USOO847645OB2

(12) United States Patent (10) Patent No.: US 8.476,450 B2 Cali et al. (45) Date of Patent: *Jul. 2, 2013

(54) LUMINOGENIC AND FLUOROGENIC 5,283,179 A 2, 1994 Wood COMPOUNDS AND METHODS TO DETECT 5,283,180 A 2f1994 Zomer et al. 5,290,684 A 3/1994 Kelly MOLECULES OR CONDITIONS 5,374,534 A 12/1994 Zomer et al. 5,498.523 A 3, 1996 Tabor et al. (75) Inventors: James J. Cali, Verona, WI (US); 5,641,641 A 6, 1997 Wood William Daily, Santa Maria, CA (US); 5,650,135 A 7/1997 Contaget al. 5,650,289 A T/1997 Wood Erika Hawkins, Pembroke (CA); Dieter 5,726,041 A 3/1998 Chrespi et al. Klaubert, Arroyo Grande, CA (US); 5,744,320 A 4/1998 Sherf et al. Jianquan Liu, Fremont, CA (US); 5,756.303 A 5/1998 Sato et al. Poncho Meisenheimer, San Lius 5,780,287 A * 7/1998 Kraus et al...... 435,236 Obispo, CA (US); Michael Scurria, San 5,814,471 A 9, 1998 Wood 5,876,946 A 3, 1999 Burbaum et al. Luis Obispo, CA (US); John Shultz, 5,976,825 A 11/1999 Hochman et al. Verona, WI (US); James Unch, Arroyo 6,143,492 A 11/2000 Makings et al. Grande, CA (US); Michael P. Valley, 6,217,847 B1 4/2001 Contag et al. Fitchburg, WI (US); Keith V. Wood, Mt. 6,291,164 B1 9/2001 Blakesly et al. Horeb, WI (US); Wenhui Zhou, Santa 6,299,858 B1 10/2001 Serbedzija et al. 6,376,208 B1 4/2002 Kajiyama Maria, CA (US) 6.420,130 B1 7/2002 Makings et al. 6,514,687 B1 2/2003 Makings et al. (73) Assignee: Promega Corporation, Madison, WI 6,638,713 B2 10/2003 Makings et al. (US) 7,118,878 B1 10/2006 Hawkins et al. 7,524,876 B2* 4/2009 Takakura et al...... 514,367 (*) Notice: Subject to any disclaimer, the term of this 2002/0076777 A1 6/2002 Merkulov et al. 2003/0211560 A1 11/2003 O'Brien et al. patent is extended or adjusted under 35 2003/0237103 A1 12/2003 Jacob et al. U.S.C. 154(b) by 0 days. 2004/014.6959 A1 7/2004 Graham et al. 2004/0171099 A1 9, 2004 Cali et al. This patent is Subject to a terminal dis 2004/0248225 A1 12/2004 Heindlet al. claimer. (Continued) (21) Appl. No.: 12/217,494 FOREIGN PATENT DOCUMENTS EP O41 1912 2, 1991 (22) Filed: Jul. 3, 2008 JP 63-5O1571 6, 1988 JP O1-5O2431 8, 1989 (65) Prior Publication Data JP 2002-080476 3, 1990 JP 08-059686 3, 1996 US 2008/O299593 A1 Dec. 4, 2008 JP 2000-505-086 4/2000 JP 2000-270894 10, 2000 Related U.S. Application Data JP 2002080476 3, 2002 JP 2006219381 8, 2006 (63) Continuation of application No. 1 1/444,145, filed on RU 22424.71 12, 2004 May 31, 2006. (Continued) (60) Provisional application No. 60/685,957, filed on May 31, 2005, provisional application No. 60/693,034, OTHER PUBLICATIONS filed on Jun. 21, 2005, provisional application No. White et al., Journal of the American Chemical Society, (1966), 60/692.925, filed on Jun. 22, 2005, provisional 88(9), pp. 2015-2018.* application No. 60/790,455, filed on Apr. 7, 2006. “5” Labelling Fluorescein and Cyanine Dyes, Biotin”, Glen Report, vol. 8, No. 2, online). Retrieved from the Internet: . (Dec. 1995), 8 pgs. A6 IK3I/428 (2006.01) “U.S. Appl. No. 10/665,314. Response to Restriction Requirement CO7D 417/04 (2006.01) filed Jul. 24, 2006', 37 pgs. “U.S. Appl. No. 10/665,314, Response filed May 19, 2008 to Final CO7D 417/4 (2006.01) Office Action mailed Mar. 19, 2008, 17. (52) U.S. Cl. USPC ...... 548/178; 514/367 (Continued) (58) Field of Classification Search USPC ...... 548/178 Primary Examiner — Laura L. Stockton See application file for complete search history. (74) Attorney, Agent, or Firm — Michael Best & Friedrich LLP (56) References Cited (57) ABSTRACT U.S. PATENT DOCUMENTS A method to detect the presence or amount of at least one 4,655,022 A 4, 1987 Natori molecule in a sample which employs a derivative of luciferin 4,665,022 A 5, 1987 Schaeffer et al. or a derivative of a fluorophore is provided. Compounds and 4,826,989 A 5, 1989 Batz et al. 4,992,531 A 2f1991 Patroni et al. compositions for carrying out the methods of the invention 5,035,999 A * 7/1991 Geiger et al...... 435/23 are also provided. 5,098,828 A 3/1992 Geiger et al. 5,114,704 A 5/1992 Spanier et al. 2 Claims, 49 Drawing Sheets US 8.476.450 B2 Page 2

U.S. PATENT DOCUMENTS “Beilstein Registry No. 30484', Database Crossfire Beilstein, 2005/OOO9098 A1 1/2005 Reymonds et al. (White, et al., J. Am. Chem. Soc., vol. 85 (1963), 337-343,(prior to 2005/0026171 A1 2/2005 Hawkins et al. May 31, 2006), 12 pgs. 2005, 0118257 A1 6, 2005 Bova “Beilstein Registry No. 3984932', Database Crossfire Beilstein, 2005. O153306 A1 7, 2005 Harris (White, et al., J. Org. Chem. vol.30 (1965), 2344-2348,(prior to May 2007, OO15790 A1 1/2007 Cali et al. 31, 2006), 2 pgs. 2007/O155806 A1 7/2007 Takakura et al. 2008. O194522 A1 8, 2008 Chen et al. “Beilstein Registry No. 4240164”. Database Crossfire Beilstein, 2009/0023173 A1 1/2009 Cali et al. (Arness, R., et al., Carbohydr. Res., vol. 1, No. 1 (1990), 225 2010.0062470 A1 3/2010 Corona et al. 233.(prior to May 31, 2006), 2 pgs. 2011/000331.6 A1 1/2011 Cali et al. “Beilstein Registry No. 926292, Database Crossfire Beilstein, (White, et al., J. Org. Chem... vol.30 (1965), 2344-2348,(prior to May FOREIGN PATENT DOCUMENTS 31, 2006), 2 pgs. WO 87,02667 5, 1987 “Chemistry 2131: Organic Chemistry for the Life Sciences (3)”. WO 88.05434 T 1988 online). Retrieved from the Internet: , (Mount Allison WO 97.36885 10, 1997 WO WO-99,60096 A2 11/1999 University),(observed Dec. 17, 2004), 3 pgs. WO OOf 16763 3, 2000 “Database Accession No. 4240164”. Dateabase Crossfire Beilstein WO WO-00/34506 A2 6, 2000 Beilstein Institute zur Foerderung der chemischen Wissenschafien, WO WO-OO,35900 A1 6, 2000 Frankfurt am Main, De, (1991), 2 pgs. WO WO-01/20002 A1 3, 2001 “European Application No. 03749715.3 Supplemental European WO WO-03/040 100 A1 5, 2003 Search Report mailed Jun. 14, 2007, 5 pgs. WO WO-03/066611 A1 8, 2003 “European Application Serial No. 06771475.8. Office Action mailed WO WO-2004/O27378 A2 4/2004 Jun. 12, 2008”, (Feb. 13, 2008), 5 pgs. WO WO-2006130551 A2 12/2006 “European Application Serial No. 08151520.7, European Search WO 2010/021686 2, 2010 Report mailed Jun. 5, 2008”, 16 pgs. OTHER PUBLICATIONS “International Search Report for corresponding PCT Application No. PCT/US2006/020731, (Mar. 13, 2007), 8 pgs. “U.S. Appl. No. 10/665,314. Restriction Requirement mailed Mar. “Japanese Application Serial No. 2004-537859, Office Action mailed 22, 2006”, 8 pgs. Aug. 13, 2008”, (w? English Translation).4 pgs. “U.S. Appl. No. 10/665,314. Supplemental Response filed Jun. 19. “Partial International Search Report for corresponding PCT Appli 2008 to Advisory Action mailed Jun. 6, 2008”, 18 pgs. cation No. PCT/US2006/020731”. (Oct. 2, 2006), 5 pgs. “PCT Application No. PCT/US03/29078 International Search “U.S. Appl. No. 10/665,314, Final Office Action mailed Mar. 19, Report mailed Oct. 8, 2004'', 4pgs. 2008”, 7 pgs. “Written Opinion in the corresponding PCT Application No. PCT/ “U.S. Appl. No. 10/665,314, Non-Final Office Action mailed Oct. 11, US2006/020731, (Mar. 13, 2007), 9 pgs. 2007”. 16 pgs. Allen, T., et al., “Cloning and Expression of the “U.S. Appl. No. 10/665,314, Amendment and Response filed Jan. 10, Phophoribosyltransferase Gene from Leishmania donovani', 2008 to Office Action mailed Oct. 11, 2007, 39 pgs. Molecular and Biochemical Parasitology, 74. (1995).99-103. “U.S. Appl. No. 10/665,314, Non-Final Office Action mailed Jul 18, Amess, R., et al., “Synthesis of luciferinglycosides as Substrates for 2008”, 14pgs. novel untrasensitive enzyme assays”. Carbohydrate Research, 205, “U.S. Appl. No. 10/665,314, Response filed Oct. 20, 2008 to Non (1990).225-233. Final Office Action mailed Jul. 18, 2008”, 25 pgs. Ben-Shlomo. Y., et al., “Using Monoamine Oxidase Type B Inhibi “U.S. Appl. No. 1 1/144,145. Non-Final Office Action mailed Mar. tors in Parkinson's Disease'. BMJ. vol. 329, (Sep. 11, 2004),581 24, 2008”, 16 pgs. 582. “U.S. Appl. No. 1 1/444,145, Responsed filed Oct. 11, 2007 to Binda, C. et al., “Structure-Function Relationships in Flavoenzyme Restriction mailed Aug. 1, 2007, 25 pgs. Dependent Amine Oxidations—A Comparison of Polyamine “U.S. Appl. No. 1 1/444, 145, Non-Final Office Action Mailed Oct. Oxidase and Monoamine Oxidase'. The Journal of Biological Chem 15, 2008”, 19 pgs. istry, 277(27), (2002).23973-23976. “U.S. Appl. No. 1 1/444,145, Preliminary Amendment mailed Aug. Black, S. D., et al., “P-450 Cytochromes: Structure and Function'. 22, 2006', 3 pgs. Adv. Enzymol. Relat. Areas Mol. Biol. 60, (1987),35-87. “U.S. Appl. No. 1 1/444,145, Response filed Jun. 24, 2008 to Non Bowie, et al., “Synthesis of a New Substrate Analog of Firefly Final Office Action mailed Mar. 24, 2008”, 53 pgs. Luciferin.”. Biochemistry, vol. 12(10), 1973, pp. 1845-1852, “U.S. Appl. No. 1 1/444,145, Restriction Requirement mailed Aug. 1, (1979), 1845-1852. 2007”, 7 pgs. Branchini, B. R., et al., “Naphthyl- and quinolylluciferin: green and “Beilstein Registry No. 1034055”. Database Crossfire Beilstein, redlight emitting firefly luciferin analogues'. Photochem Photobiol. (McCapra, F., et al., Chem. Commun. (1968), 22-23,(prior to May 31, 49(5), (1989),689-95. 2006), 1 pg. Branchini, B. R. “Naphtyl- and Quinolylluciferin: Green and Red “Beilstein Registry No. 1041968”. Database Crossfire Beilstein, Light Emitting Firefly Luciferin Analogues'. Photochemistry and (White, et al., Bioorg. Chem... vol. 1 (1971),92-116),(prior to May 31, Photobiology, 49(5), (1989),689-695. 2006), 2 pgs. Charing, Y, et al., “Molecular Cloning and Expression of the Gene “Beilstein Registry No. 11 19094'. Database Crossfire Beilstein, Encoding ADP-Glucose Pyrophosphorylase from the (Benkoe, et al., Montsh. Chem... vol. 106 (1975), 1027-1032),(prior Cyanobacterium Anabaena sp. Strain PCC 7120”. Plant Molecular to May 31, 2006), 3 pgs. Biology, 20, (1992),37-47. “Beilstein Registry No. 11 19095”. Database Crossfire Beilstein, Chen, K., et al., “R1, a Novel Repressor of the Human Monoamine (Benkoe, A., et al., Monatsh. Chem... vol. 106 (1975), 1027 Oxidase A'. The Journal of Biological Chemistry, 280(12), 1032),(prior to May 31, 2006), 2 pgs. (2005), 11552-11559. “Beilstein Registry No. 1126922. Database Crossfire Beilstein, Craig, F. F., et al., “Membrane-permeable luciferinesters for assay of (Benkoe, et al., Monatsh. Chem... vol. 106, (1975), 1027-1032.(prior firefly luciferase in live intact cells'. Biochemical Journal, 276(3), to May 31, 2006), 3 pgs. (1991),637-641. “Beilstein Registry No. 1129927. Database Crossfire Beilstein, Demir, B., et al., “Platelet Monoamine Oxidase Activity in Alcohol (Benkoe, et al., Monatsh. Chem. vol. 106 (1975), 1027-1032.(prior ism Subtypes: Relationship to Personality Traits and Executive Func to May 31, 2006), 2 pgs. tions”, Alcohol & Alcoholism, 37(6), (2002),597-602. US 8.476.450 B2 Page 3

Dukhovich, A., et al., “Time course of luciferyladenylate synthesis in Ladror, U. , et al., "Cloning, Sequencing, and Dependent the firefly luciferase reaction'. FEBS Letters, 395(2-3), (Oct. 21. Pyrophosphate-Dependent Phosphofructokinase from 1996), 188-190. Propionibacterium freudenreichii', The Journal of Biological Eriksson, J., et al., “Method for Real Time Detection of Inorganic Chemistry, 266(25), (1991), 16550-16555. Pyrophosphatase Activity”. Anal. Biochem., 293(1), (2001),67-70. Leemann, T., et al., “Cytochrome P450TB (CYP2C): A Major Eriksson, J., et al., “Method for real-time detection of inorganic Monooxygenase Catalyzing Diclofenac 4-Hydroxylation in Human pyrophosphatase activity”. Analytical Biochemistry,293, (2001),67 Liver, Life Sci., 52(1), (1993).29-34. 7O. Lembert, N., “Firefly luciferase can use L-luciferinto produce light'. Farace, C., et al., “Synthesis and characterization of a new Substrate Biochemical Journal, 317(Pt 1), (Jul. 1, 1996).273-277. of Photinus pyralis luciferase: 4-methyl-D-luciferin'. Journal of Leyh, T., et al., “The Sulfate Activation Locus of Escherichia coli Clinical Chemistry and Clinical Biochemistry, 28(7), (1990),471 K12: Cloning, Genetic, and Enzymatic Characterization'. The Jour 474. nal of Biological Chemistry, 263(5), (1988).2409-2416. Feldmann, R., et al., “Decreased Metabolism and Viability of Ludin, K. M., et al., “The Ade4 Gene of Schizosaccharomyces Mycoplasma hominis Induced by Monoclonal Antibody-Mediated pombe: Cloning, Sequence and Regulation'. Curr Genet, 25. Agglutination'. Infection and Immunitty, 60(1), (1992), 166-174. (1994),465-468. Flickinger, B., “Using Metabolism Data in Early Development”. Mancy, A., et al., “Diclofenac and its Derivatives as Tools for Study Drug Disc. Dev, 4(9), (2001),53-56. ing Human Cytochromes P450 Active Sites: Particular Efficiency and Gäbelova, A., et al., “Mutagenicity of 7H-Dibenzoc.g|Carbazole Regioselectivity of P450 2Cs”, Biochemistry, 38, (1999), 14264 and its Tissue Specific Derivatives in Genetically Engineered Chi 14270. nese Hamster V79 Cell Lines Stably Expressing Cytochrome P450”. Marolda, C., et al., “Identification, Expression and DNA Sequence of Mutation Research, 517. (2002), 135-145. the GDP-Mannose Biosynthesis Genes Encoded by the 07 rifth Gene Gandelman, O., et al., “Cytoplasmic factors that Affect the intensity Cluster of Strain VW 187 (Escherichia coli 07:K1)”,Journal of Bac and stability of Bioluminescence from firefly luciferase in living teriology, 175(1), (1993), 148-158. mammalian cells.'. Journal of Bioluminescence and Masuda-Nishimura, I., et al., “Development of a rapid positive/ab Chemiluminescence, 9(6), (1994).363-371. sent test for coliforms using sensitive bioluminescence assay'. Lett. Gómez-Lechon, M. J., et al., “Expression and Induction of a Large Appl. Microbiol.: 30, (2000), 130-135. Set of Drug-Metabolizing Enzymes by the Highly Differentiated Masuda-Nishimura, I., et al., “Development of a rapid positive/ab Human Heptoma Cell Line BC2'. Eur: J. Biochem., 268, sent test for coliforms using sensitive bioluminescence assay'. Lett (2001), 1448-1459. Appl Microbiol. 30(2), (2000), 130-135. Graham-Lorence, S., et al., “P450s: Structural Similarities and Func Miller, V. P. et al., “Fluorometric High-Throughput Screening for tional Differences”. The FASEB Journal, 10, 206-214.(1996).206 Inhibitors of Cytochrome P450”, Ann. NY Acad. Sci. 919, (2000), 214. 26-32. Guengerich, F. P. et al., “Common and Uncommon Cytochrome Miska, W., et al., “A new type of ultrasensitive bioluminogenic P-450 Reactions Related to Metabolism and Chemical Toxicity”, enzyme substrates, I. Enzyme substrates with D-Luciferin as leaving Chem. Res. Tox., 14(6), (2001),611-650. group'. Biological Chemistry Hoppe-Seyler, 369(5), (May 5, 1998), Gutierrez, M. C., et al., “The First Fluorogenic Assay for Detecting a 407-411. Baeyer-Villigerase Activity in Microbial Cells'. Org. Biomol. Chem. Miska, W., et al., “Evaluation of the Bioluminescence-enchanced 1, (2003),3500-3506. Zona binding assay'. Bioluminescence and Chemiluminescence. Hawkins, E. M., et al., "Coelenterazine derivatives for improved Molecular Reporting with Photons, Proceedings of the International solution stability'. Luminescene, 17 (Proceedings of the Interna Symposium on Bioluminescence and Chemiluminescence.(Oct. 4-8, tional Symposium on Bioluminescence and Chemiluminescence), 1996), 315-318. (Apr. 5, 2002).91-92. Miska, W., et al., “Synthesis and characterization of luciferin deriva Holt, A., “Imidazoline Binding Sites on Receptors and Enzymes: tives for use in bioluminescence enhanced enzyme immunoassays'. Emerging Targets for Novel Antidepressant Drugs?”. J. Psychiatry Journal of Clinical Chemistry and Clinical Biochemistry, vol. 25. Neuorosci, 28(6), (2003),409-414. (1987), 23-30. Hynson, R. M. G., et al., “Conformational Changes in Monoamine Mitani, M., et al., "Chemiluminescent Assay of B-D-Galactosidase Oxidase Ain Response to Ligand Binding or Reduction'. Biochimica using Cypridina Luciferin Analogue: 3-(3-D-Galactopyranosyloxy)- et Biophysica Acta, 1672, (2004),60-66. 6-(4-methoxypenyl)-2-methyl-imidazo 1.2-Opyrazine'. Analytical Inouye, S., et al., “The Use of Renilla Luciferase, Oplophorus Sciences, 10(5), (1994), 813-814. Luciferase, and Apoaequorin as Bioluminscent Reporter Protein in Monsees, T. , et al., “A Novel Bioluminogenic Assay for the Presence of Coelenterazine Analogues as Substrate'. Biochemi O-Chymotrypsin'. Journal of Biolminescene and cal and Biophysical Research Communications, 233, (1997),349 Chemiluminescence; 10(4), (Jul. 1995).213-218. 353. Monsees, T., et al., “Synthesis and Characterization of a Jagadeeswaran, P. et al., "Nucleotide Sequence and Analysis of bioluminogenic Substrate for O-chymotrypsin'. Analytic Biochemis Deletion Mutants of the Escherichia coligpt Gene in Plasmid pSV try; 221(2), (1994),329-334. gpt, Gene, 31', Gene, 31, (1996).309-313. Müller-Róber, B, et al., “Isolation and Expression Analysis of cDNA Kalmar, Gabriel, et al., “Cloning and Expression of Rat Liver CTP Clones Encoding a Small and a Large Subunit of ADP-Glucose :Phosphocholine Cytidlytransferase: An Ampipathetic Protein that Pyrophosphorylase from Sugar Beet'. Plant Molecular Biology, 27. Controls Phophatidylcholine Synthesis'. Proc. Natl. Acad. Sci USA (1995), 191-197. 87, (1990),6029-6033. Nakagawa, S., et al., “Nucleotide Sequence of the FAD Synthetase Katz, I. R., et al., “Monoamine Oxidase, an Intracellular Probe of Gene from Corynebacterium ammoniagenes and Its Expression in Oxygen Pressure in Isolated Cardiac Myocytes'. The Journal of Escherichia coli'', Biosci. Biotech Biochem. 59(4), (1995),694-702. Biological Chemistry, 259(12), (1984).7504-7509. Nelson, D.R., et al., “P450 Superfamily: Update on New Sequences, Kelly, J. H., et al., “A Fluorescent Cell-Based Assay for Cytochrome Gene Mappin, Accession Numbers and Nomenclature'. P-450 Isozyme 1A2 Induction and Inhibition”, Journal of Pharmacogenetics, 6, (1996), 1-42. Biomolecular Screening, 5(4), (2000).249-253. Nicolaus, B. J., “Symbiotic Approach to Drug Design”. Decision Kim, D.-J. , et al., “Molecular Cloning of Cucumber Making in Drug Research, (Jan. 1, 1983), 173-186. Phosphoenolpyruvate Carboxykinase and Developmental Regula Pla, J., et al., “Cloning of the Candida albicans H1 S1 Gene by Direct tion of Gene Expression'. Plant Molecular Biology, 26, (1994),423 Complementation of a C. albicans Histidine Auxotroph Using an 434. Improved Double-ARS Shuttle Vector”. Gene, 165, (1995), 115-120. Kim, J. J., et al., “Selective Enhancement of Emotional, But Not Rahman, A., et al., “Selective Biotransformation of Taxol to Motor, Learning in Monoamine Oxidase A-Deficient Mice'. Proc. 6O-Hydroxytaxol by Human Cytochrome P450 2C8”, Cancer Res., Natl. Acad. Sci. USA, 94, (May 1997),5929-5933. 54(21), (1994),5543-5546. US 8.476.450 B2 Page 4

Renwick, A. B., et al., “Evaluation of 7-benzyloxy-4- Zapata, G., et al., “Sequence of the Cloned Escherichia coli K1 trifluoromethyl-coumarin, some other 7-hydroxy-4-trifluoro CMP-N-Acetylneuraminic Acid Synthetase Gene'. The Jorunal of methylcoumarin derivatives and 7-benzyloxyquinoline as fluores Biological Chemistry, 264(25), (1989), 14769-17774. cent substrates for rat hepatic cytochrome P450 enzymes'. Zhou, M., et al., “A One-Step Fluorometric Method for the Continu Xenobiotica, vol. 31, No. 12.(2001),861-878. ous Measurement of Monoamine Oxidase Activity”. Analytical Bio Rose, A., et al., “A phosphoribosylanthranilate Transferase Gene Is chemistry, 253, (1997), 169-174. Defective in Blue Fluorescent Arabidopsis thaliana Tryptophan Zhou, M., et al., “A Stable Nonfluorescent Derivative of Resorufin for Mutants”. Plant Physiol., 100, (1992),582-592. the Fluorometric Determination of Trace Hydrogen Peroxide: Appli Sai, Y, et al., “Assessment of Specificity of Eight Chemical Inhibitors cations in Detecting the Acticity of Phagocyte NADPH Oxidase and Using cDNA-Expressed Cytochromes P450”. Xenobiotica,30(4), Other Oxidases”. Analytical Biochemistry, 253, (1997), 162-168. (2000),327-343. Zhou, W., et al., “New bioluminogenic substrates for monoamine Shanmugam, K. , et al., “Purification and Characterization oxidase assays”,Journal of the American Chemical Society, 128 (10), Nucleotidyltransferase From Lupinus albus and Functional (Mar. 15, 2006).3122-3123. Complementation of aYeast Mutation by the Corresponding cDNA'. Zhou, G., et al., “Platelet Monoamine Oxidase B and Plasma Plant Molecular Biology, 30, (1996).281-295. B-Phenylethylamine in Parkinson's disease'. J. Neurol. Neuorosurg, Shimomura, O., et al., “Semi-synthetic Aequorins with Improved Psychiatry, 70, (2001).299-231. Sensitivity to Ca' Ions”, Biochem. J. 261, (1989),913-920. “U.S. Appl. No. 10/665,314. Notice of Allowance mailed May 15, Shou, M., et al., "A Kinetic Model for the Metabolic Interaction of 2009, 9 pgs. Two Substrates at the Active Site of Cytochrome P450 3A4, J. Biol. “U.S. Appl. No. 10/665,314, Response filed Apr. 15, 2009 to Final Chem., 276(3), (2001).2256-2262. Office Action mailed Jan. 15, 2009, 21 pgs. Sussman, H. E., et al., “Choosing the Best Reporter Assay”. The “U.S. Appl. No. 1 1/444,145. Non-Final Office Action mailed Apr. 28. Scientist, 15(15), online). Retrieved from the Internet: , (Jul 23, 2001).25-27. 2009 to Office Action mailed Feb. 3, 2009, 26 pgs. Tassaneeyakul, W., et al., “Specificity of Substrate and Inhibitor “European Application Serial No. 08151520.7. Office Action mailed Probes for Human Cytochromes P450 1A1 and 1A2, J. Pharmacol. May 20, 2009, 2 pgs. Exp. Ther: 265(1), (1993).401-407. Teranishi, K., "Coelenterazine analogs as chemiluminescent probe “International Application Serial No. PCT/US2006/020731, Cor for Superoxide anion'. Analytical Biochemistry, 249(1), (Jun. 15. rected Search Report and Written Opinion mailed Apr. 12, 2007, 9 1997),37-43. pg.S. Toya, Y, et al., “Improved synthetic methods of firefly luciferin Carlile, D. J., et al., “In Vivo Clearance of Ethoxycoumarin and its derivatives for use in bioluminescent analysis of hydrolytic enzymes; Prediction from in vitro Systems'. Drug Metabolism and carboxylylic esterase and alkaline phosphatase', Bulletin of the Disposition, 26(3), (1998), 216-221. Chemical Society of Japan, vol. 65(10), (1992).2604-2610 Markaglou, N., et al., “Immobilized enzyme reactors based upon the Ubeaud, G., et al., “Estimation of flavin-containing monooxygenase flavoenzymes monoamine oxidase A and B. Journal of Chromatog activity in intake hapatoxyte monolayers or rat, hamster, rabbit, dog raphy B. vol. 804, (2004), 295-302. and human by using N-oxidation of benzidamine'. European Journal Stressor, D. M., et al., “Cytochrome P450 Flourometric of Pharmaceutical Sciences, vol. 8, (1999).255-260. Substrates: Identification of Isoform-Selective Probes for RAT Van Vleet, T.R., et al., “Metabolism and Cytotoxicity of Aflatoxin B1 CYP2D2 and Human CYP3A4, Drug and Disposition, 30(7), in Cytochrome P450 Expressing Human Lung Cells'. Journal of (2002), 845-852. Toxicological and Environmental Health, 65. (2002),853-867. “U.S. Appl. No. 10/665,314, Final Office Action mailed Jan. 15, Vinitsky, A., et al., "Cloning and Nucleic Acid Sequence of the 2009, 6 pgs. Salmonella typhimurim pncB Gene and Structur of Nicotinate “U.S. Appl. No. 10/665,314. Advisory Action mailed Jun. 6, 2008”, Phosphoribosyltransferase'. Journal of Bacteriology, 173(2), 3 pgs. (1991),536-540. “U.S. Appl. No. 1 1/444,145, Response filed Feb. 17, 2009 to Non Vonstein, V., et al., “Molecular Cloning of the pyrE Gene from the Final Office Action mailed Oct. 15, 2008”, 33 pgs. Extreme Thermophile Thermus flavus', J. Bacterio, 177(8), "Australian Application Serial No. 2003267245. Examiner's First (1995),4540-4543. Report mailed Nov. 29, 2006', 1 pg. White, E. H., et al., “Analogs of Firefly Luciferin'. Journal of "Australian Application Serial No. 2003267245. Notice of Accep Organic Chemistry, 30, (1965).2344-2348. tance mailed Jun. 29, 2007, 3 pgs. White, E. H., et al., “Analogs of firefly luciferin, III”, Journal of “Canadian Application Serial No. 2,497.560, Office Action mailed Organic Chemistry, 31. (1966), 1484-1488. Feb. 3, 2009, 2 pgs. Wood, K. A., “Engineering Luciferase enzymes and Substrates for “European Application Serial No. 03749715.3, Communication novel assay capabilities'. Proceedings of SPIE Miscroarrays and mailed Apr. 4, 2008”, 6 pgs. Combinatorial Techniques. Design, Fabrication, and Analysis II. “European Application Serial No. 03749715.3, Response filed Jan. 5328, (Jun. 2004),69-77. 30, 2009 to Communication mailed Apr. 4, 2008', 48 pgs. Wrighton, S. A., et al., “The Human Hepatic Cytochromes P450 “European Application Serial No. 03749715.3,Office Action mailed Involved in Drug Metabolism'. Critical Reviews in Toxicology, on Feb. 24, 2009, 5 pgs. 22(1), (1992), 1-21. “European Application Serial No. 06771475.8, Response filed Oct. Yang, J., et al., “An Easily Synthesized Photolyzable Luciferase for 22, 2008 to Communication mailed Jun. 12, 2008”, 22 pgs. In Vivo Luciferase Activity Measurement”. Biotechniques, vol. “International Application Serial No. PCT/US03/29078, Interna 15(5), (1993),848-850. tional Preliminary Examination Report mailed Oct. 19, 2006”. 4pgs. Yoshitomi, S., et al., “Establishment of the Transformants Express “International Application Serial No. PCT/US2006/020731, Interna ing Human Cytochrome P450 Subtypes in HepG2, and their Appli tional Preliminary Report on Patentability mailed Sep. 12, 2007, 9 cations on Drug Metabolism and Toxicology”. Toxicology in Vitro, pg.S. 15, (2001).245-246. “Japanese Application Serial No. 2004-537859, Appeal Brief mailed Youdin, M. B., et al., “Novel Substrates and Products of Amine Dec. 3, 2008”, 3 pgs. Oxidase-Catalysed Reactions'. Biochemical Society Transactions, “Japanese Application Serial No. 2004-537859, Demand for Appeal vol. 19, (1990).224-228. filed Nov. 4, 2008 Against Decision of Rejection mailed Aug. 5, Yun, C.-H... et al., “Rate-determining Steps in Phenacetin Oxidations 2008”, (w? English Translation), 12 pgs. by Human Cytochrome P450 1A2 and Selected Mutants'. Biochem “P450-GloTM Assays”, Technical Bulletin No. 325, Promega Corpo istry, 39, (2000), 11319-11329. ration, Madison, WI, (Jun. 2003), 19 pgs. US 8.476.450 B2 Page 5

