Broad-spectrum GPCR conjugates

Abstract

To provide broad-spectrum G-Protein coupled receptor (GPCR) binding agents, detectable / isolatable compounds comprising such binding agents (e.g., broad-spectrum GPCR binding agents linked to a functional element and / or solid surface), and methods of use thereof for the detection / isolation of GPCRs.SOLUTION: The present invention provides a composition comprising a broad-spectrum G-protein coupled receptor (GPCR) binding agent represented by formula (CLZP1), which is attached to a functional element or a solid surface, and a method of detecting or quantifying GPCRs in a sample, the method comprising contacting the sample with the composition and detecting or quantifying a functional element of a signal produced thereby.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Provided herein are broad-spectrum G protein-coupled receptor (GPCR) conjugates, detectable / separable compounds comprising such conjugates (e.g., broad-spectrum GPCR conjugates linked to functional elements and / or solid surfaces), and methods for using them for the detection / separation of GPCRs. [Background technology]

[0002] G protein-coupled receptors (GPCRs) are an important class of transmembrane proteins. Because they are involved in multiple diseases, they are targets in much modern medicine and are being thoroughly studied for the development of new drugs. Therefore, there is a need for tools that enable the matching of GPCR-ligand interactions in living cells. [Overview of the Initiative]

[0003] Provided herein are broad-spectrum G protein-coupled receptor (GPCR) conjugates, detectable / separable compounds comprising such conjugates (e.g., broad-spectrum GPCR conjugates linked to functional elements and / or solid surfaces), and methods for using them for the detection / separation of GPCRs.

[0004] In some embodiments, the herein provides a composition comprising a broad-spectrum G protein-coupled receptor (GPCR) conjugate bound to a functional element or a solid surface, wherein the broad-spectrum GPCR conjugate is [ka] Including, in the formula, [ka] This refers to a functional element of the broad-spectrum GPCR binder, a solid surface, or a linker binding site between the broad-spectrum GPCR binder and the functional element or solid surface.

[0005] In some embodiments, the herein provides a composition comprising a broad-spectrum G protein-coupled receptor (GPCR) conjugate bound to a functional element or a solid surface, wherein the broad-spectrum GPCR conjugate is [ka] Including, in the formula, [ka] This refers to a functional element of the broad-spectrum GPCR binder, a solid surface, or a linker binding site between the broad-spectrum GPCR binder and the functional element or solid surface.

[0006] In some embodiments, the herein provides a composition comprising a broad-spectrum G protein-coupled receptor (GPCR) conjugate bound to a functional element or a solid surface, wherein the broad-spectrum GPCR conjugate is [ka] Including, in the formula, [ka] This refers to a functional element of the broad-spectrum GPCR binder, a solid surface, or a linker binding site between the broad-spectrum GPCR binder and the functional element or solid surface.

[0007] In some embodiments, the herein provides a composition comprising a broad-spectrum G protein-coupled receptor (GPCR) conjugate bound to a functional element or a solid surface, wherein the broad-spectrum GPCR conjugate is [ka] Including, in the formula, [ka] This refers to a functional element of the broad-spectrum GPCR binder, a solid surface, or a linker binding site between the broad-spectrum GPCR binder and the functional element or solid surface.

[0008] In some embodiments, the herein provides a composition comprising a broad-spectrum G protein-coupled receptor (GPCR) conjugate bound to a functional element or a solid surface, wherein the broad-spectrum GPCR conjugate is [ka] Including, in the formula, [ka] This refers to a functional element of the broad-spectrum GPCR binder, a solid surface, or a linker binding site between the broad-spectrum GPCR binder and the functional element or solid surface.

[0009] In some embodiments, the herein provides a composition comprising a broad-spectrum G protein-coupled receptor (GPCR) conjugate bound to a functional element or a solid surface, wherein the broad-spectrum GPCR conjugate is [ka] Including, in the formula, [ka] This refers to a functional element of the broad-spectrum GPCR binder, a solid surface, or a linker binding site between the broad-spectrum GPCR binder and the functional element or solid surface.

[0010] In some embodiments, the herein provides a composition comprising a broad-spectrum G protein-coupled receptor (GPCR) conjugate bound to a functional element or a solid surface, wherein the broad-spectrum GPCR conjugate is [ka] Including, in the formula, [ka] This refers to a functional element of the broad-spectrum GPCR binder, a solid surface, or a linker binding site between the broad-spectrum GPCR binder and the functional element or solid surface.

[0011] In some embodiments, the herein provides a composition comprising a broad-spectrum G protein-coupled receptor (GPCR) conjugate bound to a functional element or a solid surface, wherein the broad-spectrum GPCR conjugate is [ka] Including, in the formula, [ka] The linker is a functional element of the broad-spectrum GPCR binder, a solid surface, or a linking site between the broad-spectrum GPCR binder and the functional element or solid surface, and a double bond may be present as a cis isomer (Z), a trans isomer (E), or a mixture of the two.

[0012] In some embodiments, the herein provides a composition comprising a broad-spectrum G protein-coupled receptor (GPCR) conjugate bound to a functional element or a solid surface, wherein the broad-spectrum GPCR conjugate is [ka] Including, in the formula, [ka] The linker is a functional element of the broad-spectrum GPCR binder, a solid surface, or a linking site between the broad-spectrum GPCR binder and the functional element or solid surface, and a double bond may be present as a cis isomer (Z), a trans isomer (E), or a mixture of the two.

[0013] In some embodiments, the herein provides a composition comprising a broad-spectrum G protein-coupled receptor (GPCR) conjugate bound to a functional element or a solid surface, wherein the broad-spectrum GPCR conjugate is [ka] Including, in the formula, [ka] The linker is a functional element of the broad-spectrum GPCR binder, a solid surface, or a linking site between the broad-spectrum GPCR binder and the functional element or solid surface, and a double bond may be present as a cis isomer (Z), a trans isomer (E), or a mixture of the two.

[0014] In some embodiments, the herein provides a composition comprising a broad-spectrum G protein-coupled receptor (GPCR) conjugate bound to a functional element or a solid surface, wherein the broad-spectrum GPCR conjugate is [ka] Including, in the formula, [ka] The linker is a functional element of the broad-spectrum GPCR binder, a solid surface, or a linking site between the broad-spectrum GPCR binder and the functional element or solid surface, and a double bond may be present as a cis isomer (Z), a trans isomer (E), or a mixture of the two.

[0015] In some embodiments, what is provided herein are compositions described herein (e.g., CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, NTRP2, etc.) which include a solid surface selected from deposited particles, films, glasses, tubes, wells, self-assembled monolayers, surface plasmon resonance chips, or solid supports having an electronically conductive surface. In some embodiments, the deposited particles are magnetic particles.

[0016] In some embodiments, what is provided herein are compositions described herein (e.g., CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, NTRP2, etc.) comprising a functional element selected from detectable elements, affinity elements, and capture elements. In some embodiments, the detectable elements include fluorescent dye molecules, chromophores, radionuclides, electron opaque molecules, MRI contrast agents, SPECT contrast agents, or mass tags.

[0017] In some embodiments, the broad-spectrum GPCR binders of the compositions described herein (e.g., CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, NTRP2, etc.) are directly bound to a functional element or solid surface. In some embodiments, the broad-spectrum GPCR binders of the compositions described herein (e.g., CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, NTRP2, etc.) are bound to a functional element or solid surface via a linker. In some embodiments, the linker is [(CH2)2O] nを In the formula, n is 1 to 20. In some embodiments, the linker is bonded to a broad-spectrum GPCR binder and / or functional element by an amide bond.

[0018] In some embodiments, what is provided herein is [ka] The composition comprises the structure shown in the formula, where n is 0 to 8 and X is a functional element or a solid surface.

[0019] In some embodiments, what is provided herein is [ka] The composition comprises the structure shown in the formula, where n is 0 to 8, m is 0 to 8, and X is a functional element or a solid surface.

[0020] In some embodiments, what is provided herein is [ka] The composition comprises the structure shown in the formula, where n is 0 to 8, m is 0 to 8, and X is a functional element or a solid surface.

[0021] In some embodiments, what is provided herein is [ka] The composition comprises the structure shown in the formula, where n is 0 to 8, m is 0 to 8, and X is a functional element or a solid surface.

[0022] In some embodiments, what is provided herein is [ka] The composition comprises the structure shown in the formula, where n is 0 to 8 and X is a functional element or a solid surface.

[0023] In some embodiments, what is provided herein is [ka] The composition comprises the structure shown in the formula, where n is 0 to 8, m is 0 to 8, and X is a functional element or a solid surface.

[0024] In some embodiments, what is provided herein is [ka] The composition comprises the structure shown in the formula, where n is 0 to 8, m is 0 to 8, and X is a functional element or a solid surface.

[0025] In some embodiments, what is provided herein is [ka] A composition comprising the structure, where n is 0 to 8, X is a functional element or a solid surface, and any geometric isomer (e.g., C=C) may exist as a cis isomer (Z), a trans isomer (E), or a mixture of the two.

[0026] In some embodiments, what is provided herein is [ka] The composition comprises the structure shown in the formula, where n is 0 to 8 and X is a functional element or a solid surface.

[0027] In some embodiments, what is provided herein is [ka] The composition comprises the structure shown in the formula, where n is 0 to 8 and X is a functional element or a solid surface.

[0028] In some embodiments, what is provided herein is [ka] The composition comprises the structure shown in the formula, where n is 0 to 8 and X is a functional element or a solid surface.

[0029] In some embodiments, the compositions provided herein include functional elements (X) being fluorescent dye molecules.

[0030] In some embodiments, what is provided herein is a composition comprising an amitriptyline system structure as shown in Figure 15, or a nortriptyline system form of the structure shown in Figure 15. In some embodiments, the amitriptyline system structure as shown in Figure 15 or the nortriptyline system form thereof is provided with alternative linkers, fluorescent dye molecules (or other X groups), or connection points on the amitriptyline or nortriptyline ring system. In some embodiments, alternative linkers and X groups are provided herein, and alternative connection points are provided, for example, by AMTRP1, AMTRP2, NTRP1, NTRP2, and NTRP2.

[0031] In some embodiments, the compositions herein contain one or more unnatural abundances of stable heavy isotopes.

[0032] In some embodiments, the foregoing provides a method for detecting or quantifying GPCRs in a sample, comprising contacting the sample with a composition described herein (e.g., a composition comprising linked GPCR binders and functional groups or solid surfaces) and detecting or quantifying the functional elements of the signal produced thereby. In some embodiments, the functional elements of the signal produced thereby are detected or quantified by fluorescence, mass spectrometry, optical imaging, magnetic resonance imaging (MRI), and energy transfer.

[0033] In some embodiments, the foregoing provides a method for isolating GPCRs from a sample, comprising contacting the sample with a composition described herein (e.g., a composition comprising linked GPCR binders and functional groups or solid surfaces) and separating the functional elements or solid surfaces, as well as the bound GPCRs, from the unbound portions of the sample. In some embodiments, characterizing the uniqueness of GPCRs in a sample includes isolating GPCRs from the sample and analyzing the isolated GPCRs by mass spectrometry.

[0034] In some embodiments, the foregoing provides a method for monitoring interactions between a GPCR and an unmodified biomolecule, comprising contacting a sample with a composition described herein (e.g., a composition comprising linked GPCR binders and functional groups or solid surfaces).

[0035] In some embodiments, any of the methods described herein may be performed using a sample selected from cells, cell lysates, body fluids, tissues, biological samples, in vitro samples, and environmental samples.

[0036] In some embodiments, what is provided herein is a system comprising a composition described herein (e.g., a composition comprising linked GPCR binders and functional groups), wherein the functional element is a fluorescent dye molecule; (b) fusion of a GPCR with a peptide component of a bioluminescent protein or bioluminescent complex, wherein the emission spectrum of the bioluminescent protein or bioluminescent complex overlaps with the excitation spectrum of the fluorescent dye molecule. In some embodiments, the system comprises a kit, cells, cell lysates, or reaction mixtures. In some embodiments, the fusion comprises a GPCR and a peptide component of a bioluminescent complex, wherein the system further comprises one or more additional components of the bioluminescent complex (e.g., polypeptide components of the bioluminescent complex) and a substrate for the bioluminescent complex.

[0037] In some embodiments, provided herein are (a) a fusion of a GPCR and a bioluminescent protein, (i) a composition described herein (e.g., a composition comprising linked GPCR binders and functional groups) in which the functional element is a fluorescent dye molecule and the emission spectrum of the bioluminescent protein overlaps with the excitation spectrum of the fluorescent dye molecule, and (ii) a substrate of the bioluminescent protein, and (b) a method comprising detecting the wavelength of light within the range of the excitation spectrum of the fluorescent dye molecule resulting from bioluminescent resonance energy transfer from the bioluminescent protein to the fluorescent dye molecule when the broad-spectrum GPCR binder binds to the GPCR.

[0038] In some embodiments, provided herein are methods comprising (a) contacting a fusion of a GPCR and the peptide component of a bioluminescent complex with (i) a composition described herein (e.g., a composition comprising linked GPCR binders and functional groups) in which the functional element is a fluorescent dye molecule and the emission spectrum of the bioluminescent protein overlaps with the excitation spectrum of the fluorescent dye molecule, (ii) a polypeptide component of a bioluminescent complex, and (iii) a substrate for the bioluminescent protein, and (b) detecting the wavelength of light within the range of the excitation spectrum of the fluorescent dye molecule resulting from bioluminescent resonance energy transfer from the bioluminescent complex to the fluorescent dye molecule when the broad-spectrum GPCR binder binds to the GPCR. [Brief explanation of the drawing]

[0039] [Figure 1] This is a schematic diagram of a BRET experiment that utilizes fluorescent ligand substitution by GPCR ligands. Cells expressing a HiBiT-GPCR fusion (left) are treated with LgBiT and a fluorescent ligand. When an LgBiT-HiBiT complex is formed and the fluorescent ligand binds to the GPCR, a BRET signal appears (center). When treated with an unlabeled ligand, the fluorescent ligand is substituted, resulting in a decrease in the BRET signal (right). [Figure 2] This is a schematic diagram illustrating the identification of GPCR-HiBiT fusions from other non-fusion GPCRs using the BRET signal. [Figure 3A] This figure shows a scheme for the chemical synthesis of an exemplary clozapine-based tracer. [Figure 3B] This figure shows a scheme for the chemical synthesis of an exemplary clozapine-based tracer. [Figure 3C] This figure shows a scheme for the chemical synthesis of an exemplary clozapine-based tracer. [Figure 3D] This figure shows a scheme for the chemical synthesis of an exemplary clozapine-based tracer. [Figure 3E] This figure shows a scheme for the chemical synthesis of an exemplary clozapine-based tracer. [Figure 3F] This figure shows a scheme for the chemical synthesis of an exemplary clozapine-based tracer. [Figure 3G] This figure shows a scheme for the chemical synthesis of an exemplary clozapine-based tracer. [Figure 3H] This figure shows a scheme for the chemical synthesis of an exemplary clozapine-based tracer. [Figure 3I] This figure shows a scheme for the chemical synthesis of an exemplary clozapine-based tracer. [Figure 4] This figure shows an example of a roxapine-based tracer chemical synthesis scheme. [Figure 5] This figure shows an example of a chemical synthesis scheme for an olanzapine-based tracer. [Figure 6] This figure shows an example of a chemical synthesis scheme for a quetiapine-based tracer. [Figure 7] This figure shows an example of a risperidone-based tracer chemical synthesis scheme. [Figure 8] This figure shows an exemplary scheme for detecting tracers that bind to GPCR / HiBiT fusions via BRET. [Figure 9]This figure shows heatmaps of exemplary BRET results generated with fluorescently tagged GPCR conjugate tracers for a diverse panel of GPCR / HiBiT fusions expressed in living cells. Assay signals were evaluated by taking the ratio of BRET signals for tracer binding in the absence and presence of competing, excessive unmodified compounds. [Figure 10A] Binding to BRET targets generates living cells containing representative GPCR / HiBiT fusions. Cells transfected with plasmid DNA encoding each GPCR / HiBiT fusion were treated with serially diluted tracers (clozapine tracer SL-1454, Figure 10A) and (respiredone tracer SL-1591, Figure 10B), resulting in a dose-dependent increase in specific BRET levels. [Figure 10B] Binding to BRET targets generates living cells containing representative GPCR / HiBiT fusions. Cells transfected with plasmid DNA encoding each GPCR / HiBiT fusion were treated with serially diluted tracers (clozapine tracer SL-1454, Figure 10A) and (respiredone tracer SL-1591, Figure 10B), resulting in a dose-dependent increase in specific BRET levels. [Figure 11] This figure shows the structure of clozapine and its modifiable core with exemplary binding sites marked on the unmodified clozapine structure. [Figure 12] This figure shows the structure of an exemplary clozapine-based tracer. [Figure 13] This figure shows the structures of exemplary roxapine, olanzapine, quetiapine, and risperidone tracers. [Figure 14] This diagram shows an exemplary linker structure that connects a "drug" (GPCR binder) to a functional element. [Figure 15A] This figure shows an exemplary structure of an amitriptyline-based tracer (A). Alternative X groups (pink), such as those of other fluorescent dye molecules, may be provided. [Figure 15B] This figure shows the structure of an exemplary (B) nortriptyline-based tracer. Alternative X groups (pink), such as other fluorescent dye molecules, may be provided. [Figure 16] This figure shows a heatmap of exemplary BRET results generated with a fluorescently tagged amitriptyline-derived GPCR conjugate for a diverse panel of GPCR / HiBiT fusions expressed in living cells. Assay signals were evaluated by taking the ratio of BRET signals for tracer binding in the absence and presence of competing excess unmodified compounds. [Modes for carrying out the invention]

[0040] definition Similar or equivalent methods and materials may be used in the practice and testing of the embodiments described herein, although several preferred methods, compositions, devices, and materials are described herein. However, before describing the materials and methods of the present invention, it should be understood that the present invention is not limited to the specific molecules, compositions, methodologies, or protocols described herein, as these may be modified according to routine experimentation and optimization. Furthermore, it should be understood that the terms used in the description are for the purpose of merely describing a particular type or embodiment and are not intended to limit the scope of the embodiments described herein.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which the present invention pertains. However, in the event of any conflict, this specification shall prevail, including the definitions. Accordingly, in the context of the embodiments described herein, the following definitions apply:

[0042] When used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. For example, a reference to "GPCR" refers to one or more GPCRs and their equivalents known to those skilled in the art.

[0043] As used herein, the term "and / or" includes any combination of the listed items that individually include any of the listed items. For example, "A, B, and / or C" includes A, B, C, AB, AC, BC, and ABC, each of which is considered to be individually described by the statement "A, B, and / or C".

[0044] As used herein, the term “comprise” and its linguistic variations mean the presence of described features, elements, and process steps, without excluding the presence of additional features, elements, and process steps. Conversely, the term “consisting of” and its linguistic variations mean the presence of described features, elements, and process steps, excluding undescribed features, elements, and process steps, except for impurities that are usually present. The expression “consisting essentially of” means described features, elements, and process steps, and additional features, elements, and process steps that do not substantially affect the fundamental nature of the composition, system, or method. Many embodiments herein are described using the open word “comprise.” Such embodiments include a number of closed embodiments consisting of and / or essentially consisting of which may be alternatively claimed or described using such language.

[0045] As used herein, the term "isomer" refers to compounds having the same composition and molecular weight but differing in physical and / or chemical properties. This structural difference may be in composition or in the ability to rotate the plane of polarization.

[0046] As used herein, the terms “stereoisomer” or “geometric isomer” refer to a set of compounds that have the same number and types of atoms and share the same bonding connectivity between those atoms, but have different three-dimensional structures. The terms “stereoisomer” or “geometric isomer” refer to any member of this set of compounds.

[0047] As used herein, the term "clozapine" refers to a compound having the following structure: [ka] The clozapine moiety or substituent of the molecular entity comprises a clozapine structure linked at any suitable bonding point to another molecular entity (e.g., a solid surface, a functional element, etc.).

[0048] As used herein, the term "roxapine" refers to a compound having the following structure: [ka] The roxapine moiety or substituent of the molecular entity comprises a roxapine structure linked at any suitable bonding point to another molecular entity (e.g., a solid surface, a functional element, etc.).

[0049] As used herein, the term "quetiapine" refers to a compound having the following structure: [ka] The quetiapine moiety or substituent of a molecular entity comprises a quetiapine structure linked at any suitable bonding point to another molecular entity (e.g., a solid surface, a functional element, etc.).

[0050] As used herein, the term "risperidone" refers to a compound having the following structure: [ka] The risperidone portion or substituent of the molecular entity comprises a risperidone structure linked at any suitable bonding point to another molecular entity (e.g., a solid surface, a functional element, etc.).

[0051] As used herein, the term "olanzapine" refers to a compound having the following structure: [ka] The olanzapine moiety or substituent of the molecular entity comprises an olanzapine structure linked at any suitable bonding point to another molecular entity (e.g., a solid surface, a functional element, etc.).

[0052] As used herein, the term "amitriptyline" refers to a compound having the following structure: [ka] The amitriptyline moiety or substituent of the molecular entity comprises an amitriptyline structure linked at any suitable bonding point to another molecular entity (e.g., a solid surface, a functional element, etc.). Amitriptyline and the amitriptyline moiety contain C=C. Although amitriptyline is symmetric, substitutions that break symmetry produce two geometric isomers of the double bond. Thus, the amitriptyline moiety may exist as a cis isomer (Z), a trans isomer (E), or a mixture of the two.

