5,5-Disubstituted luciferins and their use in luciferase-based assays

Abstract

Luciferin analogs that have improved thermostability while retaining light-generating activity in luciferase assays and in vitro methods for detecting luminescence in samples are provided. [Solution] An in vitro method for detecting luminescence in a sample includes a step of contacting the sample with a compound selected from the group consisting of, for example, 5,5-diethyl-2-(6-hydroxybenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid; (S)-2-(5-fluoro-6-hydroxybenzo[d]thiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid, or a tautomer or salt thereof.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority to U.S. Provisional Application No. 62 / 428,707, filed on December 1, 2016, the entire content of which is incorporated herein by reference.

[0002] Technical Field The present disclosure relates to luciferin analogs, methods of making luciferin analogs, and methods of using luciferin analogs in luciferase - based assays.

Background Art

[0003] Bioluminescent assays are widely used in the study of cell physiology, particularly in processes related to gene expression. In particular, luciferase reporter enzymes are very valuable tools in the art, and to date, protein engineering has been extensively used to obtain small and environmentally insensitive luciferases that can be useful in bioluminescent assays. There are multiple efficient luciferase reporters that enable whole - cell biosensor measurements, drug discovery through high - throughput screening, and in vivo imaging. This in vivo imaging also enables the study of protein - protein interactions in live cells, apoptosis, and cell viability. Luciferases that use luciferin and luciferin analogs as substrates are the most widely used systems due to their brightness and acceptability in whole - cell applications. For example, firefly luciferase and various beetle luciferases produce luminescence in the presence of luciferin, magnesium ions, oxygen, and ATP.

Summary of the Invention

[0004] In one aspect, 5,5-disubstituted luciferin or a luciferin analog is disclosed. Also disclosed are methods for making the compound, kits containing the compound, and methods for using the compound as a luciferase substrate in a luciferase-based assay. Also disclosed is a detection system comprising one or more luciferases and one or more 5,5-disubstituted luciferin analogs.

Brief Description of the Drawings

[0005]

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DETAILED DESCRIPTION OF THE INVENTION

[0006] D-Luciferin (Figure 1) is a natural substrate for firefly luciferase and Gaussia luciferase and can be used as a substrate in bioluminescence assays using luciferase reporter enzymes. However, D-luciferin is thermally unstable and decomposes over time in a stock solution at ambient temperature. Dehydroluciferin was identified as a degradation product of luciferin. This dehydroluciferin is a potent inhibitor of luciferase and may cause a decrease in light output in luciferase-based bioluminescence assays. There is a need for luciferin analogs with improved thermal stability while retaining light-generating activity in luciferase assays.

[0007] In one aspect, 5,5-disubstituted luciferin analogs are disclosed. The compounds of the present disclosure can exhibit unexpected thermal stability and can provide a luciferase detection system that is free of inhibition due to luciferin degradation products. The compounds of the present disclosure can be particularly useful for applications where it is necessary to store luciferin in solution at ambient temperature for extended periods of time. The compounds of the present disclosure can also provide a long duration in many live cell bioluminescence assays or live cell imaging methods.

[0008] The compounds of the present disclosure are thought to be substrates for luciferase enzymes (“luciferin-utilizing enzymes”) that utilize luciferin to generate light, and such enzymes include, but are not limited to, luciferases and luminescent proteins found in various organisms such as beetles (e.g., Photinus pyralis and Photuris pennsylvanica (North American fireflies)), Pyrophorus plagiophthalamus (Jamaican click beetle), Renilla reniformis (sea pansy), and several bacteria (Xenorhabdus luminescens and Vibrio spp).

[0009] 5,5-Disubstituted luciferins have historically been thought to be unable to generate light via a firefly luciferase-mediated process. In particular, 5,5-disubstituted luciferins are generally thought to generate light only via a chemiluminescent process rather than via an enzymatic process (e.g., by firefly luciferase). Surprisingly, the compounds of the present disclosure can be utilized by firefly luciferase and click beetle luciferase to generate light via an enzymatic process. Furthermore, luciferases can effectively utilize various stereoisomeric forms and mixtures of 5,5-disubstituted luciferin analogs (e.g., L-form and D-form) to generate bioluminescence and can function over a wide pH range. This enables the use of racemates in applications where racemization could potentially compromise signal stability.

[0010] 1. Definitions 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. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein can also be used in the practice and testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0011] As used herein, the terms "comprise," "include," "having," "has," "can," "contain," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. Unless the context clearly dictates otherwise, the singular forms "a," "an," and "the" include plural referents. Also, the present disclosure contemplates other embodiments that "comprise" or "consist essentially of" the embodiments or elements presented herein, whether or not explicitly shown.

[0012] The modifier "about" used in connection with a quantity includes the recited value and has the meaning determined by the context (e.g., "about" includes at least the degree of error associated with the measurement of a particular quantity). Also, the modifier "about" should be considered as disclosing a range defined by the absolute values of two endpoints. For example, the expression "about 2 to about 4" also discloses the range "2 to 4". The term "about" can refer to plus or minus 10% of the indicated number. For example, "about 10%" can indicate the range of 9% to 11%, and "about 1" can mean 0.9 to 1.1. Other meanings of "about" may become apparent from the context, such as rounding, and thus, for example, "about 1" may also mean 0.5 to 1.4.

[0013] The definitions of specific functional groups and chemical terms are described in more detail below. For the purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Ed. (front and back covers), and specific functional groups are generally defined as described in that reference. Further, general principles of organic chemistry, and specific functional moieties and reactivities are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March’s Advanced Organic Chemistry, 5 th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3 rdIt is described in Edition, Cambridge University Press, Cambridge, 1987, and the entire content of each of these is incorporated herein by reference.

[0014] As used herein, the term "alkoxy" refers to an alkyl group as defined herein that is attached 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.

[0015] As used herein, the term "alkyl" means a straight-chain or branched saturated hydrocarbon chain containing 1 to 10 carbon atoms. The term "lower alkyl" or "C1-C6 alkyl" means a straight-chain or branched-chain hydrocarbon containing 1 to 6 carbon atoms. The term "C1-C3 alkyl" means a straight-chain or branched-chain hydrocarbon containing 1 to 3 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

[0016] As used herein, the term "alkenyl" means a hydrocarbon chain containing 2 to 10 carbon atoms with at least one carbon-carbon double bond. The alkenyl group may or may not be substituted. For example, the alkenyl group may be substituted with an aryl group such as phenyl.

[0017] As used herein, the term "alkynyl" means a hydrocarbon chain containing 2 to 10 carbon atoms with at least one carbon-carbon triple bond. The alkynyl group may or may not be substituted. For example, the alkynyl group may be substituted with an aryl group such as phenyl.

[0018] As used herein, the term "alkoxyalkyl" refers to an alkoxy group as defined herein that is attached to the parent molecular moiety via an alkyl group as defined herein.

[0019] As used herein, the term "alkylene" refers to a divalent group derived from a straight-chain or branched-chain hydrocarbon of 1 to 10 carbon atoms, for example 2 to 5 carbon atoms. Representative examples of alkylene include, but are not limited to, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, and -CH2CH2CH2CH2CH2-.

[0020] The term "amino acid" refers to both natural and non-natural amino acids. It also includes protected natural and non-natural amino acids.

[0021] As used herein, the term "aryl" refers to a phenyl group, or a condensed ring system of bicyclic aryl or tricyclic aryl. Suitable examples of bicyclic condensed ring systems are phenyl groups attached to the parent molecular moiety and condensed to a phenyl group. Suitable examples of tricyclic condensed ring systems are phenyl groups attached to the parent molecular moiety and condensed to two other phenyl groups. Representative examples of bicyclic aryl include, but are not limited to, naphthyl. Representative examples of tricyclic aryl include, but are not limited to, anthracenyl. Monocyclic, bicyclic, and tricyclic aryl are connected to the parent molecular moiety via any carbon atom contained in the ring and may or may not be substituted.

[0022] As used herein, the term "cycloalkyl" refers to a carbocyclic ring system containing 3 to 10 carbon atoms, 0 heteroatoms, and zero double bonds. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, and cyclodecyl. Further, "cycloalkyl" also includes a carbocyclic ring system in which a cycloalkyl group is attached to the parent molecular moiety and is fused to an aryl group as defined herein, a heteroaryl group as defined herein, or a heterocycle as defined herein.

[0023] As used herein, the term "cycloalkenyl" means a non-aromatic monocyclic or polycyclic ring system containing at least one carbon-carbon double bond and preferably having 5 to 10 carbon atoms per ring. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, or cycloheptenyl.

[0024] As used herein, the term "fluoroalkyl" means an alkyl group as defined herein in which 1, 2, 3, 4, 5, 6, 7, or 8 hydrogen atoms are replaced by fluorine. Representative examples of fluoroalkyl include, but are not limited to, 2-fluoroethyl, 2,2,2-trifluoroethyl, trifluoromethyl, difluoromethyl, pentafluoroethyl, and trifluoropropyl such as 3,3,3-trifluoropropyl.

[0025] As used herein, the term "alkoxyfluoroalkyl" refers to an alkoxy group as defined herein that is attached to the parent molecular moiety via a fluoroalkyl group.

[0026] As used herein, the term "fluoroalkoxy" means that at least one fluoroalkyl group as defined herein is attached to the parent molecular moiety through an oxygen atom. Representative examples of fluoroalkyloxy include, but are not limited to, difluoromethoxy, trifluoromethoxy, and 2,2,2-trifluoroethoxy.

[0027] As used herein, the term "halogen" or "halo" means Cl, Br, I, or F.

[0028] As used herein, the term "haloalkyl" means an alkyl group as defined herein in which one, two, three, four, five, six, seven, or eight hydrogen atoms are replaced by halogen.

[0029] As used herein, the term "haloalkoxy" means that at least one haloalkyl group as defined herein is attached to the parent molecular moiety through an oxygen atom.

[0030] As used herein, the term "heteroalkyl" means an alkyl group as defined herein in which one or more of the carbon atoms are replaced by a heteroatom selected from S, Si, O, P, and N. This heteroatom may be oxidized. Representative examples of heteroalkyl include, but are not limited to, alkyl ether, secondary and tertiary alkylamine, amide, and alkyl sulfide.

[0031] As used herein, the term "heteroaryl" refers to an aromatic monocyclic ring, or an aromatic bicyclic ring system, or an aromatic tricyclic ring system. The aromatic monocyclic ring is a 5- or 6-membered ring containing at least 1 heteroatom independently selected from the group consisting of N, O, and S (e.g., 1, 2, 3, or 4 heteroatoms independently selected from O, S, and N). The 5-membered aromatic monocyclic ring has two double bonds, and the 6-membered aromatic monocyclic ring has three double bonds. Exemplary bicyclic heteroaryl groups are monocyclic heteroaryl rings that are attached to the parent molecular moiety and fused to a monocyclic cycloalkyl group as defined herein, a monocyclic aryl group as defined herein, a monocyclic heteroaryl group as defined herein, or a monocyclic heterocycle as defined herein. Exemplary tricyclic heteroaryl groups are monocyclic heteroaryl rings that are attached to the parent molecular moiety and fused to two of a monocyclic cycloalkyl group as defined herein, a monocyclic aryl group as defined herein, a monocyclic heteroaryl group as defined herein, or a monocyclic heterocycle as defined herein. Representative examples of monocyclic heteroaryl include, but are not limited to, pyridinyl (including pyridin-2-yl, pyridin-3-yl, pyridin-4-yl), pyrimidinyl, pyrazinyl, thienyl, furyl, thiazolyl, thiadiazolyl, isoxazolyl, pyrazolyl, and 2-oxo-1,2-dihydropyridinyl. Representative examples of bicyclic heteroaryl include, but are not limited to, chromenyl, benzothienyl, benzodioxolyl, benzotriazolyl, quinolinyl, thienopyrrolyl, thienothienyl, imidazothiazolyl, benzothiazolyl, benzofuranyl, indolyl, quinolinyl, imidazopyridine, benzoxadiazolyl, and benzopyrazolyl. Representative examples of tricyclic heteroaryl include, but are not limited to, dibenzofuranyl and dibenzothienyl. Monocyclic, bicyclic, and tricyclic heteroaryl are attached to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the ring and may or may not be substituted.

[0032] As used herein, the terms "heterocyclic ring" or "heterocyclic" mean a monocyclic heterocyclic ring, bicyclic heterocyclic ring, or tricyclic heterocyclic ring. A monocyclic heterocyclic ring is a 3-membered, 4-membered, 5-membered, 6-membered, 7-membered, or 8-membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S. A 3-membered or 4-membered ring contains zero or one double bond and one heteroatom selected from the group consisting of O, N, and S. A 5-membered ring contains zero or one double bond and one, two, or three heteroatoms selected from the group consisting of O, N, and S. A 6-membered ring contains zero, one, or two double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. A 7-membered and 8-membered ring contains zero, one, two, or three double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. Representative examples of monocyclic heterocyclic rings include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, 1,3-dimethylpyrimidine-2,4(1H,3H)-dione, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, oxetanyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, 1,2-thiadinanyl, 1,3-thiadinanyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and trithianyl.The bicyclic heterocyclic ring is a monocyclic heterocyclic ring fused to a phenyl group, or a monocyclic heterocyclic ring fused to a monocyclic cycloalkyl, or a monocyclic heterocyclic ring fused to a monocyclic cycloalkenyl, or a monocyclic heterocyclic ring fused to a monocyclic heterocyclic ring, or a spiro heterocyclic group, or a bridged monocyclic heterocyclic ring system in which two non-adjacent atoms in the ring are connected by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or by an alkenylene bridge of 2, 3, or 4 carbon atoms. Representative examples of the bicyclic heterocyclic ring include, but are not limited to, benzopyranyl, benzothiopyranyl, chromanyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothienyl, 2,3-dihydroisoquinoline, 2-azaspiro[3.3]heptan-2-yl, azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hept-2-yl), 2,3-dihydro-1H-indolyl, isoindolinyl, octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl, and tetrahydroisoquinolinyl. Suitable examples of the tricyclic heterocyclic ring are a bicyclic heterocyclic ring fused to a phenyl group, or a bicyclic heterocyclic ring fused to a monocyclic cycloalkyl, or a bicyclic heterocyclic ring fused to a monocyclic cycloalkenyl, or a bicyclic heterocyclic ring fused to a monocyclic heterocyclic ring, or a bicyclic heterocyclic ring in which two non-adjacent atoms in the bicyclic ring are connected by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or by an alkenylene bridge of 2, 3, or 4 carbon atoms. Examples of the tricyclic heterocyclic ring include, but are not limited to, octahydro-2,5-epoxypentalene, hexahydro-2H-2,5-methanocyclopenta[b]furan, hexahydro-1H-1,4-methanocyclopenta[c]furan, aza-adamantane (1-azatricyclo[3.3.1.1. 3.7 decane), and oxa-adamantane (2-oxatricyclo[3.3.1.1 3.7 decane). The monocyclic, bicyclic, and tricyclic heterocyclic rings are connected to the parent molecular moiety via any carbon atom or any nitrogen atom contained in the ring, and may or may not be substituted.

