Photoactivatable compounds and uses thereof

Abstract

Provided herein are compounds, compositions, systems, and methods for photoactivatable labeling that can be operated in biological systems. Specifically, the compounds disclosed herein contain vinyl-extended arylazide moieties that undergo photoactivation to generate reactive intermediates that can form covalent bonds with biomolecules. Photoactivation can be achieved by a variety of mechanisms, including ultraviolet (UV) irradiation, visible light irradiation, or energy transfer (e.g., from a photocatalyst). The compounds also contain functional moieties that provide useful functionality, such as fluorophores, capture elements (e.g., biotin), or reactive moieties (e.g., click handles), and detection of biomolecules, such as bifunctional moieties that contain a bioactive compound and a detection / capture element.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 338,322, filed May 4, 2022, which is incorporated by reference in its entirety.

[0002] Provided herein are compounds, compositions, systems, and methods for photoactivatable labeling that can be operated in biological systems. Specifically, the compounds disclosed herein contain vinyl-extended arylazide moieties that can undergo photoactivation to generate reactive intermediates that can form covalent bonds with biomolecules. Photoactivation can be achieved by a variety of mechanisms, including ultraviolet (UV) irradiation, visible light irradiation, or energy transfer (e.g., from a photocatalyst). The compounds also contain functional moieties that provide useful functionality, such as fluorophores, capture elements (e.g., biotin), reactive moieties (e.g., click handles), and bifunctional moieties (e.g., moieties that contain a biologically active compound and either a fluorophore or a capture element or a reactive moiety), to detect and / or concentrate biomolecules. [Background technology]

[0003] The need to study and map dynamic microenvironments, molecular networks, and small molecule interactions, as well as protein-protein and protein-nucleic acid interactions in physiologically relevant contexts, creates a demand for new functional and biological tools to enable such analyses non-destructively in living cells and complex models. Summary of the Invention

[0004] In one embodiment, a compound of formula (I) [ka] or a salt thereof, as disclosed herein. [In the formula, A is, [ka] is selected from During the ceremony, each n is independently 1, 2, 3, or 4; each R is independently selected from hydrogen, halo, C1-C4 alkyl, C2-C4 alkenyl, hydroxy, mercapto, amino, cyano, C1-C4-alkoxy, halo-C1-C4-alkyl, hydroxy-C1-C4-alkyl, amino-C1-C4-alkyl, mercapto-C1-C4-alkyl, cyano-C1-C4-alkyl, -C(O)-C1-C4-alkyl, -C(O)OH, and -C(O)NH2; R' is hydrogen or C1-C4 alkyl; L is a linker, Y is a functional moiety.

[0005] In some embodiments, A is: [ka] [In the formula, R 1 , R 2 , R 3 and R 4 are each independently selected from hydrogen, halo, hydroxy, cyano, and C1-C4 alkoxy.

[0006] In some embodiments, R 1 is hydrogen, hydroxy, or C1-C4 alkoxy; R 2 is hydrogen, halo, cyano, or C1-C4 alkoxy; R 3 is hydrogen or halo, and R 4 is hydrogen or halo.

[0007] In some embodiments, A is: [ka] wherein each R is independently selected from hydrogen, halo, cyano, and C1-C4 alkoxy.

[0008] In some embodiments, A is: [ka] wherein R is selected from hydrogen, halo, cyano, and C1-C4 alkoxy.

[0009] In some embodiments, A is: [ka] wherein R is selected from hydrogen, halo, cyano, and C1-C4 alkoxy.

[0010] In some embodiments, A has a formula selected from the following: [ka]

[0011] In some embodiments, R' is chosen from hydrogen and methyl.

[0012] In some embodiments, the linker comprises one or more moieties selected from straight or branched chain alkylene, ether (-O-), amine (-NH-), ester (-C(O)O-), amide (-C(O)NH-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), and phenylene groups.

[0013] In some embodiments, the linker has the formula: -NHCH2CH2(OCH2CH2) n NH- wherein n is 1, 2, 3, 4, 5, 6, 7, or 8.

[0014] In some embodiments, Y is a functional moiety selected from a capture element, a detectable moiety, a reactive moiety, and a bifunctional moiety.

[0015] In some embodiments, Y is a capture element selected from biotin and a haloalkane group. In some embodiments, Y has the formula: [ka]

[0016] In some embodiments, Y has the formula -(CH2) n -X, where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and X is a halogen.

[0017] In some embodiments, Y is a detectable moiety. In some embodiments, Y is a fluorescent functional group. In some embodiments, Y is a fluorescent functional group selected from xanthene, cyanine, naphthalene, oxadiazole, pyrene, oxazine, acridine, arylmethine, tetrapyrrole, coumarin, squaraine, and boron-dipyrromethene. In some embodiments, Y is a fluorogenic functional group.

[0018] In some embodiments, Y is a reactive functional group. In some embodiments, Y is a reactive functional group that includes an azide, an alkyne, an alkene, or a 1,2,4,5-tetrazinyl moiety.

[0019] In some embodiments, Y is a bifunctional moiety that includes (i) a biologically active compound and (ii) a capture element or a fluorescent moiety or a reactive moiety.

[0020] In some embodiments, the compound of formula (I) is a compound selected from the group consisting of: [ka] and salts thereof.

[0021] In another aspect, there is provided a system for photocatalytic labeling of biomolecules, comprising: (a) a compound of formula (I) (e.g., any compound of formula (I) disclosed herein); (b) a photocatalyst; The above system is disclosed herein.

[0022] In some embodiments, the photocatalyst has the following structure: [ka] [In the formula, Each series of dashed lines [ka] represents the presence or absence of a fused six-membered ring, M is a transition metal; m1, m2, m3, n1, n2, n3, p1, p2, and p3 are each independently 0, 1, or 2; R 1a , R 1b , R 1c , R 2a , R 2b , R 2c , R 3a , R 3b , and R 3c are each independently selected from halo, alkyl, haloalkyl, amino, and heteroalkyl; X 1a , X 1b , X 2a , X 2b , X 3a , and X 3b are each independently selected from N and C, 1a and X 1b At least one of them is N, and X 2a and X 2b At least one of them is N, and X 3a and X 3b at least one of is N; X 1c , X 1d , X 2c, X 2d , X 3c , and X 3d are each independently selected from CH and N; Z is an anion, [q is 0, 1, or 2].

[0023] In some embodiments, the transition metal is selected from Ru and Ir.

[0024] In some embodiments, the photocatalyst is an iridium photocatalyst selected from: [ka]

[0025] In some embodiments, the photocatalyst is a ruthenium-based photocatalyst selected from: [ka]

[0026] In some embodiments, the photocatalyst has the formula: [ka]

[0027] In one aspect, there is provided a method for labeling a biomolecule in a sample, comprising the steps of: (a) contacting the sample with a compound of formula (I) (e.g., any compound of formula (I) disclosed herein); (b) exposing the sample to light; The above method is disclosed herein, comprising:

[0028] In some embodiments, the light is selected from ultraviolet and visible light. In some embodiments, the light is visible light from a light emitting diode. In some embodiments, the light is bioluminescent light. In some embodiments, the method in step (a) further comprises contacting the sample with a photocatalyst. [Brief description of the drawings]

[0029] [Figure 1] A cartoon illustration of covalent labeling using probes containing vinyl-extended aryl-azide photoreactive groups that upon activation generate short-lived reactive intermediates capable of forming covalent bonds with adjacent biomolecules. Activation modes include (A) UV irradiation, (B) visible 455 nm LED irradiation, (C) LED-induced activation of the vinyl-extended aryl-azide photoreactive group and a light-sensitive catalyst that further participates in an energy transfer event, and (D) bioluminescence-induced activation of the vinyl-extended aryl-azide photoreactive group and a light-sensitive catalyst that further participates in an energy transfer event. [Figure 2A] 1 shows a schematic representation of a probe containing a vinyl-extended aryl-azide based photoreactive group attached to a functional moiety via a linker. [Figure 2B] 1 shows the structures of vinyl-extended aryl-azide photoreactive groups. [Figure 3A] 1 shows the absorbance profiles for a series of vinyl-extended aryl-azide photoreactive groups. [Figure 3B] 1 shows the absorbance profiles for a series of vinyl-extended aryl-azide photoreactive groups. [Figure 3C] 1 shows the absorbance profiles for a series of vinyl-extended aryl-azide photoreactive groups. [Figure 4] Data evaluation is presented to assess crosslinking efficiency for a series of vinyl-extended aryl-azide photoreactive groups. In particular, the data shows slot blot analysis of covalent protein labeling induced by direct irradiation with either UV or visible light (455 nm LED) or LED-induced activation of an iridium catalyst that further participates in an energy transfer event with vinyl-extended aryl-azide photoreactive groups. [Diagram 5]

[0023] Figure 1 shows data assessing the covalent labeling efficiency for a series of vinyl-extended aryl-azide photoreactive groups. In particular, the data shows the quantification of the slot blot analysis of Figure 4. [Figure 6A] Data are presented to assess the ability of a series of vinyl-extended aryl-azide photoreactive groups to undergo bioluminescence-triggered, photocatalytic, covalent labeling of proteins. In particular, the data show Western analysis of covalent protein labeling triggered by bioluminescence-triggered activation of an iridium catalyst that further participates in an energy transfer event with vinyl-extended aryl-azide photoreactive groups. [Figure 6B] Data are presented to assess the ability of a series of vinyl-extended aryl-azide photoreactive groups to undergo bioluminescence-triggered, photocatalytic, covalent labeling of proteins. In particular, the data show Western analysis of covalent protein labeling triggered by bioluminescence-triggered activation of an iridium catalyst that further participates in an energy transfer event with vinyl-extended aryl-azide photoreactive groups. [Figure 6C] Data are presented to assess the ability of a series of vinyl-extended aryl-azide photoreactive groups to undergo bioluminescence-triggered, photocatalytic, covalent labeling of proteins. In particular, the data show Western analysis of covalent protein labeling triggered by bioluminescence-triggered activation of an iridium catalyst that further participates in an energy transfer event with vinyl-extended aryl-azide photoreactive groups.

[0030] definition Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the embodiments described herein, some preferred methods, compositions, devices, and materials are described herein. However, before describing the materials and methods, it should be understood that the invention is not limited to the specific molecules, compositions, methodologies, or procedures described herein, as these may vary according to routine experimentation and optimization. It should also be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the embodiments described herein.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, shall control. Therefore, in the context of the embodiments described herein, the following definitions apply.

[0032] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a peptide" refers to one or more peptides and equivalents thereof known to those skilled in the art.

[0033] As used herein, the term "and / or" includes any and all combinations of the listed items, including any of the individually listed items. For example, "A, B and / or C" includes A, B, C, AB, AC, BC and ABC, each of which should be considered as being individually listed with the explicit reference "A, B and / or C."

[0034] As used herein, the term "comprising" and its linguistic variants refer to the presence of the recited feature(s), element(s), method step(s), etc., but do not exclude the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term "consisting of" and its linguistic variants refer to the presence of the recited feature(s), element(s), method step(s), etc., but excludes any feature(s), element(s), method step(s), etc. that are not recited, apart from normally accompanying impurities. The phrase "consisting essentially of" refers to the recited feature(s), element(s), method step(s), etc., plus any additional feature(s), element(s), method step(s), etc. that do not substantially affect the basic nature of the composition, system, or method. Many embodiments herein are described using the open-ended "comprising" phrase. Such embodiments include the limiting "consisting of" and / or "consisting essentially of" embodiments, which may alternatively be claimed or described using such language.

