 mg of Pd / C 5 mg was added, and the mixture was stirred at room temperature for 2.5 hours under a hydrogen atmosphere. After removing palladium carbon by filtration, the solvent was distilled off to obtain 11 mg of compound 28 (17 μmol). ¹ H NMR spectroscopy (400 MHz, CD ₃ OD) δ 7.61 (s, 1H), 7.26-7.21 (m, 1H), 6.82-6.79 (m, 2H), 6.24-6.23 (m, 1H), 5.04 (s , 2H), 4.34-4.14 (m, 4H), 4.11-4.06 (m, 1H), 3.02-2.99 (m, 4H), 2.86-2.82 (m, 2H), 1.70-1.57 (m, 2H), 1.42 (s, 9H), 1.26 (t, J = 7.2 Hz, 3H).

Compound 4 (Fluorescein)
Compound 28 (6.6 mg, 10.3 μmol) was dissolved in 0.3 ml of dry DMF. Under a nitrogen atmosphere, 5-Carboxyfluorescein diacetate N-succinimidyl ester (6.13 mg, 11.3 μmol) and TEA (4.22 μL, 30.9 μmol) were added, and the mixture was stirred at room temperature for 1.5 hours. The solvent was distilled off and the obtained compound was used for the next reaction as it was.

The obtained compound was dissolved in 0.2 ml of methanol, 0.2 ml of 0.5 M lithium hydroxide aqueous solution was added and stirred overnight at room temperature. The mixture was neutralized with 1M hydrochloric acid and then dried under reduced pressure, and the obtained compound was used as it was in the next reaction.

The crude compound was suspended in 1.0 ml dichloromethane. 0.2 ml of TFA was added and stirred at room temperature for 2 hours. After the solvent was distilled off, TFA was carefully removed by azeotropy with toluene. The obtained crude compound was purified by HPLC (acetonitrile (0.1% TFA) / water (0.1% TFA)) and dried to obtain Compound 4 (Fluorescein) (1.32 mg, 1.34 μmol). ¹ H NMR spectroscopy (400 MHz, CD ₃ OD) δ 8.78 (s, 1H), 8.24-8.22 (m, 1H), 7.72-7.66 (m, 1H), 7.60 (s, 1H), 7.25 (s, 1H ), 6.85-6.75 (m, 2H), 6.57-6.52 (m, 6H), 6.26 (s, 1H), 5.06 (s, 2H), 4.75-4.68 (m, 2H), 4.34 (s, 1H), 2.97-2.89 (m, 2H), 1.81-1.51 (m, 2H), 1.36-1.31 (m, 4H).

Scheme 6. Synthesis of Compound 4 (Alexa647)

Compound 29
Compound 28 (14.7 mg, 23 mmol) was dissolved in 0.46 ml of methanol, 0.46 ml of 0.5 M lithium hydroxide aqueous solution was added, and the mixture was stirred at room temperature for 4.5 hours. After neutralizing with 1 M hydrochloric acid, the mixture was dried under reduced pressure. The obtained compound was purified by HPLC (acetonitrile (0.1% TFA) / water (0.1% TFA)) and dried to obtain 8.1 mg of compound 29 (11.2 μmol). ¹ H NMR spectroscopy (400 MHz, CD ₃ OD) δ 7.61 (s, 1H), 7.23 (s, 1H), 6.86-6.82 (m, 2H), 6.27-6.26 (m, 1H), 5.00 (s, 2H ), 4.40-4.28 (m, 2H), 3.81 (t, J = 6.8 Hz, 1H), 3.02 (t, J = 7.2 Hz, 2H), 1.86-1.82 (m, 2H), 1.52-1.46 (m, 2H), 1.42 (s, 9H), 1.41-1.37 (m, 2H).

Compound 4 (Alexa647)
Compound 29 (0.81 mg, 1.12 μmol) was dissolved in 0.1 ml of dry DMF. Under a nitrogen atmosphere, Alexa647-NHS-ester (1 mg) and TEA 0.46 μl (3.3 μmol) were added, and the mixture was stirred at room temperature for 10.5 hours. TEA 0.46 μl (3.3 μmol) was then added to the solution and stirred at room temperature for 14 hours. The solvent was distilled off to obtain 4.1 mg of a crude compound. The obtained product was directly used for the next reaction.

