|HAO WALA NAMAN UTAMAUS010077244B2 UNA MANA TA MA TERIAL (12 ) United States Patent ( 10 ) Patent No. : US 10 , 077 ,244 B2 Cali et al. (45 ) Date of Patent: * Sep . 18 , 2018
(54 ) LUMINOGENIC AND FLUOROGENIC C09B 57 /02 (2006 . 01) COMPOUNDS AND METHODS TO DETECT CO7F 9 /6558 ( 2006 .01 ) MOLECULES OR CONDITIONS ( 52 ) U .S . CI. CPC ...... CO7D 277/ 66 ( 2013 .01 ) ; CO7D 277 /68 (71 ) Applicant: Promega Corporation , Madison , WI (2013 .01 ) ; C07D 311/ 16 (2013 . 01 ) ; C07D (US ) 417 /04 (2013 . 01 ); CO7D 417/ 14 ( 2013 . 01 ) ; ( 72 ) Inventors : James J . Cali, Verona , WI (US ); C07D 493/ 10 ( 2013 .01 ) ; C07F 9 /65583 William Daily , Santa Maria , CA ( US) ; ( 2013 . 01 ) ; CO9B 11/ 24 ( 2013 .01 ); C09B 19 / 00 Erika Hawkins, Madison , WI (US ); (2013 .01 ) ; C09B 57 /00 (2013 . 01 ) ; C09B 57 / 02 Dieter Klaubert, Arroyo Grande , CA ( 2013 .01 ) ; C12Q 1 /66 (2013 .01 ) ; GOIN (US ) ; Jianquan Liu , Fremont, CA 2500 / 00 ( 2013 . 01) (US ) ; Poncho Meisenheimer, San Luis (58 ) Field of Classification Search Obispo , CA (US ); Michael Scurria , CPC ...... C07D 417 / 04 ; CO7D 417 / 14 San Luis Obispo , CA (US ); John W . See application file for complete search history . Shultz , Verona , WI (US ) ; James Unch , Arroyo Grande, CA (US ) ; Michael P . Valley , Fitchburg , WI (US ) ; Keith V . (56 ) References Cited Wood , Mt. Horeb , WI (US ) ; Wenhui Zhou , Santa Maria , CA (US ) U . S . PATENT DOCUMENTS 4 ,612 , 302 A 9 / 1986 Szabo et al. (73 ) Assignee: PROMEGA CORPORATION , 4 ,655 ,022 A 4 / 1987 Michihiro et al . Madison , WI (US ) 4 ,665 , 022 A 5 / 1987 Schaeffer et al. 4 ,684 ,620 A 8 / 1987 Hruby et al . ( * ) Notice : Subject to any disclaimer, the term of this 4 , 826 , 989 A 5 / 1989 Batz et al . patent is extended or adjusted under 35 4 , 853 , 371 A 8 / 1989 Coy et al . 4 , 992 , 531 A 2 / 1991 Patroni et al . U . S .C . 154 (b ) by 0 days . 5 ,035 , 999 A 7 / 1991 Geiger et al . 5 , 098 , 828 A 3/ 1992 Geiger et al. This patent is subject to a terminal dis 5 , 114 , 704 A 5 / 1992 Spanier et al . claimer . 5 ,283 , 179 A 2 / 1994 Wood 5 , 283 , 180 A 2 / 1994 Zomer et al . (21 ) Appl. No. : 14/ 553, 445 5 , 290 ,684 A 3 / 1994 Kelly 5 , 374 ,534 A 12 / 1994 Zomer et al . 5 ,498 , 523 A 3 / 1996 Tabor et al. ( 22 ) Filed : Nov. 25 , 2014 5 ,641 , 641 A 6 / 1997 Wood 5 ,650 , 135 A 7 / 1997 Contag et al. (65 ) Prior Publication Data (Continued ) US 2016 /0176830 A1 Jun . 23 , 2016 FOREIGN PATENT DOCUMENTS Related U . S . Application Data EP 0411912 2 / 1991 (63 ) Continuation of application No . 13 /913 ,919 , filed on JP 63 - 501571 6 / 1988 Jun . 10 , 2013 , now abandoned , which is a ( Continued ) continuation of application No. 12 / 217 ,494 , filed on Jul. 3 , 2008 , now Pat. No. 8 ,476 ,450 , which is a OTHER PUBLICATIONS continuation of application No . 11 / 444 , 145 , filed on Chemical Abstracts Registry No . 760162 -40 - 1 , indexed in the May 31, 2006 , now Pat . No . 7 ,951 , 550 . Registry file on STN CAS Online Oct. 11, 2004. * (60 ) Provisional application No . 60 / 790 ,455 , filed on Apr. (Continued ) 7 , 2006 , provisional application No . 60 /692 ,925 , filed on Jun . 22 , 2005, provisional application No . Primary Examiner - Laura L . Stockton 60 /693 ,034 , filed on Jun . 21 , 2005 , provisional (74 ) Attorney, Agent, or Firm - Michael Best & application No. 60 /685 , 957 , filed on May 31, 2005 . Friedrich LLP (51 ) Int. CI. CO7D 417 /04 ( 2006 . 01 ) CO7D 417 / 14 ( 2006 . 01 ) (57 ) ABSTRACT C07D 277/ 66 ( 2006 .01 ) A method to detect the presence of amount of at least one CO7D 311 / 16 ( 2006 .01 ) molecule in a sample which employs a derivative of C07D 493 / 10 ( 2006 . 01 ) luciferin or a derivative of a fluorophore is provided . Com C120 1 /66 (2006 .01 ) pounds and compositions for carrying out the methods of the CO7D 277 /68 ( 2006 . 01 ) invention are also provided . C09B 11 / 24 ( 2006 . 01 ) C09B 19 /00 ( 2006 .01 ) C09B 57700 ( 2006 .01 ) 16 Claims, 49 Drawing Sheets US 10 ,077 ,244 B2 Page 2
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Patent Sep. 18 , 2018 Sheet 42 of 49 US 10 ,077 , 244 B2 3A4 PPXE assays for inhibition by nifedipine UM nifedipimPXE PPXED OXE 200 8584 7914 8244 ,667 335 .0841 100 3594 12898 12378 12056 , 67 610 . 1991 22014 21868 23066 22312 .67 647 , 8714 33136 33187 32656 3299133 291 . 3106 12 . 5 49210 47340 48470 48340 941 . 7537 6 . 25 82042 77646 81388 80358 .57 2371. 887 91696 89658 89294 .67 2502 858 0 136742 125432 129692 130622 5712 .086 M b??, . Camorex human nepatocytes conor $ 11yr old male 48 Hour induction with 10UM Rifampicin 3hr incubation with SOUM SEBE luciferin - 5FBE ??? 3174 3847 2900 5211 3783 vehicleke 1099 1197 100M rifan 34085 33834 35910 . 25 Rifamp. KE 1199 1097 941 966 1050 . 5 FIG . 51 U . S . Patent Sep . 18 , 2018 Sheet 43 of 49 US 10, 077 , 244 B2 Cambrex human hepstocytes , donoris 11 yr old male 48 Hour induction with 10UM Rifampicin 3 hr incubation with SOM PPXE 83 * * * * ? ve?? 2 ?? 3 3 % * XXX 38 3 ?? ? 4 33R 328 3 ??? f3 3 } {} {} % . * } . ? * * * 33 ??? ?? ? * 5 * * * FIG 52 ????* sors rainfar 3 * N3ase?? en 38 sefarsy ????????? " ??? ????F , 3at .arxFgsss3 ? 3 ? 18 gagggs agaisers FIG 53 USU . S . FoonPatent Sep wan. 18 , 2018 Sheet 44- of 49 IslamatusUS 10, 077 , 244 B2 ??????? ?? ????? ?????????? ? ??? ????? ?? ??????? ???? ????? ???? fwhere exy1ferini Luciferin H ???????? ?? ? ? ??????????????????? ?? ???? oooooooo??????? r?? # ?????? FE ?rr?? ??????? Luciferin 6 'mcthyi cther (Luciferin -ME } ????????????? ?? ????? ?????????? ???????? ?????????????????????Fransit ?????? ??????? Luciferin? 6 chloroethy?? ether Luciferir -CRE ?????????? ???? ?????????????? ????????????? ???? ?????????????????? ??? ?????? ??????? Lasaging & ery safer L1 iga- BE ??FIG???? 54 U . S . Patent Sep .18 ,2018 Sheet 45 of 49 | US 10. 077, 244 B2 ???????HNE # … … … … … … … . spons … … WWWWWWWWWW b4 % bb % 9A % Ethe u - 11 AMAMAHAMM * * ** HTTTTTT ????????? A MAMAMM ?nywwwMYMONCER A irsyster yyyy * * miritionimmer RELATIVELUMINESCENCE ?????” WATCHAIRIT ???????????? 11111174 " ttle LP Aventaturn UNDAL ------T LALL 144HRWAirmwwwMW ELYTESTC11KLA - WHAT ' rement tvNNEL ????? ? MUCHERS … . ? “ PES - TRY LUCIFERN MOPS WEPES TRIONE SHARM ?? U . S . Patent Sep. 18 , 2018 Sheet 46 of 49 US 10 ,077 , 244 B2 WWWWWWWWWWWWUKANAAMWWWWWWWWWWWWWWWWWWWWWWWWWWWOOOO O UNS . MoodY o ooohooooo ATP (MM )ws MAX333 NOW LOOT 3690086 7 ,53 wwwVEY 5924428wwwwwwwww 4748488 R vrywyrank W 6956226wwwww 367 1565 0 . 753 0277734 2790236 1753739Woowwwwwwwwwwwww 1061 ???????????? KV2* * * WYYYY WUMVIWY 10000000 SYLLYYYYYYYYY www $ TAL I montim LUCFONIN S - FLUOROLUCEERIN 1000000 YYYYYYYYYY 001 0. (ATP ) (MM ) FIG . 56 U . S . Patent Sep . 18 , 2018 Sheet 47 of 49 US 10, 077 , 244 B2 APPRENO UHISHULL 8 odd od Sode ) hu( ZH 224ooooooooo o 29H S 0L' 00 wwww Q & van 90€www. 9 9290 W284 , 2 wwwww 9L0 186 . 9 16 . 9 1 .00 Www 8ooooooooooooooooooooooo000000000000000oooooooooooooops 60Lwww 76 . 964 10000 1111AAAAAAAAAAAAAAAA . . . . . . LUMINESCENTINTENSITY(RLU) CASES 1000 ???????????????. texti mention HO Z Hoogland TZ met PM 6 . 7 100 from ALLA TTTTTTTTTTTTTTTM MYYYYYYYYYYYYYYYYYYYY 0 . 10 0001 (ATP ) (MM ) FIG . 57 U . S . Patent Sep . 18, 2018 Sheet 48 of 49 US 10 ,077 , 244 B2 systress ELULAR wwwwwwwww44444 wwwm 5 - 12 7. 4 ** * * * LM2 7 . 4 - ~ ~ - 7442 7. 4 w RAHN www ?????????????????? nike RELATIVEINTENSITYt%OFMAXINTENSITYFOREACHSPECTRA) Whitthe As shoesphp e hitty hom vy * * * $60 500 WAVELENGTH (nm ) FIG . 58 100000 ??????????????? ??????? * * * * Xest * KAART wewnywany * th i UMINESCEN anavyovyoteurw yyyyy 10000 w manca 6. 4 6 . 7 7. 0 7. 5 7. 6 7. 9 82 85 PH OF REAGENT FIG . 59 U . S . Patent Sep . 18 , 2018 Sheet 49 of 49 US 10 , 077, 244 B2 ???? ???? ????* ? ?? * * ay& as assays & Reg38 338 43843 ? # fazira3fy 13 psrar * * } as , his 532883 8433 syste , a32336 339ge ?? ????? ? 12 ????* 47 ,NF8 , 3 ? #sasa113533sext stage mt * * * 3xy aastafa cs53338 3 3 3 1 fiinctionality comprising any oth roxy , halo , thios Encionality uprising any of hydroxyl, halo , thio , 38 32 33334 33 3gx334 33e3a3e . ? 33333 339 38 373 34 33323 . ?????? ?? ??* 4 Xexy , * * * 11 ?? 333 33 4 335 3 * * * * . * * 3 ?? ? F35 ? 8 :28 53 888883338 333333 334 33 3gs 3 ? ? 333 33433 substituted at positions other than C1 viti 102 Substituted at positions other than C1 with or fifisa333 333333333 3y8F9y ha , #ha 33213211 333388i 3xxxx33 334 33 32 333 3, a113473 843318 , 8 , 18 , 3deye et egy step, 3g ,Rey ?? - 3 ay , 3F38 339 {{ { {{ {{ { { { A * Córry , heterdaryt optionally substituted at Risar star438 4 8 8th # 3 # statis positions cher than CI with 1 x 2 functionalities 3fy }} 3 } 2 yr3x3 ? 14 , 433 3338328 ette, sex , mprising # yarsay as , 32 31328 32G, sex, aide, aldehyde. 338 39 ??????, ???? * *? optionally substituted at positions oker tiran CI with optionally substituted at??? positions star Cher than C ) will } httlefixife thrysis 39% as, 3 ? # artsyasa 23yrsia 3assy * 8 ,43 37% 31 3, 7 , ese , 334 33 34y3 333 334 335 336 337 338 : FIG . 60 US 10 , 077 , 244 B2 LUMINOGENIC AND FLUOROGENIC of such a luciferase mediated reaction as the basis for such COMPOUNDS AND METHODS TO DETECT enzymatic or biological assays has , however, been limited . MOLECULES OR CONDITIONS Luciferase mediated reactions have been employed to detect numerous other molecules , e . g ., ATP or lactate dehydroge CROSS -REFERENCE TO RELATED 5 nase . For some of those reactions , a derivative of the APPLICATIONS naturally occurring substrate is employed . Native firefly luciferin , a polytherocyclic organic acid , D - ( - ) - 2 - ( 6 'hy This application is a continuation of U . S . application Ser . droxy - 2 ' - benzothiazolyl) -42 - thiazolin - 4 - carbozylic acid , is No. 13 /913 , 919 , filed Jun . 10 , 2013 , which is a continuation shown in FIG . 1 . For instance , methods for using luciferin of U . S . application Ser. No. 12 /217 ,494 , filed Jul. 3 , 2008 , 10 derivatives with a recognition site for an enzyme such as a now U . S . Pat. No . 8 ,476 ,450 , which is a continuation of U . S . protease as a prosubstrate were described by Miska et al. patent application Ser . No. 11/ 444 , 145 , filed May 31 , 2006 , ( Journal of Clinical Chemistry and Clinical Biochemistry , now U . S . Pat. No. 7 , 951 ,550 , which application claims the 25 : 23 (1987 ) ) . The heterogenous assays were conducted by benefit of the filing date of U . S . Application Ser. No. incubating the luciferin derivative with the appropriate 60 /685 ,957 , filed May 31, 2005 , U . S . Application Ser. No . 15 enzyme, e. g ., a protease , for a specified period of time, then 60 /693 ,034 , filed Jun . 21 , 2005 , U . S . Application Ser . No . transferring an aliquot of the mixture to a solution contain 60 /692 , 925 , filed Jun . 22 , 2005 and U . S . Application Ser . ing luciferase . Masuda - Nishimura et al. ( Letters in Applied No . 60 / 790 ,455 , filed Apr. 7 , 2006 , the disclosures of which Microbio ., 30 : 130 ( 2000 )) reported the use of a single tube are incorporated by reference herein . (homogenous ) assay which employed a galactosidase sub 20 strate -modified luciferin . In these luciferin derivatives , the BACKGROUND portion of the derivative functioning as the reactive group for the nonluciferase enzyme activity was coupled to the Luminescence is produced in certain organisms as a result D - luciferin or aminoluciferin backbone such that upon the of a luciferase -mediated oxidation reaction . Luciferase action of the nonluciferase enzyme, a D - luciferin or amino genes from a wide variety of vastly different species , par - 25 luciferin molecule was produced as the direct product of the ticularly the luciferase genes of Photinus pyralis and Pho - reaction to serve as the substrate for luciferase . A primary turis pennsylvanica ( fireflies of North America ), Pyrophorus obstacle to broadly applying luciferase mediated reactions plagiophthalamus ( the Jamaican click beetle ), Renilla reni for other enzymatic assays has been the belief that to modify formis ( the sea pansy ) , and several bacteria ( e . g ., Xenorhab - the luciferin molecule to function as a substrate for a dus luminescens and Vibrio spp ) , are extremely popular 30 nonluciferase enzyme, the activity of the nonluciferase luminescence reporter genes. Firefly luciferase is also a enzyme must directly yield a D - luciferin or aminoluciferin popular reporter for determining ATP concentrations , and , in molecule to retain its function as a substrate for luciferase . that role, is widely used to detect biomass . Luminescence is There is , therefore , a need in the field of biological assays also produced by other enzymes when those enzymes are to expand the utility of luciferase mediated reactions for mixed with certain synthetic substrate , for instance, alkaline 35 nonluciferase enzymes by identifying derivatives of phosphatase and adamantyl dioxetane phosphate , or horse - luciferin that function in a substrate for a nonluciferase radish peroxidase and luminol. enzyme or other biological molecule of interest and as a Luciferase genes are widely used as genetic reporters due prosubstrate for luciferase regardless of whether D - luciferin to the non -radioactive nature , sensitivity , and extreme linear or aminoluciferin is released as a direct result of the non range of luminescence assays. For instance , as few as 10 - 20 40 luciferase enzymatic reaction . moles of firefly luciferase can be detected . Consequently , luciferase assays of gene activity are used in virtually every SUMMARY OF THE INVENTION experimental biological system , including both prokaryotic and eukaryotic cell cultures, transgenic plants and animals , The present invention provides derivatives of luciferin and cell - free expression systems. Similarly , luciferase 45 and methods for using such derivatives in enzyme activity assays used to determine ATP concentration are highly assays or non - enzymatic biological assays where the sensitive , enabling detection to below 10 – 10 moles . luciferin derivative serves as a substrate for a desired Luciferases can generate light via the oxidation of enzyme and is a prosubstrate for luciferase or wherein the enzyme- specific substrates , e . g ., luciferins. For firefly luciferin derivative is a molecule which is modified by a luciferase and all other beetle luciferases , light generation 50 molecule of interest, which modified molecule is a substrate occurs in the presence of luciferin , magnesium ions , oxygen , for luciferin . Surprisingly , many of the luciferin derivatives and ATP . For anthozoan luciferases, including Renilla also have activity as substrates for luciferase in a light luciferase , only oxygen is required along with the substrate generating assay . Thus , by providing luciferin derivatives coelentrazine . Generally , in luminescence assays to deter- having a particular enzyme recognition site (reactive chemi mine genetic activity , reaction substrates and other lumines - 55 cal group in a molecule referred to as a substrate for a cence activating reagents are introduced into a biological particular enzyme) for a desired nonluciferase enzyme system suspected of expressing a reporter enzyme. Resultant coupled to the luciferin backbone ( or other chemical moiety luminescence , if any, is then measured using a luminometer constituting a suitable substrate for luciferase ) , such as or any suitable radiant energy -measuring device . The assay derivatives with modifications at the 6 ' hydroxy site of is very rapid and sensitive , and provides gene expression 60 luciferin or the 6 ' amino site of aminoluciferin , yielding a data quickly and easily, without the need for radioactive substrate for a desired nonluciferase enzyme and a prosub reagents strate of luciferase , numerous nonluciferase enzymes may Because most enzymatic reactions do not generate outputs be measured in a bioluminescent assay . that are as ideal as luciferase , the availability of a luciferase Modifications of luciferin within the scope of the deriva mediated assay for enzymatic reactions useful in cellular 65 tives of this invention include one or more substitutions of analysis and high - throughput screening applications would a ring atom , one or more substitutions of a substituent (atom be desirable to those working in this field . The development or group ) attached to a ring atom , and /or addition of one or US 10 ,077 , 244 B2 more atoms to the ring , e . g ., expansion or addition of rings, or a combination thereof. Numbering for some of the ring I atoms in D - luciferin is shown in FIG . 