[0035] As used herein, the term "substantially" means that the recited properties, parameters, and / or values ​​need not necessarily be achieved exactly, but deviations or variations, including, for example, tolerances, measurement errors, limits of measurement accuracy, and other factors known to those of skill in the art, may occur to an extent that does not interfere with the effect intended to be provided by the property. A substantially absent (e.g., substantially non-luminescent) property or characteristic may be within the noise, below background, below the detection capability of the assay being used, or may be a small fraction (e.g., <1%, <0.1%, <0.01%, <0.001%, <0.00001%, <0.000001%, <0.0000001%) of a significant property (e.g., luminescence intensity of a bioluminescent protein or bioluminescent complex).

[0036] As used herein, the term "biomolecule" or "biological molecule" refers to molecules and ions that are present in living organisms and are essential for biological process(es), such as cell division, morphogenesis, or development. Biomolecules include large macromolecules (or polyanions), such as proteins, carbohydrates, lipids, and nucleic acids, as well as small molecules, such as primary metabolites, secondary metabolites, and natural products. A more common name for this class of substances is biomaterials. Biomolecules are usually endogenous, but can also be exogenous. For example, drugs can be natural products, or semi-synthetic (biopharmaceuticals), or can be completely synthetic.

[0037] Definitions of certain functional groups and chemical terms are explained in more detail below. For purposes of this disclosure, chemical elements are defined as defined in the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Ed. (endpaper), and specific functional groups are generally defined as described therein. In addition, general principles of organic chemistry and specific functional moieties and reactivity are described in Sorrell, Organic Chemistry, 2001, 14th Ed., 1999, 1999-2002, 1999-2002, 1999-2002, 1999-2003, 1999-2004, 1999-2005, 1999-2006, 1999-2007, 1999-2008, 1999-2000, 1999-2001, 1999-2002, 1999-2002, 1999-2003, 1999-2004, 1999-2005, 1999-2006, nd edition,University Science Books,Sausalito,2006;Smith,March's Advanced Organic Chemistry:Reactions,Mechanism,and Structure,7 th Edition,John Wiley & Sons,Inc.,New York,2013;Larock,Comprehensive Organic Transformations,3 rd Edition, John Wiley & Sons, Inc., New York, 2018; and Carruthers, Some Modern Methods of Organic Synthesis, 3 rdEdition, Cambridge University Press, Cambridge, 1987, the contents of each of which are incorporated herein by reference in their entirety.

[0038] As used herein, the term "alkyl" refers to an alkyl group having 1 to 30 carbon atoms, e.g., 1 to 16 carbon atoms (C1-C 16 Alkyl), 1 to 14 carbon atoms (C1-C 14 Alkyl), 1 to 12 carbon atoms (C1-C 12 Alkyl), 1 to 10 carbon atoms (C1-C 10 alkyl), 1 to 8 carbon atoms (C1-C8 alkyl), 1 to 6 carbon atoms (C1-C6 alkyl), 1 to 4 carbon atoms (C1-C4 alkyl), 6 to 20 carbon atoms (C6-C 20 alkyl), or 8 to 14 carbon atoms (C8-C 14 By "alkyl" is meant a straight or branched saturated hydrocarbon chain containing the radicals (alkyl), representative examples of which include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl.

[0039] As used herein, the term "alkenyl" refers to a straight or branched hydrocarbon chain containing at least one carbon-carbon double bond. The double bond(s) can be located at any position within the hydrocarbon chain. Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, and 3-decenyl.

[0040] As used herein, the term "alkynyl" refers to a straight or branched hydrocarbon chain containing at least one carbon-carbon triple bond. The triple bond(s) can be located at any position within the hydrocarbon chain. Representative examples of alkynyl include, but are not limited to, ethynyl, propynyl, and butynyl.

[0041] As used herein, the term "alkylene" refers to a divalent alkyl radical (e.g., -CHCH-). As used herein, the term "alkenylene" refers to a divalent alkenyl radical (e.g., -CH=CH-). As used herein, the term "alkynylene" refers to a divalent alkynyl radical (e.g., -C≡C-).

[0042] The term "alkoxy" as used herein means an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, and tert-butoxy.

[0043] As used herein, the term "amino" refers to the group --NH.

[0044] The term "aminoalkyl" as used herein means an alkyl group, as defined herein, in which at least one hydrogen atom is replaced with an amino group, as defined herein. Representative examples of aminoalkyl include, but are not limited to, aminomethyl, 2-aminoethyl, 2-aminopropyl, 3-aminopropyl, and 4-aminobutyl.

[0045] As used herein, the term "cyano" refers to a -CN group.

[0046] The term "cyanoalkyl" as used herein means an alkyl group, as defined herein, in which at least one hydrogen atom is replaced with a cyano group, as defined herein. Representative examples of cyanoalkyl include, but are not limited to, cyanomethyl, 2-cyanoethyl, 2-cyanopropyl, 3-cyanopropyl, and 4-cyanobutyl.

[0047] As used herein, the term "halogen" or "halo" means F, Cl, Br or I.

[0048] As used herein, the term "haloalkyl" refers to an alkyl group, as defined herein, in which at least one hydrogen atom (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 hydrogen atoms) is replaced with a halogen. In some embodiments, each hydrogen atom of the alkyl group is replaced with a halogen. Representative examples of haloalkyl include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, and 3,3,3-trifluoropropyl.

[0049] As used herein, the term "heteroalkyl" refers to an alkyl group as defined herein in which one or more of the carbon atoms (and any associated hydrogen atoms) are each independently replaced with a heteroatom group, such as -NR-, -O-, -S-, -S(O)-, -S(O)2-, and the like, where R is H, alkyl, aryl, cycloalkyl, heteroalkyl, heteroaryl, or heterocyclyl, each of which can be optionally substituted. By way of example, one, two, or three carbon atoms can be independently replaced with the same or different heteroatom groups. Examples of heteroalkyl groups include, but are not limited to, -OCH3, -CHOCH3, -SCH3, -CH2SCH3, -NRCH3, and -CH2NRCH3, where R is hydrogen, alkyl, aryl, arylalkyl, heteroalkyl, or heteroaryl, each of which can be optionally substituted. Heteroalkyl also includes groups in which the carbon atoms of the alkyl are oxidized (i.e., -C(O)-).

[0050] As used herein, the term "hydroxy" refers to an --OH group.

[0051] The term "hydroxyalkyl" as used herein means an alkyl group, as defined herein, in which at least one hydrogen atom is replaced with a hydroxy group. Representative examples of hydroxyalkyl include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, and 4-hydroxybutyl.

[0052] As used herein, the term "mercapto" means a --SH group.

[0053] As used herein, the term "mercaptoalkyl" refers to an alkyl group, as defined herein, in which at least one hydrogen atom is replaced with a mercapto group. Representative examples of mercaptoalkyl include, but are not limited to, mercaptomethyl, 2-mercaptoethyl, 2-mercaptopropyl, 3-mercaptopropyl, and 4-mercaptobutyl.

[0054] As used herein, in a chemical structure, [ka] The designation represents the point of attachment of one moiety to another (eg, a substituent to the remainder of a compound).

[0055] For the compounds described herein, groups or substituents thereof can be selected according to the permissible valences and substituents of atoms such that selection and substitution result in stable compounds that do not spontaneously undergo transformation, e.g., by rearrangement, cyclization, elimination, and the like.

[0056] Where substituents are specified by conventional chemical formulas and written from left to right, such descriptions also encompass the substituents obtained by writing the structure from right to left. For example, where a divalent group is shown as -CHO-, such descriptions also encompass -OCH-. Similarly, -OC(O)NH- also encompasses -NHC(O)O-. Where a linker moiety is shown, the linker can be attached to the other part of the compound in either orientation.

[0057] As used herein, the term "bioactive compound" generally refers to a physiologically or pharmacologically active substance. In some embodiments, a bioactive agent is a potential therapeutic compound (e.g., a small molecule, peptide, nucleic acid, etc.) or drug molecule.

[0058] As used herein, the term "capture protein" refers to a protein or other molecular entity that forms a stable interaction (e.g., a covalent bond or a stable non-covalent interaction) when interacting with its substrate, ligand, or other molecule. The capture protein can be a receptor that forms a covalent bond when binding with its ligand, or an enzyme that forms a covalent bond with its substrate. An example of a capture protein suitable for use in embodiments of the present invention is the HALOTAG protein, described in U.S. Patent No. 7,425,436, which is incorporated herein by reference in its entirety. The capture protein can also be a protein that has a strong non-covalent interaction with its corresponding capture element, such as streptavidin.

[0059] As used herein, the term "capture element" refers to a ligand, substrate, that interacts with a corresponding capture protein (e.g., by covalent or stable non-covalent interactions). An example of a suitable capture element for use in embodiments of the present invention is the HALOTAG ligand, described, for example, in U.S. Pat. No. 7,425,436, which is incorporated herein by reference in its entirety. Moieties that find use as HALOTAG ligands include haloalkane (HA) groups (e.g., chloroalkane (CA) groups). In embodiments described herein that specify HA or CA capture elements, other suitable capture elements can be substituted unless otherwise specified. Another capture element is biotin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0060] The need to study and map dynamic microenvironments, molecular networks, small molecule interactions, as well as protein-protein and protein-nucleic acid interactions in physiologically relevant contexts creates a demand for new functional biological tools to enable such analyses in a non-destructive manner in living cells and complex models. Photoactivatable compounds, which contain functional moieties attached to photoreactive groups that can undergo activation upon covalent crosslinking with biomolecules, provide a solution to this need by allowing labeling of biomolecules with fluorophores for detection, with capture elements for enrichment and identification, as well as with bifunctional moieties that contain bioactive compounds and either fluorophores or capture elements or reactive moieties for photoaffinity labeling, as well as subsequent detection and / or enrichment, etc.

[0061] Provided herein are compounds, compositions, systems, and methods for photoactivated labeling of biomolecules that can be operated in biological systems. Specifically, the compounds disclosed herein contain photoactivatable moieties that upon exposure to light generate reactive intermediates that then form covalent bonds with biomolecules. Photoactivation can be achieved by a variety of mechanisms, including ultraviolet (UV) irradiation, visible light irradiation, or energy transfer. The compounds also contain functional moieties that provide useful functionality, such as detection and / or enrichment of biomolecules, such as fluorophores, capture elements (e.g., biotin), reactive moieties (e.g., click handles), or bifunctional moieties that contain a bioactive compound and either a fluorophore or a capture element or a reactive moiety. These bioorthogonal labeling chemistries can be cleaved for a wide range of phenotypic, proteomic, and genomic analyses.

[0062] compound Compounds of formula (I): [ka] or a salt thereof, as disclosed herein. [In the formula, A is, [ka] is selected from During the ceremony, each n is independently 1, 2, 3, or 4; each R is independently selected from hydrogen, halo, C1-C4 alkyl, C2-C4 alkenyl, hydroxy, mercapto, amino, cyano, C1-C4-alkoxy, halo-C1-C4-alkyl, hydroxy-C1-C4-alkyl, amino-C1-C4-alkyl, mercapto-C1-C4-alkyl, cyano-C1-C4-alkyl, -C(O)-C1-C4-alkyl, -C(O)OH, and -C(O)NH2; R' is hydrogen or C1-C4 alkyl; L is a linker, Y is a functional moiety.

[0063] In some embodiments, A is a group of the formula: [ka] [In the formula, R 1 , R 2 , R 3 and R 4 are each independently selected from hydrogen, halo, hydroxy, cyano, and C1-C4 alkoxy. 1 is hydrogen, hydroxy, or C1-C4 alkoxy; R 2 is hydrogen, halo, cyano, or C1-C4 alkoxy; R 3 is hydrogen or halo, and R 4 is hydrogen or halo. In some embodiments, R 1 is hydrogen, hydroxy, or methoxy, and R 2 is hydrogen, fluoro, cyano, or methoxy; R 3 is hydrogen or fluoro, and R 4 is hydrogen.