[0053] As used herein, the term "nortriptyline" refers to a compound having the following structure: [ka] The nortriptyline moiety or substituent of the molecular entity contains a nortriptyline structure linked at any suitable bonding point to another molecular entity (e.g., a solid surface, a functional element, etc.). Both nortriptyline and the nortriptyline moiety contain C=C. Although nortriptyline is symmetric, substitutions that break the symmetry result in two geometric isomers of the double bond. Thus, the nortriptyline moiety may exist as a cis isomer (Z), a trans isomer (E), or a mixture of the two.

[0054] As used herein, the term “tracer” refers to a compound or agent of interest that binds to an analyte of interest (e.g., a protein of interest (e.g., a GPCR)) and exhibits quantifiable or detectable properties (e.g., detectable or quantifiable by appropriate biochemical or biophysical methods (e.g., optically, magnetically, electrically, by resonance imaging, by mass, by radiation, etc.)). Tracers may also include compounds or agents of interest that bind to an analyte of interest (e.g., directly or via a suitable linker) and are linked to (e.g., directly or via a suitable linker) a fluorescent dye molecule, a radionuclide, a mass tag, a contrast agent for magnetic resonance imaging (MRI), planar scintigraphy (PS), positron emission tomography (PET), single-photon emission computed tomography (SPECT), and computed tomography (CT) (e.g., a metal ion chelator with bound metal ions, isotopes, or radionuclides).

[0055] As used herein, the term “sample” is used in its broadest sense. In another sense, it means specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples are obtained from animals (including humans) and may include fluids, solids, tissues, and gases. Biological samples include blood products such as plasma and serum. Samples may also refer to cell lysates, or purified forms of enzymes, peptides, and / or polypeptides as described herein. Cell lysates may include cells lysed with a solvent, or lysates such as rabbit reticulocyte lysates or wheat germ lysates. Samples may also include cell-free expression systems. Environmental samples include, for example, environmental materials such as surface materials, soil, water, crystals, and industrial samples. However, these examples should not be construed as limiting the types of samples to which the present invention can be applied.

[0056] As used herein, the term “linearly connected atom” refers to a skeletal atom of a chain or polymer, excluding pendants, side chains, or H atoms that do not form the main chain or backbone.

[0057] As used herein, the term “functional element” refers to a detectable, reactive, affinity, or otherwise bioactive agent or part that is bound (e.g., directly or via a suitable linker) to a compound or part described herein. Other additional functional elements that may be used in the embodiments described herein include, but are not limited to, “localization elements” and “detection elements.”

[0058] As used herein, the term “capture element” refers to a molecular entity that forms a covalent interaction with the corresponding “capturer.”

[0059] As used herein, the term “affinity element” refers to a molecular entity that forms a stable non-covalent interaction with the corresponding “affinity agent.”

[0060] As used herein, the term “solid support” is used in reference to any solid or stationary material to which reagents such as substrates, mutant proteins, drug-like molecules, and other test components are bound, or may be bound. Examples of solid supports include microscope slides, microtiter plate wells, coverslips, beads, particles, resins, cell culture flasks, and many other suitable ones. Beads, particles, or resins may be magnetic or paramagnetic.

[0061] When used in chemical structures, display [ka] This represents a point of connection between one part and another part.

[0062] Detailed explanation Provided herein are broad-spectrum G protein-coupled receptor (GPCR) conjugates, detectable / separable compounds comprising such conjugates (e.g., broad-spectrum GPCR conjugates linked to functional elements and / or solid surfaces), and methods for using them for the detection / separation of GPCRs.

[0063] In some embodiments, what is provided herein is a labeled GPCR ligand. Experiments were conducted during the development of the embodiments herein to demonstrate the selection of binding sites on GPCR binders that retain the binding profile of the parent drug molecule but generate a set of indiscriminate tracers including additional functionalities such as linked functional elements and solid surfaces. The labeled GPCR ligands described herein can be used in any preferred assay.

[0064] In some embodiments, the compounds provided herein bind to broad-spectrum GPCRs (e.g., specific to GPCRs but not specific between GPCRs). In some embodiments, the compounds provided herein are [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] It is a compound containing one of the structures, in the formula, [ka] The linker is a binding site to the functional element of the broad-spectrum GPCR binder, to the solid surface, or to the linker between the broad-spectrum GPCR binder and the functional element or solid surface, and the geometric isomers may exist as cis isomers (Z), trans isomers (E), or mixtures of the two.

[0065] In some embodiments, the materials provided herein are analogs or derivatives of CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2. In some embodiments, CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, or their analogs or derivatives are bonded directly (via a single covalent bond) to a functional element or solid surface. In some embodiments, CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, or their analogs or derivatives are bonded indirectly (via a linker) to a functional element or solid surface.

[0066] In some embodiments, the compounds provided herein include, for example, CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, their analogs or derivatives, where, [ka] These reactive groups are suitable for chemical bonding to functional elements (e.g., detectable elements, linkers, etc.) or solid surfaces. These reactive groups may be present on the GPCR binder or linked by a suitable linker. In some embodiments, the reactive groups are configured to react specifically (e.g., via bioorthogonal chemistry or click chemistry) with reactive partners present on or introduced on the functional element or solid surface. An exemplary click reaction is a copper-catalyzed click where the compound has an alkyne or azide and the functional element has a complementary group (e.g., azide or alkyne). Mixing these two chemical species together in the presence of a suitable copper catalyst causes the compound to covalently bond to the functional element via a triazole. Many other bioorthogonal reactions have been reported (e.g., Patterson, DM, et al. (2014) "Finding the Right (Bioorthogonal) Chemistry." ACS Chemical Biology 9(3):592-605; the whole is incorporated herein by reference), and functional elements incorporating compounds (e.g., CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, their analogs or derivatives, etc.) and complementary reactants are embodiments of the present invention.

[0067] In some embodiments, the linker provides sufficient distance between parts of the compound or composition herein (e.g., between a broad-spectrum GPCR binder and a detectable element, a solid surface, etc.) so that each can function without (or with minimal) interference from binding to the other. For example, the linker provides sufficient distance so that the GPCR binder can bind to the GPCR and the detectable part remains detectable (e.g., without or with minimal interference between the two). In some embodiments, the linker is 5 angstroms to 1000 angstroms in length (including both ends) to separate the GPCR binder herein (e.g., CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, their analogs or derivatives, etc.) from the functional element (e.g., a detectable element, a solid surface, etc.). A suitable linker separates the compounds and functional elements of this specification only within 5 Å, 10 Å, 20 Å, 50 Å, 100 Å, 150 Å, 200 Å, 300 Å, 400 Å, 500 Å, 600 Å, 700 Å, 800 Å, 900 Å, 1000 Å, and any suitable range within these (e.g., 5-100 Å, 50-500 Å, 150-700 Å, etc.). In some embodiments, the linker separates the compounds and functional elements of this specification into only 1 to 200 atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, or any preferred range therein (e.g., 2 to 20, 10 to 50, etc.)).

[0068] In some embodiments, the linker contains one or more (e.g., 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or any range between them)-(CH2)2O-(oxyethylene) groups (e.g., -(CH2)2O-(CH2)2O-(CH2)2O-(CH2)2O-, -(CH2)2O-(CH2)2O-(CH2)2O-(CH2)2O-CH2)2O-, -(CH2)2O-(CH2)2O-(CH2)2O-(CH2)2O-CH2)2O-(CH2)2O-, etc.). In some embodiments, the linker is -(CH2)2O-(CH2)2O-(CH2)2O-(CH2)2O-.

[0069] In some embodiments, the linker has two or more "linker portions" (L 1 , L 2 This includes, for example. In some embodiments, the linker has a cleavable portion (Y) and more "linker portions" (L) (e.g., enzymatically cleavable, chemically cleavable, etc.). 1 , L 2 This includes 0, 1, and 2 of (etc.). In some embodiments, the linker portion is a straight or branched chain containing any combination of alkyl chains, alkenyl chains, or alkynyl chains and main chain heteroatoms (e.g., O, S, N, P, etc.). In some embodiments, the linker portion contains one or more skeletal groups selected from -O-, -S-, -CH=CH-, =C=, carbon-carbon triple bond, C=O, NH, SH, OH, CN, etc. In some embodiments, the linker portion contains one or more substituents, pendants, side chains, etc., containing any suitable organic functional groups (e.g., OH, NH2, CN, =O, SH, halogens (e.g., Cl, Br, F, I), COOH, CH3, etc.).

[0070] In certain embodiments, the linker portion is an alkyl group of carbamate (e.g., (CH2) n OCONH, (CH2) nIncludes NHCOO, etc. In some embodiments, the alkyl carbamate is oriented such that the -NH terminus is directed toward the GPCR binder and the COO terminus is directed toward the functional element or solid surface. In some embodiments, the alkyl carbamate is oriented such that the -COO terminus is directed toward the GPCR binder and the -NH terminus is directed toward the functional element or solid surface. In some embodiments, the linker or linker moiety comprises a single alkyl carbamate. In some embodiments, the linker or linker moiety comprises two or more alkyl carbamate (e.g., 2, 3, 4, 5, 6, 7, 8, etc.).

[0071] In some embodiments, the linker portion includes more than one linearly connected C, S, N, and / or O atoms. In some embodiments, the linker portion includes one or more alkyl carbamates. In some embodiments, the linker portion includes one or more alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, etc.). In some embodiments, the linker portion includes 1 to 200 linearly connected atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, or any preferred range therein (e.g., 2 to 20, 10 to 50, 6 to 18)). In some embodiments, the linker portion is a linearly connected atom of length 1 to 200 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, or any preferred range therein (e.g., 2 to 20, 10 to 50, 6 to 18)).

[0072] An exemplary linker for connecting a "drug" (e.g., GPCR conjugates as defined herein (e.g., CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, their analogs or derivatives, etc.)) to a functional element (e.g., a detectable element, a solid surface, etc.) is shown in Figure 14. Such exemplary linkers are used with any suitable GPCR conjugate and functional element described herein.

[0073] In some embodiments, the compositions described herein (including, for example, CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, their analogs or derivatives, etc.) are biocompatible (e.g., cytocompatible) and / or permeable to cells. Therefore, in some embodiments, preferred functional elements (e.g., detectable elements, capture elements) are cytocompatible and / or permeable in the context of such compositions. In some embodiments, compositions containing additional elements, once added extracellularly, can cross the cell membrane and enter cells (e.g., via diffusion, endocytosis, active transport, passive transport, etc.). In some embodiments, preferred functional elements and linkers are selected based on cytocompatible and / or permeable to cells, in addition to their specific functions.

[0074] In certain embodiments, the functional elements have detectable properties that enable the detection of the compounds of this specification (e.g., CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, their analogs or derivatives, etc.) or analytes (e.g., GPCRs) bound thereto. Detectable functional elements include functional groups that are ferromagnetic, paramagnetic, diamagnetic, luminescent, electrochemiluminescent, fluorescent, phosphorescent, colored, antigenic, or have a distinctive mass, as well as those that have characteristic electromagnetic spectral properties such as emission or absorption, magnetism, electron spin resonance, capacitance, dielectric constant, or conductivity. Functional elements include, but are not limited to, nucleic acid molecules (e.g., DNA or RNA (e.g., oligonucleotides or nucleotides)), proteins (e.g., luminescent proteins, peptides), contrast agents (e.g., MRI contrast agents), radionuclides, affinity tags (e.g., biotin or streptavidin), haptens, amino acids, lipids, lipid bilayers, solid supports, fluorescent dye molecules, chromophores, reporter molecules, radionuclides, electron-opaque molecules, MRI contrast agents (e.g., manganese, gadolinium(III), or iron oxide particles), or their coordinators. Methods for detecting specific functional elements, or for isolating compositions containing specific functional elements and those that bind to them, are understood.

[0075] In some embodiments, the functional group is or includes a solid support. Suitable solid supports include deposited particles such as magnetic particles, Sepharose, or cellulose beads; films; glass, e.g., microscope slides; polymers prepared by cellulose, alginates, plastics, or other synthetic materials (e.g., wells in Eppendorf tubes or multiwell plates); self-assembled monolayers; surface plasmon resonance chips; or solid supports with electronically conductive surfaces.

[0076] Exemplary detectable functional elements include haptens (e.g., molecules useful for enhancing immunogenicity, such as hemacyanin derived from water oysters), cleavable labels (e.g., photocleavable biotin) and fluorescent labels (e.g., coumarin modified with N-hydroxysuccinimide (NHS) and BODIPY modified with succinimide or sulfonosuccinimide (detectable by UV and / or visible excitation fluorescence detection)), rhodamine (R110, Rhodol, CRG6, Texas Methyl Red (TAMRA), Rox5, FAM, or fluorescein), coumarin derivatives (e.g., 7-aminocoumarin and 7-hydroxycoumarin, 2-amino-4-methoxynaphthalene, 1-hydroxypyrene, resorphine, phenalenone or benzphenalenone (U.S. Patent No. 4,812,409)), acridinone (U.S. Patent No. 4,810,636), anthracene, and derivatives of alpha and beta-naphthol. Examples include fluorinated fluorescein and xanthene fluoride derivatives including rhodol (e.g., U.S. Patent No. 6,162,931), and bioluminescent molecules (e.g., luciferase (e.g., Oplophorus derivative luciferase (e.g., U.S. Patent Application No. 12 / 773,002; U.S. Patent Application No. 13 / 287,986; the whole thereof is incorporated herein by reference) or GFP or GFP derivatives)). Fluorescent (or bioluminescent) functional elements can be used to sense changes in systems such as phosphorylation in real time. Fluorescent molecules, such as metal ion chemical sensors, may be employed to label proteins that bind the composition. Bioluminescent or fluorescent functional groups such as BODIPY, rhodamine green, GFP, or infrared dyes may be used as functional elements and employed, for example, in interaction studies (e.g., using BRET, FRET, LRET, or electrophoresis).

[0077] Another class of functional elements includes molecules detectable using electromagnetic radiation, such as xanthene fluorescent dye molecules, dansyl fluorescent dye molecules, coumarin and coumarin derivatives, fluorescent acridinium moieties, benzopyrene-based fluorescent dye molecules, as well as 7-nitrobenz-2-oxa-1,3-diazole and 3-N-(7-nitrobenz-2-oxa-1,3-diazole-4-yl)-2,3-diaminopropionic acid. Preferably, the fluorescent molecules have high quantum yield fluorescence at wavelengths different from those of native amino acids, and more preferably, high quantum yield fluorescence that can be excited in the visible portion of the spectrum, or in both the UV and visible portions. When excited at a pre-selected wavelength, the molecules can be detected visually or at low concentrations using conventional fluorescence detection methods. Electrochemiluminescent molecules such as ruthenium chelates and their derivatives, or nitroxide amino acids and their derivatives, can be detected in the femtomole range or below.

[0078] In some embodiments, the functional element is a fluorescent dye molecule. Suitable fluorescent dye molecules for linking to the compounds herein (for example, for forming fluorescent tracers) include xanthene derivatives (e.g., fluorescein, rhodamine, Oregon Green, eosin, Texas Red, etc.), cyanine derivatives (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, etc.), naphthalene derivatives (e.g., dansyl and prodan derivatives), and oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole). (e.g., chlorophyll), pyrene derivatives (e.g., Cascade Blue), oxazine derivatives (e.g., Nile Red, Nile Blue, Cresil Violet, Oxazine 170, etc.), acridine derivatives (e.g., Proflavin, Acridine Orange, Acridine Yellow, etc.), arylmethine derivatives (e.g., Auramine, Crystal Violet, Malachite Green, etc.), tetrapyrrole derivatives (e.g., Porfin, Phthalocyanine, Bilirubin, etc.), CF dyes (Biotium), BODIPY (Invitrogen), ALEXA Examples include, but are not limited to, FLuoR (Invitrogen), DYLIGHT FLUOR (Thermo Scientific, Pierce), ATTO and TRACY (Sigma Aldrich), FluoProbes (Interchim), DY and MEGASTOKES (Dyomics), SULFO CY dye (CYANDYE, LLC), SETAU AND SQUARE DYES (SETA BioMedicals), QUASAR and CAL FLUOR dyes (Biosearch Technologies), SURELIGHT DYES (APC, RPE, PerCP, Phycobilisomes) (Columbia Biosciences), APC, APCXL, RPE, BPE (Phyco-Biotech), autofluorescent proteins (e.g., YFP, RFP, mCherry, mKate, etc.), and quantum dot nanocrystals.In some embodiments, the fluorescent dye molecule is a rhodamine analog (e.g., a carboxyrhodamine analog), such as that described in U.S. Patent Application No. 13 / 682,589, which is incorporated herein by reference in whole.

[0079] In addition to fluorescent molecules, various molecules with physical properties based on molecular interactions and responses to electromagnetic fields and radiation are used in the compositions and methods described herein. These properties include absorption in the UV, visible, and infrared regions of the electromagnetic spectrum, Raman activity, the presence of a chromophore which can be further enhanced by resonance Raman spectroscopy, electron spin resonance activity, nuclear magnetic resonance, and molecular weight measured by, for example, mass spectrometry.

[0080] In some embodiments, the functional element is a capture element. In some embodiments, the capture element is a substrate of a protein (e.g., an enzyme), and the captureant is that protein. In some embodiments, the capture element is a “covalent substrate,” or one that forms a covalent bond with the protein or enzyme it reacts with. The substrate may contain a reactive group (e.g., a modified substrate) that forms a covalent bond with the enzyme during interaction with the enzyme, or the enzyme may be a mutant version that cannot harmonize the covalent intermediate with the substrate. In some embodiments, the substrate is recognized by a mutant protein (e.g., a mutant dehalogenase) that forms a covalent bond with it. In such embodiments, interaction between the substrate and the wild-type version of the protein (e.g., dehalogenase) results in the product and regeneration of the wild-type protein, while interaction between the substrate (e.g., a haloalkane) and the mutant version of the protein (e.g., dehalogenase) results in the formation of a stable bond (e.g., covalent bond) between the protein and the substrate. The substrate may be any substrate suitable for any mutant protein that has been modified to form a hyperstable bond or covalent bond with a substrate that is normally only transiently bound by the protein. In some embodiments, the protein is a mutant hydrolase or dehalogenase. In some embodiments, the protein is a mutant dehalogenase and the substrate is a haloalkane. In some embodiments, the haloalkane is an alkane capped with a terminal halogen (e.g., Cl, Br, F, I, etc.) (e.g., C2-C 20 ) includes. In some embodiments, the haloalkane is of formula AX, where X is a halogen (e.g., Cl, Br, F, I, etc.) and A is an alkane containing 2 to 20 carbon atoms. In certain embodiments, A contains a linear segment of 2 to 12 carbon atoms. In certain embodiments, A is a linear segment of 2 to 12 carbon atoms. In some embodiments, the haloalkane may include any additional pendants or substitutions that do not interfere with the interaction with the mutant dehalogenase.

[0081] In some embodiments, the capture agent is a SNAP tag and the capture element is benzylguanine (see, e.g., Crivat G, Taraska JW, (January, 2012). Trends in Biotechnology 30(1):8-16; the whole is incorporated herein by reference). In some embodiments, the capture agent is a CLIP tag and the capture element is benzylcytosine (see, e.g., Gautier, et al. Chem Biol. 2008 Feb;15(2):128-36; the whole is incorporated herein by reference).

[0082] In some embodiments, the functional element is an affinity element (e.g., one that binds to an affinity agent). Examples of such pairs include an antibody as an affinity agent and an antigen as an affinity element; a His tag as an affinity element and a nickel column as an affinity agent; and proteins and small molecules with high affinity (e.g., streptavidin and biotin) as affinity agents and affinity elements, respectively. Examples of affinity molecules include, for example, immunogenic molecules (e.g., epitopes of proteins, peptides, carbohydrates, or lipids) (e.g., any molecule useful for preparing antibodies specific to that molecule); biotin, avidin, streptavidin, and their derivatives; metal-binding molecules; and molecules such as fragments and combinations of these molecules. Exemplary affinity molecules include His5(HHHHH)(SEQ ID NO: 15), HisX6(HHHHHH)(SEQ ID NO: 16), C-myc(EQKLISEEDL)(SEQ ID NO: 17), Flag(DYKDDDDK)(SEQ ID NO: 18), SteptTag(WSHPQFEK)(SEQ ID NO: 19), HA tag(YPYDVPDYA)(SEQ ID NO: 20), thioredoxin, cellulose-binding domain, chitin-binding domain, S-peptide, T7 peptide, calmodulin-binding peptide, C-terminal RNA tag, metal-binding domain, metal-binding reactive group, amino acid-reactive group, intein, biotin, streptavidin, and maltose-binding protein. Another example of an affinity molecule is dansyllysine. Antibodies that interact with the dansyl ring are commercially available (Sigma Chemical; St. Louis, Mo.) or can be prepared using known protocols such as those described in Antibodies: A Laboratory Manual (Harlow and Lane, 1988).

[0083] In some embodiments, provided herein are methods for detecting, isolating, analyzing, characterizing, etc., GPCRs within a system (e.g., a cell, cell lysate, sample, biochemical solution or mixture, tissue, organism, etc.) using the compounds provided herein (e.g., including CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, their analogs or derivatives, etc.) alone or attached (e.g., directly or via a suitable linker) to functional elements.

[0084] In some embodiments, provided herein are methods for detecting one or more GPCRs in a sample, the method comprising contacting the sample with a compound provided herein (e.g., including CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, their analogs or derivatives, etc.). In some embodiments, provided herein are methods for isolating one or more GPCRs from a sample.