Garrido-Hernandez, H., et al., “Design and Synthesis of Shinde, N. D. et al., “Synthesis of some sulfonamido and amino Phosphotyrosine Peptidomimetic Prodrugs'. J. Med. Chem., 49. alkanes and their antifungal activity.” Asian J. Chem. (1996)8(1): 85 (2006), 3368-3376. 90, Database CA Accession No. 124:232.194. Canadian Patent Office Action for Application No. 2,497.560 dated Wahler, d. et al., “Enzyme fingerprints of activity, and stereo- and Dec. 21, 2009 (2 pages). European Patent Office Action for Application No. 03749715.3 dated anantionselectivity from fluorogenic and chromogenic Substrate Jan. 20, 2010 (3 pages). arrays.” Chem. A European Journal. (2002) 8(14):3211-3228. Japanese Patent Office Action for Application No. 2004-537859 European Patent Office Action for Application No. 06771475.8 dated dated Mar. 2, 2010 (18 pages) with translation. Dec. 14, 2010 (3 pages). Beilstein Registry No. 1007 132, Database Crossfire Beilstein, White, European Patent Office Action for Application No. 08151520.7 dated E.H. et al., J. Org. Chem. (1966) 31: 1484-1488 (2 pages). Dec. 17, 2010 (8 pages). Benet, L.Z. et al., “Pharmacokinetics. The dynamics of drug absorp European Patent Office Extended Search Report for Application No. tion, distribution and elimination.” Introduction and Chapter 1 of The 10075500.8 dated Dec. 10, 2010 (8 pages). Pharmacological Basis of Therapeutics, 9th Edition, McGraw-Hill European Patent Office Extended Search report for Application No. (1996) 1-27. 10075501.6 dated Dec. 9, 2010 (5 pages). Jiang, Y. et al., “Crithidia fasciculata: isolation, sequencing and European Patent Office Extended Search Report for Application No. expression of the hypoxanthine-guanine phosphoribosyltransferase 10075502.4 dated Dec. 17, 2010 (9 pages). gene.” Exp. Parasitology (1996) 82:73-75. United States Patent Office Notice of Allowance for U.S. Appl. No. Phillips, I.R. et al., “Preface: Cytochrome P450 protocols.” Methods 1 1/444, 145 dated Jan. 24, 2011 (19 pages). in Mol. Biol. (1998) 107:v.-vi. United States Patent Office Action for U.S. Appl. No. 12/217,374 Salles, C. et al., "Biochemical characteristics of liver and brain dated Dec. 17, 2010 (9 pages). monoamine oxidase from pacu.” J. Fish Biol. (2001) 58: 1301-1310. Japanese Patent Office Action for Application No. 2008-146920 Yun, B-Set al., “Coumarins with monamine oxidase inhibitory activ dated Jul. 7, 2011 (7 pages). ity and antioxidative coumarino-lignans from hibiscus Syriacus. J. United States Patent Office Notice of Allowance for U.S. Appl. No. Natl. Prod. (2001) 64(9): 1238-1240. 12/754,164 dated Sep. 21, 2011 (5 pages). European Patent Office Examination Report for Application No. United States Patent Office Action for U.S. Appl. No. 12/754,164 06771475.8 dated Oct. 23, 2009 (3 pages). dated May 13, 2011 (5 pages). Japanese Patent Office Action for Application No. 2004537859 dated Takahashi, S. et al., “Benzimidazole N-oxide. VIII. The reactivity of Feb. 5, 2008 (8 pages) with translation. ethyl 1-methy-2-benzimidazolecarboxylate 3-oxide and related United States Patent Office Action for U.S. Appl. No. 1 1/444,145 compounds.” Chem. Pharm. Bull. (1968) 16(3):527-538. dated Dec. 3, 2009 (27 pages). European Patent Office Action for Application No. 06771475.8 dated O'Brien, M.A. et al., “Homogeneous, bioluminescent protein assays: Jun. 4, 2012 (4 pages). caspase-3 as a model.” J. Biomol. Screen. (2005) 10(2):137-148. United States Patent Office Notice of Allowance for U.S. Appl. No. Yang, X. et al., “Homogeneous enzyme immunoassay modified for 12/543,376 dated Jul. 9, 2012 (10 pages). application to luminescence-based biosensors.” Anal. Biochem. Geiger, R. et al., “A new ultrasensitive bioluminogenic enzyme Sub (2005) 336:102-107. strate for beta-galactosidase.” Biol. Chem. Hoppe-Seyler (1992) United States Patent Office Action for U.S. Appl. No. 1 1/444,145 373:11.87-1191. dated Jul. 20, 2010 (14 pages). European Patent Office Action for Application No. 10075502.4 dated United States Patent Office Action for U.S. Appl. No. 12/217,374 Jan. 11, 2012 (4 pages). dated Jul. 28, 2010 (8 pages). European Patent Office Action for Application No. 10075500.8 dated European Patent Office Action for Application No. 06771475.8 dated Jan. 11, 2012 (4 pages). Oct. 18, 2011 (3 pages). European Patent Office Action for Application No. 10075501.6 dated European Patent Office Action for Application No. 08151520.7 dated Jan. 11, 2012 (3 pages). Dec. 1, 2011 (7 pages). Japanese Patent Office Action for Application No. 2008-514754 Li, A.P., “Evaluation of luciferin-isopropyl acetal as a CYP3A4 sub dated Jan. 12, 2012 (12 pages) with English translation. strate for human hepatocytes: effects of organic solvents, chtochrome United States Patent Office Action for U.S. Appl. No. 12/543,376 P450(P450) inhibitors, and P450 inducers.” Drug Metabolism and dated Jan. 26, 2012 (7 pages). Disposition (2009) 37 (8): 1598-1603. European Patent Office Examination Report for Application No. International Search Report and Written Opinion for Application No. 09789 154.3 dated Nov. 14, 2012 (6 pages). PCT/US2009/004696 dated Nov. 19, 2009 (13 pages). European Patent Office Action for Application No. 10075502.4 dated United States Patent Office Action for U.S. Appl. No. 12/217,374 Dec. 14, 2012 (4 pages). dated May 24, 2011 (8 pages). European Patent Office Action for Application No. 10075501.6 dated "Amplex Red monoamine oxidase assay kit (A12214).” Molcular Dec. 21, 2012 (3 pages). Probes (Oct. 1, 2004) 4 pages, retrieved from the Internet: http:// Japanese Patent Office Action for Application No. 2008-514754 probes.invitrogen.com/media?pisimpl2214.pdf. dated Sep. 6, 2012 (3 pages—English Translation). Abyshev, A.Z. et al., “Preparation and antiviral effect of benzopyran Lee et al., “Synthesis of 7'-1231iodo-d-luciferin for in vivo studies 2-one derivatives.” Khimiko-Farmatsevticheskii Zhurnal (1996) of firefly luciferase gene expression.” Bioorg. Med. Chem. Lett. 30(7): 17-19: Database CAAccession No. 125:237748. 2004, v.14, pp. 1161-1163. Aparna, M.V.L. et al., “Synthesis and 5-HT2A antagonist activity of European Patent Office Action for Application No. 08151520.7 dated Some 7-(3-aminopropoxy)-4-methyl-chromen-2-ones.” Indian J. Feb. 19, 2013 (8 pages). Pharm. Sci. (2005) 67(4):467-472: Database CA Accession No. 145:62752. * cited by examiner U.S. Patent Jul. 2, 2013 Sheet 1 of 49 US 8.476.450 B2

O

N N NOH 1Y Z 5 3 2 2,3 4. HO S S — 'C' RING 'A' RING 'B' RING FIG, 1 U.S. Patent Jul. 2, 2013 Sheet 2 of 49 US 8.476.450 B2 y S S

M

L T M R Target enzyme HO- Luciferin CHOH Alcohol dehydrogenase Boc-Orn(Ac)O- COH Boc-Lys(Ac)-HN- Luciferin

Suc-LLVY-HIN Calpain substrate Z-KKR- Luciferin COH B-secretase SEVNLDAEFR- substrate HN

aSSa

2.SS2

CTL, Granzyme B toHO- N-Acetyl-2,3-dihydroluciferin CO2H HRP Substrate

FIG 2

HO S Cl F N N COOH 5-FUOROLUCIFERIN FIG 3 U.S. Patent Jul. 2, 2013 Sheet 3 of 49 US 8.476.450 B2

O N O n N C N / O REDUCING CELLULAR N N M 2 OH HO S S ENVIRONMENT 2 al-e-as-s-s HO S S LUCFERASE

FIG. 4A

O O

OH O

O N N | y - N S N S

HO 2 HO 21 P O

HO S S U.S. Patent Jul. 2, 2013 Sheet 4 of 49 US 8.476.450 B2

Y1s-Nyer's N H - 1 N )N (/ Oh HO 21 S S HO 2 S S

O O N N aN OH - || N NX (s'.N 1. 2 S S n 21 S S

O O N N N - clareas N- X-(-N HO r S S HO a S S

O FIG 5A

O 'n N 2 N HO u I. S it:3. SS HO 21n N) (2 Oh U.S. Patent Jul. 2, 2013 Sheet 5 of 49 US 8.476.450 B2

U.S. Patent Jul. 2, 2013 Sheet 6 of 49 US 8.476.450 B2

COOH ;

CF3 FIG 7A U.S. Patent Jul. 2, 2013 Sheet 7 of 49 US 8.476.450 B2 U.S. Patent Jul. 2, 2013 Sheet 8 of 49 US 8.476.450 B2

O2 u.M SUBSTRATE

PLUS UNIT PORCNEESTERASE WITHOUT ESTERASE

180000 60000 140000 20000 D 00000 - 4. 80000 60000 40000 20000 O PLUS ESTERASE MNUS ESTERASE U.S. Patent Jul. 2, 2013 Sheet 9 Of 49 US 8.476.450 B2

1 p.m. SUBSTRATE

WITH UNIT PORCINE ESTERASE WITHOUT ESTERASE FIG 9

uM SUBSTRATE

WITH UNIT PORCINE ESTERASE WITHOUT ESTERASE FIG, 10 U.S. Patent Jul. 2, 2013 Sheet 10 of 49 US 8.476.450 B2

MAO-F3

N N

- O O O O O N 1. N NH MAO-F MAO-F2 FIG 11

U.S. Patent Jul. 2, 2013 Sheet 12 of 49 US 8.476.450 B2

JC-?anN NCOOH isCOOH OESEOHN O JC-?ans'sS S 2 NO OFSEO GST-19 Q 21 N OSO Gst-20 CF Sn 2 N Sa GST-18 COOH

9 SN S o C NoS N2 s Q SNo S OFSFO GS-21 O=SFO GST-22 o oiloa Y a GS-24 or (r NO as a? O2 NO2 SCOOH COOH N N N. COOH N s COOH

Q ris O OCC- O s's2 p CCS S O-SO NO GS-25 NO2 GST-23 o=s=o GS-27 OSCOes GS-28

COOH COOH NO2 sus C-fN 2 O S S p OSEO G O O-S-O GST-29 GST-30 OSO GST-31 NO2

OCH NO2 FIG, 12B U.S. Patent Jul. 2, 2013 Sheet 13 of 49 US 8.476.450 B2

FIG 13A U.S. Patent Jul. 2, 2013 Sheet 14 of 49 US 8.476.450 B2

- N 1 H FIG, 13C

N N COOH N N COOH C Sy-r" S O Cy-rS S S-r-, MAO-3

t N N COOH CC N COOH F N N. COOH O y-rS O Sy- S ODCC) S erS S-rn, MAO-4 N-- MAO-5 N-- MAO-6 H H

COOH COOH

NS 2 S S SS Ns SS O O 21 MAo-a 12 N- O MAO-9 1 Yrh,

COOCH Cy-rN N COOH Cis-r"N. N. COOCH is s O S S O S S O 2 --- MAO-10 Yrn, MAO-11 N-- MAO-12 FIG 13D U.S. Patent Jul. 2, 2013 Sheet 15 Of 49 US 8.476.450 B2

FIG, 14 U.S. Patent Jul. 2, 2013 Sheet 16 of 49 US 8.476.450 B2

N N COOH N COOH O CC-r"C-rS S O S S ',W 2.'co r N o1 Na1 is s FMO-1 N FMO-2 N FMO-3 "O s o: COOH N N N COOH s N N COOH Cy-r" 's O CC-rS S O S S O 2 ) FMO-4 FMO-5 () FMO-6 N1 1. NH COOH H

N N COOH ra S Cy-r O N. N. COOH 2 o1 N21s is n O Cis-r"S S FMO-9 U.S. Patent Jul. 2, 2013 Sheet 17 Of 49 US 8.476.450 B2

COOH

6) O N. N. COOH y 85f YO (CC-r",S S O r'ss TE o 2 AP-1 6) TEA AP-2

COOH

O SW S r O S S O E-1 HO O AP-4 HOT O -so O Ny NCOOH

X-----.O AP-5 os-oh OH U.S. Patent Jul. 2, 2013 Sheet 18 of 49 US 8.476.450 B2

I’0IHZ U.S. Patent Jul. 2, 2013 Sheet 19 of 49 US 8.476.450 B2 U.S. Patent Jul. 2, 2013 Sheet 20 of 49 US 8.476.450 B2

6-(2,3,4,5,6 pentafluorobenzylox

6-phenyl-piperizine-2,4,5,6- w-luciferin

luciferin-6- ditrifluoromethylbenzylether

4-(O-methyl)-6-(O-methyl)- luciferin

FIG, 19A U.S. Patent Jul. 2, 2013 Sheet 21 of 49 US 8.476.450 B2

14 5-6-dimethox -luciferin Quinolylluciferin-6-methyl ether " Quinolylluciferin-6-benzyl ether 17 6-(p-aminophenyloxy)- quinolylluciferin

6-benzyloxymethoxy quinolyuciferin 19 naphthylluciferin 6-methyl ether

20 quinoxalylluciferin methyl ether

2.

N,N-Bis-benzyl-aminoluciferin

22 N-Benzyloxycarbonyl aminoluciferin 23 N-isobutoxycarbonyl aminoluciferin 24 luciferin-H ethylene glycol ester 26 Lucifern-H methyl ester 27 Luciferin-6-methylether picolinyl ester FIG, 19B U.S. Patent Jul. 2, 2013 Sheet 22 of 49 US 8.476.450 B2

6-m-picoliny luciferin methyl ester

6-p-picolinylluciferin methyl ester

Luciferin-H picolinyl ester 1A Luciferin-6-methylether methyl

lA Luciferin-6-methylether propanol Ester lA Luciferin-6-methylether ethylene divco ester

Quinolylluciferin-6-methylether methwester

1B Ester lA N O r Luciferin-6-methylether Hydrazi S-CC. e ? NH2 hydrazide de S S 4 l 1A luciferin-6-methylether-N- Methox methoxylamide amide U.S. Patent Jul. 2, 2013 Sheet 23 of 49 US 8.476.450 B2

200 uM LUCIFERIN-ME 9000 37°C/60 MINUTES 8000 7000 6000 5000 4000 O2D6 3000 CONTROL 2000 N 2019 1000 XCONTROL 5pmoles FIG, 20A

200 uM LUCIFERIN-ME 57°C/60 MINUTES

C C C) S&S 8 x&S x &XXXXXX

C) O 1 pmole O C) x35pmoles C) 8Cx x : AlA4AA 206 2C9 FIG, 20B U.S. Patent Jul. 2, 2013 Sheet 24 of 49 US 8.476.450 B2

2000000 000000 U.S. Patent Jul. 2, 2013 Sheet 25 of 49 US 8.476.450 B2

50 uM LUCIFERIN-MEPICOLINYL ESTER

CONTROL FIG, 23 U.S. Patent Jul. 2, 2013 Sheet 26 of 49 US 8.476.450 B2

50 uMD-LUCIFERIN ETHYL ESTER LDR WITHOUT PORCINE ESTERASE 1400000 1200000 1000000 800000 600000 400000

FIG, 24

600000 50 uM UCIFERIN METHYL ESTER

500000

400000

200000

FIG 25 U.S. Patent Jul. 2, 2013 Sheet 27 of 49 US 8.476.450 B2

1800000 1600000 1400000 200000

D - OOOOOO Y 800000

2D6 CONTROL p450 FIG. 26 U.S. Patent Jul. 2, 2013 Sheet 28 of 49 US 8.476.450 B2

2500000

2000000

500000

000000

500000

N-ISOPROPYL AMINO LUCIFERIN H2O FIG 27A

REACTIONS WITH A THERMOSTABLE LUCIFERASE

AMINOLUCFERIN DIMETHYLAMNOLUCIFERIN FIG 27B U.S. Patent Jul. 2, 2013 Sheet 29 Of 49 US 8.476.450 B2

50 uM 4", 6'-OMETHYLETHER LUCIFERIN

OLL\/?).CINTOR#5ÐXHOVEOLTVNSDIS

6 METHOXYQUINOLIN -2 DHYDROTHAZOLE -4- CARBOXYLIC ACID

FIG, 29 U.S. Patent Jul. 2, 2013 Sheet 30 of 49 US 8.476.450 B2

50 uM 2-(7-METHOXYQUINOAXLIN-2-YL)-4,5-DIHYDROTHAZOLE-4-CARBOXYLIC ACID U.S. Patent Jul. 2, 2013 Sheet 31 of 49 US 8.476.450 B2

50 uMLUCIFERIN 4 PPER DNEMETHYL ETHER

FIG. 32

5000 U.S. Patent Jul. 2, 2013 Sheet 32 of 49 US 8.476.450 B2

50 uM 4 -(N, N DIMETHYLAMINO) METHYL METHYL) UCFERIN

FIG. 34

50 uM 4 (N, N DIMETHYLAMINOMETHYL) METHOXYQUINOLY LUCIFERIN U.S. Patent Jul. 2, 2013 Sheet 33 of 49 US 8.476.450 B2

50 uM LUCIFERIN 2-(1-PPERAZINE) ETHYL ETHER

6000 5000

FIG, 36 U.S. Patent Jul. 2, 2013 Sheet 34 of 49 US 8.476.450 B2

& PLUS ESTERASE MINUS ESTERASE &&&&&&&&&&&&&

80000 50 uM SUBSTRATE 70000

PLUS ESTERASE MINUS ESTERASE FIG 37B U.S. Patent Jul. 2, 2013 Sheet 35 of 49 US 8.476.450 B2

50 uM 6'-(4-(2-(DIMETHYLAMINO)-1-PHENYLPROPOXY)BENZYLOXY)CARBONYLOXY)LUCIFERIN, 37°C, 30 MINUTES

U.S. Patent Jul. 2, 2013 Sheet 36 of 49 US 8.476.450 B2

50 uM N-ISOBUTOXYCARBONYL-AMINOLUCIFERIN

s s s

50 uy LUCFERIN-H METHYL ESTER PLUS PORCINE ESTERASE 400000 350000 300000 250000 O in 200000 150000 100000 U.S. Patent Jul. 2, 2013 Sheet 37 Of 49 US 8.476.450 B2

50 uM UCIFERN-MEETHYLENE CLYCOLESTER 600000 400000 200000 000000 O 800000 600000 400000 200000 : 5 s S s g s s S s . FIG, 41

50 uM METHOXYQUINOLYLLUCIFERIN METHYL ESTER + PORCINE ESTERASE 300000

250000

200000 o in 150000

100000

SS SV & Reis SSS$.SS-S-S-SN rSS &VS$sy ŠS U.S. Patent Jul. 2, 2013 Sheet 38 of 49 US 8.476.450 B2

50 uM 60-TRIFLUOROMETHYLBENZYLOXY)-LUCIFERIN 400000 1200000 1000000 B 800000 1. 600000 400000 200000 0.1 wre e o ord cd co &ad n r- N - Sn is a is a r s 5 & 3 Can Ca S & S g is se-se S s-5 she HUMAN P450s FIG 43

50u M 6'METHOXY-LUCIFERIN-HYDRAZIDE 90000 80000 70000 60000 50000 40000 30000 20000 0000 O as - - -, n - I - ssm S a Sad Sa 33coa e o a Sgsco - &a sc5 gsa v S.Cra e53 o Ska as- CU as HUMAN PA50s Cl FIG, 44 U.S. Patent Jul. 2, 2013 Sheet 39 of 49 US 8.476.450 B2

50 uM 63-(4-PHENYl-PPERIZIN-1-YL))METHYLBENZOXY-LUCFERIN O

N

20000

100000

80000

60000

40000

20000

HUMAN CYP450S FIG. 45 50 uM 6(2,4,6-TRIMETHYLBENZYLOXY)-LUCFERIN 250000

200000

150000

100000

HUMAN P450s FIG, 46 U.S. Patent Jul. 2, 2013 Sheet 40 of 49 US 8.476.450 B2

50 uM 6(4–CHLOROPHENYTHIOMETHOXY)-LUCIFERIN

c pers ce on - SYa Sca Sa & ras sh is 2 re-... a S2H e c d HUMAN P450S FIG 47 U.S. Patent Jul. 2, 2013 Sheet 41 of 49 US 8.476.450 B2

TROLEANDOMYCIN (M) FIG, 48

100000 90000 80000 70000 60000 50000 40000 30000 20000 10000

NIFEDIPINE (M) FIG, 49 U.S. Patent Jul. 2, 2013 Sheet 42 of 49 US 8.476.450 B2

3A4 PPXE assays for inhibition by nifedipine uMnifedipiPPXE PPXE PPXE 20O. 8584 8236 7914 8244,667 335.0841 100 13594 12898 12378 12956.67 610.1.191 50 22O14 21868 23056 22312.67 647.871.4 25 33136 3382 32656 32991.33 291.3166 12.5 49210 47.340 4847O 4834O. 941.7537 6.25 82042 77646 81388 80358.67 2,371887 3.125 91596 86630 89658 892.94.67 2502,858 O 136742 125432 129692 130622 5712.066 FIG 50

Cambrex human hepatocytes, donor is 11 yr old male 48 Hour induction with 10u M Rifampicin 3 hr incubation with 5OUM 5FBE

keto

luciferin-5FBE 3Ve vehicle 3174 3847 2900 5211 37.83 Vehicle.--ke 835 1099 1197 621 938 1 OuM rifan 36371 39351 34085 33834 3591 O.25 Rifamp.+ke 1199 1097 941 965 1050.5 FIG 51 U.S. Patent Jul. 2, 2013 Sheet 43 of 49 US 8.476.450 B2

Cambrex human hepatocytes; donor is 11 yr old male 48 Hour induction with 10uM Rifampicin 3 hr incubation with 50uMPPXE

luciferin-PPXE aVC sd vehicle 1297 1641 1863 2367 1792 448,513 vehicle+ke 20 196 238 224 169.5 101.1846 10M rifan 93.11 8468 6355 O540 8668.5 1761.42 rifamp-ke 428 578 356 592 488.5 15.382 FIG 52

O N N X ( OH 1. Sy (S or n--issls Y N-isopropyl-aminoluciferinA 8 N-benzyl-N-methyl-aninoluciferinPh O O N N

1NN S S ls ls Ph N,N-diethyl-aminoluciferin N-benzyl-N-ethyl-aminoluciferin FIG 53 U.S. Patent Jul. 2, 2013 Sheet 44 of 49 US 8.476.450 B2

N N COOH \ /

S S 6' deoxyluciferin Luciferin H N N COOH

C 3'N, S S

C N/No S S Luciferin 6' chloroethyl ether (Luciferin-CEE)

N N COOH Y A

O S S

Luciferin 6' benzyl ether (Luciferin-BE) FIG 54 U.S. Patent Jul. 2, 2013 Sheet 45 of 49 US 8.476.450 B2

FLUORO-LUCEFEREN -0- MOPS -- HEPES -A-TRICINE LUCFERIN -e- MOPS -e- HEPES -a-TRICINE FIG 55 U.S. Patent Jul. 2, 2013 Sheet 46 of 49 US 8.476.450 B2

0 1061 574

OOOOOOO

-O- UCFERN -- 5'-FLUOROLUCIFERIN OOOOOO 0.01 0. (ATP) (mM) O FIG 56 U.S. Patent Jul. 2, 2013 Sheet 47 of 49 US 8.476.450 B2

Luminescence (RLU mM

2O7.6 154.9

100 1262 128.9996

- 1000.0 o - 92

t 2 LU He 2 100.0 l 2 C9 t 2 e O - 100 O.O 1.00 0.00 ATP (mM) FIG. 57 U.S. Patent Jul. 2, 2013 Sheet 48 of 49 US 8.476.450 B2

5 5 120% A.

2100% ems 5'-LH2DO 7.4 g s 80% ----- LH2.747"f-LH2 7.4

D 60% f2 40% 253 20, 0% M O 9. 450 500 550 600 650 700 SS WAVELENGTH (nm)

00000

5. o 92

CD 2 2 s Z U CD c 2 s o -

OOOO 6.4 6.7 70 7.5 7.6 7.9 8.2 8.5 pH OFREAGENT FIG 59 U.S. Patent Jul. 2, 2013 Sheet 49 of 49 US 8.476.450 B2

O N N N N y ( OH C y (S OH Y optionally substituted at positions other than C1 with 1 functionality comprising any of hydroxyl, halo, thio, amino, ketone, ester, amide, aldehyde. O N N N 4' OH K ICCy (S OH --CC . Y l Y-O or CH2 ku Y=d, NH, CH. optionally substituted at positions other than Cl with 1 optionally substituted at positions other than Cl with 1 functionality comprising any of hydroxyl, halo, thio, functionality comprising any of hydroxyl, halo, thio, amino, ketone, ester, amide, aldehyde. amino, ketone, ester, amide, aldehyde. O O

O OH Rs OC)S {S H R" R=H, C-CO alkyl, alkenyl, alkynyl, with chains all R" = H, C1-C3 alkyl, alkenyl, alkynyl, with chains all being cyclic or branched or unbranched and optionally being cyclic or branched or unbranched and optionally substituted at positions other than Cl with l or 2 substituted at positions other than Cl with l or functionalities comprising hydroxyl, halo, thio, amino, functionalities comprising hydroxyl, halo, thio, amino, ketone, ester, amide, aldehyde. ketone, ester, amide, aldehyde. O O N A1 Yn O)-1-S S 1N S S - A=C6-10 aryl, heteroaryl optionally substituted at A=C6 ary, heteroaryl optionally substituted at positions other than C1 with 1 or 2 functionalities positions other than Cl with l or 2 functionalities comprising hydroxyl, halo, thio, amino, ketone, ester, comprising hydroxyl, halo, thio, amino, ketone, ester, amide, aldehyde. amide, aldehyde. e optionally substituted at positions other than Cl with optionally substituted at positions other than Cl with or 2 functionalities comprising hydroxyl, halo, thio, or 2 functionalities comprising hydroxyl halo, thio, amino, ketone, ester, amide, aldehyde. anino, ketone, ester, amide, aldehyde. -Ox-r C.J/ P. FIG 60 US 8,476,450 B2 1. 2 LUMINOGENIC AND FLUOROGENIC 1. For instance, methods for using luciferin derivatives with a COMPOUNDS AND METHODS TO DETECT recognition site for an enzyme Such as a protease as a proSub MOLECULES OR CONDITIONS strate were described by Miska et al. (Journal of Clinical Chemistry and Clinical Biochemistry, 25:23 (1987)). The CROSS-REFERENCE TO RELATED heterogenous assays were conducted by incubating the APPLICATIONS luciferin derivative with the appropriate enzyme, e.g., a pro tease, for a specified period of time, then transferring an This application is a continuation of U.S. patent applica aliquot of the mixture to a solution containing luciferase. tion Ser. No. 1 1/444,145, filed May 31, 2006, which applica Masuda-Nishimura et al. (Letters in Applied Microbio., tion claims the benefit of the filing date of U.S. Application 10 30:130 (2000)) reported the use of a single tube (homog Ser. No. 60/685,957, filed May 31, 2005, U.S. Application enous) assay which employed a galactosidase Substrate Ser. No. 60/693,034, filed Jun. 21, 2005, U.S. Application modified luciferin. In these luciferin derivatives, the portion Ser. No. 60/692,925, filed Jun. 22, 2005 and U.S. Application of the derivative functioning as the reactive group for the Ser. No. 60/790,455, filed Apr. 7, 2006, the disclosures of nonluciferase enzyme activity was coupled to the D-luciferin which are incorporated by reference herein. 15 or aminoluciferin backbone such that upon the action of the nonluciferase enzyme, a D-luciferin or aminoluciferin mol BACKGROUND ecule was produced as the direct product of the reaction to serve as the substrate for luciferase. A primary obstacle to Luminescence is produced in certain organisms as a result broadly applying luciferase mediated reactions for other of a luciferase-mediated oxidation reaction. Luciferase genes enzymatic assays has been the belief that to modify the from a wide variety of vastly different species, particularly luciferin molecule to function as a Substrate for a nonlu the luciferase genes of Photinus pyralis and Photuris penn ciferase enzyme, the activity of the nonluciferase enzyme sylvanica (fireflies of North America), Pyrophorus plagioph must directly yield a D-luciferin or aminoluciferin molecule thalamus (the Jamaican click beetle), Renilla reniformis (the to retain its function as a Substrate for luciferase. sea pansy), and several bacteria (e.g., Xenorhabdus lumine There is, therefore, a need in the field of biological assays scens and Vibrio spp), are extremely popular luminescence 25 to expand the utility of luciferase mediated reactions for reporter genes. Firefly luciferase is also a popular reporter for nonluciferase enzymes by identifying derivatives of luciferin determining ATP concentrations, and, in that role, is widely that function as a Substrate for a nonluciferase enzyme or used to detect biomass. Luminescence is also produced by other biological molecule of interest and as a prosubstrate for other enzymes when those enzymes are mixed with certain luciferase regardless of whether D-luciferin or aminolu synthetic Substrates, for instance, alkaline phosphatase and 30 ciferin is released as a direct result of the nonluciferase enzy adamantyl dioxetane phosphate, or horseradish peroxidase matic reaction. and luminol. Luciferase genes are widely used as genetic reporters due SUMMARY OF THE INVENTION to the non-radioactive nature, sensitivity, and extreme linear range of luminescence assays. For instance, as few as 10" The present invention provides derivatives of luciferin and moles of firefly luciferase can be detected. Consequently, 35 methods for using Such derivatives in enzyme activity assays luciferase assays of gene activity are used in virtually every or non-enzymatic biological assays where the luciferin experimental biological system, including both prokaryotic derivative serves as a Substrate for a desired enzyme and is a and eukaryotic cell cultures, transgenic plants and animals, prosubstrate for luciferase or wherein the luciferin derivative and cell-free expression systems. Similarly, luciferase assays is a molecule which is modified by a molecule of interest, used to determine ATP concentration are highly sensitive, 40 which modified molecule is a substrate for luciferin. Surpris enabling detection to below 10 moles. ingly, many of the luciferin derivatives also have activity as Luciferases can generate light via the oxidation of enzyme Substrates for luciferase in a light generating assay. Thus, by specific Substrates, e.g., luciferins. For firefly luciferase and providing luciferin derivatives having a particular enzyme all other beetle luciferases, light generation occurs in the recognition site (reactive chemical group in a molecule presence of luciferin, magnesium ions, oxygen, and ATP. For 45 referred to as a substrate for a particular enzyme) for a desired anthozoan luciferases, including Renilla luciferase, only oxy nonluciferase enzyme coupled to the luciferin backbone (or gen is required along with the Substrate coelentrazine. Gen other chemical moiety constituting a Suitable Substrate for erally, in luminescence assays to determine genetic activity, luciferase), such as derivatives with modifications at the 6 reaction Substrates and other luminescence activating hydroxy site of luciferin or the 6' amino site of aminolu reagents are introduced into a biological system Suspected of 50 ciferin, yielding a substrate for a desired nonluciferase expressing a reporter enzyme. Resultant luminescence, ifany, enzyme and a proSubstrate of luciferase, numerous nonlu is then measured using a luminometer or any suitable radiant ciferase enzymes may be measured in a bioluminescent assay. energy-measuring device. The assay is very rapid and sensi Modifications of luciferin within the scope of the deriva tive, and provides gene expression data quickly and easily, tives of this invention include one or more substitutions of a without the need for radioactive reagents. ring atom, one or more Substitutions of a Substituent (atom or Because most enzymatic reactions do not generate outputs 55 group) attached to a ring atom, and/or addition of one or more that are as ideal as luciferase, the availability of a luciferase atoms to the ring, e.g., expansion or addition of rings, or a mediated assay for enzymatic reactions useful in cellular combination thereof. Numbering for some of the ring atoms analysis and high-throughput Screening applications would in D-luciferin is shown in FIG.1. Native firefly luciferin has be desirable to those working in this field. The development three linked rings, a 6 membered ring having an OH group at of Such a luciferase mediated reaction as the basis for Such 60 position 6 (“ring A' or “A ring hereinafter), a 5 membered enzymatic or biological assays has, however, been limited. thiazole ring linked to the 6 membered ring (“ring B' or “B Luciferase mediated reactions have been employed to detect ring hereinafter), and a 5 membered thiazole ring that is numerous other molecules, e.g., ATP or lactate dehydroge modified with a carboxyl group at position 5 (“ring C or “C nase. For some of those reactions, a derivative of the naturally ring hereinafter). For instance, a luciferin derivative with a A occurring substrate is employed. Native firefly luciferin, a 65 ring modification may have a Substitution of a C atom in the polytherocyclic organic acid, D-(-)-2-(6'hydroxy-2-ben A ring with another atom, addition of a ring, a Substitution of Zothiazolyl)-A-thiazolin-4-carbozylic acid, is shown in FIG. a Substituent attached to a ring atom with a different atom or US 8,476,450 B2 3 4 group, or any combination thereof. Aluciferin derivative with Z and Z are independently H, OR, NHR, or NRR: a Bring modification may have an addition to or Substitution Z" is O, S, NH, NHR, or N=N; of an atom in the five membered ring, e.g., insertion of one or Q is carbonyl or CH: more atoms, thereby expanding the ring, for instance, to a six W' is H. halo, (C-C)alkyl, (C-C-o)alkenyl, hydroxyl, or membered ring, substitution of N or S in the ring with a 5 (C-C)alkoxy; or different atom, e.g., a C or O. substitution of a substituent W' and Zare both keto groups on ring A, and at least one of atom or group attached to a ring atom, or any combination the dotted lines denoting optional double bonds in ring A is thereof. Aluciferin derivative with a C ring modification may absent; have a Substitution of an atom in the ring with another atom, each W is independently H, halo, (C-C)alkyl, (C-C) a Substitution of a Substituent attached to a ring atom, with a 10 alkenyl, hydroxyl, or (C-C)alkoxy; different atom or group, or any combination thereof. In one each of K', K, K, and K" are independently CH, N,N- embodiment, a derivative of the invention is one which is oxide, or N (C-C)alkyl, and the dotted lines between K' modified at more than one position, for instance, the deriva and K, and K and K, denote optional double bonds; tive has two (or more) A ring modifications, two (or more) B A' and B' are optional aromatic rings fused to ring A, only ring modifications, two (or more) Cring modifications, or any 15 one of which is present in the compound, so as to form a fused combination thereof. In one embodiment, one modification is tricyclic system; and the substitution of a substituent on one of the rings of D-lu when B' is present, the group Z is present, and ciferin with a substrate for a nonluciferase enzyme, or a linker when A' is present, the group Z is absent; and and a substrate for the nonluciferase enzyme. the dotted line in ring B is an optional double bond; Exemplary derivatives with A ring modifications may be a each R is independently H. (C-C)alkyl, (C-C)alk substrate for a reductase, such as a cytochrome P450 reduc enyl, (C-Co.)alkynyl, (C-Co)cycloalkyl, (C-C2)alkoxy, tase, monoamine oxidase (MAO), flavin monooxygenase (C-C)aryl, heteroaryl, heterocycle, (C-C)alkylsulfoxy, (FMO), glutathione S transferase (GST), dealkylase, (Co-Co)arylsulfoxy, heteroarylsulfoxy, (C-Co)alkylsulfo deacetylase, deformylase, phosphatase, e.g., alkaline phos nyl, (C-C)arylsulfonyl, heteroarylsulfonyl, (C-C)alkyl phatase (AP), Sulfatase, beta-lactamase, alcohol dehydroge Sulfinyl, (C-C)arylsulfinyl, heteroarylsulfinyl, (C-C) nase, protease e.g., proteosome, cathepsin, calpain, beta 25 alkoxycarbonyl, amino, NH(C-C)alkyl, N(C-C)alkyl). secretase, thrombin, or granzyme, luciferase, or useful to tri(C-C)ammonium(C-Co.)alkyl, heteroaryl(C-C) detect reactive oxygen species (ROS), peroxidase, e.g., horse alkyl, heteroaryl having quaternary nitrogen, heteroarylcar radish peroxidase (HRP), and redox conditions. Exemplary bonyl having quaternary nitrogen, (C-C)arylthio, (C-C) molecules or conditions to be detected with derivatives hav alkylphosphate, (C-C)alkylphosphonate, (C-C) ing at least a Bring modification include but are not limited to 30 arylphosphate, (C-Co)arylphosphonate, phosphate, Sulfate, dealkylase, GST or luciferase, or redox conditions. Exem saccharide, or M" optionally when Z" is oxygen, wherein M plary molecules to be detected with those derivatives include is an alkali metal; a cytochrome P450 enzyme, esterase, e.g., acetylcholinest or when Z or Z is NR'R'', R'R' together with the N to erase, OH radicals, demethylase, deacetylase, deformylase, which they are attached forms a heteroaryl or heterocycle or mycoplasma carboxypeptidase. Exemplary molecules to 35 group; be detected with derivatives having C ring modifications wherein any alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, include but are not limited to esterases. amino, aryl, heteroaryl, or heterocycle group is optionally In one embodiment, derivatives of luciferin or aminolu substituted with 1, 2, 3, 4, or 5 substituents selected from ciferin have the following structure: L-X-M-Y R (com (C-Co.)alkyl, (C-Co.)alkenyl, (C-Co.)alkynyl, (C-Co) pound of formula IV), wherein L, if present, may be a sub 40 cycloalkyl, (C-C)alkoxyl, (C-C)alkylcarbonyl, (C- strate for an enzyme or another molecule which interacts with the enzyme: X may be O, NH, or a linker, e.g., a self-cleavable Co)alkylcarboxyl, halo, hydroxyl, -COOR, SOR, linker which spontaneously cleaves to yield M-Y Rafter L —SOR, nitro, amino, (C-C)alkyl-S(O)—, (C-C) has been removed from L-X-M-Y R; M may be luciferin, alkyl-SO , phosphate, (C-Co.)alkylphosphate, (C-C) quinolinyl luciferin or naphthylluciferin, or aminoluciferin alkylphosphonate, NH(C-C)alkyl, NH(C-C)alkynyl, or aminoquinolinyl luciferin, Y is O (ester), NH (amide), 45 N((C-C)alkyl), N(C-C)alkynyl), mercapto, (C-C) NH-NH (hydrazide), or S (thioester); and R, if present, may alkylthio, (C-C)aryl, (C-C)arylthio, trifluoromethyl, be alkyl, an aromatic molecule, a peptide, an oligonucleotide, =O, heteroaryl, and heterocycle, and each Substituent is or a self-cleavable linker attached to a substrate for an optionally substituted with one to three R groups: enzyme. R is H., (C-C)alkyl, or (C-C)aryl; In one embodiment, the invention provides a compound of 50 when Z or Z comprises a nitrogen moiety, one or both of formula I: the hydrogens of the Z or Z. nitrogen moiety may be replaced by (C-C)alkyl or the group L., wherein L is an amino acid radical, a peptide radical having up to 20 amino acid moieties, or any other Small molecule that is a Substrate for a nonlu 55 ciferase; with the proviso that when L is an amino acid radical B : or a peptide radical, at least one W is not H: --- as Y N Q-n -R when Z is a hydroxyl group or a nitrogen moiety, H of the K Y / Z" hydroxyl or nitrogen moiety may be replaced by (HO)2P Z--l, A KsTA, B C (O)—OCH2—, Sulfo, —POH, or by a cephalosporanic acid --K/XNx S W2 Z W A 60 attached to the group Z via a carbon chain of one to about 12 W2 carbon atoms; with the proviso that when ring B is a thiazole ring, the Sulfo or the —POH group is attached to the wherein hydroxyl oxygen via a (C-C)alkylene group: Y is N,N-oxide, N-(C-C)alkyl, or CH: when Z or Z is a hydroxyl group or a nitrogen moiety, or when Y is N, then X is not S; 65 when Z" R is a hydroxyl group, one H of the hydroxyl or X is S, O, CH-CH, N=CH, or CH-N: nitrogen moiety may be replaced by the group L'-linker, when X is S, then Y is not N: wherein L' is a group removable by an enzyme to free the US 8,476,450 B2 5 6 linker, and linker is a carbon chain that can self-cleave, wherein any alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, optionally interrupted by one or more nitrogenatoms, oxygen amino, aryl, heteroaryl, or heterocycle group is optionally atoms, carbonyl groups, optionally Substituted aromatic substituted with 1, 2, 3, 4, or 5 substituents selected from rings, or peptide bonds, (C-Co.)alkyl, (C-Co.)alkenyl, (C-Co.)alkynyl, (C-Co) linker is attached to L'via an oxygenatom oran NH group 5 cycloalkyl, (C-C)alkoxyl, (C-C)alkylcarbonyl, (C- at one terminus of the linker and the other terminus of the Co)alkylcarboxyl, halo, hydroxyl, -COOR, SOR, linker forms an ether, ester, or amide linkage with a group Z, —SO.R. nitro, amino, (C-Co.)alkyl-S(O)—, (C-C) Z", or Z" R. alkyl-SO , phosphate, (C-C)alkylphosphate, (C-C) when Z is OR, formula I is optionally a dimer connected at alkylphosphonate, NH(C-C)alkyl, NH(C-C)alkynyl, the two A rings via a linker comprisinga (C-C)alkyl diradi 10 N((C-C)alkyl), N(C-C)alkynyl), mercapto, (C-C) cal that is optionally interrupted by one to four O atoms, N alkylthio, (C-C)aryl, (C-C)arylthio, trifluoromethyl, atoms, oran optionally Substituted aryl, heteroaryl, or hetero =O, heteroaryl, and heterocycle, and each Substituent is cycle group to form a bridge between the dimer of formula I, optionally substituted with one to three R groups: and the R group of each Z group connecting the dimer of R is H., (C-C)alkyl, or (C-C)aryl; formula I is replaced by the bridge; 15 A is an anion, present when a quaternary nitrogen is A is an anion, present when a quaternary nitrogen is present; present; or a salt thereof; or a salt thereof; provided that: provided that: when rings A and B form a naphthalene or quinoline ring when rings A and B form a naphthalene or quinoline ring system, then W' is not hydrogen; system, then W' is not hydrogen; when a ring A substituent is OH, then-Q-Z" R is not when a ring A substituent is OH, then -Q-Z" R is not - C(O) NH NH: —C(O) NH NH: when Y is N or CH and X is CH=CH and W' is H, then Z when Y is N or CH and X is CH=CH and W' is H, then Z 25 is not OH attached to carbon-6 of ring A (carbon 6 as shown is not OH attached to K; and in FIG. 1); and when Y is Nor CH and X is CH=CH and Z is H, then W. when Y is N or CH and X is CH=CH and Z is H, then W. is not OH attached to K. is not OH attached to carbon-6 of ring A. In another embodiment, the invention provides a com In another embodiment, the invention provides a com pound of formula IA: 30 pound of formula II:

Ia II O

R 21 Yy WN 71 ; B' Z A B 's C --22' N N Q-R SaSás X S z-- A A. By-(c s - Ky.X XNs S 2 WI A Z K4 WI w A 40 W wherein Y is N,N-oxide, N—(C-C)alkyl, or CH: wherein when Y is N, then X is not S; Z and Z are independently OR, NHR', or NR'R'': X is S, O, CH-CH, N=CH, or CH-N: 45 Z" is O, S, NH, NHR, or N=N; when X is S, then Y is not N: Q is carbonyl or CH: Z is H, OR, NHR, or NRR: W' is H, halo, (C-C)alkyl, (C-C-o)alkenyl, hydroxyl, or Z" is O, S, NH, NHR, or N=N; (C-C)alkoxy; or W' is H. halo, hydroxyl, (C-C)alkyl, or (C-C)alkoxy: W' and Zare both keto groups on ring A, and at least one of the dotted line in ring B is an optional double bond; 50 the dotted lines denoting optional double bonds in ring A is each R is independently H. (C-C)alkyl, (C-Co.)alk absent; enyl, (C-Co.)alkynyl, (C-Co)cycloalkyl, (C-C2)alkoxy, each W is independently H, halo, (C-C)alkyl, (C-C) (Co-Co.)aryl, heteroaryl, heterocycle, (C-C)alkylsulfoxy, alkenyl, hydroxyl, or (C-C)alkoxy; (C-C)arylsulfoxy, heteroarylsulfoxy, (C-C)alkylsulfo each of K', K, K, and K are independently CH, N,N- nyl, (C-Co)arylsulfonyl, heteroarylsulfonyl, (C-C)alkyl 55 oxide, or N-(C-C)alkyl, and the dotted lines between K' sulfinyl, (C-C)arylsulfinyl, heteroarylsulfinyl, (C-C) and K, and K and K", denote optional double bonds; alkoxycarbonyl, amino, NH(C-C)alkyl, N(CC-C)alkyl). A' and B' are optional aromatic rings fused to ring A, only tri(C-C)ammonium(C-Co.)alkyl, heteroaryl(C-C) one of which is present in the compound, so as to form a fused alkyl, heteroaryl having quaternary nitrogen, heteroarylcar tricyclic system; and bonyl having quaternary nitrogen, (C-C)arylthio, (C-C) 60 when B' is present, the group Z is present, and alkylphosphate, (C-C)alkylphosphonate, (C-Co) when A' is present, the group Z is absent; and arylphosphate, (C-Co)arylphosphonate, phosphate, Sulfate, R is H. (C-Co.)alkyl, (C-C)alkenyl, (C-C)alkynyl, saccharide, or M" optionally when Z" is oxygen, wherein M (C-Co)cycloalkyl, (C-C)alkoxy, (C-Co.)aryl, het is an alkali metal; eroaryl, heterocycle, (C-Co.)alkylsulfoxy, (C-C)arylsul or when Z or Z is NR'R'', R'R' together with the N to 65 foxy, heteroarylsulfoxy, (C-C)alkoxycarbonyl, amino, which they are attached forms a heteroaryl or heterocycle NH(C-C)alkyl, N(C-C)alkyl), tri(C-C)ammonium group; (C-Co.)alkyl, heteroaryl(C-C)alkyl, heteroaryl having US 8,476,450 B2 7 8 quaternary nitrogen, heteroarylcarbonyl having quaternary provided that a saccharide is not directly attached to K: nitrogen, Saccharide, or M" optionally when Z" is oxygen, A is an anion, present when a quaternary nitrogen is wherein M is an alkali metal; present; R" is (Co-Co)aryl, heteroaryl, heterocycle, (C-Co)alky or a salt thereof. lthio, (C-C)alkyl-S(O)—, (C-C)alkyl-SO. —SO(C- In yet another embodiment, the invention provides a com Co)alkyl, saccharide, (C-C)alkylphosphate, (C-C) alkylphosphonate, (C-C)arylthio, (C-C)aryl-S(O)—, pound of formula IIA: (Co-Co.)aryl-SO. —SO(C-Co.)aryl, (C-C)arylphos phate, (C-Co)arylphosphonate, or R' is (C-C)alkyl Sub IIa stituted by R: 10 O R is (C-C-o)alkenyl, (C-C-o)alkynyl, (C-C-o)cy R cloalkyl, (C-Co.)alkoxyl, (C-C)alkylcarbonyl, (C-C) 21 N N z1 alkylcarboxyl, hydroxyl, -COOR. —SO.R., (C-C) Z-l A B X-( C alkylthio, (C-C)arylthio, (C-C)alkyl-S(O)—, (C-C) Sás S S alkyl-SO. , nitro, amino, NH(C-C)alkyl, NH(C-C) 15 WI A alkynyl, N(C-C)alkyl), or N((C-C)alkynyl), mercapto, saccharide, or trifluoromethyl: or when Z or Z is NR'R'', R'R' together with the N to wherein which they are attached forms a heteroaryl or heterocycle Z is OR, NHR', or NR'R'; group; Z" is O, S, NH, NHR, or N=N; wherein any alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, W' is H, halo, hydroxyl, (C-C)alkyl, or (C-C)alkoxy; amino, aryl, heteroaryl, or heterocycle group is optionally R is H. (C-Co.)alkyl, (C-C)alkenyl, (C-C)alkynyl, substituted with 1, 2, 3, 4, or 5 substituents selected from (C-Co)cycloalkyl, (C-C)alkoxy, (C-Co.)aryl, het (C-C-o)alkyl, (C-Co.)alkenyl, (C-Co)alkynyl, (C-Co) eroaryl, heterocycle, (C-Co.)alkylsulfoxy, (C-C)arylsul cycloalkyl, (C-Co.)alkoxyl, (C-C)alkylcarbonyl, (C- 25 foxy, heteroarylsulfoxy, (C-C)alkoxycarbonyl, amino, Co)alkylcarboxyl, halo, hydroxyl. —COOR, —SOR, NH(C-C)alkyl, N(C-C)alkyl), tri(C-C)ammonium —SO.R., (C-C)alkyl-S(O)—, (C-C)alkyl-SO , (C-Co.)alkyl, heteroaryl(C-C)alkyl, heteroaryl having phosphate, (C-C)alkylphosphate, (C-C)alkylphospho quaternary nitrogen, heteroarylcarbonyl having quaternary nate, nitro, amino, NH(C-C)alkyl, NH(C-C)alkynyl, nitrogen, Saccharide, or M" optionally when Z" is oxygen, N((C-C)alkyl), N(CC-C)alkynyl), mercapto, (C-C) 30 wherein M is an alkali metal; alkylthio, (C-C)aryl, (C-C)arylthio, trifluoromethyl, R" is (Co-Co)aryl, heteroaryl, heterocycle, (C-Co)alky —O, heteroaryl, and heterocycle, and each substituent is lthio, (C-C)alkyl-S(O)—, (C-C)alkyl-SO. —SO(C- optionally substituted with one to three R groups: Co)alkyl, saccharide, (C-Co.)alkylphosphate, (C-C) R is H or (C-C)alkyl: alkylphosphonate, (C-C)arylthio, (C-C)aryl-S(O)—, when Z or Z comprises a nitrogen moiety, a hydrogen of 35 the Z or Z. nitrogen moiety may be replaced by the group L. (Co-Co)aryl-SO. —SO(C-Co.)aryl, (C-C)arylphos wherein L is an amino acid radical, a peptide radical having phate, (Co-Co)arylphosphonate, or R' is (C-C-o)alkyl sub up to 20 amino acid moieties, or any other Small molecule that stituted by R: is a substrate for a nonluciferase; with the proviso that when R is (C-C-o)alkenyl, (C-C-o)alkynyl, (C-C-o)cy L is an amino acid radical or a peptide radical, at least one of 40 cloalkyl, (C-Co.)alkoxyl, (C-C)alkylcarbonyl, (C-C) W' or a W is not H: alkylcarboxyl, hydroxyl, -COOR, SOR', (C-C) when Z is a hydroxyl group or a nitrogen moiety, H of the alkylthio, (C-C)arylthio, (C-C)alkyl-S(O)—, (C-C) hydroxyl or nitrogen moiety may be replaced by (HO)2P alkyl-SO nitro, amino, NH(C-C)alkyl, NH(C-C) (O)—OCH2—, Sulfo, —POH, or by a cephalosporanic acid alkynyl, N((C-C)alkyl), or N((C-C)alkynyl), mercapto, attached to the group Z via a carbon chain of one to about 12 45 saccharide, or trifluoromethyl: carbon atoms; with the proviso that the sulfo or the POH or when Z or Z is NR'R'', R'R' together with the N to group is attached to the hydroxyl oxygen via a (C-C)alky which they are attached forms a heteroaryl or heterocycle lene group: group; when Z or Z is a hydroxyl group or a nitrogen moiety, or wherein any alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, when Z"—R is a hydroxyl group, one H of the hydroxyl or 50 amino, aryl, heteroaryl, or heterocycle group is optionally nitrogen moiety may be replaced by the group L'-linker, substituted with 1, 2, 3, 4, or 5 substituents selected from wherein L' is a group removable by an enzyme to free the (C-Co.)alkyl, (C-Co.)alkenyl, (C-Co.)alkynyl, (C-Co) linker, and linker is a carbon chain that can self-cleave, cycloalkyl, (C-Co.)alkoxyl, (C-C)alkylcarbonyl, (C- optionally interrupted by one or more nitrogenatoms, oxygen Co)alkylcarboxyl, halo, hydroxyl, -COOR, SOR, atoms, carbonyl groups, optionally Substituted aromatic 55 —SO.R., (C-C)alkyl-S(O)—, (C-C)alkyl-SO , rings, or peptide bonds, phosphate, (C-C)alkylphosphate, (C-C)alkylphospho linker is attached to L'via an oxygenatom oran NH group nate, nitro, amino, NH(C-C)alkyl, NH(C-C)alkynyl, at one terminus of the linker and the other terminus of the N((C-C)alkyl), N(C-C)alkynyl), mercapto, (C-C) linker forms an ether, ester, or amide linkage with a group Z, alkylthio, (C-C)aryl, (C-C)arylthio, trifluoromethyl, Z", or Z" R. 60 =O, heteroaryl, and heterocycle, and each Substituent is when Z is OR", formula II is optionally a dimer connected optionally substituted with one to three R groups: at the two A rings via linker comprising a (C-C)alkyl R is H or (C-C)alkyl; diradical that is optionally interrupted by one to four O atoms, when Z is OR", formula IIA is optionally a dimer con N atoms, or an optionally Substituted aryl, heteroaryl, or nected at the two A rings via linker comprising a (C-C) heterocycle group to form a bridge between the dimer of 65 alkyl diradical that is optionally interrupted by one to four O formula II, and the R' group of each Zgroup connecting the atoms, Natoms, or an optionally Substituted aryl, heteroaryl, dimer of formula II is replaced by the bridge; or heterocycle group to form a bridge between the dimer of US 8,476,450 B2 9 10 formula IIA, and the R' group of each Zgroup connecting the W' and Zare both keto groups on ring A, and at least one of dimer of formula II is replaced by the bridge; the dotted lines denoting optional double bonds in ring A is provided that a saccharide is not directly attached to K: absent; A is an anion, present when a quaternary nitrogen is each W is independently H, halo, (C-C)alkyl, (C-C) present; alkenyl, hydroxyl, or (C-C)alkoxy; or a salt thereof. each of K', K, K, and K" are independently CH, N,N- Other deriviates and their use in luminogenic assays is oxide, or N (C-C)alkyl, and the dotted lines between K' described hereinbelow. and K, and K and K, denote optional double bonds; The use of the luciferin derivatives described herein can A' and B' are optional aromatic rings fused to ring A, only 10 one of which is present in the compound, so as to form a fused result in an assay which produces a measurable change in tricyclic system; and optical properties upon interaction with a nonluciferase mol when B' is present, the group Z is present, and ecule, which interaction may alter the structure of the when A' is present, the group Z is absent; and luciferin derivative. As described herein, the product of a reaction between a luciferin derivative and a nonluciferase the dotted line in ring B is an optional double bond; 15 each R is independently H. (C-C)alkyl, (C-Co.)alk enzyme or other molecule of interest need not be D-luciferin enyl, (C-Co.)alkynyl, (C-C)cycloalkyl, (C-C)alkoxy, or aminoluciferin. For example, a luciferin derivative may (Co-Co)aryl, heteroaryl, heterocycle, (C-C)alkylsulfoxy, include a Substrate that includes a reactive chemical group for (Co-Co)arylsulfoxy, heteroarylsulfoxy, (C-Co)alkylsulfo a nonluciferase enzyme linked to luciferin or aminoluciferin nyl, (C-Co)arylsulfonyl, heteroarylsulfonyl, (C-C)alkyl via a chemical linker. Transformation of the reactive chemical Sulfinyl, (C-C)arylsulfinyl, heteroarylsulfinyl, (C-C) group of the derivative by the nonluciferase enzyme may alkoxycarbonyl, amino, NH(C-C)alkyl, N(C-C)alkyl). yield a product that contains (retains) a portion of the Sub tri(C-C)ammonium(C-Co.)alkyl, heteroaryl(C-C) strate, a portion of the chemical linker, the chemical linker, or alkyl, heteroaryl having quaternary nitrogen, heteroarylcar a portion of the substrate and the chemical linker, and that bonyl having quaternary nitrogen, (C-C)arylthio, (C-C) product is a substrate for luciferase. Also provided are 25 alkylphosphate, (C-C)alkylphosphonate, (C-C) luciferin derivatives which, after interaction with a nonlu arylphosphate, (C-Co)arylphosphonate, phosphate, Sulfate, ciferase enzyme or other molecule, may yield a product that saccharide, or M" optionally when Z" is oxygen, wherein M optionally undergoes one or more further reactions, e.g., is an alkali metal; O-elimination, to yield a suitable substrate for luciferase. or when Z or Z is NR'R'', R'R' together with the N to Luciferin derivatives in which the backbone of luciferin is 30 which they are attached forms a heteroaryl or heterocycle further modified in its ring structure, e.g., a quinolyl or group; napthyl luciferin, are provided, as well as advantageously wherein any alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, providing modifications at the carboxy position of the thiaz amino, aryl, heteroaryl, or heterocycle group is optionally ole ring, to provide improved characteristics to the luciferin substituted with 1, 2, 3, 4, or 5 substituents selected from derivative. Derivatives with certain modifications provide for 35 (C-Co.)alkyl, (C-Co.)alkenyl, (C-C)alkynyl, (C-C) or improve assays for certain nonluciferase enzymes or mol cycloalkyl, (C-Co.)alkoxyl, (C-C)alkylcarbonyl, (C- ecules. For instance, as described hereinbelow, a pH insensi Co)alkylcarboxyl, halo, hydroxyl, -COOR, SOR, tive derivative of luciferin was identified that is useful in —SO.R. nitro, amino, (C-Co.)alkyl-S(O)—, (C-C) biological assays that may be run at a pH other than physi alkyl-SO , phosphate, (C-Co.)alkylphosphate, (C-C) ological pH, i.e., less than about pH 7.0 and greater than about 40 alkylphosphonate, NH(C-C)alkyl, NH(C-C)alkynyl, pH 7.8. Thus, bioluminescent methods that employ a luciferin N((C-C)alkyl), N(C-C)alkynyl), mercapto, (C-C) derivative of the invention may be used to detect one or more alkylthio, (C-C)aryl, (C-C)arylthio, trifluoromethyl, molecules, e.g., an enzyme, a cofactor for an enzymatic reac =O, heteroaryl, and heterocycle, and each Substituent is tion Such as ATP, an enzyme Substrate, an enzyme inhibitor, optionally substituted with one to three R groups: an enzyme activator, or OH radicals, or one or more condi 45 R is H. (C-C)alkyl, or (Co-Co)aryl; tions, e.g., redox conditions. when Z or Z comprises a nitrogen moiety, one or both of In one embodiment, the methods employ a compound of the hydrogens of the Z or Z. nitrogen moiety may be replaced formula III: by (C-C)alkyl or the group L., wherein L is an amino acid radical, a peptide radical having up to 20 amino acid moieties, 50 or any other Small molecule that is a Substrate for a nonlu III ciferase; with the proviso that when L is an amino acid radical or a peptide radical, at least one W is not H: B : when Z is a hydroxyl group or a nitrogen moiety, H of the --2. Y. N Q-R hydroxyl or nitrogen moiety may be replaced by (HO)P 55 (O)—OCH2—, Sulfo, —POH, or by a cephalosporanic acid Z'-Hl, A Ks ...) We S-1 Á\ X S W2 attached to the group Z via a carbon chain of one to about 12 Z WI A carbon atoms; with the proviso that when ring B is a thiazole W2 ring, the Sulfo or the —POH group is attached to the hydroxyl oxygen via a (C-C)alkylene group; wherein 60 when Z or Z is a hydroxyl group or a nitrogen moiety, or Y is N,N-oxide, N-(C-C)alkyl, or CH: when Z" R is a hydroxyl group, one H of the hydroxyl or X is S, O, CH-CH, N=CH, or CH-N: nitrogen moiety may be replaced by the group L'-linker, Zand Z are independently H, OR, NHR, or NRR: Z" is O, wherein L' is a group removable by an enzyme to free the S, NH, NHR, or N=N; linker, and linker is a carbon chain that can self-cleave, Q is carbonyl or CH: 65 optionally interrupted by one or more nitrogenatoms, oxygen W' is H. halo, (C-C)alkyl, (C-C-o)alkenyl, hydroxyl, or atoms, carbonyl groups, optionally Substituted aromatic (C-C)alkoxy; or rings, or peptide bonds, US 8,476,450 B2 11 12 linker is attached to L'via an oxygenatom oran NH group and utility of a phosphatase assay Such as an alkaline phos at one terminus of the linker and the other terminus of the phatase assay is increased employing a luciferin derivative of linker forms an ether, ester, or amide linkage with a group Z, the invention. Z", or Z" R. Generally, luciferase substrates have a free hydroxyl (lu when Z is OR, formula III is optionally a dimer connected 5 ciferin) or free amino group (amino luciferin) on the benzene at the two A rings via a linker comprising a (C-C)alkyl ring. Alternate backbones for luciferin, like quinolinyl diradical that is optionally interrupted by one to four O atoms, luciferin or napthyl luciferin, with free hydroxyl or free N atoms, or an optionally Substituted aryl, heteroaryl, or amino groups, are luciferase Substrates. To expand the scaf heterocycle group to form a bridge between the dimer of folding upon which modifications can be made to luciferin formula III, and the R group of each Zgroup connecting the 10 dimer of formula III is replaced by the bridge: and result in a luciferase substrate, luciferin derivatives with A is an anion, present when a quaternary nitrogen is aryl or other chains attached through the oxygen or nitrogen present; on the A ring of luciferin were prepared. Derivatives with or a salt thereof; nitrogen on the A ring were utilized by beetle luciferase to provided that the compound of formula III is not aminolu 15 generate bright luminescence. Moreover, HPLC verified that ciferin that has been modified to include a protease substrate luciferin derivatives with aryl or alkyl chains decreased in via a peptide bond at the amino group which optionally also concentration when acted upon by a thermostable luciferase has a protected carboxyl group at position 5 on ring C, or any (as opposed to wild-type luciferin contamination causing the of luciferin 6'-methyl ether, luciferin 6'-chloroethyl ether, light and decreasing in concentration). Such luciferin deriva 6'-deoxyluciferin, 6'-luciferin-4-trifluoromethylbenzylether, tives are also shown herein to be utilized by other luciferases. luciferin 6'-phenylethylether, luciferin 6'-geranyl ether, Moreover, those scaffolds, e.g., luciferase substrates that have luciferin 6'-ethyl ether, 6'-luciferin prenyl ether, 6'-luciferin groups attached through the oxygen or nitrogen on the A ring 2-picolinylether, 6'-luciferin 3-picolinylether, 6'-luciferin of luciferin, may be modified to include a substrate for an 4-picolinyl ether, luciferin 6'-benzyl ether, D-luciferin-O-B- enzyme, a binding site for another molecule, or any reactive galacto-pyranoside, D-luciferin-O-sulfate, D-luciferin-O- 25 group useful to measure a molecule Such as a cellular bioac phosphate, D-luciferyl-L-phenylalanine, and D-luciferyl-L- tive molecule, including second messengers, e.g., a cAMP N'-arginine; or not a substrate for one or more cytochrome binding site, for instance, to measure cAMP, calmodulin, e.g., P450 enzymes but optionally may be a substrate for a non to measure calcium, or to measure IP3. cytochrome P450 enzyme. In one embodiment, a method to detect luciferase employs In one embodiment, a bioluminescent assay method to 30 detect one or more nonluciferase enzymes is provided. The a compound of formula IIIA: method includes contacting a sample suspected of having one or more nonluciferase enzymes, a Substrate or a co-factor for IIIa the reaction, with a corresponding reaction mixture that O includes a derivative of luciferin or a derivative of aminolu 35 ciferin that is a substrate for the nonluciferase enzyme. In one R embodiment, the derivative is one having a modification in Z l OA B S 7 C Z it Y the benzene ring of D-luciferin that includes a recognition site Sa for the nonluciferase enzyme, e.g., for a monoamine oxidase. ySa. X S W2 A In another embodiment, the derivative is one having a modi 40 fication in the thiazole ring (ring B) of D-luciferin which derivative is a substrate for luciferase and optionally a sub wherein strate for a nonluciferase enzyme. In another embodiment, Y is N,N-oxide, N—(C-C)alkyl, or CH: the derivative is one having a modification in the thiazole ring X is S, O, CH-CH, N=CH, or CH-N: (ring C) of luciferin that includes a recognition site for an 45 Z is H, OR, NHR, or NRR: enzyme of interest, e.g., acetylcholinesterase. In another Z" is O, S, NH, NHR, or N=N; embodiment, the derivative is one having a modification to W' is H. halo, hydroxyl, (C-C)alkyl, (C-C)alkenyl, or one of the rings that includes a recognition site for the enzyme (C-C)alkoxy; of interest, as well as a further modification to that ring or one W is H, F, or methyl: or more of the other rings. 50 the dotted line in ring B is an optional double bond; Previously, enzymes that could be tested with luciferin each R is independently H. (C-C)alkyl, (C-Co.)alk derivatives were those that interacted well close to aromatic enyl, (C-Co.)alkynyl, (C-Co)cycloalkyl, (C-C2)alkoxy, structures (D-luciferin oraminoluciferin) and interacted with (Co-Co)aryl, heteroaryl, heterocycle, (C-C)alkylsulfoxy, a recognition site (Substrate) that was stable close to aromatic (C-C)arylsulfoxy, heteroarylsulfoxy, (C-C)alkylsulfo structures. As described herein, luciferin derivatives that are 55 nyl, (C-Co)arylsulfonyl, heteroarylsulfonyl, (C-C)alkyl Substrates for nonluciferase enzymes that do not necessarily sulfinyl, (C-C)arylsulfinyl, heteroarylsulfinyl, (C-C) react with structures close to aromatic rings or those with alkoxycarbonyl, amino, NH(C-C)alkyl, N(CC-C)alkyl). recognition sites that are more stable when attached to an aryl tri(C-C)ammonium(C-Co.)alkyl, heteroaryl(C-C) chain than an aromatic structure, were identified. For alkyl, heteroaryl having quaternary nitrogen, heteroarylcar example, an assay which employed previously described 60 bonyl having quaternary nitrogen, (C-C)arylthio, (C-C) luciferin derivatives with a phosphate group attached through alkylphosphate, (C-C)alkylphosphonate, (C-C) the hydroxyl group on the benzene ring, i.e., a Substrate for arylphosphate, (C-Co)arylphosphonate, phosphate, Sulfate, alkaline phosphatase, was limited by high background saccharide, or M" optionally when Z" is oxygen, wherein M because the phosphate spontaneously hydrolyzed. Attaching is an alkali metal; the phosphate to an aryl chain is likely stabilizing, which may 65 or when Z or Z is NR'R'', R'R' together with the N to allow for a derivative that is a substrate for alkaline phos which they are attached forms a heteroaryl or heterocycle phatase and may reduce the background. Thus, the sensitivity group; US 8,476,450 B2 13 14 wherein any alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, beetle luciferase reaction mixture. Alternatively, the nonlu amino, aryl, heteroaryl, or heterocycle group is optionally ciferase and luciferase reactions may be conducted simulta substituted with 1, 2, 3, 4, or 5 substituents selected from neously in the same vessel. (C-C-o)alkyl, (C-Co.)alkenyl, (C-Co)alkynyl, (C-Co) The invention thus provides in an embodiment a method to cycloalkyl, (C-C)alkoxyl, (C-C)alkylcarbonyl, (C- detect or determine the presence or amount of a molecule for Co)alkylcarboxyl, halo, hydroxyl. —COOR, —SOR, a nonluciferase enzyme-mediated reaction in a sample. The —SO.R. nitro, amino, (C-C)alkyl-S(O)—, (C-C) method includes contacting a sample, a first reaction mixture alkyl-SO. , phosphate, (C-C)alkylphosphate, (C-C) for a nonluciferase enzyme-mediated reaction, and a deriva alkylphosphonate, NH(C-C)alkyl, NH(C-C)alkynyl, tive of luciferin which is a substrate for the nonluciferase N((C-C)alkyl), N(C-C)alkynyl), mercapto, (C-C) 10 enzyme, so as to yield a first mixture comprising a lumino alkylthio, (C-C)aryl, (C-C)arylthio, trifluoromethyl, genic product that is a Substrate for a luciferase. In one embodiment, the derivative is a compound of formula I. In =O, heteroaryl, and heterocycle, and each Substituent is another embodiment, the derivative is a compound of formula optionally substituted with one to three R groups: II. At least a portion of the first mixture is contacted with a R is H., (C-C)alkyl, or (C-C)aryl; 15 second reaction mixture for a luciferase-mediated reaction, when Z is OR, formula III is optionally a dimer connected So as to yield a second mixture. Then luminescence in the at the two A rings via a linker comprising a (C-C)alkyl second reaction is detected or determined, thereby detecting diradical that is optionally interrupted by one to four O atoms, or determining the presence or amount of a molecule for the Natoms, an optionally substituted aryl, heteroaryl, or hetero nonluciferase enzyme-mediated reaction in the sample, e.g., cycle group, or a combination thereof, to form a bridge compared to a control. In one embodiment, the derivative is a between the dimer of formula III, and the R group of each Z Substrate for a transferase, e.g., glutathione S transferase group connecting the dimer of formula III is replaced by the (GST). Exemplary derivatives useful to detect GST are shown bridge; in FIG. 12. In some embodiments, a second nonluciferase A is an anion, present when a quaternary nitrogen is enzyme may be used to further chemically transform the present; 25 product of the first nonluciferase enzyme-mediated reaction or salt thereof to yield a substrate for a luciferase. For example, the sample, provided that the compound of formula III is not aminolu nonluciferase reaction mixture or luciferase reaction mixture ciferin that has been modified to include a protease substrate may include an esterase, or the esterase may be added sepa via a peptide bond at the amino group which optionally also rately. has a protected carboxyl group at position 5 on ring C, or any 30 In one embodiment, derivatives of luciferin having an ester of luciferin 6'-methyl ether, luciferin 6'-chloroethyl ether, modification are employed in methods of the invention, Such 6'-deoxyluciferin, 6'-luciferin-4-trifluoromethylbenzylether, as those to detect nonluciferase enzymes including P450 luciferin 6'-phenylethylether, luciferin 6'-geranyl ether, enzymes or monoamine oxidases (MAOS), or other enzymes luciferin 6'-ethyl ether, 6'-luciferin prenyl ether, 6'-luciferin such as flavin monoamine oxidases (FMOs), glutathione S 2-picolinylether, 6'-luciferin 3-picolinylether, 6'-luciferin 35 transferases (GSTs), phosphatases, e.g., alkaline phos 4-picolinyl ether, luciferin 6'-benzyl ether, D-luciferin-O-B- phatases (AP), or Sulfatases, as those derivatives may have galacto-pyranoside, D-luciferin-O-sulfate, D-luciferin-O- improved properties as a nonluciferase enzyme substrate. In phosphate, D-luciferyl-L-phenylalanine, and D-luciferyl-L- particular, as shown herein, the addition of an ester group at N'-arginine; or not a substrate for one or more cytochrome the carboxy position of the thiazole ring of a luciferin deriva P450 enzymes but optionally may be a substrate for a non 40 tive provided a substrate that was recognized by two P450 cytochrome P450 enzyme. enzymes, 2D6 and 2C19, that did not react with previously Furthermore, the inclusion of a thiol compound with a described luciferin derivatives that are substrates of cyto luciferin derivative in a luciferase-mediated assay may effec chrome P450 enzymes (see U.S. published application tively stabilize the luminescence of the reaction, thereby pro 2004.0171099). viding for “glow’ kinetics, i.e., luminescent intensity of the 45 In an alternate embodiment, a method to detect or deter luciferase-mediated reaction is relatively constant over time mine the presence or amount of a molecule for a first nonlu after addition of the derivative. Such compounds may be also ciferase enzyme-mediated reaction in a sample is provided be used in bioluminogenic assays monitoring the presence or wherein the method includes contacting a sample, a reaction activity of nonluciferase enzymes or a nonenzymatic biologi mixture for a nonluciferase-mediated enzyme reaction and a cal reaction. 50 luciferase-mediated reaction, and a derivative of luciferin For derivatives that function directly as a substrate for which is a Substrate for the nonluciferase enzyme, yielding a luciferase, as well as optionally a substrate of a nonluciferase mixture. A reaction between the nonluciferase enzyme and enzyme or other molecules, the derivative may be employed the derivative yields a luminogenic product that is a Substrate to directly detect luciferase, or a co-factor, inhibitor, or acti for the luciferase. Luminescence in the mixture is detected or vator of the luciferase reaction. If the derivative is a prosub 55 determined, thereby detecting or determining the presence or strate for luciferase, i.e., the product of a reaction between the amount of a molecule for the nonluciferase-mediated reaction derivative and the nonluciferase enzyme is a substrate for in the sample. luciferase, sequential or concurrent reactions for the nonlu The invention also provides an embodiment directed to a ciferase enzyme and the luciferase may be conducted. For method to detect the presence or amount of a non-enzymatic instance, an assay for a nonluciferase enzyme that includes a 60 molecule in a sample. The method includes contacting a luciferin derivative that is a prosubstrate for luciferase may be sample, a first reaction mixture for a nonenzyme-mediated conducted in a single reaction vessel and a beetle luciferase reaction and a derivative of luciferin which in the presence of reaction mixture added to that vessel. In another embodiment, the molecule yields a luminogenic product that is a Substrate a reaction mixture for an assay for a nonluciferase enzyme for a luciferase, and contacting a portion of the first reaction that includes a luciferin derivative that is a prosubstrate for 65 and a second reaction mixture for a luciferase-mediated reac luciferin may be conducted in a single reaction vessel and a tion, to yield a second reaction, then luminescence in the portion of that reaction added to a different vessel having a second reaction is detected or determined, thereby detecting US 8,476,450 B2 15 16 or determining the presence or amount of the molecule. For and/or aldehyde intermediate which optionally undergoes a instance, a mixture is provided having a sample, a first reac noncatalytic B-elimination to yield the hydroxy fluorescent tion mixture for a nonenzyme-mediated reaction and a deriva product. Fluorescence in the mixture is detected or deter tive of luciferin which in the presence of the molecule yields mined, thereby detecting or determining the presence or a luminogenic product that is a substrate for a beetle amount of a molecule for the nonprotease enzyme mediated luciferase. At least a portion of the first mixture and a second reaction in the sample. The invention also provides for meth reaction mixture for a beetle luciferase-mediated reaction are ods of using fluorophore derivatives of the invention to iden mixed, to yield a second mixture, and then luminescence in tify inhibitor, activators, substrates or co-factors for enzy the second mixture is detected or determined, thereby detect matic reactions. ing or determining the presence or amount of the molecule. 10 For the biolumingenic assays described herein which The invention provides compositions or kits having one or employ luciferin derivatives with a lower background lumi more luciferin derivatives and/or fluorophore derivatives of nescence than D-luciferin oraminoluciferin, those assays can the invention. The kits may optionally contain other reagents, use lower amounts, e.g., because Small changes in lumines e.g., enzyme, reaction mixtures, and the like. The biolumino cence can be detected, or higher amounts, e.g., in reactions 15 genic reaction mixtures, compositions and kits of the inven that are improved by increased amounts of substrate, of the tion may optionally include an agent that slows the reaction derivative, and those derivatives may have improved reactiv rate, e.g., amino methylbenzothiazole (AMBT) or aminophe ity, e.g., with a nonluciferase enzyme. In addition, for any of nyl methylbenzothiazol (APMBT), see U.S. published appli the bioluminogenic assays described herein, other reagents cation 2004.0171099, yielding glow kinetics and/or an agent may be added to reaction mixtures, including but not limited that stabilizes light production, e.g., a thiol or coenzyme A. to those that inhibit or prevent inactivation of luciferase, or otherwise extend or enhance signal. BRIEF DESCRIPTION OF THE FIGURES Also provided is a method to identify a modulator of a nonluciferase enzyme-mediated reaction. The method FIG.1. Numbering of ring atoms in the six membered ring includes contacting one or more agents, a first reaction mix 25 (“A ring or “ring A'), five membered centerring (“Bring” or ture for a nonluciferase enzyme-mediated reaction, and a “ring B), and other five membered ring (“C ring” or “ring derivative of luciferin which is a substrate for the nonlu C”) of beetle luciferin (D-luciferin). ciferase enzyme, so as to yield a first mixture, or providing FIG. 2. Exemplary luciferin derivatives for enzymes. such a mixture. The first mixture in the absence of the one or FIG. 3. Structure of 5'-fluoroluciferin. more agents includes aluminogenic product that is a Substrate 30 FIGS. 4A-B. Derivatives of luciferin useful as a redox for a beetle luciferase. At least a portion of the first mixture SSO. and a second reaction mixture for a beetle luciferase-medi FIGS. 5A-B. Exemplary substrates. ated reaction are mixed, so as to yield a second mixture. FIG. 6. Redox substrates. Luminescence in the second mixture is compared with a FIGS. 7A-B. Other exemplary derivatives of luciferin. control mixture, thereby identifying whether one or more of 35 the agents modulates the nonluciferase enzyme-mediated FIG. 8A. A graphical representation of RLU for reactions reaction. containing an esterase and a luciferin derivative, luciferin In a further embodiment of the present invention, luciferin ethyl ester. 20 minute incubation at 37° C. derivatives that retain activity directly as a substrate for FIG. 8B. A graphical representation of RLU for reactions luciferase may be utilized to inactivate or inhibit the 40 containing an aminoluciferin derivative (isopropyl urethane luciferase enzyme, e.g., by covalent modification thereof, or aminoluciferin methyl ester; 50 uM), with and without as a competitive or noncompetitive inhibitor thereof. This esterase. 20 minute incubation at room temperature. embodiment provides a method to screen for compounds that FIG. 9. A graphical representation of RLU for reactions prevent the inactivation of luciferase. Also provided is a containing an esterase and a luciferin derivative, luciferin method to identify a modulator of a luciferase-mediated reac 45 methyl ester. 20 minute incubation at 37°C. tion using a luciferin derivative of the present invention. FIG. 10. A graphical representation of RLU for reactions The invention further provides a fluorogenic method which containing an esterase and a luciferin derivative, D-luciferin employs a derivative of a fluorophore that is modified to picolinyl ester. 20 minute incubation at 37° C. detect one or more molecules, e.g., an enzyme, a cofactor for FIG. 11. A graphical representation of derivatives of fluo an enzymatic reaction Such as ATP, an enzyme Substrate, an 50 rogenic derivatives useful as monoamine oxide (MAO) sub enzyme inhibitor, an enzyme activator, or OH radicals, or one Strates. or more conditions, e.g., redox conditions. The invention thus FIGS. 12A-B. Luciferin derivatives useful to detect glu provides for bioluminogenic and fluorogenic assays to detect tathione S transferase (GST). the amount, activity or presence of a molecule in a sample. FIGS. 13 A-D. A graphical representation of derivatives of The invention also provides further embodiments directed 55 to fluorogenic assays and derivatives of fluorophores which luciferin derivatives for MAO assays. The derivative in FIG. are a substrate for a nonluciferase nonproteolytic enzyme. 13B was not utilized by MAO. Thus, a method to detect or determine the presence or amount FIG. 14. Derivatives of luciferin useful as flavin monooxy of a molecule for a nonprotease enzyme-mediated reaction in genase (FMO) substrates. a sample by a fluorescent method is provided. The method 60 FIG. 15. Exemplary FMO substrates. includes contacting a sample, a first reaction mixture for a FIG. 16. Exemplary AP substrates. nonluciferase, nonproteolytic enzyme-mediated reaction, FIG. 17. RLU for a series of luciferin derivatives and each and a derivative of a fluorophore which includes a substrate of a panel of P450 enzymes. for the enzyme, so as to yield a first mixture, wherein if the FIG. 18. RLU for a series of luciferin derivatives and each molecule is present in the sample, the first mixture comprises 65 of a panel of P450 enzymes. a hydroxy fluorescent product. The interaction between the FIGS. 19A-C. Structures of exemplary derivatives useful enzyme and the derivative optionally produces an iminium to detect P450 enzymes. US 8,476,450 B2 17 18 FIG. 20. A graphical representation of CYP2D6 and FIG. 36. A graphical representation of RLU for reactions CYP2C19 activity against luciferin-ME.A) RLU in reactions containing one of a panel of P450 enzymes and a luciferin with luciferin-ME. B) Signal to background ratio in reactions derivative, luciferin 2-(1-piperazine)ethyl ether. Reactions with luciferin-ME. contained 1 pmol P450. FIG. 21. A graphical representation of P450 activities with 5 FIG.37A. A graphical representation of RLU for reactions luciferin-ME methyl ester. containing one of a panel of P450 enzymes and a luciferin FIG. 22. A graphical representation of CYP2D6 activity derivative, meta-N-methyl picolinyl luciferin methyl ester. with luciferin-MEpicolinyl ester with and without esterase. Reactions contained 1 pmol P450. A 30 minute incubation at Reactions contained 1 pmol CYP2D6. 37° C. was followed by a 20 minute incubation at room FIG. 23. A graphical representation of CYP2C19 activity 10 temperature with or without esterase in luciferin detection with luciferin-H-picolinylester with and without esterase. reagent. Reactions contained 1 pmol CYP2C19. FIG.37B. A graphical representation of RLU for reactions FIG. 24. A graphical representation of RLU for reactions containing a luciferin derivative, meta-N-methyl picolinyl containing one of a panel of P450 enzymes and a luciferin 15 luciferin methyl ester, with or without esterase. 20 minute derivative, D-luciferin ethyl ester with a C ring modification. incubation at room temperature. Reactions contained 1 pmol P450. FIG. 38. A graphical representation of RLU for reactions FIG. 25. A graphical representation of RLU for reactions containing one of a panel of P450 enzymes and a luciferin containing one of a panel of P450 enzymes and a luciferin derivative, methamphetamine-luciferin. Reactions contained derivative, luciferin methyl ester with a C ring modification. 1 pmol P450. Reactions contained 1 pmol P450 or 5 pmol CYP19. FIG. 39. A graphical representation of RLU for reactions FIG. 26. Graphical representation of RLU for reactions containing one of a panel of P450 enzymes and a luciferin containing one of three P450 enzymes and luciferin-ME derivative, N-isobutoxycarbonyl-aminoluciferin. Reactions ethylene glycol ester, with or without esterase. contained 1 pmol P450. FIG. 27A. A graphical representation of RLU for an ami 25 FIG. 40. A graphical representation of RLU for reactions noluciferin derivative (N-isopropylaminoluciferin) (50 uM) containing one of a panel of P450 enzymes and a luciferin in a luciferase reaction. derivative, luciferin-H methyl ester and esterase. Reactions FIG. 27B. A graphical representation of RLU for an ami contained 1 pmol P450. noluciferin derivative (dimethylaminoluciferin; 100LLM) and FIG. 41. A graphical representation of RLU for reactions aminoluciferin (100 uM) in a luciferase reaction. 30 containing one of a panel of P450 enzymes and a luciferin FIG. 28. A graphical representation of signal to back derivative, luciferin-ME ethylene glycolester. Reactions con ground ratio for a reaction between 4,6' dimethyl ether tained 1 pmol P450, 5 pmol CYP19, or 40 ug HLM. luciferin and each of a panel of P450 enzymes. Reactions FIG. 42. A graphical representation of RLU for reactions contained 1 pmol P450. containing one of a panel of P450 enzymes and a luciferin FIG. 29. A graphical representation of signal background 35 derivative, methoxyquinolylluciferin methyl ester. Reactions ratio for a reaction between a luciferin derivative, (2-(6-meth contained 1 pmol P450. oxyquinolin-2-yl)-4,5-dihydrothiazole-4-carboxylate) and FIG. 43. A graphical representation of RLU for reactions each of a panel of P450 enzymes. Reactions contained 1 pmol containing one of a panel of P450 enzymes and a luciferin P450. 40 derivative, 6'(o-trifluoromethylbenzyloxy)-luciferin. 1 pmol FIG. 30. A graphical representation of RLU for reactions P450 or 5 pmol CYP19. containing one of a panel of P450 enzymes and a luciferin FIG. 44. A graphical representation of RLU for reactions derivative, 2-(7-methyoxy-quinoxalin-2-yl)-4-5-dihydro containing one of a panel of P450 enzymes and a luciferin thiazole-4-carboxylic acid. Reactions contained 1 pmol derivative, 6'methoxy-luciferin-hydrazide. Reactions contain P450. 45 1 pmol of P450 or 5 pmol CYP19. FIG. 31. A graphical representation of RLU for reactions FIG. 45. A graphical representation of RLU for reactions containing one of a panel of P450 enzymes and a luciferin containing one of a panel of P450 enzymes and a luciferin derivative. (N-((3-chloro-4-(2-(dimethylamino)-1-phenyl derivative, 6'-(3-((4-phenylpiperizin-1-yl)methyl)benzy propoxy)benzyloxy)carbonyl)-6'-aminoluciferin). Reactions loxy)-luciferin. Reactions contained 1 pmol P450 or 20 ug 50 HLM. contained 1 pmol P450 or 5 pmol CYP19. FIG. 46. A graphical representation of RLU for reactions FIG. 32. A graphical representation of RLU for reactions containing one of a panel of P450 enzymes and a luciferin containing one of a panel of P450 enzymes and a luciferin derivative, 6'-(2,4,6-trimethylbenzyloxy)-luciferin. 1 pmol of derivative, luciferin 4-piperidinemethyl ether. Reactions con P450 or 5 pmol CYP19. tained 1 pmol P450. 55 FIG. 47. A graphical representation of RLU for reactions FIG. 33. A graphical representation of RLU for reactions containing one of a panel of P450 enzymes and a luciferin containing one of a panel of P450 enzymes and a luciferin derivative, 6'(4-chlorophenylthio)methoxyluciferin. Reac derivative, N-(2-chloroethoxycarbonyl)-aminoluciferin. tions contained 1 pmol of P450 or 5 pmol CYP19. Reactions contained 1 pmol P450. FIG. 48. Inhibition of CYP3A4 activity by troleanomycin FIG. 34. A graphical representation of RLU for reactions 60 in a reaction with luciferin-FBE (6'-(2,3,4,5,6 pentafluo containing one of a panel of P450 enzymes and a luciferin robenzyloxy)-luciferin). derivative, 4'-(N,N-dimethylamino)methyl-6'(O-methyl) FIG. 49. Inhibition of CYP3A4 activity by nifedipine in a luciferin. Reactions contained 1 pmol P450. reaction with luciferin-PPX4FE (6'-(2,3,4,6-tetrafluoro-5- FIG. 35. A graphical representation of RLU for reactions ((4-phenylpiperazin-1-yl)methyl)benzyloxy)-luciferin). containing one of a panel of P450 enzymes and a luciferin 65 FIG.50. Inhibition of CYP3A4 activity by nifedipine in a derivative, 4-(N,N-dimethylaminomethyl)-6-methox reaction with luciferin-PPXE (6'-(3-((4-phenylpiperazin-1- ycuinolylluciferin. Reactions contained 1 pmol P450. yl)methyl)benzyloxy)-luciferin. US 8,476,450 B2 19 20 FIG. 51. CYP3A4 activity in human hepatocytes hepato is independently H, alkyl, aryl, heteroaryl, heterocycle, a cytes after treatment with rifampicin, ketoconazole, or protecting group or prodrug moiety. As would be readily rifampicin and ketoconazole, in a reaction with luciferin understood by one skilled in the art, when a substituent is keto PPXE. (=O) or thioxo (=S), or the like, then two hydrogen atoms FIG. 52. CYP3A4 activity in human hepatocytes after on the Substituted atom are replaced. treatment with rifampicin, ketoconazole, or rifampicin and "Stable compound' and “stable structure' are meant to ketoconazole, in a reaction with luciferin-FBE. indicate a compound that is sufficiently robust to survive FIG. 53. Luciferin derivatives which may be luciferase isolation to a useful degree of purity from a reaction mixture. substrates: N-isopropyl aminoluciferin bisethyl aminolu Only stable compounds are contemplated by and claimed in ciferin; and benzylaminoluciferin. 