4.1 mg of crude compound was suspended in 0.2 ml of dichloromethane. 40 μl of TFA was added and stirred at room temperature for 1 hour. After the solvent was distilled off, TFA was carefully removed by azeotropy with toluene. The obtained product was purified by HPLC (acetonitrile / 10 mM ammonium acetate aqueous solution) and lyophilized to obtain the target compound 4 (Alexa647) (0.39 mg, 0.27 μmol). ESI-MS ([M-3H] = 449.4459 (obs.), 449.4475 (calc.)).

Scheme 7. Synthesis of Compound 5 (Fluorescein) and Compound 5 (Alexa647)

Compound 31
Compound 30 (4.7 mg, 13.3 μmol) (ref. 3) was dissolved in 0.1 ml of dry DMF. N- (tert-butoxycarbonyl) -2,2 ′-(ethylenedioxy) diethylamine 16.5 mg (66.7 μmol) and potassium carbonate 5.5 mg (40.0 μmol) were added, and the mixture was heated and stirred at 50 ° C. After the solvent was distilled off, purification was performed by preparative thin layer chromatography (silica gel, hexane / ethyl oxalate = ¼) to obtain 6.2 mg (11.0 μmol) of Compound 31.

Compound 32
Compound 31 (2.8 mg, 5.0 μmol) was dissolved in 0.5 ml of dichloromethane, 0.5 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 15 minutes. The crude product obtained after distilling off the solvent was dissolved in 0.1 ml of dry DMF. FmocHN-Lys (Boc) -COOH 3.5 mg (7.5 μmol), HBTU 2.8 mg (7.5 μmol), and DIEA 2.6 μl (15 μmol) were added, and the mixture was stirred at room temperature for 3 hours. After adding 10 μl of piperidine and stirring at room temperature for 10 minutes, the solvent was distilled off and purification was performed by preparative thin layer chromatography (silica gel, CHCl ₃ / MeOH = 10/1 (NH ₃ 1%)). mg (5.0 μmol) of compound 32 was obtained.

Compound 5 (Fluorescein)
Compound 32 (7.2 mg, 1.0 μmol) was dissolved in 50 μl of dry DMF. Fluorescein-NHS 1.0 mg (2.0 μmol) and DIEA 1.0 μl (6.3 μmol) were added and stirred at room temperature for 5 hours. After the solvent was distilled off, the residue was dissolved in 0.5 ml of dichloromethane, 0.5 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 15 minutes. After the solvent was distilled off, the residue was dissolved in water, purified by HPLC (acetonitrile / water (0.1% TFA)), and lyophilized to obtain the target compound 5 (Fluorescein).

Compound 5 (Alexa647)
Compound 32 (7.2 mg, 1.0 μmol) was dissolved in 50 μl of dry DMF. Alexa647-NHS 1.0 mg (1.0 μmol) and DIEA 1.0 μl (6.3 μmol) were added, and the mixture was stirred at room temperature for 5 hours. After the solvent was distilled off, the residue was dissolved in 0.5 ml of dichloromethane, 0.5 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 15 minutes. After the solvent was distilled off, the residue was dissolved in water, purified by HPLC (acetonitrile / water (0.1% TFA)), and lyophilized to obtain the target compound 5 (Alexa647).

Scheme 8. Synthesis of Compound 6 (sulfoCy3)

Compound 34
Compound 33 (20.0 mg, 69.3 μmol) (ref. 4) was dissolved in 1.0 ml of dry DMF. Under ice cooling, sodium hydride (containing 40% mineral oil) 5.5 mg (139 μmol) was added, and the mixture was stirred for 30 minutes under ice cooling. Methyl 4-bromobutyrate (10.5 μl, 83.1 μmol) and sodium iodide (10.4 mg, 69.3 μmol) were added, and the mixture was heated and stirred at 50 ° C. for 2 hours. Saturated saline was added, and extraction was performed three times with ethyl acetate. Then, the solvent of the organic layer was distilled off, and purification was performed by preparative thin layer chromatography (silica gel, hexane / ethyl oxalate = 1/1). 10.4 mg (27.0 μmol) of compound 34 was obtained.