[0033] As used herein, the term "hydroxyl" means an -OH group.

[0034] As used herein, the term "hydroxyalkyl" means an alkyl group as defined herein, wherein 1, 2, 3, 4, 5, 6, 7, or 8 hydrogen atoms are replaced by hydroxyl groups.

[0035] In some cases, the number of carbon atoms in a hydrocarbyl substituent (e.g., alkyl or cycloalkyl) is indicated by the prefix "C x -C y -", where x is the minimum number of carbon atoms in the substituent and y is the maximum number of carbon atoms in the substituent. Thus, for example, "C1-C3-alkyl" refers to an alkyl substituent containing 1 to 3 carbon atoms.

[0036] The term "substituted" refers to a group that may be further substituted with one or more non-hydrogen substituents. Substituents include, but are not limited to, halogen, =O, =S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, and acyl.

[0037] For the compounds described in this specification, their groups and substituents can be selected according to acceptable valences and substituents, and this selection and substitution are made so as to result in stable compounds, for example, compounds that do not spontaneously undergo conversions such as rearrangement, cyclization, elimination, etc.

[0038] The "ATP detection mixture" contains materials that enable the detection of ATP in a sample. The necessary materials, as well as the specific concentrations and / or amounts of the materials required for the generation of the luminescence signal, will vary depending on the luciferase enzyme used and the type of luciferase-based assay being performed. Generally, in the case of beetle luciferase, such materials include magnesium (Mg 2+ ) salts, such as magnesium sulfate, luciferase enzymes, such as UltraGlo Luciferase (Promega Corporation), and luciferase substrates, such as luciferin, luciferin derivatives, functional analogs, or novel luciferin derivatives that are capable of generating light when used as substrates for beetle luciferase (e.g., the luciferin derivatives disclosed herein). Often, other materials will also be added to the solution, including: buffers to maintain the reaction at an appropriate pH, additives such as PRIONEX or bovine serum albumin (BSA) to assist in maintaining luciferase activity, reducing agents, detergents, esterases, salts, amino acids (e.g., D-cysteine), etc. An example of a luciferase detection mixture could be one that contains a luciferase enzyme, a luciferase substrate, MgSO4, Tergitol NP-9, and tricine.

[0039] The "luciferase detection mixture" contains materials that enable the detection of luciferase enzyme in a sample. The necessary materials, as well as the specific concentrations and / or amounts of the materials required for the generation of the luminescence signal, will vary depending on the luciferase enzyme used and the type of luciferase-based assay being performed. Generally, in the case of beetle luciferase, such materials include ATP, magnesium (Mg 2+)Salts, such as magnesium sulfate, and luciferase substrates, such as luciferin, luciferin derivatives, functional analogs, or luciferase substrates (e.g., luciferin derivatives disclosed herein) can include novel luciferin derivatives capable of generating light when used as substrates for beetle luciferase. Often, other materials will also be added to the solution, including: buffers to maintain the reaction at an appropriate pH, additives such as PRIONEX or bovine serum albumin (BSA) to assist in maintaining luciferase activity, reducing agents, detergents, esterases, salts, amino acids (e.g., D-cysteine), and the like. As an example of a luciferase detection mixture, one containing a luciferase substrate, MgSO4, ATP, Tergitol NP-9, and tricine can be considered.

[0040] A "luciferase reaction mixture" contains materials that enable the generation of a light signal by the luciferase enzyme. The materials required, as well as the specific concentrations and / or amounts of the materials required for the generation of the luminescent signal, will vary depending on the luciferase enzyme used and the type of luciferase-based assay being performed. Generally, in the case of beetle luciferase, such materials can include ATP, magnesium (Mg 2+ )Salts, such as magnesium sulfate, the beetle luciferase enzyme, and luciferin or a novel luciferin derivative capable of generating light when used as a substrate for beetle luciferase. Often, other materials will also be added to the solution, including: buffers to maintain the reaction at an appropriate pH, additives such as PRIONEX or bovine serum albumin (BSA) to assist in maintaining luciferase activity, reducing agents, detergents, esterases, salts, amino acids (e.g., D-cysteine), and the like. As an example of a luciferase reaction mixture, one containing beetle luciferase, MgSO4, ATP, Tergitol NP-9, and tricine can be considered.

[0041] Regarding the enumeration of numerical ranges in this specification, each number intervening between those ranges with the same precision is explicitly contemplated. For example, for the range of 6 to 9, in addition to 6 and 9, the numbers 7 and 8 are contemplated, and for the range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0042] 2. Compound Compound of formula (I’): TIFF0007696867000001.tif3974(I’) Or its tautomer or salt is disclosed. [Wherein, R1 is hydrogen, halogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 1a , -NR 1b R 1c , -OG 1 , -NR 1x G 1 , or -NR 1x G 10 , and R2 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 2a , -NR 2b R 2c , -SR 2d , -SO2R 2e , -S(O)R 2f , -P(O)OR 2g R 2h , -OG 1 , or -NR 2x G 1 , and R3 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 3a , -NR 3b R 3c , -SR 3d, -SO2R 3e , -S(O)R 3f , -P(O)OR 3g R 3h , -OG 1 , or -NR 1x G 1 and R4 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 4a , -NR 4b R 4c , -SR 4d , -SO2R 4e , -S(O)R 4f , -P(O)OR 4g R 4h , -OG 1 , or -NR 4x G 1 and R5 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 5a , -NR 5b R 5c , -SR 5d , -SO2R 5e , -S(O)R 5f , -P(O)OR 5g R 5h , -OG 1 , or -NR 5x G 1 and Alternatively, R2 and R3 together with the atoms to which they are attached, or R3 and R4 together with the atoms to which they are attached, or R4 and R5 together with the atoms to which they are attached, form a 5- or 6-membered saturated, partially unsaturated, or fully unsaturated ring, and the 5- or 6-membered ring optionally contains, as ring members, one, two, or three heteroatoms or heteroatom groups each independently selected from the group consisting of O, N, S, NO, SO, and SO2, the 5- or 6-membered ring is optionally fused to an aryl, heteroaryl, heterocyclic, or cycloalkyl group, the 5- or 6-membered ring is substituted with zero, one, two, three, or four substituents, and the substituents are each independently halogen, =O, =S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclic, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl, acyl, -OG 1 , -NHG 1 , and -N(C1-C 10 alkyl)G 1 selected from the group consisting of, R 1a R 1b R 1c R 2a R 2b R 2c R 2d R 2e R 2f R 2g R 2h R 3a R3b , and R 3c , and R 3d , and R 3e , and R 3f , and R 3g , and R 3h , and R 4a , and R 4b , and R 4c , and R 4d , and R 4e , and R 4f , and R 4g , and R 4h , and R 5a , and R 5b , and R 5c , and R 5d , and R 5e , and R 5f , and R 5g , and R 5h is independently selected from hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl, R 1x , R2 x , R 3x , R 4x , and R 5x are each independently hydrogen or C1-C 12 alkyl, G 1 is a substrate of the first enzyme, and the biotransformation of the substrate by the first enzyme converts G 1 to H and includes the substrate of the first enzyme, -NR 1x G 10 is a group cleavable by a second enzyme to convert the -NR 1x G 10 group to -OH, and W1 and W2 are each independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl, or W1 and W2 together with the carbon to which they are attached form a cycloalkyl, cycloalkenyl, or heterocycle, In each occurrence, the alkyl, alkenyl, alkynyl, alkoxy, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl are each independently substituted with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substituents, and each of the substituents is independently selected from the group consisting of halogen, =O, =S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl, and acyl]

[0043] In some embodiments, formula (I’) is formula (I): TIFF0007696867000002.tif3974(I) or a tautomer or salt thereof. [Wherein, R1 is hydrogen, halogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 1a , or -NR 1b R 1c , and R2 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 2a , -NR 2b R 2c , -SR 2d , -SO2R2e 、 -S(O)R 2f 、 or -P(O)OR 2g R 2h and R3 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 3a 、 -NR 3b R 3c 、 -SR 3d 、 -SO2R 3e 、 -S(O)R 3f 、 or -P(O)OR 3g R 3h and R4 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 4a 、 -NR 4b R 4c 、 -SR 4d 、 -SO2R 4e 、 -S(O)R 4f 、 or -P(O)OR 4g R 4h and R5 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 5a 、 -NR 5b R 5c 、 -SR 5d 、 -SO2R 5e 、 -S(O)R 5f 、 or -P(O)OR 5g R 5h and Alternatively, R2 and R3, together with the atoms to which they are attached, or R3 and R4, together with the atoms to which they are attached, or R4 and R5, together with the atoms to which they are attached, form a 5- or 6-membered saturated, partially unsaturated, or fully unsaturated ring, and the 5- or 6-membered ring optionally contains, as ring members, one, two, or three heteroatoms or heteroatom groups each independently selected from the group consisting of O, N, S, NO, SO, and SO2, the 5- or 6-membered ring is optionally fused to an aryl, heteroaryl, heterocyclic, or cycloalkyl group, the 5- or 6-membered ring is substituted with zero, one, two, three, or four substituents, and the substituents are each independently selected from the group consisting of halogen, =O, =S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclic, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl, and acyl. R 1a 、R 1b 、R 1c 、R 2a 、R 2b 、R 2c 、R 2d 、R 2e 、R 2f 、R 2g 、R 2h 、R 3a 、R 3b 、R 3c 、R 3d 、R 3e 、R 3f 、R3g , and R 3h , and R 4a , and R 4b , and R 4c , and R 4d , and R 4e , and R 4f , and R 4g , and R 4h , and R 5a , and R 5b , and R 5c , and R 5d , and R 5e , and R 5f , and R 5g , and R 5h is independently selected from hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl, and W1 and W2 are each independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl, or W1 and W2 together with the carbon to which they are attached form a cycloalkyl, cycloalkenyl, or heterocycle, In each occurrence, the alkyl, alkenyl, alkynyl, alkoxy, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl are each independently substituted with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substituents, and each of the substituents is independently selected from the group consisting of halogen, =O, =S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl, and acyl]

[0044] In certain embodiments, the compound of formula (I’) is not 2-(6-hydroxybenzo[d]thiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid nor 2-(6-aminobenzo[d]thiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid. In certain embodiments, the compound of formula (I) is not 2-(6-hydroxybenzo[d]thiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid nor 2-(6-aminobenzo[d]thiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid.

[0045] R2 and R3, together with the atoms to which they are attached, R3 and R4, together with the atoms to which they are attached, and R4 and R5, together with the atoms to which they are attached, may be present to form a fused or unfused ring system composed of 5- or 6-membered rings.

[0046] In certain embodiments, R1 is -OR 1a In certain embodiments, R1 is -OR 1a wherein R 1a is hydrogen or C1-C4-alkyl. In certain embodiments, R1 is -OH.

[0047] In certain embodiments, R2, R3, R4, and R5 are each independently selected from the group consisting of hydrogen, halogen, cyano, nitro, C1-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl, -OH, and -NH2.

[0048] In certain embodiments, R4 is -OR 4a or -NR 4b R 4c In certain embodiments, R4 is -OH. In certain embodiments, R4 is -NR 4b R 4c wherein R 4b and R 4c are hydrogen. In certain embodiments, R4 is -NR 4b R 4c wherein R 4b is hydrogen and R 4c is hydroxyalkyl.

[0049] In certain embodiments, R3 and R4, together with the atoms to which they are attached, form a 5- or 6-membered saturated, partially unsaturated, or fully unsaturated ring, optionally substituted. In certain embodiments, R3 and R4, together with the atoms to which they are attached, form a 5-membered heterocyclic ring, optionally substituted. In certain embodiments, R3 and R4, together with the atoms to which they are attached, form a 6-membered heterocyclic ring, optionally substituted.

[0050] In certain embodiments, R4 and R5, together with the atoms to which they are attached, form a 5- or 6-membered saturated, partially unsaturated, or fully unsaturated ring, optionally substituted. In certain embodiments, R4 and R5, together with the atoms to which they are attached, form a 5-membered heterocyclic ring, optionally substituted. In certain embodiments, R4 and R5, together with the atoms to which they are attached, form a 6-membered heterocyclic ring, optionally substituted. In certain embodiments, R4 and R5, together with the atoms to which they are attached, form an optionally substituted aryl ring.

[0051] In certain embodiments, R3 and R4, together with the atoms to which they are attached, form a 6-membered heterocyclic ring, optionally substituted, and R4 and R5, together with the atoms to which they are attached, form a 6-membered heterocyclic ring, optionally substituted, where the two 6-membered rings are fused.

[0052] In certain embodiments, W1 and W2 are each alkyl. In certain embodiments, W1 and W2 are each C1-C4-alkyl. In certain embodiments, W1 and W2 together with the atom to which they are attached form cycloalkyl. In certain embodiments, W1 and W2 together with the carbon to which they are attached form a 3- to 8-membered cycloalkyl. In certain embodiments, W1 and W2 together with the carbon to which they are attached form cycloalkenyl, such as 5- to 8-membered cycloalkenyl. In certain embodiments, W1 and W2 together with the atom to which they are attached form a heterocycle, such as a 5- to 8-membered heterocycle. The cycloalkyl, cycloalkenyl, or heterocyclic group formed by W1 and W2 together with the carbon to which they are attached may be unsubstituted or substituted with one or more substituents, each of which is independently selected from the group consisting of halogen, =O, =S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl, and acyl.