10 the present invention, however, certain unstable compounds, FIG. 54. Structures of derivatives of luciferin which may be for example, those that cannot easily be isolated, can be a substrate for different luciferases. employed in the methods described herein. FIG.55. A graphical representation of RLU for luciferin or One diastereomer may display Superior properties or activ 5-fluoroluciferin in luciferase reactions at different pHs. ity compared with another. When required, separation of the FIG. 56. ATP titration of a 5' fluoroluciferin and luciferin. 15 racemic material can be achieved by HPLC using a chiral FIG. 57. ATP titration of a 7" fluoroluciferin. column or by a resolution using a resolving agent such as FIG. 58. Spectra of various fluoroluciferins. camphonic chloride as described by Thomas J. Tucker, et al., FIG. 59. pH titration of a 7" fluoroluciferin. J. Med. Chem. 1994, 37, 2437-2444. A chiral compound may FIG. 60. Luciferin derivatives useful as luciferase sub also be directly synthesized using a chiral catalyst or a chiral strates and scaffolds for luciferin derivatives useful as nonlu ligand, e.g. Mark A. Huffman, et al., J. Org. Chem. 1995, 60. ciferase Substrates. 1590-1594. As used herein, the term “alkyl refers to a branched, DETAILED DESCRIPTION OF THE INVENTION unbranched, or cyclic hydrocarbon having, for example, from 1 to 30 carbonatoms, and often 1 to 12, or 1 to about 6 carbon I. Definitions 25 atoms. Examples include, but are not limited to, methyl, ethyl, 1-propyl. 2-propyl, 1-butyl, 2-methyl-1-propyl. 2-bu As used herein, the following terms and expressions have tyl, 2-methyl-2-propyl(t-butyl), 1-pentyl, 2-pentyl, 3-pentyl, the indicated meanings. It will be appreciated that the com 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, pounds of the present invention contain asymmetrically Sub 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2- stituted carbon atoms, and may be isolated in optically active 30 pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3- or racemic forms. It is well known in the art how to prepare pentyl, 2-methyl-3-penty1, 2,3-dimethyl-2-butyl, 3.3-dim optically active forms, such as by resolution of racemic forms ethyl-2-butyl, hexyl, octyl, decyl, dodecyl, and the like. The or by synthesis from optically active starting materials. All alkyl can be unsubstituted or substituted. The alkyl can also chiral, diastereomeric, racemic forms and all geometric iso be optionally partially or fully unsaturated. As such, the reci meric forms of a structure are part of this invention. 35 tation of an alkyl group includes both alkenyl and alkynyl Specific values listed below for radicals, substituents, and groups. The alkyl can be a monovalent hydrocarbon radical, ranges, are for illustration only; they do not exclude other as described and exemplified above, or it can be a divalent defined values or other values within defined ranges for the hydrocarbon radical (i.e., alkylene). radicals and Substituents. The term “alkenyl refers to a monoradical branched or As used herein, the term "substituted” is intended to indi 40 unbranched partially unsaturated hydrocarbon chain (i.e. a cate that one or more (e.g., 1, 2, 3, 4, or 5; in Some embodi carbon-carbon, sp' double bond). In one embodiment, an ments 1, 2, or 3; and in other embodiments 1 or 2) hydrogens alkenyl group can have from 2 to 10 carbon atoms, or 2 to 6 on the group indicated in the expression using “substituted” is carbon atoms. In another embodiment, the alkenyl group has replaced with a selection from the indicated group(s), or with from 2 to 4 carbon atoms. Examples include, but are not a suitable group known to those of skill in the art, provided 45 limited to, ethylene or vinyl, allyl, cyclopentenyl, 5-hexenyl, that the indicated atoms normal valency is not exceeded, and and the like. The alkenyl can be unsubstituted or substituted. that the substitution results in a stable compound. Suitable The term “alkynyl refers to a monoradical branched or indicated groups include, e.g., alkyl, alkenyl, alkynyl, alkoxy, unbranched hydrocarbon chain, having a point of complete halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, het unsaturation (i.e. a carbon-carbon, sp triple bond). In one erocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alky 50 embodiment, the alkynyl group can have from 2 to 10 carbon lamino, dialkylamino, trifluoromethylthio, difluoromethyl, atoms, or 2 to 6 carbon atoms. In another embodiment, the acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, alkynyl group can have from 2 to 4 carbonatoms. This term is carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsul exemplified by groups such as ethynyl, 1-propynyl, 2-propy fonyl, arylsulfinyl, arylsulfonyl, heteroarylsulfinyl, heteroar nyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-hexynyl, 2-hexynyl, ylsulfonyl, heterocyclesulfinyl, heterocyclesulfonyl, phos 55 3-hexynyl, 1-octynyl, and the like. The alkynyl can be unsub phate, Sulfate, hydroxylamine, hydroxyl (alkyl)amine, and stituted or substituted. cyano. Additionally, the Suitable indicated groups can The term “cycloalkyl refers to cyclic alkyl groups of from include, e.g., —X. —R. —O. —OR, —SR, —S. —NR, 3 to 10 carbon atoms having a single cyclic ring or multiple - NR =NR, CX, CN, OCN, - SCN, condensed rings. Such cycloalkyl groups include, by way of -N=C=O, NCS, NO, NO, —N, N NC(=O) 60 example, single ring structures such as cyclopropyl, cyclobu R, C(=O)R, C(=O)NRR –S(=O).O. —S(=O), tyl, cyclopentyl, cyclooctyl, and the like, or multiple ring OH, -S(=O).R, OS(=O),OR, S(=O)NR, structures Such as adamantanyl, and the like. The cycloalkyl –S(=O)R, OP(=O)CRR, -P(=O)CRR - P(=O) can be unsubstituted or Substituted. The cycloalkyl group can (O) = P(=O)(OH), C(=O)R, C(=O)x, C(S)R, be monovalent or divalent, and can be optionally substituted C(O)CR, C(O)C, C(S)OR, C(O)SR, C(S)SR, 65 as described above for alkyl groups. The cycloalkyl group can —C(O)NRR, C(S)NRR, C(NR)NRR, where each X is optionally include one or more cites of unsaturation, for independently a halogen (“halo'): F, Cl, Br, or I; and each R example, the cycloalkyl group can include one or more car US 8,476,450 B2 21 22 bon-carbon double bonds, such as, for example, cyclohexene, selected from the group oxygen, nitrogen, and Sulfur, and 1.3-cyclohexadiene, 1,4-cyclohexadiene, and the like. optionally Substituted with one or more groups as defined The term “alkoxy' refers to the group alkyl-O , where herein under the term “substituted. A heterocycle can be a alkyl is as defined herein. In one embodiment, alkoxy groups monocyclic, bicyclic, or tricyclic group containing one or include, e.g., methoxy, ethoxy, n-propoxy, iso-propoxy, n-bu more heteroatoms. A heterocycle group also can contain an toxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dim oxo group (=O) or a thioxo (=S) group attached to the ring. ethylbutoxy, and the like. The alkoxy can be unsubstituted or Non-limiting examples of heterocycle groups include 1.3- substituted. dihydrobenzofuran, 1.3-dioxolane, 1,4-dioxane, 1,4- As used herein, “aryl refers to an aromatic hydrocarbon dithiane, 2H-pyran, 2-pyrazoline, 4H-pyran, chromanyl, imi group derived from the removal of one hydrogenatom from a 10 dazolidinyl, imidazolinyl, indolinyl, isochromanyl. single carbon atom of a parent aromatic ring system. The isoindolinyl, morpholine, piperazinyl, piperidine, piperidyl, radical can beat a saturated or unsaturated carbonatom of the pyrazolidine, pyrazolidinyl, pyrazolinyl, pyrrolidine, pyrro parent ring system. The aryl group can have from 6 to 30 line, quinuclidine, and thiomorpholine. carbon atoms. The aryl group can have a single ring (e.g., The term "heterocycle' can include, by way of example phenyl) or multiple condensed (fused) rings, wherein at least 15 and not limitation, a monoradical of the heterocycles one ring is aromatic (e.g., naphthyl, dihydrophenanthrenyl, described in Paquette, Leo A.; Principles of Modern Hetero fluorenyl, or anthryl). Typical aryl groups include, but are not cyclic Chemistry (W. A. Benjamin, New York, 1968), particu limited to, radicals derived from benzene, naphthalene, larly Chapters 1, 3, 4, 6, 7, and 9. The Chemistry of Hetero anthracene, biphenyl, and the like. The aryl can be unsubsti cyclic Compounds, A Series of Monographs” (John Wiley & tuted or optionally substituted, as described above for alkyl Sons, New York, 1950 to present), in particular Volumes 13, groups. 14, 16, 19, and 28; and J. Am. Chem. Soc. 1960, 82, 5566. In The term "halo' refers to fluoro, chloro, bromo, and iodo. one embodiment, "heterocycle' includes a “carbocycle' as Similarly, the term “halogen refers to fluorine, chlorine, defined herein, wherein one or more (e.g. 1, 2, 3, or 4) carbon bromine, and iodine. atoms have been replaced with a heteroatom (e.g. O, N, or S). The term “haloalkyl refers to alkyl as defined herein sub 25 Examples of heterocycles, by way of example and not stituted by 1 or more halo groups as defined herein, which limitation, include, dihydroypyridyl, tetrahydropyridyl (pip may be the same or different. In one embodiment, the eridyl), thiazolyl, tetrahydrothiophenyl, sulfur oxidized tet haloalkyl can be substituted with 1, 2, 3, 4, or 5 halo groups. rahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, In another embodiment, the haloalkyl can by substituted with pyrazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrroli 1, 2, or 3 halo groups. The term haloalkyl also include per 30 donyl, pyrrolinyl, tetrahydrofuranyl, tetrahydroquinolinyl, fluoro-alkyl groups. Representative haloalkyl groups include, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroiso by way of example, trifluoromethyl, 3-fluorododecyl, 12,12, quinolinyl, azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 12-trifluorododecyl 2-bromooctyl, 3-bromo-6-chloroheptyl, 2H,6H-1.5.2-dithiazinyl, thienyl, thianthrenyl, pyranyl, 1H, 1 H-perfluorooctyl, and the like. The haloalkyl can be isobenzofuranyl, chromenyl, Xanthenyl, phenoxathinyl, optionally substituted as described above for alkyl groups. 35 2H-pyrrolyl, isothiazolyl, isoxazolyl pyrazinyl, pyridazinyl, The term "heteroaryl' is defined herein as a monocyclic, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazoly, purinyl, bicyclic, or tricyclic ring system containing one, two, or three 4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, aromatic rings and containing at least one nitrogen, oxygen, quinazolinyl, cinnolinyl, pteridinyl, carbazolyl, B-carbolinyl, or Sulfur atom in an aromatic ring, and that can be unsubsti phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, tuted or substituted, for example, with one or more, and in 40 phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isoch particular one to three, substituents, as described above in the romanyl, chromanyl, imidazolidinyl, imidazolinyl, pyrazo definition of “substituted”. Typical heteroaryl groups contain lidinyl, pyrazolinyl, piperazinyl, indolinyl, isoindolinyl, qui 2-20 carbon atoms in addition to the one or more hetoeroat nuclidinyl, morpholinyl, oxazolidinyl, benzotriazolyl, oms. Examples of heteroaryl groups include, but are not benzisoxazolyl, OXindolyl, benzoxazolinyl, isatinoyl, and limited to, 2H-pyrrolyl, 3H-indolyl, 4H-quinolizinyl, acridi 45 bis-tetrahydrofuranyl. nyl, benzob thienyl, benzothiazolyl, B-carbolinyl, carba By way of example and not limitation, carbon bonded Zolyl, chromenyl, cinnolinyl, dibenzob.dfuranyl, furazanyl. heterocycles are bonded at position 2, 3, 4, 5, or 6 of a furyl, imidazolyl, imidizolyl, indazolyl, indolisinyl, indolyl, pyridine, position 3, 4, 5, or 6 of a pyridazine, position 2, 4, 5, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isox or 6 of a pyrimidine, position 2, 3, 5, or 6 of a pyrazine, azolyl, naphthyridinyl, oxazolyl, perimidinyl, phenanthridi 50 position 2, 3, 4, or 5 of a furan, tetrahydrofuran, thiofuran, nyl, phenanthrolinyl, phenarsazinyl, phenazinyl, phenothi thiophene, pyrrole or tetrahydropyrrole, position 2, 4, or 5 of azinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, an oxazole, imidazole or thiazole, position 3, 4, or 5 of an pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl pyridazi isoxazole, pyrazole, or isothiazole, position 2 or 3 of an nyl, pyridyl, pyrimidinyl, pyrimidinyl, pyrrolyl, quinazolinyl, aziridine, position 2, 3, or 4 of anaZetidine, position 2, 3, 4, 5, quinolyl, quinoxalinyl, thiadiazolyl, thianthrenyl, thiazolyl, 55 6, 7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of an thienyl, triazolyl, tetrazolyl, and Xanthenyl. In one embodi isoquinoline. Carbon bonded heterocycles include 2-pyridyl, ment the term "heteroaryl denotes a monocyclic aromatic 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, ring containing five or six ring atoms containing carbon and 1. 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 2, 3, or 4 heteroatoms independently selected from non-per 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, oxide oxygen, sulfur, and N(Z) wherein Z is absent or is H, O, 60 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiaz alkyl, aryl, or (C-C)alkylaryl. In another embodiment het olyl, 5-thiazolyl, and the like. eroaryl denotes an ortho-fused bicyclic heterocycle of about By way of example and not limitation, nitrogen bonded eight to ten ring atoms derived therefrom, particularly a benz heterocycles can be bonded at position 1 of an aziridine, derivative or one derived by fusing a propylene, trimethylene, aZetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imi or tetramethylene diradical thereto. 65 dazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyra The term "heterocycle” refers to a saturated or partially Zole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, pip unsaturated ring system, containing at least one heteroatom erazine, indole, indoline, 1H-indazole, position 2 of a US 8,476,450 B2 23 24 isoindole, or isoindoline, position 4 of a morpholine, and Co-polyhydroxy compound, typically C-pentahydroxy, and position 9 of a carbazole, or B-carboline. In one embodiment, often a cyclic glycal. The term includes the known simple nitrogen bonded heterocycles include 1-aziridyl, 1-azetedyl, Sugars and their derivatives, as well as polysaccharides with 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl. two or more monosaccaride residues. The saccharide can The term “carbocycle” refers to a saturated, unsaturated or 5 include protecting groups on the hydroxyl groups, as aromatic ring having 3 to 8 carbonatoms as a monocycle, 7 to described above in the definition of amino acids. The 12 carbonatoms as a bicycle, and up to about 30 carbonatoms hydroxyl groups of the saccharide can be replaced with one or as a polycycle. Monocyclic carbocycles typically have 3 to 6 more halo or amino groups. Additionally, one or more of the ring atoms, still more typically 5 or 6 ring atoms. Bicyclic carbonatoms can be oxidized, for example to keto or carboxyl carbocycles have 7 to 12 ring atoms, e.g., arranged as a 10 groups. bicyclo 4.5, 5.5, 5.6 or 6.6 system, or 9 or 10 ring atoms The term “interrupted indicates that another group is arranged as a bicyclo 5.6 or 6.6 system. Examples of inserted between two adjacent carbon atoms (and the hydro carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, gen atoms to which they are attached (e.g., methyl (CH), 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, methylene (CH) or methine (CH))) of a particular carbon cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclo 15 chain being referred to in the expression using the term “inter hex-3-enyl, phenyl, spiryland naphthyl. The carbocycle can rupted, provided that each of the indicated atoms normal be optionally substituted as described above for alkyl groups. Valency is not exceeded, and that the interruption results in a The term "alkanoyl or “alkylcarbonyl refers to stable compound. Suitable groups that can interrupt a carbon —C(=O)R, wherein R is an alkyl group as previously chain include, e.g., with one or more non-peroxide oxy defined. (—O—), thio (—S—), imino ( N(H)—), methylene dioxy The term “acyloxy” or “alkylcarboxy' refers to —O C (—OCHO ), carbonyl ( C(=O)—), carboxy ( C(=O) (=O)R, wherein R is an alkyl group as previously defined. O—), carbonyldioxy ( OC(=O)C ), carboxylato ( OC Examples of acyloxy groups include, but are not limited to, (=O)—), imine (C—NH), sulfinyl (SO) and sulfonyl (SO). acetoxy, propanoyloxy, butanoyloxy, and pentanoyloxy. Any Alkyl groups can be interrupted by one or more (e.g., 1, 2, 3, alkyl group as defined above can be used to form an acyloxy 25 4, 5, or about 6) of the aforementioned suitable groups. The group. site of interruption can also be between a carbon atom of an The term “alkoxycarbonyl refers to C(=O)CR (or alkyl group and a carbon atom to which the alkyl group is “COOR), wherein R is an alkyl group as previously defined. attached. The term “amino” refers to NH. The amino group can As to any of the above groups, which contain one or more be optionally substituted as defined herein for the term “sub 30 Substituents, it is understood, of course, that Such groups do stituted. The term “alkylamino” refers to —NR, wherein at not contain any Substitution or Substitution patterns that are least one Risalkyl and the second R is alkyl or hydrogen. The sterically impractical and/or synthetically non-feasible. In term “acylamino” refers to N(R)C(=O)R, wherein each Ris addition, the compounds of this invention include all stere independently hydrogen, alkyl, or aryl. ochemical isomers arising from the Substitution of these com The term "amino acid, includes a residue of a natural 35 pounds. amino acid (e.g. Ala, Arg, ASn, Asp, Cys, Glu, Gln, Gly, His, Selected substituents within the compounds described Hyl, Hyp, Ile, Leu, Lys, Met, Phe, Pro, Ser. Thr, Trp, Tyr, and herein are present to a recursive degree. In this context, Val) in D or L form, as well as unnatural amino acids (e.g. “recursive substituent’ means that a substituent may recite phosphoserine, phosphothreonine, phosphotyrosine, hydrox another instance of itself. Because of the recursive nature of yproline, gamma-carboxyglutamate; hippuric acid, octahy 40 Such substituents, theoretically, a large number may be droindole-2-carboxylic acid, statine, 1.2.3,4-tetrahydroiso present in any given claim. One of ordinary skill in the art of quinoline-3-carboxylic acid, penicillamine, ornithine, medicinal chemistry and organic chemistry understands that citruline, C.-methyl-alanine, para-benzoylphenylalanine, the total number of such substituents is reasonably limited by phenylglycine, propargylglycine, sarcosine, and tert-butylg the desired properties of the compound intended. Such prop lycine). The term also comprises natural and unnatural amino 45 erties include, by of example and not limitation, physical acids bearing a conventional amino protecting group (e.g. properties such as molecular weight, solubility or log P appli acetyl or benzyloxycarbonyl), as well as natural and unnatu cation properties such as activity against the intended target, ral amino acids protected at the carboxy terminus (e.g. as a and practical properties such as ease of synthesis. (C-C)alkyl, phenyl or benzyl ester oramide; or as an O-me Recursive substituents are an intended aspect of the inven thylbenzyl amide). Other suitable amino and carboxyprotect 50 tion. One of ordinary skill in the art of medicinal and organic ing groups are known to those skilled in the art (See for chemistry understands the versatility of such substituents. To example, Greene, T. W.; Wutz, P. G. M. Protecting Groups. In the degree that recursive Substituents are present in an claim Organic Synthesis, 2" edition, John Wiley & Sons, Inc., New of the invention, the total number will be determined as set York (1991) and references cited therein). forth above. The term "peptide' describes a sequence of 2 to 35 amino 55 The term “linker” as used herein is a carbon chain that acids (e.g. as defined hereinabove) or peptidyl residues. The covalently attaches two chemical groups together and option sequence may be linear or cyclic. For example, a cyclic pep ally can self-cleave or if covalently bonded to a substrate for tide can be prepared or may result from the formation of an enzyme, may be cleaved by that enzyme or another mol disulfide bridges between two cysteine residues in a ecule, which chain is optionally interrupted by one or more sequence. Preferably a peptide comprises 3 to 20, or 5 to 15 60 nitrogenatoms, oxygenatoms, carbonyl groups, (Substituted) amino acids. Peptide derivatives can be prepared as disclosed aromatic rings, or peptide bonds. in U.S. Pat. Nos. 4,612,302; 4.853,371; and 4,684,620, or as The term “luciferase.” unless specified otherwise, refers to described in the Examples herein below. Peptide sequences a naturally occurring or mutant luciferase. The luciferase, if specifically recited herein are written with the amino termi naturally occurring, may be obtained easily by the skilled nus on the left and the carboxy terminus on the right. 65 from an organism. If the luciferase is one that occurs naturally The term “saccharide' refers to a sugar or other carbohy or is a mutant, which retains activity in the luciferase-lu drate, especially a simple Sugar. The Saccharide can be a ciferin reaction, of a naturally occurring luciferase, it can be US 8,476,450 B2 25 26 obtained readily from a culture of bacteria, yeast, mammalian ciferin or a derivative of a fluorophore to detect one or more cells, insect cells, plant cells, or the like, transformed to molecules, e.g., an enzyme, a cofactor for an enzymatic reac express a cDNA encoding the luciferase, or from an in vitro tion Such as ATP, an enzyme Substrate, an enzyme inhibitor, cell-free system for making the luciferase from a nucleic acid an enzyme activator, or OH radicals, or one or more condi encoding same. Luciferases are available from Promega Cor tions, e.g., redox conditions. The invention thus provides for poration, Madison, Wis. bioluminogenic and fluorogenic assays to detect the amount, As used herein, a “fluorophore' includes a molecule which activity or presence of a molecule in a sample. is capable of absorbing energy at a wavelength range and The methods may be used, for example, to determine the releasing energy at a wavelength range other than the absor presence or amount of at least one molecule, e.g., a nonlu bance range. The term “excitation wavelength” refers to the 10 ciferase enzyme, a regulator of a nonluciferase enzyme, a range of wavelengths at which a fluorophore absorbs energy. nonluciferase enzyme substrate, and/or cofactors of the reac The term "emission wavelength” refers to the range of wave tion, or a condition in a sample including but not limited to an lengths that the fluorophore releases energy or fluoresces. animal, e.g., vertebrate, physiological fluid, e.g., blood, As used herein, a “bioluminogenic assay” or “biolumino plasma, urine, mucous secretions and the like, a cell, cell genic reaction' includes a reaction in which a product of a 15 lysate, cell Supernatant, or purified fraction of a cell (e.g., a reaction between a nonluciferase enzyme and a derivative of subcellular fraction). In one embodiment, the methods luciferin or aminoluciferin is a substrate for luciferase or a according to the present invention provide a rapid method for product of a nonenzymatic reaction having a derivative of detecting one or more molecules in a single sample Such as an luciferin or aminoluciferin is a substrate for luciferase, or a aliquot of cells or a lysate thereof. In one embodiment, the reaction between a luciferase and a derivative of luciferin or method includes quantifying the presence, amount or specific aminoluciferin, is bioluminogenic, i.e., produces a measur activity of a molecule Such as an enzyme, Substrate or cofac able amount of light. tor in a bioluminogenic assay or quantifying the presence or As used herein, “bioluminescence' is light produced as a amount of an enzyme, Substrate or cofactor in a fluorogenic result of a reaction between an enzyme and a substrate that assay. The intensity of the bioluminogenic or fluorogenic generates light. Examples of Such enzymes (bioluminescent 25 signal is a function of the presence or amount of the respective enzymes) include firefly luciferase, click beetle luciferase, molecule. In addition, the reaction may contain one or more Renilla luciferase, cypridina luciferase, Aequorin photopro test agents, e.g., enzyme inhibitors or activators, and/or dif tein, obelin photoprotein and the like. ferent concentrations of inhibitors or activators. In one As used herein, a “bioluminogenic assay reagent may embodiment, the method employs at least two different reac include a Substrate, as well as a cofactor(s) or other mole 30 tions, where the first reaction is a nonluciferase enzyme cule(s) such as a protein, e.g., an enzyme, for a biolumino mediated reaction and the second reaction is a beetle genic reaction. luciferase-mediated reaction. In another embodiment, the A “reaction mixture' may contain all reagents for a par first reaction is a nonenzymatic reaction and the second reac ticular reaction, or may lack at least one of the reagents for the tion is a beetle luciferase-mediated reaction. In yet another reaction. For example, a luciferase reaction mixture may con 35 embodiment, the method employs a single reaction, e.g., a tain reagents for the reaction except for a Substrate for the beetle luciferase-mediated reaction or a fluorogenic reaction. luciferase, e.g., a reaction mixture useful to determine Thus, a bioluminogenic assay may directly or indirectly whether a test sample has a luciferase Substrate. A reaction detect, e.g., measure, the amount, presence or specific activity mixture for a nonluciferase enzyme may include all reagents of for example, a cofactor for an enzyme-mediated reaction, for that reaction except for a molecule to be detected, e.g., the 40 an enzyme, an enzyme Substrate, an inhibitor of the enzyme, mixture contains all reagents except for a cofactor for the an activator of the enzyme, or a condition. For instance, in one nonluciferase enzyme, and so the mixture is useful to detect embodiment, a beetle luciferase and a derivative of luciferin the presence of the cofactor in a test sample. that is a substrate of the beetle luciferase may be employed in As used hereina"derivative of luciferin' ora"derivative of a bioluminogenic assay to detect ATP concentration. In aminoluciferin' is a molecule that is a substrate for a nonlu 45 another embodiment, a derivative of luciferin which is a sub ciferase enzyme and a proSubstrate of a luciferase, a substrate strate for a nonluciferase enzyme, for instance, a derivative for a luciferase, a Substrate for a nonluciferase enzyme and a which is a Substrate of a monoamine oxidase, yields a product substrate for a luciferase, or is useful to detect molecules which is a substrate for a beetle luciferase, and so may be generated in nonenzymatic reactions. The derivatives of the employed in a bioluminogenic assay to detect the oxidase. In invention have one or more modifications to one or more of 50 one embodiment, the derivative is a prosubstrate of a beetle the three rings and/or substituents attached to one or more of luciferase, which yields a product that is a substrate of the rings of the D-luciferin or aminoluciferin backbone (see luciferase but does not itself yield a substantial amount of FIG. 1). light in a reaction with the beetle luciferase. In some embodi A “fluorogenic assay” or “fluorogenic reaction' includes a ments, the derivative is a Substrate for a nonluciferase enzyme reaction in which a product of a reaction between a nonlu 55 or useful to detect another molecule, and a substrate for ciferase, nonproteolytic enzyme and a derivative of a fluoro luciferase which yields a substantial amount of light. In this phore is fluorescent. A “fluorogenic assay reagent may embodiment, the derivative is altered by luciferase but is include a Substrate, as well as a cofactor(s) or other mole generally inefficient in a light generating reaction. cule(s) Such as a protein, e.g., an enzyme, for a fluorogenic In one embodiment, the invention provides a biolumines reaction. Thus, the invention provides a method to detect or 60 cent assay method to detect one or more nonluciferase determine the presence or amount of a molecule for a non enzymes. The method includes contacting a sample Suspected protease enzyme-mediated reaction in a sample. of having one or more nonluciferase enzymes, or a substrate or a co-factor for the nonluciferase-mediated reaction, with a II. Methods of the Invention corresponding reaction mixture that includes a derivative of 65 luciferin or a derivative of aminoluciferin that is a substrate The invention provides a bioluminogenic or fluorogenic for the nonluciferase enzyme. In one embodiment, the deriva method which employs a derivative of luciferin or aminolu tive is one having a modification in the A ring of D-luciferin US 8,476,450 B2 27 28 that includes a recognition site for the nonluciferase enzyme, enzyme-mediated reaction in a sample. The method includes e.g., for a phosphatase. In another embodiment, the derivative contacting a sample, a reaction mixture for a nonluciferase is one having a modification in the Bring of D-luciferin which mediated enzyme reaction and for a luciferase-mediated reac derivative is a substrate for luciferase or a prosubstrate for tion, and a derivative of luciferin which is a substrate for the luciferase. In another embodiment, the derivative is one hav nonluciferase enzyme, yielding a mixture. A reaction ing a modification in the C ring of luciferin that includes a between the nonluciferase enzyme and the derivative yields a recognition site for an enzyme of interest, e.g., acetylcho luminogenic product that is a Substrate for the luciferase. In linesterase. In another embodiment, the derivative is one hav one embodiment, the derivative is a compound of formula I. ing a modification in one of the rings that includes a recog In another embodiment, the derivative is a compound of for nition site for the enzyme of interest, as well as a further 10 mula II. Luminescence in the mixture is detected or deter modification in that ring or one or more of the other rings. mined, thereby detecting or determining the presence or For derivatives that area substrate for luciferase, as well as amount of a molecule for the nonluciferase-mediated reaction optionally a substrate of a nonluciferase enzyme or other in the sample. molecules, the derivative may be employed to detect The invention further provides a method to detect or deter luciferase, or a co-factor, inhibitor, or activator of the 15 mine the presence or amount of a molecule for a luciferase luciferase reaction. If the derivative is a prosubstrate for mediated reaction in a sample. The method includes contact luciferase, i.e., the product of a reaction between the deriva ing a sample, a reaction mixture for a beetle luciferase, and a tive and the nonluciferase enzyme is a Substrate for luciferase, derivative of luciferin which is a substrate for the luciferase, sequential or concurrent reactions for the nonluciferase to yield a reaction. enzyme and the luciferase may be conducted. For instance, a The invention also provides a method to detect the presence reaction for a nonluciferase enzyme that contains the proSub or amount of a molecule in a sample. The method includes strate may be conducted in a single well and a beetle contacting a sample, a first reaction mixture for a nonenzyme luciferase reaction mixture added to that well. In another mediated reaction and a derivative of luciferin which in the embodiment, a reaction mixture for a nonluciferase enzyme presence of the molecule yields a luminogenic product that is that contains the proSubstrate is conducted in a single well and 25 a Substrate for a luciferase, and then contacting at least a a portion of that reaction added to a different well having a portion of the first reaction and a second reaction mixture for beetle luciferase reaction mixture. Alternatively, reactions a luciferase-mediated reaction, to yield a second reaction. may be conducted simultaneously in the same well. Luminescence in the second reaction is detected or deter The invention thus provides a method to detect or deter mined, thereby detecting or determining the presence or mine the presence oramount of a molecule for a nonluciferase 30 amount of the molecule. For instance, a mixture is provided enzyme-mediated reaction in a sample. The method includes having a sample, a first reaction mixture for a nonenzyme contacting a sample, a first reaction mixture for a nonlu mediated reaction and a derivative of luciferin which in the ciferase enzyme-mediated reaction, and a derivative of presence of the molecule yields a a luminogenic product that luciferin which is a substrate for the nonluciferase enzyme, so is a substrate for a beetle luciferase. At least a portion of the as to yield a first mixture or providing Such a first mixture 35 first mixture and a second reaction mixture for a beetle comprising a luminogenic product that is a Substrate for a luciferase-mediated reaction are mixed, to yield a second luciferase, or providing such a first mixture. In one embodi mixture, and then luminescence in the second mixture is ment, the derivative is a compound of formula I. In another detected or determined, thereby detecting or determining the embodiment, the derivative is a compound of formula II. At presence or amount of the molecule. least a portion of the first mixture is contacted with a second 40 For the biolumingenic assays described herein which reaction mixture for a beetle luciferase-mediated reaction, so employ luciferin derivatives with a lower background, those as to yield a second mixture. Then luminescence in the second assays can use lower (or higher) amounts of the derivative, mixture is detected or determined, thereby detecting or deter and those derivatives may have improved reactivity, e.g., with mining the presence or amount of a molecule for the nonlu a nonluciferase enzyme. In addition, for any of the biolumi ciferase enzyme-mediated reaction in the sample. In some 45 nogenic assays described herein, other reagents may be added embodiments, the nonluciferase reaction mixture or to reaction mixtures, including but not limited to those that luciferase reaction mixture may include an esterase, e.g., if inhibit or prevent inactivation of luciferase, or otherwise the product of the reaction between the derivative and the extend or enhance luminescent signal. nonluciferase enzyme has an ester group and that product is a Also provided is a method to identify or measure the proluciferase substrate. The esterase may be included with 50 potency of a modulator of a nonluciferase enzyme-mediated the first reaction mixture, added prior to initiation of the reaction. The method includes contacting one or more agents, luciferase reaction mixture, or included in the luciferase reac a first reaction mixture for a nonluciferase enzyme-mediated tion mixture. In some embodiments, e.g., a derivative with a reaction, and a derivative of luciferin which is a substrate for picolinyl ester, the product of the reaction between the deriva the nonluciferase enzyme, so as to yield a first mixture, or tive and the nonluciferase enzyme is a substrate for luciferase 55 providing Such a mixture, wherein the derivative includes an in the absence of an exogenously added esterase. In one A or B ring modification relative to D-luciferin. The first embodiment, derivatives of luciferin having an ester modifi mixture in the absence of the one or more agents includes a cation are employed in methods of the invention, Such as luminogenic product that is a Substrate for a beetle luciferase. those to detect nonluciferase enzymes including cytochrome At least a portion of the first mixture and a second reaction P450 enzymes, as those derivatives may have improved prop 60 mixture for a beetle luciferase-mediated reaction are mixed, erties as a nonluciferase Substrate. Although not intending to So as to yield a second mixture. Luminescence in the second be bound by any mechanism, the inclusion of the ester modi mixture is compared with a control mixture, thereby identi fication at position 5 in ring C may block negative charges or fying whether one or more of the agents modulates the non add a lipophilic quality to the derivative, rendering it an luciferase enzyme-mediated reaction and/or to what extent improved substrate. 65 and with what potency. Further provided is a method to detect or determine the In one embodiment of the invention, test compounds can be presence or amount of a molecule for a nonluciferase screened and evaluated for their activities as substrates or US 8,476,450 B2 29 30 cofactors of, or regulators, either inhibitors or activators, of an embodiment, a luciferin derivative is contacted with and dif enzymatic or nonenzymatic reaction by using the luciferin fuses into a cell and, if the appropriate molecule is present, and fluorophore derivatives of the present invention. A can yields a product, which is a substrate for luciferase. If a didate compound may be determined to be regulator or a luciferase is present in the cell, luminescence can be detected. Substrate of a reaction by contacting a reaction mixture with Alternatively, in a cell which lacks luciferase, the product a derivative and the test compound, under conditions that passes out of the cell into the medium and that medium is would, in the absence of the test compound, yield biolumi added to a luciferase reaction mixture. Thereafter, the activity nescence, fluorescence, or a bioluminogenic product. resulting from the interaction of the cell with the compound is In one aspect of the invention, a method is provided to determined by measuring luminescence of the reaction mix distinguish between a Substrate and an inhibitor of a reaction. 10 ture relative to a control (minus test compound) reaction For example, the compound is incubated with at least one mixture. enzyme under conditions which allow for metabolism of the In one aspect of the invention, the compound is preferably compound prior to providing a luciferin derivative under con contacted first with the cell for a predetermined time period. ditions that, in the absence of an inhibitor or substrate of the Thereafter, the cell is contacted with the luciferin derivative enzyme, would be suitable for interaction between the 15 and luciferase simultaneously or contemporaneously and the luciferin derivative and the enzyme. In one embodiment, the mixture allowed to incubate for a second predetermined time product of that reaction is a substrate of luciferase and in the period. Enzyme activity is determined by measuring the presence of luciferase yields a light emitting second reaction. amount of luminescence generated from the reaction mixture The resulting light emitting reaction is compared to the one relative to a control reaction mixture (e.g., minus test com obtained from contacting the enzyme with the compound and pound). In another aspect of the invention, the test compound the derivative, under conditions that would, in the absence of is preferably contacted first with the cell for a predetermined an inhibitor of the enzyme, be suitable for interaction between time period. Thereafter, the exposed cell is then contacted the luciferin derivative and the enzyme. Metabolism of the with the luciferin derivative and incubated for a second pre compound by the enzyme reduces its concentration in the determined time period. The cell is then contacted with assay medium and may lead to an apparent loss of inhibitory 25 luciferase to formathird mixture which is allowed to incubate activity compared to conditions without metabolism of the for a third predetermined time period. Thereafter, the activity compound which would indicate it was a substrate for the of the cell resulting from the interaction of the cell with the enzyme. An inhibitory compound that was not metabolized test compound(s) is determined by measuring luminescence would show equal potency, irrespective of the time of addition of the reaction mixture relative to a control reaction mixture of the substrate. 30 (e.g., minus test compound). Detergent addition can rupture In one aspect of the invention, the compound is preferably the cells and release cell content. contacted first with the enzyme for a first predetermined time A cell-based luminescence detection assay for molecules period. Thereafter, the mixture is contacted with a luciferin present in the cell medium, e.g., molecules which actively or derivative and bioluminescent enzyme, e.g., luciferase, via inactive mechanisms are present in the cell medium, can simultaneously or contemporaneously, and the mixture is 35 include adding a reaction mixture with the luciferin derivative allowed to incubate for a second predetermined time period. to the cell medium, or adding the cell medium to a reaction In another aspect of the invention, the compound is incu mixture with the luciferin derivative, and detecting lumines bated with the enzyme for a first predetermined time period to CCCC. form a first mixture. Thereafter, the first mixture is contacted In yet another embodiment of the cell-based assay of the with the luciferin derivative, to form a second mixture that is 40 invention, the cells may be lysed in an appropriate lysis allowed to incubate for a second predetermined time period. buffer. For animal cells, a buffer with 0.1-1.0% non-ionic The second mixture is then contacted with a bioluminescent detergents such as Triton X 100 or Tergitol is typically suffi enzyme, e.g., luciferase, to form a third mixture, which is cient. Bacteria, plant, fungal or yeast cells are usually more allowed to incubate for a third predetermined time period. difficult to lyse. Detergents, freeze/thaw cycles, hypotonic Thereafter, the activity resulting from the interaction of the 45 buffers, Sonication, cavitation or combinations of these meth enzyme with the compound is determined by measuring ods may be used. The method of lysis that produces a lysate is luminescence during and/or after the third predetermined compatible with luciferase or other enzyme activity, or the time period relative to a control (e.g., no compound) reaction. detection of other molecules or conditions. In this way, for example, mechanism based inhibitors of the The presence or activity of nonluciferase enzymes may be first enzyme can be identified and distinguished from non 50 measured in cells grown in culture medium or in cells within mechanism based inhibitors because the first incubation with animals, e.g., living animals. For measurements in cells in the test compound but without the luciferin derivative will animals, a luciferin derivative may be administered to the lead to a more profound inhibition by a mechanism based animal, e.g., injected into the animal or added to an aqueous inhibitor than would be observed without the first incubation Solution, e.g., water, or food consumed by the animal. Con or substrates of the first reaction will show reduced inhibition. 