Compound 35
Compound 34 (10.4 mg, 26.8 μmol) was dissolved in 0.5 ml of THF and 0.5 ml of water, 3.4 mg (80.3 μmol) of monohydrated lithium hydroxide was added, and the mixture was stirred at room temperature overnight. After neutralizing with 1N hydrochloric acid, extraction was performed three times with ethyl acetate. The crude product obtained after distilling off the solvent of the organic layer was dissolved in 0.3 ml of dry DMF. N- (tert-butoxycarbonyl) -2,2 '-(ethylenedioxy) diethylamine 6.6 mg (32.1 μmol), HBTU 15.2 mg (40.1 μmol), DIEA 14 μl (80.2 μmol) were added, and the mixture was stirred at room temperature. . Saturated brine was added, and the mixture was extracted three times with ethyl acetate. The solvent in the organic layer was distilled off, and the residue was purified by preparative thin layer chromatography (silica gel, ethyl oxalate). 7.2 mg (12.9 μmol) Compound 35 was obtained.

Compound 36
Compound 35 (7.2 mg, 12.8 μmol) was dissolved in 0.5 ml of dichloromethane, 0.5 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 15 minutes. The crude product obtained after distilling off the solvent was dissolved in 0.15 ml of dry DMF. FmocHN-Lys (Boc) -COOH 7.2 mg (15.4 μmol), HBTU 7.3 mg (19.3 μmol), DIEA 6.7 μl (38.5 μmol) were added, and the mixture was stirred at room temperature for 3 hours. After adding 15 μl of piperidine and stirring at room temperature for 10 minutes, the solvent was distilled off, and purification was performed by preparative thin layer chromatography (silica gel, CHCl ₃ / MeOH = 10/1 (NH ₃ 1%)) to obtain 6.7 mg (9.7 μmol) of compound 36 was obtained.

Compound 6 (sulfoCy3)
Compound 36 (1.0 mg, 1.5 μmol) was dissolved in 0.1 ml of dry DMF. SulfoCy3-NHS 1.0 mg (1.3 μmol) and DIEA 0.8 μl (4.0 μmol) were added, and the mixture was stirred at room temperature for 5 hours. After the solvent was distilled off, the residue was dissolved in 0.5 ml of dichloromethane, 0.5 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 15 minutes. After the solvent was distilled off, the residue was dissolved in water, purified by HPLC (acetonitrile / water (0.1% TFA)), and lyophilized to obtain the target compound 6 (sulfoCy3).

Reference (ref)
1. Coleman, S. K. et al. J. Biol. Chem. 291, 8784-8794 (2016)
2. Brunet, A. et al. Bioorg. Med. Chem. Lett. 24, 3186-3188 (2014)
3. Satoh, A. et al. Bioorg. Med. Chem. Lett. 19, 5464-5468 (2009)
4. Yamaura. K. et al. Nat. Chem. Biol. 12, 822-830 (2016)

Biochemical experiment: LSM800 (Carl Zeiss) was used as the confocal laser microscope.

Cell culture and gene transfer
(1) HEK293T cells 10% FBS, penicillin (100 units / mL) and streptomycin (100 μg / mL) Dulbecco's Modified Eagle Medium supplemented with (DMEM, glucose 4.5 g / L) medium, in 5% CO ₂ For all experiments cultured in a humid atmosphere, cells were harvested from subconfluent (<80%) with trypsin-EDTA solution and resuspended in fresh medium. Subculture was performed every 2-3 days. The AMPA glutamate receptor (GluR2) was introduced using lipofectamine 2000 (Invitrogen) according to the attached manual. For labeling, cells 36 to 48 hours after gene introduction were used.
(2) HEK293T cells transfected with mGlu1 were prepared in the same manner as described above except that the metabotropic glutamate receptor (mGlu1) gene was used instead of the AMPA type glutamate receptor (GluR2) gene.
(3) HEK293T cells into which GABA _A R was introduced were prepared in the same manner as described above except that the GABA _A receptor (GABA _A R) gene was used in place of the AMPA type glutamate receptor (GluR2) gene.