[0085] In some embodiments, methods are provided for characterizing a sample by analyzing the presence, amount, or population of GPCRs (e.g., which GPCRs are present and / or what amounts) in the sample by contacting the sample with a compound provided herein (e.g., including CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, their analogs or derivatives, etc.).

[0086] In some embodiments, provided herein is a method for diagnosing a disease state comprising detecting the presence or amount of one or more GPCRs in a sample from a subject by contacting the sample with a compound provided herein (e.g., including CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, their analogs or derivatives, etc.), wherein the presence or amount of one or more GPCRs in the sample indicates the disease, condition, or predisposition thereto.

[0087] In some embodiments, provided herein is a method of monitoring a subject's response to a therapeutic treatment, comprising: (a) detecting the presence or amount of one or more GPCRs in a sample from the subject by contacting the sample with a compound herein (e.g., including CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, their analogs or derivatives, etc.) prior to the administration of the therapeutic treatment; and (b) detecting the presence or amount of one or more GPCRs in a sample from the subject by contacting the sample with a compound herein (e.g., including CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, their analogs or derivatives) subsequent to the administration of the therapeutic treatment, wherein a change in the presence or amount of one or more GPCRs indicates the subject's response to the therapeutic treatment.

[0088] In some embodiments, the GPCRs bound by the compounds herein are detected, quantified, and / or isolated by utilizing the unique properties of the compounds and / or the functional elements bound thereto by any means including electrophoresis, gel filtration, high pressure or high-speed liquid chromatography, mass spectrometry, affinity chromatography, ion exchange chromatography, chemical extraction, magnetic bead separation, precipitation, hydrophobic interaction chromatography (HIC), or any combination thereof. The isolated GPCR(s) may be employed in structural and functional studies for diagnostic use, for the preparation of biological or pharmaceutical reagents, as tools for drug development, and for the isolation and characterization of protein complexes, etc., for the study of protein interactions.

[0089] In some embodiments, methods are provided for detecting and / or quantifying a compound (e.g., including CC CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, their analogs or derivatives, etc.) and / or an analyte (e.g., GPCR) bound thereto in a sample. In some embodiments, techniques for the detection and / or quantification of a compound (e.g., including CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, its analogs or derivatives, etc.) and / or an analyte (e.g., GPCR) bound thereto herein rely on the uniqueness of functional elements (e.g., capture elements, affinity elements, detectable elements (e.g., fluorescent dye molecules, luciferase, chelated radionuclides, chelated contrast agents, etc.)) bound to the compound and / or specific modifications to the compound (e.g., mass tags (e.g., heavy isotopes (e.g., 13 C, 15 N, 2 H, etc.)). For example, if a compound (e.g., including CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, their analogs or derivatives, etc.) herein is linked to a fluorescent dye molecule or other luminescent functional element, a system, device, and / or apparatus provided for detecting, quantifying, or monitoring the amount of light (e.g., fluorescence) emitted or changes thereto may be used to detect / quantify the compound and / or an analyte (e.g., GPCR) bound thereto in a sample. In some embodiments, the detection, quantification, and / or monitoring is provided by a device, system, or apparatus including one or more of a spectrophotometer, fluorometer, luminometer, photomultiplier tube, photodiode, turbidimeter, photon counter, electrode, ammeter, voltmeter, capacitance sensor, flow cytometer, CCD, etc.

[0090] In addition to fluorescent functional elements, various functional elements with physical properties based on the interaction and response of functional elements to electromagnetic fields and radiation can be used to detect the compounds of this specification (e.g., CLZP1, CLZP2, CLZP3, QTP, RSPD, LXP, OLZP, AMTRP1, AMTRP2, NTRP1, NTRP2, their analogs or derivatives, etc.) and / or conjugated GPCRs. These properties include absorption in the UV, visible, and infrared regions of the electromagnetic spectrum, the presence of a chromophore with Raman activity that can be further enhanced by resonance Raman spectroscopy, electron spin resonance activity, nuclear magnetic resonance, and molecular weight, for example, via mass spectrometry.

[0091] In some embodiments, the compounds herein bind broad-spectrum GPCRs, including Class A, Class B, Class C, Class Frizzled, adhesion class protein GPCRs, and other seven-transmembrane proteins. In some embodiments, the binding agents herein are, for example, 5-hydroxytryptamine receptors, acetylcholine receptors (muscarinic), adenosine receptors, adrenaline receptors, angiotensin receptors, apelin receptors, bile acid receptors, bombesin receptors, bradykinin receptors, cannabinoid receptors, chemerin receptors, chemokine receptors, cholecystokinin receptors, Class A orphan receptors, complement peptide receptors, dopamine receptors, endothelin receptors, formyl peptide receptors, free fatty acid receptors, galanin receptors, ghrelin receptors, glycoprotein hormone receptors, gonadotropin-releasing hormone receptors, histamine receptors, hydroxycarboxylic acid receptors, leukotriene receptors, lysophospholipid (LPA) receptors, lysophospholipid (S1P) receptors, melanin-concentrating hormone receptors, melanocortin receptors, melatonin receptors Receptors include: neuromedin U receptor, neuropeptide FF / neuropeptide AF receptor, neuropeptide W / neuropeptide B receptor, neuropeptide Y receptor, neurotensin receptor, opioid receptor, opsin receptor, orexin receptor, P2Y receptor, prokinethicin receptor, prolactin-releasing peptide receptor, prostanoid receptor, proteinase-activating receptor, QRFP receptor, relaxin family peptide receptor, somatostatin receptor, succinate receptor, tachykinin receptor, thyrotropin-releasing hormone receptor, trace amine receptor, urotensin receptor, vasopressin and oxytocin receptor, calcitonin receptor, corticotropin-releasing factor receptor, glucagon receptor family, parathyroid hormone receptor, VIP and PACAP receptor, calcium-sensing receptor, class C orphan receptor, GABA. BThe compounds conjugate to multiple diverse GPCRs and / or GPCRs of multiple GPCR families, such as receptors, metabolite glutamate receptors, taste 1 receptors, Class Frizzled GPCRs, and adhesion class GPCRs. In some embodiments, the compounds herein conjugate to GPCRs of any suitable organism. In some embodiments, the compounds herein conjugate to human GPCRs and / or homologs and analogs derived from other organisms.

[0092] In some embodiments, the binders of this specification are broad-spectrum GPCR binders. Thus, the binders of this specification may bind to GPCRs of multiple (e.g., 2, 3, 4, 5, 10, 20, 30, 40, or more) GPCR classes or GPCR families. In some embodiments, the binders of this specification bind to multiple (e.g., 2, 3, 4, 5, 10, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 400, 500, or more) distinct GPCRs.

[0093] In some embodiments, the GPCR conjugates and tracers described herein are used in systems and methods using such systems that further include bioluminescent proteins (or bioluminescent complexes) to generate bioluminescent resonance energy transfer (BRET) for the detection, characterization, and monitoring of GPCRs. Accordingly, this disclosure includes bioluminescent polypeptides, bioluminescent complexes and their components, as well as materials and methods related to bioluminescent resonance energy transfer (BRET).

[0094] In some embodiments, provided herein are assays, devices, methods, and systems incorporating bioluminescent polypeptides and / or bioluminescent complexes (of peptides and / or polypeptide components) based (e.g., structurally, functionally, etc.) on NanoLuc luciferase (Promega Corporation; U.S. Patent No. 8,557,970; U.S. Patent No. 8,669,103; the whole thereof is incorporated herein by reference), which is a luciferase of Oplophorus gracilirostris, and / or NanoBiT (US.9,797,889; the whole thereof is incorporated herein by reference), or NanoTrip (U.S. Provisional Patent Application No. 62 / 684,014). As described below, in some embodiments, the assays, devices, methods, and systems of this specification incorporate commercially available NanoLuc-based technologies (e.g., NanoLuc luciferase, NanoBRET, NanoBiT, NanoTrip, NanoGlo, etc.), while in other embodiments, various combinations, modifications, or derivatives derived from commercially available NanoLuc-based technologies are employed.

[0095] PCT applications PCT / US2010 / 033449, U.S. Patent No. 8,557,970, PCT applications PCT / 2011 / 059018, and U.S. Patent No. 8,669,103 (each of which is incorporated herein in whole and for all purposes by reference) describe compositions and methods comprising bioluminescent polypeptides. Such polypeptides can be used in the embodiments herein and in conjunction with the assays and methods described herein.

[0096] In some embodiments, the assays, methods, devices, and systems herein include the bioluminescent polypeptide of SEQ ID NO: 5, or a bioluminescent polypeptide having at least 60% sequence identity with SEQ ID NO: 5 (e.g., 0.6%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or in between). In some embodiments, the bioluminescent polypeptide is fused to a GPCR or otherwise linked to a component of the assays, methods, devices, and / or systems described herein.

[0097] U.S. Patent Applications PCT / US14 / 26354 and U.S. Patent No. 9,797,889 (each incorporated herein in whole and for all purposes by reference) describe compositions and methods for organizing bioluminescent complexes. Such complexes, as well as their peptide and polypeptide components, can be used in the embodiments herein and in conjunction with the assays, methods, devices, and / or systems described herein. In some embodiments, what is provided herein is a polypeptide having at least 60% sequence identity with SEQ ID NO: 9 (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or a range between those), but less than 100% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 6 (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%). In some embodiments, what is provided herein is a peptide having at least 60% sequence identity with SEQ ID NO: 10 (e.g., 0.6%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or a range between those), but less than 100% sequence identity with SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 8 (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%). In some embodiments, what is provided herein is a peptide having at least 60% sequence identity with SEQ ID NO: 11 (e.g., 0.6%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or a range between those), but less than 100% sequence identity with SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 8 (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%).In some embodiments, one of the aforementioned NanoBiT peptides or polypeptides is fused to a GPCR or otherwise linked to a component of the assay, method, device, and / or system described herein (e.g., fused, chemically linked, etc.).

[0098] U.S. Provisional Patent Application No. 62 / 684,014 (which is incorporated herein by reference in whole and for all purposes) describes compositions and methods for organizing bioluminescent complexes. Such complexes, as well as their peptide and polypeptide components, can be used in the embodiments herein and in conjunction with the assays and methods described herein. In some embodiments, provided herein are polypeptides having at least 60% sequence identity with SEQ ID NO: 12 (e.g., 0.6%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or a range between these), but less than 100% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 9 (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%). In some embodiments, what is provided herein is a peptide having at least 60% sequence identity with SEQ ID NO: 11 (e.g., 0.6%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or a range between those), but less than 100% sequence identity with SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 8 (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%). In some embodiments, what is provided herein is a peptide having at least 60% sequence identity with SEQ ID NO: 13 (e.g., 0.6%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or a range in between), but less than 100% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7 (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%).In some embodiments, what is provided herein is a peptide having at least 60% sequence identity with SEQ ID NO: 14 (e.g., 0.6%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or a range between those), but less than 100% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 8 (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%). In some embodiments, any of the aforementioned NanoTrip peptides or polypeptides are fused to a GPCR or otherwise linked to components of the assays, methods, devices, and / or systems described herein (e.g., fused, chemically linked, etc.).

[0099] PCT application PCT / US13 / 74765 and U.S. Patent Application 15 / 263,416 (which are incorporated herein by reference in whole and for all purposes) describe bioluminescent resonance energy transfer (BRET) systems and methods (e.g., incorporating NanoLuc system technology). Such systems and methods, as well as bioluminescent polypeptides and their fluorescent dye molecule-conjugated components, are used in embodiments herein and can be used in conjunction with assays, methods, devices, and systems described herein.

[0100] In some embodiments, any NanoLuc, NanoBiT, and / or NanoTrip peptides, polypeptides, complexes, fusions, etc., may be used in BRET-based applications in conjunction with the assays, methods, devices, and systems described herein.

[0101] As used herein, the term “energy acceptor” refers to any small molecule (e.g., a chromophore), a polymer (e.g., an autofluorescent protein, phycobiliprotein, nanoparticle, surface, etc.), or a molecular complex that generates an readily detectable signal in response to energy absorption (e.g., resonance energy transfer). In certain embodiments, the energy acceptor is a fluorescent dye molecule or other detectable chromophore (e.g., any fluorescent dye molecule or other detectable chromophore described herein or understood in the art).Suitable fluorescent dye molecules include xanthene derivatives (e.g., fluorescein, rhodamine, Oregon Green, eosin, Texas Red, etc.), cyanine derivatives (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, etc.), naphthalene derivatives (e.g., dansyl and prodan derivatives), oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzooxadiazole, benzooxadiazole, etc.), pyrene derivatives (e.g., Cascade Blue), oxazine derivatives (e.g., Nile Red, Nile Blue, Cresyl Violet, Oxazine 170, etc.), acridine derivatives (e.g., proflavin, acridine orange, acridine yellow, etc.), arylmethine derivatives (e.g., auramine, crystal violet, malachite green, etc.), tetrapyrrole derivatives (e.g., porfin, phthalocyanine, bilirubin, etc.), CF dyes (Biotium), BODIPY (Invitrogen), ALEXA Examples include, but are not limited to, FLuoR (Invitrogen), DYLIGHT FLUOR (Thermo Scientific, Pierce), ATTO and TRACY (Sigma Aldrich), FluoProbes (Interchim), DY and MEGASTOKES (Dyomics), SULFO CY dye (CYANDYE, LLC), SETAU AND SQUARE DYES (SETA BioMedicals), QUASAR and CAL FLUOR dyes (Biosearch Technologies), SURELIGHT DYES (APC, RPE, PerCP, Phycobilisomes) (Columbia Biosciences), APC, APCXL, RPE, BPE (Phyco-Biotech), autofluorescent proteins (e.g., YFP, RFP, mCherry, mKate, etc.), and quantum dot nanocrystals. In some embodiments, the fluorescent dye molecule is a rhodamine analog (e.g., a carboxyrhodamine analog), such as the one described in U.S. Patent Application No. 13 / 682,589, which is incorporated entirely herein by reference.

[0102] In some embodiments, a system is provided comprising (a) a fusion of a GPCR and a bioluminescent protein (or a component of a bioluminescent complex); and (b) a broad-spectrum GPCR binding moiety linked to a fluorescent dye molecule, wherein when the broad-spectrum GPCR binding moiety binds to the GPCR, the emission spectrum of the bioluminescent protein overlaps with the excitation spectrum of the fluorescent dye molecule, so that BRET can be detected between the bioluminescent protein and the fluorescent dye molecule. A similar BRET system (e.g., utilizing NANOLUC luciferase) is described, for example, in International Patent Application No. PCT / US13 / 74765 (which is incorporated herein in its entirety by reference), and embodiments thereof will be used in the systems and methods herein.

[0103] U.S. Patents 10,107,800; 9,869,670; and 9,797,890 (both incorporated herein by reference) describe two-component systems for organizing bioluminescent complexes from peptide and polypeptide components. U.S. Provisional Patent Application 62 / 684,014 (both incorporated herein by reference) describes a three-component system for organizing bioluminescent complexes from three peptide and polypeptide components. In some embodiments, such systems (and associated methods) are used in the embodiments herein. For example, the peptide component of the bioluminescent complex is provided as a fusion with one or more GPCRs. When such a fusion is contacted with a broad-spectrum GPCR fluorescence tracer and the polypeptide component of the bioluminescent complex as described herein, a BRET signal is detectable. However, a non-target GPCR not fused to the peptide component of the bioluminescent complex will not produce a BRET signal, even if bound to a broad-spectrum GPCR fluorescence tracer. In some embodiments, the peptide tag (e.g., fused to a GPCR) and other components of the bioluminescent complex system (e.g., polypeptide components, substrates, etc.) are described in the above patent / patent application and / or are commercially available as NanoBiT and / or NanoTrip technologies (Promega Corp., Madison, WI). In some embodiments, the peptide tag fused to the GPCR for BRET application exhibits high affinity for the polypeptide components of the bioluminescent complex (e.g., and / or additional peptide components), so that the bioluminescent complex is formed without facilitating the introduction of appropriate components.

[0104] In some embodiments, the BRET application of the techniques described herein relies on a GPCR protein structure that is minimally destabilized by gene fusion to the peptide component of the bioluminescent complex. In some embodiments, the peptide exhibits high affinity for the polypeptide component and / or other peptide components of the bioluminescent complex (e.g., HiBiT). In some embodiments, fusion occurs within the GPCR at the N-terminus, C-terminus, and internally. In some embodiments, the small size of the peptide tag allows for minimal genetic manipulation of the protein. Experiments performed during the development of the embodiments herein demonstrated that the peptide tag (e.g., a component of the bioluminescent complex (e.g., HiBiT)) can be easily inserted into the GPCR of interest via CRISPR-Cas, thus enabling matching of GPCR-ligand interactions without the need for GPCR overexpression and / or the use of membrane preparations. In some embodiments, the polypeptide component of the bioluminescent complex (e.g., LgBiT) is not cell-permeable, so the signal is detected only on the cell surface. This feature of the detection method allows for monitoring of both the cell surface expression level and internalization of the GPCR.

[0105] Figure 1 shows an exemplary embodiment of the BRET composition and method described herein. The fluorescent signal is generated via energy transfer between the HiBiT-tagged GPCR protein (via the formation of a bioluminescent HiBiT-LgBiT complex) and the fluorescent ligand, enabling real-time monitoring of GPCR-fluorescent ligand interactions. Substitution of the fluorescent ligand with an unlabeled ligand results in loss of the BRET signal, allowing determination of both kinetic and binding data for the unlabeled compound. The advantage of this approach is the detection of interactions only between the fluorescent ligand and the HiBiT-GPCR fusion. Other interactions of the fluorescent ligand are not detected, resulting in significantly reduced background and improved sensitivity of BRET signal detection (Figure 2).

[0106] In some embodiments, provided in certain embodiments herein are systems that include, for example, mutant proteins (e.g., mutant hydrolases (e.g., mutant dehalogenases)) that covalently bind their substrates (e.g., haloalkane substrates), such as those described in U.S. Patent No. 7,238,842; U.S. Patent No. 7,425,436; U.S. Patent No. 7,429,472; U.S. Patent No. 7,867,726, each of which is hereby incorporated by reference in its entirety. Such proteins may be provided as fusions with GPCRs. In other embodiments, such proteins are used to capture GPCRs bound to the agents described herein (e.g., when the functional group is the substrate of the mutant protein).

Example

[0107]

Chem.

[0108]

Chem.

[0109] [ka] A 10 mL microwave vial equipped with a stirring bar was filled with SL-1202 (56 mg, 0.21 mmol), tert-butyl (3-(piperazin-1-yl)propyl)carbamate (104 mg, 126 μmol), K2CO3 (74 mg, 0.53 mmol), and dioxane (4 mL). The vial was placed in a microwave reactor and heated at 120 °C for 1 hour. Consumption of the starting materials was confirmed by HPLC analysis, and the solution was then filtered and concentrated under reduced pressure. The crude residue was purified by flash chromatography (gradient elution, 0 → 20% MeOH / DCM) to obtain 72 mg (73% yield) of amidine SL-1236 as a yellow solid. 1 H-NMR (400MHz, DMSO-d6) δ7.33(td,J=7.8,1.5Hz,1H),7.26-7.10(m,2H),7.09-6.92(m,2H),6.87-6 .71(m,3H),2.94(app.q,J=6.6Hz,2H),2.42(s,3H,partially overlaps with DMSO-d5),2.30(t,J=7.3Hz,2H,DMSO-d5- 12 (partially overlaps with C), 1.66-1.46(m,2H),1.37(s,9H);HRMS(ESI)C 25 H 33 ClN5O2[M+H] + Calculated value: 470.2300; Measured value: 470.2300.

[0110] [ka] A 50 mL flask equipped with a stirring bar was packed with amidine SL-1236 (72 mg, 0.15 mmol) and a cutting cocktail (10 mL, 85:15:1 DCM / TFA / TIPS). The resulting pale yellow solution was stirred at 22°C for 20 hours, at which point HPLC showed complete consumption of the starting material, and the solvent was removed under reduced pressure. The residue was dissolved in 10 mL of MeOH, and the solvent was removed under reduced pressure to obtain 72 mg (97% yield) of primary amine SL-1239 as a yellow oil. 1H NMR (400MHz, DMSO-d6) δ7.81(s,2H),7.50-7.36(m,2H),7.32(dd,J=7.8,1.6Hz,1H),7.14-6.99(m,2H),6.99-6.76(m,3H),3 HRMS(ESI)C 20 H 25 ClN5[M+H] + Calculated value: 370.1798; Measured value: 370.1790.

[0111] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1239 (12 mg, 25 μmol), 2,2-dimethyl-4 oxo-3,8,11,14,17-pentaoxa-5-azaicosan-20-euic acid (14 mg, 37 μmol), HATU (12 mg, 31 μmol), NEt3 (24 μL, 0.17 mmol), and DMF (6 mL). The resulting pale yellow solution was stirred at 22 °C for 1 hour, at which point HPLC showed complete consumption of the starting materials, and the solvent was removed under reduced pressure. The crude residue was purified by preparative HPLC (C18, 5 → 95% MeCN / H2O, 0.05% TFA) to obtain 18 mg (quantitative yield) of amide SL-1448 as a yellow oil. 1H-NMR(400MHz,MeOD)δ7.58-7.51(m,1H),7.47(dd,J=7.8,1.6Hz,1H),7-22 7.20(m1H),7.20-7.08(m,3H),6.97(d,J=8.5Hz,1H),3.94(br.s,4H),3.76 (t,J=5.8Hz,2H),3.61(m,12H),3.49(m,6H),3.38(t,J=6.3Hz,2H),3.28-3 .03(m,4H),2.50(t,J=5.8Hz,2H),2.11-1.86(m,2H),1.43(s,9H).MS(ESI)C 36 H 54 ClN6O7[M+H] + Calculated value: 717.37; Measured value: 717.58.