1 . Native firefly luciferin has three linked rings, a 6 membered ring having an B ' OH group at position 6 (“ ring A ” or “ A ring ” hereinafter ), a 5 5 membered thiazole ring linked to the 6 membered ring ZA | ALB Y ZR ( “ ring B ” or “ B ring " hereinafter ) , and a 5 membered thiazole ring that is modified with a carboxyl group at SW2 A position 5 (“ ring C ” or “ C ring ” hereinafter ). For instance , Z w W2 a luciferin derivative with a A ring modification may have a 10 substitution of a C atom in the A ring with another atom , wherein addition of a ring, a substitution of a substituent attached to Y is N , N -oxide , N - (CZ - C .) alkyl, or CH ; a ring atom with a different atom or group , or any combi when Y is N , then X is not S ; nation thereof. A luciferin derivative with a4 B 115ring modifi11 - 1615 cation may have an addition to or substitution of an atom in X is S , O , CH = CH , N = CH , or CH = N ; the five membered ring , e . g . , insertion of one or more atoms, when X is S , then Y is not N ; thereby expanding the ring , for instance , to a six membered Z and Z are independently H , OR , NHR , or NRR ; ring , substitution of Nor S in the ring with a different atom , Z " is O , S , NH , NHR , or N = N ; e . g ., a C or O , substitution of a substituent atom or group 20 O is carbonyl or CH . . attached to a ring atom , or any combination thereof. A luciferin derivative with a C ring modification may have a W is H , halo , (C1 - C6 )alkyl , (C2 -C20 ) alkenyl, hydroxyl, substitution of an atom in the ring with another atom , a or ( C - C . ) alkoxy ; or substitution of a substituent attached to a ring atom , with a W and Z are both keto groups on ring A , and at least one different atom or group , or any combination thereof. In one 25 of the dotted lines denoting optional double bonds in ring A embodiment, a derivative of the invention is one which is is absent; modified at more than one position , for instance , the deriva - each W2 is independently H , halo , ( C , - CoJalkyl, (C2 - C2 ) tive has two (or more ) A ring modifications, two (or more ) alkenyl , hydroxyl , or ( C . - C .) alkoxy ; Bring modifications , two ( or more ) C ring modifications , or each of Kl. K2 K3. and K4 are independently CH , N . any combination thereof. In one embodiment, one modifi - 30 N - oxide , or N - ( C . - C . Jalkyl, and the dotted lines between cation is the substitution of a substituent on one of the rings Kl and K ? , and K3 and K4, denote optional double bonds ; of D - luciferin with a substrate for a nonluciferase enzyme, or a linker and a substrate for the nonluciferase enzyme. A ' and B ' are optional aromatic rings fused to ring A , only Exemplary derivatives with A ring modifications may be one of which is present in the compound , so as to form a a substrate for a reductase , such as a cytochrome P450 35 fused tricyclic system ; and reductase , monoamine oxidase (MAO ) , flavin monooxy when B ' is present, the group Z is present, and genase (FMO ) , glutathione S transferase (GST ) , dealkylase , when A ' is present , the group Z is absent; and deacetylase , deformylase , phosphatide, e . g . , alkaline phos phatase ( AP ) , sulfatase, beta - lactamase , alcohol dehydroge the dotted line in ring B is an optional double bond ; nase , protease e . g . , proteosome, cathepsin , calpain , beta 40 each R is independently H , (C , -C20 )alkyl , (C2 -C20 )alk secretase , thrombin , or granzyme, luciferase , or useful to enyl, (C2 - C20 )alkynyl , (C3 -C20 )cycloalkyl , ( C1 -C12 )alkoxy , detect reactive oxygen species (ROS ) , peroxidase e . g ., (Co -C30 ) aryl, heteroaryl, heterocycle , (C1 - C20 )alkylsulfoxy , horseradish peroxidase (HRP ) , and redox conditions . Exem (Co -C30 )arylsulfoxy , heteroarylsulfoxy , (C , -C20 )alkylsulfo plary molecules or conditions to be detected with derivatives nyl, (C -C30 ) arylsulfonyl, heteroarylsulfonyl, ( C ,- C20 ) having at least a B ring modification include but are not 45 alkylsulfinyl, (Co -C30 ) arylsulfinyl, heteroarylsulfinyl, ( C , limited to dealkylase , GST or luciferase , or redox condi C20 ) alkoxycarbonyl, amino , NH ( C . - C . ) alkyl, N ( C , - C . ) tions. Exemplary molecules to be detected with those alkyl2 , tri( C , -C20 ) ammonium ( C , -C20 ) alkyl, heteroaryl( C derivatives include a cytochrome P450 enzyme , esterase , C20 ) alkyl, heteroaryl having quaternary nitrogen , e . g ., acetylcholinesterase, OH radicals , demethylase , heteroarylcarbonyl having quaternary nitrogen , ( C .- C30 ) deacetylase , deformylase , or mycoplasma carboxypeptidase . 50 arylthio , ( C , - C20 )alkylphosphate , ( C . - C20 )alkylphospho Exemplary molecules to be detected with derivatives having nate. , (Co - C30 )arylphosphate , (Co - C30 ) arylphosphonate , C ring modifications include but are not limited to esterases . phosphate , sulfate , saccharide, or M * optionally when Z " is In one embodiment, derivatives of luciferin or aminolu oxygen , wherein M is an alkali metal; ciferin have the following structure ; L - X - M - Y - R ( compound or when Z or Z ' is NR 'R ', R ' Rl together with the N to of formula IV ) , wherein L , if present, may be a substrate for 55 which they are attached forms a heteroaryl or heterocycle an enzyme or another molecule which interacts with the group ; enzyme; X may be O , NH , or a linker, e .g ., a self -cleavable wherein any alkyl, alkenyl , alkynyl, cycloalkyl, alkoxy, linker which spontaneously cleaves to yield M - Y - R afterL a mino , aryl, heteroaryl, or heterocycle group is optionally has been removed from L - X - M - Y -R ; M may be luciferin , substituted with 1 , 2 , 3 , 4 , or 5 substituents selected from quinolinyl luciferin or naphthyl luciferin , or aminoluciferin 60 (C1 - C20 )alkyl , (C2 - C20 ) alkenyl, (C2 - C20 Jalkynyl, (C2 -C20 ) or aminoquinolinyl luciferin , Y is O ( ester ) , NH ( amide ), cycloalkyl, ( C , - C20 ) alkoxyl, ( C , -C20 ) alkylcarbonyl, ( C , NH - NH (hydrazide ), or S (thioester ), and R , if present, C20 ) alkylcarboxyl, halo , hydroxyl , COORT, SO , R , may be alkyl, an aromatic molecule , a peptide, an oligo SO3R , nitro , amino , (C , -C20 )alkyl - S (O ) - , (C ,- C20 ) nucleotide, or a self -cleavable linker attached to a substrate alkyl- S02 — , phosphate , ( C , -C20 ) alkylphosphate , (C1 - C20 ) for an enzyme. 65 alkylphosphonate , NH (C2 - C6) alkyl, NH (C7 - C ) alkynyl , In one embodiment, the invention provides a compound N ( ( C / - C . ) alkyl ) 2 , N ( C , - C . ) alkynyl) 2 , mercapto , (C1 - C20 ) of formula I : alkylthio , (C6 - C30Jaryl, (C6 -C30 ) arylthio , trifluoromethyl, US 10 ,077 ,244 B2 5 = O , heteroaryl, and heterocycle , and each substituent is Z is H , OR , NHR , or NRR ; optionally substituted with one to three R groups ; Z " is O , S , NH , NHR , or N = N ; R * is H , (C ,- C .) alkyl , or (C6 -C30 ) aryl; w is H halo , hydroxyl, ( C . - C . ) alkyl, or (C1 - C . ) alkoxy ; the dotted line in ring B is an optional double bond ; when Z or Z ' composes a nitrogen moiety , one or both of each R is independently H , ( C -C20 ) alkyl, (C2 -C20 )alk the hydrogens of the Z or Z ' nitrogen moiety may be 5 enyl, (C2 - C20 ) alkynyl, (Cz - C20 )cycloalkyl , ( C , - C12) alkoxy , replaced by ( C , - C20 ) alkyl or the group L , wherein L is an (Co - C30 )aryl , heteroaryl, heterocycle , (C2 - C20 Jalkylsulfoxy, amino acid radical, a peptide radical having up to 20 ammo (C6 -C30 ) arylsulfoxy, heteroarylsulfoxy , ( C , -C20 )alkylsulfo acid moieties, or any other small molecule that is a substrate nyl, (Co -C30 Jarylsulfonyl, heteroarylsulfonyl, (C ,- C20 ) for a nonluciferase ; with the proviso that when L is an amino 10 alkylsulfinyl, (C6 - C30 Jarylsulfinyl, heteroarylsulfinyl, (C1 acid radical or a peptide radical, at least one W2 is not H ; 1 C20 Jalkoxycarbonyl, amino NH ( C , - C ) alkyl, N ( ( C / - C ) when Z is a hydroxyl group or a nitrogen moiety , H of the alkyl) 2, tri (C1 -C20 )ammonium ( C2 - C20 )alkyl , heteroaryl (C1 hydroxyl or nitrogen moiety may be replaced by (HO )2P C20 ) alkyl, heteroaryl having quaternary nitrogen , ( O ) - OCH — , sulfo , - POZH2, or by a cephalosporanic heteroarylcarbonyl having quaternary nitrogen ( C . - C ) acid attached to the group Z via a carbon chain of one to 15 arylthio , ( C , -C20 )alkylphosphate , ( C , - Cz0Jalkylphospho about 12 carbon atoms; with the proviso that when ring B is nate , (Co - C30 )arylphosphate , (Co - C30 )arylphosphonate , a thiazole ring , the sulfo or the - POZH2 group is attached phosphate , sulfate , saccharide, or M * optionally when Z " is to the hydroxyl oxygen via a ( C , - C . )alkylene group ; oxygen , wherein M is an alkali metal; when Z or Z ' is a hydroxyl group or a nitrogen moiety , or o r when Z or Z ' is NR ' R ', R ' R ! together with the N to when Z " R is a hydroxyl group , one H of the hydroxyl or 20 which they are attached forms a heteroaryl or heterocycle nitrogen moiety may be replaced by the group L '- linker, group ; wherein L ' is a group removable by an enzyme to free the wherein any alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy , linker, and linker is a carbon chain that can self -cleave , amino , aryl, heteroaryl , or heterocycle group is optionally optionally interrupted by one or more nitrogen atoms, oxy - substituted with 1to , 2 , 3 , 4 , or 5 substituents selected from gen atoms, carbonyl groups, optionally substituted aromatic 25 (C1 - C20 ) alkyl, ( C2- C20 )alkenyl , ( C2 -C20 )alkynyl , (C2 - C . ) cycloalkyl, ( C1 - C20 )alkoxyl , (C1 - C20 )alkylcarbonyl , (C3 rings , or peptide bonds, C20 ) alkylcarboxyl, halo , hydroxyl, COOR “ , SO , R * , linker is attached to L ' via an oxygen atom or an NH group SO3R *, nitro , amino , (C , -C20 )alkyl - S ( O ) - , ( C , - C20 ) at one terminus of the linker and the other terminus of the alkyl -S02 — , phosphate , (C1 - C20 )alkylphosphate , (C2 -C20 ) linker forms an ether, ester, or amide linkage with a groupP30 alkylphosphonate , NH (C1 -C .) alkyl , NH (C1 - C .) alkynyl , Z , Z ' or Z " - R ; N ( ( C / - C . ) alkyl ) 2 , N ( C , - C . ) alkynyl) 2 , mercapto , (C1 - C20 ) when Z is OR , formula I is optionally a dimer connected alkylthio , (Co -C30 )aryl , (Co - C3 . ) arylthio , trifluoromethyl, at the two A rings via a linker comprising a (C2 -C12 ) alkyl = O , heteroaryl, and heterocycle , and each substituent is diradical that is optionally interrupted by one to four O optionally substituted with one to three R groups; atoms, N atoms, or an optionally substituted aryl, heteroaryl, 35 R * is H , ( C , - Cs) alkyl , or ( C . - C . )aryl ; or heterocycle group to form a bridge between the dimer of A is an anion , present when a quaternary nitrogen is formula I , and the R group of each Z group connecting the present; dimer of formula I is replaced by the bridge ; or a salt thereof, A is an anion , present when a quaternary nitrogen is provided that: present; 40 when rings A and B form a naphthalene or quinoline ring or a salt thereof, system , then Wl is not hydrogen . provided that: 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 W1 is H , then when a ring A substituent is OH , then - Q - R " - R is not 45 Z is not OH attached to carbon -6 of ring A (carbon 6 as C ( O ) _ NH _ NH ; shown in FIG . 1 ) ; and when Y is N or CH and X is CH = CH and W ' is H , then when Y is N or CH and X is CH = CH and Z is H , then Z is not OH attached to K ’ ; and W is not OH attached to carbon - 6 of ring A . when Y is N or CH and X is CH = CH and Z is H , then In another embodiment, the invention provides a com wl is not OH attached to K ? . 50 pound of formula II: In another embodiment, the invention provides a com pound of formula IA : IIII IA 55 Lot KN 2- R Z A T AB 1 7 AL 647 s - w2 W2 60 wi W2W2A WI OLDE wherein wherein Z and Z ' are independently OR !, NHR ' , or NR ' R '; Y is N , N - oxide , N - (C1 - C . ) alkyl, or CH ; Z " is O , S , NH , NHR , r N = N ; when Y is N , then X is not S ; 65 Q is carbonyl or CH , . X is S , O , CH = CH , N = CH , or CH = N ; Wl is H , halo , ( C .- C .) alkyl , (C2 - C20 )alkenyl , hydroxyl, when X is S , then Y is not N ; or (C1 -C .) alkoxy ; or US 10 ,077 , 244 B2 wl and Z are both keto groups on ring A , and at least one when Z or Z ' is a hydroxyl group or a nitrogen moiety , or of the dotted lines denoting optional double bonds in ring A when Z " - R is a hydroxyl group , one H of the hydroxyl or is absent; nitrogen moiety may be replaced by the group L '- linker , each W2 is independently H , halo , (C - C )alkyl , (C2 - C4 ) wherein L ' is a group removable by an enzyme to free the alkenyl, hydroxyl, or (C1 - C . ) alkoxy ; 5 linker , and linker is a carbon chain that can self -cleave , each of K ' , K™, K , and K * are independently CH , N , optionally interrupted by one or more nitrogen atoms, oxy N -oxide , or N - (C - C . )alkyl , and the dotted lines between K and K ? , and Kº and K4, denote optional double bonds; gen 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.. only a 10 it linker is attached to L ' via an oxygen atom or an NH group fused tricyclic system ; and it one terminus of the linker and the other terminus of the when B ' is present, the group Z is present, and linker forms an ether, ester , or amide linkage with a group when A ' is present, the group Z is absent; and Z , Z ', or Z " - R ; R is H , ( C , -C20 )alkyl , (C2 -C20 )alkenyl , (C2 - C20 )alkynyl up, when Z is OR ! , formula II is optionally a dimer connected ( C3- C20 )cycloalkyl , (C1 - C12 ) alkoxy, (Co - C30Jarylarul , hethet - 15is at the two A rings via linker comprising a (C7 -C12 )alkyl eroaryl, heterocycle , ( C , - C , Jalkylsulfoxy , (C . - C . Jarylsul- diradical that is optionally interrupted by one to four 0 foxy , heteroarylsulfoxy , (C -C20 )alkoxycarbonyl , amino , atoms, N atoms, or an optionally substituted aryl, heteroaryl , NH ( C . - C . ) alkyl, N ( ( C / - C ) alkyl) 2 , tri (C1 - C20 ) ammonium or heterocycle group to form a bridge between the dimer of ( C , - C20) alkyl, heteroaryl( C , -C20 )alkyl , heteroaryl having formula II , and the R ' group of each Z group connecting the quarternary nitrogen , heteroarylcarbonyl having quaternary 20 dimer of formula II is replaced by the bridge ; nitrogen , saccharide , of M * optionally when Z " is oxygen , provided that a saccharide is not directly attached to K ’ ; wherein M is an alkali metal; A is an anion , present when a quaternary nitrogen is Rl is (Co -C30 )aryl , heteroaryl , heterocycle , (C ,- C20 Jalky - present; Ithio , (C1 - C20 )alkyl - S ( O ) - , ( C , -C20 ) alkyl- SO2 , — SO3 (C1 or a salt thereof. C20 )alkyl , saccharide, ( C - C20 ) alkylphosphate , ( C , -C20 ) 25 In yet another embodiment, the invention provides a alkylphosphonate , ( C -C30 )arylthio , (Co - C30 ) aryl- S ( O ) - , compound of formula IIA : (C6 -C30 ) aryl- SO2, SO3 (Co - C30Jaryl, (C6 -C30 ) arylphos phate , (C6 - C30 )arylphosphonate , or R is ( C , -C20 )alkyl sub stituted by R ? ; IIA R2 is (C2 - C20 )alkenyl , (C2 - C20 ) alkynyl , (C3 -C20 )cy - 30 cloalkyl, (C -C20 )alkoxyl , (C2 -C20 )alkylcarbonyl , ( C -C20 ) alkylcarboxyl , hydroxyl, COORT, SO3R " , ( C . -C20 ) an- R alkylthio , (Co - C3. ) arylthio , ( C , - C20 ) alkyl- S ( O ) - , ( C , - C20 ) N alkyl -S02 — , nitro , amino , NH ( C , - C . ) alkyl, NH (C1 - C6) alkynyl, N ( C . - C . ) alkyl) 2 or N ( C - C . )alkynyl ) 2 , 35 mercaptom , saccharide, or trifluoromethyl; or when Z or Z ' is NR 'RI , R ' Rl together with the N to which they are attached forms a heteroaryl or heterocycle wherein group ; Z is ORP, NHR ' , or NR ' R ' ; wherein any alkyl1. , alkenyl , alkynyl, , cycloalkyl, alkoxy , 40 Z " is O , S , NH , NHR , or N = N ; amino , aryl, heteroaryl, or heterocycle group is optionally Wl is H , halo , hydroxyl , ( C , -C ) alkyl , or ( C . -C .) alkoxy ; substituted with 1 , 2 , 3 , 4 , or 5 substitutes selected from R is H , ( C1- C20 ) alkyl, (C2 -C20 ) alkenyl, (C2 -C20 )alkynyl , (C1 - C20 )alkyl , (C2 - C20 )alkenyl , (C2 - C20 )alkynyl , (Cz - C20 ) ( C3 -C20 )cycloalkyl , ( C , -C12 )alkoxy , (Co - C30 )aryl , het cycloalkyl, ( C , -C20 ) alkoxyl, (C ,- C20 Jalkylcarbonyl, (C - eroaryl, heterocycle , (C1 - C20 ) alkylsulfoxy, (Co -C30 )arylsul C20) alkylcarboxyl, halo , hydroxyl, COORT, — SO R +, 45 foxy, heteroarylsulfoxy, ( C , -C20alkoxycarbonyl , amino - SO3R * , (C , -C20 )alkyl - S ( O ) , ( C , -C20 )alkyl - S02 — , NH (C3 - C . )alkyl , N ( C , -C .) alkyl ) 2 , tri (C -C20 )ammonium phosphate , ( C , -C20 ) alkylphosphate , ( C -C20 ) alkylphospho - ( C -C20 ) alkyl , heteroaryl( C -C20 ) alkyl, heteroaryl having nate , nitro , amino , NH (C7 - C . ) alkyl , NH ( C , - C . ) alkynyl, quaternary nitrogen , heteroarylcarbonyl having quaternary N ( ( C - C . )alkyl ) 2 , N (( CZ - C6) alkynyl) 2 , mercapto , (C , -C20 ) nitrogen , saccharide , or M + optionally when is oxygen , alkylthio , ( Co- C30Jaryl , (Co - C30 Jarylthio , trifluoromethyl, 50 wherein M is in alkali metal ; = O , heteroaryl, and heterocycle , and each substituent is Rl is (C6 - C30 ) aryl , heteroaryl, heterocycle , ( C -C20 ) alky optionally substituted with one to three R groups ; Ithio , ( C , -C20 ) alkyl- S ( O ) - , ( C -C20 ) alkyl- SO2, SO3( C1 R * is H or ( C . - C . ) alkyl; C20 ) alkyl, saccharide , (C1 - C20 )alkylphosphate , (C2 -C20 ) when Z or Z " comprises a nitrogen moiety , a hydrogen of alkylphosphonate , (Co - C30 ) arylthio , ( Co- C30 Jaryl- S ( O ) - , the Z or Z ' nitrogen moiety may be replaced by the group L , 55 (Co - C30 )aryl - SO2 , — SO3( Co - C30 )aryl , (C6 -C30 ) arylphos wherein L is an amino acid radical, a peptide radical having phate , (Co - C30 Jarylphosphonate , or R is ( C . - C20 ) alkyl sub up to 20 amino acid moieties, or any other small molecule stituted by R ?; that is a substrate for a nonluciferase ; with the proviso that R2 is (C2 - C20 ) alkenyl, (C2 - C20 ) alkynyl , (C3 - C20 ) cy when L is an amino acid radical or a peptide radical, at least cloalkyl, (C , -C20 )alkoxyl , (C , -C20 )alkylcarbonyl , (C ,- C20 ) one of W ' or a W ' is not H ; 60 alkylcarboxyl, hydroxyl, COOR , SORT, ( C , - C ) when Z is a hydroxyl group or a nitrogen moiety , H of the alkylthio , (Co -C30 ) arylthio , ( C , -C20 ) alkyl- S ( O ) - , ( C , -C20 ) hydroxyl or nitrogen moiety may be replaced by (HO P) alkyl -S02 — , nitro , amino , NH (C , -Co - alkyl, NH ( C , -C6 ) ( 0 ) OCH — , sulfo , - POZH2, or by a cephalosporanic alkynyl, N ( C , - C . )alkyl ) 22 or N ( C - C . ) alkynyl) 2 , acid attached to the group Z via a carbon chain of one 10 mercapto , saccharide , or trifluoromethyl ; about 12 carbon atoms; with the proviso that the sulfo or the 65 or when Z or Z ' is NR ' R , R ' R together with the N to - POZH2 group is attached to the hydroxyl oxygen via a which they are attached forms a heteroaryl or heterocycle ( C1- C . ) alkylene group ; group ; US 10 ,077 , 244 B2 wherein any alkyl , alkenyl, alkynyl, cycloalkyl, alkoxy, amino , aryl, heteroaryl, or heterocycle group is optionally III substituted with 1 , 2 , 3 , 4 , or 5 substituents selected from ( C2- C20 ) alkyl , (C2 - C20 ) alkenyl , ( C2- C20 ) alkynyl, (C3 -C20 ) B cycloalkyl, (C , -C20 ) alkoxyl, ( C , - C20 )alkylcarbonyl , ( C , - 5 R2K C20 )alkylcarboxyl , halo , hydroxyl, COOR ", — SOR" , Z ' - A ALB 2nR SO3R *, (C2 -C20 ) alkyl- S (O ) , (C ,- C20 )alkyl - SO2 , KY SW2 phosphate , ( C , - C20 )alkylphosphate , ( C , -C20 )alkylphospho wi A nate , nitro , amino NH ( C . - C . ) alkyl, NH (C2 - C ) alkynyl, W2 N (( C / - C .) alkyl) 2, N ( C2 - C6) alkynyl) 2 , mercapto , ( C , - C20 ) alkylthio , (Co - C30 )aryl , ( C6- C30Jarylthio , trifluoromethyl, wherein = O , heteroaryl , and heterocycle , and each substituent is Y is N , N - oxide, N - (C1 - C . )alkyl , or CH ; optionally substituted with one to three R groups ; X is S , O , CH = CH , N — CH , or CH = N ; R * is H or ( C , - C ) alkyl; 15 Z and Z are independently H , OR , NHR , or NRR ; when Z is ORT, formula IIA is optionally a dimer con 15 Z " is O , S , NH , NHR , or N = N ; nected at the two A rings via linker comprising a (C , -C12 ) Q is carbonyl or CH , ; alkyl diradical that is optionally interrupted by one to four 0 W is H , halo , ( C , - C . ) alkyl, (C2 -C20 ) alkenyl, hydroxyl, atoms, N atoms, or an optionally substituted aryl, heteroaryl, or (C1 - C )alkoxy ; or or heterocycle group to form a bridge between the dimer of 20 W ' and Z are both keto groups on ring A , and at least one formula IIA , and the Rl group of each Z group connecting of the dotted lines denoting optional double bonds in ring A the dimer of formula II is replaced by the bridge ; is absent; provided that a saccharide is not directly attached to K ; each W2 is independently H , halo , ( C , - C . ) alkyl, (C2 - CA ) A is an anion , present when a quaternary nitrogen is alkenyl, hydroxyl, or (C1 - C . ) alkoxy ; present; 25 each of K ' , K², K ” , and K4 are independently CH , N , or a salt thereof. N -oxide , or N - ( C .