[0053] G 1 is a substrate of the first enzyme, and the in vivo conversion of the substrate by the first enzyme converts G 1 to H and includes a substrate of the first enzyme. In some embodiments, G 1 is G 2 -L1 - is (G 2 is an enzyme substrate, L 1 is G 2 is a linker that connects 2 to the remainder of the compound of formula (I') (i.e., the parent molecular moiety). G

[0054] In some embodiments, L 1 is a divalent group composed of a bond or an array of atoms stable under neutral ambient conditions, and the atoms are selected from carbon, hydrogen, nitrogen, oxygen, sulfur, phosphorus, and silicon. The divalent group can include a single bond (e.g., CH2-CH2, CH2-O), a double bond (e.g., C=O), or a triple bond (e.g., C≡C), and can also contain or include a ring structure (e.g., cycloalkyl). In some embodiments, the divalent group is -C 1-10 alkylene-, -C 2-10 alkylene-O-, C 3-8 cycloalkylene, -C(O)-, -O-, -S-, -S(O)-, -S(O)2-, -NH-, -N(C 1-4 alkyl)-, -N(COC 1-4 alkyl)-, an amino acid moiety, a protected amino acid moiety, and one or more sequences of phenylene, where C 3-8 cycloalkylene and phenylene are independently optionally substituted with 1 to 4 substituents independently selected from C 1-4 alkyl, C 1-4 haloalkyl, C 1-4 alkoxy, halo, cyano, or hydroxy. In some embodiments, L 1 is C1-C 10 alkylene (e.g., C2-C3 alkylene).

[0055] L 1 can be a linker between a luciferin analog and an enzyme substrate. In some embodiments, the linker L 1can be a traceless linker as described in U.S. Patent Application No. 62 / 541,350 (Encell et al., “COMPOSITIONS AND METHODS FOR STABILIZING BENZOTHIAZOLE LUCIFERIN ANALOGS” (filed Aug. 4, 2017)), such as trimethyloxonium, quinone methide, dipeptidyl, para-aminobenzyloxycarbonyl, or an alkylenediaminocarbonyl linker, shown below. JPEG0007696867000003.jpg65161

[0056] G 2 The enzymatic in vivo conversion of G 2 results in cleavage of the bond to the heteroatom to which G is attached, releasing a linker that can spontaneously self-sacrifice to release a benzothiazole luciferin analog. Some traceless linkers (e.g., alkylene linkers) can be spontaneously cleaved by β-elimination as described in WO2006 / 130551.

[0057] Enzyme substrate G 2 Representative examples of G include substrates for proteases, cytochrome (CYP) P450 reductase, monoamine oxidase (MAO), flavin monooxygenase (FMO), glutathione S-transferase (GST), dealkylase (e.g., demethylase), deacetylase, deformylase, sulfatase, phosphatase (e.g., alkaline phosphatase (AP)), beta-lactamase, and alcohol dehydrogenase, as described in WO2006 / 130551 or US2007 / 0015790 (which are hereby incorporated by reference in their entirety).

[0058] Representative protease substrates include, but are not limited to, peptides such as Z-DEVD-, Z-LETD-, GP-, Suc-LLVY-, Z-LRR-, Z-nLPnLD-, Z-QEVY-, VP-, Z-VDVAD-, Z-VEID-, Z-ATAD-, Z-IEPD-, Z-IETD-, Z-TSAVLQ-, and Z-VNSTLQ- as described by Cosby et al. in Cell Notes (2007) 18, pp. 9-11 (which is hereby incorporated by reference in its entirety). In the case of these protease substrates, the enzyme substrate is directly bound to the luciferin analog and is directly cleaved, so L 1 is a bond.

[0059] Other suitable linkers and G containing linkers 1 moieties include those described in U.S. Patent Application No. 62 / 541,350 (which is hereby incorporated by reference in its entirety).

[0060] In some embodiments, R1 is -NR 1x G 10 is. -NR 1x G 10 The group is a group cleavable by a second enzyme to convert the -NR 1x G 10 group to -OH. Therefore, after cleavage of the -NR 1x G 10 group, the -CO-R1 moiety becomes a carboxy group (-CO-OH). In some embodiments, the -NR 1x G 10 group is TIFF0007696867000004.tif3258, wherein R AA at each occurrence is independently hydrogen, alkyl, or alkyl substituted with a substituent selected from the group consisting of -OH, -NH2, -SH, -SCH3, phenyl, -COOH, -CO-NH2, TIFF0007696867000005.tif31164 and t is from 1 to 10. In some embodiments, t is 1, 2, 3, 4, or 5. In some embodiments, -NR1x G 10 The radical is TIFF0007696867000006.tif4840.

[0061] The second enzyme catalyzes the cleavage of the -NR 1x G 10 radical from the remainder of the compound, and this cleavage converts the -NR 1x G 10 radical to -OH. In some embodiments, the second enzyme is a protease. In some embodiments, the second enzyme is a carboxypeptidase (e.g., carboxypeptidase B).

[0062] In some embodiments, the -NR 1x G 10 radical is TIFF0007696867000007.tif4840, and carboxypeptidase B can cleave this from the remainder of the compound and convert the -NR 1x G 10 radical to -OH. Thus, after cleavage of the -NR 1x G 10 radical, the -CO-R1 moiety of the compound becomes a carboxy group (-CO-OH) as shown below. JPEG0007696867000008.jpg4696

[0063] In certain embodiments, the compound of formula (I) has the structure of formula TIFF0007696867000009.tif4182 (I-a) or a tautomer or salt thereof. [Wherein, A and B are each independently an optionally selected 5- or 6-membered ring selected from the group consisting of aryl, heteroaryl, and heterocycle, and A, when present, is optionally substituted with one or more R A 's, B, when present, is substituted with one or more R B 's, R A and R BEach of them, when present, is independently alkyl, halo, haloalkyl, hydroxyalkyl, -OH, -NH2, or alkyl-NH-, When both A and B are absent, Q is -OR Q1 or -NR Q1 R Q1 wherein R at each occurrence Q1 is independently hydrogen, alkyl, or hydroxyalkyl; or when at least one of A and B is present, Q is C, CR Q2 , CR Q2 R Q2 , N, NR Q2 , or O, and R at each occurrence Q2 is independently hydrogen, alkyl, or hydroxyalkyl. R is alkyl, alkoxy, halo, haloalkyl, hydroxyalkyl, hydroxy, cyano, nitro, amino, alkylamino, dialkylamino, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, or cycloalkenyl, p is 0, 1, 2, or 3, R1, W1, and W2 are as defined above.

[0064] In certain embodiments, ring A is present and ring B is absent. In certain embodiments, ring A is absent and ring B is present. In certain embodiments, both ring A and ring B are present. In certain embodiments, both ring A and ring B are absent. In certain embodiments, both ring A and ring B are absent and Q is -NHR Q1 .

[0065] In certain embodiments, the compound has the structure of formula (I-b) or (I-c): TIFF0007696867000010.tif2874(I-b) TIFF0007696867000011.tif3177(I-c) having, wherein m is 0, 1, 2, or 3 and p is 0, 1, or 2.

[0066] In certain embodiments, the compound has the structure of formula (I-d): TIFF0007696867000012.tif3674(I-d) having, wherein n is 0, 1, 2, or 3 and p is 0, 1, or 2.

[0067] In certain embodiments, the compound has the structure of formula (I-e): TIFF0007696867000013.tif3679(I-e) having, wherein m is 0, 1, 2, or 3, n is 1, 2, or 3, and p is 0 or 1.

[0068] In certain embodiments, the compound has the structure of formula (I-f): TIFF0007696867000014.tif3195(I-f) having, wherein p is 0, 1, 2, or 3 and n is 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0069] In certain embodiments, the compound has the structure of formula (I-g): TIFF0007696867000015.tif2869(I-g) having, wherein p is 0, 1, 2, or 3, TIFF0007696867000016.tif1825 is TIFF0007696867000017.tif26164, and each of these is optionally substituted by W1 and W2 as defined above. In certain embodiments, R1 is -OH. In certain embodiments, Q is -OH. In certain embodiments, R1 is -OH and Q is -OH, TIFF0007696867000018.tif1825 is TIFF0007696867000019.tif is 26164, and each of these is optionally replaced by W1 and W2 as defined above.

[0070] In some embodiments, a compound of formula (I’) wherein R1 is -OG 1 or -NR 1x G 1 is disclosed. In some embodiments, a compound of formula (I’) wherein R2 is -OG 1 or -NR 2x G 1 is disclosed. In some embodiments, a compound of formula (I’) wherein R3 is -OG 1 or -NR 3x G 1 is disclosed. In some embodiments, a compound of formula (I’) wherein R4 is -OG 1 or -NR 4x G 1 is disclosed. In some embodiments, a compound of formula (I’) wherein R5 is -OG 1 or -NR 5x G 1 is disclosed.

[0071] In some embodiments, a compound of formula (I’) wherein R2 and R3, together with the atom to which they are attached, form a 5- or 6-membered saturated, partially unsaturated, or fully unsaturated ring, and the 5- or 6-membered ring is selected from the group consisting of -OG 1 , -NHG 1 , and -N(C1-C 10 alkyl)G 1 and the 5- or 6-membered ring is further substituted with 0, 1, 2, or 3 other substituents disclosed herein, a compound of formula (I’) is disclosed.

[0072] In some embodiments, a compound of formula (I’) wherein R3 and R4, together with the atom to which they are attached, form a 5- or 6-membered saturated, partially unsaturated, or fully unsaturated ring, and the 5- or 6-membered ring is -OG 1 , -NHG1 and -N(C1-C 10 alkyl)G 1 selected from the group consisting of, wherein the 5- or 6-membered ring is further substituted with 0, 1, 2, or 3 other substituents disclosed herein, a compound of formula (I') is disclosed.

[0073] In some embodiments, a compound of formula (I'), wherein R4 and R5, together with the atoms to which they are attached, form a 5- or 6-membered saturated, partially unsaturated, or fully unsaturated ring, and the 5- or 6-membered ring is -OG 1 , -NHG 1 and -N(C1-C 10 alkyl)G 1 selected from the group consisting of, wherein the 5- or 6-membered ring is further substituted with 0, 1, 2, or 3 other substituents disclosed herein, a compound of formula (I') is disclosed.

[0074] In some embodiments, a compound of formula (I') contains a G 1 group and can be a non-luciferase enzyme substrate disclosed herein. For example, the G 1 group of a compound of formula (I') (e.g., -OG 1 or -NHG 1 group) can be subjected to non-luciferase enzyme-mediated in vivo conversion that converts G 1 to H, which then results in the appearance of the corresponding hydroxyl-luciferin analog or amino-luciferin analog. In some embodiments, the G 1 group can contain a linker between the portion that acts as a non-luciferase substrate and the remaining structure of the luciferin analog disclosed herein. In some embodiments, G 1 is G 2 -L 1 -, wherein G 2 is a non-luciferase enzyme substrate and L 1 is a linker as described above. In some embodiments, G 2 is a group removable by a non-luciferase enzyme and L 1is a group that spontaneously hydrolyzes after the action of a non-luciferase enzyme. For example, L 1 The linker is a traceless or self-sacrificial linker as described herein. Exemplary non-luciferase enzymes, enzyme substrate moieties, and linkers include those described in WO2006 / 130551 (which is hereby incorporated by reference in its entirety).

[0075] In some embodiments, formula (I') is formula (II): TIFF0007696867000020.tif3275(II) or a tautomer or salt thereof, wherein R4 is -OG 1 , -NHG 1 , or -N(C1-C 12 alkyl)G 1 and R1, W1, and W2 are as described above.

[0076] In some embodiments, compounds of formula (II) wherein R4 is -NH-CO-G2 are disclosed. In some embodiments, formula (II)) is formula (II-a): TIFF0007696867000021.tif3389(II-a) or a tautomer or salt thereof, wherein G 2 , W1, and W2 are as described above.

[0077] In some embodiments, compounds of formula (II-a) are disclosed wherein G 2 is a peptide (e.g., one composed of natural amino acids). In some embodiments, G 2 is a peptide that is a substrate for a non-luciferase enzyme of interest. For example, G 2 can be a peptide containing the amino acids of DEVD, wherein the non-luciferase enzyme of interest is caspase 3. Thereby, the proluciferase substrate can report caspase 3 activity in the presence of an appropriate luciferase.

[0078] Representative compounds of formula (I’) or formula (I) include, but are not limited to, the following: 5-(6-Hydroxybenzo[d]thiazol-2-yl)-4-thia-6-azaspiro[2.4]hept-5-ene-7-carboxylic acid; 6-(6-Hydroxybenzo[d]thiazol-2-yl)-5-thia-7-azaspiro[3.4]oct-6-ene-8-carboxylic acid; 2-(6-Hydroxybenzo[d]thiazol-2-yl)-1-thia-3-azaspiro[4.4]nona-2-ene-4-carboxylic acid; 2-(6-Hydroxybenzo[d]thiazol-2-yl)-1-thia-3-azaspiro[4.5]deca-2-ene-4-carboxylic acid; 5,5-Diethyl-2-(6-hydroxybenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid; (S)-2-(5-Fluoro-6-hydroxybenzo[d]thiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid; (R)-2-(5-Fluoro-6-hydroxybenzo[d]thiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid; (S)-2-(7-Aminonaphtho[2,1-d]thiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid; (S)-5,5-Dimethyl-2-(6-(pyrrolidin-1-yl)benzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid; (S)-2-(6,7-Dihydro-5H-thiazolo[4,5-f]indol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid; (S)-2-(6-((3-Hydroxypropyl)amino)benzo[d]thiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid; 2-(5-Fluoro-6-hydroxybenzo[d]thiazol-2-yl)-1-thia-3-azaspiro[4.5]deca-2-ene-4-carboxylic acid; 5,5-Dibenzyl-2-(6-hydroxybenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid; 5,5-Diethyl-2-(5-fluoro-6-hydroxybenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid; 2-(6-Hydroxybenzo[d]thiazol-2-yl)-1-thia-3-azaspiro[4.6]undec-2-ene-4-carboxylic acid; 2-(6-Hydroxybenzo[d]thiazol-2-yl)-8-methyl-1-thia-3-azaspiro[4.5]dec-2-ene-4-carboxylic acid; 2-(6-Hydroxybenzo[d]thiazol-2-yl)-8-methyl-1-thia-3,8-diazaspiro[4.5]dec-2-ene-4-carboxylic acid; 2-(6-Hydroxybenzo[d]thiazol-2-yl)-5,5-dipropyl-4,5-dihydrothiazole-4-carboxylic acid; and 2-(6-Hydroxybenzo[d]thiazol-2-yl)-8-oxa-1-thia-3-azaspiro[4.5]dec-2-ene-4-carboxylic acid, or a tautomer or salt thereof.

[0079] The compound names are assigned by using the Struct=Name naming algorithm as part of CHEMDRAW® ULTRA v.12.0.