[0112] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1248 (18 mg, 25 μmol) and a cutting cocktail (10 mL, 80:20:1 DCM / TFA / TIPS). The resulting pale yellow solution was stirred at 22°C for 1 hour. At this point, HPLC showed complete consumption of the starting material, and the solvent was removed under reduced pressure. The residue was dissolved in 10 mL of MeOH, and the solvent was removed under reduced pressure to obtain 18 mg (quantitative yield) of the primary amine SL-1451 as a yellow oil. This substance was used further without further purification. HRMS(ESI)C 31 H 46 ClN5O5[M+H] + Calculated value: 617.32; Measured value: 617.38.

[0113] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1239 (5.7 mg, 12 μmol), BODIPY576 / 589SE (5.0 mg, 12 μmol), DIPEA (11 μL, 59 μmol), and DMF (8 mL). The resulting dark purple solution was stirred at 22°C for 1 hour, at which point HPLC showed complete consumption of the starting materials. The reaction mixture was purified by preparative HPLC (C18, 5 → 95% MeCN / H2O, 0.05% TFA) to obtain 3 mg (38% yield) of amide SL-1425 as a dark purple thin film. 1 H-NMR(400MHz,MeOD)δ7.40(td,J=7.8,1.5Hz,1H),7.25(dd,J=8.2,1.5Hz,1H),7.23-7 .20(m,2H),7.19(s,1H),7.16(d,J=4.6Hz,1H),7.05-6.98(m,4H),6.98-6.90(m,2H),6. 85(d,J=8.4Hz,1H),6.39(d,J=3.9Hz,1H),6.36(dd,J=3.9,2.5Hz,1H),3.45-3.01(br. s.12H, overlaps with CD2HOD), 3.00 (t, J = 7.5 Hz, 2 H), 2.73 (t, J = 7.2 Hz, 2 H), 1.91 (p, J = 6.8 Hz, 2 H).

[0114] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1239 (5.7 mg, 12 μmol), BODIPY630 / 650SE (7.8 mg, 12 μmol), NEt3 (8 μL, 60 μmol), and DMF (6 mL). The resulting dark purple solution was stirred at 22 °C for 1.5 hours, at which point HPLC showed complete consumption of the starting materials. The reaction mixture was purified by preparative HPLC (C18, 5 → 95% MeCN / H2O, 0.05% TFA) to obtain 6.5 mg (60% yield) of amide SL-1444 as a dark blue-green thin film. 1¹H-NMR (400MHz, MeOD) δ 8.19 (t, J=5.9Hz, 1H, peak of partially replaced NH amide), 8.12 (dd, J=3.8, 1.1Hz, 1H), 7.72-7.58 (m, 3H), 7.55 (d, J=4.4Hz, 2H), 7.53-7.40 (m, 1H), 7.39-7.33 (m, 2H), 7.25-7.16 (m, 2H), 7.16-6.95 (m, 8H), 6.90 (d, J=8.5Hz, 1H),6.85(d,J=4.3Hz,1H),4.04-3.52(br.s,4H),3.45-3.32(br.s,4H),3.29-3.26(m,2H),3.24(t,J=6.5Hz,2H),3. 10(t,J=7.6Hz,2H),2.19(t,J=7.4Hz,2H),1.90(p,J=6.6Hz,2H),1.65-1.49(m,4H),1.32-1.25(m,2H));HRMS(ESI)C 49 H 51 BClF2N8O5S[M+H] + Calculated value: 915.3554; Measured value: 915.3544.

[0115] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1451 (7.0 mg, 11 μmol), BODIPY576 / 589SE (4.9 mg, 11 μmol), DIPEA (14 μL, 79 μmol), and DMF (6 mL). The resulting dark purple solution was stirred at 22°C for 1 hour, at which point HPLC showed complete consumption of the starting materials. The reaction mixture was purified by preparative HPLC (C18,5 → 95% MeCN / H2O, 0.05% TFA) to obtain 1.8 mg (17% yield) of amide SL-1453 as a dark purple thin film. 1H-NMR(400MHz,MeOD)δ7.43(ddd,J=8.0,7.4,1.6Hz,1H),7.33(dd,J=7.8,1.5Hz,1H),7.24(s,1H),7.20(m,3H),7.09(dd,J =7.5,1.2Hz,1H),7.07-7.03(m,2H),7.02(d,J=4.6Hz,1H),6.99(dd,J=8.5,2.4Hz,1H),6.93(d,J=4.0Hz,1H),6.87(d,J=8. 4Hz,1H),6.35(m,2H),4.20-3.72(br.s,2H),3.71(t,J=5.8Hz,2H),3.64-3.55(m,14H),3.53(m,3H),3.37(t,J=5.5Hz,4H) ,3.28(m,4H),3.14(d,J=14.3Hz,3H),2.65(dd,J=8.3,7.1Hz,2H),2.45(t,J=5.8Hz,2H),1.92(p,J=6.8Hz,2H).HRMS(ESI)C 47 H 57 BClF2N9O6Na[M+Na] + The calculated value for this is: 950.4079; the measured value is: 9050.4050.

[0116] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1451 (7.0 mg, 11 μmol), BODIPY630 / 650SE (7.5 mg, 11 μmol), DIPEA (14 μL, 79 μmol), and DMF (6 mL). The resulting dark purple solution was stirred at 22 °C for 1.5 hours, at which point HPLC showed complete consumption of the starting materials. The reaction mixture was purified by preparative HPLC (C18, 5 → 95% MeCN / H2O, 0.05% TFA) to obtain 5.7 mg (43% yield) of amide SL-1454 as a dark blue-green thin film. 1H-NMR(400MHz,MeOD)δ8.12(dd,J=3.9,1.1Hz,1H),7.70-7.59(m,3H),7.56(d,J=4.0Hz,2H),7.45(ddd,J=8.1,7.4,1.6Hz,1H),7.36(m,2H),7.2 1 (m, 2H), 7.14 (br. 4.3Hz,1H),4.58(s,2H),4.00-3.73(br.s.2H),3.72(t,J=5.8Hz,2H),3.61-3.52(m,13H),3.47(d,J=5.6Hz,3H),3.38(br.s,6H,overlaps with CD2HOD),3.2 7(m,2H),3.21-3.06(m,2H),2.46(t,J=5.8Hz,2H),2.16(t,J=7.4Hz,2H) ,1.93(p,J=6.7Hz,2H),1.65-1.48(m,4H),1.33-1.25(m,2H)HRMS(ESI)C 60 H 71 BClF2N9O8SNa[M+Na] + Calculated value: 1184.4794; Measured value: 1184.4794.

[0117] [ka] A 10 mL microwave vial equipped with a stirring bar was filled with SL-1202 (18 mg, 68 μmol), Cu(hfacac)2 (3.3 mg, 6.9 μmol), and DCE (2 mL). The vial was sealed, and tert-butyl diazoacetate (28 μL, 0.21 mmol) was slowly added to the stirred solution (gas generation may occur). The vial was placed in a microwave reactor and heated at 120°C for 1 minute (the gradient to 120°C took 2 minutes). The cooled solution was purified by flash chromatography (gradient elution, 0 → 20% siRNA / heptane) to obtain 10 mg (39% yield) of ester SL-1427 as a yellow solid. 1H-NMR(400MHz,CDCl3)δ7.65(dd,J=7.8,1.6Hz,1H),7.42(ddd,J=8.2,7.4,1.6Hz,1H),7.22(d,J=2.5Hz,1H),7.19-7.08(m,2H) ),6.91(dd,J=8.2,1.0Hz,1H),6.82(d,J=8.6Hz,1H),4.39(d,J=16.4Hz,1H),4.26(d,J=16.3Hz,1H),1.30(s,9H);HRMS(ESI)C 19 H 19 Cl2N2O2[M+H] + Calculated value: 377.0818; Measured value: 377.0809.

[0118] [ka] A 20 mL microwave vial equipped with a stirring bar was filled with SL-1427 (54 mg, 0.14 mmol), 1-methylpiperazine (80 μL, 0.72 mmol), K2CO3 (50 mg, 0.36 mmol), and dioxane (10 mL). The vial was placed in a microwave reactor and heated at 120 °C for 2 hours. The cooled solution was filtered, and the solvent was removed under reduced pressure. The crude residue was purified by flash chromatography (gradient elution, 0 → 30% MeOH / DCM) to obtain 50 mg (79% yield) of amidine SL-1428 as a gray solid. 1 H-NMR(400MHz,MeOD)δ7.42(ddd,J=8.2,7.3,1.6Hz,1H),7.36-7.23(m,1H),7.23-7.04(m,2H),6.98(dd,J=1.9,1.0Hz,1H),6.94-6.8 0(m,2H),4.53(d,J=16.8Hz,1H),4.25(d,J=16.7Hz,1H),3.61-3.40(m,4H),2.69-2.44(m,4H),2.34(s,3H),1.38(s,9H);HRMS(ESI)C 24 H 30 ClN4O2[M+H] + Calculated value: 441.2057; Measured value: 441.2042.

[0119] [ka] A 250 mL flask equipped with a stirring bar was packed with amidine SL-1428 (5.4 mg, 12 μmol) and a cutting cocktail (10 mL, 75:25:1 DCM / TFA / TIPS). The resulting pale yellow solution was stirred at 22°C for 3 hours. At this point, HPLC showed complete consumption of the starting material, and the solvent was removed under reduced pressure. The residue was dissolved in 10 mL of MeOH, and the solvent was removed under reduced pressure to obtain carboxylic acid SL-1447 as a yellow oil, which was used in the following steps without further purification. 1 H-NMR(400MHz,DMSO-d6)7.62(ddd,J=8.7,7.3,1.6Hz,1H),7.50(dd,J=7.8,1.6Hz,1H),7.38(dd,J=8.3,1.0Hz,1H),7.29(td,J=7.6,1.1Hz,1H ),7.26-7.19(m,2H),7.16(d,J=8.7Hz,1H),4.79(d,J=17.5Hz,1H),4.4 8(d,J=17.6Hz,1H),4.29-3.71(m,4H),3.68-3.38(m,4H),2.98(s,3H).

[0120] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1447 (20 mg, 52 μmol), tert-butyl (2-aminoethyl)carbamate (10 mg, 65 μmol), HATU (25 mg, 65 μmol), DIPEA (65 μL, 0.36 mmol), and DMF (6 mL). The resulting pale yellow solution was stirred at 22 °C for 3 hours, at which point HPLC showed complete consumption of the starting materials, and the solvent was removed under reduced pressure. The crude residue was purified by flash chromatography (gradient elution, 0 → 20% MeOH / DCM) to obtain 3 mg (11% yield) of amide SL-1450 as yellow oil. 1H-NMR(400MHz,MeOD)δ7.47(ddd,J=8.6,7.3,1.6Hz,1H),7.35(dd,J=7.7,1.6 Hz,1H),7.28-7.12(m,2H),7.03(d,J=2.2Hz,1H),7.01-6.88(m,2H),4.40(d, J=15.8Hz,1H),4.31(d,J=15.7Hz,1H),3.82-3.38(m,4H),3.19(m,2H),3.08- 2.95(m,2H),2.68(s,2H),2.59(m,2H),2.40(s,3H),1.39(s,9H);HRMS(ESI)C 27 H 36 ClN6O3[M+H] + Calculated value: 527.2537; Measured value: 527.2528.

[0121] [ka] A 25 mL flask equipped with a stirring bar was packed with amide SL-1450 (6 mg, 11 μmol) and a cutting cocktail (10 mL, 80:20:1 DCM / TFA / TIPS). The resulting pale yellow solution was stirred at 22°C for 1 hour, at which point HPLC showed complete consumption of the starting material, and the solvent was removed under reduced pressure. The residue was dissolved in 10 mL of MeOH, and the solvent was removed under reduced pressure to obtain primary amine SL-1432 as a yellow oil, which was used in the following steps without further purification. 1 H-NMR(400MHz,MeOD)δ7.98(s,3H),7.63(ddd,J=8.6,7.4,1.6Hz,1H),7.52(dd,J=7.8 ,1.6Hz,1H),7.39-7.29(m,2H),7.27(d,J=2.4Hz,1H),7.22(dd,J=8.7,2.4Hz,1H),7.1 4(d,J=8.7Hz,1H),4.60(d,J=15.7Hz,1H),4.45(d,J=15.8Hz,1H),3.97(br.s,4H),3. 70-3.47(m,4H),3.44(td,J=6.2,4.6Hz,2H),3.05-3.01(m,2H),3.00(s,4H);MS(ESI)C 22 H 28 ClN6O[M+H]+ Calculated value: 427.20; Measured value: 427.09.

[0122] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1447 (43 mg, 0.11 mmol), (14-amino-3,6,9,12-tetraoxatetradecyl)carbamate tert-butyl (56 mg, 0.17 mmol), HATU (53 mg, 0.14 mmol), DIPEA (140 μL, 0.78 mmol), and DMF (8 mL). The resulting pale yellow solution was stirred at 22 °C for 21 hours, at which point HPLC showed complete consumption of the starting materials, and the solvent was removed under reduced pressure. The crude residue was purified by preparative HPLC (C18, 5 → 95% MeCN / H2O, 0.05% TFA) to obtain 47 mg (60% yield) of the carbamate salt SL-1461 as a clear oil. 1 H-NMR(400MHz,MeOD)δ7.54(ddd,J=8.2,7.3,1.6Hz,1H),7.42(dd,J=7.7,1.6Hz,1H),7.30-7.21(m,2H),7.15-7.06(m,2H),7.04(d,J=8.6Hz,1H), 4.54(d,J=15.5Hz,1H),4.31(d,J=15.6Hz,1H),3.73-3.56(m,11H),3.56 -3.39(m,10H),3.21(t,J=5.7Hz,2H),2.99(s,3H),1.43(s,9H);MS(ESI)C 35 H 52 ClN6O7[M+H] + Calculated value: 703.36; Measured value: 703.44.

[0123] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1461 (47 mg, 67 μmol) and a cutting cocktail (10 mL, 80:20:1 DCM / TFA / TIPS). The resulting pale yellow solution was stirred at 22°C for 1 hour. At this point, HPLC showed complete consumption of the starting material, and the solvent was removed under reduced pressure. The residue was dissolved in 10 mL of MeOH, and the solvent was removed under reduced pressure to obtain 56 mg (quantitative yield) of the primary amine SL-1451 as a yellow oil. The substance was used further without further purification. HRMS(ESI)C 30 H 44 ClN6NH2O5[M+H] + Calculated value: 603.31; Measured value: 603.24.

[0124] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1432 (4.8 mg, 11 μmol), BODIPY576 / 589SE (4.8 mg, 11 μmol), DIPEA (14 μL, 79 μmol), and DMF (10 mL). The resulting dark purple solution was stirred at 22°C for 3 hours, at which point HPLC showed complete consumption of the starting materials. The reaction mixture was purified by preparative HPLC (C18, 5 → 95% MeCN / H2O, 0.05% TFA) to obtain 4.3 mg (52% yield) of amide SL-1434 as a dark purple thin film. 1H-NMR(400MHz,MeOD)δ7.42(ddd,J=8.3,7.3,1.6Hz,1H),7.34(dd,J=7.7,1.6Hz,1H),7.28-7.16(m,4H),7. 16-7.10(m,3H),7.10-7.01(m,2H),6.98(d,J=8.7Hz,1H),6.88(d,J=4.0Hz,1H),6.37(dd,J=3.9,2.5Hz,1H ),6.30(d,J=4.0Hz,1H),4.23(s,2H),4.09-3.55(br.s,4H),3.55-3.40(br.s,4H),3.40-3.32(m,2H),3.27 (t,J=7.2Hz,1H),3.20(t,J=7.2Hz,1H),3.17-3.08(m,2H),2.93(s,3H),2.59(t,J=7.4Hz,2H);HRMS(ESI)C 38 H 40 Calculated value for BClF2N9O2[M+H]+: 738.3055; Measured value: 738.3055.

[0125] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1432 (3.0 mg, 7 μmol), BODIPY630 / 650SE (4.6 mg, 7 μmol), DIPEA (9 μL, 50 μmol), and DMF (8 mL). The resulting dark purple solution was stirred at 22°C for 1 hour, at which point HPLC showed complete consumption of the starting materials. The reaction mixture was purified by preparative HPLC (C18, 5 → 95% MeCN / H2O, 0.05% TFA) to obtain 3.3 mg (48% yield) of amide SL-1460 as a dark blue-green thin film. 1H-NMR(400MHz,MeOD)δ8.12(dd,J=3.8,1.1Hz,1H),7.66-7.58(m,3H),7.55(m,2H),7.53-7.48(m,1H),7.42-7.38(m,1H),7.38(s ,1H),7.21(ddd,J=8.9,6.4,4.4Hz,4H),7.14(t,J=4.3Hz,2H),7.11(d,J=2.4Hz,1H),7.08-7.02(m,3H),6.99(d,J=8.7Hz,1H),6 .86(d,J=4.3Hz,1H),4.58(s,2H),4.44(d,J=15.6Hz,1H),4.25(d,J=15.6Hz,1H),4.10-3.54(m,4H),3.38(d,J=26.4Hz,5H),3.2 6(td,J=6.9,2.1Hz,3H),3.21-3.14(m,3H),2.94(s,3H),2.09(t,J=7.4Hz,2H),1.63-1.49(m,4H),1.39-1.06(m,2H);HRMS(ESI)C 51 H 54 BClF2N9O4S[M+H] + Calculated value: 972.3769; Measured value: 972.3769.

[0126] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1463 (10 mg, 17 μmol), BODIPY576 / 589SE (7.1 mg, 17 μmol), DIPEA (20 μL, 0.12 mmol), and DMF (6 mL). The resulting dark purple solution was stirred at 22 °C for 18 hours, at which point HPLC showed complete consumption of the starting materials. The reaction mixture was purified by preparative HPLC (C18, 5 → 95% MeCN / H2O, 0.05% TFA) to obtain 4.8 mg (32% yield) of amide SL-1464 as a dark purple thin film. 1H-NMR(400MHz,MeOD)δ7.53(ddd,J=8.2,7.3,1.6Hz,1H),7.40(dd,J=7.7,1.6Hz,1H),7 .29-7.17(m,6H),7.15-7.06(m,2H),7.06-6.97(m,2H),6.93(d,J=4.0Hz,1H),6.34(td ,J=4.3,3.8,1.8Hz,2H),4.48(d,J=15.6Hz,1H),4.28(d,J=15.6Hz,1H),4.14-3.65(m, 4H),3.64-3.56(m,8H),3.56-3.33(m,14H),2.95(s,3H),2.72-2.58(m,2H);HRMS(ESI)C 46 H 56 BClF2N9O6[M+H] + Calculated value: 914.4103; Measured value: 914.4111.

[0127] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1463 (10 mg, 17 μmol), BODIPY630 / 650SE (11 mg, 17 μmol), DIPEA (20 μL, 0.12 mmol), and DMF (6 mL). The resulting dark purple solution was stirred at 22 °C for 18 hours, at which point HPLC showed complete consumption of the starting materials. The reaction mixture was purified by preparative HPLC (C18, 5 → 95% MeCN / H2O, 0.05% TFA) to obtain 12 mg (62% yield) of amide SL-1465 as a dark blue-green thin film. 1H-NMR(400MHz,MeOD)δ8.12(dd,J=3.9,1.1Hz,1H),7.66-7.56(m,4H),7.55(dd,J=4.1,2.7Hz,2H),7.45(dd,J=7.8 ,1.6Hz,1H),7.37(s,1H),7.31-7.22(m,2H),7.22-7.17(m,3H),7.17-7.11(m,3H),7.10-6.99(m,3H),6.86(d,J=4. 3Hz,1H),4.57(s,2H),4.51(d,J=15.5Hz,1H),4.32(d,J=15.5Hz,1H),3.89(d,J=41.1Hz,4H),3.64-3.36(m,20H), 3.30-3.21(m,2H),2.96(s,3H),2.16(t,J=7.4Hz,2H),1.56(dp,J=21.9,7.3Hz,4H),1.37-1.14(m,2H);HRMS(ESI)C 59 H 70 BClF2N9O8S[M+H] + Calculated value: 1148.4818; Measured value: 1148.4887.

[0128] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1188 (29 mg, 0.12 mmol), toluene (4 mL), and DMSO (10 mg, 0.13 mmol). Precipitation occurred upon addition of HBr (33 wt% in H2O, 58 mg, 0.24 mmol). The resulting suspension was heated at 50°C for 1 hour, at which point HPLC showed complete consumption of the starting material, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (0 → 60% toluene / hexane) to obtain 3 mg (78% yield) of aryl bromide SL-1433 as a yellow solid. 1H-NMR(400MHz,DMSO-d6)δ10.04(s,1H),8.17(s,1H),7.75(d,J=2.6Hz,1H),7.51(dd,J=8.7,2.6Hz,1 H),7.02(dd,,J=8.3,2.3Hz,1H),7.00(d,J=2.2Hz,1H),6.97(d,J=8.4Hz,1H),6.93(d,J=8.6Hz,1H); 13 C-NMR(100MHz,DMSO)δ166.2,149.0,137.9,136.0,134.2,130.8,126.6,124.2,123.9,121.3,121.2,120.6,112.1;HRMS(ESI)C 13 H9BrClN2O[M+H] + Calculated value: 322.9587; Measured value: 322.9585.