- C . )alkyl and the dotted lines between Other deriviates and their use in luminogenic assays is K and K ? , and K and K4, denote optional double bonds ; described hereinbelow . A ' and B ' are optional aromatic rings fused to ring A , only The use of the luciferin derivatives described herein can one of which is present in the compound , so as to form a result in an assay which produces a measurable change in 30 fused tricyclic system ; and optical properties upon interaction with a nonluciferase when B ' is present, the group Z is present, and molecule , 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 the dotterdotted line in ring B is an optional double bond ; reaction between a luciferin derivative and a nonluciferase each R is independently H , ( C , - C20 ) alkyl , (C2 - C20 ) alk enzyme or othermolecule of interest need not be D - luciferin 35 enyl, (C2 - C20 ) alkynyl, ( C3- C20 ) cycloalkyl , ( C , - C12 ) alkoxy , or aminoluciferin . For example , a luciferin derivative may (C6 -C30 ) aryl, heteroaryl , heterocycle , (C2 - C20 ) alkylsulfoxy, include a substrate that includes a reactive chemical group (C6 - C30 ) arylsulfoxy, heteroarylsulfoxy, (C2 - C20 ) alkylsulfo for a nonluciferase enzyme linked to luciferin or aminolu - nyl , (Co -C30 ) arylsulfonyl, heteroarylsulfonyl , ( C , -C20 ) ciferin via a chemical linker . Transformation of the reactive alkylsulfinyl, (Co - C30 Jarylsulfinyl , heteroarylsulfinyl , (C chemical group of the derivative by the nonluciferase 40 C20 ) alkoxycarbonyl, amino , NH (CZ -C6 ) alkyl; N ( C2 -C6 ) enzyme may yield a product that contains (retains ) a portion alkyl) 2 , tri ( C / -C20 )ammonium (C1 - C20 )alkyl , heteroaryl (C1 of the substrate , a portion of the chemical linker, the C20 ) alkyl, heteroaryl having quaternary nitrogen , chemical linker, or a portion of the substrate and the chemi- heteroarylcarbonyl having quaternary nitrogen , (Co - C30 ) cal linker , and that product is a substrate for luciferase . Also arylthio , ( C , -C20 ) alkylphosphate , ( C , - C20 ) alkylphospho provided are luciferin derivatives which , after interaction 45 nate , (Co -C30 )arylphosphate , (Co - C30 ) arylphosphonate , with a nonluciferase enzyme or other molecule , may yield a phosphate , sulfate , saccharide, or M ' optionally when Z " is product that optionally undergoes one or more further reac - oxygen , wherein M is an alkali metal; tions, e . g ., B - elimination , to yield a suitable substrate for o r when Z or Z ' is NR ' R , R ' R together with the N to luciferase . Luciferin derivatives in which the backbone of which they are attached forms a heteroaryl or heterocycle luciferin is further modified in its ring structure , e. g. , a 50 group ; quinolyl or napthyl luciferin , are provided , as well as wherein any alkyl, alkenyl , alkynyl, cycloalkyl, alkoxy, advantageously providing modifications at the carboxy posi - amino , aryl, heteroaryl, or heterocycle group is optionally tion of the thiazole ring, to provide improved characteristics substituted with 1, 2 , 3 , 4 , or 5 substituents selected from to the luciferin derivative . Derivatives with certain modifi- (C1 - C20 ) alkyl, (C2 - C20 )alkenyl , ( C2- C20 )alkynyl , (C3 -C20 ) cations provide for or improve assays for certain nonlu - 55 cycloalkyl, (C ,- C20 ) alkoxyl, (C , -C20 )alkylcarbonyl , (C , ciferase enzymes or molecules . For instance , as described C20 ) alkylcarboxyl , halo , hydroxyl , COOR " , SO , R " , hereinbelow , a pH insensitive derivative of luciferin was SOBRT, nitro , amino , (C , -C20 )alkyl - S (O ) - , (C , -C20 ) identified that is useful in biological assays that may be run alkyl- S02 — , phosphate , ( C , - C20 Jalkylphosphate , (C1 - C20 ) at a pH other than physiological pH , i. e ., less than about pH alkylphosphonate , NH (C7 - C . ) alkyl , NH ( C . - C . ) alkynyl , 7 .0 and greater than about 7 . 9. Thus, bioluminescent meth - 60 N (( C1- C6 ) alkyl ) 2 , N ( (C / -C ) alkynyl ) 2, mercapto , ( C , -C20 ) ods that employ a luciferin derivative of the invention may alkylthio , (Co -C30 ) aryl, (Co -C30 ) arylthio , trifluoromethyl , be used to detect one or more molecules, e . g . , an enzyme, a = O , heteroaryl, and heterocycle , and each substituent is cofactor for an enzymatic reaction such as ATP, an enzyme optionally substituted with one to three R groups ; substrate , an enzyme inhibitor, an enzyme activator, or OH R * is H , ( C , - C .) alkyl , or (C6 -C3 . )aryl ; radicals , or one or more conditions, e . g . , redox conditions. 65 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 formula III: replaced by (CH , C20 ) alkyl or the group L , wherein L is an US 10 ,077 , 244 B2 12 amino acid radical, a peptide radical having up to 20 amino site for the enzyme of interest, as well as a further modifi acid moieties , or any other small molecule that is a substrate cation to that ring or one or more of the other rings. for a nonluciferase ; with the proviso that when Lis an amino acid radical or a peptide radical, at least one W ? is not H ; Previously, enzymes that could be tested with luciferin when Z is a hydroxyl group or a nitrogen moiety , H of the 5 derivatives were those that interacted well close to aromatic hydroxyl or nitrogen moiety may be replaced by (HO )2P structures ( D - luciferin or aminoluciferin ) and interacted ( 0 ) OCH — , sulfo , - POZH , , or by a cephalosporanic with a recognition site (substrate ) that was stable close to acid attached to the group Z via a carbon chain of one to aromatic structures . As described herein , luciferin deriva about 12 carbon atoms; with the proviso that when ring B is tives that are substrates for nonluciferase enzymes that do a thiazole ring , the sulfo or the PO , H , group is attached 10 not necessarily react with structures close to aromatic rings to the hydroxyl oxygen via a (C - C ) alkylene group ; or those with recognition sites that are more stable when when Z or Z ' is a hydroxyl group or a nitrogen moiety , or attached to an aryl chain than an aromatic structure , were when Z " - R is a hydroxyl group , one H of the hydroxyl or identified . For example , an assay which employed previ nitrogen moiety may be replaced by the group L ' - linker, ously described luciferin derivatives with a phosphate group wherein L ' is a group removable by an enzyme to free the 15 attached through the hydroxyl group on the benzene ring , linker , and linker is a carbon chain that can self -cleave , i. e . , a substrate for alkaline phosphatase , was limited by high optionally interrupted by one or more nitrogen atoms, oxy background because the phosphate spontaneously hydro gen atoms, carbonyl groups , optionally substituted aromatic lyzed . Attaching the phosphate to an aryl chain is likely rings , or peptide bonds, stabilizing , which may allow for a derivative that is a linker is attached to L ' via an oxygen atomatom or an NH group 20 substrate for alkaline phosphatase and may reduce the at one terminus of the linker and the other terminus of the background . Thus, the sensitivity and utility of a phos linker forms an ether , ester, or amide linkage with a group phatase assay such as an alkaline phosphatase assay is Z , Z ', or Z " — R ; increased employing a luciferin derivative of the invention . when Z is OR , formula III is optionally a dimer connected Generally , luciferase substrates have a free hydroxyl at the two A rings via a linker comprising a ( C C - alkyl 25 ( luciferin ) or free amino group (amino luciferin ) on the diradical that is optionally interrupted by one to four 0 benzene ring . Alternate backbones for luciferin , like quino atoms, N atoms, or an optionally substituted aryl, heteroaryl, linyl luciferin or napthyl luciferin , with free hydroxyl or free or heterocycle group to form a bridge between the dimer of amino groups , are luciferase substrates . To expand the formula III, and the R group of each Z group connecting the scaffolding upon which modifications can be made to dimer of formula III is replaced by the bridge ; 30 luciferin and result in a luciferase substrate , luciferin deriva A ' is an anion , present when a quaternary nitrogen is tives with aryl or other chains attached through the oxygen present; or nitrogen on the A ring of luciferin were prepared . Deriva or a salt thereof; tives with nitrogen on the A ring were utilized by beetle provided that the compound of formula III is not aminolu - luciferase to generate bright luminescence . Moreover, ciferin that has been modified to include a protease substrate 35 HPLC verified that luciferin derivatives with aryl or alkyl via a peptide bond at the amino group which optionally also chains decreased in concentration when acted upon by a has a protected carboxyl group at position 5 on ring C , or any thermostable luciferase ( as opposed to wild - type luciferin of luciferin 6 '- methyl ether , luciferin 6 - chloroethyl ether, contamination causing the light and decreasing in concen 6 ' -deoxyluciferin , 6 '- luciferin - 4 - trifluoromethylbenzylether , tration ) . Such luciferin derivatives are also shown herein to luciferin 6 '- phenytethylether , luciferin 6 - geranyl ether, 40 be utilized by other luciferases . Moreover, those scaffolds, luciferin 6 -ethyl ether, 6 '- luciferin prenyl ether, 6 '- luciferin e. g . , luciferase substrates that have groups attached through 2 -picolinylether , 6 '- luciferin 3 - picolinylether , 6 ' - luciferin the oxygen or nitrogen on the A ring of luciferin , may be 4 -picolinyl ether , luciferin 6 -benzyl ether, D - luciferin - O -B - modified to include a substrate for an enzyme, a binding site galacto -pyranoside , D - luciferin - O - sulfate , D - luciferin - O - for another molecule , or any reactive group useful to mea phosphate , D - luciferyl- L -phenylalanine , and D -luciferyl - L - 45 sure a molecule such as a cellular bioactive molecule , N '- arginine ; or not a substrate for one or more cytochrome including second messengers, e . g . , a cAMP binding site , for P450 enzymes but optionally may be a substrate for a instance , to measure CAMP, calmodulin , e . g . , to measure non -cytochrome P450 enzyme. calcium , or to measure IP3 . In one embodiment, a bioluminescent assay method to In one embodiment. a method to detect luciferase detect one or more nonluciferase enzymes is provided . The 3050 employsemplo 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 the reaction , with a corresponding reaction mix ????IIIA ture that includes a derivative of luciferin or a derivative of aminoluciferin that is a substrate for the nonluciferase 55 enzyme. In one embodiment , the derivative is one having a 29- R modification in the benzene ring of D - luciferin that includes - A a recognition site for the nonluciferase enzyme, e . g . , for a monoamine oxidase . In another embodiment , the derivative W2 A is one having a modification in the thiazole ring ( ring B ) of 60 D - luciferin which derivative is a substrate for luciferase and optionally a substrate for a nonluciferase enzyme. In another wherein embodiment, the derivative is one having a modification in Y is N , N -oxide . N - (C2 - C . )alkyl , or CH ; the thiazole ring ( ring C ) of luciferin that includes a recog nition site for on enzyme of interest , e . g . , acetylcholinest- 65 X is S , O , CH = CH , N = CH , or CH = N ; erase . In another embodiment , the derivative is one having Z is H , OR , NHR , or NRR ; a modification to one of the rings that includes a recognition Z " is O , S , NH , NHR , or N = N ; US 10 , 077 , 244 B2 13 14 Wl is H , halo , hydroxyl, (C . -C .) alkyl , (C2 - C20 ) alkenyl, compounds may be also be used in bioluminogenic assays ( C , - C . )alkoxy ; monitoring the presence or activity of nonluciferase W ? is H , F , or methyl; enzymes or a nonenzymatic biological reaction . the dotted line in ring B is an optional double bond ; For derivatives that function directly as a substrate for each R is independently H , ( C , -C20 ) alkyl , ( C , - C20 )alk - 5 luciferase, as well as optionally a substrate of a nonlu enyl, (C2 -C20 )alkynyl , (Cz - C20 )cycloalkyl , ( C , -C12 )alkoxy , ciferase enzyme or other molecules , the derivative may be ( Co- C30 ) aryl, heteroaryl, heterocycle , ( C , -C20 )alkylsulfoxy , employed to directly detect luciferase , or a co - factor, inhibi ( C6- C30 ) arylsulfoxy , heterarylsulfoxy, (C2 - C20 Jalkylsulfo tor , or activator of the luciferase reaction . If the derivative is nyl, (Co -C30 )arylsulfonyl , heteroarylsulfonyl , ( C , -C20 ) a prosubstrate for luciferase , i. e ., the product of a reaction alkylsulfinyl, (Co - C30 )arylsulfinyl , heteroarylsulfinyl, ( C - 10 between the derivative and the nonluciferase enzyme is a C20 )alkoxycarbonyl , amino , NH ( C .- C . ) alkyl , N ( C2 -C6 ) substrate for luciferase , sequential or concurrent reactions alkyl) 2, tri (C1 - C20 )ammonium (C -C20 )alkyl , heteroaryl( C1 for the nonluciferase enzyme and the luciferase may be C20 ) alkyl, heteroaryl having quaternary nitrogen , conducted . For instance, an assay for a nonluciferase heteroarylcarbonyl having quaternary nitrogen , (Co - C30 ) enzyme that includes a luciferin derivative that is a prosub arylthio , (C1 - C20 )alkylphosphate , ( C , - C20 ) alkylphospho - 15 strate for luciferase may be conducted in a single reaction nate , ( C . -C30 ) arylphosphate , (C6 - C30 ) arylphosphonate , vessel and a beetle luciferase reaction mixture added to that phosphate , sulfate , saccharide, or M * optionally when Z " is vessel . In another embodiment, a reaction mixture for an oxygen , wherein M is an alkali metal ; assay for a nonluciferase enzyme that includes a luciferin or when Z or Z ' is NR ' R ' , R ' R together with the N to derivative that is a prosubstrate for luciferin may be con which they are attached forms a heteroaryl or heterocycle 20 ducted in a single reaction vessel and a portion of that group ; reaction added to a different vesselhaving a beetle luciferase wherein any alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy , reaction mixture . Alternatively , the nonluciferase and amino , aryl, heteroaryl, or heterocycle group is optionally luciferase reactionsmay be conducted simultaneously in the substituted with 1, 2 , 3 , 4 , or 5 substituents selected from same vessel. (C2 -C20 )alkyl , (C2 - C20 ) alkenyl , (C2 - C20 ) alkynyl , (C3 - C20 ) 25 The invention thus provides in an embodiment a method cycloalkyl, (C1 - C20 )alkoxyl , (C1 - C20 Jalkylcarbonyl, (C1 - to detect or determine the presence or amount of a molecule C20 ) alkylcarboxyl, halo , hydroxyl, COOR ", — SOR , for a nonluciferase enzyme-mediated reaction in a sample. - SO3R , nitro , amino , ( C , - C20) alkyl - S (O ) - , (C , -C20 ) The method includes contacting a sample , a first reaction alkyl- S02 — , phosphate , (C ,- C20 )alkylphosphate , (C ,- C20 ) mixture for a nonluciferase enzyme- mediated reaction , and alkylphosphonate , NH (C7 - C ) alkyl , NH ( C , - C . ) alkynyl, 30 a derivative of luciferin which is a substrate for the nonlu N ( ( C / - C . )alkyl ) 2 , N ( C - C . ) alkynyl) 2 , mercapto , ( C , -C20 ) ciferase enzyme, so as to yield a first mixture comprising a alkylthio , (Co - C30 )aryl , (Co -C30 Jarylthio , trifluoromethyl, luminogenic product that is a substrate for a luciferase . In = O , heteroaryl, and heterocycle, and each substituent is one embodiment, the derivative is a compound of formula I . optionally substituted with one to three R groups; In another embodiment, the derivative is a compound of R * is H , ( C , - C .) alkyl , or (Co - C30 )aryl ; 35 formula II . At least a portion of the first mixture is contacted when Z is OR , formula III is optionally a dimer connected with a second reaction mixture for a luciferase -mediated at the two A rings via a linker comprising a (C7 - C12Jalkyl reaction , so as to yield a second mixture . Then luminescence diradical that is optionally interrupted by one to four 0 in the second reaction is detected or determined , thereby atoms, N atoms, an optionally substituted aryl, heteroaryl, or detecting or determining the presence or amount of a mol heterocycle group , or a combination thereof, to form a 40 ecule for the nonluciferase enzyme-mediated reaction in the bridge between the dimer of formula III, and the R group of sample , e . g . , compared to a control. In one embodiment, the each Z group connecting the dimer of formula III is replaced derivative is a substrate for a transferase , e . g . glutathione S by the bridge; transferase (GST ) . Exemplary derivatives useful to detect A is an anion , present when a quaternary nitrogen is GST are shown in FIG . 