[0064] In some embodiments, A is a group of the formula: [ka] wherein n is 1, 2, or 3, and each R is independently selected from hydrogen, halo, cyano, and C1-C4 alkoxy. In some embodiments, n is 1 and R is hydrogen or cyano. In some embodiments, R is hydrogen. In some embodiments, R is cyano.

[0065] In some embodiments, A is a group of the formula: [ka] wherein each R is independently selected from hydrogen, halo, cyano, and C1-C4 alkoxy. In some embodiments, R is selected from hydrogen and cyano. In some embodiments, R is hydrogen. In some embodiments, R is cyano.

[0066] In some embodiments, A is a group of the formula: [ka] wherein n is 1, 2, or 3, and each R is independently selected from hydrogen, halo, cyano, and C1-C4 alkoxy. In some embodiments, n is 1 and R is hydrogen or cyano. In some embodiments, R is hydrogen.

[0067] In some embodiments, A is a group of the formula: [ka] wherein R is hydrogen, halo, cyano, or C1-C4 alkoxy. In some embodiments, R is hydrogen or cyano. In some embodiments, R is hydrogen. In some embodiments, R is cyano.

[0068] In some embodiments, A is a group of the formula: [ka] wherein n is 1, 2, or 3, and each R is independently selected from hydrogen, halo, cyano, and C1-C4 alkoxy. In some embodiments, n is 1 and R is hydrogen or cyano. In some embodiments, R is hydrogen.

[0069] In some embodiments, A is a group of the formula: [ka] wherein R is hydrogen, halo, cyano, or C1-C4 alkoxy. In some embodiments, R is hydrogen or cyano. In some embodiments, R is hydrogen. In some embodiments, R is cyano.

[0070] In some embodiments, A has a formula selected from the following: [ka]

[0071] In some embodiments, R' is hydrogen. In some embodiments, R' is C1-C4 alkyl. In some embodiments, R' is methyl. In some embodiments, R' is selected from hydrogen and methyl.

[0072] The linker may include one or more groups independently selected from methylene (-CH-), ethylene (-CH=CH-), ethynylene (-C≡C-), ether (-O-), amine (-NR-, where R is hydrogen or an alkyl group), thioether (-S-), carbonyl (-C(O)-), thiocarbonyl (-C(S)-), sulfonyl (-S(O)-), arylene, heteroarylene, and heterocyclylene moieties, or any combination thereof. For example, the above moieties can be combined to form additional groups that can be included in a linker, e.g., a carbonyl group and an ether group can together provide an ester moiety (-C(O)O-), a carbonyl group and two ether groups can together provide a carbonate moiety (-OC(O)O-), a carbonyl group and an unsubstituted amine group can together provide an unsubstituted amide moiety (-C(O)NH-), a carbonyl group and two unsubstituted amine groups can together provide an unsubstituted urea moiety (-NHC(O)NH-), a carbonyl group can together with an unsubstituted amine group and an ester group to provide an unsubstituted carbamate moiety (-OC(O)NH-), a carbonyl group can together with a thioether and an unsubstituted amine group to provide an S-thiocarbamate moiety, a thiocarbonyl group can together with an ether and an unsubstituted amine group to provide an O-thiocarbamate moiety, multiple methylene groups can together form an alkylene chain, and so forth. In some embodiments, the linker comprises one or more methylene, ether, ester, amide, carbamate, carbonate, urea, thioether, thioester, thioamide, thiocarbamate, thiocarbonate, thiourea, arylene, heteroarylene, or heterocyclylene moieties, or any combination thereof. In some embodiments, the linker comprises one or more -CH2-, -O-, -C(O)O-, -C(O)NH-, -NHC(O)O-, -OC(O)O-, -NHC(O)NH-, -S-, -C(O)S-, -C(S)NH-, -NHC(S)O-, -OC(S)O-, -NHC(S)NH-, arylene, heteroarylene, or heterocyclylene moieties, or any combination thereof.

[0073] In some embodiments, the linker comprises one or more moieties selected from linear or branched alkylene, -O-, -NH-, -C(O)O-, -C(O)NH-, -NHC(O)O-, -NHC(O)NH-, and phenylene groups. In some embodiments, the linker comprises one or more moieties selected from linear or branched alkylene, -O-, and -NH- groups. In some embodiments, the linker comprises one or more ethylene glycol units (-CH2CHO-).

[0074] In some embodiments, the linker has the formula: -NHCH2CH2(OCH2CH2) n NH- wherein n is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, n is 3, 4, 5, or 6. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8.

[0075] The group Y in the compound of formula (I) is a functional moiety such as a capture element, a detectable moiety, a reactive moiety, or a bifunctional moiety (e.g., a moiety that includes a biologically active compound and a reactive moiety or a capture element or a fluorophore). In some embodiments, Y is a capture element, which is a group such as a ligand or substrate that upon interaction with a protein ("capture protein") forms a covalent or non-covalent bond with the protein. In some embodiments, the capture element is a HALOTAG ligand, which is described, for example, in U.S. Pat. No. 7,425,436, which is incorporated herein by reference in its entirety. Moieties that find use as HALOTAG ligands include haloalkane (HA) groups (e.g., chloroalkane (CA) groups). For example, in some embodiments, Y is a group of the formula -(CH2) n-X, where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and X is a halogen (e.g., chloro). In such embodiments, the corresponding capture protein is a HALOTAG protein, which is described, for example, in U.S. Patent No. 7,425,436. Another example of a capture element is biotin. For example, in some embodiments, Y has the formula: [ka] In such an embodiment, the corresponding capture protein is, for example, streptavidin.

[0076] In some embodiments, Y is a detectable moiety, such as a fluorescent moiety. Suitable fluorescent functionalities include, but are not limited to, 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, nitrobenzoxadiazole, benzoxadiazole, etc.), pyrene derivatives (e.g., cascade, cyclopentadiazole, cyclohex ... blue), oxazine derivatives (e.g., Nile red, Nile blue, cresyl violet, oxazine 170, etc.), acridine derivatives (e.g., proflavine, acridine orange, acridine yellow, etc.), arylmethine derivatives (e.g., auramine, crystal violet, malachite green, etc.), tetrapyrrole derivatives (e.g., porphine, phthalocyanine, bilirubin, etc.), CF dyes (Biotium), boron dipyrromethene dyes (BODIPY dyes, Invitrogen), ALEXA Examples of dyes include FLUOR (Invitrogen), DYLIGHT FLUOR (Thermo Scientific, Pierce), ATTO and TRACY (Sigma Aldrich), FluoProbes (Interchim), DY and MEGASTOKES (Dyomics), SULFO CY dyes (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), and quantum dot nanocrystals.

[0077] In some embodiments, Y comprises a fluorogenic functional group that produces an enhanced fluorescent signal upon association with a target (e.g., upon binding of a protein to a moiety attached to the fluorogenic functional group). Upon target binding, background signal is reduced by producing significantly increased fluorescence (e.g., 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 100-fold, or more). Exemplary fluorogenic dyes for use in embodiments herein include the JANELIA FLUOR family of fluorophores, such as: JANELIA FLUOR 549: [ka] JANELIA FLUOR 646: [ka] JANELIA FLUOR 585: [ka] JANELIA FLUOR 635: [ka] JANELIA FLUOR 669: [ka]

[0078] In some embodiments, Y comprises a reactive functional group that can undergo further reaction with a corresponding reactive moiety on another molecule to affect a covalent bond. For example, in some embodiments, Y comprises a group selected from an azide, an alkyne, an alkene, or a 1,2,4,5-tetrazinyl moiety, all of which are commonly known as "click handles" that can undergo copper-catalyzed or copper-free "click" reactions (e.g., reaction of an azide with an alkyne, reaction of an azide with a difluorinated cyclooctyne, or reaction of a 1,2,4,5-tetrazinyl group with a trans-cyclooctene moiety).

[0079] In some embodiments, Y comprises a bifunctional moiety that includes a biologically active compound and either a fluorophore or a capture element or a reactive moiety for photoaffinity labeling and subsequent detection and / or enrichment, etc. In some embodiments, the biologically active compound is a small molecule, e.g., a therapeutic agent.

[0080] In some embodiments, the compound of formula (I) is a compound selected from the following: [ka] and salts thereof.

[0081] The present disclosure also includes isotopically labeled compounds that are identical to those recited in formula (I) except for the fact that one or more atoms have been replaced with an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Examples of isotopes suitable for incorporation into the compounds of the present invention include, but are not limited to, 2 H, 3 H, 13 C. 14 C. 15 N, 18 O. 17 O. 31 P, 32 P, 35 S, 18 F, and 36The elements are hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as Cl. Deuterium, i.e. 2 Substitution with heavier isotopes such as H may confer certain benefits, such as increased half-life in vivo, resulting from greater metabolic stability, and therefore may be preferred in some cases. Isotopically labeled compounds of formula (I) may generally be prepared by conventional techniques known to those skilled in the art, or by processes similar to those described in the accompanying examples, using appropriate isotopically labeled reagents in place of non-isotopically labeled reagents.

[0082] The compounds disclosed herein can be in the form of salts, which may be prepared during the final isolation and purification of the compounds or separately, for example, by reacting a basic group (e.g., an amino group) of the compound with a suitable acid or an acidic group (e.g., a carboxylic acid group) of the compound with a suitable base.

[0083] Acid salts may be prepared during the final isolation and purification of the compound, or may be prepared separately by reacting a suitable group of the compound, such as an amino group, with a suitable acid. For example, the compound may be dissolved in a suitable solvent, such as, but not limited to, methanol and water, and treated with at least one equivalent of an acid, such as hydrochloric acid. The resulting salt may precipitate and be isolated by filtration and dried under reduced pressure. Alternatively, the solvent and excess acid may be removed under reduced pressure to provide the salt. Representative salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, isethionate, fumarate, lactate, maleate, methanesulfonate, naphthylenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, oxalate, maleate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, glutamate, para-toluenesulfonate, undecanoate, hydrochloride, hydrobromide, sulfate, phosphate, and the like. The amino groups of the compounds may also be quaternized with alkyl chlorides, bromides, and iodides, such as methyl, ethyl, propyl, isopropyl, butyl, lauryl, myristyl, stearyl, and the like.

[0084] Basic addition salts can be prepared during the final isolation and purification of the disclosed compounds by reaction of the carboxyl group with a suitable base, for example, hydroxides, carbonates or bicarbonates of metal cations such as lithium, sodium, potassium, calcium, magnesium, or aluminum, or organic primary, secondary or tertiary amines. Quaternary amine salts can be prepared, for example, salts derived from methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine, and N,N'-dibenzylethylenediamine, ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like.

[0085] The compounds disclosed herein can be synthesized by a variety of methods, including those illustrated in the Examples. Routine experimentation, including appropriate manipulation of reaction conditions, reagents and sequences of synthetic routes, protection of any chemical functionality that may not be compatible with the reaction conditions, and deprotection at appropriate points in the reaction sequence of the method, is within the scope of this disclosure. Suitable protecting groups and methods for protecting and deprotecting different substituents using such suitable protecting groups are well known to those skilled in the art, and examples can be found in the book by Greens, entitled Protective Groups in Organic Synthesis (4 th ed.), John Wiley & Sons, NY (2006), which is incorporated herein by reference in its entirety. Synthesis of the compounds of the present disclosure can be accomplished by methods analogous to those described in the synthetic schemes and specific examples described herein.