[0129] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1433 (145 mg, 593 μmol), N,N-dimethylaniline (0.30 mL, 2.4 mmol), POCl3 (166 μL, 1.78 mmol), and toluene (5 mL). The resulting suspension was heated at 95 °C for 2.5 hours, forming a dark brown solution. The solvent was removed under reduced pressure, and the residue was dissolved in a mixture of dioxane (5 mL) and 2 M Na2CO3 aqueous solution (7 mL). The resulting solution was heated at 80 °C for 45 minutes, the dioxane was removed under reduced pressure, and the residue was extracted with HCl (3 × 25 mL). The combined HCl solution was dried over MgSO4, filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (0 → 30% HCl / hexane) to obtain 107 mg (69% yield) of SL-1438 chloride as a yellow solid. 1 H-NMR(400MHz,DMSO-d6)δ7.80(s,1H),7.64-7.49(m,2H),7.19(dd,J=8.5,2.5Hz,1H) ,7.07(d,J=2.4Hz,1H),6.87(d,J=8.6Hz,1H),6.84(dt,J=8.7,1.1Hz,1H;HRMS(ESI)C 13H8BrClN2[M+H] + Calculated value for: 340.9248; measured value: 340.9244.

[0130]

Chem.

[0131]

Chem.

[0132]

Chem.

[0133]

Chem.

[0134] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1441 (3.5 mg, 7.5 μmol), BODIPY630 / 650SE (4.9 mg, 7.5 μmol), DIPEA (7 μL, 37 μmol), and DMF (8 mL). The resulting dark purple solution was stirred at 22°C for 1.5 hours, at which point HPLC showed complete consumption of the starting materials. The reaction mixture was purified by preparative HPLC (C18, 5 → 95% MeCN / H2O, 0.05% TFA) to obtain 6 mg (89% yield) of amide SL-1460 as a dark blue-green thin film. 1H-NMR(400MHz,MeOD)δ8.12(dd,J=3.8,1.1Hz,1H),7.65-7.56(m,3H),7.55(s,1H),7.51(d,J=16.3Hz,1H),7.37(s,1H),7.35(d d,J=8.3,2.1Hz,1H),7.29(d,J=2.1Hz,1H),7.26-7.16(m,2H),7.14(dd,J=7.7,4.4Hz,2H),7.10(d,J=2.4Hz,1H),7.03(td,J=8. 8,2.6Hz,4H),6.88(d,J=8.5Hz,1H),6.86(d,J=4.3Hz,1H),4.56(s,2H),4.24(s,2H),3.76(s,4H),3.42(s,4H),3.25(t,J=6.9H) HRMS(ESI)C 48 H 49 BClF2N8O3S[M+H] + Calculated value: 901.3398; Measured value: 901.3386.

[0135] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1441 (7.0 mg, 15 mmol), 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azaicosan-20-euic acid (8 mg, 20 μmol), HATU (7 mg, 0.02 mmol), DIPEA (15 μL, 0.10 mmol), and DMF (6 mL). The resulting pale yellow solution was stirred at 22 °C for 1.5 hours, at which point HPLC showed complete consumption of the starting materials, and the solvent was removed under reduced pressure. The crude residue was purified by preparative HPLC (C18, 5 → 95% MeCN / H2O, 0.05% TFA) to obtain 11 mg (quantitative) of carbamate SL-1449 as a yellow oil. 1H-NMR(400MHz,MeOD)δ7.45(dd,J=8.3,2.0Hz,1H),7.36(d,J=2.0Hz,1H),7.18(d,J =2.4Hz,1H),7.14-7.03(m,2H),6.94(d,J=8.6Hz,1H),4.32(s,2H),3.85(s,3H),3.7 5(dt,J=15.5,6.0Hz,4H),3.68-3.43(m,26H),3.21(dt,J=11.2,5.6Hz,3H),3.01(s ,3H),2.55(t,J=6.3Hz,1H),2.49(t,J=5.9Hz,2H),1.43(d,J=6.8Hz,14H);MS(ESI)C 35 H 52 ClN6O7[M+H] + Calculated value: 703.36; Measured value: 703.59.

[0136] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1449 (16 mg, 23 μmol) and a cutting cocktail (10 mL, 80:20:1 DCM / TFA / TIPS). The resulting pale yellow solution was stirred at 22°C for 1 hour, at which point HPLC showed complete consumption of the starting material, and the solvent was removed under reduced pressure. The residue was dissolved in 10 mL of MeOH, and the solvent was removed under reduced pressure to obtain 16 mg of primary amine SL-1452 as a yellow oil. This substance was used further without further purification.

[0137] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1452 (7 mg, 12 μmol), BODIPY576 / 589SE (5.0 mg, 12 μmol), DIPEA (15 μL, 82 μmol), and DMF (6 mL). The resulting dark purple solution was stirred at 22 °C for 18 hours, at which point HPLC showed complete consumption of the starting materials. The reaction mixture was purified by preparative HPLC (C18, 5 → 95% MeCN / H2O, 0.05% TFA) to obtain 2.3 mg (23% yield) of amide SL-1456 as a dark purple thin film. 1 H-NMR(400MHz,MeOD)δ7.38(dd,J=8.2,2.1Hz,1H),7.29(d,J=2.0Hz,1H),7.24(s,1H),7.23-7.13 (m,3H),7.10(d,J=2.4Hz,1H),7.06-6.97(m,3H),6.92(d,J=4.0Hz,1H),6.89(d,J=8.4Hz,1H),6.5 4-6.21(m,2H),4.29(s,2H),3.96-3.58(m,6H),3.58-3.45(m,15H),3.43(s,3H),3.36(t,J=5.4Hz ,3H),3.27(d,J=7.7Hz,2H),2.95(s,3H),2.64(t,J=7.7Hz,2H),2.45(t,J=5.8Hz,2H);HRMS(ESI)C 46 H 56 BClF2N9O6[M+H] + Calculated value: 914.4103; Measured value: 914.4089.

[0138] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1452 (7.0 mg, 12 μmol), BODIPY630 / 650SE (7.7 mg, 12 μmol), DIPEA (15 μL, 81 μmol), and DMF (8 mL). The resulting dark purple solution was stirred at 22 °C for 1.5 hours, at which point HPLC showed complete consumption of the starting materials. The reaction mixture was purified by preparative HPLC (C18, 5 → 95% MeCN / H2O, 0.05% TFA) to obtain 3.1 mg (23% yield) of amide SL-1459 as a dark blue-green thin film.1 H-NMR(400MHz,MeOD)δ8.12(dd,J=3.9,1.1Hz,1H),7.71-7.58(m,3H),7.55(m,2H),7.40(dd,J=8.3,2.1Hz,1H),7.38 (s,1H),7.31(d,J=2.0Hz,1H),7.27-7.18(m,2H),7.15(d,J=2.3Hz,2H),7.14(s,1H),7.10-7.00(m,4H),6.91(d,J=8 .5Hz,1H),6.86(d,J=4.2Hz,1H),4.57(s,2H),4.29(s,2H),3.74(t,J=5.8Hz,5H),3.62-3.39(m,17H),3.27(t,J=5.6 HRMS(ESI)C 59 H 69 BClF2N9O8S[M+H] + Calculated value: 1148.4818; Measured value: 1148.4829.

[0139] [ka] A 25 mL flask equipped with a stirring bar was packed with 2-chlorodibenzo[b,f][1,4]oxazepine-11(10H)-one (100 mg, 0.4 mmol), N,N-dimethylaniline (0.21 mL, 1.6 mmol), POCl3 (114 μL, 1.14 mmol), and toluene (4 mL). The resulting suspension was heated at 95 °C for 2.5 hours to form a dark brown solution. The solvent was removed under reduced pressure, and the residue was dissolved in a mixture of dioxane (2 mL) and 2 M Na2CO3 aqueous solution (3 mL). The resulting solution was heated at 80 °C for 50 minutes, the dioxane was removed under reduced pressure, and the residue was extracted with butyl (3 × 10 mL). The combined butyl solution was dried over MgSO4, filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (0-30% Â / hexane) to obtain 35 mg (33% yield) of chloride midomyl SL-1511 as a white solid.1 H-NMR(400MHz,CDCl3)δ7.71(d,J=2.6Hz,1H),7.47(dd,J=8.7,2.5Hz,1H),7.33(dd,J=7.5,2.1Hz,1H),7.26(td,J= 7.5,1.7Hz,1H,overlapping with CHCl3),7.21(td,J=7.5,1.7Hz,1H),7.15(dd,J=7.6,1.7Hz,1H),7.13(d,J=8.7Hz,1H);MS(ESI)C 13 H8Cl2NO[M+H] + Calculated value: 264.00; Measured value: 263.87.

[0140] [ka] A 10 mL microwave vial equipped with a stirring bar was filled with SL-1511 (35 mg, 0.13 mmol), tert-butyl (3-(piperazin-1-yl)propyl)carbamate (65 mg, 0.27 mmol), K2CO3 (46 mg, 0.33 mmol), and dioxane (3 mL). The vial was placed in a microwave reactor and heated at 120 °C for 7 hours. Consumption of the starting materials was confirmed by HPLC analysis, the solution was filtered, and concentrated under reduced pressure. The crude residue was purified by flash chromatography (gradient elution, 0 → 20% MeOH / DCM) to obtain 32 mg (51% yield) of amidine SL-1513 as a yellow solid. 1 H-NMR(400MHz,MeOD)δ7.51(dd,J=8.7,2.6Hz,1H),7.40(d,J=2.6Hz,1H),7.29(d,J=8.7Hz,1H),7.17-7.05(m,3H),7.01(ddd,J=7.8,6. 7,2.4Hz,1H),3.53(br.s,4H),3.10(d,J=6.8Hz,2H),2.62(br.s,4H),2.54-2.40(m,2H),1.72(p,J=6.9Hz,2H),1.44(s,9H);HRMS(ESI)C 25 H 32 ClN4O 3NN [M+H] + Calculated value: 471.2163; Measured value: 471.2152.

[0141] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1513 (32 mg, 68 μmol) and a cutting cocktail (10 mL, 80:20:1 DCM / TFA / TIPS). The resulting pale yellow solution was stirred at 22°C for 1 hour. At this point, HPLC showed complete consumption of the starting material, and the solvent was removed under reduced pressure. The residue was dissolved in 10 mL of MeOH, and the solvent was removed under reduced pressure to obtain 16 mg of primary amine SL-1452 as a yellow oil. This substance was used further without further purification. MS(ESI)C 20 H 24 ClN4O[M+H] + Calculated value: 371.16; Measured value: 371.22.

[0142] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1519 (25 mg, 37 μmol), 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azaicosan-20-euic acid (31 mg, 84 μmol), HATU (32 mg, 84 μmol), DIPEA (66 μL, 0.47 mmol), and DMF (6 mL). The resulting pale yellow solution was stirred at 22 °C for 18 hours, at which point HPLC showed complete consumption of the starting materials, and the solvent was removed under reduced pressure. The crude residue was purified by preparative HPLC (C18, 5 → 95% MeCN / H2O, 0.05% TFA) to obtain 41 mg (85% yield) of carbamate SL-1520 as a clear oil. 1H-NMR(400MHz,DMSO-d6)δ9.58(br.s,1H),8.05(t,J=5.8Hz,1H),7.68(dd,J=8.7,2.6Hz,1H),7.5 7(d,J=2.6Hz,1H),7.44(d,J=8.7Hz,1H),7.23(dd,J=7.8,1.5Hz,1H),7.18-6.90(m,3H),6.75(t,J =5.7Hz,1H),3.61(t,J=6.4Hz,2H),3.57-3.44(m,16H),3.36(t,J=6.2Hz,2H),3.26(s,2H),3.17-3 .09(m,4H),3.05(q,J=6.0Hz,2H),2.35(d,J=6.4Hz,2H),1.97-1.73(m,2H),1.37(s,9H);MS(ESI)C 36 H 53 ClN5O8[M+H] + Calculated value: 718.36; Measured value: 718.41.

[0143] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1520 (41 mg, 57 μmol) and a cutting cocktail (10 mL, 80:20:1 DCM / TFA / TIPS). The resulting pale yellow solution was stirred at 22°C for 1 hour. At this point, HPLC showed complete consumption of the starting material, and the solvent was removed under reduced pressure. The residue was dissolved in 10 mL of MeOH, and the solvent was removed under reduced pressure to obtain 35 mg (99% yield) of the primary amine SL-1528 as a yellow oil. This substance was used further without further purification. MS(ESI)C 31 H 45 ClN5O6[M+H] + Calculated value: 618.31; Measured value: 618.16.

[0144] [ka] The container was packed with ODIPY576 / 589SE (8.0 mg, 19 μmol), DIPEA (50 μL, 0.28 mmol), and DMF (8 mL). The resulting dark purple solution was stirred at 22°C for 20 hours, at which point HPLC showed complete consumption of the starting material. The reaction mixture was purified by preparative HPLC (C18, 5 → 95% MeCN / H2O, 0.05% TFA) to obtain 16.4 mg (94% yield) of amide SL-1529 as a dark purple thin film. 1 H-NMR(400MHz,MeOD)δ10.74(s,1H),7.56(dd,J=8.7,2.6Hz,1H),7.47(d,J=2.6Hz,1H),7.32(d,J=8.7Hz,1H),7 .24(s,1H),7.22-7.18(m,3H),7.18-7.12(m,3H),7.12-7.05(m,1H),6.92(d,J=3.9Hz,1H),6.42-6.27(m,2H),4. 19(s,2H),3.72(t,J=5.8Hz,2H),3.63-3.55(m,14H),3.52(t,J=5.5Hz,2H),3.37(t,J=5.5Hz,2H),3.28(d,J=7. HRMS(ESI)C 47 H 57 BClF2N8O7[M+H] + Calculated value: 929.4100; Measured value: 929.4094.

[0145] [ka] A 25 mL flask equipped with a stirring bar was packed with 2-methyl-5,10-dihydro-4H-benzo[b]thieno[2,3-e][1,4]diazepine-4-one (166 mg, 721 μmol), N,N-dimethylaniline (0.37 mL, 2.9 mmol), POCl3 (200 μL, 2 mmol), and toluene (8 mL). The resulting suspension was heated at 95 °C for 2.5 hours to form a dark brown solution. The solvent was removed under reduced pressure, and the residue was dissolved in a mixture of dioxane (4 mL) and 2 M Na2CO3 aqueous solution (6 mL). The resulting solution was heated at 80 °C for 50 minutes, the dioxane was removed under reduced pressure, and the residue was extracted with ethyl acetate (3 × 25 mL). The combined ethyl acetate solution was dried over MgSO4, filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (0-20% alkylammonium hexane) to obtain 7 mg (4% yield) of imidoyl chloride SL-1533 as a solid. 1 H-NMR(400MHz,DMSO-d6)δ8.22(s,1H),7.03(td,J=7.6,1.6Hz,1H),6.92(td,J=7.6,1.6Hz,1H),6.80(d MS(ESI)C 12 H 10 ClN2S[M+H] + Calculated value: 249.03; Measured value: 249.02.

[0146] [ka] A 10 mL microwave vial equipped with a stirring bar was filled with SL-1533 (7.0 mg, 28 μmol), tert-butyl (3-(piperazin-1-yl)propyl)carbamate (14 mg, 56 μmol), K2CO3 (10 mg, 70 μmol), and dioxane (2 mL). The vial was placed in a microwave reactor and heated at 120 °C for 2 hours. Consumption of the starting materials was confirmed by HPLC analysis, the solution was filtered, and concentrated under reduced pressure. The crude residue was purified by preparative HPLC (C18, 5 → 95% MeCN / H2O, 0.05% TFA) to obtain 5.5 mg (43% yield) of SL-1536 carbamate as a yellow oily solid. 1 H-NMR(400MHz,MeOD)δ7.29(td,J=7.6,1.6Hz,1H),7.25(dd,J=8.0,1.6Hz,1H),7.21-7.11(m,1H),6.92(dd,J=8.0,1.3Hz,1H),6.62(q,J=1.3Hz,1H) ),4.08(br.s,4H),3.74-3.50(rs,4H),3.29-3.22(m,2H),3.19(t,J=6.6H z,2H),2.38(d,J=1.3Hz,3H),2.07-1.89(m,2H),1.45(s,9H);HRMS(ESI)C 24 H 34 N5O2S[M+H] + Calculated value: 456.2433; Measured value: 456.2427.

[0147] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1536 (5.5 mg, 12 μmol) and a cutting cocktail (7 mL, 80:20:1 DCM / TFA / TIPS). The resulting pale yellow solution was stirred at 22°C for 1 hour. At this point, HPLC showed complete consumption of the starting material, and the solvent was removed under reduced pressure. The residue was dissolved in 10 mL of MeOH, and the solvent was removed under reduced pressure to obtain 4.2 mg (98% yield) of primary amine SL-1540 as a yellow oil. This substance was used further without further purification. MS(ESI)C 19 H 26 N5S[M+H]+ Calculated value: 356.19; Measured value: 356.08.

[0148] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1540 (4.2 mg, 12 mmol), 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azaicosan-20-euic acid (5.4 mg, 15 μmol), HATU (5.6 mg, 15 μmol), DIPEA (16 μL, 11 μmol), and DMF (6 mL). The resulting pale yellow solution was stirred at 22 °C for 3 hours, at which point HPLC showed complete consumption of the starting materials, and the solvent was removed under reduced pressure. The crude residue was purified by preparative HPLC (C18, 5 → 95% MeCN / H2O, 0.05% TFA) to obtain 9 mg (quantitative) of carbamate SL-1542 as a yellow oil. 1 H-NMR(400MHz,MeOD)δ7.39-7.27(m,1H),7.25(dd,J=8.0,1.5Hz,1H),7.22-7.07(m,1H),6 .93(dd,J=8.0,1.3Hz,1H),6.63(q,J=1.2Hz,1H),4.10(br.s,4H),3.77(t,J=5.9Hz,2H),3 .62-3.59(m,16H),3.56(q,J=5.5Hz,4H),3.40(dd,J=7.0,5.6Hz,2H),3.25(d,J=7.1Hz,2H ),2.51(t,J=5.8Hz,2H),2.39(d,J=1.2Hz,3H),2.09-1.95(m,2H),1.43(s,9H);HRMS(ESI)C 35 H 54 N6O7SNa[M+Na] + Calculated value: 725.3672; Measured value: 725.3661.

[0149] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1542 (8 mg, 12 μmol) and a cutting cocktail (7 mL, 80:20:1 DCM / TFA / TIPS). The resulting pale yellow solution was stirred at 22°C for 1 hour, at which point HPLC showed complete consumption of the starting material, and the solvent was removed under reduced pressure. The residue was dissolved in 10 mL of MeOH, and the solvent was removed under reduced pressure to obtain 6 mg (85% yield) of the primary amine SL-1528 as a yellow oil. This substance was used further without further purification. MS(ESI)C 30 H 47 N6O5S[M+H] + Calculated value: 603.33; Measured value: 603.08.

[0150] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1545 (6 mg, 10 μmol), BODIPY576 / 589SE (3.5 mg, 8 μmol), DIPEA (14 μL, 82 μmol), and DMF (8 mL). The resulting dark purple solution was stirred at 22 °C for 3 hours, at which point HPLC showed complete consumption of the starting materials. The reaction mixture was purified by preparative HPLC (C18, 5 → 95% MeCN / H2O, 0.05% TFA) to obtain 2 mg (27% yield) of amide SL-1516 as a dark purple thin film. 1H-NMR(400MHz,MeOD)δ7.28(ddd,J=8.0,7.2,1.7Hz,1H),7.25(s,1H),7.24-7.18(m,4H),7.15(ddd,J=8.0,7.2,1.3Hz, 1H),7.01(d,J=4.6Hz,1H),6.96-6.85(m,2H),6.55(q,J=1.2Hz,1H),6.34(td,J=4.2,1.8Hz,2H),4.03(s,4H),3.74(t,J =5.8Hz,2H),3.59(d,J=3.7Hz,12H),3.53(t,J=5.6Hz,2H),3.46(br.s,4H),3.41-3.34(m,4H),3.27(d,J=7.6Hz,2H),3. 18(t,J=7.0Hz,2H),2.64(t,J=7.6Hz,2H),2.48(t,J=5.7Hz,2H),2.34(d,J=1.2Hz,3H),1.95(p,J=6.8Hz,2H);MS(ESI)C 46 H 59 BF2N9O6S[M+H] + Calculated value: 914.44; Measured value: 914.26.

[0151] [ka] A 50 mL flask equipped with a stirring bar was packed with quetiapine (263 mg, 686 μmol), 4-nitrophenyl chloroformate (200 mg, 1 mmol), and DCM (30 mL). The resulting solution was cooled to 0°C under Ar and pyridine (166 μL, 2.06 mmol) was added dropwise. The solution was heated to 22°C and stirred for 20 hours. At this point, the solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (0 → 50% MeOH / DCM) to obtain 121 mg (32% yield) of carbonate SL-1530 as a yellow oily solid. 1H-NMR(400MHz,CDCl3)δ8.35-8.10(m,2H),7.51(dt,J=7.3,1.2Hz,1H),7.44-7.35(m,3H),7.35-7.27(m,3H),7.18(t,J=7.7Hz,1H),7.06(dd, MS(ESI)C 28 H 29 N4O6S[M+H] + Calculated value: 549.18; Measured value: 549.03.