12 . In some embodiments , a second present; 45 nonluciferase enzyme may be used to further chemically or salt thereof, transform the product of the first nonluciferase enzyme provided that the compound of formula III is not aminolu - mediated reaction to yield a substrate for a luciferase. For ciferin that has been modified to include a protease substrate example , the sample , nonluciferase reaction mixture or via a peptide bond at the amino group which optionally also luciferase reaction mixture may include an esterase , or the has a protected carboxyl group at position 5 on ring C , or any 50 esterase may be added separately . of luciferin 6 -methyl ether, luciferin 6 - chloroethyl ether , In one embodiment, derivatives of luciferin having on 6 ' - deoxyluciferin , 6 '- luciferin - 4 - trifluoromethylbenzylether , ester modification are employed in methods of the invention , luciferin 6 -phenylethylether , luciferin 6 '- geranyl ether , such as those to detect nonluciferase enzymes including luciferin 6 '- ethyl ether, 6 - luciferin prenyl ether, 6 ' - luciferin P450 enzymes or monoamine oxidases (MAOs ) , or other 2 - picolinylether, 6 ' - luciferin 3 - picolinylether, 6 - luciferin 55 enzymes such as flavin monoamine oxidases ( FMOs) , glu 4 -picolinyl ether , luciferin 6 '- benzyl ether , D - luciferin - O - B tathione S transferases (GSTs ) , phosphatases, e . g ., alkaline galacto - pyranoside , D - luciferin - O - sulfate , D - luciferin - o - phosphatases (AP ) , or sulfatases , as those derivatives may phosphate , D - luciferyl- L -phenylalanine , and D - luciferyl- L - have improved properties as a non luciferase enzyme sub N '- arginine ; or not a substrate for one or more cytochrome strate . In particular, as shown herein , the addition of an ester P450 enzymes but optionally may be a substrate for a 60 group at the carboxy position of the thiazole ring of a non - cytochrome P450 enzyme. luciferin derivative provided a substrate that was recognized Furthermore , the inclusion of a thiol compound with a by two P450 enzymes, 2D6 and 2C19 , that did not reactwith luciferin derivative in a luciferase - mediated assay may previously described luciferin derivatives that are substrates effectively stabilize the luminescence of the reaction , of cytochrome P450 enzymes (see U . S . published applica thereby providing for “ glow ” kinetics, i. e ., luminescent 65 tion 20040171099) . intensity of the luciferase -mediated reaction is relatively In an alternate embodiment, a method to detect or deter constant overtime after addition of the derivative. Such mine the presence or amount of a molecule for a first US 10 ,077 , 244 B2 15 16 nonluciferase enzyme-mediated reaction in a sample is a method to identify a modulator of a luciferase -mediated provided wherein the method includes contacting a sample , reaction using a luciferin derivative of the present invention . a reaction mixture for a nonluciferase -mediated enzyme The invention further provides a fluorogenic method reaction and a luciferase -mediated reaction , and a derivative which employs a derivative of a fluorophore that is modified of luciferin which is a substrate for the nonluciferase 5 to detect one or more molecules , e . g . , an enzyme, a cofactor enzyme yielding a mixture . A reaction between the nonlu for an enzymatic reaction such as ATP , an enzyme substrate , ciferase enzyme and the derivative yields a luminogenic an enzyme inhibitor, an enzyme activator, or OH radicals , or product that is a substrate for the luciferase . Luminescence in the mixture is detected or determined , thereby detecting one or more conditions, e . g ., redox conditions. The inven or determining the presence or amount of a molecule for the 10 tion thus provides for bioluminogenic and fluorogenic nonluciferase -mediated reaction in the sample . assays to detect the amount, activity or presence of a The invention also provides an embodiment directed to a molecule in a sample . method to detect the presence or amount of a non - enzymatic The invention also provides further embodiments directed molecule in a sample . The method includes contacting a to fluorogenic assays and derivatives of fluorophores which sample , a first reaction mixture for a nonenzyme -mediated dinted 15 are a substrate for a nonluciferase nonproteolytic enzyme. reaction and a derivative of luciferin which in the presence Thus, a method to detect or determine the presence or of the molecule yields a luminogenic product that is a amount of a molecule for a nonprotease enzyme -mediated substrate for a luciferase , and contacting a portion of the first reaction in a sample by a fluorescent method is provided . reaction and a second reaction mixture for a luciferase The method includes contacting a sample , a first reaction mediated reaction , to yield a second reaction , then lumines - 20 mixture for a nonluciferase , nonproteolytic enzyme- medi cence in the second reaction is detected or determined , ated reaction , and a derivative of a fluorophore which thereby detecting or determining the presence or amount of includes a substrate for the enzyme, so as to yield a first the molecule. For instance , a mixture is provided having a mixture , wherein if the molecule is present in the sample , the sample , a first reaction mixture for a nonenzyme -mediated first mixture composes a hydroxy fluorescent product . The reaction and a derivative of luciferin which in the presence 25 interaction between the enzyme and the derivative option of the molecule yields a a luminogenic product that is a ally produces an minimum and / or aldehyde intermediate substrate for a beetle luciferase. Atleast a portion of the first which optionally undergoes a noncatalytic B - elimination to mixture and a second reaction mixture for a beetle yield the hydroxy fluorescent product. Fluorescence in the luciferase -mediated reaction are mixed , to yield a second mixture is detected or determined , thereby detecting or mixture , and then luminescence in the second mixture is 30 dete detected or determined , thereby detecting or determining the determining the presence or amount of a molecule for the presence or amount of the molecule . nonprotease enzymemediated reaction in the sample . The For the biolumingenic assays described herein which invention also provides for methods of using fluorophore employ luciferin derivatives with a lower background lumi derivatives of the invention to identify inhibitor, activators , nescence than D - luciferin or aminoluciferin , those assays 35 substratesSu or co - factors for enzymatic reactions . can use lower amounts , e . g . , because small changes in The invention provides compositions or kits having one or luminescence can be detected , or higher amounts , e . g ., in more luciferin derivatives and/ or fluorophore derivatives of reactions that are improved by increased amounts of sub the invention . The kits may optionally contain other strate , of the derivative, and those derivatives may have reagents , e . g . , enzyme , reaction mixtures, and the like . The improved reactivity , e . g ., with a nonluciferase enzyme. In 40 bioluminogenic reaction mixtures , compositions and kits of addition , for any of the bioluminogenic assays described the invention may optionally include an agent that slows the herein , other reagents may be added to reaction mixtures , reaction rate, e . g ., amino methyl benzothiazole (AMBT ) or including but not limited to those that inhibit or prevent aminophenylmethyl benzothiazol (APMBT ) , see U .S . pub inactivation of luciferase , or otherwise extend or enhance lished application 20040171099 , yielding glow kinetics and / signal. 45 or an agent that stabilizes light production , e . g ., a thiol or Also provided is a method to identify a modulator of a coenzyme A . nonluciferase enzyme- mediated reaction . The method includes contacting one or more agents , a first reaction BRIEF DESCRIPTION OF THE FIGURES mixture for a nonluciferase enzyme- mediated reaction , and a derivative of luciferin which is a substrate for the nonlu - 50 FIG . 1 . Numbering of ring atoms in the six membered ring ciferase enzyme, so as to yield a first mixture , or providing (" A ring” or “ ring A ” ) , five mannered center ring (“ B ring” such a mixture . The first mixture in the absence of the one or " ring B ” ), and other five membered ring (“ C ring ” or “ ring or more agents includes a luminogenic product that is a C ” ) of beetle luciferin ( D - luciferin ) . substrate for a beetle luciferase . At least a portion of the first FIG . 2 . Exemplary luciferin derivatives for enzymes. mixture and a second reaction mixture for a beetle 55 FIG . 3 . Structures of 5 - fluoroluciferin . luciferase -mediated reaction are mixed , so as to yield a FIGS. 4A - B . Derivatives of luciferin useful as a redox second mixture . Luminescence in the second mixture is sensor. compared with a control mixture , thereby identifying FIGS . 5A - 8 . Exemplary substrates . whether one or more of the agents modulates the nonlu - FIG . 6 . Redox substrates . ciferase enzyme- mediated reaction . 60 FIGS . 7A - B . Other exemplary derivatives of luciferin . In a further embodiment of the present invention , luciferin FIG . 8A . A graphical representation of RLU for reactions derivatives that retain activity directly as a substrate for containing an esterase and a luciferin derivative, luciferin luciferase may be utilized to inactivate or inhibit the ethyl ester . 20 minute incubation at 37° C . luciferase enzyme, e .g . ,by covalentmodification thereof, or FIG . 8B . A graphical representation of RLU for reactions as a competitive or noncompetitive inhibitor thereof. This 65 containing an aminoluciferin derivative ( isopropyl urethane embodiment provides a method to screen for compounds aminoluciferin methyl ester ; 50 uM ) , with and without that prevent the inactivation of luciferase . Also provided is esterase . 20 minute incubation at room temperature . US 10 ,077 ,244 B2 17 18 FIG . 9 . A graphical representation of RLU for reactions derivative, ( N - ( 3 - chloro - 4 - ( 2 - ( dimethylamino ) - 1 -phenyl containing an esterase and a luciferin derivative , luciferin propoxy )benzyloxy )carbonyl ) -6 '- aminoluciferin ) . Reactions methyl ester. 20 minute incubation at 37° C . contained 1 pmol P450 or 5 pmol CYP19 . FIG . 10 . A graphical representation of RLU for reactions FIG . 32 . A graphical representation of RLU for reactions containing an esterase and a luciferin derivative , O - luciferin 5 containing one of a panel of P450 enzymes and a luciferin picolinyl ester. 20 minute incubation at 37° C . derivative , luciferin 4 - piperidinemethyl ether . Reactions FIG . 11 . A graphical representation of derivatives of contained 1 pmol P450 . fluorogenic derivatives useful us monoamine oxide (MAO ) FIG . 33 . A graphical representation of RLU for reactions substrates . containing one of a panel of P450 enzymes and a luciferin FIGS. 12A - B . Luciferin derivatives useful to detect glu - 10 derivative, N - ( 2 -chloroethoxycarbonyl ) - aminoluciferin . tathione S transferase (GST ) . Reactions contained 1 pmol P450 . FIGS . 13A - D . A graphical representation of derivatives of FIG . 34 . A graphical representation of RLU for reactions luciferin derivatives for MAO assays. The derivative in FIG . containing one of a panel of P450 enzymes and a luciferin 13B was not utilized by MAO . derivative, 4 ' - ( N , N - dimethylamino )methyl - 6 - ( 0 -methyl ) FIG . 14 . Derivatives of luciferin useful as flavin 15 luciferin . Reactions contained 1 pmol P450 . monooxygenase (FMO ) substrates . FIG . 35 . A graphical representation of RLU for reactions FIG . 15 . Exemplary FMO substrates . containing one of a panel of P450 enzymes and a luciferin FIG . 16 . Exemplary AP substrates . derivative, 4 - ( N , N - dimethylaminomethyl) - 6 -methoxyqui FIG . 17 . RLU for a series of luciferin derivatives and each nolyl luciferin . Reactions contained 1 pmol P450 . of a panel of P450 enzymes. 20 FIG . 36 . A graphical representation of RLU for reactions FIG . 18 . RLU for a series of luciferin derivatives and each containing one of a panel of P450 enzymes and a luciferin of a panel of P450 enzymes . derivative , luciferin 2 - ( 1 -piperazine ) ethyl ether. Reactions FIGS . 19A - 19C . Structures of exemplary derivatives use contained 1 pmol P450 . ful to detect P450 enzymes . FIG . 37A . A graphical representation of RLU for reac FIG . 20 . A graphical representation of CYP2D6 and 25 tions containing one of a panel of P450 enzymes and a CYP2C19 activity against luciferin -ME . luciferin derivative, meta- N -methyl picolinyl luciferin FIG . 21. A graphical representation of P430 activities with methyl ester . Reactions contained 1 pmol P450 . A 30 minute luciferin -ME methyl ester. incubation at 37° C . was followed by a 20 minute incubation FIG . 22 . A graphical representation of CYP2U6 activity at room temperature with or without esterase in luciferin with luciferin -ME picolinyl ester with and without esterase . 30 detection reagent . Reactions contained 1 pmol CYP2D6 . FIG . 37B . A graphical representation of RLU for reactions FIG . 23 . A graphical representation of CYP2C19 activity containing a luciferin derivative , meta - N -methyl picolinyl with luciferin - H - picolinylester with and without esterase . luciferin methyl ester , with or without esterase . 20 minute Reactions contained 1 pmol CYP2C19. incubation at room temperature . FIG . 24 . A graphical representation of RLU for reactions 35 FIG . 38 . 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 . D - luciferin ethyl ester with a C ringmodification . derivative , methamphetamine - luciferin Reactions contained Reactions contained 1 pmol P450 . 1 pmol P450 . FIG . 25 . A graphical representation of RLU for reactions FIG . 39 . A graphical representation of RLU for reactions containing one of a panel of P450 enzymes and a luciferin 40 containing one of a panel of P450 enzymes and a luciferin derivative , luciferin methyl ester with a C ring modification . derivative . N - isobutoxycarbonyl - aminoluciferin . Reactions Reactions contained 1 pmol P450 or 5 pmol CYP19 . contained 1 pmol P450 . FIG . 26 . Graphical representation of RLU for reactions FIG . 40 . A graphical representation of RLU for reactions containing one of three P450 enzymes and luciferin -ME - containing one of a panel of P450 enzymes and a luciferin ethylene glycol ester, with or without esterase . 45 derivative , luciferin - H -methyl ester and esterase . Reactions FIG . 27A . A graphical representation of RLU for an contained 1 pmol P450 . aminoluciferin derivative ( N - isopropylaminoluciferin ) (50 FIG . 41. A graphical representation of RLU for reactions uM ) in a luciferase reaction . containing one of a panel of P450 enzymes and a luciferin FIG . 27B . A graphical representation of RLU for an derivative , luciferin -ME ethylene glycolester . Reactions aminoluciferin derivative ( dimethyl aminoluciferin ; 100 50 contained 1 pmol P450 , 5 pmol CYP19 , or 40 ug HLM . uM ) and aminoluciferin ( 100 uM ) in a luciferase reaction . FIG . 42 . A graphical representation of RLU for reactions FIG . 28 . A graphical representation of signal to back containing one of a panel of P450 enzymes and a luciferin ground ratio for a reaction between 4' , 6 ' dimethyl ether derivative , methoxyquinolyl luciferin methyl ester . Reac luciferin and each of a panel of P450 enzymes. Reactions tions contained 1 pmol P450 . contained 1 pmol P450 . 55 FIG . 43 . A graphical representation of RLU for reactions FIG . 29 . A graphical representation of signal background containing one of a panel of P450 enzymes and a luciferin ratio for a reaction between a luciferin derivative , ( 2 - ( 6 - derivative, 6 '( 0 - trifluoromethylbenzyloxy ) - luciferin 1 pmol methoxyquinolin -2 -yl ) -4 , 5 -dihydrothiazole -4 -carboxylate ) P450 or 5 pmol CYP19 . and each of a panel of P450 enzymes. Reactions contained FIG . 44 . A graphical representation of RLU for reactions 1 pmol P450 . 60 containing one of a panel of P450 enzymes and a luciferin FIG . 30 . A graphical representation of RLU for reactions derivative, 6 'methyoxy - luciferin - hydrazide . Reactions con containing one of a panel of P450 enzymes and a luciferin tain 1 pmol of P450 or 5 pmol CYP19 . derivative , 2 -( 7 -methyoxy - quinoxalin - 2 -yl ) - 4 ,5 -dihydro FIG . 45 . A graphical representation of RLU for reactions thiazole - 4 -carboxylic acid . Reactions contained 1 pmol containing one of a panel of P450 enzymes and a luciferin P450 . 