[0080] The compound may exist as stereoisomers having an asymmetric center or a chiral center. The stereoisomers are either "R" or "S" depending on the configuration of the substituents around the chiral carbon atom. The terms "R" and "S" as used herein have the configuration defined in IUPAC 1974 Recommendations for Section E, Fundamental Stereochemistry, in Pure Appl. Chem., 1976, 45:13-30. The present disclosure contemplates various stereoisomers and mixtures thereof, which are expressly included within the scope of the invention. Stereoisomers include enantiomers, diastereomers, and mixtures of enantiomers or diastereomers. The individual stereoisomers of the compound can preferably be prepared by synthesis from commercially available starting materials containing an asymmetric center or a chiral center, or by using resolution methods well known to those skilled in the art after preparing a racemic mixture. Exemplary such resolution methods are (1) a method of binding a mixture of enantiomers to a chiral auxiliary, separating the resulting mixture of diastereomers by recrystallization or chromatography, and optionally liberating the optically pure product from the auxiliary (described in Furniss, Hannaford, Smith, and Tatchell, "Vogel’s Textbook of Practical Organic Chemistry", 5 th edition (1989), Longman Scientific & Technical, Essex CM20 2JE, England), or (2) a method of directly separating a mixture of optical enantiomers on a chiral chromatography column, or (3) a fractional recrystallization method.

[0081] It should be understood that the compound may also have tautomeric forms and further geometric isomers, which also constitute an aspect of the present invention.

[0082] In addition, the present disclosure also includes isotope-labeled compounds that are identical to the compounds listed in formula (I) except that one or more atoms are replaced by atoms having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Examples of isotopes suitable for inclusion in the compounds of the present invention are hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, and include, for example, but 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 36 Cl. Substitution with heavier isotopes (e.g., deuterium, i.e., 2 H) may provide certain therapeutic advantages due to greater metabolic stability (e.g., increased in vivo half-life or reduced dosing requirements) and may therefore be preferred in some situations. Compounds can incorporate positron-emitting isotopes for medical imaging and positron emission tomography (PET) studies to determine receptor distribution. Suitable positron-emitting isotopes that can be incorporated into the compounds of formula (I) are 11 C, 13 N, 15 O, and 18 F. Generally, the isotope-labeled compounds of formula (I) can be prepared by conventional techniques known to those skilled in the art or by processes similar to those described in the attached examples using appropriate isotope-labeled reagents in place of non-isotope-labeled reagents.

[0083] A. Characteristics of the Compounds (i) Thermal Stability The compounds of the present disclosure can exhibit better thermal stability than known luciferins. Dehydroluciferin was identified as the main product resulting from the time-dependent degradation of D-luciferin in solution (Figures 1 and 2). Since dehydroluciferin inhibits luciferase and causes a decrease in light output, it can have a significant impact on luciferase assays (Figure 2).

[0084] The 5,5-disubstituted luciferin analogs of the present disclosure can show improved thermal stability in solution (Figure 3). As shown in Figure 3B, there is substantially no decomposition in 5,5-dimethyl luciferin III-a (6-OH compound) and III-b (6-NH2 compound), while significant decomposition with corresponding dehydroluciferin formation is observed in unsubstituted luciferin. The 5,5-disubstitution is thought to improve the overall thermal stability of the luciferin analog compounds by eliminating dehydroluciferin formation.

[0085] As used herein, "thermal stability" can refer to the degree to which a luciferin analog compound remains stable in solution over a particular time period such that it maintains its ability to generate light in the presence of luciferase (e.g., including live cell assays using live cell luciferase). Examples of solutions of luciferin analogs can include liquid media in which luciferase is present, such as aqueous buffer systems in which luciferase assays are performed. The stability of the compounds of the present disclosure can be demonstrated by the percentage of compound degradation over time in a particular environment. The percentage of purity for a particular compound can be determined by various techniques known to those skilled in the art. Such techniques include, for example, nuclear magnetic resonance (NMR) and high performance liquid chromatography (HPLC).

[0086] In some embodiments, the thermal stability of the disclosed compounds can be determined after a particular compound solution is stored at a particular temperature for a particular time period in the absence or presence of luciferase. The temperature during storage can be 20°C or higher, 30°C or higher, 40°C or higher, 50°C or higher, 60°C or higher, or 70°C or higher. In some embodiments, the disclosed compounds are combined with luciferase (or a biological sample containing luciferase) in solution and maintained at a certain temperature (e.g., 20 - 70°C) for a certain time period.

[0087] In some embodiments, the inventive compounds can provide a stable luminescence signal in solution at ambient or elevated temperatures (e.g., 30 - 70°C) for a period of at least 24 hours, at least 48 hours, at least 60 hours, at least 80 hours, at least 100 hours, at least 120 hours, at least 150 hours, at least 200 hours, at least 250 hours, at least 300 hours, at least 350 hours, or at least 400 hours. In some embodiments, the inventive compounds can provide a stable luminescence signal in a luciferase assay medium at ambient temperature for a period of 100 - 400 hours. The disclosed compounds with improved thermal stability in solution may enable applications that require storing luciferin at ambient temperature over time for extended periods. The disclosed compounds can be particularly useful for applications where the formation of dehydro luciferin is significant and harmful (e.g., reactive oxygen species (ROS) detection assays or P450 assays).

[0088] The compounds of the present invention demonstrate superior thermal stability compared to luciferin analogs lacking 5,5-disubstitution over the same time period. In some embodiments, the compounds of the present invention exhibit at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% improvement in thermal stability compared to luciferin analogs lacking 5,5-disubstitution. The remarkable stability of the compounds of the present invention allows for longer storage without degradation of the luciferin analogs, which can be used for various enhancements of luminescence assays.

[0089] In some embodiments, the thermal stability of the compounds of the present invention can be enhanced by storing the compounds of the present invention in solution with an additive. Such an additive can enhance the stability of the compounds of the present invention by preventing oxidative degradation or other forms of degradation. The enhancement of stability can be achieved by converting oxidized compounds back to the original compounds through reduction, or by removing reactive oxygen species such as oxygen or hydrogen peroxide through other mechanisms. In some embodiments, such an additive is compatible with the reaction of the compounds of the present invention with luciferase and does not cause a decrease in light production. In some embodiments, such an additive is azathiothymidine (ATT) or an analog thereof, as described in U.S. Patent Application No. 62 / 541,350, which is incorporated herein by reference in its entirety. In some embodiments, the additive is thiourea, or another carbon-sulfur double bond-containing compound.

[0090] (ii) Light production Surprisingly, the compounds of the present disclosure can generate sufficient light to serve as suitable substrates for luciferase assays using various luciferases. In some embodiments, the compounds of the present disclosure provide a much stronger light signal than 5,5-dimethyl luciferin in luciferase assays under various conditions. For example, both firefly luciferase and Gaussia luciferase can effectively utilize various stereoisomeric forms (e.g., both D-form and L-form in a mixture) of the 5,5-disubstituted luciferin analogs of the present disclosure to generate bioluminescence over a wide pH range (Figures 4A - 6B). From past studies, it was not clear whether certain 5,5-disubstituted luciferin analogs generated sufficient light to be useful in luciferase assays. Furthermore, 5,5-disubstituted luciferins were previously thought to generate light only via a chemiluminescence process, rather than via an enzymatic process by firefly luciferase. For example, Compound III-a was thought to be unable to generate light via a process mediated by firefly luciferase or Gaussia luciferase (Branchini et al., J. Am. Chem. Soc. 2002, 124, 2112 - 2113). On the other hand, only the D-form of Compound III-b was shown to generate light when tested using recombinant beetle luciferase (Viviani et al., Biochemistry, 2014, 53, 5208 - 5220). The decreased activity of 5,5-disubstituted luciferin substrates compared to luciferin seems to suggest to some extent that any substrate with more steric volume at the 5-position of the luciferin structure results in insufficient activity for luciferase assays. However, surprisingly, the compounds of the present disclosure show that the steric hindrance imparted by the substituents at the 5-position does not necessarily reduce the activity of the substrate. In certain embodiments, the compounds of the present disclosure having substituents larger than the 5,5-dimethyl compound (e.g., 5,5-diethyl or cyclohexyl substituents) show higher luciferase activity than the 5,5-dimethyl compound, contrary to the expectation that higher steric hindrance reduces activity.Accordingly, the disclosed compounds provide unexpected advantages over known 5,5-disubstituted luciferins due to their unprecedented activity in the luciferase assay.

[0091] "Luminescence" refers to the light output of luciferase under appropriate conditions, e.g., in the presence of a suitable substrate such as a luciferin analog. The light output can be measured as the instantaneous or nearly instantaneous light output at the start of the luminescence reaction (sometimes referred to as "T = 0" luminescence or "flash"), which can be initiated upon addition of the luciferin substrate. In various embodiments, the luminescence reaction is carried out in solution. In other embodiments, the luminescence reaction is carried out on a solid support. The solution can contain a lysate (e.g., a lysate from cells in a prokaryotic or eukaryotic expression system). In other embodiments, expression occurs in a cell-free system, or the luciferase protein is secreted into an extracellular medium, in which case it is not necessary to generate a lysate. In some embodiments, the reaction is initiated by injecting appropriate materials (e.g., luciferin analog, buffer, etc.) into a reaction chamber (e.g., a well of a multiwell plate such as a 96-well plate) containing the luminescent protein. In still other embodiments, the luciferase and / or luciferin analog (e.g., a compound of formula (I)) is introduced into a host, and the measurement of luminescence is carried out in the host or a part thereof. The host can include an entire organism, or its cells, tissues, explants, or extracts. The reaction chamber can be located within a reading device capable of measuring the light output using, for example, a luminometer or a photomultiplier tube. Also, the light output or luminescence can be measured over time, e.g., within the same reaction chamber, for periods such as seconds, minutes, hours, etc. The light output or luminescence can be reported as a time average, the half-life of signal decay, the sum of the signal over a certain time period, or the peak output. Luminescence can be measured in relative light units (RLU).

[0092] The compound of formula (I) can be used in a bioluminescence assay to provide 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more RLU relative to luciferin or a known luciferin analog.

[0093] B. Synthetic Method The compound of formula (I) can be synthesized as shown in Scheme 1. Abbreviations used in the following description of the scheme are as follows: DMF represents dimethylformamide. Scheme 1. Synthesis of the compound of formula (I) TIFF0007696867000022.tif34158

[0094] As shown in Scheme 1, the compound of formula (I) can be prepared by treating the benzothiazol-2-yl intermediate A with a β,β-disubstituted cysteine derivative. The reaction can be carried out at a pH of 8 - 9 in a solution of dimethylformamide and water.

[0095] The optimal reaction conditions and reaction times in each individual step can vary depending on the specific reactants used and the substituents present in the reactants. Specific procedures are shown in the Examples section. The reaction can be worked up in a conventional manner, for example, by removing the solvent from the residue and further purifying it according to methodologies generally known in the art (e.g., but not limited to crystallization, distillation, extraction, trituration, and chromatography). Unless otherwise stated, starting materials and reagents are either commercially available or can be prepared from commercially available materials by methods described in the chemical literature by those skilled in the art. If the starting material is not commercially available, the starting material can be prepared by a procedure selected from standard organic chemical techniques, techniques similar to the synthesis of known compounds with a similar structure, or techniques similar to the procedures described in the Schemes or Synthesis Examples section above.

[0096] Routine experimental operations are included within the scope of the present invention, including appropriate manipulation of reaction conditions, reagents, and the order of synthetic routes, protection of any chemical functionality that may not be compatible with the reaction conditions, and deprotection at suitable points in the reaction sequence of the method. Suitable protecting groups, and methods for protecting and deprotecting various substituents using such suitable protecting groups, are well known to those skilled in the art, and examples of such methods can be found in Greene's book, Protective Groups in Organic Synthesis (4 th ed.) by PGM Wuts and TW Greene (John Wiley & Sons, NY (2006)), which is hereby incorporated by reference in its entirety. The synthesis of the compounds of the present invention can be accomplished by methods similar to those described in the above-described synthetic schemes or specific examples.

[0097] When optically active forms of the disclosed compounds are required, such forms can be obtained by performing one of the procedures described herein using optically active starting materials (e.g., prepared by asymmetric induction in a suitable reaction step), or by resolving a mixture of stereoisomers of a compound or intermediate using standard procedures such as chromatographic separation, recrystallization, or enzymatic resolution.

[0098] Similarly, when pure geometric isomers of a compound are required, such isomers can be obtained by performing one of the above procedures using pure geometric isomers as starting materials, or by resolving a mixture of geometric isomers of a compound or intermediate using standard procedures such as chromatographic separation.

[0099] It is understood that the described synthetic schemes and specific examples are illustrative and should not be construed as limiting the scope of the present invention as defined in the appended claims. All alternative methods, variations, and equivalents in the synthetic methods and specific examples are included within the scope of the claims.

[0100] Base addition salts can be prepared by reacting the carboxyl groups of the compounds of the present disclosure with suitable bases (e.g., hydroxides, carbonates, or bicarbonates) of metal cations (e.g., lithium, sodium, potassium, calcium, magnesium, or aluminum), or with organic primary, secondary, or tertiary amines during the final isolation and purification of the compounds. Quaternary amine salts, for example, those 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, etc. can be prepared.

[0101] 3. Methods of Use and Kits The luciferin analogs of the present invention can act as substrates for the desired enzyme in an enzyme activity assay or a non-enzyme bioassay (e.g., a reporter gene assay). In some embodiments, the desired enzyme may be a non-luciferase, and the luciferin analogs of the present invention can be used as non-luciferase enzyme substrates and as prosubstrates for luciferase. In other embodiments, the luciferin analogs of the present invention may be modified by the target molecule, and this modified molecule is a substrate for luciferin. Surprisingly, the luciferin analogs of the present invention can have activity as substrates for luciferase in a light generation assay. Therefore, in a bioluminescence assay using the luciferin analogs of the present invention, many non-luciferase enzymes or luciferase enzymes can be measured.

[0102] The compounds of the present disclosure can be used in any method in which a luciferase substrate (e.g., a luciferin analog) is used. For example, the compounds of the present disclosure can be used in a bioluminescence method for detecting one or more molecules (e.g., an enzyme, a cofactor for an enzymatic reaction, an enzyme substrate, an enzyme inhibitor, an enzyme activator, or an OH radical) or one or more states (e.g., a redox state) in a sample using an analog of luciferin. Examples of the sample include an animal (e.g., a vertebrate), a plant, a fungus, a physiological fluid (e.g., blood, plasma, urine, mucus secretion), a cell, a cell lysate, a cell supernatant, or a purified cell fraction (e.g., an intracellular fraction). The presence, amount, spectral distribution, release kinetics, or specific activity of such a molecule can be detected or quantified. The molecule can be detected or quantified in a solution including a multiphase solution (e.g., an emulsion or a suspension) or on a solid support (e.g., a particle, a capillary, or an assay vessel).