[0086] If an optically active form of a disclosed compound is required, it may be obtained by carrying out one of the procedures described herein using optically active starting materials (prepared, for example, by asymmetric induction of an appropriate reaction step), or by resolving a stereoisomeric mixture of the compound or intermediate using standard procedures (such as chromatographic separation, recrystallization, or enzymatic resolution).

[0087] Similarly, if a pure geometric isomer of a compound is required, it can be obtained by carrying out one of the procedures described above using a pure geometric isomer as a starting material, or by resolving a geometric isomeric mixture of the compound or intermediate using standard procedures such as chromatographic separation.

[0088] photocatalyst In some embodiments, the compounds disclosed herein are used (e.g., in the systems and methods described herein) together with a photocatalyst that can absorb light and then activate a photoactivatable aryl-azide moiety on the compound of formula (I). Any compound or moiety that can receive light energy and transfer that energy to activate a photoactivatable moiety can find use in the embodiments herein. In some embodiments, the excited photocatalyst transfers energy to the compound of formula (I) via Forster resonance energy transfer, Dexter energy transfer, single electron transfer, singlet oxygen, or any other suitable energy or electron transfer mechanism.

[0089] In some embodiments, the photocatalyst is an iridium- or ruthenium-based photocatalyst (Bevernaegie et al. “A Roadmap Towards Visible Light Mediated Electron Transfer Chemistry with Iridium(III) Complexes.” ChemPhotoChem 2021, 5, 217; incorporated by reference in its entirety). In some embodiments, the photocatalyst is a compound having the formula: [ka] [In the formula, Each series of dashed lines [ka] represents the presence or absence of a fused six-membered ring, M is a transition metal; m1, m2, m3, n1, n2, n3, p1, p2, and p3 are each independently 0, 1, or 2; R 1a , R 1b , R 1c , R 2a , R 2b , R 2c , R 3a , R 3b , and R 3c are each independently selected from halo, alkyl, haloalkyl, amino, and heteroalkyl; X 1a , X 1b , X 2a , X 2b , X 3aZ , and X 3b are each independently selected from N and C, 1a and X 1b At least one of them is N, and X 2a and X 2b At least one of them is N, and X 3a and X 3b at least one of is N; X 1c , X 1d , X 2c , X 2d , X 3c , and X 3d are each independently selected from CH and N; Z is an anion, [q is 0, 1, or 2].

[0090] In some embodiments, the photocatalyst comprises a transition metal selected from Ru and Ir.

[0091] In some embodiments, the photocatalyst is an iridium-based photocatalyst selected from: [ka]

[0092] In some embodiments, the photocatalyst is a ruthenium-based photocatalyst selected from: [ka]

[0093] In some embodiments, m2, n2, and p2 are each 0 and each series of dashed lines represents the absence of a fused 6-membered ring, i.e., the compound has the formula: [ka] In some embodiments, X 1a is N and X 1b is C and X 2a is N and X 2b is C and X 3a is C and X 3b is N. In some embodiments, X 1a is N and X 1b is C and X 2a is N and X 2b is C and X 3a is N and X 3b is N.

[0094] In some embodiments, X 1c , X 1d , X 2c , X 2d , X 3c , and X 3d Each is CH. In some embodiments, X 1c , X 1d , X 2c , X 2d , X 3c , and X 3d are N respectively.

[0095] In some embodiments, R 1a , R 1b , R 1c , R 2a , R 2b , R 2c , R 3a , R 3b , and R 3c are each independently selected from fluoro, methyl, tert-butyl, and trifluoromethyl. In some embodiments, M is Ru. In some embodiments, M is Ir.

[0096] System and method of use In some embodiments, the present disclosure provides systems and methods for labeling biomolecules with compounds of formula (I). As described above, compounds of formula (I) include an azide moiety substituted on a phenyl, naphthyl, or quinolinyl group. The aryl azide can undergo photoinduced activation to form a reactive nitrene group that can react with and covalently modify a biomolecule.

[0097] Thus, the present disclosure provides systems and methods for labeling biomolecules, comprising a compound of formula (I). For example, in some embodiments, systems for labeling biomolecules are provided herein, comprising a compound of formula (I) and a photocatalyst. Upon exposure to light, the activated photocatalyst can facilitate the transfer of energy to a photoactivatable moiety on the compound of formula (I), facilitating the generation of a reactive intermediate that covalently labels the biomolecule. Exemplary photocatalysts that can be used in the systems disclosed herein are those disclosed above (e.g., the ruthenium or iridium catalysts described above).

[0098] Also disclosed herein is a method of labeling a biomolecule in a sample, comprising contacting the sample with a compound of formula (I) and exposing the sample to light. In some embodiments, the light is ultraviolet (UV) light. In some embodiments, the light is visible light. In some embodiments, the light is provided by a light emitting diode. In some embodiments, the method further comprises contacting the sample with a photocatalyst, such as a photocatalyst disclosed herein, prior to exposing the sample to light.

[0099] In some embodiments, the compounds and systems disclosed herein are used to carry out various methods (e.g., in cells). In addition to other applications, various functional proteosome and genomic analyses are enabled by the compounds, methods, and systems disclosed herein. Exemplary proteosome-focused applications of the system methods herein include proximity-based protein labeling and photoaffinity labeling using bioactive compounds modified with capture elements and photoreactive groups for dynamic microenvironment and subsequent detection, protein-protein and cell-cell interactions under relevant physiological conditions, subsequent enrichment, and identification of their cellular targets; ligand-directed protein labeling using fluorophores or click handles for subsequent attachment of various functional groups, such as PROTACs, small molecule drugs, for downstream manipulation of proteins of interest; and small molecule drug profiling.

[0100] Additional systems and methods using the compounds disclosed herein include systems and methods for bioluminescence-triggered catalysis, such as those disclosed in U.S. Provisional Patent Application No. 63 / 338,322, filed May 4, 2022, entitled "BIOLUMINESCENCE-TRIGGERED PHOTOCATALYTIC LABELING," which is incorporated by reference in its entirety. EXAMPLES

[0101] Example 1 Experiments conducted during the development of embodiments herein evaluated the ability of vinyl-aryl-azides, vinyl-naphthyl-azides, and vinyl-quinoline-azides to undergo activation and subsequent crosslinking by UV irradiation, visible 455 nm irradiation, as well as LED-induced iridium catalyzed activation, which further involves energy transfer events with azide-based photoreactive groups (Figures 2-5). The structures and absorbance profiles of vinyl-aryl-azides, vinyl-naphthyl-azides, and vinyl-quinoline-azides are included in Figures 2 and 3, respectively, and their syntheses are described in Example 3.

[0102] The absorbance profiles of 200 μM biotin probes containing vinyl-extended aryl-azide photoreactive groups in 2% DMSO were monitored on a SPARK multimode plate reader (Figure 3). In general, all vinyl-extended aryl-azide photoreactive groups showed red-shifted absorbance compared to simple phenyl-azides (λmax = ca. 260 nm), suggesting easier activation by visible blue light and photocatalytic energy transfer.

[0103] The ability of these vinyl-extended aryl-azide photoreactive groups to undergo activation by either UV or visible 455 nm light in the presence or absence of a catalyst for subsequent covalent cross-linking with adjacent proteins was further evaluated by labeling efficiency of model proteins (Figures 4 and 5). To this end, reactions containing either (1) 0.8 μM acetylated BSA (Promega) and 50 μM vinyl-extended aryl-azide-biotin probe or (2) 0.8 μM acetylated BSA (Promega), 50 μM vinyl-extended aryl-azide-biotin probe, and 100 μM iridium catalyst (Ir-9049) were assembled in TE buffer (pH 7.5) in the wells of a UV-transparent 96-well plate. Plates were either kept in the dark (control) or irradiated using an Efficiency Aggregators bioreactor for 20 min at either 365 nm (100% UV = approx. 80 W) or 455 nm (100% = approx. 80 W or 2% = approx. 2 W). To assess subsequent covalent labeling efficiency, reactions were passed through a Zeba column cleanup (ThermoFisher) to remove uncrosslinked vinyl-extended aryl-azide-biotin probes and then spotted onto nitrocellulose membranes using a slot blot apparatus. The membranes were then blocked with 5% BSA (Promega) in TBST for 1 h at room temperature and then incubated with anti-biotin antibody (Invitrogen) in TBST overnight at 4°C. The next day, the membranes were washed three times with TBST and then incubated with secondary anti-goat HRP antibody (Jackson laboratories) for 1 h. After three washes with TBST, the membrane was treated with ECL substrate (Promega) and scanned in the chemiluminescence channel to detect biotin-labeled BSA. Western analysis and quantification of band volumes using Image J software revealed that all vinyl-extended aryl-azide photoreactive groups were directly activated by either UV or 455 nm, albeit with different efficiencies.Of the 15 vinyl-extended aryl-azide photoreactive groups tested, eight showed greater UV activation efficiency, five showed equal activation efficiency at UV and 455 nm, and two (9616 and 9582) showed greater 455 nm activation efficiency. Interestingly, both are vinyl-naphthyl-azides with methyl substitution on the vinyl group. Furthermore, iridium catalysts activated by either 100% or 2% LED were able to participate in photocatalytic energy transfer events, resulting in greater labeling efficiency compared to direct LED activation (i.e., in the absence of catalyst) for all 15 vinyl-extended aryl-azide photoreactive groups tested. Quantified band volumes were further normalized to 9157 UV-induced labeling (common across all membranes), revealing a wide range of activation and labeling efficiencies.

[0104] Example 2 Experiments conducted during the development of embodiments herein evaluated the ability of vinyl-aryl-azides, vinyl-naphthyl-azides, and vinyl-quinoline-azides to undergo bioluminescence-triggered photocatalytic activation and subsequent covalent cross-linking with nearby proteins (Figure 6). The structures and absorbance profiles of vinyl-aryl-azides, vinyl-naphthyl-azides, and vinyl-quinoline-azides are included in Figures 2 and 3, and their syntheses are described in Example 3.

[0105] The ability of the bioluminescence-triggered photocatalytic complex to undergo an energy transfer event with a vinyl-extended arylazide-biotin probe for subsequent cross-linking with nearby proteins was further investigated. 178 -cpNLuc- 179 The HaloTag (Ir-9049) comprises a circularly permuted NanoLuc (i.e., cpNLuc) inserted into a surface loop of HaloTag (between residues 178 and 179) near the ligand binding site, and a chloroalkane-iridium catalytic complex (Ir-9049) that is bound to the chimera and is activatable by bioluminescence energy transfer. 178-cpNLuc- 179 Briefly, the kit consisted of 100 μM vinyl-extended arylazide-biotin probe, 0.1 mg / mL K562 cell lysate depleted of biotinylated proteins, and 60 nM HT 178 -cpNLuc- 179 Reactions containing the Ir-9049 conjugate were assembled in TBS (pH 7.5) in the wells of a white 96-well plate. Bioluminescence was induced upon treatment with 100 μM fluorofurimazine for 20 min, whereas control wells were left untreated (light-independent background). In some cases, HT 178 -cpNLuc- 179 An additional light-dependent background control was included in which the Ir-9049 conjugate was replaced with NanoLuc (no catalyst). To assess labeling efficiency, samples were collected, resolved by SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were blocked with 5% BSA (Promega) in TBST for 1 h at room temperature, and then incubated overnight at 4° C. with streptavidin-HRP (Invitrogen) in TBST supplemented with 5% BSA. After three washes with TBST, the membranes were treated with ECL substrate (Promega) and scanned in the chemiluminescence channel to detect biotin-labeled proteins. Western analysis demonstrated that these vinyl-extended aryl-azide photoreactive groups can undergo activation by bioluminescence-triggered photocatalytic energy transfer for subsequent crosslinking to nearby proteins, despite a wide range of efficiencies and light-dependent and light-independent backgrounds. Notably, the degree of red-shifted absorbance for these vinyl-azide photoreactive groups was associated with an increased ability to undergo activation by bioluminescence-triggered photocatalytic energy transfer. Furthermore, some substitutions increased the specificity of labeling to different degrees, presumably by interfering with the rate of generation and / or lifetime of reactive intermediates and resulting in an overall smaller labeling radius.