65 derivative , 6 -( 3 - ( (4 -phenylpiperizin - 1 -yl ) )methyl ) benzy FIG . 31 . A graphical representation of RLU for reactions loxy ) - luciferin . Reactions contained 1 pmol P450 or 20 ug containing one of s panel of P450 enzymes and a luciferin HLM . US 10 , 077 , 244 B2 19 20 FIG . 46 . A graphical representation of RLU for reactions alkenyl, alkynyl, alkoxy, halo , haloalkyl, hydroxy , hydroxy containing one of a panel of P450 enzymes and a luciferin alkyl, aryl, heteroaryl , heterocycle , cycloalkyl, alkanoyl , derivative , 6 '- ( 2 , 4 , 6 - trimethylbenzyloxy ) - luciferin . 1 pmol alkoxycarbonyl, amino , alkylamino , dialkylamino , trifluo of P450 or 5 pmol CYP19 . romethylthio , difluoromethyl, acylamino , nitro , trifluorom FIG . 47. A graphical representation of RLU for reactions 5 ethyl, trifluoromethoxy , carboxy, carboxyalkyl, keto , thioxo , containing one of a panel of P450 enzymes and a luciferin alkylthio , alkylsulfinyl, alkylsulfonyl , arylsulfinyl, arylsul derivative , 6 ' (4 -chlorophenylthio )methoxyluciferin . Reac fonyl, heteroarylsulfinyl, heteroarylsulfonyl, heterocycle tions contained 1 pmol of P450 or 5 pmol CYP19 . sulfinyl, heterocyclesulfonyl , phosphate , sulfate , hydroxyl FIG . 48. Inhibition of CYP3A4 activity by troleanomycin amine, hydroxyl ( alkyl ) amine, and cyano . Additionally , the in a reaction with luciferin - F3BE (6 -( 2 ,3 , 4, 5 ,6pentafluo - 10 suitable indir robenzyloxy ) - luciferin ) . suitable indicated groups can include, e . g . ; - X , - R , - O , FIG . 49 . Inhibition of CYP3A4 activity by nifedipine in OR , SR , S , - NR2, - NR3, = NR , CX3, CN , a reaction with luciferin - PPX4FE ( 6 - ( 2 , 3 , 4 , 6 - tetrafluoro - 5 - OCN , SCN , — N — C — O , NCS , NO , NO2, ( ( 4 - phenylpiperazin - 1 - yl) methyl ) benzyloxy ) - luciferin ) . = N2, N3, NCC= O ) R , COR, CEO) NRR FIG . 50 . Inhibition of CYP3A4 activity by nifedipine in 155 S ( 0 ) 20 - , - S ( O ) 2OH , - S ( O ) R , a reaction with luciferin -PPXE ( 6 - (( 4 -phenylpiperazin - 1- yl ) - OS ( O ) 2OR , S ( 0 ) 2NR , S ( O ) R , OPO ) methyl)benzyloxy ) - luciferin . O , RR , — P ( O ) O , RR — P ( O ) ( 0 ) 2 , — P ( O ) (OH ) 2 , FIG . 51 . CYP3A4 activity in human hepatocytes hepato - CIOR , - CEO) X , - C ( S ) R , - C ( O )OR , - C ( O ) O - , cytes after treatment with rifampicin , ketoconazole , or C ( S )OR , - C (O )SR , C ( O )SR , C ( S ) SR , C ( O ) rifampicin and ketoconazole , in a reaction with luciferin - 20 NRR , — C ( S )NRR , - C (NR )NRR , where each X is inde PPXE . pendently a halogen (“ halo ” ) : F , CI, BR , or I; and each R is FIG . 52 . CYP3A4 activity in human hepatocytes after independently H , alkyl, aryl, heteroaryl, heterocycle , a pro treatment with rifampicin , ketoconazole , or rifampicin and tecting group or prodrug moiety . As would be readily ketoconazole , in a reaction with luciferin - F -BE . understood by one skilled in the art , when a substituent is FIG . 53 . Luciferin derivatives which may be luciferase 25 keto ( O ) or thioxo ( S ) , or the like , then two hydrogen substrates: N - isopropyl aminoluciferin bisethyl aminolu - atoms on the substituted atom are replaced . ciferin ; and benzylaminoluciferin . " Stable compound ” and “ stable structure ” are meant to FIG . 54 . Structures of derivatives of luciferin which may indicate a compound that is sufficiently robust to survive be a substrate for different luciferases . isolation to a useful degree of purity from a reaction mixture . FIG . 55 . A graphical representation of RLU for luciferin 30 Only stable compounds are contemplated by and claimed in or 5 - fluoroluciferin in luciferase reactions at different pHs. the present invention , however , certain unstable compounds , FIG . 56 . ATP titration of a 5 ' fluoroluciferin and luciferin . for example , those that cannot easily be isolated , can be FIG . 57 . ATP titration of a 7 ' fluoroluciferin . employed in the methods described herein . FIG . 58 . Spectra of various fluoroluciferins . One diastereomer may display superior properties or FIG . 59 . pH titration of a 7 ' fluoroluciferin . 35 activity compared with another. When required , separation FIG . 60 . Luciferin derivatives useful as luciferase sub - of the racemic material can be achieved by HPLC using a strates and scaffolds for luciferin derivatives useful as non chiral column or by a resolution using a resolving agent such luciferase substrates. as camphonic chloride as described by Thomas S . Tucker , et al. , J . Med. Chem . 1994 , 37 ,2437 - 2444 . A chiral compound DETAILED DESCRIPTION OF THE 40 may also be directly synthesized using a chiral catalyst or a INVENTION chiral ligand , e . g . Mark A . Huffman , et al. , J . Org . Chem . 1995 , 60 , 1590 - 1594 . I. Definitions As used herein , the term “ alkyl” refers to a branched , unbranched , or cyclic hydrocarbon having , for example , As used herein , the following termsand expressions have 45 from 1 to 30 carbon atoms, and often 1 to 12 , or 1 to about the indicated meanings . It will be appreciated that the 6 carbon atoms. Examples include , but are not limited to , compounds of the present invention contain asymmetrically methyl, ethyl, 1 - propyl, 2 -propyl , 1 -butyl , 2 -methyl - 1 - pro substituted carbon atoms, and may be isolated in optically pyl, 2 -butyl , 2 -methyl - 2 - propyl ( t -butyl ) , 1 -pentyl , 2 -pentyl , active or racemic forms. It is well known to the art how to 3 -pentyl , 2 -methyl - 2 -butyl , 3 -methyl - 2 - butyl , 3 -methyl - 1 prepare optically active forms , such as by resolution of 50 butyl , 2 -methyl - 1 -butyl , 1 - hexyl , 2 - hexyl, 3 -hexyl , racemic forms or by synthesis from optically active starting 2 -methyl - 2 - pentyl, 3 -methyl - 2 -pentyl , 4 -methyl - 2 -pentyl , materials . All chiral, diastereomeric , racemic forms and all 3 -methyl - 3 -pentyl , 2 -methyl - 3 -pentyl , 2 ,3 -dimethyl - 2 -bu geometric isomeric forms of a structure are part of this tyl, 3 ,3 -dimethyl - 2- butyl , hexyl, octyl, decyl, dodecyl , and invention . the like . The alkyl can be unsubstituted or substituted . The Specific values listed below for radicals , substituents , and 55 alkyl can also be optionally partially or fully unsaturated . As ranges, are for illustration only ; they do not exclude other such , the recitation of an alkyl group includes both alkenyl defined values or other values within defined ranges for the and alkynyl groups. The alkyl can be a monovalent hydro radicals and substituents . carbon radical, as described and exemplified above , or it can As used herein , the term " substituted ” is intended to be a divalent hydrocarbon radical ( i . e . , alkylate ) . indicate that one or more ( e . g ., 1 , 2 , 3 , 4 , or 5 ; in some 60 The term " alkenyl” refers to a monoradical branched or embodiments 1 , 2 , or 3 ; and in other embodiments 1 or 2 ) unbranched partially unsaturated hydrocarbon chain ( i . e . a hydrogens on the group indicated in the expression using carbon -carbon , sp² double bond ) . In one embodiment, an " substituted ” is replaced with a selection from the indicated alkenyl group can have from 2 to 10 carbon atoms, or 2 to group ( s ) , or with a suitable group known to those of skill in 6 carbon atoms. In another embodiment, the alkenyl group the art , provided that the indicated atom ' s normal valency is 65 has from 2 to 4 carbon atoms Examples include , but are not not exceeded , and that the substitution results in a stable limited to , ethylene or vinyl, allyl, cyclopentenyl, 5 - hexenyl , compound . Suitable indicated groups include , e . g ., alkyl, and the like . The alkenyl can be unsubstituted or substituted . US 10 ,077 , 244 B2 21 22 The term “ alkynyl” refers to a monoradical branched or but are not limited to , 2H -pyrrolyl , 3H -indolyl , 4H -quino unbranched hydrocarbon chain , having a point of complete lizinyl, acridinyl, benzo [ b ] thienyl, benzothiazolyl , ß -car unsaturation ( i. e . a carbon -carbon , sp triple bond ). In one bolinyl, carbazolyl, chromenyl, cinnolinyl, dibenzo [ b ,d ] embodiment, the alkynyl group can have from 2 to 10 furanyl, furazanyl, furyl, imidazolyl, imidizolyl, indazolyl , carbon atoms, or 2 to 6 carbon atoms. In another embodi - 5 indolisinyl, indolyl, isobenzofuranyl, isoindolyl , isoqui ment, the alkynyl group can have from 2 to 4 carbon atoms. nolyl, isothiazolyl, isoazolyl, naphthyridinyl, oxazolyl, per This term is exemplified by groups such as ethynyl, 1 - pro imidinyl , phenanthridinyl, phenanthrolinyl, phenarsazinyl, pynyl, 2 - propynyl, 1 - butynyl , 2 - butynyl, 3 - butynyl, 1 -hexy phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, nyl, 2 -hexynyl , 3- hexynyl , 1 -octynyl , and the like . The phthalazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyra alkynyl can be unsubstituted or substituted . 10 zolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrimidinyl, pyr The term " cycloalkyl” refers to cyclic alkyl groups of roloyl, quinazolinyl, quinolyl, quinoxalinyl, thiadiazolyl , from 3 to 10 carbon atoms having a single cyclic ring or thianthrenyl, thiazolyl , thienyl , triazolyl, tetrazolyl, and xan multiple condensed rings . Such cycloalkyl groups include thenyl. In one embodiment the term “ heteroaryl” denotes a by way of example , single ring structures such as cyclopro monocyclic aromatic ring containing five or six ring atoms pyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like , or 15 containing carbon and 1, 2 , 3 , or 4 heteroatoms indepen multiple ring structures such as adamantanyl, and the like . dently selected from non - peroxide , oxygen , sulfur, and N ( Z ) The cycloalkyl can be unsubstituted or substituted . The wherein Z is absent or is H , O , alkyl, aryl, or ( C , - Co ) cycloalkyl group can be monovalent or divalent, and can be alkylaryl. In another embodiment heteroaryl denotes an optionally substituted as described above for alkyl groups . ortho - fused bicyclic heterocycle of about eight to ten ring The cycloalkyl group can optionally include one or more 20 atoms derived therefrom , particularly a benz -derivative or cites of unsaturation , for example , the cycloalkyl group can one derived by fusing a propylene , trimethylene , or tetram include one or more carbon - carbon double bonds, such as, ethylene diradical thereto . for example , cyclohexene , 1, 3 -cyclohexadiene , 1, 4 -cyclo The term " heterocycle ” refers to a saturated or partially hexadiene , and the like. unsaturated ring system , containing at least one heteroatom The term “ alkoxy ” refers to the group alkyl - 0 — , where 25 selected from the group oxygen , nitrogen , and sulfur, and alkyl is as defined herein . In one embodiment, alkoxy groups optionally substituted with one or more groups as defined include , e . g ., methoxy , ethoxy , n - propoxy , iso - propoxy , herein under the term “ substituted ” . A heterocycle can be a n -butoxy , sec butoxy , n - pentoxy, n -hexoxy , 1 , 2 -dimethylbu - monocyclic , bicyclic , or tricyclic group containing one or toxy , and the like. The alkoxy can be unsubstituted or more heteroatoms. A heterocycle group also can contain an substituted . 30 Oxo group BO( ) or a thioxo group attached to the ring . As used herein , " aryl” refers to an aromatic hydrocarbon Non - limiting examples of heterocycle groups , include 1 , 3 group derived from the removal of one hydrogen atom from dihydrobenzofuran , 1 , 3 -dioxolane , 1 , 4 -dioxane , 1 , 4 -dithi a single carbon atom of a parent aromatic ring system . The ane , 2H -pyran , 2 - pyrazoline , 4H -pyran , chromanyl, imida radical can be at a saturated or unsaturated carbon atom of zolidinyl, imidazolinyl, indolinyl, isochromanyl, the parent ring system . The aryl group can have from 6 to 30 35 isoindolinyl , morpholine . piperazinyl, piperidine, piperidyl , carbon atoms. The aryl group can have a single ring ( e. g ., pyrazolidino , pyrazolidinyl, pyrazolinyl , pyrrolidine, pyrro phenyl) or multiple condensed ( fused ) rings, wherein at least line , quinuclidine , and thiomorpholine . one ring is aromatic ( e .g ., naphthyl , dihydrophenanthrenyl, The term " heterocycle ” can include, by way of example fluorenyl , or anthryl) . Typical aryl groups include , but are and not limitation , a monoradical of the heterocycles not limited to , radicals derived from benzene, naphthalene, 40 described in Paquette , Leo A . ; Principles of Modern Het anthracene , biphenyl, and the like . The aryl can be unsub - erocyclic Chemistry ( W . A . Benjamin , New York , 1968 ) , stituted or optionally substituted , as described above for particularly Chapters 1 , 3 , 4 ,6 , 7 , and 9 ; “ The Chemistry of alkyl groups . Heterocyclic Compounds A Series of Monographs” (John The term “ halo ” refers to fluoro , chloro , bromo, and iodo . Wiley & Sons, New York , 1950 to present) , in particular Similarly , the term " halogen ” refers to fluorine , chlorine, 45 Volumes 13 , 14 , 16 , 19 , and 28 ; and J . Am . Chem . Soc. 1960 , bromine , and iodine . 82 , 5566 . In one embodiment, " heterocycle ” includes a The term “ haloalkyl” refers to alkyl as defined herein " carbocycle ” as defined herein , wherein one or more ( e . g . 1 , substituted by 1 or more halo groups as defined herein , 2 , 3 , or 4 ) carbon atoms have been replaced with a heteroa which may be the same or different. In one embodiment, the tom ( e . g . O , N , or S ) . haloalkyl can be substituted with 1 , 2 , 3 , 4 , or 5 halo groups . 50 Examples of heterocycles , by way of example and not In another embodiment, the haloalkyl can by substituted limitation , include , dihydroypyridyl, tetrahydropyridyl (pip with 1 , 2 , or 3 halo groups. The term haloalkyl also include eridyl) , thiazolyl , tetrahydrothiophenyl, sulfur oxidized tet perfluoro -alkyl groups. Representative haloalkyl groups rahydrothiophen , pyrimidinyl, furanyl, thienyl, pyrrolyl, include , by way of example , trifluoromethyl, 3 - fluorodode pyrazolyl , piperidinyl, 4 -piperidonyl , pyrrolidinyl, 2 -pyr cyl , 12 , 12 , 12 -trifluorododecyl , 2 - bromooctyl , 3 -bromo - 6 - 55 rolidonyl , pyrrolinyl, tetrahydrofuranyl, tetrahydroquinoli chloroheptyl, 1H , 1H - perfluorooctyl, and the like . The nyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahy haloalkyl can be optionally substituted as described above droisoquinolinyl, azocinyl, triazinyl , 6H - 1 ,2 ,5 -thiadiazinyl , for alkyl groups. 2H ,6H - 1 , 5 , 2 - dithiazinyl, thienyl, thianthrenyl, pyranyl, The term " heteroaryl” is defined herein as a monocyclic , isobenzofuranyl, chromenyl, xanthenyl, phenoxathinyl, bicyclic , or tricyclic ring system containing one , two , or 60 2H -pyrrolyl , isothiazolyl , isoxazolyl, pyrazinyl, pyridazinyl, three aromatic rings and containing at least one nitrogen , indolizinyl, isoindolyl, 3H - indolyl, 1H - indazoly , purinyl, oxygen , or sulfur atom in an aromatic ring , and that can be 4H - quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl , unsubstituted or substituted , for example , with one or more , quinazolinyl, cinnolinyl, pteridinyl, carbazolyl, ß - carboli and in particular one to three , substituents , as described nyl , phenanthridinyl, acridinyl, pyrimidinyl, phenanthroli above in the definition of “ substituted ” . Typical heteroaryl 65 nyl , phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, groups contain 2 - 20 carbon atoms in addition to the one or isochromanyl, chromanyl, imidazolidinyl, imidazolinyl, more heteroatoms. Examples of heteroaryl groups include , pyrazolidinyl , pyrazolinyl, piperazinyl, indolinyl , isoindoli US 10 ,077 ,244 B2 23 24 nyl, quinuclidinyl, morpholinyl, oxazolidinyl , benzotriaz octahydroindole - 2 - carboxylic acid , statine, 1 , 2 , 3 , 4 - tetrahy olyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, isatinoyl, droisoquinoline - 3 - carboxylic acid , penicillamine, ornithine , and bis - tetrahydrofuranyl. citruline , a -methyl - alanine, para - benzoylphenylalanine , By way of example and not limitation , carbon bonded phenylglycine , propargylglycine , sarcosine , and tert -butyl heterocycles are bonded at position 2 , 3 , 4 , 5 , or 6 of a 5 glycine ) The term also comprises natural and unnatural pyridine , position 3 , 4 , 5 , or 6 of a pyridazine, position 2 , 4 , amino acids hearing a conventional amino protecting group 5 , or 6 of a pyrimidine , position 2 , 3 , 5 , or 6 of a pyrazine , (e . g . acetyl or benzyloxycarbonyl) , as well as natural and position 2 , 3 , 4 , or 5 of a furan , tetrahydrofuran , thiofuran , unnatural amino acids protected at the carboxy terminus thiophene , pyrrole or tetrahydropyrrole, position 2 , 4 , or 5 of ( e . g . as a ( C , - C Jalkyl, phenyl or benzyl ester or amide ; or an oxazole , imidazole or thiazole , position 3 , 4 , or 5 of an 10 as an a -methylbenzyl amide ) . Other suitable amino and isoxazole , pyrazole , or isothiazole , position 2 or 3 of an carboxy protecting groups are known to those skilled in the aziridine position 2 , 3 , or 4 of an azetidine, position 2 , 3 , 4 , art (See for example . Greene, T . W . ; Wutz , P . G . M . 5 , 6 , 7 , or 8 of a quinoline or position 1, 3, 4 , 5 , 6 , 7 , or 8 Protecting Groups In Organic Synthesis . 2nd edition , John of an isoquinoline . Carbon bonded heterocycles include Wiley & Sons, Inc ., New York ( 1991) and references cited 2 -pyridyl , 3 -pyridyl , 4 -pyridyl , 5 -pyridyl , 6 -pyridyl , 15 therein ). 