Test example 1
Labeling and visualization of AMPAR (AMPA glutamate receptor) in HEK293T cells (Figure 3)
The above-described HEK293T cells into which the GluR2 gene had been introduced were washed twice with PBS (−). Compound 1 (1 μM) was added to PBS (−), added to the cells, and incubated at room temperature for 10 minutes. A 10% volume of 4% PFA phosphate buffer solution of the above-mentioned Compound 1-added PBS (-) was added to the cells and incubated at room temperature for 30 minutes. The plate was washed 3 times with PBS (-) and observed with confocal microscopy.

Test example 2
Labeling and visualization of mGlu1 in HEK293T cells (Figure 4a)
HEK293T cells into which mGlu1 gene had been introduced were washed twice with PBS (−). Compound 5 (0.1 μM) was added to PBS (−), added to the cells, and incubated at room temperature for 10 minutes. A 10% volume of 4% PFA phosphate buffer solution of the above-mentioned compound 5 added PBS (−) was added to the cells, and incubated at room temperature for 30 minutes. The plate was washed 3 times with PBS (-) and observed with a confocal microscope.

Test example 3
Labeling and visualization of GABA _A R in HEK293T cells (Figure 4b)
HEK293T cells into which GABA _A R gene had been introduced were washed twice with PBS (−). Compound 6 (1 μM) was added to PBS (−), added to the cells, and incubated at room temperature for 10 minutes. A 10% volume of 4% PFA phosphate buffer solution of the above-described compound 6-added PBS (−) was added to the cells, and incubated at room temperature for 30 minutes. The plate was washed 3 times with PBS (-) and observed with a confocal microscope.

Test example 4
Labeling and visualization of mGlu1 in brain slices (Figure 6)
ACSF solution (125 mM NaCl, 2.5 mM KCl, 2 mM CaCl ₂ , 1 mM MgCl ₂ , 1.25 mM NaH ₂ PO ₄ ) with compound 5 (0.1 μM) added to acute brain slices isolated from 3-week-old ICR mice , 26 mM NaHCO ₃ , 10 mM D-glucose) and incubated in a 95% O ₂ /5% CO ₂ atmosphere at room temperature for 30 minutes. A 10% volume of 4% PFA phosphate buffer solution of the above-mentioned Compound 5 added ACSF solution was added to the cerebellar slice and incubated at room temperature for 1 hour. The plate was washed 3 times with PBS (-) and observed with a confocal microscope.

Protocol 1 (Figure 5)
The above-described HEK293T cells into which the GluR2 gene had been introduced were washed twice with PBS (−). Labeling agent (1 μM) was added to 1% PFA phosphate buffer solution, added to the cells and incubated for 1 hour at room temperature. The plate was washed 3 times with PBS (-) and observed with a confocal microscope.

Protocol 2 (Figure 5)
The above-described HEK293T cells into which the GluR2 gene had been introduced were washed twice with PBS (−). 1% PFA phosphate buffer solution was added to the cells and incubated at room temperature for 30 minutes. The cells were washed 3 times with PBS (−), 1% PFA phosphate buffer solution containing a labeling agent (1 μM) added to the cells, and incubated at room temperature for 1 hour. The plate was washed 3 times with PBS (-) and observed with a confocal microscope.

Test Example 5
1. Immobilization drive labeling in hippocampal neurons (Fig. 8)
Experimental method Conventional method (Wakayama, S .; Kiyonaka, S .; Arai, I .; Kakegawa, W .; Matsuda, S .; Ibata, K .; Nemoto, YL; Kusumi, A .; Yuzaki, M .; Hamachi , I. Nat. Commun. 2017, 8, 14850.), cultured hippocampal neurons were washed twice with PBS (−). Compound (1) (Alexa647) (2 μM), the labeling agent obtained in Scheme 2, was added to PBS (−), added to the cells, and incubated at room temperature for 5 minutes. The same amount of 2% PFA phosphate buffer solution as PBS (−) containing the labeling agent described above was added to the cells and incubated at room temperature for 30 minutes. After washing 3 times with PBS (-), immunostaining was performed and confocal microscopy was performed. The results are shown in FIG.