[0106] Example 3: Synthesis of Compounds Synthesis of aryl azide intermediates A-1 Synthesis of (E)-3-(6-azidonaphthalen-2-yl)acrylic acid [ka]

[0107] Step 1: To a 20 mL vial was added tert-butyl 2-(diethoxyphosphoryl)acetate (0.475 mL, 2.13 mmol) and THF (6 mL). The mixture was stirred under nitrogen and 1M LHMDS in THF (2.13 mL, 2.13 mmol) was added dropwise over 5 min. To the mixture was added 6-bromo-2-naphthaldehyde (500 mg, 2.13 mmol) over 1 min. After 20 min, the mixture was adsorbed onto Celite and purified by silica gel chromatography using 0-30% EtOAc in heptane as eluent to give tert-butyl (E)-3-(6-bromonaphthalen-2-yl)acrylate. LRMS [M+H-C4H4] + 277.

[0108] Step 2: To a 20 mL vial was added tert-butyl (E)-3-(6-bromonaphthalen-2-yl)acrylate (200 mg, 0.600 mmol), Pd(dppf)Cl2 (22.0 mg, 0.030 mmol), B2pin2 (183 mg, 0.720 mmol), potassium acetate (118 mg, 1.20 mmol), and dioxane (4 mL). The mixture was degassed with nitrogen for 1 min. The mixture was stirred and heated at 100 °C for 1 h. The mixture was cooled to room temperature. The mixture was diluted with EtOAc and filtered through Celite. The solvent was evaporated to give tert-butyl (E)-3-(6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-2-yl)acrylate, which was carried on to the next step without further purification. LRMS [M+H-C4H4] + 325.

[0109] Step 3: To a 20 mL vial was added tert-butyl (E)-3-(6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-2-yl)acrylate (225 mg, 0.592 mmol), NaN3 (57.8 mg, 0.889 mmol), Cu(OAc)2 (215 mg, 1.18 mmol), and MeOH (3 mL). The mixture was vigorously stirred at 65 °C for 90 min. The mixture was diluted with EtOAc and washed with 10% ammonia. The organic layer was dried over sodium sulfate, filtered, and the solvent was evaporated. The residue was purified by silica gel chromatography using 0-30% EtOAc in heptane as eluent to give tert-butyl (E)-3-(6-azidonaphthalen-2-yl)acrylate. LRMS [M+H-C4H4] + 240.

[0110] Step 4: To a 20 mL vial was added tert-butyl (E)-3-(6-azidonaphthalen-2-yl)acrylate (100 mg, 0.339 mmol), DCM (2 mL), and formic acid (1 mL). The mixture was stirred for 14 h. A solid precipitated. The solid was collected by filtration and washed with DCM to give (E)-3-(6-azidonaphthalen-2-yl)acrylic acid of A-1. LRMS [MH] - 238.

[0111] Synthesis of A-2(E)-3-(6-azidonaphthalen-2-yl)but-2-enoic acid [ka]

[0112] Step 1: To a 20 mL vial was added 6-bromo-2-naphthaldehyde (500 mg, 2.13 mmol) and THF (10 mL). The mixture was stirred under nitrogen at 0° C. To the mixture was added a 1.4 M solution of MeMgBr (1.82 mL, 2.55 mmol) over 5 min. The mixture was stirred for 10 min. The mixture was quenched with saturated ammonium chloride (ca. 0.5 mL) and filtered through Celite. The organic layer was diluted with diethyl ether and washed with water. The organic layer was dried over magnesium sulfate, filtered and the solvent was evaporated. The residue was purified by silica gel chromatography using 0-30% EtOAc in heptane as eluent to give 1-(6-bromonaphthalen-2-yl)ethan-1-ol. LRMS [M+H-H2O] + 233.

[0113] Step 2: To a 100 mL flask was added PCC (1.09 g, 5.07 mmol), Celite (2.5 g), and DCM (20 mL). To the stirred mixture was added 1-(6-bromonaphthalen-2-yl)ethan-1-ol (424 mg, 1.69 mmol). The mixture was stirred at room temperature for 1 h. The mixture was filtered through Celite and washed with DCM. The filtrate's solvent was evaporated. The residue was purified by silica gel chromatography using 0-30% EtOAc in heptane as eluent to give 1-(6-bromonaphthalen-2-yl)ethan-1-one. LRMS [M+H] + 249.

[0114] Step 3: To a 20 mL vial was added tert-butyl 2-(diethoxyphosphoryl)acetate (0.307 mL, 1.38 mmol) and THF (5 mL). The mixture was stirred under nitrogen. To the mixture was added 1M LHMDS in THF (1.38 mL, 1.38 mmol) dropwise over 5 min. To this mixture was added a THF solution of 1-(6-bromonaphthalen-2-yl)ethan-1-one (343 mg, 1.38 mmol) dropwise over 5 min. After 5 min, the vial was sealed and then stirred and heated at 70 °C for 3 h. The mixture was adsorbed onto Celite and purified by silica gel chromatography using 0-20% EtOAc in heptane as eluent to give tert-butyl (E)-3-(6-bromonaphthalen-2-yl)but-2-enoate. LRMS [M+H-C4H4] + 291.

[0115] Step 4: To a 20 mL vial was added tert-butyl (E)-3-(6-bromonaphthalen-2-yl)but-2-enoate (350 mg, 1.01 mmol), Pd(dppf)Cl2 (73.8 mg, 0.101 mmol), B2pin2 (307 mg, 1.21 mmol), potassium acetate (198 mg, 2.02 mmol), and dioxane (5 mL). The mixture was degassed with nitrogen for 1 min. The mixture was stirred and heated at 120 °C for 2.5 h. The mixture was cooled to room temperature. The mixture was diluted with EtOAc and filtered through Celite. The solvent was evaporated to give tert-butyl (E)-3-(6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-2-yl)but-2-enoate, which was carried on to the next step without further purification. LRMS [M+H-C4H4] + 339.

[0116] Step 5: To a 20 mL vial was added tert-butyl (E)-3-(6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-2-yl)but-2-enoate (145 mg, 0.368 mmol), NaN3 (35.9 mg, 0.552 mmol), Cu(OAc)2 (134 mg, 0.736 mmol), and MeOH (8 mL). The mixture was vigorously stirred at 65 °C for 90 min. The mixture was diluted with EtOAc and washed with 10% ammonia. The organic layer was dried over sodium sulfate, filtered, and the solvent was evaporated. The residue was purified by silica gel chromatography using 0-50% EtOAc in heptane as eluent to give tert-butyl (E)-3-(6-azidonaphthalen-2-yl)but-2-enoate. LRMS [M+H-C4H4] + 254.

[0117] Step 6: To a 20 mL vial was added tert-butyl (E)-3-(6-azidonaphthalen-2-yl)but-2-enoate (30.0 mg, 0.0970 mmol), DCM (2 mL), and formic acid (1 mL). The mixture was stirred for 14 h. The solvent was evaporated to give A-2, (E)-3-(6-azidonaphthalen-2-yl)but-2-enoic acid. LRMS [MH] - 252.

[0118] A-3 Synthesis of (E)-3-(7-azidoquinolin-3-yl)acrylic acid [ka]

[0119] Step 1: To a 100 mL round bottom flask was added quinoline-3-carbaldehyde (2.00 g, 12.7 mmol), p-toluenesulfonic acid monohydrate (219 mg, 1.27 mmol), MeOH (15 mL), and trimethyl orthoformate (13.9 mL, 127 mmol). The mixture was stirred and heated at 70 °C for 3 h. The mixture was concentrated and purified by silica gel chromatography using 0-70% EtOAc in heptane as eluent to give 3-(dimethoxymethyl)quinoline. LRMS [M+H] + 204.

[0120] Step 2: To a 20 mL vial was added 3-(dimethoxymethyl)quinoline (500 mg, 2.46 mmol), [Ir(COD)OMe]2 (81.5 mg, 0.123 mmol), B2pin2 (937 mg, 3.69 mmol), and 4,4'-di-tert-butylbipyridine (dtbpy, 66.0 mg, 0.246 mmol). The vial was purged with nitrogen. To this mixture was added anhydrous THF (5 mL). The mixture was sparged with nitrogen for 1 min. The mixture was stirred at room temperature for 14 h. The solvent was evaporated to give crude 3-(dimethoxymethyl)-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quinoline, which was carried on to the next step without purification. LRMS [M+H] + 330.

[0121] Step 3: To a 20 mL vial was added 3-(dimethoxymethyl)-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quinoline (810 mg, 2.46 mmol), NaN3 (240 mg, 3.69 mmol), Cu(OAc)2 (894 mg, 4.62 mmol), and MeOH (8 mL). The mixture was vigorously stirred at 55 °C for 90 min. The mixture was diluted with EtOAc and washed with 10% ammonia. The organic layer was dried over sodium sulfate, filtered, and the solvent was evaporated. The residue was purified by silica gel chromatography using 0-40% EtOAc in heptane as eluent to give 7-azido-3-(dimethoxymethyl)quinoline. LRMS [M+H] +245.

[0122] Step 4: To a 20 mL vial was added 7-azido-3-(dimethoxymethyl)quinoline (26.9 mg, 0.110 mmol), TFA (1 mL), and water (0.1 mL). The mixture was stirred for 10 min. The solvent was evaporated to give 7-azidoquinoline-3-carbaldehyde. LRMS [M+H] + 199.

[0123] Step 5: To a 20 mL vial was added tert-butyl 2-(diethoxyphosphoryl)acetate (0.0278 mL, 0.110 mmol), 7-azidoquinoline-3-carbaldehyde (21.8 mg, 0.110 mmol), and MeOH (1 mL). To the stirred mixture was added tetramethylguanidine (TMG, 0.055 mL, 0.441 mmol). After 20 min, the solvent was evaporated and the residue was purified by silica gel chromatography using 0-50% EtOAc in heptane as eluent to give tert-butyl (E)-3-(7-azidoquinolin-3-yl)acrylate. LRMS [M+H] + 297.

[0124] Step 6: To a 20 mL vial was added tert-butyl (E)-3-(7-azidoquinolin-3-yl)acrylate (23.7 mg, 0.0800 mmol) and TFA (1 mL). The mixture was stirred for 15 min. The solvent was evaporated to give A-3, (E)-3-(7-azidoquinolin-3-yl)acrylic acid. LRMS [M+H] + 241.

[0125] A-4 Synthesis of (E)-3-(6-azidoquinolin-3-yl)acrylic acid [ka]

[0126] Step 1: To a 20 mL vial was added tert-butyl 2-(diethoxyphosphoryl)acetate (0.378 mL, 1.69 mmol) and THF (6 mL). The mixture was stirred under nitrogen. To the mixture was added 1M LHMDS in THF (1.69 mL, 1.69 mmol) dropwise over 5 min. To the mixture was added 6-bromoquinoline-3-carbaldehyde (Cheng, Yuan et al. WO2011063233 A1) (400 mg, 1.69 mmol) over 1 min. After 20 min, the mixture was adsorbed onto Celite and purified by silica gel chromatography using 0-50% EtOAc in heptane as eluent to give tert-butyl (E)-3-(6-bromoquinolin-3-yl)acrylate. LRMS [M+H] + 334.