55 version of the derivative to a product that is a luciferase In another embodiment of the invention, a cell-based substrate may be detected by luminescence mediated by method is provided for Screening a compound to determine its luciferase expressed in cells in the animal, e.g., transgenic effect on enzyme activity of the cell. The test compound is cells, by luciferase administered to the animal, e.g., injected contacted with a cell having the enzyme, either naturally or into the animal, or by collecting physiological fluids, e.g., via recombinant expression, the luciferin derivative, and 60 blood, plasma, urine, and the like, or tissue samples, and bioluminescent enzyme, e.g., luciferase, or contacted with a combining those with a luciferase reagent. cell having the enzyme and luciferase, and the derivative, for In one embodiment, the derivative employed in the meth a predetermined period of time. Thus, in one embodiment, a ods is not aminoluciferin which is modified to include a cell that either transiently or stably expresses a recombinant protease Substrate via a peptide bond at the amino group enzyme Such as a bioluminescent enzyme, e.g., luciferase, 65 which optionally also has a protected carboxyl group at posi may be employed. Any conventional method for creating tion 5 in the C ring, or any of luciferin 6' methyl ether, transient or stable transfected cells may be used. In one luciferin 6' chloroethyl ether, 6' deoxyluciferin, 6' luciferin US 8,476,450 B2 31 32 4-trifluoromethyl benzylether, luciferin 6' phenylethylether, human, simian, murine, canine, bovine, equine, feline, Ovine, luciferin 6' geranyl ether, luciferin 6' ethyl ether, 6' luciferin caprine or Swine cells, or prokaryotic cells, or cells from two prenyl ether, 6' luciferin2-picolinylether, 6' luciferin 3-picoli or more different organisms, or cell lysates or Supernatants nylether, 6' luciferin 4-picolinyl ether, luciferin 6' benzyl thereof, or a sample which includes a purified form of the ether, D-luciferin-O-B-galactopyranoside, D-luciferin-O- molecule, e.g., purified nonluciferase enzyme which is useful sulfate, D-luciferin-O-phosphate, D-luciferyl-L-phenylala to prepare a standard curve. The cells may not have been nine, and D-luciferyl-L-N'-arginine. In one embodiment, the genetically modified via recombinant techniques (nonrecom derivative employed in the methods is not a luciferin deriva binant cells), or may be recombinant cells which are tran tive disclosed in U.S. published application 2004.0171099, siently transfected with recombinant DNA and/or the genome the disclosure of which is incorporated by reference herein. 10 of which is stably augmented with a recombinant DNA, or Assays which employ two reactions may be conducted which genome has been modified to disrupt a gene, e.g., simultaneously (one step) or sequentially (two step) to detect disrupt a promoter, intron or open reading frame, or replace one or more moieties including proteins (peptides or polypep one DNA fragment with another. The recombinant DNA or tides), e.g., enzymes, Substrates, cofactors, inhibitors or acti replacement DNA fragment may encode a molecule to be vators for enzymatic reactions, or conditions, e.g., redox con 15 detected by the methods of the invention, a moiety which ditions. A sequential reaction may be conducted in the same alters the level or activity of the molecule to be detected, vessel, e.g., a well of a multiwell plate. For a two step assay, and/or a gene product unrelated to the molecule or moiety that the first reaction mixture may contain all of the reagents or alters the level or activity of the molecule. less than all of the reagents for a nonluciferase enzyme The present methods can be employed to detect a molecule mediated reaction, where one of the reagents that is absent is for an enzyme-mediated reaction, a nonenzymatic-mediated the one to be detected in a sample, e.g., a cell lysate. For reaction or condition. For instance, molecules or conditions instance, a nonluciferase enzyme-mediated reaction is per to be detected by the method include but are not limited to formed under conditions effective to convert a luciferin enzymes, e.g., demethylases, oxidases (e.g., a MAO), derivative that is a substrate for the nonluciferase and a pro deacetylases, deformylases, proteases (proteosome, calpain, substrate of luciferase, to a product that is a substrate of 25 beta-secretase, cathepsin, calpain, thrombin, granzyme B), luciferase. The first reaction may be quenched at the time, or phosphatases, kinases, peroxidases, transferases, e.g., GST. prior to addition, of a luciferase reaction mixture. For Sulfotases, beta-lactamases, cytochrome P450 enzymes, instance, a quencher of the first reaction may be present in the esterase, e.g., acetylcholinesterase, dehydrogenase, luciferase reaction mixture. The luciferase reaction mixture luciferase, Substrates, inhibitors, co-factors, activators of preferably substantially lacks a substrate for the luciferase, 30 enzyme mediated reactions, reactive oxygen species, reduc e.g., the only source of substrate for the luciferase is provided ing conditions and transcriptional regulators or regulators of by a reaction between the nonluciferase enzyme and the gene transcription. The enzymes employed in the methods, derivative. When all the reagents for the first reaction are either enzymes to be detected or enzymes which are useful to present in the first reaction mixture, the assay may be detect a Substrate or cofactor, can be selected from any com employed to identify moieties that alter the reaction, e.g., 35 bination of enzymes including recombinant and endogenous inhibitors or enhancers of the reaction. After performing the (native) enzymes. In one embodiment, the enzyme to be reactions, either simultaneously or sequentially, the presence detected is an endogenous enzyme. In another embodiment, or amount of one or more molecules, or one or more inhibitors the enzyme is a recombinant enzyme. Other combinations or activators of the reaction(s) is/are detected or determined apparent to one of ordinary skill in the art can be used in the and/or to what extent and with what potency. 40 present assays and methods according to the teachings herein. For a one step assay, a reaction mixture may contain The enzymes include but are not limited to proteases, phos reagents for two reactions, such as reagents for a nonlu phatases, peroxidases, Sulfatases, peptidases, oxidases, ciferase enzyme-mediated reaction and a luciferase-mediated dealkylases, deformylases and glycosidases. The enzyme reaction or for a nonenzymatic reaction and a luciferase may be a hydrolase, oxidoreductase, lyase, transferase, e.g., mediated reaction, or a reaction mixture for a single reaction, 45 glutathione S transferase, isomerase, ligase, or synthase. Of e.g., for a reaction between a derivative of a fluorophore particular interest are classes of enzymes that have physi which is a Substrate for an enzyme and the enzyme or a ological significance. These enzymes include protein pepti luciferase-mediated reaction, e.g., a luciferase is Suspected in dases, esterases, protein phosphatases, glycosylases, pro a sample to be tested. teases, dehydrogenases, oxidases, oxygenases, reductases, For assays which employ two reactions, the order of adding 50 methylases and the like. Exemplary cleavage sites for some the molecules for the assays can vary. If initiated and con proteases are set forth in FIG. 2. Enzymes of interest include ducted sequentially (whether in the same vessel or not), those involved in making or hydrolyzing esters, both organic adjustments to reaction conditions, e.g., reagent concentra and inorganic, glycosylating, and hydrolyzing amides. In any tion, temperatures or additional reagents, may be performed. class, there may be further subdivisions. For instance, a quenching agent or enhancing agent may be 55 In particular, enzymes that are useful in the present inven added between reactions (see, e.g., U.S. Pat. Nos. 5,774.320 tion include any protein that exhibits enzymatic activity, e.g., and 6,586,196, the disclosures of which are specifically incor lipases, phospholipases, Sulphatases, ureases, peptidases, porated by reference herein). In one embodiment, the two or proteases and esterases, including acid phosphatases, glu more reactions are carried out simultaneously in a single cosidases, glucuronidases, galactosidases, carboxylesterases, reaction mixture. Optionally, the assays are a homogeneous 60 and luciferases. In one embodiment, the enzyme is a hydro assay, e.g., the components are mixed prior to adding the lytic enzyme. Examples of hydrolytic enzymes include alka mixture to the sample. Results may be read without additional line and acid phosphatases, esterases, decarboxylases, phos transfer of reagents. pholipase D, P-Xylosidase, B-D-fucosidase, thioglucosidase, The assays of the present invention thus allow the detection B-D-galactosidase, C-D-galactosidase, C-D-glucosidase, of one or more molecules or conditions in a sample, e.g., a 65 B-D-glucosidase, B-D-glucuronidase, C.-D-mannosidase, sample which includes eukaryotic cells, e.g., yeast, avian, B-D-mannosidase, B-D-fructofuranosidase, and B-D-gluco plant, insect or mammalian cells, including but not limited to siduronase. US 8,476,450 B2 33 34 In one embodiment, the invention provides the use of a each W is independently H, halo, (C-C)alkyl, (C-C) compound of formula III or IIIA as described herein. alkenyl, hydroxyl, or (C-C)alkoxy; In one embodiment, an enzyme, for instance a nonpro each of K', K, K, and K are independently CH, N,N- teolytic enzyme, is detected using a Substrate which is oxide, or N-(C-C)alkyl, and the dotted lines between K' covalently linked to a fluorophore. In one embodiment, the 5 and K, and K and K", denote optional double bonds; Substrate includes a recognition site for the enzyme. In the A' and B' are optional aromatic rings fused to ring A, only absence of the appropriate enzyme or cofactor, a mixture one of which is present in the compound, so as to form a fused including Such a Substrate generates minimal light at the tricyclic system; and emission wavelength as the fluorescent properties of the fluo when B' is present, the group Z is present, and rophore are quenched, e.g., by the proximity of the quenching 10 when A' is present, the group Z is absent; and group. In the presence of the appropriate enzyme, cleavage of the dotted line in ring B is an optional double bond; the conjugate yields the fluorophore. each R is independently H. (C-C)alkyl, (C-Co.)alk enyl, (C-Co.)alkynyl, (C-Co)cycloalkyl, (C-C2)alkoxy, III. Luciferin Derivatives 15 (C-C)aryl, heteroaryl, heterocycle, (C-C)alkylsulfoxy, (Co-Co)arylsulfoxy, heteroarylsulfoxy, (C-Co)alkylsulfo In one embodiment, derivatives of luciferin or aminolu nyl, (C-Co)arylsulfonyl, heteroarylsulfonyl, (C-C)alkyl ciferin have the following structure: L-X-M-Y R (com sulfinyl, (C-C)arylsulfinyl, heteroarylsulfinyl, (C-C) pound of formula IV), wherein L, if present, may be a sub alkoxycarbonyl, amino, NH(C-C)alkyl, N(CC-C)alkyl). strate for an enzyme or another molecule which interacts with tri(C-C)ammonium(C-Co.)alkyl, heteroaryl(C-C) the enzyme: X may be O, NH, or a linker, e.g., a self-cleavable alkyl, heteroaryl having quaternary nitrogen, heteroarylcar linker which spontaneously cleaves to yield M-Y Rafter L bonyl having quaternary nitrogen, (C-C)arylthio, (C-C) has been removed from L-X-M-Y R; M may be luciferin, alkylphosphate, (C-C)alkylphosphonate, (C-C) quinolinyl luciferin or naphthyl luciferin (X—O), or ami arylphosphate, (C-C)arylphosphonate, phosphate, sulfate, noluciferin or aminoquinolinyl luciferin (X—NH); Y is O 25 saccharide, or M" optionally when Z" is oxygen, wherein M (ester), NH (amide), NH NH (hydrazide), or S (thioester); is an alkali metal; and R, if present, may be alkyl, an aromatic molecule, a or when Z or Z is NR'R'', R'R' together with the N to peptide, an oligonucleotide, or a self-cleavable linker which they are attached forms a heteroaryl or heterocycle attached to a substrate for an enzyme. In one embodiment, the group; derivative may be modified at L or R to include a substrate for 30 wherein any alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, an enzyme such as a P450 enzyme, protease, MAO, FMO, or amino, aryl, heteroaryl, or heterocycle group is optionally GST. In one embodiment, a derivative of the invention is a substituted with 1, 2, 3, 4, or 5 substituents selected from Substrate for luciferase, including 6-aminoquinolinyl (C-Co.)alkyl, (C-Co.)alkenyl, (C-Co.)alkynyl, (C-Co) luciferin, which is a substrate of luciferase having substantial cycloalkyl, (C-Co.)alkoxyl, (C-C)alkylcarbonyl, (C- light output. 35 Co)alkylcarboxyl, halo, hydroxyl, -COOR, SOR, Bioluminescent Substrates according to the present inven —SO.R. nitro, amino, (C-Co.)alkyl-S(O)—, (C-C) tion are derivatives of luciferin or aminoluciferin, i.e., are alkyl-SO , phosphate, (C-Co.)alkylphosphate, (C-C) luminescent Substrates other than luciferin oraminoluciferin, alkylphosphonate, NH(C-C)alkyl, NH(C-C)alkynyl, and include compounds having the general formulas 40 N((C-C)alkyl), N(C-C)alkynyl), mercapto, (C-C) described below including, e.g., formulas I and II. alkylthio, (C-C)aryl, (C-C)arylthio, trifluoromethyl, In one embodiment, the invention provides a compound of =O, heteroaryl, and heterocycle, and each Substituent is formula I: optionally substituted with one to three R groups: R is H. (C-C)alkyl, or (Co-Co)aryl; when Z or Z comprises a nitrogen moiety, one or both of the hydrogens of the Z or Z. nitrogen moiety may be replaced by (C-C)alkyl or the group L., wherein L is an amino acid B ; radical, a peptide radical having up to 20 amino acid moieties, -'-2' Y. N Q-R or any other Small molecule that is a Substrate for a nonlu Z-H A A By 4 c 50 ciferase; with the proviso that when L is an amino acid radical t s Ksy^\ X S W2 or a peptide radical, at least one W is not H: Z W1 A W2 when Z is a hydroxyl group or a nitrogen moiety, H of the hydroxyl or nitrogen moiety may be replaced by (HO)P wherein 55 (O)—OCH2—, Sulfo, —POH, or by a cephalosporanic acid Y is N,N-oxide, N-(C-C)alkyl, or CH: attached to the group Z via a carbon chain of one to about 12 when Y is N, then X is not S; carbon atoms; with the proviso that when ring B is a thiazole X is S, O, CH-CH, N=CH, or CH-N: ring, the Sulfo or the —POH group is attached to the when X is S, then Y is not N: hydroxyl oxygen via a (C-C)alkylene group; Z and Z are independently H, OR, NHR, or NRR: 60 when Z or Z is a hydroxyl group or a nitrogen moiety, or Z" is O, S, NH, NHR, or N=N; when Z" R is a hydroxyl group, one H of the hydroxyl or Q is carbonyl or CH: nitrogen moiety may be replaced by the group L'-linker, W' is H. halo, (C-C)alkyl, (C-Co.)alkenyl, hydroxyl, or wherein L' is a group removable by an enzyme to free the (C-C)alkoxy; or linker, and linker is a carbon chain that can self-cleave, W' and Zare both keto groups on ring A, and at least one of 65 optionally interrupted by one or more nitrogenatoms, oxygen the dotted lines denoting optional double bonds in ring A is atoms, carbonyl groups, optionally Substituted aromatic absent; rings, or peptide bonds, US 8,476,450 B2 35 36 linker is attached to L'via an oxygenatom oran NH group —SO.R. nitro, amino, (C-Co.)alkyl-S(O)—, (C-C) at one terminus of the linker and the other terminus of the alkyl-SO , phosphate, (C-C)alkylphosphate, (C-C) linker forms an ether, ester, or amide linkage with a group Z, alkylphosphonate, NH(C-C)alkyl, NH(C-C)alkynyl, Z", or Z" R. N((C-C)alkyl), N(C-C)alkynyl), mercapto, (C-C) when Z is OR, formula I is optionally a dimer connected at 5 alkylthio, (C-C)aryl, (C-C)arylthio, trifluoromethyl, the two A rings via a linker comprisinga (C-C)alkyl diradi =O, heteroaryl, and heterocycle, and each Substituent is cal that is optionally interrupted by one to four O atoms, N optionally substituted with one to three R groups: atoms, oran optionally Substituted aryl, heteroaryl, or hetero R is H., (C-C)alkyl, or (C-C)aryl; cycle group to form a bridge between the dimer of formula I, A is an anion, present when a quaternary nitrogen is and the R group of each Z group connecting the dimer of 10 present; formula I is replaced by the bridge; or a salt thereof; A is an anion, present when a quaternary nitrogen is provided that: present; when rings A and B form a naphthalene or quinoline ring or a salt thereof; system, then W' is not hydrogen; provided that: 15 when a ring A substituent is OH, then-Q-Z" R is not when rings A and B form a naphthalene or quinoline ring —C(O) NH NH: system, then W' is not hydrogen; when Y is N or CH and X is CH=CH and W' is H, then Z when a ring A substituent is OH, then -Q-Z" R is not is not OH attached to carbon-6 of ring A; and —C(O) NH NH: when Y is N or CH and X is CH=CH and Z is H, then W. when Y is N or CH and X is CH=CH and W' is H, then Z is not OH attached to carbon-6 of ring A. is not OH attached to K; and As illustrated by formulas I and IA, the core structure of when Y is N or CH and X is CH=CH and Z is H, then W. rings A and B can be a number of different ring systems. is not OH attached to K. Modification of ring Ballows the core structure to include In another embodiment, the invention provides a com benzofuran, benzothiophene, benzoxazole, naphthalene, pound of formula IA: 25 quinoline, isoquinoline, quinazoline, and quinoxyline ring systems, including the corresponding N-oxide and N-alkyl derivatives. Ring B modification also allows for access to Ia O N-oxide and N-alkyl derivatives of benzodthiazole. Fur 30 thermore, by Substituting a carbon atom of ring A in formula R I with N,N-oxide, or N-alkyl (e.g., substituting one value of K', K, K, or K for another value), other ring systems can be Z-l OA B )-(7 C 71 obtained. Such as 1.8-naphthyridine, 1.7-naphthyridine, 1.6- Sás X S naphthyridine, and 1.5-naphthyridine ring systems; pyrido3. WI A 2-bipyrazine, pyrido4.3-bipyrazine, pyrido3.4-bipyrazine, 35 and pyrido 2,3-bipyrazine ring systems; pyrido 2,3-dipyri wherein midine, pyrido3,4-dipyrimidine, pyrido4.3-dipyrimidine, Y is N,N-oxide, N—(C-C)alkyl, or CH: and pyrido3.2-dpyrimidine ring systems; benzodoxazole, when Y is N, then X is not S; oxazolo 4,5-b]pyridine, oxazolo 4.5-cpyridine, oxazolo.5. X is S, O, CH-CH, N=CH, or CH-N: 4-cpyridine, and oxazolo.5,4-bipyridine ring systems; and when X is S, then Y is not N: 40 thiazolo 4,5-b]pyridine, thiazolo 4.5-cpyridine, thiazolo.5, Z is H, OR, NHR, or NRR: 4-cpyridine, and thiazolo 5.4-bipyridine ring systems. Z" is O, S, NH, NHR, or N=N; Formula I also illustrates that by substituting a second W' is H, halo, hydroxyl, (C-C)alkyl, or (C-C)alkoxy; carbon atom of ring A with N,N-oxide, or N-alkyl, the corre the dotted line in ring B is an optional double bond; sponding pyrazine, pyramidine, and pyridazine ring A ana each R is independently H. (C-C)alkyl, (C-Co.)alk 45 enyl, (C-Co.)alkynyl, (C-Co)cycloalkyl, (C-C2)alkoxy, logs can be obtained for each of the above described ring (C-C)aryl, heteroaryl, heterocycle, (C-C)alkylsulfoxy, systems. The Substitution of a third carbon atom in ring A of (Co-Co.)arylsulfoxy, heteroarylsulfoxy, (C-Co)alkylsulfo formula I with N.N-oxide, or N-alkyl provides access to the nyl, (C-C)arylsulfonyl, heteroarylsulfonyl, (C-C)alkyl corresponding 1,2,4-triazine ring system derivatives. Sulfinyl, (C-C)arylsulfinyl, heteroarylsulfinyl, (C-C) 50 Formulas I and IA further include the various dihydro, alkoxycarbonyl, amino, NH(C-C)alkyl, N(CC-C)alkyl). tetrahydro, and hexahydro derivatives of each of its ring sys tri(C-C)ammonium(C-Co.)alkyl, heteroaryl(C-C) temS. alkyl, heteroaryl having quaternary nitrogen, heteroarylcar In another embodiment, the invention provides a com bonyl having quaternary nitrogen, (C-C)arylthio, (C-C) pound of formula II: alkylphosphate, (C-Co.)alkylphosphonate, (C-C so) 55 arylphosphate, (C-C)arylphosphonate, phosphate, sulfate, saccharide, or M" optionally when Z" is oxygen, wherein M II is an alkali metal; or when Z or Z is NR'R'', R'R' together with the N to B ; which they are attached forms a heteroaryl or heterocycle 60 --2. N N Q-R group; Z-Ht A' Ks| A b) (c wherein any alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, se (\ S S W2 Z W1 A amino, aryl, heteroaryl, or heterocycle group is optionally W2 substituted with 1, 2, 3, 4, or 5 substituents selected from (C-C-o)alkyl, (C-Co.)alkenyl, (C-Co)alkynyl, (C-Co) 65 wherein cycloalkyl, (C-Co.)alkoxyl, (C-C)alkylcarbonyl, (C- Z and Z are independently OR, NHR', or NR'R'': Co)alkylcarboxyl, halo, hydroxyl. —COOR, —SOR, Z" is O, S, NH, NHR, or N=N; US 8,476,450 B2 37 38 Q is carbonyl or CH: carbon atoms; with the proviso that the sulfo or the POH W' is H. halo, (C-C)alkyl, (C-Co.)alkenyl, hydroxyl, or group is attached to the hydroxyl oxygen via a (C-C)alky (C-C)alkoxy; or lene group: W' and Zare both keto groups on ring A, and at least one of when Z or Z is a hydroxyl group or a nitrogen moiety, or the dotted lines denoting optional double bonds in ring A is 5 when Z" R is a hydroxyl group, one H of the hydroxyl or absent; nitrogen moiety may be replaced by the group L'-linker, each W is independently H, halo, (C-C)alkyl, (C-C) wherein L' is a group removable by an enzyme to free the alkenyl, hydroxyl, or (C-C)alkoxy; linker, and linker is a carbon chain that can self-cleave, each of K', K, K, and K are independently CH, N,N- optionally interrupted by one or more nitrogenatoms, oxygen oxide, or N-(C-C)alkyl, and the dotted lines between K' 10 and K, and K and K, denote optional double bonds; atoms, carbonyl groups, optionally Substituted aromatic A' and B' are optional aromatic rings fused to ring A, only rings, or peptide bonds, one of which is present in the compound, so as to form a fused linker is attached to L'via an oxygenatom oran NH group tricyclic system; and at one terminus of the linker and the other terminus of the when B' is present, the group Z is present, and 15 linker forms an ether, ester, or amide linkage with a group Z, when A' is present, the group Z is absent; and Z", or Z" R. R is H. (C-Co.)alkyl, (C-Co.)alkenyl, (C-C)alkynyl, when Z is OR', formula II is optionally a dimer connected (Cs-Co)cycloalkyl, (C-C2)alkoxy, (C-Cao)aryl, het at the two A rings via linker comprising a (C-C)alkyl eroaryl, heterocycle, (C-Co.)alkylsulfoxy, (C-C)arylsul diradical that is optionally interrupted by one to four Oatoms, foXy, heteroarylsulfoxy, (C-C)alkoxycarbonyl, amino, N atoms, or an optionally Substituted aryl, heteroaryl, or NH(C-C)alkyl, N(CC-C)alkyl), tri(C-C)ammonium heterocycle group to form a bridge between the dimer of (C-Co.)alkyl, heteroaryl(C-C)alkyl, heteroaryl having formula II, and the R' group of each Z group connecting the quaternary nitrogen, heteroarylcarbonyl having quaternary dimer of formula II is replaced by the bridge; nitrogen, saccharide, or M" optionally when Z" is oxygen, provided that a saccharide is not directly attached to K: wherein M is an alkali metal; 25 A is an anion, present when a quaternary nitrogen is R" is (Co-Co)aryl, heteroaryl, heterocycle, (C-C-o)alky present; lthio, (C-C)alkyl-S(O)—, (C-C)alkyl-SO. —SO(C- or a salt thereof. Co)alkyl, saccharide, (C-C)alkylphosphate, (C-C) In yet another embodiment, the invention provides a com alkylphosphonate, (C-Co)arylthio, (C-Co)aryl-S(O)—, pound of formula IIA: (C-C)aryl-SO. —SO(C-C)aryl, (C-C)arylphos 30 phate, (Co-Co)arylphosphonate, or R' is (C-C-o)alkyl sub stituted by R: IIa R is (C-Co.)alkenyl, (C-C)alkynyl, (C-C)cy O cloalkyl, (C-Co.)alkoxyl, (C-C)alkylcarbonyl, (C-C) R alkylcarboxyl, hydroxyl, -COOR, SOR, (C-C) 35 21 N N 71 alkylthio, (C-C)arylthio, (C-C)alkyl-S(O)—, (C-C) Z-l A B X-( C alkyl-SO. , nitro, amino, NH(C-C)alkyl, NH(C-C) SÁs S S alkynyl, N(C-C)alkyl), or N((C-C)alkynyl), mercapto, WI A saccharide, or trifluoromethyl: or when Z or Z is NR'R'', R'R' together with the N to 40 which they are attached forms a heteroaryl or heterocycle wherein group; Z is OR, NHR', or NR'R'; wherein any alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, Z" is O, S, NH, NHR, or N=N; amino, aryl, heteroaryl, or heterocycle group is optionally W' is H. halo, hydroxyl, (C-C)alkyl, or (C-C)alkoxy: substituted with 1, 2, 3, 4, or 5 substituents selected from 45 R is H. (C-Co.)alkyl, (C-C)alkenyl, (C-C)alkynyl, (C-C-o)alkyl, (C-Co.)alkenyl, (C-Co)alkynyl, (C-Co) (Cs-Co)cycloalkyl, (C-C2)alkoxy, (C-Cao)aryl, het cycloalkyl, (C-C)alkoxyl, (C-C)alkylcarbonyl, (C- eroaryl, heterocycle, (C-C)alkylsulfoxy, (C-C)arylsul Co)alkylcarboxyl, halo, hydroxyl. —COOR, —SOR, foxy, heteroarylsulfoxy, (C-C)alkoxycarbonyl, amino, —SO.R., (C-C)alkyl-S(O)—, (C-C)alkyl-SO , NH(C-C)alkyl, N(C-C)alkyl), tri(C-C)ammonium phosphate, (C-Co.)alkylphosphate, (C-C)alkylphospho 50 (C-Co.)alkyl, heteroaryl(C-C)alkyl, heteroaryl having nate, nitro, amino, NH(C-C)alkyl, NH(C-C)alkynyl, quaternary nitrogen, heteroarylcarbonyl having quaternary N((C-C)alkyl), N(C-C)alkynyl), mercapto, (C-C) nitrogen, Saccharide, or M" optionally when Z" is oxygen, alkylthio, (C-C)aryl, (C-C)arylthio, trifluoromethyl, wherein M is an alkali metal; =O, heteroaryl, and heterocycle, and each Substituent is R" is (Co-Co)aryl, heteroaryl, heterocycle, (C-Co)alky optionally substituted with one to three R groups: 55 lthio, (C-C)alkyl-S(O)—, (C-C)alkyl-SO. —SO(C- R is H or (C-C)alkyl: Co)alkyl, saccharide, (C-C)alkylphosphate, (C-C) when Z or Z comprises a nitrogen moiety, a hydrogen of alkylphosphonate, (C-Co.)arylthio, (C-C-o)aryl-S(O)—, the Z or Z. nitrogen moiety may be replaced by the group L. (Co-Co)aryl-SO. —SO(C-Co.)aryl, (C-C)arylphos wherein L is an amino acid radical, a peptide radical having phate, (Co-Co)arylphosphonate, or R' is (C-Co)alkyl Sub up to 20 amino acid moieties, or any other Small molecule that 60 stituted by R: is a substrate for a nonluciferase; with the proviso that when R is (C-Co.)alkenyl, (C-C)alkynyl, (C-C)cy L is an amino acid radical or a peptide radical, at least one of cloalkyl, (C-Co.)alkoxyl, (C-C)alkylcarbonyl, (C-C) W or a W is not H: alkylcarboxyl, hydroxyl, -COOR, SOR', (C-C) when Z is a hydroxyl group or a nitrogen moiety, H of the alkylthio, (C-C)arylthio, (C-C)alkyl-S(O)—, (C-C) hydroxyl or nitrogen moiety may be replaced by (HO)2P 65 alkyl-SO nitro, amino, NH(C-C)alkyl, NH(C-C) (O)—OCH2—, Sulfo, —POH, or by a cephalosporanic acid alkynyl, N(C-C)alkyl), or N((C-C)alkynyl), mercapto, attached to the group Z via a carbon chain of one to about 12 saccharide, or trifluoromethyl: US 8,476,450 B2 39 40 or when Z or Z is NR'R'', R'R' together with the N to product is a substrate for luciferase. Also provided are which they are attached forms a heteroaryl or heterocycle luciferin derivatives which, after interaction with a nonlu group; ciferase enzyme or other molecule, may yield a product that wherein any alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, optionally undergoes one or more further reactions, e.g., amino, aryl, heteroaryl, or heterocycle group is optionally B-elimination, to yield a suitable substrate for luciferase. substituted with 1, 2, 3, 4, or 5 substituents selected from Luciferin derivatives in which the backbone of luciferin is (C-Co.)alkyl, (C-Co.)alkenyl, (C-Co.)alkynyl, (C-C) further modified in its ring structure, e.g., a quinolyl or cycloalkyl, (C-Co.)alkoxyl, (C-C)alkylcarbonyl, (C- napthyl luciferin, are provided, as well as advantageously Co)alkylcarboxyl, halo, hydroxyl, -COOR. -SOR, providing modifications at the carboxy position of the thiaz —SO.R., (C-C)alkyl-S(O)—, (C-C)alkyl-SO , 10 ole ring, to provide improved characteristics to the luciferin phosphate, (C-Co.)alkylphosphate, (C-C)alkylphospho derivative. Derivatives with certain modifications provide for nate, nitro, amino, NH(C-C)alkyl, NH(C-C)alkynyl, or improve assays for certain nonluciferase enzymes or mol N((C-C)alkyl), N(C-C)alkynyl), mercapto, (C-C) ecules. For instance, as described hereinbelow, a pH insensi alkylthio, (C-C)aryl, (C-C)arylthio, trifluoromethyl, tive derivative of luciferin was identified that is useful in =O, heteroaryl, and heterocycle, and each Substituent is 15 biological assays that may be run at a pH other than physi optionally substituted with one to three R groups: ological pH, i.e., less than about pH 7.0 and greater than about R is H or (C-C)alkyl: pH 7.8. Thus, bioluminescent methods that employ a luciferin when Z is OR", formula IIA is optionally a dimer con derivative of the invention may be used to detect one or more nected at the two A rings via linker comprising a (C-C) molecules, e.g., an enzyme, a cofactor for an enzymatic reac alkyl diradical that is optionally interrupted by one to four O tion Such as ATP, an enzyme Substrate, an enzyme inhibitor, atoms, Natoms, or an optionally Substituted aryl, heteroaryl, an enzyme activator, or OH radicals, or one or more condi or heterocycle group to form a bridge between the dimer of tions, e.g., redox conditions. formula IIA, and the R' group of each Zgroup connecting the In one embodiment, the invention provides a compound of dimer of formula II is replaced by the bridge; formula (V): provided that a saccharide is not directly attached to K: 25 A is an anion, present when a quaternary nitrogen is present; V or a salt thereof. As illustrated by formulas II and IIA, the core structure of B : ringSA and B can also be a variety of ring systems. In addition 30 --- a's Y N Qa -R to benzodthiazole, Substituting a carbon atom of ring A of K Y W Z formula II with N,N-oxide, or N-alkyl allows access to thia Z--t A Ks A || B) C Zolo4,5-b]pyridine, thiazolo 4.5-cpyridine, thiazolo 5,4-c. ser y\\ X s-A pyridine, and thiazolo.54-bipyridine ring systems, and their Z WI (W)n A corresponding N-oxides and N-alkyl derivatives. 35 Formula II also illustrates that by substituting a second wherein carbon atom of ring A with N,N-oxide, or N-alkyl, the corre Y is N,N-oxide, N-loweralkyl, or CH: sponding pyrazine, pyramidine, and pyridazine ring A ana X is S, CH-CH, or N–C, logs can be obtained for each of the above described ring Z and Z are H, OR, NHR, or NRR: systems. The Substitution of a third carbon atom in ring A of 40 Z" is O, S, NH, NHR, or N=N; formula II provides access to the corresponding 1,2,4-triazine each W is independently H, halo, Calkyl, Coalkenyl, derivatives. hydroxyl, or Formulas II and IIA include the various dihydro and tet Coalkoxy; or rahydro derivatives of each of its ring systems. W and Z on ring A are both keto groups; All ring systems described above can be substituted as 45 each of K', K, K, and K are independently CH, N,N- illustrated in the formulas described herein, for example, oxide, or N-loweralkyl: formulas I and/or II. Each of the above-described individual R is H. Coalkyl, Substituted Coalkyl, Coalkenyl, ring systems and their derivatives, Substituted as described in Substituted Coalkenyl, halogenated Coalkenyl, Substi the formulas herein, are separate embodiments of the inven tuted halogenated Coalkenyl, Coalkynyl, substituted tion. 50 C-2alkynyl, C2-20alkenylC-2alkyl, Substituted C2-20alk Other deriviates and their use in luminogenic assays is enylC-2calkyl, C-2alkynylC2-20alkenyl, Substituted described hereinbelow. C-2alkynylC2-20alkenyl, C-2 cycloalkyl, Substituted The use of the luciferin derivatives described herein can Cs-20cycloalkyl, Co-soaryl, heteroaryl, Co-soarylC-20alkyl, result in an assay which produces a measurable change in Substituted Caryl, substituted heteroaryl, substituted optical properties upon interaction with a nonluciferase mol 55 Co-soarylC-2alkyl, alkylsulfoxyC-20alkyl, C-2 oalkoxycar ecule, which interaction may alter the structure of the bonyl, C-soarylCoalkoxycarbonyl, CoarylthioCo luciferin derivative. As described herein, the product of a alkyl, hydroxyC-2alkyl, triC-2ammoniumC-2alkyl, het reaction between a luciferin derivative and a nonluciferase eroarylCoalkyl, Substituted heteroarylCoalkyl, het enzyme or other molecule of interest need not be D-luciferin eroaryl having quaternary nitrogen, heteroarylcarbonyl hav or aminoluciferin. For example, a luciferin derivative may 60 ing quaternary nitrogen, and N-methyl-tetrahydropyridinyl: include a Substrate that includes a reactive chemical group for and M' when Z" is oxygen, wherein M is an alkali metal; a nonluciferase enzyme linked to luciferin or aminoluciferin wherein the alkyl, cycloalkyl, alkenyl, and/or alkynyl groups via a chemical linker. Transformation of the reactive chemical may be optionally substituted by one more Coalkyl, halo. group of the derivative by the nonluciferase enzyme may hydroxyl, acetyl, amino, lower alkylamino, lower alkyny yield a product that contains (retains) a portion of the Sub 65 lamino, imidazolinylmethylamino, di-lower alkylamino, di strate, a portion of the chemical linker, the chemical linker, or lower alkynylamino, piperidino, pyrrolidino, aZetidino, aziri a portion of the substrate and the chemical linker, and that dino, US 8,476,450 B2 41 42 di-imidazolinylmethylamino, mercapto, Coalkylthio. C-2 cycloalkoxy, Substituted C-2 ocycloalkoxy, C-2cy Carylthio, or trifluoromethyl groups, substituted cloalkylamino, substituted Cocycloalkylamino, Coalky CoarylCoalkyl carbonyl; and each group R is defined lamino, Substituted Coalkylamino, diCoalkylamino, independently if more than one is present; Substituted diCoalkylamino, Coalkenylamino, Substi Q is (C=O), or (CH): tuted Coalkenylamino, diCoalkenylamino, Substituted n is 0, 1, or 2: diC2-20alkenylamino, C2-20alkenylC-2alkylamino, Substi and wherein tuted C2-2alkenylC-2 alkylamino, C-2alkynylamino, Sub when Z or Z" is amino, one or both of the hydrogens may stituted Coalkynylamino, diCoalkynylamino, Substi be replaced by Coalkyl, or the group L., wherein tuted diC-2alkynylamino, C-2 oalkynylC2-20alkenylamino, L is an amino acid radical, a peptide radical having up to 20 10 or Substituted CoalkynylCoalkenylamino; amino acid moieties, or may be any other Small molecule that R represents CH-OH: COR'' wherein R'' represents H, OH, is a substrate for a nonluciferase; with the proviso that when Coalkoxide, Coalkenyl, or NR'R' wherein R'' and L is an amino acid radical or a peptide radical, W is not H; R" are independently Hor Coalkyl; or OM" wherein M" and wherein is an alkali metal; or a salt; and when Z is hydroxyl oramino, H may be replaced by (HO) 15 R" represents H, C-alkyl, C-2alkenyl, halogen, or P(O) OCH , sulfo, C-galkoxide, or —POH, or by cephalosporanic acid attached to the with the proviso that when R is OH or NH, R7 is not H. R. group Z via a carbon chain of one or more carbonatoms; with is not COR'' wherein R'' is OH or OMe formula VI does not the proviso that when ring B is a thiazole ring, the sulfo or the —POH group is attached to the hydroxyl oxygen via a include luciferin, luciferin methyl ester, and aminoluciferin. loweralkylene chain; and Further derivatives of luciferin or aminoluciferin have the when Z is hydroxyl or amino or when Z"—R is hydroxyl, general formula VII: H may be replaced by the group L'-linker, wherein L' is a group removable by an enzyme to free the linker, and the VII linker is a carbon chain that may optimally self-cleave, 25 optionally interrupted by one or more nitrogenatoms, oxygen atoms, carbonyl groups, (Substituted)aromatic rings, or pep tide bonds, and linker is attached to L'via an oxygenatom oran NH group at one terminus of the linker and the other terminus of the 30 linkerforms an ether, ester, oramide linkage with the group Z: (W)n A and when Z is hydroxyl, formula Vincludes a luciferin dimer connected at the two A rings via an—OCHO—bridge; and wherein wherein 35 Y is N-oxide, N-loweralkyl, or CH: A' and B' are optional aromatic rings fused to ring A, only X is S or CH-CH; or one of which may be present at a time, so as to form a fused Y is N and X is N–C or CH-CH: tricyclic system; and Z and Z are H, OR, NHR, or NRR; or when B' is present, the group Z is present, and Z is a cyclic dietherified dihydroxyborane group attached to 40 ring A via the boronatom; when A' is present, the group Z is absent; and Z" is O, S, NH, NHR, or N=N; wherein each W is independently H, halo, Calkyl, Coalkenyl, one carbon of ring A may be replaced by an N-oxide hydroxyl, or Calkoxy; moiety; each of K', K, K, and K" are independently CH, N,N- the dotted line in ring B is an optional double bond; 45 oxide, or N-loweralkyl: if X is N=C, ring C is attached at the carbon atom of the R is H. C.20alkyl, Substituted C-2calkyl, C2-20alkenyl, N=C moiety; and Substituted Coalkenyl, halogenated Coalkenyl, Substi A is an anion, present when a quaternary nitrogen is tuted halogenated C2-20alkenyl, C2-20alkenylC-2alkyl, Sub present; or a salt thereof, with the proviso that W is not stituted CoalkenylCoalkyl, Coalkynyl. Substituted hydrogen when the compound to which W is attached is 50 Cs-20alkynyl, Cs-20alkynylC2-20alkenyl, Substituted luciferin, luciferin methyl ester, or aminoluciferin or when C-2alkynylC2-20alkenyl, C-2 cycloalkyl, Substituted rings A and B form a naphthalene or quinoline ring system. Cs-20cycloalkyl, Co-soaryl, heteroaryl, Co-soarylC-20alkyl, Further derivatives of luciferin or aminoluciferin have the Substituted Coaryl, Substituted heteroaryl, Substituted general formula VI: Co-soarylC-2oalkyl, C-20alkoxycarbonyl, Co-soarylC1-20 55 alkoxycarbonyl, C-soarylthioC-20alkyl, hydroxyC-20alkyl, triCoammoniumCoalkyl, heteroarylCoalkyl, Substi VI tuted heteroarylCoalkyl, heteroaryl having quaternary 'Xa-N, N nitrogen, heteroarylcarbonyl having quaternary nitrogen, N-methyl-tetrahydropyridinyl, pentafluorophenylsulphonyl, CI)2 S ()S 60 and M' when Z" is oxygen, wherein M is an alkali metal; RI wherein the alkyl, cycloalkyl, alkenyl, and/or alkynyl groups may be optionally Substituted by one more Coalkyl, halo, wherein hydroxyl, acetyl, amino, lower alkylamino, lower alkyny R" represents hydrogen, hydroxyl, amino, Coalkoxy, Sub lamino, imidazolinylmethylamino, di-lower alkylamino, di stituted C-2 oalkoxy, C2-20alkenyloxy, Substituted C2-20alk 65 lower alkynylamino, piperidino, pyrrolidino, aZetidino, aziri enyloxy, halogenated Coalkoxy, Substituted halogenated dino, di-imidazolinylmethylamino, mercapto, Co C-2alkoxy, C-2 alkynyloxy, Substituted C-2alkynyloxy, alkylthio. US 8,476,450 B2 43 44 Coarylthio, or trifluoromethyl groups; and each group R R=H, F, or OH: is defined independently if more than one is present; R—H. methyl, ethyl, propyl, butyl, benzyl, hydroxyethyl, Q is (C=O), or (CH), or an ester of hydroxyethyl: n is 0, 1, or 2: R and Rare independently H. methyl, ethyl, propyl, allyl, and wherein imidazolinylmethyl, or when Z is amino, one or both of the hydrogens may be RandR together with the nitrogenatom to which they are replaced by Coalkyl, or the group L., wherein attached form a piperidino, pyrrolidino, aZetidino, or aziri L is an amino acid radical, a peptide radical having up to 20 dino ring; amino acid moieties, or may be any other Small molecule that Rs and Rare independently H or methyl: is a Substrate for a nonluciferase; 10 R, is H or methyl: and wherein Rs is H. methyl, hydroxyl, or acetyl; and when Z is hydroxyl oramino, H may be replaced by (HO) R is H or methyl. P(O)—OCH2—, sulfo, or Compounds of formula VIII may be useful as MAO sub —POH, or by a cephalosporanic acid attached to the 15 Strates. group Z via a carbon chain of one or more carbonatoms; and Yet other derivatives include a compound of formula IX: when Z is hydroxyl or amino or when Z"—R is hydroxyl, H may be replaced by the group L'-linker, wherein L' is a group removable by an enzyme to free the linker, and linker is carbon chain that can self-cleave, optionally interrupted by one or more nitrogenatoms, oxygenatoms, carbonyl groups, (Substituted)aromatic rings, or peptide bonds, and linker is attached to L'via an oxygenatom oran NH group at one terminus of the linker and the other terminus of the linkerforms an ether, ester, oramide linkage with the group Z: 25 wherein R' is H, OR, NH-C(O)-O-benzyl, or NH-O-iso and butyl: when Z is hydroxyl, formula VII includes a luciferin dimer R is lower alkyl, benzyl, 2,4,6-trimethylbenzyl, phenylpip corrected at the two A rings via an—OCHO-bridge; and erazinobenzyl, o-trifluoromethylbenzyl, or 3-picolinyl: wherein R is a carboxyl group esterified by lower alkyl, 3-picoli A' and B' are optional aromatic rings fused to ring A, only 30 nyl, ethylene glycol when R' is H or OR, or R is carboxyl one of which may be present at a time, so as to form a fused when R is NH C(O) O-benzyl or NH-O-iso-butyl or tricyclic System; and when R' is OR wherein R is 2,4,6-trimethylbenzyl, phe when B' is present, the group Z is present, and nylpiperazinobenzyl, o-trifluoromethylbenzyl. Such deriva when A' is present, the group Z is absent; and tives may be useful as P450 substrates. wherein 35 one carbon of ring A may be replaced by an N-oxide Also provided is a compound of formula X: moiety; the dotted line in ring B is an optional double bond; X if X is N=C, ring C can optionally be attached at the carbon atom of the N=C moiety; and 40 B : A is an anion, present when a quaternary nitrogen is --- a's Y N Qa - R present; or a salt thereof; with the proviso that W is not r K Y / Z" hydrogen when rings A and B form a naphthalene ring sys Z--l, A KsTA, B 3 C tem. s y^\ X S Z W (W)n A Other derivatives include a compound of formula VIII: 45