[0152] [ka] A 250 mL flask equipped with a stirring bar was packed with SL-1530 (60 mg, 110 μmol), 2,2'-(ethane-1,2-diylbis(oxy))bis(ethane-1-amine) (81 mg, 0.55 mmol), DIPEA (180 μL, 1.0 mmol), and DMF (100 mL). The resulting yellow solution was stirred at 22 °C for 18 hours, at which point HPLC showed complete consumption of the starting material. The solvent was removed under reduced pressure, and the residue was purified by preparative HPLC (C18, 5 → 95% MeCN / H2O, 0.05% TFA) to obtain 86 mg (quantitative) of amide SL-1532 as a dark purple thin film. 1 H-NMR(400MHz,MeOD)δ7.71(dd,J=7.8,1.3Hz,1H),7.68-7.46(m,4H),7.42- 7.28(m,2H),7.21(ddd,J=7.7,6.8,2.1Hz,1H),4.22(t,J=4.6Hz,2H),4.17-3 .79(m,6H),3.79-3.62(m,10H),3.57(d,J=9.9Hz,2H),3.52(t,J=5.7Hz,2H) ,3.51-3.40(m,2H),3.27(t,J=5.7Hz,2H),3.12(t,J=5.1Hz,2H);HRMS(ESI)C 27 H 39 N5O5S[M+H] +Calculated value: 558.2750; Measured value: 558.2746.

[0153] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1532 (8 mg, 10 μmol), BODIPY576 / 589SE (4 mg, 9 μmol), DIPEA (16 μL, 94 μmol), and DMF (8 mL). The resulting dark purple solution was stirred at 22 °C for 16 hours, at which point HPLC showed complete consumption of the starting materials. The reaction mixture was purified by preparative HPLC (C18, 5 → 95% MeCN / H2O, 0.05% TFA) to obtain 5.1 mg (63% yield) of amide SL-1534 as a dark purple thin film. 1 H-NMR(400MHz,MeOD)δδ7.57(dd,J=7.7,1.2Hz,1H),7.51-7.35(m,4H),7.28-7.22( m,2H),7.22-7.16(m,3H),7.09(dd,J=8.0,1.4Hz,1H),7.04-6.87(m,3H),6.39-6.2 2(m,2H),4.20(t,J=4.6Hz,2H),3.83-3.78(m,2H),3.69(t,J=4.5Hz,3H),3.62-3.4 2(m,12H),3.42-3.35(m,6H),3.29-3.16(m,4H),2.66(t,J=7.7Hz,2H);HRMS(ESI)C 44 H 52 BF2N8O6S[M+H] + Calculated value: 869.3792; Measured value: 869.3784.

[0154] [ka] A 50 mL flask equipped with a stirring bar was packed with paliperidone (220 mg, 516 μmol), pyridine (1 mL), and DCM (10 mL). 4-nitrophenyl chloroformate (200 mg, 1 mmol) was slowly added to the resulting solution. The solution was stirred at 22°C for 20 hours. At this point, the solution was purified by silica gel chromatography (0 → 50% MeOH / DCM) to obtain carbonate SL-1586 as a yellow solid. MS(ESI)C 30 H 31 FN5O7[M+H] + Calculated value: 592.22; Measured value: 592.11.

[0155] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1586 (19 mg, 32 μmol), tert-butyl (2-aminoethyl)carbamate (6.2 mg, 39 μmol), DIPEA (70 μL, 97 μmol), and MeCN (10 mL). The resulting yellow solution was stirred at 22 °C for 2 hours, at which point HPLC showed complete consumption of the starting materials. The solvent was removed under reduced pressure, and the residue was purified by preparative HPLC (C18, 5 → 95% MeCN / H2O, 0.05% TFA) to obtain 12 mg (60% yield) of SL-1588 carbamate as a clear oil. 1 H-NMR(400MHz,MeOD)δ7.92(dd,J=8.8,5.1Hz,1H),7.45(dd,J=8.7,2.2Hz,1H),7.22(td,J=9.0,2.2Hz,1H),5.72-5.53(m,1H),4.07(dt,J=14 .3,5.1Hz,1H),3.96-3.77(m,3H),3.75-3.38(m,2H),3.23-3.10(m,4H) ,3.10-2.89(m,2H),2.63-2.33(m,6H),2.33-1.91(m,6H),1.43(s,9H).

[0156] [ka] A 25 mL flask equipped with a stirring bar was packed with carbamate SL-1590 (12 mg, 19 μmol) and a cutting cocktail (7 mL, 80:20:1 DCM / TFA / TIPS). The resulting pale yellow solution was stirred at 22 °C for 1.5 hours. At this point, HPLC showed complete consumption of the starting material, and the solvent was removed under reduced pressure. The residue was dissolved in 10 mL of MeOH, and the solvent was removed under reduced pressure to obtain 10 mg (quantitatively) of primary amine SL-1590 as a yellow oil, which was used further without further purification. MS(ESI)C 26 H 34 FN6O4[M+H] + Calculated value: 513.26; Measured value: 513.13.

[0157] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1590 (9 mg, 18 μmol), BODIPY576 / 589SE (5 mg, 12 μmol), DIPEA (15 μL, 82 μmol), and DMF (7 mL). The resulting dark purple solution was stirred at 22°C for 2 hours, at which point HPLC showed complete consumption of the starting materials. The reaction mixture was purified by preparative HPLC (C18, 5 → 95% MeCN / H2O, 0.05% TFA) to obtain 3.2 mg (33% yield) of amide SL-1592 as a dark purple thin film. 1H-NMR(400MHz,MeOD)δ7.88(dd,J=8.8,5.1Hz,1H),7.44(dd,J=8.7,2.2Hz,1H),7.24(d,J=3.9Hz,1H),7.20 (t,J=4.2Hz,4H),7.09-6.95(m,1H),6.92(d,J=3.9Hz,1H),6.52-6.15(m,2H),5.62(d,J=5.6Hz,1H),4.03( dd,J=14.5,5.7Hz,1H),3.83(t,J=13.4Hz,3H),3.49(d,J=24.6Hz,1H),3.42-3.32(m,2H),3.26-3.06(m,5H) ),3.06-2.78(m,2H),2.69-2.49(m,2H),2.42(t,J=15.6Hz,2H),2.32(m,3H),2.24-1.85(m,5H);HRMS(ESI)C 42 H 46 BF3N9O5[M+H] + Calculated value: 824.3667; Measured value: 824.3677.

[0158] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1586 (19 mg, 32 μmol), (14-amino-3,6,9,12-tetraoxatetradecyl)carbamate tert-butyl (13 mg, 39 μmol), DIPEA (17 μL, 97 μmol), and MeCN (10 mL). The resulting yellow solution was stirred at 22 °C for 1 hour, at which point HPLC showed complete consumption of the starting materials. The solvent was removed under reduced pressure, and the residue was purified by preparative HPLC (C18,5 → 95% MeCN / H2O, 0.05% TFA) to obtain 13 mg (51% yield) of carbamate SL-1587 as a clear oil. 1H-NMR(400MHz,MeOD)δ8.01-7.75(m,1H),7.45(dd,J=8.8,2.2Hz,1H),7.22(td,J=9.0 ,2.2Hz,1H),5.63(t,J=4.6Hz,1H),4.17-4.00(m,1H),4.00-3.79(m,3H),3.79-3.52( m,16H),3.50(t,J=5.7Hz,3H),3.26-3.14(m,3H),3.11-2.85(m,2H),2.56-2.42(m,2H ),2.38(d,J=10.8Hz,3H),2.31-2.15(m,2H),2.15-1.90(m,4H),1.43(s,9H)MS(ESI)C 39 H 58 FN6O 10 [M+H] + Calculated value: 789.42; Measured value: 789.29.

[0159] [ka] A 25 mL flask equipped with a stirring bar was packed with carbamate SL-1587 (13 mg, 16 μmol) and a cutting cocktail (7 mL, 80:20:1 DCM / TFA / TIPS). The resulting pale yellow solution was stirred at 22°C for 1 hour. At this point, HPLC showed complete consumption of the starting material, and the solvent was removed under reduced pressure. The residue was dissolved in 10 mL of MeOH, and the solvent was removed under reduced pressure to obtain 12 mg (quantitatively) of primary amine SL-1589 as a yellow oil, which was used further without further purification. MS(ESI)C 34 H 50 FN6O8[M+H] + Calculated value: 689.37; Measured value: 689.34.

[0160] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1589 (12 mg, 17 μmol), BODIPY576 / 589SE (6 mg, 14 μmol), DIPEA (18 μL, 99 μmol), and DMF (7 mL). The resulting dark purple solution was stirred at 22 °C for 2.5 hours, at which point HPLC showed complete consumption of the starting materials. The reaction mixture was purified by preparative HPLC (C18, 5 → 95% MeCN / H2O, 0.05% TFA) to obtain 3.6 mg (26% yield) of amide SL-1591 as a dark purple thin film. 1 H-NMR(400MHz,MeOD)δ7.86(dd,J=8.8,5.0Hz,1H),7.52-7.37(m,1H),7.29-7.09(m,5H),7.00(d,J=4.6Hz,1 H),6.92(d,J=4.0Hz,1H),6.40-6.15(m,2H),5.63(d,J=3.9Hz,1H),4.05(dd,J=14.3,4.8Hz,1H),3.85(t,J=1 5.4Hz,3H),3.53(td,J=5.4,2.2Hz,5H),3.37(td,J=5.2,2.6Hz,3H),3.28-3.07(m,6H),2.97(dq,J=7.8,3.9 HRMS(ESI)C 50 H 626 BF3N9O9[M+H] + Calculated value: 1001.4773; Measured value: 1000.4716.

[0161] Synthesis of amitriptyline fluorescent tracer: [ka] A 50 mL round-bottom flask equipped with a stirring bar and rubber diaphragm was packed with 1-bromo-4-chloro-2-iodobenzene (3.17 g, 10 mmol), CuI (38 mg, 0.2 mmol), and PdCl2(PPh3)2 (140 mg, 0.2 mmol) under an argon atmosphere. Degassed diethylamine (18 mL) was added via syringe, followed by 3-buty-1-ol. The reaction mixture was stirred at 23 °C for 72 hours, at which point HPLC showed complete consumption of the starting materials, and the solvent was removed under reduced pressure. The crude residue was purified by flash chromatography (gradient elution, 0 → 50% siRNA / heptane) to obtain 2.15 g (83% yield) of alkyne SL-1809 as a yellow solid. 1 H-NMR(400MHz,CDCl3)δ7.49(d,J=8.6Hz,1H),7.43(d,J=2.6Hz,1H),7.13(dd,J=8.6,2.5Hz,1H),3.85(t,J=6.1Hz,2H),2.74(t,J=6.1Hz,2H); 13 C-NMR(100MHz,CDCl3)δ133.3,133.0,132.9,129.3,126.8,123.6,93.1,80.4,77.3,77.0,76.7,60.9,24.0;HRMS(ESI)C 10 H9BrClO[M+H] + Calculated value: 258.9525; Measured value: 258.9520.

[0162] [ka] A 50 mL pressure vessel equipped with a stirring bar was packed with SL-1809 (146 mg, 560 μmol), potassium trifluoro(phenethyl)borate (125 mg, 590 μmol), K2CO3 (206 mg, 1.69 mmol), and Pd(dppf)Cl2 (8 mg, 11 μmol). Argon was flowed into the space created at the top, and degassed toluene (5 mL) and degassed water (1 mL) were added. The reaction mixture was stirred at 95 °C for 21 hours. The reaction mixture was cooled to room temperature, diluted with toluene (40 mL), dried over MgSO4, filtered, and the solvent was removed under reduced pressure. The crude residue was purified by preparative HPLC (C18,5 → 95% MeCN / H2O, 0.05% TFA) to obtain 60 mg (38% yield) of alkyne SL-1801 as a clear oil. 1 H-NMR(400MHz,CDCl3)δ7.39(d,J=2.3Hz,1H),7.33-7.26(m,2H),7.24-7.19(m,1H),7.16(dd,J=8.4,2.0Hz,3H),7 .04(d,J=8.2Hz,1H),3.83(t,J=6.3Hz,2H),3.13-2.95(m,2H),2.90(dd,J=9.7,6.2Hz,2H),2.74(t,J=6.3Hz,2H); 13 C-NMR(100MHz,CDCl3)δ13C-NMR(101MHz,CDCl3)δ142.0,141.4,132.0,131.4,130.1 ,128.4,128.4,128.1,126.0,124.4,91.2,79.8,61.2,36.8,36.2,23.9;HRMS(ESI)C 18 H 18 ClO[M+H] + Calculated value: 285.1046; Measured value: 285.1044.

[0163] [ka] A 25 mL round-bottom flask equipped with a stirring bar was packed with SL-1812 (72 mg, 0.25 mmol), EtN(iPr)2 (90 μL, 0.51 mmol), and DCM (7 mL). The resulting solution was cooled to 0°C under argon, and then mesyl chloride (29 μL, 0.38 mmol) was added. The reaction mixture was stirred at 0°C for 1 hour, at which point HPLC showed complete consumption of the starting materials, and the solvent was removed under reduced pressure. The crude residue was used in the next step without further purification.

[0164] A 25 mL round-bottom flask equipped with a stirring bar was packed with a solution of SL-1822 (90 mg, 0.25 mmol) and 2 M diethylamine in THF (12 mL, 25 mmol). The reaction mixture was stirred at 50 °C for 20 hours, at which point HPLC showed complete consumption of the starting material, and the solvent was removed under reduced pressure. The crude residue was purified by flash chromatography (gradient elution, 0 → 100% Â / heptane) to obtain 26 mg (35% yield) of amine SL-1823 as a clear oil. 1 H-NMR(400MHz,CDCl3)δ7.37(d,J=2.3Hz,1H),7.28(dd,J=8.0,6.6Hz,2H),7.23-7.16(m,3H),7.14(dd,J =8.2,2.3Hz,1H),7.02(d,J=8.2Hz,1H),3.07-2.95(m,2H),2.95-2.81(m,2H),2.64(s,4H),2.31(s,6H); 13 C NMR(100MHz,CDCl3)δ13C-NMR(101MHz,CDCl3)δ142.0,141.6,131.9,131.3,130.0,128.4,1 28.3,127.9,126.0,124.8,78.8,77.3,77.0,76.7,58.3,45.1,36.7,36.2,18.5;HRMS(ESI)C 20 H 23 CLN[M+H] + Calculated value: 312.1519; Measured value: 312.1516.

[0165] [ka] A 10 mL round-bottom flask equipped with a stirring bar was filled with a 3 mL solution of SL-1823 (25 mg, 80 μmol) in DCM. The solution was cooled to 0°C, and trifluic acid (39 μL, 0.44 mmol) was added all at once. The resulting brown solution was stirred at 0°C for 10 minutes, at which point the reaction was stopped by adding a saturated aqueous solution of K2CO3 (3 mL). The organic layer was separated, and the aqueous solution was extracted (2 × 3 mL of DCM). The organic compounds were combined, dried over MgSO4, filtered, and concentrated under vacuum. The crude residue was used in the next step without further purification. 1 ¹H-NMR (400MHz, CDCl3, reported for a mixture of E / Z isomers): δ 7.25-6.85 (m,7H), 5.86-5.80 (m,1H), 3.41-3.18 (m,2H), 3.00-2.86 (m,1H), 2.81-2.64 (m,3H), 2.44 (s,8H); MS(ESI)C 10 H 23 ClN[M+H] + Calculated value: 312.15; Measured value: 312.11; Single peak of HPLC at 254 nm.

[0166] A 50 mL round-bottom flask equipped with a stirring bar and diaphragm was packed with SL-1824 (25 mg, 80 μmol), K2CO3 (33 mg, 0.24 mmol), Pd(OAc)2 (1.8 mg, 8.0 μmol), and [dcpp2BF4] (9.8 mg, 16 μmol). The flask was degassed and refilled with argon (repeated 3 times). Degassed DMSO (2 mL) and H2O (0.2 mL) were added, the reaction vessel was degassed, and the flask was refilled with carbon monoxide (repeated 3 times). CO was bubbled through the solution for 5 minutes. The resulting yellow suspension was heated at 110°C for 18 hours under a CO balloon, at which point HPLC analysis showed complete consumption of the starting materials. The reaction mixture was diluted with MeOH (3 mL), passed through a syringe filter, and purified by preparative HPLC (C18,5 → 95% MeCN / H2O, 0.05% TFA) to obtain 25 mg (97% yield) of an E / Z mixture of carboxylic acid SL-1825 as a clear oil. 1¹H-NMR (400MHz, MeOD, reported for a mixture of E / Z isomers): δ 8.06-7.67 (m, 2H), 7.49-6.92 (m, 5H), 5.89 (m, 1H), 3.49-3.34 (m, 2H), 3.25 (m, 2H), 3.09-2.90 (m, 1H), 2.81 (d, J=12.1Hz, 7H), 2.66-2.40 (m, 2H); MS(ESI)C 21 H 24 NO2 [M+H] + Calculated value: 322.18; Measured value: 322.15.

[0167] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1825 (25 mg, 78 μmol), tert-butyl (2-aminoethyl)carbamate (37 mg, 97 μmol), HATU (16 mg, 97 μmol), EtN(iPr)2 (70 μL, 0.39 mmol), and DMF (8 mL). The resulting pale yellow solution was stirred at 22 °C for 2.5 hours, at which point HPLC showed complete consumption of the starting materials, and the solvent was removed under reduced pressure. The crude residue was purified by preparative HPLC (C18,5 → 95% MeCN / H2O, 0.05% TFA) to obtain 10.6 mg (29% yield) of the E isomer of amide SL-1826-E as a clear oil and 4.7 mg (13% yield) of the Z isomer of amide SL-1826-Z as a clear oil (combined yield 42%). SL-1826-E: 1 H-NMR(400MHz,MeOD)δ7.80(d,J=1.9Hz,1H),7.62(dd,J=8.0,2.0Hz,1H),7.34-7.20(m,3H),7.20-7.12(m,2H),5.92(t,J=7.3Hz,1H),3. MS(ESI)C 28 H 38 N3O3[M+H] +Calculated value for 464.3: 464.3; Measured value: 464.4. SL-1826-Z: 1 H-NMR(400MHz,MeOD)δ7.82-7.69(m,1H),7.63(d,J=1.9Hz,1H),7.40(d,J=7.9H z,1H),7.31(dd,J=6.8,2.4Hz,1H),7.19(m,2H),7.10(dd,J=7.2,1.8Hz,1H),5. 89(t,J=7.4Hz,1H),3.45(t,J=6.0Hz,3H),3.39(s,1H),3.31-3.17(m,3H),2.93 (d,J=13.3Hz,2H),2.88-2.71(m,6H),2.71-2.31(m,2H),1.41(s,9H);MS(ESI)C 28 H 38 N3O3[M+H] + Calculated value: 464.3; Measured value: 464.4.

[0168] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1826-E (10.6 mg, 22.9 μmol) and a cutting cocktail (7 mL, 80:20:1 DCM / TFA / TIPS). The resulting pale yellow solution was stirred at 22°C for 35 minutes, at which point HPLC showed complete consumption of the starting material, and the solvent was removed under reduced pressure. The residue was dissolved in 10 mL of MeOH, and the solvent was removed under reduced pressure. The reaction mixture was purified by preparative HPLC (C18, 5 → 95% MeCN / H2O, 0.05% TFA) to obtain 6 mg (72% yield) of amine SL-1827 as a clear oil. 1H-NMR(400MHz,MeOD)δ7.85(d,J=2.0Hz,1H),7.67(dd,J=8.0,2.0Hz,1H),7.33-7.26(m,2H),7.24(dt,J=6.6,3.4Hz,1H),7.22-7.14(m,2H),5. 92(t,J=7.3Hz,1H),3.66(m,2H),3.40(s,2H),3.28-3.20(m,2H),3.17( t,J=6.0Hz,2H),3.05-2.93(m,1H),2.80(m,6H),2.60(m,2H);MS(ESI)C 23 H 30 N3O3 + [M+H] + Calculated value: 364.2; Measured value: 364.3.

[0169] [ka] A 10 mL flask equipped with a stirring bar was packed with SL-1826-Z (4.7 mg, 10.1 μmol) and a cutting cocktail (4 mL, 80:20:1 DCM / TFA / TIPS). The resulting pale yellow solution was stirred at 22°C for 40 minutes, at which point HPLC showed complete consumption of the starting material, and the solvent was removed under reduced pressure. The residue was dissolved in 10 mL of MeOH, and the solvent was removed under reduced pressure. The crude residue was used in the next step without further purification. 1 H-NMR(400MHz,MeOD)δ7.78(dd,J=7.9,1.9Hz,1H),7.68(d,J=1.9Hz,1H),7.41(d, J=7.9Hz,1H),7.33-7.22(m,1H),7.21-7.12(m,2H),7.08(dd,J=7.2,1.9Hz,1H),5 .88(t,J=7.3Hz,1H),3.66(t,J=5.9Hz,2H),3.49-3.34(m,2H),3.28-3.20(m,2H), 3.16(t,J=6.0Hz,2H),2.98-2.87(m,2H),2.81(m,6H),2.70-2.49(m,2H);MS(ESI)C 23 H 30 N3O3 + [M+H] + Calculated value: 364.2; Measured value: 364.5.