[0127] Step 2: To a 20 mL vial was added tert-butyl (E)-3-(6-bromoquinolin-3-yl)acrylate (64.0 mg, 0.191 mmol), Pd(dppf)Cl2 (7.0 mg, 0.0096 mmol), B2pin2 (58.4 mg, 0.230 mmol), potassium acetate (37.6 mg, 0.383 mmol), and dioxane (4 mL). The mixture was degassed with nitrogen for 1 min. The mixture was stirred and heated at 100 °C for 2 h. The mixture was cooled to room temperature. The mixture was diluted with EtOAc and filtered through Celite. The solvent was evaporated to give tert-butyl (E)-3-(6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quinolin-3-yl)acrylate, which was carried on to the next step without further purification. LRMS [M+H] + 382.

[0128] Step 3: To a 20 mL vial was added tert-butyl (E)-3-(6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quinolin-3-yl)acrylate (73.0 mg, 0.191 mmol), NaN3 (18.7 mg, 0.287 mmol), Cu(OAc)2 (69.6 mg, 0.383 mmol), and MeOH (3 mL). The mixture was vigorously stirred at 65° C. for 90 min. The mixture was diluted with EtOAc and washed with 10% ammonia. The organic layer was dried over sodium sulfate, filtered, and the solvent was evaporated. The residue was purified by silica gel chromatography using 0-50% EtOAc in heptane as eluent to give tert-butyl (E)-3-(6-azidoquinolin-3-yl)acrylate. LRMS [M+H] + 297.

[0129] Step 4: To a 20 mL vial was added tert-butyl (E)-3-(6-azidoquinolin-3-yl)acrylate and 4M HCl in dioxane (2 mL). The mixture was stirred and heated at 70° C. for 2 h. The solvent was concentrated to give A-4, (E)-3-(6-azidoquinolin-3-yl)acrylic acid. LRMS [M+H] + 241.

[0130] A-5 Synthesis of (E)-3-(6-azidoquinolin-3-yl)but-2-enoic acid [ka]

[0131] Step 1: To a 20 mL vial was added 6-bromoquinoline-3-carbaldehyde (Cheng, Yuan et al. WO2011063233 A1) (334 mg, 1.42 mmol) and THF (8 mL). The mixture was stirred under nitrogen at 0° C. To the mixture was added a 1.4 M solution of MeMgBr (1.52 mL, 2.12 mmol) over 5 min. The mixture was stirred for 10 min. The mixture was quenched with saturated ammonium chloride (1 mL). The mixture was diluted with EtOAc and filtered through Celite. The filtrate was concentrated and the residue was purified by silica gel chromatography using 0-70% EtOAc in heptane as eluent to give 1-(6-bromoquinolin-3-yl)ethan-1-ol. LRMS [M+H] + 252.

[0132] Step 2: To a 20 mL vial was added 1-(6-bromoquinolin-3-yl)ethan-1-ol (260 mg, 1.03 mmol), PCC (668 g, 3.10 mmol), Celite (1 g), and DCM (6 mL). The mixture was stirred at room temperature for 2 h. The mixture was filtered through Celite and washed with DCM. The filtrate's solvent was evaporated. The residue was purified by silica gel chromatography using 0-70% EtOAc in heptane as eluent to give 1-(6-bromoquinolin-3-yl)ethan-1-one. LRMS [M+H] + 250.

[0133] Step 3: To a 20 mL vial was added tert-butyl 2-(diethoxyphosphoryl)acetate (0.185 mL, 0.829 mmol) and THF (6 mL). The mixture was stirred under nitrogen. To the mixture was added 1M LHMDS in THF (1.13 mL, 1.13 mmol) dropwise over 5 min. To this mixture was added a THF solution of 1-(6-bromoquinolin-3-yl)ethan-1-one (188 mg, 0.753 mmol) dropwise over 5 min. The mixture was stirred for 20 min. The mixture was adsorbed onto Celite and purified by silica gel chromatography using 0-50% EtOAc in heptane as eluent to give tert-butyl (E)-3-(6-bromoquinolin-3-yl)but-2-enoate. LRMS [M+H] + 348.

[0134] Step 4: To a 20 mL vial was added tert-butyl (E)-3-(6-bromoquinolin-3-yl)but-2-enoate (55.0 mg, 0.158 mmol), Pd(dppf)Cl2 (5.8 mg, 0.0079 mmol), B2pin2 (48.1 mg, 0.190 mmol), potassium acetate (31.0 mg, 0.316 mmol), and dioxane (1 mL). The mixture was degassed with nitrogen for 1 min. The mixture was stirred and heated at 100 °C for 2 h. The mixture was cooled to room temperature. The mixture was diluted with EtOAc and filtered through Celite. The solvent was evaporated to give tert-butyl (E)-3-(6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quinolin-3-yl)but-2-enoate, which was carried on to the next step without further purification. LRMS [M+H] + 396.

[0135] Step 5: To a 20 mL vial was added tert-butyl (E)-3-(6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quinolin-3-yl)but-2-enoate (62.4 mg, 0.158 mmol), NaN3 (15.4 mg, 0.237 mmol), Cu(OAc)2 (57.3 mg, 0.316 mmol), and MeOH (3 mL). The mixture was vigorously stirred at 65 °C for 90 min. The mixture was diluted with EtOAc and washed with 10% ammonia. The organic layer was dried over sodium sulfate, filtered, and the solvent was evaporated. The residue was purified by silica gel chromatography using 0-50% EtOAc in heptane as eluent to give tert-butyl (E)-3-(6-azidoquinolin-3-yl)but-2-enoate. LRMS [M+H] + 311.

[0136] Step 6: To a 20 mL vial was added tert-butyl (E)-3-(6-azidoquinolin-3-yl)but-2-enoate (43.5 mg, 0.140 mmol) and 4M HCl in dioxane (2 mL). The mixture was stirred and heated at 70° C. for 2 h. The solvent was evaporated to give A-5, (E)-3-(6-azidoquinolin-3-yl)but-2-enoic acid. LRMS [M+H] + 255.

[0137] A-6 Synthesis of (E)-3-(6-azido-7-cyanonaphthalen-2-yl)acrylic acid [ka]

[0138] Step 1: To a 100 mL flask was added 2-bromo-6-fluoronaphthalene (1.00 g, 4.44 mmol), [Ir(COD)OMe]2 (147 mg, 0.222 mmol), B2pin2 (1.69 g, 6.66 mmol), and 4,4'-di-tert-butylbipyridine (dtbpy, 119 mg, 0.444 mmol). The vial was purged with nitrogen. To this mixture was added anhydrous THF (10 mL). The mixture was sparged with nitrogen for 1 minute. The mixture was stirred at room temperature for 14 hours. The solvent was evaporated. The residue was purified by silica gel chromatography to give 2-(7-bromo-3-fluoronaphthalen-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane. 1 H NMR (400 MHz, DMSO-d6) δ 8.54 (d, J = 2.2 Hz, 1H), 8.38 (dd, J = 4.3, 2.2 Hz, 2H), 8.33 (d, J = 6.0 Hz, 1H), 8.21 (d, J = 11.6 Hz, 1H), 8.03 (d, J = 2.2 Hz, 1H), 7.89 (d, J = 8.8 Hz, 1H), 7.76 - 7.65 (m, 2H), 1.39 (s, 12H).

[0139] Step 2: To a 100 mL flask was added 2-(7-bromo-3-fluoronaphthalen-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.18 g, 3.35 mmol), Cu(NO3)2·3H2O (1.62 g, 6.70 mmol), Zn(CN)2 (1.18 g, 10.1 mmol), CsF (509 mg, 3.35 mmol), MeOH (20 mL), and water (8 mL). The reaction was stirred and heated at reflux for 1 h. The mixture was cooled to room temperature. The mixture was diluted in EtOAc and washed with 1 M aqueous ammonia. The organic layer was dried over sodium sulfate, filtered, and the filtrate's solvent was evaporated. The residue was purified by silica gel chromatography using 0-20% EtOAc in heptane to give 7-bromo-3-fluoro-2-naphthonitrile. 1H NMR (400 MHz, DMSO-d6) δ 8.71 (d, J = 6.6 Hz, 1H), 8.39 (d, J = 1.9 Hz, 1H), 8.11 (d, J = 10.6 Hz, 1H), 8.02 (d, J = 8.9 Hz, 1H), 7.91 (dd, J = 8.8, 2.0 Hz, 1H).

[0140] Step 3: To a 20 mL vial was added 7-bromo-3-fluoro-2-naphthonitrile (199 mg, 0.797 mmol), t-butyl acrylate (0.146 mL, 0.996 mmol), triethylamine (0.222 mL, 1.59 mmol), Pd(OAc)2 (1.8 mg, 0.0080 mmol), tri-(o-tolyl)phosphine (9.7 mg, 0.032 mmol), and toluene (8 mL). The mixture was purged with nitrogen for 1 min. The mixture was stirred and heated at 100° C. under nitrogen for 3 h. The solvent was evaporated and the residue was purified by silica gel chromatography using 0-20% EtOAc in heptane to give tert-butyl (E)-3-(7-cyano-6-fluoronaphthalen-2-yl)acrylate. LRMS [M+H+MeCN] + 339.

[0141] Step 4: To a 20 mL vial was added tert-butyl (E)-3-(7-cyano-6-fluoronaphthalen-2-yl)acrylate (64.7 mg, 0.218 mmol), NaN3 (15.5 mg, 0.239 mmol), and DMSO (1 mL). The mixture was stirred and heated at 100 °C for 2 h. The mixture was diluted in 1:2 EtOAc / Et2O (12 mL), filtered through Celite, and rinsed with Et2O. The filtrate was washed with water (3 x 10 mL). The organic layer was dried over sodium sulfate, filtered, and the filtrate's solvent was evaporated. The mixture was purified by silica gel chromatography using 0-30% EtOAc in heptane as eluent to give tert-butyl (E)-3-(6-azido-7-cyanonaphthalen-2-yl)acrylate. LRMS [M+H-H2O] + 293.

[0142] Step 5: To a 20 mL vial was added tert-butyl (E)-3-(6-azido-7-cyanonaphthalen-2-yl)acrylate (37.4 mg, 0.117 mmol) and TFA (1 mL). The mixture was stirred for 15 min. The solvent was evaporated to give A-6, (E)-3-(6-azido-7-cyanonaphthalen-2-yl)acrylic acid. LRMS [MH] - 263.

[0143] A-7 Synthesis of (E)-3-(6-azido-7-cyanonaphthalen-2-yl)but-2-enoic acid [ka]

[0144] Step 1: To a solution of 7-bromo-3-fluoro-2-naphthonitrile (from step 2 of A-6) (266 mg, 1.07 mmol) in dioxane (2 mL) was added tributyl(1-ethoxyvinyl)tin (0.396 mL, 1.17 mmol) and Pd(PPh3)2Cl2 (37.4 mg, 0.0533 mmol). The mixture was purged with nitrogen for 2 min. The mixture was stirred and heated at 130° C. for 30 min. The mixture was cooled to room temperature, the mixture was diluted with EtOAc and filtered through Celite. The filtrate solvent was evaporated. The residue was purified by silica gel chromatography using 0-30% EtOAc in heptane as eluent to give 7-(1-ethoxyvinyl)-3-fluoro-2-naphthonitrile. LRMS [M+H] + 242.

[0145] Step 2: 7-(1-ethoxyvinyl)-3-fluoro-2-naphthonitrile (154 mg, 0.637 mmol) and a 10% v / v aqueous solution of TFA (2 mL) were added to a 20 mL vial and stirred for 10 min. The solvent was evaporated to give 7-acetyl-3-fluoro-2-naphthonitrile.