wherein VIII R3 Y is N,N-oxide, N-loweralkyl, or CH: Ro \ X is S, CH-CH, or N–C, YR 50 Z and Z are independently H, OR, NHR, or NRR: Rs Z" is O, S, NH, NHR, or N=N; each W is independently H, halo, Calkyl, Coalkenyl, hydroxyl, or Calkoxy; or W and Zare both keto groups on ring A, and the dottedlines O ^SÁs 55 in ring A are absent; each of K', K, K, and K are independently CH, N,N- oxide, or N-loweralkyl, and the dotted lines between K' and K, and K and K, denote optional double bonds; C N COOR2 R is H. amino, C2alkyl, C2-2alkenyl, halogenated ks! { 3. 60 C2-20alkenyl, Cs-20alkynyl, C2-20alkenylC-20alkyl, S R6 Cs-oalkynylC2-20alkenyl, C-2cycloalkyl, Co-soaryl, het eroaryl, Co-soarylC-2alkyl, C-12alkoxy, C-2alkylsulfoxy, wherein Coarylsulfoxy, CoarylsulfoxyCoalkyl, Coalkylsul n=0 or 1 and when foxyC-20alkyl, C-2alkoxycarbonyl, Co-soarylC-2alkoxy 65 carbonyl, Co-soarylthioC-2alkyl, hydroxyC-20alkyl, n=0, then X—S, and T is a single bond; or when triCoammoniumCoalkyl, heteroarylsulfoxy, het n=1, then X—CH, and T is a double bond; eroarylCoalkyl, heteroaryl having quaternary nitrogen, US 8,476,450 B2 45 46 heteroarylcarbonyl having quaternary nitrogen, N-methyl hydrogen when the compound to which W is attached is tetrahydropyridinyl; or M" when Z" is oxygen, wherein M is luciferin, luciferin methyl ester, or aminoluciferin or when an alkali metal; rings A and B form a naphthalene or quinoline ring system, wherein any alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, with the proviso that-Q-Z" R is not—C(O)—NH NH aryl, or heteroaryl groups of R can be optionally Substituted when a ring A substituent is OH. by one or more, e.g., 1,2,3,4, or 5, Coalkyl, halo, hydroxyl, In one embodiment, the W group attached to ring C is acetyl, -COOR', SOR', amino, nitro, lower alkylamino, absent (i.e., the value of “n” is 0). In another embodiment, the lower alkynylamino, imidazolinylmethylamino, di-lower W group attached to ring C is H or F. alkylamino, di-lower alkynylamino, piperidino, pyrrolidino, Further provided is a compound of formula XI: aZetidino, aziridino, di-imidazolinylmethyl-amino, mer 10 capto, Coalkylthio, Carylthio, trifluoromethyl, C-2alkylcarboxyl, Co-soaryl, Substituted Co-soaryl, XI Co-soarylC-2alkoxyl, heterocycle C-2calkyl, Substituted CoarylCoalkoxyl, C-soarylCoalkyl carbonyl, Substi B : tuted CoarylCoalkyl carbonyl or additional unsubsti 15 --- a's Y N Qa - R tuted R groups; and wherein each group R is defined inde K YS / Z" pendently if more than one is present; Z--t A .K.TA, B 3 C wherein heterocycle Coalkyl is optionally substituted s y^\ X S with one or more, e.g., 1.2.3.4, or 5, R groups; Z W (W)n A R" is hydrogen or C-calkyl; Q is C(=O) or CH: wherein n is 0, 1, or 2: Y is N-oxide, N-loweralkyl, or CH: and wherein X is S or CH-CH; or when Z or Z" is amino, one or both of the hydrogens of the Y is N and X is N–C or CH-CH: amino group may be replaced by Coalkyl, or the group L. 25 Z and Z are H, OR, NHR, or NRR; or wherein Z is a cyclic dietherified dihydroxyborane group attached L is an amino acid radical, a peptide radical having up to 20 to ring A via the boron atom; amino acid moieties, or any other Small molecule that is a Z" is O, S, NH, NHR, or N=N; substrate for a nonluciferase; with the proviso that when L is each W is independently H, halo, Calkyl, Coalkenyl, an amino acid radical or a peptide radical, W is not H; 30 hydroxyl, or Calkoxy; and wherein each of K', K, K, and K are independently CH, N,N- when Z is hydroxyl or amino, H of the hydroxyl or amino oxide, or N-loweralkyl, and the dotted lines between K' and may be replaced by (HO), P(O)—OCH , sulfo, K. and K and K, denote optional double bonds; —POH, or by a cephalosporanic acid attached to the R is H. C.20alkyl, C-2alkenyl, halogenated C2-20alkenyl, group Z via a carbon chain of one or more carbonatoms; with 35 Cs-oalkynyl, C2-20alkenylC-2oalkyl, C.20alkynylC2-20alk the proviso that when ring B is a thiazole ring, the sulfo or the enyl, C-20cycloalkyl, Co-soaryl, heteroaryl, Co-soarylC1-20 —POH group is attached to the hydroxyl oxygen via a alkyl, C-2alkylsulfoxy, Co-soarylsulfoxy, Co-soarylsul loweralkylene chain; and foxyC-20alkyl, C-2 oalkylsulfoxyC-2alkyl, C-2alkoxy when Z or Z is hydroxyl or amino or when Z" R is carbonyl, Co-soarylC-2calkoxycarbonyl, C-soarylthioC-2 hydroxyl, one Hofthehydroxyl or amino may be replaced by 40 alkyl, hydroxyCoalkyl, triCoammoniumCoalkyl, het the group L'-linker, wherein L' is a group removable by an eroaryl-sulfoxy, heteroarylCoalkyl, heteroaryl having qua enzyme to free the linker, and linker is carbon chain that can ternary nitrogen, heteroarylcarbonyl having quaternary nitro self-cleave, optionally interrupted by one or more nitrogen gen, N-methyl-tetrahydropyridinyl: or M" when Z" is atoms, oxygenatoms, carbonyl groups, (Substituted)aromatic oxygen, wherein M is an alkali metal; rings, or peptide bonds, and 45 wherein the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heteroaryl groups of R can be optionally substituted by one or linker is attached to L'via an oxygenatom oran NH group more, e.g., 1, 2, 3, 4, or 5. Coalkyl, halo, hydroxyl, acetyl, at one terminus of the linker and the other terminus of the —COOR', SOR', amino, nitro, lower alkylamino, lower linkerforms an ether, ester, oramide linkage with the group Z, alkynylamino, imidazolinylmethylamino, di-lower alky Z", or Z" R. and lamino, di-lower alkynylamino, piperidino, pyrrolidino, aze when Z is OR, formula X can optionally be a dimer con 50 tidino, aziridino, di-imidazolinylmethyl-amino, mercapto, nected at the two A rings via a CH or CH-CH CH Coalkylthio, Carylthio, trifluoromethyl, Substituted bridge, and the R group of each Zgroup connecting the dimer Co-soaryl, C-20alkylcarboxyl, Substituted Co-soaryl, Substi of formula X is replaced by the bridge; and tuted C-soarylC-2alkoxyl, Substituted Co-soarylC-2alkyl wherein carbonyl or additional unsubstituted R group; and each group A' and B' are optional aromatic rings fused to ring A, only 55 R is defined independently if more than one is present; one of which may be present at a time, so as to form a fused R" is hydrogen or C-calkyl; tricyclic system; and Q is C(=O) or CH: when B' is present, the group Z is present, and each n is independently 0, 1, or 2; when A' is present, the group Z is absent; and and wherein wherein 60 when Z is amino, one or both of the hydrogens of the amino one carbon of ring A may be replaced by an N-oxide group may be replaced by Coalkyl, or the group L., wherein moiety; L is an amino acid radical, a peptide radical having up to 20 the dotted line in ring B is an optional double bond; amino acid moieties, or may be any other Small molecule that if X is N=C, ring C can optionally be attached at the is a Substrate for a nonluciferase; carbon atom of the N=C moiety; and 65 and wherein A is an anion, present when a quaternary nitrogen is when Z or Z is hydroxyl or amino, H of the hydroxyl or present; or a salt thereof, with the proviso that W is not amino may be replaced by (HO) P(O)—OCH , sulfo, US 8,476,450 B2 47 48 —POH, or by a cephalosporanic acid attached to the group wherein the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and Z via a carbon chain of one or more carbon atoms; and heteroaryl groups of R can be optionally substituted by one or when Z is hydroxyl or amino or when Z"—R is hydroxyl, more, e.g., 1, 2, 3, 4, or 5. Coalkyl, halo, hydroxyl, acetyl, one H of the hydroxyl or amino may be replaced by the group —COOR', -SOR', amino, nitro, lower alkylamino, lower L'-linker, wherein L' is a group removable by an enzyme to alkynylamino, imidazolinylmethylamino, di-lower alky free the linker, and linker is carbon chain that can self-cleave, lamino, di-lower alkynylamino, piperidino, pyrrolidino, aze optionally interrupted by one or more nitrogenatoms, oxygen tidino, aziridino, di-imidazolinylmethyl-amino, mercapto, atoms, carbonyl groups, (Substituted)aromatic rings, or pep Coalkylthio, Carylthio, trifluoromethyl, Coalkylcar tide bonds, and boxyl. Co-soaryl, Substituted Co-soaryl, Co-soarylC-20 linker is attached to L'via an oxygenatom oran NH group 10 alkoxyl. Substituted Co-soarylC-2alkoxyl, Co-soarylC-2 at one terminus of the linker and the other terminus of the alkyl carbonyl, Substituted CoarylCoalkyl carbonyl or linkerforms an ether, ester, oramide linkage with the group Z, additional unsubstituted R groups; and wherein each group R Z", or Z" R. and is defined independently if more than one is present; when Z is hydroxyl, formula XI includes a luciferin dimer wherein substituted aryl groups are substituted with one or connected at the two A rings via an—OCHO—bridge; and 15 alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclic, Substi wherein tuted heterocyclic, and heteroaryl groups of R can be option A' and B' are optional aromatic rings fused to ring A, only ally substituted by one or more Coalkyl, C-Caryl, halo, one of which may be present at a time, so as to form a fused hydroxyl, acetyl, -COOR', amino, nitro, lower alkylamino, tricyclic system; and lower alkynylamino, imidazolinylmethylamino, di-lower when B' is present, the group Z is present, and alkylamino, di-lower alkynylamino, piperidino, pyrrolidino, when A' is present, the group Z is absent; and aZetidino, aziridino, di-imidazolinylmethylamino, mercapto, wherein Coalkylthio, Carylthio, or trifluoromethyl groups: the dotted line in ring B is an optional double bond; R" is hydrogen or C-calkyl; if X is N=C, ring C can optionally be attached at the carbon atom of the N=C moiety; and 25 Q is C(=O) or CH: A is an anion, present when a quaternary nitrogen is n is 0, 1, or 2: present; or a salt thereof; with the proviso that W is not and wherein hydrogen when rings A and B form a naphthalene ring sys when Z or Z" is amino, one or both of the hydrogens of the tem. In one embodiment, the W group attached to ring C is amino group may be replaced by Coalkyl, or the group L. absent (i.e., the value of “n” is 0). In another embodiment, the 30 wherein W group attached to ring C is H or F. L is an amino acid radical, a peptide radical having up to 20 Also provided is a compound of formula XII: amino acid moieties, or any other Small molecule that is a substrate for a nonluciferase; with the proviso that when L is XII 35 an amino acid radical or a peptide radical, W is not H; and wherein B : when Z is hydroxyl or amino, H may be replaced by (HO), --- as Y N Q-n -R P(O)—OCH2—, sulfo, or—POH, or by a cephalosporanic K Y / Z" acid attached to the group Zvia a carbon chain of one or more Z-- A TA, B C 40 carbon atoms; with the proviso that when ring B is a thiazole l, 1 KsÁ\ X s-A ring, the Sulfo or the —POH group is attached to the Z W (W)n A hydroxyl oxygen via a loweralkylene chain; and when Z or Z is hydroxyl or amino or when Z" R is wherein hydroxyl, one H may be replaced by the group L'-linker, Y is N,N-oxide, N-loweralkyl, or CH: 45 wherein L' is a group removable by an enzyme to free the X is S, CH-CH, or N–C, linker, and linker is carbon chain that can self-cleave, option Z and Z are independently H, OR, NHR, or NRR: ally interrupted by one or more nitrogen atoms, oxygen Z" is O, S, NH, NHR, or N=N; atoms, carbonyl groups, (Substituted)aromatic rings, or pep each W is independently H, halo, Calkyl, Coalkenyl, tide bonds, and hydroxyl, or Calkoxy; or 50 linker is attached to L'via an oxygenatom oran NH group W and Zare both keto groups on ring A, and the dotted lines at one terminus of the linker and the other terminus of the in ring A are absent; linker forms an ether, ester, oramide linkage with the group Z, each of K', K, K, and K" are independently CH, N,N- Z", or Z" R. and oxide, or N-loweralkyl, and the dotted lines between K' and when Z is hydroxyl, formula XII includes a luciferin dimer K, and K and K, denote optional double bonds; 55 R is H. Coalkyl, Coalkenyl, halogenated Coalkenyl, connected at the two A rings via an—OCHO—bridge; and Cs-20alkynyl, C2-20alkenylC1-20alkyl, C-20alkynylC2-20alk wherein enyl, C-20 cycloalkyl, Co-soaryl, heteroaryl, heterocyclic, A' and B' are optional aromatic rings fused to ring A, only Substituted heterocyclic, Co-soarylC-2alkyl, C-2alkylsul one of which may be present at a time, so as to form a fused foxy, Co-soarylsulfoxy, Co-soarylsulfoxyC-20alkyl, 60 tricyclic system; and CoalkylsulfoxyCoalkyl, Coalkoxycarbonyl, when B' is present, the group Z is present, and Co-soarylC-2alkoxycarbonyl, Co-soarylthioC1-20alkyl, hydroxyCoalkyl, triCoammoniumCoalkyl, heteroar when A' is present, the group Z is absent; and ylsulfoxy, heteroarylCoalkyl, heteroaryl having quaternary wherein nitrogen, heteroarylcarbonyl having quaternary nitrogen, 65 the dotted line in ring B is an optional double bond; if X is N-methyl-tetrahydropyridinyl; or M" when Z" is oxygen, N=C, ring C can optionally be attached at the carbonatom of wherein M is an alkali metal; the N=C moiety; and US 8,476,450 B2 49 50 A is an anion, present when a quaternary nitrogen is Derivatives of the invention include fluorinated luciferin, present; or a salt thereof, with the proviso that W is not quinolinyl luciferin, aminoquinolinyl luciferin, and naphthyl hydrogen when the compound to which W is attached is luciferin. For instance, the optical properties of fluorinated luciferin, luciferin methyl ester, or aminoluciferin or when derivatives of aminoquinolinyl luciferin may be altered due to rings A and B form a naphthalene or quinoline ring system. the electron withdrawing power of fluorine. Fluorinated In one embodiment, the W group attached to ring C is absent (i.e., the value of “n” is 0). In another embodiment, the derivatives include a compound of formula XXII and XXIII: W group attached to ring C is H or F. IV. Exemplary Luciferin Derivatives 10 XXII Derivatives having a six membered Bring include:

compound of formula (XIX) 15 L-X-HN

wherein X=O or NH;Y—N or CH; and F may be at 3', 4',5', 7", or 8

XXIII

wherein L. may be any structure for an enzyme. Such as a 25 peptide sequence or small molecules: X is O, NH, or a self cleavable linker; Z is N (aminoquinolinyl luciferin) or CH (naphthyl luciferin); Y is O (ester), NH (amide), NH NH (hydrazide), or S (thioester); and R may be alkyl, aromatic, a peptide, an oligonucleotide, or a self-cleavable linker 30 attached to a substrate for an enzyme. In one embodiment, a derivative may be employed to detect wherein X=O or NH; and F may be at 4, 5’, or 7. N-dealkylation, e.g., by cytochrome (CYP) P450 isozymes. An exemplary fluorinated derivative is 5-fluoro-6-ami In the case of an ester (R-alkyl chain), an esterase may be 35 noluciferin (FIG. 3). added after the dealkylation reaction is commenced, or at the In one embodiment, the invention provides for quinolinyl time the dealkylation reaction is initiated, and before a derivatives (compounds of formula XXIV-XXVI): luciferase-mediated reaction is initiated.

40 HO L w e N -N N 1) CYP N-Dealkylation 2) Esterase N 2 O Z aN 45 S N2 e S O-R S L = alkyl Z = N or CH R = H or alkyl compound of formula XX N 50 N2 eN HN S

55 HN 2 N

compound of formula XXI 60 Although light output with 6-aminoquinolinyl luciferin (formula V) is pH sensitive, reactions including control reac tions may be conducted at the same pH. Alternatively, 6-ami Derivatives of the invention may be employed as sensors noquinolinyl luciferin may be modified so as to alter electron 65 for reactive oxygen species (ROS) or a redox sensor for density distribution of the aromatic ring, for example, with horseradish peroxidase (HRP) and other enzymes. Those halogenation. derivatives include compounds of formula XXVII-XXXII: US 8,476,450 B2

OH

O NB He

O

O

N N O O y ( OH - NH S S He1) H2O2

O NB

O

compound of formula XXVII

O Ny (N OH -->2) Luciferase Light

HN S S aminoluciferin

O

HX N N 1) ROS

O S S compound of formula XXVIII

O \ 7 OH 2) Luciferase Light

HO S S luciferin

O

NH N N OH 2 He- Y-( 1) H2O2 He- ON S S I O

compound of formula XXIX

O N 7 OH 2)) LucifLuciferase Light

HO S S luciferin US 8,476,450 B2 53 54 -continued

N N F O O N 7 OH suyuNo CCS S He1) H2O2

F F

F compound of formula XXX

O \ y OH 2) Luciferase Light

HO S S luciferin

O

N N N N N W OH N W OH He1) OH radical 2) Luciferase Light S S HO S S compound of formula XXXI luciferin

O

O N N HO N N OH 2) Lucif y ( OH He1) reduction y ( ) Luciferase Light S S O HO S S compound of formula XXXII 5'hydroxyluciferin

Exemplary bioluminogenic MAO substrates include: Exemplary bioluminogenic FMO substrates include formula XXXIV 45

compound of formula XXXIII R R O / \ N N COOR2 R8 O / \ N N COOR2 50 R- X ))n {S RsR6 v s N )-(N-(R X=/( )-(3. )n S wherein n = 0 and X = S or wherein n = 1 and X = CH: / wherein R = H, F, or OH: 55 wherein R = H, CH, CH2CH3, CH2CH2CH3, CHCH(CH3)2, CHAr, CH2CH2OH, or any ester; wherein n = 0 and X = S or n = 1 and X = CH: wherein R3 = H, CH3, CH2CH3, CH2CH2CH3, any alkyl chain, wherein R = H, F, or OH: O wherein R, R2': H, F, Cl, Br, CN, NO2, CH, OCH, wherein R = H, CH, CH2CH3, CH2CH2CH3, CHCH(CH3)2, CHAr, CH2CH2OH, or any ester; R wherein R3, R = H, CH3, CH2CH3, CH2CH2CH3, H2C-CFCH, 60 R" --KD NH2, or N(CH3): or R3 = (CH) R4:R3 = R4 = CH and n = 0, 1, 2, or 3: wherein R5 and R6 are independently Hor CH3. wherein R5 and R6 are independently Hor CH3: wherein R = H or CH3: 65 The A ring of D-luciferin may also be modified to include wherein R3 = H, CH3, OH, or COCH3. another ring structure (compound of formulas XXXV XXXVI): US 8,476,450 B2 55 56

compound of formula XXXV compound of formula XXXVIII COOH O F G1 5 x^ NY ( 1- NQ 5 - N S S Crs 10 wherein X = O or NH

compound of formula XXXVI compound of formula XXXIX

15 O (1 COOH s 7 1S-1N lu Y

wherein X = O or NH, and Y = N or CH

25 O el Other A ring modifications include a stable substrate for N N 1n-NS 1) AChE-Ser-OH phosphatase which may also be a luciferase Substrate (com pound of formula XXXVII): 30 compound of formula XXXX wherein X = O or NH compound of formula XXXVII N O N COOH 35 2) Hydrolysis HON P y - N N O1 Ser-AChE 3) Luciferase HO1 No1 No S S X-( ---

compound of formula XXXXI The C ring of luciferin may be modified to include a sub 40 Light strate for acetylcholinesterase (ACh), which optionally has a modified A ring or a modified C and A ring. ACh has a very Derivatives useful to detect B-lactamase-mediated reac high turnover number (25,000 s) and has attained kinetic tions include quinolinyl luciferin, aminoquinolinyl luciferin perfection as indicated by its ki/K value of 2x10 M'S'. 45 and naphthyl luciferin derivatives, optionally also modified Two types of derivatives which are substrates for ACh include with a halogen, e.g., a A ring modification that replaces a H (compounds of formula XXXIX-XXXX): linked to a C ring atom with a F.

R S DC- Z C. r" beta-lactamase O OH 2NX2 (S compound of formula XXXXII

R S F HZ Y N COOH N -- O 21 / r O OH compound of formula XXXXIII US 8,476,450 B2 57 58 wherein R=different group, X=N or CH, Y—S or CH=CH, -continued and Z-O or NH O Derivatives which include esters of luciferin (a C ring N N 1. modification) optionally with a A ring modification or a B 5 N W O ring modification, or a A and a C ring modification, include compound of formulas XXXXIV-LVIII: N O S S 2 N O 10 compound of formula LII N N 1-0

No OC)-(S S 'Nan1s o 15 N compound of formula XXXXIV 4. 2 1. O S OH compound of formula LIII NN /N 1N1 p O 2O S S N N 1. compound of formula XXXXV N 7( O O N O S S N N 1n 1a 25 || O N /( 2.O OH N 2 compound of formula LIV No S S p compound of formula XXXXVI 30 O O N N N N N / O N Y / O N 2 35 No S S GE)1. G No S S N compound of formula XXXXVII compound of formula LV O O NN 7N O N 40 N N o1 S S N2 Y M compound of formula XXXXIII 21 O S S O 45 1. N O N N No N O compound of formula LVII S 2 50 N O compound of formula XXXXIX O N N N N 2N 55 usO OC \)-( / o1 X-( O Sr. NH S S HO S S s compound of formula LVIII compound of formula L O O 60 N N N s ls -CH3 N 1n-OH Ya A O N2 W O HO S S S 65 L-luciferin methyl ester compound of formula LI US 8,476,450 B2 59 To prepare esters for a derivative of luciferin that are sub -continued strates for enzymes including P450. MAO and GST, a O cyclization procedure, may be employed with TCEP to HC1 N reduce ethylene glycol ester D-cysteine before adding to a cyano-containing molecule, is utilized. For instance, to a 4. S solution of D-cysteine ester neutralized with potassium bicar bonate, addTECP. After a few minutes, add the solution to the N appropriate nitrile dissolved in methanol. Also, methyl ester COOH luciferin derivatives are made by direct cyclization of ben Zothiole derivatives with methyl ester D-cysteine or methy 10 O lation of carboxylic acids of luciferin with diazomethane. HC1 S

O 15 N

N N COOH ) ( J. " NMe2 HO S S luciferin 1. O N formula LIX O 2 N COOH GE N 7 HN OH 3 1N1 25 S

S BO N S 30

HS 9N-1aOH O formula LX 35 NH3 HS N. w O NCH,

40 COOH Derivatives of luciferin with A, B and/or C ring modifica tions include: (compounds of formula LXI-LXX below) 45

OCH N N COOH N N COOH 50 X-( X-( HO S S HCO S S

55 O N N COOH X-( MeO S S 21 N OyS (1"S NMe2 60 H N NG N N COOH

X-( 65 MeO S S Yet further derivatives include compounds of formula LXXI, LXXII and LXXIII (below)

US 8,476,450 B2 63 -continued -continued wherein n = 0 and X = S or n = 1 and X = CH: LXXXVI wherein R = H, F, or OH: wherein R2 H, CH3, CHCH3, CHCH2CH3, CH2CH(CH3)2, CH2Ar, CH2CH2OH, or any ester; wherein R3, R3, R4 are indepenently NO, CF, or H COOR2 Rs Exemplary phosphatase and Sulfatase Substrates include R6 formulas LXXX-LXXXIII: 10 LXXXVII

COOR2 15 Rs R6

other derivatives of the invention include compounds of formula LXXXVIII-LXXXIX:

25 LXXXVIII

30 X / 35 wherein n = 0 and X = S or n = 1 and X = CH: wherein R = H, F, or OH: wherein R2 H, CH3, CHCH3, CHCH2CH3, CH2CH(CH3)2, CH2Ar, CH2CH2OH, or any ester; wherein R = H, F, Cl, Br, CN, NO2, CH, OCH, NH, or N(CH3)2; or wherein R5 and R6 are independently Hor CH3. 40 LXXXIX

wherein n = 0 and X = S or n = 1 and X = CH: wherein R = H, F, or OH: wherein R = H, CH, CH2CH3, CH2CH2CH3, CHCH(CH3)2, CHAr, 45 CH2CH2OH, or any ester. LXXXIV

50 Derivatives for use in redox or dealkylase bioluminescent OOR assays include compounds of formulas LXXXX-LXXXXI: 5

6 55 LXXXX

LXXXV A E N

60 2 y & OOR Cn D S S wherein A is modified from CH to NO, NMe or N; or 5 wherein B is modified from CH to NO, NMe or N; or wherein C is modified from COH or CNH2 to NO, NMe, or N: 6 wherein D is modified from CH to NO, NMe, or N; or 65 wherein E is modified from N to NO or NMe; or any combination thereof. US 8,476,450 B2 65 66 -continued -continued LXXXXI O O OR N NN /N OH 5 -- Z N Y % S S

n S Z 10 LXXXXV Y 2 O wherein Z = O or Me: N N N wherein Y = OH or NH2: N W OH wherein R = H or alkyl 2 15 Y N S S Specific derivatives useful as reduction sensors or a dealky Y = OH, NH, lase sensor include compounds of formula LXXXXII- Z O LXXXXVI: f 2O N Ny (N OH --> Z O Y % S S for Redox sensor; Z = O Nn \ y OH for dealkylase;ky Z = Me LXXXXVI O ) { 25 21Ns S Y LXXXXII N-Ny -(N OH 2 Y N S S O 30 Y = OH, NH, N n N N OH O X-( OH 2Ns S Y 35 Z N Y = OH, NH, He N S O n

Z Y-1N N y OH -- 40 Y 21 X-( LXXXXVII Y 21Ns S O OH LXXXXIII

45 N O s S N N N N N 7 OH 21 50 Y Y S S Y = OH, NH2 Y = OH, NH,

O 55 Other examples of redox sensors are shown in FIGS. 4 and 6. N-N N Otheresterase substrates are shown in FIGS. 8-10, 22-23, 26, y ( OH --> 37B, 40 and 42. al N 21 S S Z V. Derivatives of Fluorophores LXXXXIV 60 O In one embodiment, derivatives of a fluorophore of the N N invention have the following structure L-F, where F is a fluo N N / OH rophore and L is a substrate linked via O to the fluorophore. In N 65 one embodiment, fluorescent Substrates according to the 2Ns S present invention are benzopyran derivatives having the gen eral formula XIII: US 8,476,450 B2 68 -continued C XIII

O wherein 10 MeO O r the dashed ring is an optionally present benzo ring; NH2 the dashed bond in the Bring is present only when the dashed ring is absent; 15 Fluorogenic FMO substrates include formulas CI-CIII: X is CH when the dashed ring is absent; X is NH when the dashed ring is present; CI X is a carbon atom when the dashed ring is present and X" R R forms part of a spiro ring system which is a Y-butyrolactone ring having an optionally Substituted benzo ring fused at the C. and B carbons of the lactone ring and attached to X' at the Y O s4\, l O carbon; Rs W', W, and Wareindependently H, halo, carboxyl, carboxy 25 R ester, loweralkyl, hydroxyloweralkyl, Coaryl, or Substi tuted C2aryl; W is hydroxyl, loweralkoxy, or amino, R wherein one or both amino hydrogens may be replaced by s Ro lower alkyl; R4 CII Z' is a loweralkylene chain terminated by an amino group, a 30 loweralkylamino group, a diloweralkylamino group, a thiol group, or a lower alkylthio group; and W’ is alkoxy, or OCH (R7)CH(Rs)CH(R)N(RR); and Z is a keto group present only when the benzo ring is absent. 35 Exemplary fluorogenic MAO substrates include formulas LXXXXVIII-C:

40 R8 LXXXXVIII R R N s Ro 45 R4 O O O CIII R H R2 XN N SAS

50 N N O 4\, 4N R8 R R 55 LXXXXIX s Ro R4 R1,R2, R2 = H, F, Cl, Br, I, COOH, COOR (any ester) N R, R = H, CH, CH2CHCH2CH2CH, HC - CECH, 60 O O O o HC -() or R-(CH2)R. RRFCH2, n = 0, 1,2,3, R8 = H, CH, OH, COCH3 Ro = H, CH 65 R = Alkoxyl (any), or symetric chain OCH(R7)CH(R8)CH(R9)N(R3R4) HN US 8,476,450 B2 69 70 See FIGS. 5 and 11 for exemplary fluorogenic substrates for wherein R is as defined for any one of formulas I-III or V-XII, redox and MAO reactions. which is a group removable by an enzyme, e.g., an enzyme Any fluorophore may be employed to prepare a fluorogenic that is being assayed; the trimethyl lock linker replaces a substrate, fluorophores including but not limited to fluoros hydrogen atom of one of the groups Z, Z, or Z"—R; and cein, Texas Red, DAPI, PI, acridine orange, Alexafluors, e.g., leaving group is the remainder of the structure of formula Alexa 350, Alexa 405 or Alexa 488, cyanine dyes, e.g., Cy3, I-III or V-XII. See Wang et al., J. Org. Chem., 62:1363 (1997) coumarin, ethidium bromide, fluorescein, BODIPY, a rhodol, and Chandran et al., J. Am. Chem. Soc., 127:1652 (2005) for Rox. 5-carboxyfluorescein, 6-carboxyfluorescein, an the use of trimethyl lock linkers. anthracene, 2-amino-4-methoxynapthalene, a phenalenone, 10 A quinine methide linker can be illustrated as follows: an acridone, fluorinated Xanthene derivatives, C.-naphtol, B-napthol, 1-hydroxypyrene, coumarins, e.g., 7-amino-4- methylcoumarin (AMC) or 7-amino-4-trifluoromethylcou leaving group marin (AFC), rhodamines, e.g., tetramethylrhodamine, rhodamine-1 10, or carboxyrhodamine, cresyl violet, or 15 resorufin, as well as fluorophores disclosed in U.S. Pat. No. 6.420,130, the disclosure of which is incorporated by refer O ence herein. R VI. Linkers wherein R is as defined for any one of formulas I-III or V-XII, A linker strategy may be employed for either the A or C which is a group removable by an enzyme, e.g., an enzyme ring modified luciferins, or with fluorophore containing that is being assayed; the quinine methide linker replaces a derivatives, to introduce a substrate for an enzyme of interest 25 hydrogen atom of one of the groups Z, Z, or Z"—R; and Such as a deacetylase, deformylase, demethylase or other leaving group is the remainder of the structure of formula enzyme that can remove the L. group of formula I (a Substrate I-III or V-XII. See Greenwald et al., J. Med. Chem., 42:3657 for that enzyme) to free the linker, yielding a substrate of (1999) and Greenwald et al., Bioconjugate Chem., 14:395 (2003) for the use of quinine methide linkers. luciferase, or a proSubstrate, where the remaining linker may 30 optionally be removed by a nonenzymatic reaction. A diketopiperazine linker can be illustrated as follows: Linkers can be alkyl oralkoxy chains, such as (C-C)alkyl or (C-C)alkoxy groups. The chain can have one or more CVI electron withdrawing group Substituents R. Such as an alde R 35 H hyde, acetyl, Sulfoxide, Sulfone, nitro, cyano group, or a com R N bination thereof. Other linkers include trimethyllock, quinine leaving group methide and diketopiperazine linkers, and their derivatives. A trimethyl lock linker can be illustrated as follows: R R' 40 wherein R is as defined for any one of formulas I-III or V-XII, CIV which is a group removable by an enzyme, e.g., an enzyme that is being assayed; each R' of the diketopiperazine linker is independently H or an alkyl chain optionally interrupted by leaving group 45 O, S, or NH, preferably a methyl group; the diketopiperazine linker replaces a hydrogenatom of one of the groups Z, Z, or Z" R. and leaving group is the remainder of the structure of formula I-III or V-XII. See Wei et al., Bioorg. Med. Chem. Lett., 10: 1073 (2000) for the use of diketopiperazine linkers. Other linker containing derivatives include:

compound of formula CVII

HN X S COOH

O N X = O or NH compound of formula CVIII usu-OfO N N COOH X = O or NH US 8,476,450 B2 71 72 -continued compound of formula CIX

N N COOH O X-O/

1N compound of formula CX C us S N COOH W O )-OS

25 B-elimination of a product of a bioluminogenic or fluoro -continued genic reaction with a substrate having a group R that in the product is an electron withdrawing group R. Such as alde hyde, acetyl, Sulfoxide, Sulfone, nitro, or cyano, may yield a substrate for luciferase or a fluorophore. 30

Any fluorogenic chromophore or luminophore B-elimination 35 Specific embodiments of reactions and derivatives having formulas CXI-CXV which employ a linker include:

Z 1. 40 L-Linker X 1) Enzyme cleavage Any fluorogenic chromophore or luminophore 2 N Hess For instance, to detect MAO, the derivative may undergo Y e COOH the following reaction. 45

50 Linker X 2) self-elimination 2 N Y e COOH

55 Any fluorogenic chromophore or luminophore To detect FMO, the derivative may undergo the following reaction:

60

B-elimination A.S=O)n R 65 wherein L-Me for demethylase, Ac for deacetylase, or CHO n = 1, 2 for deformylase, X=S or CH=CH, Y=N or CH, and Z=O or NH; US 8,476,450 B2 73 74 -continued HZ HZ X O 1) Enzyme X cleavage 2 N He- 2 N Y e 5 Y e COOH O-Linker-R S S compound of formula CXII HZ X O 2) self-elimination 10 a N 1N He Y e O-Link- Ke wherein R=Me for demethylase, Ac for deacetylase, or CHO S for deformylase, X=S or CH=CH, Y=N or CH, and Z=O or NH

L Ya O N X r-r 1) Enzyme cleavage O 2 N He Y S e yCOOH compound of formula CXIII /1N (, HN X 2) self-elimination He O 2 N Y S e yCOOH

HO X

2 N Y Se yCOOH

wherein L-Me for demethylase, Ac for deacetylase, or CHO for deformylase, X=S or CH=CH, and Y=N or CH:

11 Z

O X 1) Enzyme cleavage 2 N Y S e yCOOH compound of formula CXIV

/ HZ

X 2) self-elimination

2 Y eN COOH S y US 8,476,450 B2 75 76 -continued X

2 N Y e y1 COOH wherein L-Me for demethylase, Ac for deacetylase, or CHO for deformylase, X=S or CH=CH, Y=N or CH, and Z=O 10 or NH; or

L N Z n 1N1 X 1) Enzyme cleavage O 2 N He Y S e yCOOH compound of formula CXV /N 1) self-elimination O 2 N -- rouY e yCOOH HZ X