[0103] In certain embodiments, the compounds of formula (I') or formula (I) can be used to quantify a target molecule. In some embodiments, the compounds of formula (I') or formula (I) can be used as a probe for a specific biochemical activity (e.g., apoptosis or drug metabolism). In some embodiments, the compounds of formula (I') or formula (I) can be conjugated to a specific enzyme activity, and at this time, the compound is subject to the action of the specific enzyme of interest. In some embodiments, this approach can be used for enzymes used in, for example, drug metabolism (e.g., cytochrome P450 enzymes, monoamine oxidase, and glutathione S-transferase) and enzymes used in apoptosis (e.g., caspases). In some embodiments, the luciferin analog can provide a single reagent and a homogeneous assay in combination with other components necessary to support luminescence (e.g., a luminescent protein such as luciferase). For example, when the luciferin analog and luciferase are added to a sample, luminescence can be generated.

[0104] This method can be used, for example, to determine the presence, amount, or state of at least one molecule (e.g., a non-luciferase enzyme, a regulator of a non-luciferase enzyme, a non-luciferase enzyme substrate, and / or a cofactor of a reaction) in a sample. In certain embodiments, the disclosed method provides a rapid method for detecting one or more molecules from a single sample (e.g., an aliquot of cells or a lysate thereof). In certain embodiments, the method includes quantifying the presence, amount, or specific activity of a molecule (e.g., an enzyme, a substrate, or a cofactor) in a bioluminescence assay or quantifying the presence or amount of an enzyme, a substrate, or a cofactor in a fluorogenic assay. The intensity of the bioluminescence signal or the fluorogenic signal is a function of the presence or amount of the respective molecule. Additionally, the reaction may contain one or more test agents, e.g., an enzyme inhibitor or an enzyme activator, and / or inhibitors or activators at different concentrations. In certain embodiments, the method uses at least two different reactions. The first reaction is a non-luciferase enzyme-mediated reaction, and the second reaction is a reaction mediated by beetle luciferase. In another embodiment, the first reaction is a non-enzymatic reaction, and the second reaction is a reaction mediated by beetle luciferase. In yet another embodiment, the method uses a single reaction, e.g., a reaction mediated by beetle luciferase or a fluorogenic reaction.

[0105] Accordingly, bioluminescence assays can directly or indirectly detect (e.g., measure) cofactors, enzymes, enzyme substrates, enzyme inhibitors, enzyme activators, or the amount, presence, or specific activity, or state, for example, of enzyme-mediated reactions. For example, in certain embodiments, beetle luciferase and a luciferin analog that is a substrate for beetle luciferase can be used in a bioluminescence assay to detect ATP concentration. In another embodiment, a luciferin analog that is a substrate for a non-luciferase enzyme, for example, a derivative that is a monoamine oxidase substrate, results in a product that is a substrate for beetle luciferase and can therefore be used in a bioluminescence assay to detect the oxidase. In certain embodiments, the luciferin analog is a pro-substrate of beetle luciferase that produces a product that is a luciferase substrate but does not itself produce a significant amount of light in reaction with beetle luciferase. In some embodiments, the analog is a substrate for a non-luciferase enzyme or is useful for detecting another molecule and is a substrate for luciferase that produces a significant amount of light. In this embodiment, the analog is modified by luciferase but is generally inefficient for the light-producing reaction. In some embodiments, novel luciferin derivatives that are capable of light production in the case of luciferase and luciferase substrates, for example, luciferin, luciferin derivatives, functional analogs, or luciferase substrates (e.g., luciferin derivatives disclosed herein), have enhanced stability to assay conditions and / or lysis reagents, for example, assay stability, storage stability, to individual chemical components (alone or as a multi-component mixed solution in which it is included).

[0106] In certain embodiments, a bioluminescence assay method for detecting one or more non-luciferase enzymes is disclosed. The method includes contacting a suspect sample having one or more non-luciferase enzymes, or a substrate or cofactor for a non-luciferase-mediated reaction, with a corresponding reaction mixture that includes a luciferin analog that is a substrate for the non-luciferase enzyme. In certain embodiments, the analog is an analog having a modification that includes a discrimination site for a non-luciferase enzyme (e.g., phosphatase) within the G1 ring of D-luciferin (Figure 1). In another embodiment, the analog is an analog having a modification within the G2 ring of D-luciferin and is a substrate for luciferase or a pro-substrate for luciferase. In another embodiment, the derivative is a derivative having a modification within the G3 ring of luciferin that includes a discrimination site for a target enzyme (e.g., acetylcholinesterase). In another embodiment, the derivative is a derivative having a discrimination site for a target enzyme and further modifications within the ring or within one or more other rings.

[0107] An analog that is a substrate for luciferase and optionally also a substrate for a non-luciferase enzyme or other molecule can be used to detect luciferase, or a cofactor, inhibitor, or activator of the luciferase reaction. If the analog is a luciferase pro-substrate, i.e., the reaction product between the analog and the non-luciferase enzyme is a substrate for luciferase, sequential or simultaneous reactions regarding the non-luciferase enzyme and luciferase can be performed. For example, the reaction regarding the non-luciferase enzyme containing the pro-substrate can be carried out in a single well, and a beetle luciferase reaction mixture is added to this well. In another embodiment, the reaction mixture for the non-luciferase enzyme containing the pro-substrate is carried out in a single well, and a portion of this reaction product is added to another well having a beetle luciferase reaction mixture. As an alternative, the reactions can be carried out in the same well.

[0108] Thus, the present disclosure provides a method for determining the presence or amount of a molecule for a non-luciferase enzyme-mediated reaction in a sample. The method involves contacting the sample with a first reaction mixture for a non-luciferase enzyme-mediated reaction and a luciferin analog (e.g., a compound of formula (I’) or formula (I) which is a substrate for a non-luciferase enzyme) to produce a first mixture, or providing such a first mixture that contains a luminescent product which is a substrate for luciferase, or providing such a first mixture. At least a portion of this first mixture is contacted with a second reaction mixture for a beetle luciferase-mediated reaction to produce a second mixture. Next, the luminescence in the second mixture is detected or determined by detecting or determining the presence or amount of a molecule for a non-luciferase enzyme-mediated reaction in the sample. In some embodiments, the non-luciferase reaction mixture or the luciferase reaction mixture can include an esterase, for example, when the reaction product between the analog and the non-luciferase enzyme has an ester group and this product is a proluciferase substrate. The esterase can be included with the first reaction mixture, added prior to the initiation of the luciferase reaction mixture, or included in the luciferase reaction mixture. In some embodiments, for example, an analog containing a picolinyl ester, i.e., the reaction product between this analog and the non-luciferase enzyme, is a substrate for luciferase in the absence of an exogenously added esterase. In certain embodiments, a luciferin analog having an ester modification is used in the method of the present disclosure, for example, for detecting a non-luciferase enzyme including a cytochrome P450 enzyme, because it is considered that the properties as a non-luciferase enzyme substrate are improved. Without intending to be bound by any mechanism, it is considered that including an ester modification at the 4-position in the G3 ring blocks the negative charge or induces lipophilic properties in the derivative, making the derivative an improved substrate.

[0109] Further provided is a method for determining the presence or amount of a molecule in a sample for a non-luciferase enzyme-mediated reaction. The method includes contacting the sample with a reaction mixture for a non-luciferase-mediated enzyme reaction and a luciferase-mediated reaction, and a luciferin analog that is a substrate for the non-luciferase enzyme to produce a mixture. A luminescent product that is a substrate for luciferase is produced by the reaction between the non-luciferase enzyme and the luciferin analog of the present invention. Luminescence in the mixture is detected or determined by detecting or determining the presence or amount of a molecule in the sample for a non-luciferase-mediated reaction.

[0110] Further, the present disclosure provides a method for detecting or determining the presence or amount of a molecule in a sample for a luciferase-mediated reaction. The method includes contacting the sample with a reaction mixture for beetle luciferase and a luciferin analog that is a substrate for luciferase to produce a reaction product.

[0111] Also, the present disclosure provides a method for detecting the presence or amount of a molecule in a sample. The method includes contacting the sample with a first reaction mixture for a non-enzyme-mediated reaction and a luciferin analog that produces a luminescent product that is a substrate for luciferase in the presence of the molecule, and then contacting at least a portion of the first reaction product with a second reaction mixture for a luciferase-mediated reaction to produce a second reaction product. The presence or amount of the molecule is detected or determined by detecting or determining luminescence in the second reaction product. For example, a mixture is provided that includes the sample, a first reaction mixture for a non-enzyme-mediated reaction, and a luciferin analog that produces a luminescent product that is a substrate for beetle luciferase in the presence of the molecule. At least a portion of the first mixture is mixed with a second reaction mixture for a beetle luciferase-mediated reaction to produce a second mixture, and then the presence or amount of the molecule is detected or determined by detecting or determining luminescence in the second mixture.

[0112] The bioluminescence assays using luciferin analogs having a lower background as described herein can use a lower (or higher) amount of the analog, and such analogs are thought to have improved reactivity (e.g., towards non-luciferase enzymes). Additionally, for any of the bioluminescence assays described herein, other reagents can be added to the reaction mixture, and such reagents include, but are not limited to, reagents that inhibit or prevent the inactivation of luciferase, or reagents that extend or enhance the luminescence signal.

[0113] Also provided is a method for identifying or measuring the efficacy of a modulator in a non-luciferase enzyme-mediated reaction. The method involves contacting one or more agents with a first reaction mixture for a non-luciferase enzyme-mediated reaction and a luciferin analog that is a substrate for the non-luciferase enzyme to produce or provide such a mixture, wherein the analog comprises a modification of the G1 or G2 ring relative to D-luciferin. The first mixture in the absence of the one or more agents comprises a luminescent product that is a substrate for beetle luciferase. At least a portion of the first mixture is mixed with a second reaction mixture for a beetle luciferase-mediated reaction to produce a second mixture. By comparing the luminescence in the second mixture with a control mixture, it is determined whether and / or to what extent and with what efficacy one or more of the agents modulate the non-luciferase enzyme-mediated reaction.

[0114] In certain embodiments, test compounds can be screened and evaluated using the luciferin and fluorophore analogs of the invention for activity as a substrate or cofactor in an enzymatic or non-enzymatic reaction, or for a regulatory factor that is either an inhibitor or activator. A candidate compound can be determined to be a regulatory factor or substrate in the reaction by contacting the reaction mixture with the analog and the test compound under conditions that produce bioluminescence, fluorescence, or a bioluminescence product in the absence of the test compound.

[0115] In one aspect, a method for differentiating a substrate and an inhibitor of a reaction is provided. For example, a compound is incubated with at least one enzyme under conditions that allow metabolism of the compound, and then a luciferin analog is provided under conditions that are believed to be suitable for the interaction between the luciferin analog and the enzyme in the absence of an inhibitor or substrate of the enzyme. In certain embodiments, the product of this reaction is a substrate for luciferase and, in the presence of luciferase, results in a second reaction that emits light. The resulting light emission reaction is compared to a reaction obtained by contacting the enzyme with the compound and the analog under conditions that are believed to be suitable for the interaction between the luciferin analog and the enzyme in the absence of an inhibitor of the enzyme. Metabolism of the compound by the enzyme can reduce the concentration of the compound in the assay medium and may result in an apparent loss of inhibitory activity compared to conditions without metabolism of the compound, in which case it is believed that this compound is a substrate for the enzyme. An inhibitory compound that is not metabolized is believed to exhibit the same potency regardless of the time of addition of the substrate.

[0116] In one aspect, it is preferred that the compound first be contacted with the enzyme for a first predetermined time period. Thereafter, the mixture is contacted simultaneously or concomitantly with a luciferin analog and a bioluminescent enzyme (e.g., luciferase), and the mixture is incubated for a second predetermined time period.

[0117] In another aspect, the compound is incubated with the enzyme for a first predetermined time period to form a first mixture. Thereafter, the first mixture is contacted with a luciferin analog to form a second mixture, which is incubated for a second predetermined time period. Next, the second mixture is contacted with a bioluminescent enzyme (e.g., luciferase) to form a third mixture, which is incubated for a third predetermined time period. Thereafter, the activity resulting from the interaction between the enzyme and the compound is determined by measuring the luminescence during and / or after the third predetermined time period and comparing it to a control (e.g., no compound) reaction. In this way, for example, a first incubation with a test compound but without a luciferin analog may result in a deeper inhibition than the inhibition that would be observed without the first incubation in the first reaction by a mechanism-based inhibitor, or the substrate of the first reaction shows a reduction in inhibition, thus identifying a mechanism-based inhibitor of the first enzyme and distinguishing it from a non-mechanism-based inhibitor.

[0118] In another aspect, a cell-based method is provided for screening a compound to determine the effect of the compound on the enzymatic activity of a cell. The test compound is contacted with a cell having an enzyme either naturally or through recombinant expression, a luciferin analog, and a bioluminescent enzyme (e.g., luciferase) for a predetermined time period, or with a cell having the enzyme and luciferase and the analog. Thus, in certain embodiments, cells that transiently or stably express a recombinant enzyme such as a bioluminescent enzyme (e.g., luciferase) can be used. Any conventional method for creating transiently or stably transfected cells can be used. In certain embodiments, the luciferin analog is contacted with the cell, dispersed within the cell, and produces a product that is a substrate for luciferase when appropriate molecules are present. If luciferase is present within the cell, luminescence can be detected. As an alternative method, in cells lacking luciferase, the product migrates from the cell to the medium, and the medium is added to the luciferase reaction mixture. The activity resulting from the interaction of the cell and the compound is then determined by measuring the luminescence of the reaction mixture in comparison to a control (no test compound) reaction mixture.

[0119] In one aspect, it is preferred that the compound is first contacted with the cells for a predetermined time period. Thereafter, the cells are contacted with the luciferin analog and luciferase simultaneously or concurrently, and the mixture is incubated for a second predetermined time period. The enzyme activity is determined by measuring the amount of luminescence generated from the reaction mixture in comparison to a control reaction mixture (e.g., without the test compound). In another aspect, it is preferred that the test compound is first contacted with the cells for a predetermined time period. Thereafter, the exposed cells are then contacted with the luciferin analog and incubated for a second predetermined time period. Next, the cells are contacted with luciferase to form a third mixture, which is incubated for a third predetermined time period. Thereafter, the activity of the cells resulting from the interaction of the cells with the test compound(s) is determined by measuring the luminescence of the reaction mixture in comparison to a control (e.g., without the test compound) reaction mixture. By adding a detergent, the cells can be lysed and the cell contents can be released.