[0146] Step 3: To a 20 mL vial was added tert-butyl 2-(diethoxyphosphoryl)acetate (0.152 mL, 0.679 mmol) and THF (3 mL). The mixture was stirred under nitrogen. To the mixture was added 1M LHMDS in THF (0.679 mL, 0.679 mmol) dropwise over 5 min. To this mixture was added a THF solution of 7-acetyl-3-fluoro-2-naphthonitrile (145 mg, 0.679 mmol) dropwise over 5 min. After 5 min, the vial was sealed and then stirred and heated at 70° C. for 14 h. The mixture was adsorbed onto Celite and purified by silica gel chromatography using 0-30% EtOAc in heptane as eluent to give tert-butyl (E)-3-(7-cyano-6-fluoronaphthalen-2-yl)but-2-enoate. LRMS [M+H+MeCN] + 353.

[0147] Step 4: To a 20 mL vial was added tert-butyl (E)-3-(7-cyano-6-fluoronaphthalen-2-yl)but-2-enoate (40.5 mg, 0.130 mmol), NaN3 (9.3 mg, 0.14 mmol), and DMSO (1 mL). The mixture was stirred and heated at 100 °C for 2 h. The mixture was diluted in 1:2 EtOAc / Et2O (12 mL) and filtered through Celite, rinsing with Et2O. The filtrate was washed with water (3 x 10 mL). The organic layer was dried over sodium sulfate, filtered, and the filtrate's solvent was evaporated. The mixture was purified by silica gel chromatography using 0-30% EtOAc in heptane as eluent to give tert-butyl (E)-3-(6-azido-7-cyanonaphthalen-2-yl)but-2-enoate. LRMS [M+H-H2O] + 307.

[0148] Step 5: To a 20 mL vial was added tert-butyl (E)-3-(6-azido-7-cyanonaphthalen-2-yl)but-2-enoate (13.5 mg, 0.0404 mmol) and formic acid (1 mL). The mixture was stirred and heated at 40° C. for 15 min. The solvent was evaporated to give A-7, (E)-3-(6-azido-7-cyanonaphthalen-2-yl)but-2-enoic acid. LRMS [MH] - 277.

[0149] Synthesis of A-8: [ka]

[0150] Step 1: To a solution of ethyl 2-(diethoxyphosphoryl)acetate (0.60 mL, 3.0 mmol) was added a 1M solution of THF (3.3 mL, 3.3 mmol) in LHMDS dropwise over 10 min. The mixture was stirred at room temperature for 30 min, after which 4-bromo-2-methoxybenzaldehyde (645 mg, 3.00 mmol) was added in one portion. The reaction was then stirred at room temperature for 16 h and quenched by the addition of saturated aqueous NH4Cl (20 mL). The quenched reaction was extracted with EtOAc (30 x 3 mL). The combined organic layers were dried over Na2SO4 and concentrated in vacuo to give the crude product. Ethyl (E)-3-(4-bromo-3-methoxyphenyl)acrylate was isolated using silica gel chromatography. LRMS [M+H] + 285.

[0151] Step 2: Ethyl (E)-3-(4-bromo-3-methoxyphenyl)acrylate (285 mg, 1.00 mmol), CuI (9.5 mg, 0.050 mmol), and sodium ascorbate (20 mg, 0.10 mmol) were charged to a N2 purged vial. To the mixture was added DMSO (5 mL) and DMEDA (17 μL, 0.15 mmol). The mixture was then stirred at room temperature for 15 min before NaN3 (98 mg, 1.5 mmol) was added. The reaction was stirred at 100 °C for 16 h. The reaction was then stirred at room temperature for 16 h and quenched by the addition of saturated aqueous NH4Cl (20 mL). The quenched reaction was extracted with EtOAc (50 × 3 mL). The combined organic layers were dried over Na2SO4 and concentrated in vacuo to give the crude product. Ethyl (E)-3-(4-azido-2-methoxyphenyl)acrylate was isolated using silica gel chromatography. LRMS [M+H] + 248.

[0152] Step 3: To a solution of ethyl (E)-3-(4-azido-2-methoxyphenyl)acrylate (170 mg, 0.690 mmol) in THF (4 mL) was added LiOH (32.0 mg, 1.38 mmol) predissolved in HO (2 mL). The reaction mixture was stirred at room temperature for 3 h. The reaction was concentrated under reduced pressure and diluted with HO (20 mL). The pH of the aqueous suspension was adjusted to 4 and extracted with EtOAc (20×3 mL). The combined organic layers were washed with HO (30 mL) and brine (30 mL), dried over NaSO, and concentrated under reduced pressure to give the crude product. A-8 was isolated using silica gel chromatography. LRMS [MH] - 218.

[0153] Synthesis of A-9 [ka]

[0154] Similar to the synthesis of A-8, 4-bromo-3-methoxybenzaldehyde was converted to A-9 in three steps. LRMS [MH] - 218.

[0155] Synthesis of A-10 [ka]

[0156] Similar to the synthesis of A-8, 1-(4-bromophenyl)ethan-1-one was converted to A-10 in three steps. LRMS [MH] - 202.

[0157] Synthesis of A-11 [ka]

[0158] Step 1: To a solution of ethyl 2-(diethoxyphosphoryl)acetate (0.32 mL, 1.7 mmol) was added a 1M solution of THF (1.7 mL, 1.7 mmol) in LHMDS dropwise over 10 min. The mixture was stirred at room temperature for 30 min, after which 3,5-difluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde (402 mg, 1.50 mmol) was added in one portion. The reaction was then stirred at room temperature for 16 h and quenched by the addition of saturated aqueous NH4Cl (20 mL). The quenched reaction was extracted with EtOAc (30 x 3 mL). The combined organic layers were dried over Na2SO4 and concentrated in vacuo to give the crude product. Ethyl (E)-3-(3,5-difluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acrylate was isolated using silica gel chromatography. LRMS [M+H] + 339.

[0159] Step 2: To a solution of ethyl (E)-3-(3,5-difluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acrylate (73 mg, 0.22 mmol) in MeOH (4 mL) was added Cu(OAc)2 (4.0 mg, 0.022 mmol) and NaN3 (14 mg, 0.22 mmol). The reaction was heated at 60 °C for 30 min. The reaction was diluted with EtOAc (50 mL) and then quenched by the addition of saturated aqueous NH4Cl (10 mL). The aqueous layer was extracted with EtOAc (10 x 3 mL). The combined organic layers were washed with H2O (30 mL) and brine (30 mL), dried over Na2SO4, and concentrated in vacuo. The desired product was purified by silica gel chromatography to give ethyl (E)-3-(4-azido-3,5-difluorophenyl)acrylate. LRMS [M+H] + 254.

[0160] Step 3: To a solution of ethyl (E)-3-(4-azido-3,5-difluorophenyl)acrylate (30 mg, 0.12 mmol) in THF (4 mL) was added LiOH (80 mg, 2.0 mmol) predissolved in HO (2 mL). The reaction mixture was stirred at room temperature for 3 h. The reaction was concentrated under reduced pressure and diluted with HO (20 mL). The pH of the aqueous suspension was adjusted to 4 and extracted with EtOAc (3×20 mL). The combined organic layers were washed with HO (30 mL) and brine (30 mL), dried over NaSO, and concentrated under reduced pressure to give the crude product. A-11 was isolated using silica gel chromatography. LRMS [MH] - 224.

[0161] Synthesis of A-12 [ka]

[0162] Analogous to the synthesis of A-11, 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde was converted to A-12 in three steps. LRMS [MH] - 206.

[0163] Synthesis of A-13 [ka]

[0164] Step 1: To a solution of ethyl 2-(diethoxyphosphoryl)acetate (0.57 mL, 2.9 mmol) was added a 1M solution of THF (3.1 mL, 3.1 mmol) in LHMDS dropwise over 10 min. The mixture was stirred at room temperature for 30 min, after which 2-fluoro-5-formylbenzonitrile (425 mg, 2.85 mmol) was added in one portion. The reaction was then stirred at room temperature for 16 h and quenched by the addition of saturated aqueous NH4Cl (20 mL). The quenched reaction was extracted with EtOAc (30 x 3 mL). The combined organic layers were dried over Na2SO4 and concentrated in vacuo to give the crude product. Ethyl (E)-3-(3-cyano-4-fluorophenyl)acrylate was isolated using silica gel chromatography. LRMS [M+H] + 220.

[0165] Step 2: To a solution of ethyl (E)-3-(3-cyano-4-fluorophenyl)acrylate (149 mg, 0.68 mmol) in DMF (3 mL) was added NaN3 (66 mg, 1.0 mmol) in one portion. The mixture was heated at 70 °C overnight. The reaction was cooled, diluted with EtOAc (50 mL) and poured onto crushed ice. After partitioning in a separatory funnel, the aqueous layer was extracted with EtOAc (3 x 20 mL). The combined organic layers were washed with H2O (50 mL) and brine (50 mL), dried over Na2SO4 and concentrated in vacuo. Ethyl (E)-3-(4-azido-3-cyanophenyl)acrylate was isolated using silica gel chromatography. LRMS [M+H] + 243.

[0166] Step 3: To a solution of ethyl (E)-3-(4-azido-3-cyanophenyl)acrylate (25 mg, 0.10 mmol) in THF (4 mL) was added LiOH (40 mg, 1.0 mmol) predissolved in HO (2 mL). The reaction mixture was stirred at room temperature for 3 h. The reaction was concentrated under reduced pressure and diluted with HO (20 mL). The pH of the aqueous suspension was adjusted to 4 and extracted with EtOAc (3×20 mL). The combined organic layers were washed with HO (30 mL) and brine (30 mL), dried over NaSO, and concentrated under reduced pressure to give the crude product. A-13 was isolated using silica gel chromatography. LRMS [MH] - 213.

[0167] Synthesis of A-14 [ka]

[0168] Similar to the synthesis of A-13, 5-acetyl-2-fluorobenzonitrile was converted to A-14 in three steps. LRMS [MH] - 227.

[0169] Synthesis of vinyl-extended aryl-azido-biotins Synthesis of compound 9575 [ka]

[0170] To a 20 mL vial was added A-1, biotin-PEG-4-amine, DMF (1 mL), and DIPEA (0.161 mL, 0.920 mmol). To this mixture was added HATU (69.9 mg, 0.184 mmol). This mixture was purified by RP HPLC (MeCN / water w / 0.1% TFA) to give compound 9575. LRMS [M+H] + 684. 1H NMR (400 MHz, DMSO-d6) δ 8.22 (t, J = 5.7 Hz, 1H), 8.07 (s, 1H), 8.00 (d, J = 8.8 Hz, 1H), 7.94 (d, J = 8.7 Hz, 1H), 7.83 (t, J = 5.7 Hz, 1H), 7.74 (dd, J = 8.7, 1.7 Hz, 1H), 7.70 (d, J = 2.2 Hz, 1H), 7.57 (d, J = 15.7 Hz, 1H), 7.31 (dd, J = 8.7, 2.3 Hz, 1H), 6.79 (d, J = 15.7 Hz, 1H), 6.42 (s, 1H), 6.36 (s, 1H), 4.30 (dd, J = 7.8, 5.0 Hz, 1H), 4.12 (dd, J = 7.8, 4.4 Hz, 1H), 3.56 - 3.45 (m, 16H), 3.40 - 3.35 (m, 4H), 3.12 - 3.03 (m, 1H), 2.81 (dd, J = 12.5, 5.1 Hz, 1H), 2.57 (d, J = 12.4 Hz, 1H), 2.06 (t, J = 7.4 Hz, 2H), 1.67 - 1.40 (m, 4H), 1.37 - 1.22 (m, 3H).