[0170] [ka] To a solution of SL-1827 (1.3 mg, 3.5 μmol) in DMF (8 mL), DIPEA (3.0 μL, 18 μmol) was added, followed by NanoBRET590 SE (1.5 mg, 3.5 μmol, Promega). The resulting solution was reacted at 22°C for 2 hours, at which point HPLC analysis showed complete consumption of the starting material. The solvent was removed under vacuum, and the crude residue was purified by preparative RP HPLC (5 → 95% MeCN / H2O buffered with 0.5% TFA) to obtain 0.9 mg (36% yield) of SL-1830 as a purple thin film. HPLC: 98% purity at 254 nm; 1 H-NMR(400MHz,MeOD)1δ7.71(d,J=2.0Hz,1H),7.58(dd,J=8.0,2.0Hz,1H),7.33-7.23(m,2H),7. 23-7.16(m,3H),7.16-7.09(m,3H),7.08(s,1H),7.01(d,J=4.6Hz,1H),6.72(d,J=4.0Hz,1H),6.3 4(dd,J=3.9,2.5Hz,1H),6.26(d,J=4.0Hz,1H),5.82(t,J=7.3Hz,1H),3.63-3.43(m,4H),3.17-3. 05(m,2H),3.01-2.86(m,1H),2.83-2.75(m,1H),2.73-2.69(m,8H),2.56-2.38(m,2H);HRMS(SI)C 39 H 42 BF2N6O2 + Calculated value for [M+H]+: 675.3430; Measured value: 675.3413.

[0171] [ka] To a solution of SL-1827 (1.6 mg, 4.5 μmol) in DMF (8 mL), DIPEA (6.0 μL, 31 μmol) was added, followed by NanoBRET590 PEG SE (3.0 mg, 4.5 μmol). The resulting solution was reacted at 22°C for 24 hours, at which point HPLC analysis showed complete consumption of the starting material. The solvent was removed under vacuum, and the crude residue was purified by preparative RP HPLC (5 → 95% MeCN / H2O buffered with 0.5% TFA) to obtain 1.1 mg (27% yield) of SL-1831 as a purple thin film. HPLC: 99% purity at 254 nm; 1 H-NMR(400MHz,MeOD)δ7.78(d,J=2.0Hz,1H),7.61(dd,J=8.0,2.0Hz,1H),7.33-7.08(m,9H),7.01(d,J =4.6Hz,1H),6.91(d,J=4.0Hz,1H),6.40-6.32(m,1H),6.32(d,J=4.0Hz,1H),5.89(t,J=7.3Hz,1H),3. 66(t,J=6.0Hz,2H),3.55-3.52(m,4H),3.52-3.40(m,12H),3.37-3.34(m,2H),3.29-3.25(m,2H),3.23 -3.15(m,2H),2.82-2.73(m,6H),2.63(t,J=7.7Hz,2H),2.56(s,2H),2.42(t,J=6.0Hz,2H);HRMS(SI)C 50 H 63 BF2N7O7 + Calculated value for [M+H]+: 922.4850; Measured value: 922.4835.

[0172] [ka] To a solution of SL-1833 (2.1 mg, 5.9 μmol) in DMF (6 mL), DIPEA (5.0 μL, 29 μmol) was added, followed by NanoBRET590 SE (2.5 mg, 5.9 μmol, Promega). The resulting solution was reacted at 22°C for 16 hours, at which point HPLC analysis showed complete consumption of the starting material. The solvent was removed under vacuum, and the crude residue was purified by preparative RP HPLC (5 → 95% MeCN / H2O buffered with 0.5% TFA) to obtain 1.6 mg (41% yield) of SL-1835 as a purple thin film. HPLC: 99% purity at 254 nm; 1 H-NMR(400MHz,MeOD)δ7.68(dd,J=7.9,2.0Hz,1H),7.58(d,J=1.9Hz,1H),7.34(d,J=7.9Hz,1H),7.26(dd,J=7. 5,1.6Hz,1H),7.23-7.16(m,4H),7.16-7.10(m,2H),7.08(t,J=7.4Hz,1H),7.02(d,J=4.6Hz,1H),6.80(d,J=4.0 Hz,1H),6.35(dd,J=3.9,2.5Hz,1H),6.26(d,J=4.0Hz,1H),5.82(t,J=7.4Hz,1H),3.47-3.42(m,2H),3.35-3.33 (m,2H),3.25-3.19(m,2H),2.94-2.81(m,2H),2.77(s,6H),2.65(t,J=7.7Hz,2H),2.61-2.42(m,2H);HRMS(SI)C 39 H 42 BF2N6O2 + Calculated value for [M+H]+: 675.3430; Measured value: 675.3429.

[0173] [ka] To a solution of SL-1833 (1.0 mg, 3.0 μmol) in DMF (6 mL), DIPEA (4.0 μL, 22 μmol) was added, followed by NanoBRET590-PEG SE (2.0 mg, 3.0 μmol). The resulting solution was reacted at 22°C for 24 hours, at which point HPLC analysis showed complete consumption of the starting material. The solvent was removed under vacuum, and the crude residue was purified by preparative RP HPLC (5 → 95% MeCN / H2O buffered with 0.5% TFA) to obtain 1.5 mg (55% yield) of SL-1836 as a purple thin film. HPLC: 99% purity at 254 nm; 1 H-NMR(400MHz,MeOD)δ7.70(dd,J=7.8,1.9Hz,1H),7.58(d,J=1.9Hz,1H),7.36(d,J=7.9Hz,1H),7.28(dd,J=7.1,2.0Hz,1H),7. 24-7.10(m,6H),7.07(dd,J=7.1,2.0Hz,1H),7.01(d,J=4.6Hz,1H),6.91(d,J=3.9Hz,1H),6.35(dt,J=4.1,2.4Hz,1H),6.32(d,J =3.9Hz,1H),5.83(t,J=7.3Hz,1H),3.63(t,J=5.9Hz,2H),3.53(s,4H),3.51-3.46(m,4H),3.45-3.36(m,10H),3.36-3.33(m,4H) ),3.28-3.16(m,4H),2.97-2.84(m,2H),2.81(s,6H),2.63(t,J=7.7Hz,2H),2.60-2.43(m,2H),2.39(t,J=6.0Hz,2H);HRMS(SI)C 50 H 63 BF2N7O7 + Calculated value for [M+H]+: 922.4850; Measured value: 922.4871.

[0174] [ka] A 50 mL round-bottom flask equipped with a stirring bar and rubber diaphragm was packed with 2-bromo-4-chloro-1-iodobenzene (2.14 g, 6.74 mmol), CuI (25.7 mg, 0.135 mmol), and PdCl2(PPh3)2 (95 mg, 0.13 mmol) under an argon atmosphere. Degassed diethylamine (12 mL) was added via syringe, followed by 3-buty-1-ol. The reaction mixture was stirred at 23 °C for 48 hours, at which point HPLC showed complete consumption of the starting materials, and the solvent was removed under reduced pressure. The crude residue was purified by flash chromatography (gradient elution, 0 → 40% siRNA / heptane) to obtain 1.57 g (90% yield) of alkyne SL-1808 as a yellow solid. 1 H-NMR(400MHz,CDCl3)δ7.59(d,J=2.1Hz,1H),7.37(d,J=8.3Hz,1H),7.23(dd,J=8.4,2.1Hz,1H),3.85(t,J=6.1Hz,2H),2.74(t,J=6.1Hz,2H); 13 C-NMR(100MHz,CDCl3)δ134.3,133.7,132.1,127.5,126.0,124.0,92.7,80.5,60.9,24.0;HRMS(ESI)C 10 H9BrClO[M+H] + Calculated value: 258.9525; Measured value: 258.9521.

[0175] [ka] A 500 mL round-bottom flask equipped with a stirring bar and reflux condenser was packed with SL-1808 (1.57 g, 6.05 mmol), potassium trifluoro(phenethyl)borate (1.35 g, 6.35 mmol), K2CO3 (2.22 g, 18.2 mmol), and Pd(dppf)Cl2 (220 mg, 0.30 mmol). The flask was de-aired and refilled with argon (repeated 3 times). De-aeroded toluene (50 mL) and H2O (10 mL) were added, and the reaction mixture was stirred at 95 °C for 24 hours. The reaction mixture was cooled to room temperature, and the solvent was removed under reduced pressure. The crude residue was separated into DCM (100 mL) and water (100 mL), the aqueous layer was extracted into DCM (2 × 100 mL), the organic matter was combined, dried over MgSO4, filtered, and the solvent was removed under reduced pressure. The crude residue was first purified by flash chromatography (gradient elution, 0→100% RINKAN / heptane), and then further purified by preparative HPLC (C18, 5→95% MeCN / H2O, 0.05% TFA) to obtain 392 mg (23% yield) of alkyne SL-1821 as a white solid. 1 H-NMR(400MHz,CDCl3)δ7.38-7.27(m,3H),7.24-7.16(m,3H),7.16-7.08(m,2H),3 .82(t,J=6.3Hz,2H),3.10-2.97(m,2H),2.96-2.81(m,2H),2.74(t,J=6.3Hz,2H); 13 C-NMR(100MHz,CDCl3)δ113C-NMR(101MHz,CDCl3)δ145.4,141.4,133.7,133.5,128. 9,128.4,128.4,126.2,126.1,121.3,90.9,80.0,61.2,36.7,36.7,24.0;HRMS(ESI)C 18 H 18 ClO[M+H] + Calculated value: 285.1046; Measured value: 285.1044.

[0176] [ka] A 50 mL round-bottom flask equipped with a stirring bar was packed with SL-1821 (210 mg, 0.74 mmol), EtN(iPr)2 (263 μL, 1.47 mmol), and DCM (20 mL). The resulting solution was cooled to 0°C under argon, and then mesyl chloride (86 μL, 1.1 mmol) was added. The reaction mixture was stirred at 0°C for 4 hours, at which point HPLC showed complete consumption of the starting materials. The reaction was stopped by adding saturated K2CO3 aqueous solution (20 mL), and the aqueous phase was further extracted with DCM (3 × 20 mL). The organic compounds were combined, dried over MgSO4, filtered, and the solvent was removed under reduced pressure. The crude residue was used in the next step without further purification. 1 H-NMR (400MHz, CDCl3) δ7.37-7.27(m,3H),7.25-7.07(m,5H),4.37(t,J=6.8Hz,2H),3.12-2.99(m,2H),2.98(s,3H),2.96-2.84(m,4H).

[0177] A 50 mL round-bottom flask equipped with a stirring bar was packed with SL-1828 (270 mg, 0.74 mmol) and a 2 M diethylamine THF (37 mL, 74 mmol) solution. The reaction mixture was stirred at 50°C for 17 hours, at which point HPLC showed complete consumption of the starting material, and the solvent was removed under reduced pressure. The crude residue was purified by flash chromatography (gradient elution, 0 → 20% MeOH / DCM) to obtain 125 mg (54% yield) of amine SL-1829 as a clear oil. 1 H-NMR(400MHz,CDCl3)δ7.39-7.27(m,3H),7.24-7.18(m,3H),7.16-7.03(m,2H),3.08-2.95(m,2H),2.95-2.83(m,2H),2.63(s,4H),2.31(s,6H); 13 C-NMR(100MHz,CDCl3)δ145.4,141.6,133.4,128.8,128.4,126.1,126.0,121.7,58.4,45.1,36.7,36.7,18.5;HRMS(ESI)C 20 H 23 ClN[M+H] +Calculated value: 312.1519; Measured value: 312.1516.

[0178] [ka] A 50 mL round-bottom flask equipped with a stirring bar was filled with a 15 mL solution of SL-1829 (120 mg, 0.38 mmol) in DCM. The solution was cooled to 0°C, and trifluic acid (170 μL, 1.9 mmol) was added all at once. The resulting brown solution was stirred at 0°C for 10 minutes, at which point the reaction was stopped by adding a saturated aqueous solution of K2CO3 (15 mL). The organic layer was separated, and the aqueous solution was extracted (2 × 15 mL DCM). The organic compounds were combined, dried over MgSO4, filtered, and concentrated under vacuum. The crude residue was purified by flash chromatography (gradient elution, 0 → 30% MeOH / DCM) to obtain 101 mg (99% yield) of amine SL-1832 as a clear oil. 1 ¹H-NMR (400 MHz, CDCl3, reported for a mixture of E / Z isomers): δ 7.27-7.23 (m,1H), 7.23-6.98 (m,6H), 5.86 (m,1H), 3.65-3.12 (m,2H), 2.94 (s,1H), 2.75 (s,1H), 2.50-2.34 (m,2H), 2.29 (m,2H), 2.19 (s,6H); 13 ¹³C NMR (100MHz, CDCl3, reported for a mixture of E / Z isomers) δ 142.6, 142.5, 141.2, 140.8, 139.7, 139.6, 139.0, 138.8, 138.5, 136.7, 132.8, 132.5, 130.0, 129.9, 129.8, 129.6, 129.6, 128.6, 128.1, 128.0, 127.6, 127.2, 126.1, 126.0, 125.9, 125.8, 59.2, 45.2, 45.2, 33.7, 33.4, 31.9, 31.7, 27.8, 27.7. HRMS (ESI) C 10 H 23 ClN[M+H] + Calculated value: 312.1519; Measured value: 312.1510; Single peak of HPLC at 254 nm.

[0179] [ka] A 50 mL round-bottom flask equipped with a stirring bar and diaphragm was packed with SL-1832 (100 mg, 0.32 mmol), K2CO3 (130 mg, 0.96 mmol), Pd(OAc)2 (7.2 mg, 32 μmol), and [dcpp2BF4] (39 mg, 64 μmol). The flask was degassed and refilled with argon (repeated 3 times). Degassed DMSO (8 mL) and H2O (0.8 mL) were added, the reaction vessel was degassed, and the flask was refilled with carbon monoxide (repeated 3 times). CO was bubbled through the solution for 5 minutes. The resulting yellow suspension was heated at 110 °C for 22 hours under a CO balloon, at which point HPLC analysis showed complete consumption of the starting materials. The reaction mixture was diluted with MeOH (8 mL), passed through a syringe filter, and purified by preparative HPLC (C18,5 → 95% MeCN / H2O, 0.05% TFA) to obtain 54 mg (51% yield) of SL-1834-E as a clear oil and 51 mg (48% yield) of SL-1834-Z as a clear oil. The E isomer had a shorter retention time than the Z isomer. Characteristic data for SL-1834-E: 1 H-NMR(400MHz,MeOD)δ7.82(dd,J=8.0,1.8Hz,1H),7.78(d,J=1.8Hz,1H),7.43(d,J=8.0Hz,1H),7.36-7.24(m,3H),7.23-7.16(m,1H),5.93 (t,J=7.3Hz,1H),3.39(t,J=9.0Hz,2H),3.26(q,J=7.4Hz,2H),3.02(d,J=14.9Hz,1H),2.82(d,J=8.1Hz,7H),2.60(dd,J=16.9,8.3Hz,2H); 13 C NMR(100MHz,MeOD)δ169.6,147.7,146.1,140.6,139.7,138.7,132.8,131.1,129 .7,129.6,129.6,129.1,128.5,127.4,126.5,58.0,34.8,32.7,26.2;HRMS(ESI)C 21 H 24 NO2 [M+H] +Calculated value: 322.1807; Measured value: 322.1807. Characteristic data for SL-1834-Z: 1 H-NMR(400MHz,MeOD)δ7.97(d,J=1.7Hz,1H),7.92(dd,J=7.8,1.8Hz,1H),7.37-7.25(m,2H),7.25-7.13(m,2H),7.10(dd,J=7.2,1.9Hz ,1H),5.89(t,J=7.3Hz,1H),3.55-3.36(m,2H),3.26(q,J=8.6Hz,2H),2.97(d,J=25.6Hz,2H),2.82(d,J=6.3Hz,6H),2.68-2.45(m,2H); 13 C NMR(100MHz,MeOD)δ169.5,147.6,145.4,141.2,140.8,138.2,131.7,131.3,130 .7,129.5,129.1,129.0,128.8,127.4,125.9,58.0,34.5,32.8,26.2;HRMS(ESI)C 21 H 24 NO2 [M+H] + Calculated value: 322.1807; Measured value: 322.1807.

[0180] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1834-E TFA salt (36 mg, 83 μmol), tert-butyl (2-aminoethyl)carbamate (39 mg, 103 μmol), HATU (17 mg, 0.10 mmol), EtN(iPr)2 (74 μL, 0.41 mmol), and DMF (8 mL). The resulting pale yellow solution was stirred at 22 °C for 18 hours, at which point HPLC showed complete consumption of the starting materials, and the solvent was removed under reduced pressure. The crude residue was purified by preparative HPLC (C18, 5 → 95% MeCN / H2O, 0.05% TFA) to obtain 47 mg (99% yield) of amide SL-1829 as a clear oil. MS(ESI)C 28 H 38 NO3 [M+H] +Calculated value: 464.3; Measured value: 464.4; Single peak of HPLC at 254 nm.

[0181] A 10 mL flask equipped with a stirring bar was packed with SL-1839 (20 mg, 35 μmol) and a cutting cocktail (4 mL, 80:20:1 DCM / TFA / TIPS). The resulting pale yellow solution was stirred at 22°C for 75 minutes, at which point HPLC showed complete consumption of the starting material, and the solvent was removed under reduced pressure. The residue was dissolved in 10 mL of MeOH, and the solvent was removed under reduced pressure. The crude residue was used in the next step without further purification. MS(ESI)C 23 H 30 N3O3 + [M+H] + Calculated value: 364.2; Measured value: 364.3.254 nm single peak of HPLC.

[0182] [ka] A 25 mL flask equipped with a stirring bar was packed with SL-1834-Z TFA salt (26 mg, 81 μmol), tert-butyl (2-aminoethyl)carbamate (39 mg, 0.10 mmol), HATU (16 mg, 0.10 mmol), EtN(iPr)2 (72 μL, 0.40 mmol), and DMF (8 mL). The resulting pale yellow solution was stirred at 22 °C for 17 hours, at which point HPLC analysis showed complete consumption of the starting materials, and the solvent was removed under reduced pressure. The crude residue was purified by preparative HPLC (C18, 5 → 95% MeCN / H2O, 0.05% TFA) to obtain 33 mg (89% yield) of amide SL-1840 as a clear oil. MS(ESI)C 28 H 38 NO3 [M+H] + Calculated value: 464.3; Measured value: 464.4; Single peak of HPLC at 254 nm.

[0183] A 10 mL flask equipped with a stirring bar was packed with SL-1840 (15 mg, 35 μmol) and a cutting cocktail (4 mL, 80:20:1 DCM / TFA / TIPS). The resulting pale yellow solution was stirred at 22°C for 85 minutes, at which point HPLC showed complete consumption of the starting material, and the solvent was removed under reduced pressure. The residue was dissolved in 10 mL of MeOH, and the solvent was removed under reduced pressure. The crude residue was used in the next step without further purification. MS(ESI)C 23 H 30 N3O3 + [M+H] + Calculated value: 364.2; Measured value: 364.4; Single peak of HPLC at 254 nm.

[0184] [ka] To a solution of SL-1842 TFA salt (4 mg, 7 μmol) in DMF (6 mL), DIPEA (9.0 μL, 49 μmol) was added, followed by NanoBRET590 SE (3.0 mg, 7.0 μmol, Promega). The resulting solution was reacted at 22 °C for 17 hours, at which point HPLC analysis showed complete consumption of the starting material. The solvent was removed under vacuum, and the crude residue was purified by preparative RP HPLC (5 → 95% MeCN / H2O buffered with 0.5% TFA) to obtain 3.6 mg (76% yield) of SL-1834 as a purple thin film. HPLC: 99% purity at 254 nm. 11H-NMR (400 MHz, MeOD) δ 7.55 (dd, J = 8.1, 1.9 Hz, 1H), 7.50 (d, J = 1.9 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.30 - 7.18 (m, 3H), 7.22 - 7.12 (m, 4H), 7.10 (s, 1H), 7.00 (d, J = 4.5 Hz, 1H), 6.78 (d, J = 4.0 Hz, 1H), 6.35 (dt, J = 4.0, 2.3 Hz, 1H), 6.27 (d, J = 4.0 Hz, 1H), 5.83 (t, J = 7.3 Hz, 1H), 3.51 - 3.40 (m, 4H), 3.37 - 3.34 (m, 2H), 3.29 - 3.24 (m, 2H), 3.23 - 3.15 (m, 2H), 3.02 - 2.88 (m, 2H), 2.84 - 2.72 (m, 7H), 2.65 (t, J = 7.7 Hz, 2H), 2.59 - 2.46 (m, 2H); HRMS (SI) C 39 H 42 BF2N6O2 + Calculated value for [M + H]+: 675.3430; Measured value: 675.3426.

[0185]

Chem.

[0186] A 10 mL flask equipped with a stirring bar was packed with SL-1846 (3.8 mg, 4.6 μmol) and a cutting cocktail (4 mL, 80:20:1 DCM / TFA / TIPS). The resulting pale yellow solution was stirred at 22°C for 60 minutes, at which point HPLC showed complete consumption of the starting material, and the solvent was removed under reduced pressure. The residue was dissolved in 10 mL of MeOH, and the solvent was removed under reduced pressure. The crude residue was used in the next step without further purification. MS(ESI)C 34 H 52 N4O6 2+ [M+H] 2+Calculated value for / 2: 306.2; Measured value: 306.4; Single peak of HPLC at 254 nm.