[0120] A cell-based luminescence detection assay for molecules present in cell culture medium (e.g., molecules present actively or via an inactive mechanism in cell culture medium) involves adding a reaction mixture containing a luciferin analog to the cell culture medium, or adding the cell culture medium to a reaction mixture containing a luciferin analog, and detecting luminescence.

[0121] In yet another embodiment of the cell-based assay, the cells can be lysed in a suitable lysis buffer. For animal cells, typically a buffer containing 0.1 - 1.0% non-ionic detergent (e.g., Triton X 100 or Tergitol) is sufficient. Usually, cells of bacteria, plants, fungi, or yeast are more difficult to lyse. Detergents, freeze-thaw cycles, hypotonic buffers, sonication, cavitation, or combinations of these methods can be used. The lysis method for generating the lysate is compatible with luciferase or other enzyme activities, or for the detection or status of other molecules.

[0122] The presence or activity of a non-luciferase enzyme can be measured in cells growing in a medium or in cells of an animal (e.g., a living animal). For measurements in cells within an animal, a luciferin analog can be administered to the animal, for example, by injecting the luciferin analog into the animal or adding it to an aqueous solution (e.g., water or food consumed by the animal). The conversion of the analog to the product that is the luciferase substrate can be detected by luminescence mediated by luciferase expressed in the cells of the animal (e.g., transfected cells), by luciferase administered to the animal (e.g., injected into the animal), or by collecting a physiological fluid (e.g., blood, plasma, urine, etc.) or tissue sample and combining it with a luciferase reagent.

[0123] Assays using two reactions can be performed simultaneously (1 step) or sequentially (2 steps) to detect one or more moieties, such as an enzyme, substrate, cofactor, inhibitor or activator for an enzymatic reaction, or a state (e.g., redox state), including a protein (peptide or polypeptide). The sequential reactions can be performed in the same vessel (e.g., one well of a multiwell plate). For a two-step assay, the first reaction mixture may contain all, less than all, or one reagent absent among the reagents for a non-luciferase enzyme-mediated reaction, where the absent reagent is the reagent to be detected in the sample (e.g., cell lysate). For example, the non-luciferase enzyme-mediated reaction is carried out under conditions effective to convert a luciferin analog, which is a substrate for the non-luciferase, and a pro-substrate of luciferase into a product that is a substrate for luciferase. The first reaction can be quenched upon or prior to the addition of the luciferase reaction mixture. For example, the quencher of the first reaction may be present in the luciferase reaction mixture. The luciferase reaction mixture preferably lacks a substrate for luciferase, e.g., the only source of substrate for luciferase is provided by the reaction between the non-luciferase enzyme and the analog. If all the reagents for the first reaction are present in the first reaction mixture, the assay can be used to identify a moiety that modifies the reaction, e.g., an inhibitor or enhancer of the reaction. After performing the reaction, either simultaneously or sequentially, the presence or amount of one or more molecules, or one or more inhibitors or activators in the reaction(s), is detected or determined, and / or to what extent and by what potency is detected or determined.

[0124] For a one-step assay, the reaction mixture may contain reagents for two reactions, for example, reagents for a non-luciferase enzyme-mediated reaction and a luciferase-mediated reaction or a non-enzymatic reaction, or for a single reaction, for example, for a reaction between an analog of a fluorophore that is a substrate for an enzyme and the enzyme, or for a luciferase-mediated reaction (e.g., where luciferase is suspected in a sample to be tested).

[0125] For an assay using two reactions, the order in which assay molecules are added may vary. When starting sequentially (whether in the same container or not), adjustments to the reaction conditions (e.g., reagent concentration, temperature, or additional reagents) may be made. In certain embodiments, two or more reactions are carried out simultaneously in a single reaction mixture. Optionally, the assay is a homogeneous assay, for example, the components are mixed and then the mixture is added to the sample. The results can be read without further transfer of reagents.

[0126] The assays of the present disclosure thus enable the detection of one or more molecules or states in a sample, which can be, for example, eukaryotic cells such as yeast, avian, plant, insect, or mammalian cells (including, but not limited to, human, monkey, mouse, dog, cow, horse, cat, sheep, goat, or pig cells), or prokaryotic cells, a sample containing cells from two or more different organisms, or a cell lysate or its supernatant, or a sample containing molecules in purified form (e.g., a purified non-luciferase enzyme useful for preparing a standard curve). The cells may not be genetically recombinant (non-recombinant cells) via recombinant techniques, or may be transiently transfected recombinant cells with a genome stably enhanced with recombinant DNA and / or recombinant DNA, or a genome modified to disrupt a gene (e.g., disrupt a promoter, intron, or open reading frame), or replace one DNA fragment with another. The recombinant DNA or replacement DNA fragment can encode a molecule to be detected by the methods of the present disclosure, a moiety that modifies the level or activity of the molecule to be detected, and / or a gene product unrelated to the molecule or moiety that modifies the level or activity of the molecule.

[0127] The method of the present invention can be used to detect enzyme-mediated reactions, molecules or states for non-enzyme-mediated reactions. For example, the molecules or states to be detected by the present method include, but are not limited to, enzymes such as demethylase, oxidase (e.g., MAO), deacetylase, deformylase, protease (proteasome, calpain, beta-secretase, cathepsin, calpain, thrombin, granzyme B), phosphatase, kinase, peroxidase, transferase such as GST, sulfatase, beta-lactamase, cytochrome P450 enzyme, esterase such as acetylcholinesterase, dehydrogenase, luciferase, substrates, inhibitors, cofactors, activators, reactive oxygen species, reduction states, and transcription regulatory factors or gene transcription regulatory factors in enzyme-mediated reactions. The enzymes used in the present method (enzymes to be detected or enzymes useful for the detection of substrates or cofactors) can be selected from any combination of enzymes including recombinant enzymes and endogenous (natural) enzymes. In certain embodiments, the enzyme to be detected is an endogenous enzyme. In another embodiment, the enzyme is a recombinant enzyme. Other combinations apparent to those skilled in the art can be used in accordance with the teachings herein in the assays and methods of the present invention. Enzymes include, but are not limited to, protease, phosphatase, peroxidase, sulfatase, peptidase, oxidase, dealkylase, deformylase, and glycosidase. The enzyme may be a hydrolase, oxidoreductase, lyase, transferase such as glutathione S-transferase, isomerase, ligase, or synthetase. Of particular interest are classes of enzymes of physiological importance. Such enzymes include protein peptidase, esterase, protein phosphatase, glycosylase, protease, dehydrogenase, oxidase, oxygenase, reductase, methylase, and the like. Enzymes of interest include those involved in making or hydrolyzing both organic and inorganic esters, glycosylation, and hydrolyzing amides. In any case, further subdivision may exist.

[0128] In particular, enzymes useful in the present disclosure include any protein exhibiting enzyme activity, such as lipase, phospholipase, sulfatase, urease, peptidase, protease, and esterase, and acid phosphatase, glucosidase, glucuronidase, galactosidase, carboxylesterase, and luciferase are included therein. In certain embodiments, the enzyme is a hydrolase. Examples of hydrolases include alkaline phosphatase and acid phosphatase, esterase, decarboxylase, phospholipase D, P-xylosidase, β-D-fucosidase, thioglucosidase, β-D-galactosidase, α-D-galactosidase, α-D-glucosidase, β-D-glucosidase, β-D-glucuronidase, β-D-mannosidase, β-D-mannosidase, β-D-fructofuranosidase, and β-D-glucuronosyltransferase.

[0129] In certain embodiments, the compounds of formula (I') or formula (I) can be used, for example, in vivo to detect luminescence in living cells. In some embodiments, luciferase can be expressed (as a reporter or otherwise) in cells treated with a luciferin analog (e.g., a compound of formula (I') or formula (I)) that permeates the cells in culture medium and reacts with the luciferase to produce luminescence. In addition to being cell permeable, the compounds of formula (I') or formula (I) can exhibit biocompatibility comparable to that of natural luciferin in terms of cell viability. In some embodiments, compounds of formula (I') or formula (I) containing chemical modifications known to increase the stability of natural luciferin or proluciferin in the medium can be synthesized and used for more robust reporter assays of the cell-based luciferase system. In yet other embodiments, samples containing luciferase and a compound of formula (I') or formula (I) (including cells, tissues, animals, etc.) can be assayed using various microscopy and imaging techniques (e.g., in vivo imaging). In yet other embodiments, a secretable luciferase is expressed intracellularly as part of a cell-based reporter system.

[0130] In certain embodiments, the compounds of the invention can be used in a method for detecting luminescence in a transgenic animal. The method comprises administering one of the compounds of the invention to a transgenic animal that expresses a luciferin-utilizing luciferase and detecting the luminescence.

[0131] In certain embodiments, the compounds of formula (I’) or formula (I) disclosed herein may be provided as part of a kit. In some embodiments, the kit can include one or more luciferases (polypeptide, polynucleotide, or both forms) and a luciferin analog of formula (I’) or formula (I), along with suitable reagents and instructions for use, such that a user can perform an assay as disclosed herein. The kit may also include one or more buffers such as those buffers disclosed herein.

[0132] In some embodiments, an assay system for detecting or quantifying ATP in a sample is provided herein, the assay system comprising: (a) a reagent composition comprising a compound of formula (I’) or formula (I) as described herein; and (b) a sample that contains or is suspected of containing ATP. In some embodiments, the assay system further includes a device for detecting and / or measuring luminescence (e.g., a luminometer; however, other light detection devices or apparatuses may be used). In some embodiments, the sample is a cell lysate.

[0133] In some embodiments, provided herein is a method for detecting or quantifying ATP in a sample, the method comprising: (a) adding to the sample a reagent composition comprising a compound of formula (I’) or formula (I) described herein; and (b) detecting luminescence. In some embodiments, the sample comprises cells, and the method further comprises lysing the cells to produce a cell lysate. In some embodiments, provided herein is a method for quantifying the amount or concentration of ATP in a sample, the method comprising: (a) adding to the sample a reagent composition comprising a compound of formula (I’) or formula (I) described herein; (b) quantifying luminescence from the sample; and (c) comparing the luminescence to a control value to determine the amount or concentration of ATP in the sample. In some embodiments, the control value is determined from separate quantifications of luminescence produced by a control sample comprising ATP at a known concentration. In some embodiments, the method further comprises adding ATP at a known concentration to the sample. In some embodiments, the luminescence is quantified at a plurality of time points. In some embodiments, the luminescence is quantified in real time.

[0134] In some embodiments, provided herein are methods, compositions, and kits for use in effectively and accurately detecting and quantifying cellular ATP levels. In some embodiments, luciferase and reagent compositions are used to detect surface ATP in cell-free samples (e.g., water) for purposes such as hygiene monitoring. In some embodiments, the method includes adding a single reagent composition comprising luciferase and luciferin or a luciferin analog (and optionally dehydro-luciferin) to a sample (e.g., a sample containing or suspected of containing ATP). In some embodiments, additional components and / or reagents (see above) are included with or added separately to the reagent composition (e.g., a kinase inhibitor, which is a compound that prevents the accumulation of ATP, a cell lysing agent (e.g., polyoxyethylene such as THESIT), an ATP extractant, magnesium, a buffer, salts, etc.). In some embodiments, including luciferase and luciferin or a luciferin analog in a single reagent speeds up ATP detection, simplifies assay and handling, and improves reproducibility.

[0135] As taken up throughout, the methods, compositions, and kits herein are particularly useful for the qualitative and quantitative detection of ATP (or an ATP analog that can function as a luciferase substrate) in a sample. In some embodiments, the presence of ATP is indicated from a simple qualitative experiment in which luminescence is generated in a sample using a reagent composition (e.g., a reagent composition comprising luciferase and luciferin). In some embodiments, an assay is provided for quantifying the amount of ATP in a sample. ATP can be detected (e.g., qualitatively) and / or quantified using the luciferase, reagent compositions, and / or kits herein at a single time point, at multiple time points, or in real time.

[0136] In some embodiments, the sample is any sample that contains or is suspected of containing ATP or a suitable ATP analog, such as a cell lysate, intact cells, a biopsy, food, beverage, water, a swab taken from a surface (e.g., the surface of an animal, plant, or inanimate object), etc. Other examples of samples include compositions of known ATP concentration. The cell or cell lysate can be from any organism (prokaryotic or eukaryotic). Eukaryotic cells can be from plants, animals, fungi, insects, etc., or cultured cells from such organisms. These examples are merely illustrative and are not intended to be limiting.

[0137] A cell lysate contains cell components that are no longer organized into a recognizable and intact cell structure. A cell lysate can contain both soluble and insoluble components, and either of these can be removed prior to use of the lysate. The lysate can be prepared by any means, including sonication, Dounce, mortar and pestle, freeze-thaw cycles, or physical disruption using any other device or process that breaks the physical integrity of the cells, or lysis with a detergent (e.g., a detergent that maintains luciferase activity, such as zwitterionic and non-ionic detergents, or the cationic detergent DTAB or CTAB). Preferably, the cell lysate is generated in a manner such that the integrity of the ATP concentration is maintained upon collection of the cells.

[0138] In some embodiments, to accurately detect ATP in the sample, enzymes that are thought to break down cellular ATP or enzymes that are thought to generate ATP are preferably inhibited or removed. Inhibitors of ATP-generating enzymes, i.e., enzymes that have ATP as a product or byproduct (e.g., kinase activity), can be incorporated into a reagent composition (e.g., a reagent composition containing luciferase and luciferin or a luciferin analog) or into a kit containing the reagent composition.

[0139] The luciferases, reagent compositions, methods, and kits herein enable a user to quantify the amount of ATP by quantifying the amount of luminescence. In some embodiments, the luciferase and luciferin or a luciferin analog are applied (in a single composition) to a test sample of interest. In some embodiments, the luciferase and luciferin or a luciferin analog are also applied (in a single composition) to a sample (control) containing a known amount of ATP. The magnitude of the signal generated from the test sample correlates with the ATP concentration in the sample. In some embodiments, the magnitude of the luminescence signal from a sample of unknown ATP concentration is correlated with the signal generated by either an internal control (adding a known amount of ATP to the sample and measuring the subsequent luminescence) or an external standard curve generated by measuring the luminescence of several samples of known ATP concentration and plotting them graphically.

[0140] 4. Examples Example 1. Synthesis Method I Generally, 5,5-disubstituted luciferin analogs were synthesized as described in Scheme 1. The 2-cyanobenzothiazole (CBT) derivative (A) was either a commercial product or synthesized according to a protocol described previously (Woodroofe, et al., Biochemistry, 2008, 47, 10383-10393; Woodroofe, et al., WO2014 / 159044 A1; Mofford, et al., J. Am. Chem. Soc. 2014, 136, 13277-13282). The β,β-disubstituted cysteine analog (B) was synthesized according to a protocol described previously (Stanfield, C.F. et al. J. Org. Chem. 1986, 51, 5153-5156).