It was clarified that the spine containing the endogenous AMPA-type glutamate receptor can be selectively stained and can be used in combination with a normal immunostaining method.

2. Multiple simultaneous staining in cerebellar slices (Figure 9)
According to the conventional method (Tamura, T .; Song, Z .; Amaike, K .; Lee, S .; Yin, S .; Kiyonaka, S .; Hamachi, IJ Am. Chem. Soc. 2017, 139, 14181.) Mouse cerebellar slices were prepared. Labeling agents obtained in Schemes 2 and 7 on ACSF (125 mM NaCl, 2.5 mM KCl, 2 mM CaCl ₂ , 1 mM MgCl ₂ , 1.25 mM NaH ₂ PO ₄ , 26 mM NaHCO ₃ , 10 mM D-glucose) Compound 1 (Alexa647) (2 μM) and Compound 5 (Fluorescein) (0.2 μM), which are added to the mouse cerebellar slice, at room temperature under 95% O ₂ /5% CO ₂ atmosphere Incubated for 30 minutes. The same amount of 2% PFA phosphate buffer solution as ACSF containing the labeling agent described above was added to the cells and incubated at room temperature for 60 minutes. Washed 3 times with PBS (-) and observed with confocal microscopy. The results are shown in FIG.

It became clear that multiple receptors (mGlu1 and AMPAR) in the cerebellar slice can be visualized simultaneously. Inhibitors (FITM: 4-Fluoro-N- [4- [6- (isopropylamino) pyrimidin-4-yl] -1,3-thiazol-2-yl] -N-methylbenzamide (4 μM) and NBQX: 2 , 3-dioxo-6-nitro-1,2,3,4- tetrahydrobenzo [f] quinoxaline-7-sulfonamide (40 μM)), compound 1 (Alexa647) (2 μM), and compound 5 (Fluorescein) (0.2 When combined with μM), it was revealed that the binding of fluorescein (Fl) to mGlu1 and the binding of Alexa647 to AMPAR were inhibited (see the image in the lower column of FIG. 7).

3. Voltage-dependent calcium channels in cerebellar slices ^{(VDCC: Voltage-dependent Ca 2+} channel) for labeling (Fig. 10)
(1) Synthesis of labeling agent for VDCC visualization

Scheme 9. Synthesis of Fluorophore-AgTx [1-48]

N ₃ -AgTx [1-24] -CONHNH ₂ (AgTX: ω-agatoxin IVA from Funnel Web Spider, Agelenopsis aperta)
2-chlorotrityl chloride resin (1.51 mmol / g, 750 mg, 1.13 mmol) was added to 5% H ₂ NNH ₂ / NMP (v / v) (4 mL) and stirred for 30 minutes. After removing the solution and washing the resin with NMP, 5% H ₂ NNH ₂ / NMP (v / v) (4 mL) was allowed to act on the resin for 30 minutes. After removing the solution and washing the resin with NMP, the corresponding peptide fragment was synthesized by solid phase synthesis and purified by reverse phase HPLC to obtain N ₃ -AgTx [1-24] -CONHNH ₂ . AgTx [1-24] is shown in SEQ ID NO: 2.

Cys-AgTx [26-48]
Using NovaSyn (registered trademark) TGA resin, the corresponding peptide fragment was synthesized by solid phase synthesis and purified by reverse phase HPLC to obtain Cys-AgTx [26-48]. Cys-AgTx [26-48] shown in SEQ ID NO: 3 is the 25th to 48th peptides of SEQ ID NO: 1.