[0171] The compounds in the following table were synthesized from the corresponding aryl azide intermediate (or commercially available aryl azide) and biotin-PEG4-amine in a manner similar to the synthesis of 9575. [Table 1]

[0172] Example 4 Synthesis of Ir catalyst [ka]

[0173] Intermediate 2: bi-Ir-Cl complex 1 (0.1 mmol, 1.0 equiv.) was combined with AgOTf (53 mg, 0.2 mmol, 2.0 equiv.) in CH3CN (5 mL). This mixture was stirred at room temperature overnight in the dark. The resulting suspension was then filtered through Celite and concentrated. The residue was redissolved in DCM / MeOH (1 / 1, 10 mL), filtered through Celite, and concentrated to give intermediate 2 as a yellow film, which was used without further purification.

[0174] Intermediate 4: To a solution of 3 (94 mg, 0.5 mmol, 1.0 equiv) in DMF (5 mL) was added NaI (7.5 mg, 0.05 mmol, 0.1 equiv), 1-chloro-2-(2-(2-methoxyethoxy)ethoxy)ethane (110 mg, 0.6 mmol, 1.2 equiv), and K2CO3 (207 mg, 1.5 mmol, 3.0 equiv). The suspension was heated at 60 °C overnight. After cooling, the mixture was diluted with EtOAc (50 mL), filtered through Celite, and the filtrate was concentrated under reduced pressure to give the crude product. The desired product was isolated by silica gel chromatography. 1 H NMR (400 MHz, methanol-d4) δ 8.63 (d, J = 5.9 Hz, 1H), 8.46 (dd, J = 6.7, 2.1 Hz, 1H), 7.90 (s, 1H), 7.85 (s, 1H), 7.32 - 7.23 (m, 1H), 7.20 (d, J = 6.7 Hz, 1H), 4.43 (t, J = 4.6 Hz, 2H), 3.93 (p, J = 2.2 Hz, 2H), 3.76 - 3.47 (m, 8H), 3.35 (s, 3H).LRMS [M+H] + 335.4.

[0175] To a solution of intermediate 5:4 (25 mg, 75 μmol, 1.0 equiv) in DMF (5 mL) was added bromoethanol (47 mg, 374 μmol, 5.0 equiv), NaI (1.2 mg, 7.5 μmol, 0.1 equiv), and K2CO3 (31 mg, 224 μmol, 3.0 equiv). The mixture was stirred at 60 °C overnight. After cooling, the mixture was diluted with EtOAc (50 mL), filtered through Celite, and the filtrate was concentrated under reduced pressure to give the crude product. The desired product was isolated by silica gel chromatography. 1 H NMR (400 MHz, chloroform-d) δ 8.66 - 8.44 (m, 2H), 8.13 - 7.75 (m, 2H), 7.00 - 6.85 (m, 2H), 6.79 (brs, 1H), 4.37 - 4.20 (m, 4H), 4.04 - 3.58 (m, 12H), 3.34 (s, 3H).LRMS [M+H] + 379.4.

[0176] To a solution of intermediate 6:5 (16 mg, 0.04 mmol, 1.0 equiv) in DMF (4 mL) was added pyridine (0.5 mL) and p-nitrophenyl chloroformate (10 mg, 0.05 mmol, 1.2 equiv). The solution was stirred at room temperature overnight. The reaction was diluted with DCM (10 mL), filtered through Celite, and the filtrate was concentrated under reduced pressure to give the crude product, which was used in the next step without further purification.

[0177] To a solution of the crude product from the previous step in ACN (2 mL) was added chloroalkaneamine reactant (39 mg, 150 μmol, 3 equiv.) and NEt3 (0.2 mL). The solution was stirred at room temperature overnight and concentrated onto Celite. The desired product was isolated using silica gel chromatography. 1H NMR (400 MHz, methanol-d4) δ 8.48- 8.46 (m, 2H), 7.95 - 7.74 (m, 2H), 7.17 - 6.92 (m, 2H), 4.47 - 4.25 (m, 6H), 3.95 - 3.42 (m, 20H), 3.35 (s, 3H), 3.29 - 3.17 (m, 2H), 1.77 - 1.50 (m, 2H), 1.63 - 1.50 (m, 2H), 1.47 - 1.21 (m, 4H).LRMS [M+H] + 628.3.

[0178] Ir-9049: Intermediate 2 (14 mg, 15 μmol, 1.0 equiv.) and 6 (9.4 mg, 15 μmol, 1.0 equiv.) were dissolved in DCM / MeOH (1 / 1, 2 mL). The solution was stirred at room temperature overnight. The desired product was isolated by silica column using DCM / MeOH as eluent. 1 H NMR (400 MHz, methanol-d4) δ 8.58 (d, J = 8.9 Hz, 2H), 8.39 (s, 2H), 8.33 (d, J = 8.9 Hz, 2H), 7.84 (d, J = 6.9 Hz, 4H), 7.27 (s, 2H), 6.81 (t, J = 10.9 Hz, 2H), 5.79 (d, J = 8.0 Hz, 2H), 4.47 - 4.25 (m, 4H), 3.92- 3.78 (m, 2H), 3.75 - 3.58 (m, 20H), 3.32 (s, 3H), 1.78 - 1.70 (m, 2H), 1.63 - 1.50 (m, 2H), 1.47 - 1.37 (m, 4H).LRMS [M+H] + 1336.7.

Claims

1. Compounds of formula (I): 【Chemistry 1】 or its salt [In the formula: A is, 【Chemistry 2】 is selected from During the ceremony, each n is independently 1, 2, 3, or 4; Each R is independently hydrogen, halo, C 1 -C 4 Alkyl, C 2 -C 4 Alkenyl, hydroxy, mercapto, amino, cyano, C 1 -C 4 -Alkoxy, halo-C 1 -C 4 -Alkyl, hydroxy-C 1 -C 4 -Alkyl, amino-C 1 -C 4 -Alkyl, mercapto-C 1 -C 4 -Alkyl, cyano-C 1 -C 4 -alkyl, -C(O)-C 1 -C 4 -alkyl, -C(O)OH, and -C(O)NH 2 is selected from R' is hydrogen or C 1 -C 4 is alkyl, L is a linker, Y is a functional moiety.

2. The compound of claim 1, wherein A is: 【Chemistry 3】 [In the formula, R 1 , R 2 , R 3 and R 4 are each independently hydrogen, halo, hydroxy, cyano, and C 1 -C 4 alkoxy.

3. R 1 is hydrogen, hydroxy, or C 1 -C 4 is an alkoxy; R 2 is hydrogen, halo, cyano, or C 1 -C 4 is an alkoxy; R 3 is hydrogen or halo, R 4 is hydrogen or halo; The compound according to claim 2, or a salt thereof.

4. The compound of claim 1, wherein A is: 【Chemistry 4】 wherein each R is independently hydrogen, halo, cyano, or C 1 -C 4 alkoxy.

5. The compound of claim 1, wherein A is: 【Chemistry 5】 [In the formula, R is hydrogen, halo, cyano, and C 1 -C 4 alkoxy.

6. The compound of claim 1, wherein A is: 【Chemistry 6】 [In the formula, R is hydrogen, halo, cyano, and C 1 -C 4 alkoxy.

7. 2. The compound of claim 1, wherein A has a formula selected from the following: 【Chemistry 7】

8. The compound or salt thereof according to any one of claims 1 to 7, wherein R' is selected from hydrogen and methyl.

9. 9. The compound or salt thereof according to any one of claims 1 to 8, wherein the linker comprises one or more moieties selected from a straight or branched chain alkylene, an ether (-O-), an amine (-NH-), an ester (-C(O)O-), an amide (-C(O)NH-), a carbamate (-NHC(O)O-), a urea (-NHC(O)NH-), and a phenylene group.

10. The compound of any one of claims 1 to 9, wherein the linker has the formula: -NHCH 2 CH 2 (OCH) 2 CH 2 ) n H [In the formula, n is 1, 2, 3, 4, 5, 6, 7, or 8].

11. 11. The compound or salt thereof of any one of claims 1 to 10, wherein Y is a functional moiety selected from a capture element, a detectable moiety, a reactive moiety, and a bifunctional moiety.

12. 12. The compound or salt thereof according to any one of claims 1 to 11, wherein Y is a capture element selected from biotin and a haloalkane group.

13. The compound according to any one of claims 1 to 12, wherein Y has the formula: 【Chemistry 8】

14. The compound according to any one of claims 1 to 12, wherein Y has the formula: -(CH 2 ) n -X wherein n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and X is a halogen.

15. 12. The compound or salt thereof according to any one of claims 1 to 11, wherein Y is a detectable moiety.

16. The compound or salt thereof according to claim 15 , wherein Y is a fluorescent functional group.

17. 17. The compound or salt thereof according to claim 16, wherein Y is a fluorescent functional group selected from xanthene, cyanine, naphthalene, oxadiazole, pyrene, oxazine, acridine, arylmethine, tetrapyrrole, coumarin, squaraine, and boron-dipyrromethene.

18. 16. The compound or salt thereof according to claim 15, wherein Y is a fluorogenic functional group.

19. The compound or salt thereof according to any one of claims 1 to 11, wherein Y is a reactive functional group.

20. 20. The compound of claim 19, or a salt thereof, wherein Y is a reactive functional group that includes an azide, an alkyne, an alkene, or a 1,2,4,5-tetrazinyl moiety.

21. 12. The compound of any one of claims 1 to 11, or a salt thereof, wherein Y is a bifunctional moiety comprising (i) a biologically active compound and (ii) a capture element or a fluorescent moiety or a reactive moiety.

22. The compound of claim 1 selected from the group consisting of: 【Chemistry 9】 and their salts.

23. 1. A system for photocatalytic labeling of biomolecules, comprising: (a) a compound according to any one of claims 1 to 22; (b) a photocatalyst; and The system comprising:

24. 24. The system of claim 23, wherein the photocatalyst has the following structure: 【Chemistry 10】 [In the formula, Each series of dashed lines 【Chemistry 11】 represents the presence or absence of a fused 6-membered ring, M is a transition metal; m1, m2, m3, n1, n2, n3, p1, p2, and p3 are each independently 0, 1, or 2; R 1a , R 1b , R 1c , R 2a , R 2b , R 2c , R 3a , R 3b , and R 3c are each independently selected from halo, alkyl, haloalkyl, amino, and heteroalkyl; X 1a , X 1b , X 2a , X 2b , X 3a , and X 3b are each independently selected from N and C, 1a and X 1b At least one of X is N; 2a and X 2b At least one of X is N; 3a and X 3b at least one of is N; X 1c , X 1d , X 2c , X 2d , X 3c , and X 3d are each independently selected from CH and N; A is an anion, q is 0, 1, or 2.

25. 25. The system of claim 24, wherein the transition metal is selected from Ru and Ir.

26. 24. The system of claim 23, wherein the photocatalyst is an iridium photocatalyst selected from: 【Chemistry 12】

27. 24. The system of claim 23, wherein the photocatalyst is a ruthenium-based photocatalyst selected from: 【Chemistry 13】

28. 25. The system of claim 24, wherein the photocatalyst has the formula: 【Chemistry 14】

29. 1. A method for labeling a biomolecule in a sample, comprising the steps of: (a) contacting said sample with a compound according to any one of claims 1 to 22; (b) exposing the sample to light; The method comprising:

30. 30. The method of claim 29, wherein the light is selected from ultraviolet and visible light.

31. 30. The method of claim 29, wherein the light is visible light from a light emitting diode.

32. 30. The method of claim 29, wherein the light is bioluminescent light.

33. 30. The method of claim 29, further comprising contacting the sample with a photocatalyst in step (a).

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