[0141] The hydrochloride salt of β,β-disubstituted cysteine analog (B, 0.15 mmol, 1.5 equiv) dissolved in H2O (1 mL) under N2 was neutralized with 1 N NaOH (aqueous, 0.30 mmol, 300 μL, 3.0 equiv). The neutralized solution of the β,β-disubstituted cysteine analog was added to a solution of the CBT derivative (A) (0.1 mmol, 1.0 equiv) in DMF (2 mL) at room temperature under N2. Next, the solution was stirred for 30 minutes under N2. LC-MS indicated complete consumption of the CBT substrate (A). Pure product (I) was obtained by preparative HPLC (mobile phase A: 10 mM aqueous NH4OAc solution; mobile phase B: CH3CN; gradient conditions: from 5% B to 95% B over 30 minutes).

[0142] Table 1 below lists representative compounds prepared using the general procedure of Scheme 1. Table 1 JPEG0007696867000023.jpg113164 JPEG0007696867000024.jpg216164 JPEG0007696867000025.jpg216164 JPEG0007696867000026.jpg159164 * RLU was normalized based on that generated by 1 mM racemic luciferin under the same conditions.

[0143] Example 2. Thermal Stability Representative thermal stability profiling in luciferin: Luciferin stock solutions (pH = 6.0, [LH2] 最終 = 7.0 mM) containing various amounts of detergent were used with ULTRA-GLO™ luciferase ([enzyme] 最終= 0.1 mg / mL; Promega) with or without incubation at 60°C. Aliquots (20 μL) were taken at various time points, diluted with H2O (180 μL), and analyzed by RP-HPLC. The percentage of the components was calculated based on the UV absorbance at 330 nm for 6'-OH-luciferin and 295 nm for 6'-NH2-luciferin. As shown in Figure 2A, dehydro-luciferin was the main degradation product of luciferin, and luciferase had little effect on the degradation of luciferin.

[0144] Inhibition of luciferase activity by dehydro-luciferin: ULTRA-GLO™ luciferase (Promega, 0.1 mg / mL) + 0.1% PRIONEX® in the detection reagent buffer was prepared. The solution was divided into two parts, and 0.25 mM racemic H-luciferin was added to one part, and 0.25 mM racemic NH2-luciferin was added to the other part. H-dehydro-luciferin (0.25 mM) was added to an aliquot of the racemic H-luciferin solution, and NH2-dehydro-luciferin (0.25 mM) was added to an aliquot of the racemic NH2-luciferin solution. Serial 2× dilutions of each dehydro-luciferin sample were prepared using the racemic solution as a diluent (500 μL of the dehydro-luciferin solution was added to 500 μL of ULTRA-GLO™ + racemic luciferin). Next, 50 μL of each titration series was added to 50 μL of 0.1 mM ATP. The samples were incubated for 1 minute, and luminescence was measured on a GLOMAX®-Multi+ plate luminometer (n = 6). As shown in Figure 2B, dehydro-luciferin was a potent inhibitor of luciferase, which could explain the decrease in light output after storing the luciferin stock solution at ambient temperature for a long time.

[0145] Comparison of thermal stability: Various amounts of detergent and ULTRA-GLO™ luciferase ([enzyme] 最終A stock solution of luciferin (LH2) or 5,5-disubstituted luciferin (Luc-III, Figure 3A) containing 最終 = 7.0 mM) was incubated at 60 °C. Aliquots (20 μL) were taken at various time points, diluted with H2O (180 μL), and analyzed by RP-HPLC. The percentage of the components was calculated based on the UV absorbance at 330 nm for 6'-OH-luciferin and at 295 nm for 6'-NH2-luciferin. The results showed that 5,5-dimethyl-luciferin III-a (6-OH compound) and III-b (6-NH2 compound) were thermally more stable than unsubstituted luciferin (Figure 3B).

[0146] Example 3. Luminescence properties The compounds of the present disclosure were tested to determine the activity of the compounds of the present disclosure as substrates for various luciferase enzymes, including firefly green luciferase, firefly red luciferase, ULTRA-GLO™ luciferase, and Renilla luciferase. A solution of each enzyme at 0.05 mg / mL was prepared in BRIGHT-GLO™ assay buffer (Promega E263A) containing 3 mM ATP. Next, 3× serial dilutions of each enzyme were prepared in BRIGHT-GLO™ assay buffer containing 3 mM ATP, and 300 μL of each stock was serially diluted into 700 μL of buffer. A solution of each substrate (0.5 mM) was prepared in luciferin-free water, and 50 μL of each substrate solution was combined with 50 μL of the enzyme dilution. The samples were incubated at room temperature for 1 minute, and luminescence was measured on a GLOMAX®-Multi+ plate luminometer. As shown in Figures 4A-4D, the enzymes tested (firefly green, firefly red, ULTRA-GLO™, and Renilla luciferase) were able to generate light using the dimethyl substrates of the present disclosure. In these results, the firefly enzymes generated a stronger signal using the dimethyl substrates than ULTRA-GLO™ and Renilla luciferase.

[0147] As a means of comparing the binding affinity of the substrates, further tests were conducted at various substrate concentrations in the presence of sub-saturated D-luciferin. A solution of 0.005 mg / ml of firefly green luciferase (CBG) or UltoraGlo luciferase was prepared in Bright-Glo™ assay buffer (Promega E263A) containing 3 mM ATP. Next, a 2 mM solution of each substrate was prepared in water containing D-luciferin (sub-saturated). Each substrate + D-luciferin was serially diluted 2-fold with luciferin-free water. 50 μL of each sample of the substrate series was combined with 50 μL of the enzyme solution. The samples were incubated at room temperature for 1 minute and luminescence was measured on a GLOMAX®-Multi+ plate luminometer. Substrates having larger side chains at the 5,5 positions have higher apparent RLUmax values. This indicates that this substrate is less inhibitory to D-luciferin compared to the case of smaller 5,5-disubstituted side chains. From the apparent Km values, it is suggested that analogs having smaller 5,5-position side chains bind more tightly to the luciferase enzyme in the presence of D-luciferin compared to larger 5,5-position substitutions (FIGS. 5A-F). The RLU values (normalized relative to racemic luciferin under the same conditions) for certain representative compounds are shown in Table 1 above. For certain representative compounds, substrate titration tests were performed using the procedures described herein without comparison to luciferin. As a result, RLU values that are not normalized to the RLU value of luciferin were obtained.

[0148] The activity of the compounds of the present disclosure at various pHs was also tested. A stock buffer containing 25 mM of one of the following buffers: citric acid, MES, PIPES, HEPES, and TAPS was prepared. This buffer further contained 0.5% (v / v) Tergitol, 0.05% Mazu DF204, and 10 mM MgSO4. Aliquots of the buffer were taken and NaOH was added in various amounts to achieve a pH series. The exact pH of each solution was determined with a pH meter. ATP (1 mM) and firefly luciferase (CBG, 0.01 mg / mL) were added to each buffer in the pH series. A racemic mixture of each dimethyl substrate (0.2 mM) was prepared in luciferin-free water. In triplicate, each diluted substrate (50 μL) and each buffer in the pH series (50 μL) were combined and luminescence was measured immediately (t = 0) and after a 15-minute incubation at room temperature (t = 15) using a GLOMAX®-Multi+ plate luminometer. The results indicated that the racemic mixture of the dimethyl substrates was an active luciferase substrate over a pH range from approximately pH 5.6 to approximately pH 8.3 (Figures 6A - 6B). Thus, the compounds of the present disclosure can be utilized by firefly luciferase or Cypridina luciferase in both the L and D forms to produce bioluminescence over a wide range of pHs (Figures 6A - 6B).

[0149] To determine whether luciferin analogs having substitutions at the 5,5-position can be substrates for UltraGlo™ luciferase, assays were performed in a luminescence assay using analogs containing various disubstitutions of alkyl groups and a closed-ring structure. A solution of 0.005 mg / ml of UltraGlo™ luciferase (Promega Corp) prepared with 1×TBS + 1% Prionex was combined with 0.5 mM ATP in Bright-Glo™ assay buffer (Promega E263A). All substrates were diluted separately to 20 mM in DMSO. After incubation for 30 minutes at room temperature, the diluted substrates were added to a final concentration of 1 mM in the reaction and mixed by pipetting. The reaction was incubated for an additional 1 minute at room temperature and luminescence was measured on a GLOMAX®-Multi+ plate luminometer. Some of the luciferin analogs having larger substitutions at the 5,5-position (e.g., cyclohexyl (CS0392), dibenzyl (CS0396), and diethyl (CS0388)) all showed measurable luminescence values, demonstrating that UltraGlo can utilize these as substrates (Figure 7).

[0150] The embodiments and accompanying examples for carrying out the above invention are merely illustrative and should not be construed as limiting the scope of the invention. The scope of the invention is defined only by the appended claims and their equivalents.

[0151] Various changes and modifications to the embodiments of the present disclosure will be apparent to those skilled in the art. Such changes and modifications (including, but not limited to, changes and modifications regarding chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use in the present invention) can be made without departing from the spirit and scope of the present invention. Another aspect of the present invention may be as follows. [1] Formula (I’) TIFF0007696867000027.tif3974 (I’) [wherein, R 1 is hydrogen, halogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 1a , -NR 1b R 1c , -OG 1 , -NR 1x G 1 , or -NR 1x G 10 , R 2 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 2a , -NR 2b R 2c , -SR 2d , -SO 2 R 2e , -S(O)R 2f , -P(O)OR 2g R 2h , -OG 1 , or -NR 2x G 1 , R 3 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 3a , -NR 3b R 3c , -SR 3d , -SO 2 R 3e , -S(O)R 3f , -P(O)OR 3g R 3h , -OG 1 , or -NR 1x G 1 , R 4 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 4a, -NR 4b R 4c , -SR 4d , -SO 2 R 4e , -S(O)R 4f , -P(O)OR 4g R 4h , -OG 1 , or -NR 4x G 1 , R 5 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 5a , -NR 5b R 5c , -SR 5d , -SO 2 R 5e , -S(O)R 5f , -P(O)OR 5g R 5h , -OG 1 , or -NR 5x G 1 , or, R 2 and R 3 together with the atom to which they are attached, R 3 and R 4 together with the atom to which they are attached, or R 4 and R 5 together with the atom to which they are attached, form a 5- or 6-membered saturated ring, partially unsaturated ring, or fully unsaturated ring, and the 5- or 6-membered ring is O, N, S, NO, SO, and SO 2 Optionally containing, as ring members, one, two, or three heteroatoms or heteroatom groups independently selected from the group consisting of, wherein the 5- or 6-membered ring is optionally fused to aryl, heteroaryl, heterocyclic, or cycloalkyl, and the 5- or 6-membered ring is substituted with 0, 1, 2, 3, or 4 substituents, and the substituents are each independently halogen, =O, =S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclic, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl, acyl, -OG 1 , -NHG 1 , and -N(C 1 -C 10 alkyl)G 1 selected from the group consisting of, R 1a 、R 1b 、R 1c 、R 2a 、R2b 、R 2c 、R 2d 、R 2e 、R 2f 、R 2g 、R 2h 、R 3a 、R 3b 、R 3c 、R 3d 、R 3e 、R 3f 、R 3g 、R 3h 、R 4a 、R 4b 、R 4c 、R 4d 、R 4e 、R 4f 、R 4g 、R 4h 、R 5a 、R 5b 、R 5c 、R 5d 、R 5e 、R 5f 、R 5g , and R 5h are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, and cycloalkenyl, R 1x 、R 2x 、R 3x 、R 4x , and R 5x are each independently hydrogen or C 1 -C 12 alkyl, G 1 contains a substrate of a first enzyme, and the in vivo conversion of the substrate by the first enzyme is to convert G 1 to H, -NR 1x G 10 is a group cleavable by a second enzyme to convert the -NR 1x G 10 group to -OH, and W 1 and W 2 are each independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, and cycloalkenyl, or W 1 and W 2 together with the carbon to which they are attached form cycloalkyl, cycloalkenyl, or heterocyclic, In each occurrence, the alkyl, alkenyl, alkynyl, alkoxy, heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, and cycloalkenyl are independently substituted with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substituents, and each of the substituents is independently selected from the group consisting of halogen, =O, =S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclic, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl, and acyl] or a tautomer or salt thereof (provided that the compound is not 2-(6-hydroxybenzothiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid or 2-(6-aminobenzothiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid). [2] Formula (I) TIFF0007696867000028.tif3974 (I) [wherein, R 1 is hydrogen, halogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, cycloalkenyl, -OR 1a , or -NR 1b R 1c , R 2 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, cycloalkenyl, -OR 2a , -NR 2b R 2c , -SR 2d , -SO 2 R 2e , -S(O)R 2f , or -P(O)OR 2g R 2h , R 3 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, cycloalkenyl, -OR 3a , -NR 3b R 3c , -SR3d , -SO 2 R 3e , -S(O)R 3f 、or -P(O)OR 3g R 3h wherein R 4 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 4a , -NR 4b R 4c , -SR 4d , -SO 2 R 4e , -S(O)R 4f , or -P(O)OR 4g R 4h wherein R 5 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 5a , -NR 5b R 5c , -SR 5d , -SO 2 R 5e , -S(O)R 5f , or -P(O)OR 5g R 5h wherein alternatively, R 2 and R 3 together with the atom to which they are attached form 3 and R 4 together with the atom to which they are attached form, or R 4 and R 5 together with the atom to which they are attached form a 5- or 6-membered saturated, partially unsaturated, or fully unsaturated ring, said 5- or 6-membered ring optionally containing, as ring members, 1, 2, or 3 heteroatoms or heteroatom groups each independently selected from the group consisting of O, N, S, NO, SO, and SO 2 , and said 5- or 6-membered ring is optionally fused to aryl, heteroaryl, heterocycle, or cycloalkyl, and said 5- or 6-membered ring is substituted with 0, 1, 2, 3, or 4 substituents, said substituents each independently being selected from the group consisting of halogen, =O, =S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl, and acyl R 1a 、R 1b 、R 1c 、R 2a 、R 2b 、R 2c 、R 2d 、R2e 、R 2f 、R 2g 、R 2h 、R 3a 、R 3b 、R 3c 、R 3d 、R 3e 、R 3f 、R 3g 、R 3h 、R 4a 、R 4b 、R 4c 、R 4d 、R 4e 、R 4f 、R 4g 、R 4h 、R 5a 、R 5b 、R 5c 、R 5d 、R 5e 、R 5f 、R 5g , and R 5h is independently selected from hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl, and W 1 and W 2 are each independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl, or W 1 and W 2 together with the carbon to which they are attached form a cycloalkyl, cycloalkenyl, or heterocycle, in each occurrence, the alkyl, alkenyl, alkynyl, alkoxy, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl are independently substituted with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substituents, and each of the substituents is independently halogen, =O, =S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl, and acyl selected from the group consisting of] a compound or a tautomer or salt thereof (provided that the compound is not 2-(6-hydroxybenzothiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid or 2-(6-aminobenzothiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid). 〔3〕R 1 The compound according to [2] above, wherein 〔4〕R 2 、R 3 、R 4 is -OH. 5and R 1 -C 4 are each independently hydrogen, halogen, cyano, nitro, C 2 -C 4 -alkyl, C -alkenyl, C 2 -C 4 -alkynyl, -OH, and -NH 2 The compound according to [2] above, which is selected from the group consisting of 〔5〕R 4 is -OR 4a or -NR 4b R 4c The compound according to [2] above, which is 〔6〕R 3 and R 4 The compound according to [2] above, wherein together with the atom to which they are attached, they form a 5- or 6-membered, optionally substituted, saturated, partially unsaturated, or fully unsaturated ring 〔7〕R 4 and R 5 The compound according to [2] above, wherein together with the atom to which they are attached, they form a 5- or 6-membered, optionally substituted saturated, partially unsaturated, or fully unsaturated ring 〔8〕R 3 and R 4 The compound according to [2] above, wherein together with the atom to which they are attached, they form a 6-membered optionally substituted heterocyclic ring, and R 4 and R 5 The compound according to [2] above, wherein together with the atom to which they are attached, they form a 6-membered optionally substituted heterocyclic ring, and at this time, the two 6-membered rings are fused 〔9〕W 1 and W 2 The compound according to [2] above, wherein each is alkyl 〔10〕W 1 and W 2 The compound according to [2] above, wherein together with the atom to which they are attached, they form cycloalkyl