N ₃ -AgTx [1-48]
Add N ₃ -AgTx [1-24] -CONHNH ₂ (11.2 mg, 3 μmol) to 0.2 M phosphate buffer / 6 M guanidinium chloride (pH 3.15) (500 μL), and cool to -20 ° C. 0.5 M NaNO ₂ aqueous solution (60 μL, 30 μmol, 10 eq.) Was added, and the mixture was stirred at −20 ° C. for 30 minutes. Then, 4-mercaptophenylacetic acid (MPAA) (51 mg, 0.3 mmol, 100 eq.) In 0.2 M phosphate buffer / 6 M guanidinium chloride (pH 6.90) (500 μL), and Cys-AgTx [26- 48] (8.4 mg, 3 μmol, 1.0 eq.) In 0.2 M phosphate buffer / 6 M guanidinium chloride (pH 6.90) (500 μL) was added, and the pH was adjusted to 6 with 5 M and 1 M aqueous sodium hydroxide. Adjusted to 8-7.0. After reacting at room temperature for 5 hours, add 100 mM DTT in 0.2 M phosphate buffer / 6 M guanidinium chloride (pH 6.90) (500 μL) and stir for 30 minutes, then add 30% acetic acid (500 μL). Purification by reverse phase HPLC yielded N ₃ -AgTx [1-48] _linear .

Obtained N ₃ -AgTx [1-48] _linear (2.0 mg, 0.375 μmol), reduced glutathione (11.5 mg, 37.5 μmol, 100 eq.), Oxidized glutathione (2.3 mg, 3.75 μmol, 10 eq.) Was dissolved in 1 M ammonium acetate buffer (pH 7.9) / 20 v / v% glycerol / 0.1 M EDTA (37.5 mL) and stirred at 4 ° C. overnight. Thereafter, purification was performed by reverse phase HPLC to obtain N ₃ -AgTx [1-48]. The amino acid sequence of AgTx [1-48] is shown in SEQ ID NO: 1.

Fluorophore-AgTx [1-48]
106.8 μM N ₃ -AgTx [1-48] (50 mM HEPES buffer (pH 7.4)) solution with 1.5 eq. Of Click-iT ^™ Alexa Fluor ^™ 555 sDIBO alkyne (Thermo Fisher Scientific) or Click- iT ^™ Alexa Fluor ^™ 647 DIBO alkyne (Thermo Fisher Scientific) was added and stirred at room temperature for 3 hours. Thereafter, acetic acid was added to prepare a 30% acetic acid solution, followed by purification by reverse phase HPLC to obtain Fluorophore-AgTx [1-48].
ESI-MS: Alexa Fluro 555-AgTx [1-48]: [M + 2Na ⁺ + 2H ⁺ ] ⁴⁺ = 1705.24, Alexa Fluro 647-AgTx [1-48]: [M-3H ⁺ ] ^3- = 1160.1

(2) VDCC visualization in cerebellar slices Conventional method (Tamura, T .; Song, Z .; Amaike, K .; Lee, S .; Yin, S .; Kiyonaka, S .; Hamachi, IJ Am. Chem. Soc. Mouse cerebellar slices were prepared according to 2017, 139, 14181.). ACSF (125 mM NaCl, 2.5 mM KCl, 2 mM CaCl ₂ , 1 mM MgCl ₂ , 1.25 mM NaH ₂ PO ₄ , 26 mM NaHCO ₃ , 10 mM D-glucose) labeling agent Fluorophore-AgTx [1-48] (0.2 μM) was added and added to mouse cerebellar slices and incubated for 30 minutes at room temperature in a 95% O ₂ /5% CO ₂ atmosphere. The same amount of 2% PFA phosphate buffer solution as ACSF containing the labeling agent described above was added to the cells and incubated at room temperature for 60 minutes. Washed 3 times with PBS (-) and observed with confocal microscope. Alternatively, in the same system as described above, instead of the labeling agent (0.2 μM) alone, a competitive inhibitor (ω-agatoxion (P / Q type voltage-dependent calcium channel Cac2.1 blocker): concentration 4 μM)) The confocal microscope was also observed in the combined system. The results are shown in FIG.

It became clear that it can be applied not only to cell surface receptors but also to ion channels, and not only small molecules but also middle molecules (peptides) can be used as ligands.

When a peptide such as the present disclosure is used as a ligand, if the peptide contains a nucleophilic amino acid residue such as lysine, it is not necessary to incorporate an amine for cross-linking into the labeling agent. This is because the nucleophilic amino acid residue present in the peptide ligand is used instead for cross-linking with the receptor.