3 -pyridazinyl , 4 -pyridazinyl , 5 -pyridazinyl , 6 -pyridazinyl , The term “ peptide” describes a sequence of 2 to 35 amino 2 - pyrimidinyl, 4 -pyrimidinyl , 5 -pyrimidinyl , 6 -pyrimidinyl , acids ( e . g . as defined hereinabove) or peptidyl residues . The 2 -pyrazinyl , 3 -pyrazinyl , 5 -pyrazinyl , 6 - pyrazinyl, 2 -thiaz - sequence may be linear or cyclic . For example , a cyclic olyl, 4 - thiazolyl, 5 - thiazolyl, and the like . peptide can be prepared ormay result from the formation of By way of example and not limitation , nitrogen bonded 20 disulfide bridges between two cysteine residues in a heterocycles can be bonded at position 1 of an aziridine , sequence . Preferably a peptide comprises 3 to 20 , or 5 to 15 azetidine, pyrrole , pyrrolidine, 2 -pyrroline , 3 -pyrroline , imi amino acids. Peptide derivatives can be prepared as dis dazole , imidazolidine, 2 - imidazoline, 3 -imidazoline , pyra closed in U .S . Pat . Nos. 4 ,612 , 302 ; 4 ,853 ,371 ; and 4 ,684 , zole , pyrazoline, 2 -pyrazoline , 3 -pyrazoline , piperidine , pip - 620 , or as described in the Examples herein below . Peptide erazine , indole , indoline , 1H - indazole , position 2 of a 25 sequences specifically recited herein are written with the isoindole , or isoindoline , position 4 of a morpholine , and amino terminus on the left and the carboxy terminus on the position 9 of a carbazole , or B - carboline . In one embodi- right . ment, nitrogen bonded hetetocycles include 1 - aziridyl, The term " saccharide” refers to a sugar or other carbo 1 -azetedyl , 1 - pyrrolyi, 1 - imidazolyl, 1 -pyrazolyl , and 1 - pi- hydrate , especially a simple sugar . The saccharide can be a peridinyl. 30 Co - polyhydroxy compound , typically Co - pentahydroxy , and The term “ carbocycle” refers to a saturated , unsaturated or often a cyclic glycal. The term includes the known simple aromatic ring having 3 to 8 carbon atoms as a monocycle , 7 sugars and their derivatives, as well as polysaccharides with to 12 carbon atoms as a bicycle , and up to about 10 carbon two or more monosaccaride residues . The saccharide can atoms as a polycycle . Monocyclic carbocycles typically include protecting groups on the hydroxyl groups, as have 3 to 6 ring atoms, still more typically 5 or 6 ring atoms. 35 described above in the definition of amino acids . The Bicyclic carbocycles have 7 to 12 ring atoms, e . g ., arranged hydroxy groups of the saccharide can be replaced with one as a bicyclo [4 , 5 ] , [5 , 5 ] , [ 5 ,6 ] or [6 , 6 ] system , or 9 or 10 ring or more halo or amino groups. Additionally , one or more of atoms arranged as a bicyclo [ 5 , 6 ] or [ 6 , 6 ] system . Examples the carbon atoms can be oxidized , for example to keto or of carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, carboxyl groups. 1 - cyclopent - 1- enyl, 1 -cyclopent - 2 -enyl , 1 -cyclopent - 3 -enyl , 40 The term “ interrupted ” indicates that another group is cyclohexyl, 1 - cyclohex - 1 - enyl , 1 - cyclohex - 2 -enyl , 1 - cyclo - inserted between two adjacent carbon atoms ( and the hydro hex - 3 - enyl, phenyl, spiryl and naphthyl. The carbocycle can gen atoms to which they are attached ( e . g ., methyl (CH3 ) be optionally substituted as described above for alkyl (methylene (CH2 ) or methine (CH ) ) ) of a particular carbon groups . chain being referred to in the expression using the term The term " alkanoyl” or “ alkylcarboxy ” refers to — 0 C 45 “ interrupted , provided that each of the indicated atoms” ( O ) R , wherein R is an alkyl group as previously defined . normal valency is not exceeded , and that the interruption The term “ acyloxy ” or “ alkylcarboxy” refers to 40C results in a stable compound . Suitable groups that can GOR , wherein R is an alkyl group as previously defined . interrupt a carbon chain include, e . g ., with one or more Examples of acyloxy groups include , but are not limited to , non - peroxide oxy ( 0 - ) , thio ( - 5 ) , imino ( N ( H ) — ) , acetoxy , propanoyloxy , butanoyloxy, and pentanoyloxy . Any 50 methylene dioxy ( OCH , 0 ) , carbonyl ( CFO) ) , alkyl group as defined above can be used to form an acyloxy carboxy ( 40 ) O ) , carbonyldioxy GOCO ) , group . Carlcarboxylato ( OCCO) — ) , imine ( C = NH ), sulfinyl (SO ) The term “ alkoxycarbonyl” refers to — CEO )OR ( or and sulfonyl (SO2 ) . Alkyl groups can be interrupted by one “ COOR ” ) , wherein R is an alkyl group as previously ore more ( e . g . , 1 , 2 , 3 , 4 , 5 , or about 6 ) of the aforementioned defined 55 suitable groups . The site of interruption can also be between The term “ amino ” refers to — NH ,. The amino group can a carbon atom of an alkyl group and a carbon atom to which be optionally substituted as defined herein for the term the alkyl group is attached . " substituted ” . The term “ alkylamino ” refers to — NR ; As to any of the above groups , which contain one or more wherein at least one R is alkyl and the second R is alkyl or substituents , it is understood , of course, that such groups do hydrogen . The term “ acylamino ” refers to N ( R ) CEOR , 60 not contain any substitution or substitution patterns that are wherein each R is independently hydrogen , alkyl, or aryl. sterically impractical and /or synthetically non - feasible . In The term “ amino acid ,” includes a residue of a natural addition , the compounds of this invention include all ste amino acid ( e . g . Ala , Arg , Asn , Asp , Cys, Glu ,Gln , Gly , His , reochemical isomers arising from the substitution of these Hyl , Hyp , He, Leu , Lys , Met , Phe , Pro , Ser , Thr, Trp , Tyr, compounds. and Val ) in D or L form , as well as unnatural amino acids 65 Selected substituents within the compounds described ( e . g . phosphoserine , phosphothreonine, phosphotyrosine , herein are present to a recursive degree . In this context, hydroxyproline, gamma- carboxyglutamate ; hippuric acid , “ recursive substituent” means that a substituent may recite US 10 ,077 ,244 B2 25 26 another instance of itself . Because of the recursive nature of may contain reagents for the reaction except for a substrate such substituents, theoretically , a large number may be for the luciferase , e . g ., a reaction mixture useful to deter present in any given claim . One of ordinary skill in the art mine whether a test sample has a luciferase substrate. A of medicinal chemistry and organic chemistry understands reaction mixture for a nonluciferase enzyme may include all that the total number of such substituents is reasonably 5 reagents for that reaction except for a molecule to be limited by the desired properties of the compound intended detecteddet , e . g . , the mixture contains all reagents except for a Such properties include , by of example and not limitation , cofactor for the nonluciferase enzyme, and so the mixture is physical properties such as molecular weight, solubility or log P , application properties such as activity against the useful to detect the presence of the cofactor in a test sample . intended target , and practical properties such as ease of 10 As used herein a “ derivative of luciferin ” or a “ derivative synthesis . of aminoluciferin ” is a molecule that is a substrate for a Recursive substituents are an intended aspect of the nonluciferase enzyme and a prosubstrate of a luciferase , a invention . One of ordinary skill in the art of medicinal and substrate for a luciferase , a substrate for a nonluciferase organic chemistry understands the versatility of such sub enzyme and a substrate for a luciferase , or is useful to detect stituents . To the degree that recursive substituents are pres - 15is molmolecules generated in nonenzymatic reactions. The deriva ent in an claim of the invention , the total number will be tives of the invention have one or more modifications to one determined as set forth above . ormore of the three rings and / or substituents attached to one The term " linker” as used herein is a carbon chain that or more of the rings of the D - luciferin or aminoluciferin covalently attaches two chemical groups together and backbone (see FIG . 1 ) . optionally can self- cleave or if covalently bonded to a 20 A “ fluorogenic assay ” or “ fluorogenic reaction ” includes substrate for an enzyme, may be cleaved by that enzyme or a reaction in which a product of a reaction between a another molecule, which chain is optionally interrupted by nonluciferase, nonproteolytic enzyme and a derivative of a one or more nitrogen atoms, oxygen atoms, carbonyl groups, fluorophore is fluorescent. A “ fluorogenic assay reagent ” ( substituted ) aromatic rings , or peptide bonds . may include a substrate , as well as a cofactor( s ) or other The term “ luciferase , " unless specified otherwise , refers 25 molecule ( s ) such as a protein , e . g . , an enzyme, for a fluo to a naturally occurring or mutant luciferase . The luciferase , rogenic reaction . Thus , the invention provides a method to if naturally occurring , may be obtained easily by the skilled detect or determine the presence or amount of a molecule for from an organism . If the luciferase is one that occurs a nonprotease enzyme- mediated reaction in a sample . naturally or is a mutant, which retains activity in the luciferase - luciferin reaction , of a naturally occurring 30 II. Methods of the Invention luciferase , it can be obtained readily from a culture of bacteria , yeast , mammalian cells , insect cells , plant cells , or The invention provides a bioluminogenic or fluorogenic the like, transformed to express a cDNA encoding the method which employs a derivative of luciferin or amino luciferase , or from an in vitro cell - free system for making luciferin or a derivative of a fluorophore to detect one or the luciferase from a nucleic acid encoding same. 35 more molecules , e . g . , an enzyme, a cofactor for an enzy Luciferases are available from Promega Corporation . Madi - matic reaction such as ATP, an enzyme substrate , an enzyme son , Wis . inhibitor, an enzyme activator, or OH radicals , or one or As used herein , a “ fluorophore” includes a molecule more conditions, e. g ., redox conditions. The invention thus which is capable of absorbing energy at a wavelength range provides for bioluminogenic and fluorogenic assays to detect and releasing energy at a wavelength range other than the 40 the amount, activity or presence of a molecule in a sample . absorbance range . The term “ excitation wavelength ” refers The methodsmay be used , for example , to determine the to the range of wavelengths at which a fluorophore absorbs presence or amount of at least one molecule , e . g . , a nonlu energy . The term “ emission wavelength ” refers to the range ciferase enzyme, a regulator of a nonluciferase enzyme, a of wavelengths that the fluorophore releases energy or nonluciferase enzyme substrate , and / or cofactors of the fluoresces . 45 reaction , or a condition in a sample including but not limited As used herein , a “ bioluminogenic assay " or " biolumi- to an animal, e . g . , vertebrate , physiological fluid , e . g ., blood , nogenic reaction ” includes a reaction in which a product of plasma, urine , mucous secretions and the like , a cell, cell a reaction between a nonluciferase enzyme and a derivative lysate , cell supernatant , or purified fraction of a cell ( e . g . , a of luciferin or aminoluciferin is a substrate of luciferase or subcellular fraction ) . In one embodiment, the methods a product of a nonenzymatic reaction having a derivative of 50 according to the present invention provide a rapid method luciferin or aminoluciferin is a substrate for luciferase , or a for detecting one or more molecules in a single sample such reaction between a luciferase and a derivative of luciferin or a s an aliquot of cells or a lysate thereof. In one embodiment , aminoluciferin , is bioluminogenic , i. e . , produces a measur the method includes quantifying the presence , amount or able amount of light. specific activity of a molecule such as an enzyme, substrate As used herein , " bioluminescence ” is light produced as a 55 or cofactor in a bioluminogenic assay or quantifying the result of a reaction between an enzyme and a substrate that presence or amount of an enzyme, substrate or cofactor in a generates light. Examples of such enzymes (bioluminescent fluorogenic assay . The intensity of the bioluminogenic or enzymes ) include firefly luciferase , click beetle luciferase . fluorogenic signal is a function of the presence or amount of Renilla luciferase , cypridina luciferase , Aequorin photopro the respective molecule . In addition , the reaction may con tein , obelin photoprotein and the like . 60 tain one or more test agents . e . g . , enzyme inhibitors or As used herein , a “ bioluminogenic assay reagent” activators , and / or different concentrations of inhibitors or may include a substrate , as well as a cofactor ( s ) or other activators . In one embodiment, the method employs at least molecule ( s ) such as a protein , e . g . , an enzyme, for a biolu two different reactions, where the first reaction is a nonlu minogenic reaction . ciferase enzyme- mediated reaction and the second reaction A “ reaction mixture ” may contain all reagents for a 65 is a beetle luciferase -mediated reaction . In another embodi particular reaction , or may lack at least one of the reagents ment, the first reaction is a nonenzymatic reaction and the for the reaction . For example , a luciferase reaction mixture second reaction is a beetle luciferase -mediated reaction . In US 10 ,077 , 244 B2 27 28 yet another embodiment , the method employs a single ciferase enzyme- mediated reaction in a sample . The method reaction , e . g . , a beetle luciferase -mediated reaction or a includes contacting a sample , a first reaction mixture for a fluorogenic reaction . nonluciferase enzyme -mediated reaction , and a derivative of Thus, a bioluminogenic assay may directly or indirectly luciferin which is a substrate for the nonluciferase enzyme, detect , e . g . , measure , the amount, presence , or specific 5 so as to yield a first mixture or providing such a first mixture activity of, for example , a cofactor for an enzyme -mediated comprising a luminogenic product that is a substrate for a reaction , and enzyme, an enzyme substrate , an inhibitor of luciferase , or providing such a first mixture . In one embodi the enzyme, an activator of the enzyme, or a condition . For ment, the derivative is a compound of formula I . In another instance , in one embodiment, a beetle luciferase and a embodiment, the derivative is a compound of formula II. At derivative of luciferin that is a substrate of the beetle 10 least a portion of the first mixture is contacted with a second luciferase may be employed in a bioluminogenic assay to reaction mixture for a beetle luciferase -mediated reaction , so detect ATP concentration . In another embodiment, a deriva - as to yield a second mixture . Then luminescence in the tive of luciferin which is a substrate for a nonluciferase second mixture is detected or determined , thereby detecting enzyme, for instance , a derivative which is a substrate of a or determining the presence or amount of a molecule for the monoamine oxidase , yields a product which is a substrate for 15 nonluciferase enzyme- mediated reaction in the sample . In a beetle luciferase , and so may be employed in a biolumi- some embodiments, the nonluciferase reaction mixture or nogenic assay to detect the oxidase . In one embodiment, the luciferase reaction mixture may include an esterase , e . g . , if derivative is a prosubstrate of a beetle luciferase , which the product of the reaction between the derivative and the yields a product that is a substrate of luciferase but does not nonluciferase enzyme has an ester group and that product is itself yield a substantial amount of light in a reaction with 20 a proluciferase substrate . The esterase may be included with the beetle luciferase . In some embodiments, the derivative is the first reaction mixture , added prior to initiation of the a substrate for a nonluciferase enzyme or useful to detect luciferase reaction mixture , or included in the luciferase another molecule , and a substrate for luciferase which yields reaction mixture . In some embodiments , e. g. , a derivative a substantial amount of light. In this embodiment , the with a picolinyl ester , the product of the reaction between the derivative is altered by luciferase but is generally inefficient 25 derivative and the nonluciferase enzyme is a substrate for in a light generating reaction . luciferase in the absence of an exogenously added esterase . In one embodiment, the invention provides a biolumines - In one embodiment, derivatives of luciferin having an ester cent assay method to detect one or more nonluciferase modification are employed in methods of the invention , such enzymes . The method includes contacting a sample sus - as those to detect nonluciferase enzymes including pected of having one or more nonluciferase enzymes , or a 30 cytochrome P450 enzymes, as those derivatives may have substrate or a co -factor for the nonluciferase -mediated reac - improved properties as a nonluciferase substrate . Although tion , with a corresponding reaction mixture that includes a not intending to be bound by any mechanism , the inclusion derivative of luciferin or a derivative of aminoluciferin that of the ester modification at position 5 in ring C may block is a substrate for the nonluciferase enzyme. In one embodi - negative charges or add a lipophilic quality to the derivative , ment, the derivative is one having a modification in the A 35 rendering it an improved substrate . ring of D - luciferin that includes a recognition site for the Further provided is a method to detect or determine the nonluciferase enzyme , e . g . , for a phosphatase . In another presence or amount of a molecule for a nonluciferase embodiment, the derivative is one having a modification