2 N Y e yCOOH wherein L-Me for demethylase, Ac for deacetylase, or CHO wherein X is S or Se; R and R are each independently for deformylase, X=S or CH=CH, Y=N or CH, and Z=O hydrogen, (C-Co.)alkyl, (C-Cs)cycloalkyl, (C-Co) or NH. alkoxy, (C-C)alkenyl, (C-C)alkynyl, aryl, heteroaryl, or 40 NRR: or R and R together with the carbon to which they VII. Agents Useful to Stabilize Light Production in are attached form a 5, 6, 7, or 8 membered saturated or Luciferase-Mediated Reactions unsaturated ring comprising carbon and optionally compris ing 1, 2, or 3 heteroatoms selected from oxy ( O—), thio Agents useful to stabilize light production in a luciferase (—S—), or nitrogen ( NR)—, wherein said ring is option mediated reaction include organic compounds (i.e., com 45 ally substituted with 1, 2, or 3 halo, hydroxy, oxo, thioxo, pounds that comprise one or more carbon atoms). Such an carboxy, (C-C)alkyl, (C-C)cycloalkyl, (C-Co)alkoxy, agent may be added prior to, at the initiation of and/or during (C-C)alkanoyl, (C-C)alkoxycarbonyl, (C-C)alk a nonluciferase enzyme-mediated reaction or a luciferase enyl, (C-Co.)alkynyl, aryl, or heteroaryl; and R. R., and R. mediated reaction. Suitable organic compounds can comprise 50 are each independently hydrogen, (C-C)alkyl, (C-C)cy a carbon-sulfur bond or a carbon-selenium bond, for example cloalkyl, (C-C)alkenyl, (C-Co.)alkanoyl (C-Co) Suitable organic compounds can comprise a carbon-sulfur alkoxycarbonyl, (C-Co.)alkynyl, aryl, heteroaryl; wherein double bond (C=S), a carbon selenium double bond any (C-Co)alkyl, (C-Cs)cycloalkyl, (C-Co.)alkoxy, (C2 (C—Se), a carbon-sulfur single bond (C S), or carbon-se Co)alkenyl (C-C)alkanoyl, (C-C)alkoxycarbonyl, or lenium single bond (C-Se). Suitable organic compounds 55 (C-Co.)alkynyl of R. R. R. R. and R is optionally Sub can also comprise a carbon bound mercapto group (C-SH) stituted with one or more (e.g. 1, 2, 3, or 4) halo, hydroxy, or a sulfur atom bound to two carbon atoms (C. S. C). In mercapto, oxo, thioxo, carboxy, (C-C)alkanoyl, (C-C) one embodiment, compounds are lipophilic in nature. alkoxycarbonyl, aryl, or heteroaryl; and wherein any aryl or Suitable compounds that comprise a carbon sulfur double heteroaryl is optionally substituted with one or more (1, 2, 3, bond or a carbon selenium double bond include for example 60 or 4) halo, hydroxy, mercapto, carboxy, cyano, nitro, trifluo compounds of formula (XIV): romethyl, trifluoromethoxy, (C-C)alkanoyl, (C-C)al kanoyloxy, Sulfo or (C-C)alkoxycarbonyl: orasalt thereof. XIV Suitable compounds that comprise a mercapto group include for example compounds of the formula RSH 65 wherein: R is (C-Co.)alkyl, (C-C)cycloalkyl, (C-C) Rus R alkenyl, (C-Co.)alkynyl, aryl, or heteroaryl; wherein any (C-Co.)alkyl, (C-C)cycloalkyl, (C-Co.)alkenyl, or (C- US 8,476,450 B2 77 78 Co)alkynyl of R is optionally Substituted with one or more 4-hexynyl, or 5-hexynyl; (C-C)alkanoyl can be acetyl, (e.g. 1, 2, 3, or 4) halo, hydroxy, mercapto oxo, thioxo, car propanoyl or butanoyl: (C-C)alkoxycarbonyl can be meth boxy, (C-C)alkanoyl, (C-C)alkoxycarbonyl, aryl, het oxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopro eroaryl, or NRR, wherein RandR are each independently poxycarbonyl, butoxycarbonyl, pentoxycarbonyl, or hexy hydrogen, (C-Co)alkyl, (C-C)cycloalkyl, (C-Co.)alk loxycarbonyl: (C-C)alkanoyloxy can be acetoxy, enyl, (C-Co.)alkynyl, (C-Co.)alkanoyl, (C-Co.)alkoxy propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy, carbonyl aryl, or heteroaryl; and wherein any aryl or het or hexanoyloxy: aryl can be phenyl, indenyl, or naphthyl; and eroaryl is optionally substituted with one or more (1, 2, 3, or heteroaryl can be furyl, imidazolyl, triazolyl, triazinyl, 4) halo, mercapto, hydroxy, OXO, carboxy, cyano, nitro, trif oxazoyl, isoxazoyl, thiazolyl, isothiazoyl pyrazolyl pyrro luoromethyl, trifluoromethoxy, (C-C)alkanoyl, (C-C) 10 alkanoyloxy, Sulfo or (C-C)alkoxycarbonyl; or a salt lyl, pyrazinyl, tetrazolyl pyridyl, (or its N-oxide), thienyl, thereof. pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or its N-ox Other suitable compounds include for example compounds ide) or quinolyl (or its N-oxide). of the formula RNCS wherein: R is (C-C)alkyl, (C-C) Specifically, R and R can each independently be hydro cycloalkyl, (C-C)alkenyl, (C-Co.)alkynyl, aryl, or het 15 gen, (C-Co.)alkyl, (C-C)cycloalkyl, (C-Co.)alkenyl, (C- eroaryl; wherein any (C-C)alkyl, (C-C)cycloalkyl, (C- Co)alkynyl, aryl, heteroaryl, or NRR, wherein R, and R. Co)alkenyl, or (C-C)alkynyl of R is optionally are each independently hydrogen, (C-C)alkyl, (C-C)cy Substituted with one or more (e.g. 1, 2, 3, or 4) halo, hydroxy, cloalkyl, (C-C)alkenyl, (C-Co.)alkanoyl (C-Co) mercapto Oxo, thioXo, carboxy, (C-C)alkanoyl, (C-C) alkoxycarbonyl, (C-C)alkynyl, aryl, or heteroaryl; alkoxycarbonyl, aryl, heteroaryl, or NRR, wherein R, and wherein any (C-Co.)alkyl, (C-C)cycloalkyl, (C-C) Rare each independently hydrogen, (C-Co.)alkyl, (C-Cs) alkoxy, (C-C)alkenyl (C-C)alkanoyl, (C-C)alkoxy cycloalkyl, (C-Co.)alkenyl, (C-Co)alkynyl, (C-Co.)al carbonyl, or (C-Co)alkynyl of R. R. R., and R, is option kanoyl, (C-C)alkoxycarbonyl aryl, or heteroaryl; and ally Substituted with 1 or 2 halo, hydroxy, mercapto, OXo, wherein any aryl or heteroaryl is optionally substituted with thioxo, carboxy, (C-C)alkanoyl, (C-C)alkoxycarbonyl, one or more (1, 2, 3, or 4) halo, mercapto, hydroxy, OXo, 25 aryl, or heteroaryl; and wherein any aryl or heteroaryl is carboxy, cyano, nitro, trifluoromethyl, trifluoromethoxy, (C- optionally Substituted with one or more halo, hydroxy, mer Co)alkanoyl, (C-Co.)alkanoyloxy, Sulfo or (C-C) capto, carboxy, cyano, nitro, trifluoromethyl, trifluo alkoxycarbonyl: or a salt thereof. romethoxy, (C-C)alkanoyl (C-Co.)alkanoyloxy, Sulfo or Other Suitable compounds that comprise a carbon-sele (C-C)alkoxycarbonyl. nium single bond or a carbon Sulfur single bond include 30 Specifically, R and R can each independently be hydro compounds of formula Rs X-R wherein: gen, (C-Co.)alkyl, (C-Co)alkenyl, (C-Co.)alkynyl, aryl, X is —S or —Se—; Rs is (C-C)alkyl, (C-C)cycloalkyl, (C-C)alkenyl, or NRR. (C-Co.)alkynyl, aryl, or heteroaryl; and R is hydrogen, (C- Specifically, R and R together with the carbon to which Co)alkyl, (C-Cs)cycloalkyl, (C-Co.)alkenyl, (C-Co) 35 they are attached can form a 5 or 6 membered saturated or alkynyl, aryl, or heteroaryl; unsaturated ring comprising carbon and optionally compris or Rs and R together with X form a heteroaryl; ing 1 or 2 heteroatoms selected from oxy (—O—), thio wherein any (C-C)alkyl, (C-C)cycloalkyl, (C-C) (—S—), or nitrogen ( NR)—, wherein said ring is option alkenyl, or (C-C)alkynyl of Rs or R is optionally Substi ally substituted with 1, 2, or 3 halo, hydroxy, oxo, thioxo, tuted with one or more (e.g. 1, 2, 3, or 4) halo, hydroxy, 40 carboxy, (C-C)alkyl, (C-C)cycloalkyl, (C-Co)alkoxy, mercapto Oxo, thioXo, carboxy, (C-C)alkanoyl, (C-C) (C-Co.)alkanoyl (C-C)alkoxycarbonyl, (C-C)alk alkoxycarbonyl, aryl, heteroaryl, or NRR: enyl, (C-C)alkynyl, aryl, or heteroaryl; wherein R is wherein RandR are each independently hydrogen, (C- hydrogen, (C-Co)alkyl, (C-C)cycloalkyl, (C-Co.)alk Co)alkyl, (C-Cs)cycloalkyl, (C-Co)alkenyl, (C-Co) enyl, (C-Co)alkanoyl, (C-Co.)alkoxycarbonyl, (C-Co) alkynyl, (C-C)alkanoyl, (C-Co.)alkoxycarbonyl aryl, or 45 alkynyl, aryl, heteroaryl; wherein any (C-C)alkyl, (C- heteroaryl; and Co)cycloalkyl, (C-Co.)alkoxy, (C-Co.)alkenyl (C-Co) wherein any aryl or heteroaryl is optionally substituted alkanoyl, (C-C)alkoxycarbonyl, or (C-C)alkynyl of R, with one or more (1, 2, 3, or 4) halo, mercapto, hydroxy, OXo, R, and R is optionally substituted with one or more halo, carboxy, cyano, nitro, trifluoromethyl, trifluoromethoxy, (C- hydroxy, mercapto, Oxo, thioxo, carboxy, (C-C)alkanoyl. Co)alkanoyl, (C-Co.)alkanoyloxy, Sulfo or (C-C) 50 (C-Co.)alkoxycarbonyl, aryl, or heteroaryl; and wherein any alkoxycarbonyl: or a salt thereof. aryl or heteroaryl is optionally substituted with one or more Specific and preferred values listed below for radicals, halo, hydroxy, mercapto, carboxy, cyano, nitro, trifluorom Substituents, and ranges, are for illustration only; they do not ethyl, trifluoromethoxy, (C-Co.)alkanoyl, (C-C)alkanoy exclude other defined values or other values within defined loxy, Sulfo or (C-C)alkoxycarbonyl. ranges for the radicals and Substituents 55 Specifically, R and R can each independently be NRR: Specifically, (C-C)alkyl can be methyl, ethyl, propyl. wherein R, and R, are each independently hydrogen, (C- isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or Co)alkyl, (C-Cs)cycloalkyl, (C-Co.)alkenyl, (C-Co.)al hexyl, (C-C)cycloalkyl can be cyclopropyl, cyclobutyl, kanoyl, (C-Co.)alkoxycarbonyl, (C-C)alkynyl, aryl, het cyclopentyl, or cyclohexyl, (C-C)alkoxy can be methoxy, eroaryl; wherein any (C-C)alkyl, (C-C)cycloalkyl, (C- ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, 60 Co)alkenyl (C-C)alkanoyl, (C-C)alkoxycarbonyl, or pentoxy, 3-pentoxy, or hexyloxy; (C-C)alkenyl can be (C-C)alkynyl is optionally substituted with one or more vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, halo, hydroxy, mercapto, oxo, thioxo, carboxy, aryl, or het 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, eroaryl; and wherein any aryl or heteroaryl is optionally Sub 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or 5-hexenyl: stituted with one or more halo, hydroxy, mercapto, carboxy, (C-Co.)alkynyl can be ethynyl, 1-propynyl, 2-propynyl, 65 cyano, nitro, trifluoromethyl, trifluoromethoxy, (C-Co.)al 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, kanoyl, (C-C)alkanoyloxy, Sulfo or (C-C)alkoxycarbo 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, nyl. US 8,476,450 B2 79 80 Specifically, R and R can each independently be amino, Specifically, the assay can be carried out in a solvent com (C-C)alkyl, (C-C)alkylamino, allylamino, 2-hydroxy prising at least about 10% water. More specifically, the inven ethylamino, phenylamino, or 4-thiazoylamino. tion can be carried out in a solvent comprising at least about Specifically, R and R can each independently be amino, 25% water, or at least about 40% water. methyl, allylamino, 2-hydroxyethylamino, phenylamino, or 4-thiazoylamino. VII. Kits A specific value for R is (C-C)alkyl optionally substi tuted with one or more halo, mercapto Oxo, thioxo, carboxy, The present invention also provides kits for detecting the (C-C)alkanoyl, (C-C)alkoxycarbonyl, aryl, heteroaryl, presence or activity of one or more molecules which are or NRR. 10 reagents for a reaction, enhance or inhibit a reaction, or for A specific value for R is 2-aminoethyl, 2-amino-2-car detecting a condition, in a sample such as a sample including boxyethyl, or 2-acylamino-2-carboxyethyl. intact cells, a cell lysate, e.g., a lysate which is at least par A specific value for R is aryl, optionally substituted with tially purified, or a cellular Supernatant. In one embodiment, one or more halo, mercapto, hydroxy, OXO, carboxy, cyano, the kit includes a derivative of the invention. For kits of the nitro, trifluoromethyl, trifluoromethoxy, (C-C)alkanoyl, 15 (C-C)alkanoyloxy, sulfo or (C-C)alkoxycarbonyl. invention that include two or more of the following, a deriva Specifically, Rs is (C-Co.)alkyl, (C-C)cycloalkyl, (C- tive of luciferin or aminoluciferin, a derivative of a fluoro Co)alkenyl, (C-Co.)alkynyl, aryl, or heteroaryl; and R is phore, another Substrate, enzyme, or reaction mixture, each hydrogen, (C-Co)alkyl, (C-C)cycloalkyl, (C-Co.)alk can be contained in a separate container, some components enyl, (C-C)alkynyl, aryl, or heteroaryl. may be combined in Some containers, or they can be con Specifically, Rs and R together with X form a heteroaryl. tained in a single container. The kit can optionally comprise a In one embodiment, the organic compound is not a buffer solution suitable for use in an assay, and the derivative polypeptide or protein with one or more mercapto (C SH) or enzyme, and the buffer Solution can optionally be con groups. tained in a single container. Additionally, the derivative and In one embodiment, the organic compound is not a com 25 the buffer Solution can optionally be contained in a single pound that includes one or more mercapto (C-SH) groups. container. The kits can also optionally include a quenching Preferred compounds are coenzyme A and DTT. agent for a nonbioluminescent reaction. The compounds described hereinabove are available from commercial sources or can be prepared from commercially IX. Exemplary Synthesis available starting materials using procedures that are known 30 in the field of synthetic chemistry. For example, see Jerry A. Experimental Procedures for Luciferin Modifications March, Advanced Organic Chemistry, 4th ed. Wiley-Inter Included below are experimental procedures for syntheses science, John Wiley and Sons, New York, 1992. of various luciferin derivatives: In cases where compounds are sufficiently basic or acidic I. General procedures for synthesizing esters of luciferins to form stable salts, use of the compounds as salts in the 35 II. D-cysteine or D-cystine esters methods of the invention may be appropriate. Examples of III. Esters of luciferin methyl ether or luciferin-H Suitable salts include organic acid addition salts, for example, IV. Quinolinyl luciferin derivatives tosylate, methanesulfonate, acetate, citrate, malonate, tart V. Miscellaneous arate. Succinate, benzoate, ascorbate, C.-ketoglutarate, and I. General Procedures for Synthesizing Luciferin Esters C-glycerophosphate salts. Suitable inorganic salts may also 40 There are two ways of synthesizing esters of D-luciferin be formed, including hydrochloride, sulfate, nitrate, bicar and derivatives, including luciferin-H and quinolinyl bonate, and carbonate salts. luciferin: a) by direct cyclization of the corresponding cyano Salts can be obtained using standard procedures well group with D-cysteine or D-cystine esters, and b) by CsF known in the art, for example by reacting a Sufficiently basic promoted esterification with halogenated organic com compound with a suitable acid. Alkali metal (for example, 45 pounds, such as 3-iodomethyl pyridine hydriodide (picolinyl Sodium, potassium or lithium) or alkaline earth metal (for iodide) or 3-iodopropanol. example calcium) salts can also be used. A. Direct Cyclization When used inaccord with the methods of the invention, the Under this category, two D-cysteine esters are used, one is compounds described herein can be present in a lumines D-cysteine methyl ester and the other is D-cystine 2-hydroxy cence reaction at any effective concentration. The optimum 50 ethyl ester. The latter requires a reducing agent before concentration of a given compound will depend on the lumi cyclization. nescent reagent(s) employed, and on the specific conditions i) D-Cysteine Methyl Ester under which a given assay is carried out. However, Suitable concentrations can be determined using standard techniques that are available in the art. 55 N Specifically, the compound can be present in a lumines cence reaction at a concentration of at least about 0.01 uM, or X-CN -- at a concentration of at least about 0.1 uM, e.g., at least about R X 0.1 mM. More specifically, the compound can be present in the luminescence reaction at a concentration in the range 60 formula CXVI from about 0.1 M to about 500 mM (inclusive), or in the O MeOH, range from about 0.001 uM, 0.01 uM, or 0.1 uM to about 250 GE) K2CO3, HN H7 mM (inclusive). Preferably, the compound is present at a 3 O1. p concentration in the range from about 0.001 uM, 0.01 uM, 0.1 uM, 1 uM or 10 uM to about 100 mM (inclusive). 65 Specifically, the assay can be performed in the presence of HS purified enzymes, cell lystates or whole cells. US 8,476,450 B2 81 82 -continued The procedure of cyclization with D-cystine 2-hydroxyethyl O ester is very similar to D-cysteine methyl ester except 1 / equivalent of tris(2-carboxyethyl)phosphine hydroxhloride (TCEP) was added before pH adjustment. y-g O B. CSF-Promoted Esterification R X S formula CXVII O R = OH, OCHH X = S, CHFCH 10 N N A derivative of 2-cyanobenzothiazole or 2-cyanoquinoline (1 ) { OH + equivalent) was dissolved in methanol and was degassed with R X S a gentle stream of nitrogen for 10 minutes. Meanwhile, formula CXXI D-cysteine methyl ester (1.5 equivalents) was dissolved in 15 DMF CSF water and its pH was adjusted to about 7. This solution was I-R -- also degassed with a gentle stream of nitrogen for 5 minutes. O Then, the aqueous Solution was added to above organic solu tion dropwise and the resultant solution was stirred under N N 1 R nitrogen for 30 minutes to a few hours depending on Sub strates. The reaction was monitored by TLC with methanol in )-( " dichloromethane. After the completion of the reaction, the R X S mixture was filtered and was purified by HPLC. Typical formula CXXII R = alkyl, aromatic HPLC conditions are the following. R = OMe, OH, H Column: Dynamax 60A, 1 inch, 250 mm, reversed phase 25 X = S, CHFCH C-18 column Mobile phase A: water or 100 mMammonium acetate, pH To a solution of luciferin or a derivative (1 equivalent) in DMF 7, or 0.1% TFA in water was added cesium fluoride (1.5 equivalents), followed by Mobile phase B: acetonitrile 30 3-iodomethylpyridine hydriodide or 3-iodopropanol (1.5 Flow rate: 20 mL/min equivalents). After the resultant mixture was stirred at room Detection wavelength: 300 nm temperature for a few hours, monitored by TLC with metha Gradient: from 100% A to 100% B, 30 minutes, and keep nol in dichloromethane, the solution was concentrated down 100% B for 30 minutes. and water was added. The mixture was then filtered through a The purified compound was then characterized by NMR, MS, 35 0.2 um filter and was subjected to HPLC purification using UV-Vis, and HPLC. similar condition described above. ii) D-Cystine 2-Hydroxyethyl Ester II. D-Cysteine or D-cystine esters 1. Synthesis of 2-hydroxyethyl Ester of D-Cystine

40 HS X-CN -- R X H2O OH HO HCl (g) formula CXIII HN -- N1 No O 45 G GE C O H3 e OH 1N1 formula CXXIII O S 1) TCEP, H2O H e OH 2) MeOH, K2CO3, 50 3 1N1 S - Pi—-

S O HN N1 No S GE) 55 O formula CXIX HN O N1 No GE) O -/." O 60 N s O formula CXXIV R X S To anhydrous ethylene glycol solution (10 mL) was formula CXX charged D-cysteine hydrochloride monohydrate (1 g). A R = OH, OCHH 65 gentle stream of hydrochloride gas was introduced to bubble X = S, CHFCH the solution for 1 hour. The solution was then let stand over night. Cold isopropyl alcohol (12.5 mL) was added slowly US 8,476,450 B2 83 84 and the resultant mixture was put at -20°C. for 1 hour. The -continued white precipitates were then filtered, washed with cold iso propyl alcohol, and pumped by maintaining the Suction of the O -/." filtrate cake. White crystals (595 mg) were obtained (yield: 52%). 5 "H NMR (DMSO-d6): 8.6-88 ppm (broad, 6H), 4.35 ppm No OCS sS O (broad singlet, 2H), 4.20 ppm (m, 4H), 3.63 ppm (m, 4H), 3.05 ppm (broad singlet, 4H) ES' 330.53 (M.W. 330.44) formula CXXVIII 2. Synthesis of Methyl Ester of D-Cysteine 10 2-cyano-6-methoxybenzothiazole (100 mg 0.526 mmol) was dissolved in methanol (20 mL) and the resultant solution HS was degassed with agentle stream of nitrogen for 10 minutes. HO To a solution of 2-hydroxyethyl ester of D-cysteine (87 mg, OH HCl(g) 15 HN + HO-CH - - - 0.263 mmol) in water (10 mL) was added tris(2-carboxy e8 ethyl)phosphine hydroxhloride (TCEP) (77 mg, 0.263 C O mmol). The pH of this solution was adjusted to about 7 with 1 M potassium carbonate. The resultant solution was added to formula CXXV aforementioned solution dropwise and the mixture was HS stirred for 30 minutes. The precipitates were filtered out through a 0.2 um filter and the filtrate was purified by semi O preparative HPLC (see general procedure: Mobile phase A: HN N GE) water). 102 mg of pure compound was obtained (Yield: 52%). O 25 H NMR (CD3CN): 8.0 ppm (d. 1H), 7.55 ppm (d. 1H), formula CXXVI 7.20 ppm (dd. 1H), 5.42 ppm (t, 1H), 4.25 ppm (m. 2H), 3.75 ppm (m, 4H) MS: ES' 338.58 (M.W. 338.40) To a methanol solution (100 mL) was charged D-cysteine hydrochloride monohydrate (6 g). A gentle stream of hydro Extinction coefficient: 16,630 (at 325 nm in acetonitrile) chloride gas was introduced to bubble the solution for 105 30 b. By Ethylene Glycol minutes. The solution was then stirred over night. Then the solvent was then removed under vacuum. The white residue was dissolved in hot methanol (25 mL) and 25 mL of isopro pyl alcohol was added. The mixture was put on a rotavap to remove some of the methanol until white precipitates crashed 35 OH Ethylene glycol out of the solution. The precipitates were put on ice briefly HCl (g) and then filtered under vacuum, washed with cold isopropyl alcohol, and pumped by maintaining the Suction of the filtrate formula CXXIX cake. 40 O H NMR (CD3OD): 4.35 ppm (t, 1H), 3.85 ppm (s.3H), 3.10 ppm (d. 2H) N N OH III. Luciferin Methyl Ether Esters No O SX (S - 3. Synthesis of 2-hydroxyethyl Ester of Luciferin Methyl 45 Ether formula CXXX a. By 2-hydroxyethyl Ester of D-Cystine To a solution of luciferin methyl ether (100 mg, 0.34 mmol) N in anhydrous ethylene glycol (4 mL) in a 5 mL Schlenk flask \ -- was introduced a gentle stream of hydrochloride gas. After 5 minutes of bubbling, the flask was sealed and put into an oil su X- CN bath of 62° C. for 3 hours. The HCl bubbling was repeated for formula CXXVII another 5 minutes and put back into the oil bath for another 1 O 55 hour. After the solution was cooled to room temperature, GE) water (6 mL) was added and the resultant mixture was filtered HN OH and purified with semi-preparative HPLC (see general proce 1N1 dure: Mobile phase A: water). 4. Synthesis of 2-hydroxy Ethyl Ester of Luciferin-H S 1) TCEP, H2O 2) MeOH, K2CO3, 60 S pH7

O X-CN -- HN N-1 No S GE) 65 formula CXXXI US 8,476,450 B2 86 -continued 6. Synthesis of M-Picolinyl Ester of Luciferin O GE) HN 1N1 OH

S 1) TCEP, H2O N / OH 2) MeOH, K2CO3, S pH7 y-g HO 10 formula CXXXVI N HN 9N-1N OH DMF GE) CSF O 2 He N formula CXXXII O OH 15 formula CXXXVIII ~ O y & N N X S HO S S ro4. formula CXXXIII formula CXXXIX This compound was synthesized in a similar way to procedure described in section 3a. To a solution of luciferin mono potassium salt (51 mg, 0.16 H NMR (CD3CN): 8.15 ppm (m, 2H), 7.60 ppm (m, 2H), 25 mmol) in DMF (10 mL) was added cesium fluoride (24 mg. 5.45 ppm (t, 1H), 4.25 ppm (m. 2H), 3.75 ppm (m, 4H), 1.95 0.16 mmol), followed by 3-bromomethyl pyridine hydrobro ppm (t, 1H) mide (44 mg., 0.17 mmol). After 16 hours of reaction at room MS: ES 308.58 (M.W. 308.38) temperature, the solvent was stripped away under vacuum. Extinction coefficient: 14,050 (at 294 nm in acetonitrile) The residue was then dissolved in 10 mL of 60% DMF in 5. Synthesis of M-Picolinyl Ester of Luciferin Methyl Ether 30 water. The resultant solution was filtered through an 0.2 um filter and was subjected to HPLC purification (see general procedure: Mobile phase A: water). 8.6 mg of pure compound was obtained. 35 H NMR (CD3OD-D2O): 8.58 ppm (s, 1H), 8.50 ppm (d. N N 1H), 7.90 ppm (d. 1H, 7.85 ppm (dd. 1H), 7.42 ppm (dd. 1H), y ( OH 7.40 ppm (s, 1H), 7.10 ppm (dd. 1H), 5.45 ppm (t, 1H), 5.25 NICC S S ppm (s. 2H), 3.75 ppm (m. 2H) formula CXXXIV MS: ES": 371.65 (M.W. 371) 40 UV-Vis: 263 nm and 334 nm I N DMF 7. Synthesis of M-Picolinyl Ester of Luciferin-H CSF 2 -- N O formula CXXXV 45 O N N X-( OH -- S S ( 2 formula CXXXX No OC)S S r N 50 formula CXXXVI I S. DME CSF 2 Her To a solution of luciferin methyl ether (215 mg, 0.73 mmol) in N formula CXXXXI DMF (35 mL) was added cesium fluoride (170 mg, 11.1 55 mmol), followed by 3-iodomethylpyridine hydriodide (380 O mg, 1.1 mmol). After the resultant mixture was stirred at room temperature for 3 hours and twenty minutes, the Solution was N N O N concentrated down to about 10 mL and 6 mL of water was 2 added. The mixture was filtered through a 0.2 um filter and 60 S S N was subjected to HPLC purification (see general procedure: Mobile phase A: 0.1%TFA in water). formula CXXXXII 57 mg of pure compound was obtained. H NMR (CDCN): 8.62 ppm (s, 1H), 8.54 ppm (d. 1H), This compound was synthesized and purified in a similar 7.96 ppm (d. 1H), 7.84 ppm (d. 1H), 7.56 ppm (d. 1H), 7.41 65 fashion to the synthesis of m-picolinyl ester of luciferin ppm (m. 1H), 7.20 ppm (dd. 1H) methyl ether using luciferin-H as starting material. 13 mg of MS: ES 385.77 (M.W. 385.46) pure compound was obtained. US 8,476,450 B2 87 88 H NMR (CD3CN-D2O): 8.60 ppm (s, 1H), 8.52 ppm (d. -continued 1H), 8.10 ppm (m. 2H), 7.85 ppm (m. 1H), 7.59 ppm (m. 2H), N O OH 7.42 ppm (m. 1H), 5.50 ppm (t, 1H), 5.28 ppm (s. 2H), 3.79 ppm (m, 2H) N O/N/ MS: ES" 355.54 and 711.36 (dimer) (M.W. 355.43) 4N/ 8. Synthesis of 3-Hydroxypropyl Ester of Luciferin Methyl S Ether formula CXXXXVIII

10 A Solution of 2-cyanoquinoline (50 mg, 0.32 mmol) in 1,4- dioxane (5 mL) was degassed for 5 minutes by passing N N through a gentle stream of nitrogen gas. y ( OH Cystine 2-hydroxyethyl ester hydrochloride (53 mg, 0.16 15 mmol) in water (5 mL) was treated with tris(2-carboxyethyl) NICC S S phosphine hydrochloride (46 mg, 0.16 mmol). The pH of this formula CXXXXIII solution was adjusted to about 7 with 1 M potassium carbon ate. It was then added dropwise to the solution of 2-cyano DMF quinoline. The resultant solution was stirred at room tempera CSF ture for 7.5 hours and was purified in a similar way to 11N1 Noi - - m-picolinyl ester of luciferin (see general procedure: Mobile formula CXXXIV phase A: water). 13 mg of pure compound was obtained (yield: 26.7%) H NMR (CD3-OD-D2O): 8.40 ppm (d. 1H), 8.15 ppm (d. O 25 1H), 8.10 ppm (d. 1H), 7.95 ppm (d. 1H), 7.82 ppm (m. 1H), 7.68 ppm (m. 1H), 5.53 ppm (t, 1H), 4.25 ppm (m. 2H), 3.75 N N 1N1N ppm (m. 2H), 3.65 pppm (m. 2H) y ( 1. OH MS: ES": 302.35,324.47 (M.W. 302.35) NICC S S UV-Vis: 247 nm and 295 nm. 30 Extinction coefficient: 7,500 (at 288 nm in acetonitrile) formula CXXXXV 10. Synthesis of Quinolinyl Luciferin-H This compound was synthesized in a similar way to m-picoli nyl ester of luciferin methyl ether using luciferin methyl ether 35 N BZ-Cl, and 3-iodopropanol as starting materials. KCN "H NMR (CD3OD): 7.95 ppm (d. 1H), 7.55 ppm (d. 1H), e - - 7.18 ppm (dd. 1H), 5.41 ppm (t, 1H), 4.35 ppm (t, 2H), 3.92 e ppm (s.3H), 3.75 ppm (dd, 2H), 3.65 ppm (t, 2H), 1.92 ppm Oe 40 (m. 2H) formula CXXXXIX MS: ES: 352.70 (M.W. 352.43) UV-Vis: 330 nm and 264 nm. e D-Cys IV. Quinolinyl Luciferin Derivatives 45 9. Synthesis of 2-Hydroxy Ester of Quinolinyl Luciferin-H formula CL N

2 N N N e COOH 50 2 -- S y N CN formula CXXXXVI formula CLI O GE) 55 a. 2-cyanoquinoline HN 1N1 OH To a solution of quinoline N-oxide (2 g, 13.8 mmol) was added benzoyl chloride (2.6 mL, 22.0 mmol), followed by S 1) TCEP, H2O water (20 mL). Then, a solution of potassium cyanide (3.6 g. 2) MeOH, K2CO3, 55.1 mmol) in water (80 mL) was added slowly over a period S - Pi—- 60 of 15 minutes with vigorous stirring. Dioxane (15 mL) was added to help the solubility. After the mixture was stirred at RT for 2.5 hours, it was extracted with equal volume of HN O N1,No. dichloromethane three times. The organic layer was dried GE) over anhydrous sodium sulfate and the solvent was removed O 65 under vacuum. The residue was then purified by flash chro formula CXXXXVII matography with 30% of ethyl acetate in hexane. 2.1 g of product was obtained (yield: 100%). US 8,476,450 B2 89 NMR (CDC13): 8.32 ppm (d. 1H), 8.18 ppm (d. 1H), 7.85 -continued ppm (m. 2H), 7.65 ppm (m. 2H) O ES": 152.44 (M.W. 15416) N O Diazomethane b. Quinolinyl Luciferin-H 5 Her 2 N (Generated in 2-cyanoquinoline (51 mg, 0.33 mmol) was dissolved in 10 N 2 OH situ) mL of methanol and the resultant solution was degassed with agentle stream of nitrogen for 10 minutes. Meanwhile D-cys S teine hydrochloride monohydrate (90 mg, 0.51 mmol) was formula CLV dissolved in 5 mL of water and the pH of the resultant solution 10 was adjusted to about 7. It was then degassed with a gentle O stream of nitrogen for 5 minutes. The aqueous solution was N O then added to the organic Solution and the resultant mixture 2 N was stirred under nitrogen for 4 hours. It was then purified by N 2 1. semipreparative HPLC (see general procedure: Mobile phase 15 A: 0.1%TFA in water). S NMR: characteristic peaks: 5.45 ppm (t, 1H), 3.68 ppm (m, formula CLVI 2H) ES": 257.83 (M.W. 256.28) a. 6'-hydroxyquinolinyl Luciferin Methyl Ether This compound was synthesized in a similar fashion to quino UV-Vis: 295 nm. linyl luciferin-H starting with 2-cyano-6-methoxyquinoline Extinction coefficient: 28,530 (at 242 nm in methanol) and D-cysteine. 11. Synthesis of Naphthyl Luciferin Methyl Ether NMR (CD3CN): 8.35 ppm (d. 1H), 8.16 ppm (d. 1H), 8.03 25 ppm (d. 1H), 7.52 ppm (dd. 1H), 7.38 ppm (d. 1H), 5.45 ppm (t, 1H), 3.97 ppm (s.3H), 3.75 ppm (m, 2H) ES": 289.33 (288.32) O UV-Vis: 343 nm, 329 mm, 261 nm. D-cysteine 30 Extinction coefficient -es N b. Methyl Ester of 6'-Hydroxyquinolinyl Luciferin Methyl 2 Ether (881-92) formula CLI To a mixture of 3 mL of 40% KOH solution and 10 mL of ethyl ether on ice was added N-nitroso-N-methylurea (1 g, 9.7 35 mmol) in portions over a period of 5 minutes. Once the bubbling died down, the yellow top layer was carefully O decanted into an Erlenmyer flask with a few potassium O hydroxide pellets. The mixture was let stand on ice for 1 hour. N To a solution of 6-hydroxyquinolinylluciferin methyl ether 2 OH 40 (19.5 mg 0.068 mmol) in 10 mL of anhydrous THF was S added dropwise the diazomethane Solution made above using formula CLII a fire-polished glass pipet until the yellow color persisted. 5 drops of acetic acid was added to quench the excess diaz omethane. The solvent was removed under reduced pressure Starting with commercially available 6-methoxy-2-naph 45 and the residue was dissolved in 40% water inacetonitrile and thonitrile, this compound was synthesized in one step similar was purified by semipreparative HPLC (see general proce to quinolinyl luciferin-H. dure: Mobile phase A: water). NMR (CD3CN): 8.29 ppm (s.1H),8.05 ppm (dd. 1H), 7.94 NMR (CD3CN): 8.25 ppm (d. 1H), 8.14 ppm (d. 1H), 7.96 ppm (d. 1H), 7.86 ppm (d. 1H), 7.36 ppm (d. 1H), 7.25 ppm 50 ppm (d. 1H), 7.45 ppm (dd. 1H), 7.36 ppm (d. 1H), 5.42 ppm (dd. 1H), 5.35 ppm (t, 1H), 3.95 ppm (s.3H), 3.75 ppm (m, (t, 1H), 3.95 ppm (s.3H), 3.80 ppm (s.3H), 3.65 ppm (m,3H) 2H) ES":303.49 (M.W. 302.35) UV-Vis: 249 mm, 265=n, 326 nm, 341 mm ES": 288.23 (M.W. 287.33) 13. Synthesis of 6-hydroxyquinolinyl Luciferin (Standard UV-Vis: 253 nm, 279 mm, 315 nm, 339 mm. 55 Quinolinyl Luciferin) 12. Synthesis of Methyl Ester of 6'-Hydroxyquinolinyl This compound was synthesized according to literature pro Luciferin Methyl Ether cedures.

60

O N 1) mCPBA D-cysteine 2) Bz-Cl, 2 KCN 65 N formula CLIV formula CLVII US 8,476,450 B2

-continued -continued HN N O D-Cys N MeOH Pyridine HCI 2 -- --- N CN K2CO3 2 N N 2 formula CLXXI formula CLVIII HN N HO N 10 2 N D-Cys N e COOH N 2 a N --- S y formula CLIX formula CLXXII HO N 15

N COOH a. 6-amino-2-cyanoquinoline 4. - This compound was synthesized according to a literature S procedure (see WO 03/096.980 A2). formula CLX b. 6-aminoquinolinyl luciferin 6-amino-2-cyanoquinoline (100 mg, 0.59 mmol) was dis 14. Synthesis of 8-hydroxyquinolinyl Luciferin solved in 35 mL of methanol and the resultant solution was degassed with a gentle stream of nitrogen for 10 minutes. 25 Meanwhile, D-cysteine hydrochloride monohydrate (130 mg, 0.74 mmol) was dissolved in 15 mL of water and the N resultant solution was degassed with a gentle stream of nitro gen for 5 minutes. The aqueous Solution was then added to the 4. N D-cysteine 2 He 30 organic Solution and the resultant mixture was stirred under nitrogen for 4 hours. The volume of the solution was reduced OH under vacuum and it was then purified by semipreparative formula CLXI HPLC with and acetonitrile (see general procedure: Mobile phase A: 100 mM NHAc, pH7). 35 NMR (CD3CN): 8.25 ppm (d. 1H), 7.95 ppm (m, 2H), 7.46 ppm (dd. 1H), 7.06 ppm (s, 1H), 5.53 ppm (t, 1H), 3.96 ppm (m. 2H) OH ES": 274.67 (M.W. 273.31) formula CLXII 40 UV-Vis: 268 nm, 330 nm, and 373 nm. Extinction coefficient: 15, 320 (at 224 nm in MeOH), This compound was synthesized in a similar way to quinoli 13,020 (at 274 nm in MeOH) nyl luciferin-H with commercially available 2-cyano-8-hy 16. Synthesis of Quinolinyl Luciferin Benzyloxymethyl droxyquinoline and D-cysteine as starting materials. 45 Ether 15. Synthesis of 6-aminoquinolinyl Luciferin

ON N 50 mCPBA Crs N2 DCM formula CLXXIII N N formula CLXIII N A. ON K2CO3 N 55 Acetone TMS-CN HO 2 --

% HerTEA GE) ACN formula CLXXIVI Oc 75 C. N N 60 formula CLIX

ON H2 Her N Po?C O O 2 Her EtOAc 2 MeOH N CN 65 formula CLXX formula CLXXV