4. Labeling to mGlu1 in the whole cerebellum (Figure 11)
Mice are comatose with a triple mixed anesthetic, the scalp is incised, and a hole is made in the skull of the cerebellum on the extension of the longitudinal cerebral fissure. Inject 6 μL of PBS (−) containing Compound 5 (Alexa647) (10 μM) as a labeling agent using a microinjector. The scalp is then sutured, the antagonist is driven, and the mouse is returned to the cage. Three hours later, 4% PFA was used to perform reflux fixation (4% PFA perfusion for fixation). After removing the brain, the brain was immersed in 4% PFA for 1 hour. The brain was washed with PBS (−), frozen into sections, and observed with a confocal microscope. The results are shown in FIG.

It was revealed that a target receptor can be labeled at the timing of reflux fixation by applying a labeling agent in an animal in a live state.

Claims (11)

1. A method for labeling a biological sample, the method comprising:
   (A) applying a fixing agent to the cell-containing biological sample, and (b) applying a labeling agent to the cell-containing biological sample,
   Including
   here,
   Either step (a) and step (b) may be performed first, step (a) and step (b) may be performed simultaneously,
   The labeling agent has at least one reactive group that reacts with a ligand for a cell surface receptor, a labeling substance, and an immobilizing agent;
   Some cells contained in the cell-containing biological sample include the cell surface receptor,
   The cell surface receptor has at least one reactive group that reacts with the immobilizing agent and a ligand binding site,
   The immobilizing agent reacts with a reactive group of the labeling agent and a reactive group of the cell surface receptor to form a covalent bond, thereby linking the labeling agent and the cell surface receptor; A method for labeling biological samples.
2. The method for labeling a biological sample according to claim 1, wherein the number of reactive groups of the labeling agent involved in the covalent bond with the immobilizing agent is the same as the number of reactive groups of the cell surface receptor.
3. The reactive group of the cell surface receptor is selected from the group consisting of NH ₂ , OH and SH, and the reactive group of the labeling agent is selected from the group consisting of NH ₂ , NHNH ₂ , CONHNH ₂ , OH and SH. The method for labeling a biological sample according to any one of claims 1 to 2, which is selected.
4. The method for labeling a biological sample according to any one of claims 1 to 3, wherein the step (a) is performed simultaneously with the step (b).
5. Either by providing a cell-containing biological sample pre-fixed with the immobilizing agent, or by further coexisting the immobilizing agent with a cell-containing biological sample pre-fixed with the immobilizing agent. (a) is achieved,
   The method for labeling a biological sample according to any one of claims 1 to 4, wherein in the step (b), the biological sample immobilized with the immobilizing agent reacts with the labeling agent.
6. The labeling of a biological sample according to any one of claims 1 to 5, wherein a reaction between the reactive group of the cell surface receptor, the reactive group of the labeling agent, and the immobilizing agent is reversible. Method.
7. The method for labeling a biological sample according to any one of claims 1 to 6, wherein the cell surface receptor is a neurotransmitter receptor.
8. The biological sample according to any one of claims 1 to 7, wherein the immobilizing agent is at least one selected from the group consisting of formaldehyde, paraformaldehyde, glutaraldehyde, glyoxal, methylglyoxal, and dimethyl suberimidate. Labeling method.
9. The cell-containing biological sample is brain, heart, liver, gallbladder, kidney, adrenal gland, pancreas, lung, thyroid, esophagus, stomach, duodenum, small intestine, large intestine, ureter, bladder, prostate, uterus, breast, bronchi, blood vessel, The method for labeling a biological sample according to any one of claims 1 to 8, comprising at least one selected from the group consisting of lymphatic vessels, skin, bones, joints, muscles, oral mucosa and nasal mucosa.
10. A labeled cell-containing biological sample labeled with a labeling agent, wherein the labeling agent is covalently linked by a cell surface receptor of the biological sample and a fixing agent. .
11. Having at least one reactive group that reacts with a ligand for a cell surface receptor, a labeling substance, and an immobilizing agent, and is used for linking to the cell surface receptor of a cell-containing biological sample by the immobilizing agent. Labeling agent.

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