[0187] To a solution of SL-1848 (4 mg, 5 μmol) in DMF (6 mL), DIPEA (6.0 μL, 33 μmol) was added, followed by NanoBRET590 SE (2.0 mg, 4.7 μmol, Promega). The resulting solution was reacted at 22°C for 23 hours, at which point HPLC analysis showed complete consumption of the starting material. The solvent was removed under vacuum, and the crude residue was purified by preparative RP HPLC (5 → 95% MeCN / H2O buffered with 0.5% TFA) to obtain 3.0 mg (70% yield) of SL-1850 as a purple thin film. HPLC: 99% purity at 254 nm. 1 H-NMR(400MHz,MeOD)δ7.60(dd,J=8.1,1.9Hz,1H),7.55(d,J=1.9Hz,1H),7.37(d,J=8.0Hz,1H),7.28(td,J=4.5,4.1,2.7Hz,2H ),7.26-7.17(m,5H),7.17-7.10(m,1H),7.01(d,J=4.6Hz,1H),6.91(d,J=4.0Hz,1H),6.34(t,J=3.1Hz,1H),6.31(d,J=4.0Hz,1 H),5.86(t,J=7.3Hz,1H),3.65(t,J=6.0Hz,2H),3.56-3.43(m,16H),3.41(d,J=5.6Hz,2H),3.35(m,4H),3.27(d,J=7.7Hz,2H), 3.19(s,2H),2.97(s,1H),2.77(d,J=13.9Hz,7H),2.63(t,J=7.7Hz,2H),2.55(t,J=8.8Hz,2H),2.40(t,J=6.0Hz,2H);HRMS(SI)C 50 H 63 BF2N7O7 + Calculated value for [M+H]+: 922.4850; Measured value: 922.4847.

[0188] [ka] To a solution of SL-1843 TFA salt (4 mg, 7 μmol) in DMF (6 mL), DIPEA (9.0 μL, 49 μmol) was added, followed by NanoBRET590 SE (3.0 mg, 7.0 μmol, Promega). The resulting solution was reacted at 22°C for 17 hours, at which point HPLC analysis showed complete consumption of the starting material. The solvent was removed under vacuum, and the crude residue was purified by preparative RP HPLC (5 → 95% MeCN / H2O buffered with 0.5% TFA) to obtain 3.6 mg (76% yield) of SL-1844 as a purple thin film. HPLC: 99% purity at 254 nm; 1 H-NMR(400MHz,MeOD)δ10.76(s,1H),7.74(d,J=1.9Hz,1H),7.60(dd,J=7.9,1.8Hz,1H),7.37-7.27(m,1H) ,7.21(tq,J=5.5,2.7,2.3Hz,5H),7.16(d,J=4.6Hz,1H),7.12(s,1H),7.10-7.05(m,1H),7.03(d,J=4.6Hz, 1H),6.82(d,J=3.9Hz,1H),6.37(dt,J=4.2,2.4Hz,1H),6.31(d,J=4.0Hz,1H),5.83(t,J=7.3Hz,1H),3.52 (m,4H),3.16(t,J=7.8Hz,2H),2.94(d,J=23.2Hz,2H),2.82-2.60(m,8H),2.49(t,J=8.8Hz,2H);HRMS(SI)C 39 H 42 BF2N6O2 + Calculated value for [M+H]+: 675.3430; Measured value: 675.3421.

[0189] [ka] To a solution of SL-1843 (8.0 mg, 14 μmol) in DMF (6 mL), DIPEA (12 μL, 68 μmol) was added, followed by NanoBRET590-PEG SE (5.5 mg, 8.1 μmol, Promega). The resulting solution was reacted at 22°C for 2 hours, at which point HPLC analysis showed complete consumption of the starting material. The solvent was removed under vacuum, and the crude residue was purified by preparative RP HPLC (5 → 95% MeCN / H2O buffered with 0.5% TFA) to obtain 2.3 mg (19% yield) of SL-1894 as a purple thin film. HPLC: 99% purity at 254 nm; 1 H-NMR(400MHz,MeOD)δ7.74(d,J=1.9Hz,1H),7.68(dd,J=7.8,1.9Hz,1H),7.38-7.11(m,8H),7.07(dd,J=7.2,1.9Hz,1H),7.0 1(d,J=4.5Hz,1H),6.91(d,J=4.0Hz,1H),6.35(dt,J=4.0,2.3Hz,1H),6.32(d,J=4.0Hz,1H),5.83(t,J=7.3Hz,1H),3.66(t,J= 6.0Hz,2H),3.53(s,4H),3.50-3.45(m,10H),3.45-3.38(m,2H),3.35(d,J=5.4Hz,2H),3.26(d,J=7.7Hz,2H),3.23-3.14(m,2 HRMS(SI)C 50 H 63 BF2N7O7 + Calculated value for [M+H]+: 922.4850; Measured value: 922.4859. Another aspect of the present invention may be as follows: [1] A composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binder bound to a functional element or a solid surface, wherein the broad-spectrum GPCR binder is JPEG0007871443000131.jpg53170 Including, in the formula, JPEG0007871443000132.jpg11170 The composition wherein the broad-spectrum GPCR binder has a binding site to the functional element, to a solid surface, or to a linker between the broad-spectrum GPCR binder and the functional element or solid surface. [2] A composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binder bound to a functional element or a solid surface, wherein the broad-spectrum GPCR binder is JPEG0007871443000133.jpg52170 Including, in the formula, JPEG0007871443000134.jpg11170 The composition wherein the broad-spectrum GPCR binder has a binding site to the functional element, to a solid surface, or to a linker between the broad-spectrum GPCR binder and the functional element or solid surface. [3] A composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binder bound to a functional element or a solid surface, wherein the broad-spectrum GPCR binder is JPEG0007871443000135.jpg52170 Including, in the formula, JPEG0007871443000136.jpg11170 The composition wherein the broad-spectrum GPCR binder has a binding site to the functional element, to a solid surface, or to a linker between the broad-spectrum GPCR binder and the functional element or solid surface. [4] A composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binder bound to a functional element or a solid surface, wherein the broad-spectrum GPCR binder is JPEG0007871443000137.jpg63170 Including, in the formula, JPEG0007871443000138.jpg11170 The composition wherein the broad-spectrum GPCR binder has a binding site to the functional element, to a solid surface, or to a linker between the broad-spectrum GPCR binder and the functional element or solid surface. [5] A composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binder bound to a functional element or a solid surface, wherein the broad-spectrum GPCR binder is JPEG0007871443000139.jpg59170 Including, in the formula, JPEG0007871443000140.jpg11170 The composition wherein the broad-spectrum GPCR binder has a binding site to the functional element, to a solid surface, or to a linker between the broad-spectrum GPCR binder and the functional element or solid surface. [6] A composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binder bound to a functional element or a solid surface, wherein the broad-spectrum GPCR binder is JPEG0007871443000141.jpg48170 Including, in the formula, JPEG0007871443000142.jpg11170 The composition wherein the broad-spectrum GPCR binder has a binding site to the functional element, to a solid surface, or to a linker between the broad-spectrum GPCR binder and the functional element or solid surface. [7] A composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binder bound to a functional element or a solid surface, wherein the broad-spectrum GPCR binder is JPEG0007871443000143.jpg54170 Including, in the formula, JPEG0007871443000144.jpg11170 The composition wherein the broad-spectrum GPCR binder has a binding site to the functional element, to a solid surface, or to a linker between the broad-spectrum GPCR binder and the functional element or solid surface. [8] A composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binder bound to a functional element or a solid surface, wherein the broad-spectrum GPCR binder is JPEG0007871443000145.jpg58170 Including, in the formula, JPEG0007871443000146.jpg11170 The composition wherein the broad-spectrum GPCR binder is a binding site to the functional element, a solid surface, or a linker between the broad-spectrum GPCR binder and the functional element or solid surface, and the broad-spectrum GPCR binder may exist as a cis isomer (Z), a trans isomer (E), or a mixture of the two. [9] A composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binder bound to a functional element or a solid surface, wherein the broad-spectrum GPCR binder is JPEG0007871443000147.jpg60170 Including, in the formula, JPEG0007871443000148.jpg11170 The composition wherein the broad-spectrum GPCR binder is a binding site to the functional element, a solid surface, or a linker between the broad-spectrum GPCR binder and the functional element or solid surface, and the broad-spectrum GPCR binder may exist as a cis isomer (Z), a trans isomer (E), or a mixture of the two.

[10] A composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binder bound to a functional element or a solid surface, wherein the broad-spectrum GPCR binder is JPEG0007871443000149.jpg57170 Including, in the formula, JPEG0007871443000150.jpg11170 The composition wherein the broad-spectrum GPCR binder is a binding site to the functional element, a solid surface, or a linker between the broad-spectrum GPCR binder and the functional element or solid surface, and the broad-spectrum GPCR binder may exist as a cis isomer (Z), a trans isomer (E), or a mixture of the two.

[11] A composition comprising a broad-spectrum G protein-coupled receptor (GPCR) binder bound to a functional element or a solid surface, wherein the broad-spectrum GPCR binder is JPEG0007871443000151.jpg66170 Including, in the formula, JPEG0007871443000152.jpg11170 The composition wherein the broad-spectrum GPCR binder is a binding site to the functional element, a solid surface, or a linker between the broad-spectrum GPCR binder and the functional element or solid surface, and the broad-spectrum GPCR binder may exist as a cis isomer (Z), a trans isomer (E), or a mixture of the two.

[12] The composition according to any one of the above [1] to

[11] , wherein the solid surface is selected from deposited particles, films, glass, tubes, wells, self-assembled monolayers, surface plasmon resonance chips, or solid supports having an electronically conductive surface.

[13] The composition according to

[12] , wherein the deposited particles are magnetic particles.

[14] The composition according to any one of [1] to

[11] , wherein the functional element is selected from a detectable element, an affinity element, and a capture element.

[15] The composition according to

[14] , wherein the detectable element comprises a fluorescent dye molecule, a chromophore, a radionuclide, an electron opaque molecule, an MRI contrast agent, a SPECT contrast agent, or a mass tag.

[16] The composition according to any one of [1] to

[11] , wherein the broad-spectrum GPCR binder is directly bound to the functional element or a solid surface.

[17] The composition according to any one of [1] to

[11] , wherein the broad-spectrum GPCR binder is bound to the functional element or solid surface via a linker.

[18] The linker is [(CH 2 ) 2 O] n The composition according to

[17] , comprising n being 1 to 20.

[19] The composition according to

[17] , wherein the linker is bound to the broad-spectrum GPCR binder and / or the functional element by an amide bond.

[20] Structure below: JPEG0007871443000153.jpg96170 The composition according to [1], comprising, where n is 0 to 8 and X is a functional element or a solid surface.

[21] The following structure: JPEG0007871443000154.jpg159170 The composition according to [2], comprising, where n is 0 to 8, m is 0 to 8, and X is a functional element or a solid surface.

[22] The following structure: JPEG0007871443000155.jpg195170 The composition according to [3], comprising, where n is 0 to 8, m is 0 to 8, and X is a functional element or a solid surface.

[23] The following structure: JPEG0007871443000156.jpg51170 The composition according to [4], wherein n is 0 to 8, m is 0 to 8, and X is a functional element or a solid surface.

[24] The following structure: JPEG0007871443000157.jpg103170 The composition according to [5], comprising, where n is 0 to 8 and X is a functional element or a solid surface.

[25] The following structure: JPEG0007871443000158.jpg41170 The composition according to [6], comprising, where n is 0 to 8, m is 0 to 8, and X is a functional element or a solid surface.

[26] The following structure: JPEG0007871443000159.jpg45170 The composition according to [7], comprising, where n is 0 to 8, m is 0 to 8, and X is a functional element or a solid surface.

[27] The following structure: JPEG0007871443000160.jpg187170 The composition according to [8] or [9], comprising, where n is 0 to 8 and X is a functional element or a solid surface.

[28] The following structure: JPEG0007871443000161.jpg179170 The composition according to

[10] or

[11] , comprising, where n is 0 to 8 and X is a functional element or a solid surface.

[29] The following structure: JPEG0007871443000162.jpg149170 The composition according to claim 10 or 11, comprising, where n is 0 to 8 and X is a functional element or a solid surface.

[30] Structure below: JPEG0007871443000163.jpg164170 The composition according to [8] or [9], comprising, where n is 0 to 8 and X is a functional element or a solid surface.

[31] The composition according to any one of the above

[15] to

[30] , wherein X is a fluorescent dye molecule.

[32] The composition according to any one of the above items [1] to

[31] , comprising one or more stable heavy isotopes in unnatural abundance.

[33] A method for detecting or quantifying a GPCR in a sample, comprising contacting the sample with a composition described in any one of [1] to

[32] above, and detecting or quantifying the functional elements of the signal produced thereby.

[34] The method according to

[33] , wherein the functional elements of the signal generated thereby are detected or quantified by fluorescence, mass spectrometry, optical imaging, magnetic resonance imaging (MRI), and energy transfer.

[35] A method for isolating a GPCR from a sample, comprising contacting the sample with a composition described in any one of [1] to

[32] above, and separating the bound GPCR from the unbound portion of the sample, as well as from a functional element or a solid surface.

[36] A method for characterizing the uniqueness of a GPCR in a sample, comprising isolating the GPCR from the sample by the method described in

[35] , and analyzing the isolated GPCR by mass spectrometry.

[37] A method for monitoring interactions between a GPCR and an unmodified biomolecule, comprising contacting the sample with a composition described in any one of [1] to

[32] above.

[38] The method according to any one of the above

[33] to

[37] , wherein the sample is selected from cells, cell lysates, body fluids, tissues, biological samples, in vitro samples, and environmental samples.

[39] A system, (a) A composition according to any one of the above [1] to

[32] , wherein the functional element is a fluorescent dye molecule; (b) The system comprising a fusion of a GPCR and a peptide component of a bioluminescent protein or bioluminescent complex, wherein the emission spectrum of the bioluminescent protein or bioluminescent complex overlaps with the excitation spectrum of the fluorescent dye molecule.

[40] The system according to

[39] , comprising a kit, cells, cell lysates, or reaction mixture.

[41] The system according to

[39] , wherein the fusion comprises a GPCR and a peptide component of the bioluminescent complex, and the system further comprises one or more additional components of the bioluminescent complex and a substrate of the bioluminescent complex.

[42] A method, (a) A fusion of a GPCR and a bioluminescent protein, (i) A composition according to any one of the above items [1] to

[32] , wherein the functional element is a fluorescent dye molecule and the emission spectrum of the bioluminescent protein overlaps with the excitation spectrum of the fluorescent dye molecule, and (ii) bringing the bioluminescent protein into contact with a substrate; (b) The method comprising detecting the wavelength of light within the range of the excitation spectrum of the fluorescent dye molecule, which is produced as a result of bioluminescent resonance energy transfer from the bioluminescent protein to the fluorescent dye molecule when the broad-spectrum GPCR binder binds to the GPCR.

[43] A method, (a) A fusion of a GPCR and the peptide component of a bioluminescent complex, (i) The composition according to any one of the above [1] to

[32] , wherein the functional element is a fluorescent dye molecule and the emission spectrum of the bioluminescent protein overlaps with the excitation spectrum of the fluorescent dye molecule. (ii) The polypeptide component of the bioluminescent complex, and (iii) bringing the bioluminescent protein into contact with a substrate; (b) The method comprising detecting the wavelength of light within the range of the excitation spectrum of the fluorescent dye molecule, which is generated as a result of bioluminescence resonance energy transfer from the bioluminescent complex to the fluorescent dye molecule when the broad-spectrum GPCR binder binds to the GPCR.

[0190] array WT OgLuc(Sequence ID 1) JPEG0007871443000164.jpg20164

[0191] WT OgLuc Lg (Sequence No. 2) JPEG0007871443000165.jpg20164

[0192] WT OgLuc β9 (SEQ ID NO: 3) JPEG0007871443000166.jpg1236

[0193] WT OgLuc β10 (Sequence No. 4) JPEG0007871443000167.jpg745

[0194] NanoLuc (SEQ ID NO: 5) JPEG0007871443000168.jpg19164

[0195] NanoLuc Lg (SEQ ID NO: 6) JPEG0007871443000169.jpg18164

[0196] NanoLuc β9 (SEQ ID NO: 7) JPEG0007871443000170.jpg741

[0197] NanoLuc β10 (SEQ ID NO: 8) JPEG0007871443000171.jpg746

[0198] LgBiT (Sequence ID 9) JPEG0007871443000172.jpg18164

[0199] SmBiT(Sequence ID 10) JPEG0007871443000173.jpg837

[0200] HiBiT (Sequence ID 11) JPEG0007871443000174.jpg937

[0201] LgTrip(3546)(Sequence ID 12) JPEG0007871443000175.jpg18164

[0202] SmTrip9 (SEQ ID NO: 13) JPEG0007871443000176.jpg837

[0203] β9 / β10 dipeptide (SEQ ID NO: 14) JPEG0007871443000177.jpg968

[0204] His5 (Sequence ID 15) JPEG0007871443000178.jpg620

[0205] HisX6 (sequence number 16) JPEG0007871443000179.jpg723

[0206] C-myc (SEQ ID NO: 17) JPEG0007871443000180.jpg731

[0207] Flag (Sequence ID 18) JPEG0007871443000181.jpg732

[0208] SteptTag(Sequence ID 19) JPEG0007871443000182.jpg630

[0209] HA tag (sequence number 20) JPEG0007871443000183.jpg733

Claims

1. A composition comprising a broad-spectrum G protein-coupled receptor (GPCR) conjugate bound to a fluorescent dye molecule, wherein the broad-spectrum GPCR conjugate is 【Chemistry 1】 Including, in the formula, 【Chemistry 2】 The composition for use in a method for detecting or quantifying a GPCR, which includes binding the broad-spectrum GPCR binder to the fluorescent dye molecule, or to a linker between the broad-spectrum GPCR binder and the fluorescent dye molecule.

2. The composition according to claim 1, wherein the broad-spectrum GPCR binder directly binds to the fluorescent dye molecule.

3. The composition according to claim 1, wherein the broad-spectrum GPCR binder is bound to the fluorescent dye molecule via a linker.

4. The linker is [(CH 2 ) 2 O] n The composition according to claim 3, comprising n being 1 to 20.

5. The composition according to claim 3, wherein the linker is bound to the broad-spectrum GPCR binder and / or the fluorescent dye molecule by an amide bond.

6. The following structure: 【Transformation 3】 The composition according to claim 1, comprising, where n is 0 to 8, m is 0 to 8, and X is a fluorescent dye molecule.

7. The following structure: 【Chemistry 4】 The composition according to claim 1, comprising, where n is 0 to 8, m is 0 to 8, and X is a fluorescent dye molecule.

8. The composition according to any one of claims 1 to 7, comprising one or more stable heavy isotopes in unnatural abundance.

9. A method for detecting or quantifying a GPCR in a sample, comprising contacting the sample with a composition according to any one of claims 1 to 8, and detecting or quantifying a fluorescent dye molecule or a signal generated thereby.

10. The method according to claim 9, wherein the fluorescent dye molecule or the signal generated thereby is detected or quantified by fluorescence, mass spectrometry, optical imaging, magnetic resonance imaging (MRI), and energy transfer.

11. A method for isolating a GPCR from a sample, comprising contacting the sample with a composition according to any one of claims 1 to 8, and separating the GPCR bound to the broad-spectrum GPCR binder from the unbound portion of the sample.

12. A method for characterizing the uniqueness of a GPCR in a sample, comprising isolating the GPCR from the sample by the method of claim 11, and analyzing the isolated GPCR by mass spectrometry.

13. A method for monitoring the interaction between a GPCR and an unmodified biomolecule, comprising contacting a sample with a composition according to any one of claims 1 to 8.

14. The method according to any one of claims 9 to 13, wherein the sample is selected from cells, cell lysates, body fluids, tissues, biological samples, in vitro samples, and environmental samples.

15. It is a system, (a) the composition according to any one of claims 1 to 8; (b) The system for use in a method for detecting or quantifying a GPCR, comprising conjugating a broad-spectrum GPCR binder in the composition to the GPCR, wherein the broad-spectrum GPCR binder in the composition comprises a fusion of the GPCR and a peptide component of a bioluminescent protein or bioluminescent complex, wherein the emission spectrum of the bioluminescent protein or bioluminescent complex overlaps with the excitation spectrum of the fluorescent dye molecule.

16. The system according to claim 15, comprising a kit, cells, cell lysate, or reaction mixture.

17. The system according to claim 15, wherein the fusion comprises a GPCR and a peptide component of a bioluminescent complex, and the system further comprises one or more additional components of the bioluminescent complex and a substrate of the bioluminescent complex.

18. It is a method, (a) A fusion of a GPCR and a bioluminescent protein, (i) A composition according to any one of claims 1 to 8, wherein the emission spectrum of the bioluminescent protein overlaps with the excitation spectrum of the fluorescent dye molecule, and (ii) Substrate of the bioluminescent protein To bring into contact with; (b) The method comprising detecting the wavelength of light within the range of the excitation spectrum of the fluorescent dye molecule, which is produced as a result of bioluminescent resonance energy transfer from the bioluminescent protein to the fluorescent dye molecule when the broad-spectrum GPCR binder binds to the GPCR.

19. It is a method, (a) A fusion of the GPCR and the peptide component of the bioluminescent complex, (i) A composition according to any one of claims 1 to 8, wherein the emission spectrum of the bioluminescent complex overlaps with the excitation spectrum of the fluorescent dye molecule, (ii) polypeptide components of the bioluminescent complex, and (iii) Substrate of the bioluminescent complex To bring into contact with; (b) The method comprising detecting the wavelength of light within the range of the excitation spectrum of the fluorescent dye molecule, which is generated as a result of bioluminescence resonance energy transfer from the bioluminescent complex to the fluorescent dye molecule when the broad-spectrum GPCR binder binds to the GPCR.

Images