[11] Formula (I-a) TIFF0007696867000029.tif4182 (I-a) [wherein, A and B are each independently, optionally, a 5- or 6-membered ring selected from the group consisting of aryl, heteroaryl, and heterocyclic ring, and A, if present, is optionally substituted with one or more R A and B, if present, is optionally substituted with one or more R B , and each of R A and R B , if present, is independently alkyl, halo, haloalkyl, hydroxyalkyl, -OH, -NH 2 , or alkyl-NH-, When both A and B are absent, Q is -OR Q1 or -NR Q1 R Q1 , and R Q1 in each occurrence is independently hydrogen, alkyl, or hydroxyalkyl, or when at least one of A and B is present, Q is C, CR Q2 , CR Q2 R Q2, N, NR Q2 , or O, and R Q2 in each occurrence is independently hydrogen, alkyl, or hydroxyalkyl, R is alkyl, alkoxy, halo, haloalkyl, hydroxyalkyl, hydroxy, cyano, nitro, amino, alkylamino, dialkylamino, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclic ring, cycloalkyl, or cycloalkenyl, p is 0, 1, 2, or 3] The compound according to [2] above having the same, or its tautomer or salt

[12] Formula (I-b) or (I-c) TIFF0007696867000030.tif2874 (I-b) TIFF0007696867000031.tif3177 (I-c) [wherein, m is 0, 1, 2, or 3, p is 0, 1, or 2] The compound according to the above

[11] , having the same.

[13] Formula (I-d) TIFF0007696867000032.tif3674 (I-d) [wherein, n is 0, 1, 2, or 3, p is 0, 1, or 2] The compound according to the above

[11] , having the same.

[14] Formula (I-e) TIFF0007696867000033.tif3679 (I-e) [wherein, m is 0, 1, 2, or 3, n is 0, 1, 2, or 3, p is 0 or 1] The compound according to the above

[11] , having the same.

[15] Formula (I-f) TIFF0007696867000034.tif3195 (I-f) [wherein n is 0, 1, 2, 3, 4, 5, 6, 7, or 8] The compound according to the above

[11] , having the same.

[16] Formula (I-g) TIFF0007696867000035.tif2869 (I-g) [wherein, TIFF0007696867000036.tif1825 is, TIFF0007696867000037.tif26164 and each of these is optionally substituted]

[17] The following compounds: 5-(6-Hydroxybenzothiazol-2-yl)-4-thia-6-azaspiro[2.4]hept-5-ene-7-carboxylic acid; 6-(6-Hydroxybenzothiazol-2-yl)-5-thia-7-azaspiro[3.4]oct-6-ene-8-carboxylic acid; 2-(6-Hydroxybenzothiazol-2-yl)-1-thia-3-azaspiro[4.4]nona-2-ene-4-carboxylic acid; 2-(6-Hydroxybenzothiazol-2-yl)-1-thia-3-azaspiro[4.5]deca-2-ene-4-carboxylic acid; 5,5-Diethyl-2-(6-hydroxybenzothiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid; (S)-2-(5-Fluoro-6-hydroxybenzothiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid; (R)-2-(5-Fluoro-6-hydroxybenzothiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid; (S)-2-(7-Aminonaphtho[2,1-d]thiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid; (S)-5,5-Dimethyl-2-(6-(pyrrolidin-1-yl)benzothiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid; (S)-2-(6,7-Dihydro-5H-thiazolo[4,5-f]indol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid; (S)-2-(6-((3-Hydroxypropyl)amino)benzo[d]thiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid; 2-(5-Fluoro-6-hydroxybenzo[d]thiazol-2-yl)-1-thia-3-azaspiro[4.5]deca-2-ene-4-carboxylic acid; 5,5-Dibenzyl-2-(6-hydroxybenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid; 5,5-Diethyl-2-(5-fluoro-6-hydroxybenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid; 2-(6-Hydroxybenzo[d]thiazol-2-yl)-1-thia-3-azaspiro[4.6]undec-2-ene-4-carboxylic acid; 2-(6-Hydroxybenzo[d]thiazol-2-yl)-8-methyl-1-thia-3-azaspiro[4.5]deca-2-ene-4-carboxylic acid; 2-(6-Hydroxybenzo[d]thiazol-2-yl)-8-methyl-1-thia-3,8-diazaspiro[4.5]deca-2-ene-4-carboxylic acid; 2-(6-Hydroxybenzo[d]thiazol-2-yl)-5,5-dipropyl-4,5-dihydrothiazole-4-carboxylic acid; and 2-(6-Hydroxybenzo[d]thiazol-2-yl)-8-oxa-1-thia-3-azaspiro[4.5]deca-2-ene-4-carboxylic acid, The compound according to [2] above, or a tautomer or salt thereof, selected from the group consisting of.

[18] Formula (II) TIFF0007696867000038.tif3275 (II) [Wherein, R 4 is -OG 1 , -NHG 1 , or -N(C 1 -C 12 alkyl)G 1 The compound according to [1] above, or a tautomer or salt thereof, which is a compound of.

[19] Formula (II-a) (II-a) TIFF0007696867000039.tif3389 [Wherein, G is a peptide] 2 The compound according to

[18] above, or a tautomer or salt thereof, which is a compound of. The compound according to

[19] above, or a tautomer or salt thereof, which is a substrate for a non-luciferase enzyme. 〔20〕G 2

[21] A kit containing a 5,5-disubstituted luciferin analog.

[22] The kit according to

[21] above, wherein the 5,5-disubstituted luciferin analog is the compound according to any one of [1] and

[18] to

[20] . ​ [

[23] ] The kit according to [

[21] ], wherein the 5,5-disubstituted luciferin analog is the compound according to any one of [[2]] to [

[17] ], 2-(6-hydroxybenzo[d]thiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid, or 2-(6-aminobenzo[d]thiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid. [

[24] ] The kit according to [

[21] ], further comprising luciferase. [

[25] ] The kit according to [

[21] ], further comprising a buffer reagent. [

[26] ] A method for detecting luminescence in a sample, comprising: contacting the sample with a 5,5-disubstituted luciferin analog; when luciferin-utilizing luciferase does not exist in the sample, contacting the sample with luciferin-utilizing luciferase; and detecting luminescence. [

[27] ] The method according to [

[26] ], wherein the 5,5-disubstituted luciferin analog is the compound according to any one of [[1]] and [

[18] ] to [

[20] ]. [

[28] ] The method according to [

[26] ], wherein the 5,5-disubstituted luciferin analog is the compound according to any one of [[2]] to [

[17] ], 2-(6-hydroxybenzo[d]thiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid, or 2-(6-aminobenzo[d]thiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid. [

[29] ] The method according to [

[26] ], wherein the sample contains living cells. [

[30] ] The method according to [

[26] ], wherein the sample contains luciferin-utilizing luciferase. [

[31] ] A method for detecting luminescence in a transgenic animal, comprising: administering a 5,5-disubstituted luciferin analog to the transgenic animal; and detecting luminescence, wherein the transgenic animal expresses luciferin-utilizing luciferase. [

[32] ] The method according to [

[31] ], wherein the 5,5-disubstituted luciferin analog is the compound according to any one of [[1]] and [

[18] ] to [

[20] ]. ​ 〔33〕The method according to the above

[31] , wherein the 5,5-disubstituted luciferin analog is the compound according to any one of the above [2] to

[17] , 2-(6-hydroxybenzothiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid, or 2-(6-aminobenzothiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylic acid. 〔34〕A method for detecting ATP in a sample, comprising: (a) adding a reagent composition containing the compound according to any one of the above [1] to

[20] to the sample; and (b) detecting luminescence.

Claims

1. An in vitro method for detecting luminescence in a sample containing luciferin-utilizing firefly luciferase or luciferin-utilizing click beetle luciferase, the method comprising contacting the sample with a compound of formula (I) (I) [wherein, R 1 is -OR 1a and R 2 is hydrogen R 3 is hydrogen or halogen R 4 is -OR 4a and R 5 is hydrogen or R 3 and R 4 together with the atom to which they are attached, or R 4 and R 5 together with the atom to which they are attached form a 5- or 6-membered saturated, partially unsaturated, or fully unsaturated ring, the 5- or 6-membered ring optionally containing one heteroatom selected from the group consisting of O, N, and S, the 5- or 6-membered ring being substituted with 0 or 1 amino substituent, R 1a and R 4a are each independently selected from the group consisting of hydrogen and alkyl, and W 1 and W 2 are each independently selected from the group consisting of C2-C4 alkyl and arylalkyl]] and a step of contacting with a tautomer or salt thereof.

2. The method according to claim 1, wherein R 1 is -OH.

3. R 3 and R 4are, together with the atoms to which they are attached, forming a 5- or 6-membered, saturated, partially unsaturated, or fully unsaturated ring, or R 4 and R 5 are, together with the atoms to which they are attached, forming a 5- or 6-membered, saturated, partially unsaturated, or fully unsaturated ring, the method according to claim 1. **Claim 4** W 1 and W 2 are each C2-C4 alkyl, the method according to claim 1. **Claim 5** The compound is the following compound: 5,5-diethyl-2-(6-hydroxybenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid; 5,5-dibenzyl-2-(6-hydroxybenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid; 5,5-diethyl-2-(5-fluoro-6-hydroxybenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid; and 2-(6-hydroxybenzo[d]thiazol-2-yl)-5,5-dipropyl-4,5-dihydrothiazole-4-carboxylic acid, a compound selected from the group consisting of or a tautomer or salt thereof, the method according to claim 1. **Claim 6** The sample contains living cells, the method according to claim 1. **Claim 7** An in vitro method for detecting ATP in a sample containing luciferin-utilizing firefly luciferase or luciferin-utilizing Gaussia luciferase, (a) To the sample, a compound of formula (I) (I) [wherein, R 1 is -OR 1a and R 2 is hydrogen, R 3 is hydrogen or halogen, R 4 is -OR 4a wherein R 5 is hydrogen, or alternatively, R 3 and R 4 together with the atom to which they are attached, or R 4 and R 5 together with the atom to which they are attached, form a 5- or 6-membered saturated, partially unsaturated, or fully unsaturated ring, said 5- or 6-membered ring optionally containing one heteroatom selected from the group consisting of O, N, and S, and said 5- or 6-membered ring being substituted with 0 or 1 amino substituent, R 1a and R 4a are each independently selected from the group consisting of hydrogen and alkyl, and W 1 and W 2 are each independently selected from the group consisting of C2-C4 alkyl and arylalkyl]], adding a reagent composition comprising a compound or a tautomer or salt thereof; and (b) detecting luminescence; A method comprising:

8. R 1 is -OH, the method according to claim 7.

9. R 3 and R 4 together with the atom to which they are attached, form a 5- or 6-membered saturated, partially unsaturated, or fully unsaturated ring, or R 4 and R 5 together with the atom to which they are attached, form a 5- or 6-membered saturated, partially unsaturated, or fully unsaturated ring, the method according to claim 7.

10. W 1 and W 2The method according to claim 7, wherein each is C2-C4 alkyl. **Claim 11** The compound is the following compound: 5,5-Diethyl-2-(6-hydroxybenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid; 5,5-Dibenzyl-2-(6-hydroxybenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid; 5,5-Diethyl-2-(5-fluoro-6-hydroxybenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid; and 2-(6-Hydroxybenzo[d]thiazol-2-yl)-5,5-dipropyl-4,5-dihydrothiazole-4-carboxylic acid, The method according to claim 7, which is a compound selected from the group consisting of or a tautomer or salt thereof. **Claim 12** The method according to claim 7, wherein the sample contains living cells. **Claim 13** (a) Formula (I) (I) [Wherein, R 1 is -OR 1a and R 2 is hydrogen, R 3 is hydrogen or halogen, R 4 is -OR 4a and R 5 is hydrogen, or R 3 and R 4 together with the atom to which they are attached, or R 4 and R 5together with the atoms to which they are attached form a 5- or 6-membered saturated, partially unsaturated, or fully unsaturated ring, said 5- or 6-membered ring optionally containing one heteroatom selected from the group consisting of O, N, and S, said 5- or 6-membered ring being substituted with 0 or 1 amino substituents, R 1a and R 4a are each independently selected from the group consisting of hydrogen and alkyl, and W 1 and W 2 are each independently selected from the group consisting of C2-C4 alkyl and arylalkyl]; a compound thereof or a tautomer or salt thereof; and (b) luciferin-utilizing firefly luciferase or luciferin-utilizing click beetle luciferase A kit for detecting luminescence, comprising

14. The kit according to claim 13, further comprising a buffer reagent.

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