in enzyme -mediated reaction in a sample . The method includes the B ring of D - luciferin which derivative is a substrate for contacting a sample , a reaction mixture for a nonluciferase luciferase or a prosubstrate for luciferase . In another 40 mediated enzyme reaction and for a luciferase -mediated embodiment, the derivative is one having a modification in reaction , and a derivative of luciferin which is a substrate for the C ring of luciferin that includes a recognition site for an the nonluciferase enzyme, yielding a mixture . A reaction enzyme of interest, e . g . , acetylcholinesterase . In another between the nonluciferase enzyme and the derivative yields embodiment, the derivative is one having a modification in a luminogenic product that is a substrate for the luciferase . one of the rings that includes a recognition site for the 45 In one embodiment, the derivative is a compound of formula enzyme of interest , as well as a further modification in that I. In another embodiment, the derivative is a compound of ring or one or more of the other rings . formula II . Luminescence in the mixture is detected or For derivatives that are a substrate for luciferase , as well determined , thereby detecting or determining the presence as optionally a substrate of a nonluciferase enzyme or other or amount of a molecule for the nonluciferase -mediated molecules , the derivative may be employed to detect 50 reaction in the sample . luciferase , or a co - factor , inhibitor, or activator of the The invention further provides a method to detect or luciferase reaction . If the derivative is a prosubstrate for determine the presence or amount of a molecule for a luciferase , i . e . , the product of a reaction between the deriva - luciferase -mediated reaction in a sample . The method tive and the nonluciferase enzyme is a substrate for includes contacting a sample , a reaction mixture for a beetle luciferase , sequential or concurrent reactions for the nonlu - 55 luciferase , and a derivative of luciferin which is a substrate ciferase enzyme and the luciferase may be conducted . For for the luciferase , to yield a reaction . instance , a reaction for a nonluciferase enzyme that contains The invention also provides a method to detect the the prosubstrate may be conducted in a single well and a presence or amount of a molecule in a sample . The method beetle luciferase reaction mixture added to that well . In includes contacting a sample, a first reaction mixture for a another embodiment, a reaction mixture for a nonluciferase 60 nonenzyme- mediated reaction and a derivative of luciferin enzyme that contains the prosubstrate is conducted in a which in the presence of the molecule yields a luminogenic single well and a portion of that reaction added to a different product that is a substrate for a luciferase , and then contact well having a beetle luciferase reaction mixture . Alterna - ing at least a portion of the first reaction and a second tively, reactions may be conducted simultaneously in the reaction mixture for a luciferase -mediated reaction , to yield same well . 65 a second reaction . Luminescence in the second reaction is The invention thus provides a method to detect or deter - detected or determined , thereby detecting or determining the mine the presence or amount of a molecule for a nonlu - presence or amount of the molecule . For instance , a mixture US 10 , 077 , 244 B2 29 30 is provided having a sample , a first reaction mixture for a not metabolized would show equal potency, irrespective of nonenzyme -mediated reaction and a derivative of luciferin the time of addition of the substrate . which in the presence of the molecule yields a a luminogenic In one aspect of the invention , the compound is preferably product that is a substrate for a beetle luciferase . At least a contacted first with the enzyme for a first predetermined portion of the first mixture and a second reaction mixture for 5 time period . Thereafter, the mixture is contacted with a a beetle luciferase -mediated reaction are mixed , to yield a luciferin derivative and bioluminescent enzyme, e . g ., second mixture , and then luminescence in the second mix luciferase , simultaneously or contemporaneously , and the mixture is allowed to incubate for a second predetermined ture is detected or determined , thereby detecting or deter time period . mining the presence or amount of the molecule . ch 10 In another aspect of the invention , the compound is For the biolumingenic assays described herein which incubated with the enzyme for a first predetermined time employ luciferin derivatives with a lower background , those period to form a first mixture . Thereafter, the firstmixture is assays can use lower ( or higher ) amounts of the derivative , contacted with the luciferin derivative, to form a second and those derivatives may have improved reactivity , e . g ., mixture that is allowed to incubate for a second predeter with a nonluciferase enzyme. In addition , for any of the 15 mined time period . The second mixture is then contacted bioluminogenic assays described herein , other reagents may with a bioluminescent enzyme, e . g . , luciferase , to form a be added to reaction mixtures , including but not limited to third mixture , which is allowed to incubate for a third those that inhibit or prevent inactivation of luciferase , or predetermined time period . Thereafter, the activity resulting otherwise extend or enhance luminescent signal. from the interaction of the enzyme with the compound is Also provided is a method to identify or measure the 20 determined by measuring luminescence during and /or after potency of a modulator of a nonluciferase enzyme- mediated the third predetermined time period relative to a control reaction . The method includes contacting one or more ( e . g ., no compound ) reaction . In this way, for example , agents , a first reaction mixture for a nonluciferase enzyme- mechanism based inhibitors of the first enzyme can be mediated reaction , and a derivative of luciferin which is a identified and distinguished from nonmechanism based substrate for the nonluciferase enzyme, so as to yield a first 25 inhibitors because the first incubation with the test com mixture , or providing such a mixture, wherein the derivative pound but without the luciferin derivative will lead to a more includes an A or B ring modification relative to D -luciferin . profound inhibition by a mechanism based inhibitor than The first mixture in the absence of the one or more agents would be observed without the first incubation or substrates includes a luminogenic product that is a substrate for a beetle of the first reaction will show reduced inhibition . luciferase . At least a portion of the first mixture and a second 30 In another embodiment of the invention , a cell -based reaction mixture for a beetle luciferase -mediated reaction method is provided for screening a compound to determine are mixed , so as to yield a second mixture . Luminescence in its effect on enzyme activity of the cell. The test compound the second mixture is compared with a control mixture , is contacted with a cell having the enzyme, either naturally thereby identifying whether one or more of the agents or via recombinant expression , the luciferin derivative , and modulates the nonluciferase enzyme -mediated reaction and 35 bioluminescent enzyme, e . g . , luciferase , or contacted with a or to what extent and with what potency . cell having the enzyme and luciferase , and the derivative , for In one embodiment of the invention , test compounds can a predetermined period of time. Thus , in one embodiment, a be screened and evaluated for their activities as substrates or cell that either transiently or stably expresses a recombinant cofactors of, or regulators , either inhibitors or activators , of enzyme such as a bioluminescent enzyme, e . g ., luciferase , an enzymatic or nonenzymatic reaction by using the 40 may be employed . Any conventional method for creating luciferin and fluorophore derivatives of the present inven - transient or stable transfected cells may be used . In one tion . A candidate compound may be determined to be embodiment, a luciferin derivative is contacted with and regulator or a substrate of a reaction by contacting a reaction diffuses into a cell and , if the appropriate molecule is mixture with a derivative and the test compound , under present , yields a product , which is a substrate for luciferase . conditions that would , in the absence of the test compound , 45 If a luciferase is present in the cell, luminescence can be yield bioluminescence , fluorescence , or a bioluminogenic detected . Alternatively , in a cell which lacks luciferase , the product. product passes out of the cell into the medium and that In one aspect of the invention , a method is provided to medium is added to a luciferase reaction mixture . Thereafter , distinguish between a substrate and an inhibitor of a reac the activity resulting from the interaction of the cell with the tion . For example , the compound is incubated with at least 50 compound is determined by measuring luminescence of the one enzyme under conditions which allow for metabolism of reaction mixture relative to a control (minus test compound ) the compound prior to providing a luciferin derivative under reaction mixture . conditions that , in the absence of an inhibitor or substrate of In one aspect of the invention , the compound is preferably the enzyme, would be suitable for interaction between the contacted first with the cell for a predetermined time period . luciferin derivative and the enzyme . In one embodiment , the 55 Thereafter, the cell is contacted with the luciferin derivative product of that reaction is a substrate of luciferase and in the and luciferase simultaneously or contemporaneously and the presence of luciferase yields a light emitting second reac mixture allowed to incubate for a second predetermined time tion . The resulting light emitting reaction is compared to the period . Enzyme activity is determined by measuring the one obtained from contacting the enzyme with the com - amount of luminescence generated from the reaction mix pound and the derivative , under conditions that would , in the 60 ture relative to a control reaction mixture ( e . g . , minus test absence of an inhibitor of the enzyme, be suitable for compound ) . In another aspect of the invention , the test interaction between the luciferin derivative and the enzyme. compound is preferably contacted first with the cell for a Metabolism of the compound by the enzyme reduces its predetermined time period . Thereafter, the exposed cell is concentration the assay medium and may lead to an apparent then contacted with the luciferin derivative and incubated loss of inhibitory activity compared to conditions without 65 for a second predetermined time period . The cell is then metabolism of the compound which would indicate it was a contacted with luciferase to form a third mixture which is substrate for the enzyme. An inhibitory compound that was allowed to incubate for a third predetermined time period . US 10 , 077 , 244 B2 31 32 Thereafter , the activity of the cell resulting from the inter - luciferase. The first reaction may be quenched at the time, or action of the cell with the test compound ( s ) is determined by prior to addition , of a luciferase reaction mixture . For measuring luminescence of the reaction mixture relative to instance , a quencher of the first reaction may be present in a control reaction mixture ( e . g . , minus test compound ) . the luciferase reaction mixture . The luciferase reaction mix Detergent addition can rupture the cells and release cell 5 ture preferably substantially lacks a substrate for the content. luciferase , e . g . , the only source of substrate for the luciferase A cell -based luminescence detection assay for molecules is provided by a reaction between the non -luciferase enzyme present in the cell medium , e . g . , molecules which actively or and the derivative . When all the reagents for the first reaction via inactive mechanisms are present in the cell medium , can are present in the first reaction mixture , the assay may be include adding a reaction mixture with the luciferin deriva - 10 employed in identify moieties that alter the reaction , e . g . , tive to the cell medium , or adding the cell medium to a inhibitors or enhancers of the reaction . After performing the reaction mixture with the luciferin derivative , and detecting reactions , either simultaneously or sequentially , the presence luminescence . or amount of one or more molecules , or one or more In yet another embodiment of the cell- based assay of the inhibitors or activators of the reaction ( s ) is / are detected or invention , the cells may be lysed in an appropriate lysis 15 determined and/ or to what extent and with what potency . buffer . For animal cells , a buffet with 0 . 1 - 1 . 0 % non - ionic For a one step assay , a reaction mixture may contain detergents such as Triton X 100 or Tergitol is typically reagents for two reactions, such as reagents for a nonlu sufficient . Bacteria , plant, fungal or yeast cells are usually ciferase enzyme -mediated reaction and a luciferase -medi more difficult to lyse . Detergents , freeze / thaw cycles, hypo - ated reaction or for a nonenzymatic reaction and a tonic buffers , sonication , cavitation or combinations of these 20 luciferase -mediated reaction , or a reaction mixture for a methods may be used . The method of lysis that produces a single reaction , e . g ., for a reaction between a derivative of a lysate is compatible with luciferase or other enzyme activity , fluorophore which is a substrate for an enzyme and the or the detection of other molecules or conditions. enzyme or a luciferase -mediated reaction , e . g. , a luciferase The presence or activity of nonluciferase enzymes may be is suspected in a sample to be tested . measured in cells grown in culture medium or in cells within 25 For assays which employ two reactions , the order of animals , e . g . , living animals . For measurements in cells in adding the molecules for the assays can vary if initiated and animals , a luciferin derivative may be administered to the conducted sequentially (whether in the same vessel or not ) , animal, e . g . , injected into the animal or added to an aqueous adjustments to reaction conditions, e . g ., reagent concentra solution , e . g . , water, or food consumed by the animal. tion , temperatures or additional reagents , may be performed . Conversion of the derivative to a product that is a luciferase 30 For instance , a quenching agent or enhancing agent may be substrate may be detected by luminescence mediated by added between reactions ( see , e . g ., U . S . Pat. Nos . 5 ,774 , 320 luciferase expressed in cells in the animal, e . g ., transgenic and 6 ,586 , 196 , the disclosures of which are specifically cells , by luciferase administered to the animal, e . g . , injected incorporated by reference herein ) . In one embodiment, the into the animal, or by collecting physiological fluids, e . g . , two or more reactions are carried out simultaneously in a blood , plasma , urine, and the like , or tissue samples , and 35 single reaction mixture . Optionally , the assays are a homo combining those with a luciferase reagent. geneous assay, e .g . , the components are mixed prior to In one embodiment, the derivative employed in the meth - adding the mixture to the sample . Results may be read ods is not aminoluciferin which is modified to include a without additional transfer of reagents . protease substrate via a peptide bond at the amino group The assays of the present invention thus allow the detec which optionally also has a protected carboxyl group at 40 tion of one or more molecules or conditions in a sample , e . g . , position 5 in the C ring, or any of luciferin 6 ' methyl ether, a sample which includes eukaryotic cells , e . g . , yeast, avian , luciferin 6 ' chloroethyl ether, 6 ' deoxyluciferin , 6 ' luciferin plant, insect or mammalian cells , including but not limited 4 -trifluoromethyl benzylether, luciferin 6 ' phenylethylether, to human , simian , murine, canine, bovine, equine , feline , luciferin 6 " geranyl ether , luciferin 6 ' ethyl ether , 6 ' luciferin ovine , caprine or swine cells . or prokaryotic cells , or cells prenyl ether, 6 luciferin 2 - picolinylether, 6 ' luciferin 3 - pi- 45 from two or more different organisms, or cell lysates or colinylether, 6 ' luciferin 4 - picolinyl ether, luciferin 6 benzyl supernatants thereof, or a sample which includes a purified ether , D - luciferin - O - B - galactopyranoside , D - luciferin - O - form of the molecule , e . g ., purified nonluciferase enzyme sulfate , D - luciferin - O -phosphate , D - luciferyl- L - phenylala - which is useful to prepare a standard curve . The cells may nine , and D - luciferyl- L - N -arginine . In one embodiment, the not have been genetically modified via recombinant tech derivative employed in the methods is not a luciferin deriva - 50 niques ( nonrecombinant cells ) , or may be recombinant cells tive disclosed in U . S . published application 20040171099 , which are transiently transfected with recombinant DNA the disclosure of which is incorporated by reference herein . and /or the genome of which is stably augmented with a Assays which employ two reactions may be conducted recombinant DNA , or which genome has been modified to simultaneously ( one step ) or sequentially ( two step ) to detect disrupt a gene , e . g . , disrupt a promoter, intron or open one or more moieties including proteins (peptides or poly - 55 reading frame, or replace one DNA fragment with another . peptides ), e . g ., enzymes, substrates, cofactors , inhibitors or The recombinant DNA or replacement DNA fragment may activators for enzymatic reactions, or conditions, e .g ., redox encode a molecule to be detected by the methods of the conditions. A sequential reaction may be conducted in the invention , a moiety which alters the level or activity of the same vessel, e . g . , a well of a multiwell plate . For a two step molecule to be detected , and /or a gene product unrelated to assay , the first reaction mixture may contain all of the 60 the molecule or moiety that alters the level or activity of the reagents or less than all of the reagents for a nonluciferase molecule . enzyme- mediated reaction , where one of the reagents that is The present methods can be employed to detect a mol absent is the one to be detected in a sample , e . g . , a cell lysate . ecule for an enzyme- mediated reaction , a nonenzymatic For instance , a nonluciferase enzyme -mediated reaction is mediated reaction or condition . For instance , molecules or performed under conditions effective to convert a luciferin 65 conditions to be detected by the method include but are not derivative that is a substrate for the nonluciferase and a limited to enzymes , e . g . , demethylases, oxidases ( e . g ., a prosubstrate of luciferase , to a product that is a substrate of MAO ), deacetylases, deformylases , proteases (proteosome , US 10 ,077 , 244 B2 33 34 calpain , beta - secretase , cathepsin , calpain , thrombin , gran noluciferin or aminoquinolinyl luciferin (X = NH ) ; Y is O zyme B ) . phosphatases , kinases , peroxidases , transferases , ( ester ), NH (amide ) , NH?NH (hydrazide ) , or S ( thioester ) ; e . g . , GST, sulfotases , beta - lactamases , cytochrome P450 and R , if present, may be alkyl, an aromatic molecule , a enzymes , esterase , e. g ., acetylcholinesterase, dehydroge peptide , an oligonucleotide , or a self -cleavable linker nase , luciferase , substrates , inhibitors , co - factors, activators 5 attached to a substrate for an enzyme. In one embodiment, of enzyme mediated reactions, reactive oxygen species the derivative may be modified at L or R to include a reducing conditions and transcriptional regulators or regu substrate for an enzyme such as a P450 enzyme, protease , lators of gene transcription . The enzymes employed in the MAO , FMO , or GST . In one embodiment, a derivative of the methods , either enzymes to be detected or enzymes which invention is a substrate for luciferase, including 6 -amino are useful to detect a substrate or cofactor, can be selected 1010 quinolinyl luciferin , which is a substrate of luciferase having from any combination of enzymes including recombinant substantial light output. and endogenous ( native ) enzymes . In one embodiment, the enzyme to be detected is an endogenous enzyme . In another Bioluminescent substrates according to the present inven embodiment, the enzyme is a recombinant enzyme. Other tion are derivatives of luciferin or aminoluciferin , i. e ., are combinations apparent to one of ordinary skill in the art can 155 luminescent substrates other than luciferin or aminolu be used in the present assays and methods according to the ciferin , and include compounds having the general formulas teachings herein . The enzymes include but are not limited to described below including , e . g . , formulas I and II. proteases, phosphatases, peroxidases, sulfatases , peptidases, In one embodiment, the invention provides a compound oxidases, dealkylases, deformylases and glycosidases . The of formula 1 : enzyme may be a hydrolase , oxidoreductase , lyase , trans - 20 ferase , e . g ., glutathione S transferase , isomerase , ligase , or synthase . Of particular interest are classes of enzymes that I have physiological significance . These enzymes include protein peptidases, esterases, protein phosphatases, glyco sylases , proteases, dehydrogenases , oxidases , oxygenases, 25 i reductases , methylases and the like . Exemplary cleavage Z ' - - A oL 1IdA sites for some proteases are set forth in FIG . 2 . Enzymes of WC A interest include those involved in making or hydrolyzing AlWA RE W2 esters , both organic and inorganic , glycosylating, and hydro Nato lyzing amides . In any class , there may be further subdivi- 30 sions. wherein In particular, enzymes that are useful in the present Y is N , N - oxide, N - ( C . - C . )alkyl , or CH ; invention include any protein that exhibits enzymatic activ when Y is N , then X is not S ; ity , e . g ., lipases, phospholipases, sulphatases, ureases , pep X is S , O , CH = CH , N = CH , or CH = N . tidases , proteases and esterases , including acid phos - 35 when X is S . then Y is not N : phatases, glucosidases, glucuronidases, galactosidases, Z and Z are independently H , OR , NHR , or NRR ; carboxylesteraes, and luciferases . In one embodiment, the enzyme is a hydrolytic enzyme. Examples of hydrolytic Z " is O , S , NH , NHR , N = N ; enzymes include alkaline and acid phosphatases , esterases, Q is carbonyl or CH2; decarboxylases, phospholipase D , P - xylosidase , B - fucosi- 40 Wl is H , halo , ( C ,- C . ) alkyl, (C2 -C20 ) alkenyl, hydroxyl , dase , thioglucosidase , B - D - galactosidase , a - D - galactosi or ( C , -C ) alkoxy ; or dase , a - D - glucosidase , B - D - glucosidase , B - D - glucuroni W and X are both keto groups on ring A , and at least one dase , a - D -mannosidase , B - D -mannosidase , B - D of the dotted lines denoting optional double bonds in ring A fructofuranosidase , and B - D - glucosiduronase. is absent; In one embodiment, the invention provides the use of a 45 each W ? is independently H , halo , ( C , - C . )alkyl , (C2 -C4 ) 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 ', K², K ’ , and K4 are independently CH , N , teolytic enzyme, is detected using a substrate which is N -oxide , or N - (C1 - C . )alkyl , and the dotted lines between covalently linked to a fluorophore . In one embodiment, the K and K ? , and Kº and K4, denote optional double bonds; substrate includes a recognition site for the enzyme. In the 50 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 including such a substrate generates minimal light at the fused tricyclic system ; and emission wavelength as the fluorescent properties of the when B ' is present, the group Z is present, and fluorophore are quenched , e. g. , by the proximity of the when A ' is present, the group Z is absent; and quenching group . In the presence of the appropriate enzyme, 55 the dotted line in ring B is an optional double bond ; cleavage of the conjugate yields the fluorophore . each R is independently H , (C , -C20 ) alkyl , ( C2 -C20 )alk enyl, (C2 - C20 ) alkynyl, (Cz - C20 ) cycloalkyl, ( C , - C12) alkoxy, III . Luciferin Derivatives (Co -C30 )aryl , heteroaryl, heterocycle , (C1 -C20 )alkylsulfoxy , (Co -C30 )arylsulfoxy , heteroarylsulfoxy , (C1 -C20 ) alkylsulfo In one embodiment, derivatives of luciferin or aminolu - 60 nyl , (Co -C30 Jarylsulfonyl, heteroarylsulfonyl , ( C -C20 ) ciferin have the following structure; L - X - M - Y -R (compound alkylsulfinyl , (Co -C30 )arylsulfinyl , heteroarylsulfinyl, (C , of formula IV ) . wherein L , if present, may be a substrate for C20 ) alkoxycarbonyl, amino , NH ( C - C . ) alkyl, N ( C , - C . ) an enzyme or another molecule which interacts with the alkyl ) 2 , tri ( C -C20 ) ammonium (C2 -C20 ) alkyl, heteroaryl( C enzyme; X may be O , NH , or a linker, e .g ., a self -cleavable C20 )alkyl , heteroaryl having quarternary nitrogen , linker which spontaneously cleaves to yield M - Y - R after L 65 heteroarylcarbonyl having quaternary nitrogen , (C6 - C30 ) has been removed from L - X - M - Y - R ; M may be luciferin , arylthio , ( C , -C20 ) alkylphosphate , ( C - C20 ) alkylphospho quinolinyl luciferin or naphthyl luciferin ( X = O ) , or ami nate , (C6 -C30 ) arylphosphate , (Co - C30 ) arylphosphonate , US 10 , 077 ,244 B2 35 3636 phosphate , sulfate , saccharide, or M + optionally when Z " is oxygen , wherein M is an alkali metal ; IA or when Z or Z ' is NR ' R ' , R ' R ' together with the N to which they are attached forms a heteroaryl or heterocycle group ; 5 O ZR wherein any alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy , ZA | BD amino , aryl, heteroaryl, or heterocycle group is optionally substituted with 1 , 2 , 3 , 4 , or 5 substituents selected from (C , -C20 )alkyl , (C2 - C20 )alkenyl , (C2 - C20Jalkynyl , (Cz - C20 ) cycloalkyl, (C , -C20 ) alkoxyl , (C1 - C20 )alkylcarbonyl , (C1 - " wherein C20 ) alkylcarboxyl, halo , hydroxyl, COOR " , — SO , R * , Y is N , N - oxide, N - ( C , - C ) alkyl, or CH ; - SO3R *, nitro , amino, (C2 - C20 )alkyl - S ( O ) , ( C , - C20 ) when Y is H , then X is not S ; alkyl- S02 — , phosphate , (C , -C20 )alkylphosphate , (C ,- C20 ) X is S , O , CH = CH , N = CH , or CH = N ; alkylphosphonate , NH (C , -C . ) alkyl, NH (C , -C .) alkynyl , 15 when X is S , then Y is not N ; N ( C1 -C6 ) alkyl) 2, N ( C1- C .) alkynyl) 2 , mercapto , (C1 - C20 ) Z is H , OR , NHR , or NRR ; alkylthio , ( C . - C . Jaryl , ( C . -C20 Jarylthio , trifluoromethyl, Z " is O , S , NH , NHR , or N = N : = O , heteroaryl, and heterocycle , and each substituent is W is H , halo , hydroxyl, (C ,- C .) alkyl, or (C ,- C .) alkoxy ; optionally substituted with one to three R groups ; the dotted line in ring B is an optional double bond ; R * is H , ( C - C . )alkyl , or (C6 -C20 )aryl ; 20 each R is independently H , ( C , -C20 )alkyl , (C2 - C20 ) alk when Z or Z ' comprises a nitrogen moiety , one or both of enyl, (C2 - C20 ) alkynyl, (Cz - C20 ) cycloalkyl , ( C , -C12 ) aryl , the hydrogens of the Z or Z ' nitrogen moiety may be heteroaryl, heterocycle , ( C , -C20Jalkylsulfoxy , (C6 - C20 )aryl replaced by (CZ -C20 ) alkyl or the group L , wherein L is an sulfoxy , heteroarylsulfoxy, (C2 - C20 Jalkylsulfonyl , (Co -C30 ) ammo acid radical, a peptide radical having up to 20 amino arylsulfonyl, heteroarylsulfonyl, (C1 - C20 ) alkylsulfinyl, (Co acid moieties, or any other smallmolecule that is a substrate 25 C30 )arylsulfinyl heteroarylsulfinyl, ( C , -C20 ) for a nonluciferase ; with the proviso that when L is an amine alkoxycarbonyl , amino, NH (C7 - C . ) alkyl, N ( ( C -C6 ) 2 , tri acid radical or a peptide radical, at least one W2 is not H ; (C1 - C20 ) ammonium (C1 - C20 )alkyl , heteroaryl( C1 - C20 )alkyl , when Z is a hydroxyl group or a nitrogen moiety , H of the heteroaryl having quaternary nitrogen , heteroarylcarbonyl hydroxyl or nitrogen moiety may be replaced by (HO ) 2P having quaternary nitrogen , ( Co - C30 )arylthio , (C , -C20 )alky ( 0 ) OCH — , sulfo , - POZH2, or by a cephalosporanic 30 iphosphate , ( C -C20 ) alkylphosphonate , (C6 -C30 ) arylphos acid attached to the group Z via a carbon chain of one to phate , ( Co -C30 ) arylphosphonate , phosphate , sulfate , saccha about 12 carbon atoms; with the proviso that when ring B is ride, or M * optionally when Z " is oxygen , wherein M is an a thiazole ring , the sulfo or the — PO H , group is attached alkali metal; to the hydroxyl oxygen via a ( C , - C . ) alkylene group ; or when Z or Z ' is NR ' R , RR together with the N to when Z or Z ' is a hydroxyl group or a nitrogen moiety , or 35 which they are attached forms a heteroaryl or heterocycle when Z " - R is a hydroxyl group , one H of the hydroxyl or group ; nitrogen moiety may be replaced by the group L ' -linker , wherein any alkyl , alkenyl, alkynyl, cycloalkyl , alkoxy , wherein L ' is a group removable by an enzyme to free the amino , aryl, heteroaryl, or heterocyclic group is optionally linker, and linker is a carbon chain that can self -cleave , substituted with 1 , 2 , 3 , 4 , or 5 substituents selected from optionally interrupted by one or more nitrogen atoms, oxy - 40 ( C , -C20 ) alkyl, (C2 - C20 ) alkenyl, (C2 - C20 ) alkynyl , (Cz - C20 ) gen atoms, carbonyl groups, optionally substituted aromatic cycloalkyl, (C ,- C20 ) alkoxyl, (C , -C20 )alkylcarbonyl , (C , rings, or peptide bonds, C20 ) alkylcarboxyl , halo , hydroxyl , COOR " , SO , R " , linker is attached to L ' via an oxygen atom or an NH group SO3R , nitro , amino , (C1 - C20 ) alkyl- S ( O ) - , ( C , -C20 ) at one terminus of the linker and the other terminus of the alkyl- SO2 - , phosphate , (C2 - C20 ) alkylphosphate , ( C , - C20 ) linker forms an ether, ester, or amide linkage with a group 45 alkylphosphonate , NH (CZ - C . ) alkyl , NH (C1 - C . ) alkynyl , Z , Z ', or Z " - R ; N (( (C . -C . ) alkyl) 2, N ( (C / -C .) alkynyl) 2 ,mercapto , ( C ,- C20 ) when Z is OR , formula I is optionally a dimer connected alkylthio , (Co - C3. ) aryl, (C6 -C30 )arylthio , trifluoromethyl , at the two A rings via a linker comprising a ( C , -C12 )alkyl = O , heteroaryl, and heterocycle , and each substituent is diradical that is optionally interrupted by one to four O optionally substituted with one to three R groups ; atoms, N atoms, or an optionally substituted aryl, heteroaryl, 50 R * is H , ( C , - C . ) alkyl, or (C6 - C30Jaryl; or heterocycle group to form a bridge between the dimer of A is an anion , present when a quarternary nitrogen is formula I, and the R group of each Z group connecting the present; dimer of formula I is replaced by the bridge ; or a salt thereof; A is an anion , present when a quaternary nitrogen is provided that present; 55 when rings A and B form a naphthalene or quinoline ring or a salt thereof; system , then Wl is not hydrogen ; provided that: 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 - NH2; system , then W is not hydrogen ; when Y is N or CH and X is CH = CH and Wl is H , then when a ring A substituent is OH , then - Q - Z " - R is not 60 Z 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 when Y is N or CH and X is CH = CH and Wl is H , then Wl is not OH attached to carbon - 6 of ring A . Z 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 rings A and B can be a number of different ring systems . wl is not OH attached to K " . 65 Modification of ring B allows the core structure to include In another embodiment, the invention provides a com - benzofuran , benzothiophene, benzoaxazole , naphthalene , pound of formula IA : quinoline, isoquinoline , quinazoline, and quinoxyline ring US 10 ,077 ,244 B2 37 38 systems, including the corresponding N -oxide and N - alkyl nitrogen , saccharide , or M * optionally when Z " is oxygen , derivatives. Ring B modification also allows for access to wherein M is an alkali metal; N -oxide and N - alkyl derivatives of benzo [d ] thiazole . Fur- Riis (Co - C30Jaryl , heteroaryl, heterocycle , (C -C20 ) alky thermore , by substituting a carbon atom of ring A in formula Ithio , (C1 - C20 ) alkyl- S ( O ) , ( C , -C20 )alkyl - SO2 , SO3 (C - I with N ,N - oxide, or N - alkyl ( e. g. , substituting one value of 5 C20 ) alkyl, saccharide, (C ,- C20 ) alkylphosphate , (C2 -C20 ) K ' , K², K ’, or K4 for another value ) , other ring systems can alkylphosphonate , (C6 - C30 ) arylthio , ( Co- C30 Jaryl- S( O ) - , be obtained , such as 1, 8 -naphthyridine , 1 , 7 -naphthyridine (C6 -C30 ) aryl- SO2, S03 (Co -C30 ) aryl, (Co - C30 ) arylphos 1 ,6 -naphthyridine , and 1 , 5 -naphthyridine ring systems; phate , ( Co- C30Jarylphosphonate , or R is (C1 - C20 ) alkyl sub pyrido [ 3 ,2 - b ]pyrazine , pyrido [ 4 ,3 -b ]pyrazine , pyrido [ 3 ,4 - b ] . stituted by R ? ; pyrazine , and pyrido [ 2 , 3 -b ] pyrazine ring systems; pyrido [ 2 , R² is (C2 -C20 )alkenyl , (C2 - C20 Jalkynyl , (Cz - C20 )cy 3 -d ]pyrimidine , pyrido [3 ,4 - d ]pyrimidine , pyrido [4 , 3 - d ]py - cloalkyl, (C ,- C20) alkoxyl , ( C , - C20 )alkylcarbonyl , (C , -C20 ) rimidine , and pyrido [3 ,2 - d ]pyrimidine ring systems; benzo alkylcarboxyl, hydroxyl, COOR ", SO3R , (C , -C20 ) [ d ] oxazole , oxazole [4 , 5 - b ]pyridine , oxazolo [4 , 5 -c ]pyridine , alkylthio , (Co -C20 )arylthio , (C , -C20 )alkyl - S ( O ) - , (C , -C20 ) oxazolo [ 5 , 4 - c ] pyridine , and oxazole [ 5 , 4 - b ]pyridine ring 15 alkyl- Soz - , nitro , amino , NH ( C , - C . ) alkyl, NH ( C , - C ) systems; and thiazolo [ 4 , 5 - b ]pyridine , thiazolo [ 4 , 5 -c ]pyri alkynyl, N ( C1- C . )alkyl ) 2, or N ( C1- C .) alkynyl ) 2 , dine , thiazolo [5 , 4 -c ]pyridine , and thiazolo [5 , 4 -b ]pyridine mercapto , saccharide , or trifluoromethyl; ring systems. or when Z or Z ' is NR ' R ' , R ' R ! together with the N to Formula I also illustrates that by substituting a second which they are attached forms a heteroaryl or heterocycle carbon atom of ring A with N , N -oxide , or N -alkyl , the 20 group ; corresponding pyrazine, pyramidine , and pyridazine ring A wherein any alkyl, alkenyl , alkynyl, cycloalkyl, alkoxy, analogs can be obtained for each of the above described ring amino, aryl , heteroaryl, or heterocycle group is optionally systems. The substitution of a third carbon atom in ring A of substituted with 1 , 2 , 3 , 4 , or 5 substituents selected from formula I with N , N -oxide , or N - alkyl provides access to the ( C - C20 ) alkyl , (C2 - C20 ) alkenyl, ( C2- C20 )alkynyl , ( C3- C20 ) corresponding 1 , 2 , 4 - triazine ring system derivatives . 2525 cycloalkyl, (C ,- C20 ) alkoxyl, ( C , -C20 )alkylcarbonyl , ( C , Formulas I and IA further include the various dihydro , C20 ) alkylcarboxyl , halo , hydroxyl, COOR' , SO , R " , tetrahydro , and hexahydro derivatives of each of its ring SO3R *, (C2 -C20 )alkyl - S (O ) , (C , -C20 )alkyl -SO2 - , systems. phosphate , ( C , - C20 ) alkylphosphate , ( C , - C20 ) alkylphospho In another embodiment, the invention provides a com nate , nitro , amino , NH ( C , - C ) alkyl, NH (C2 - C6) alkynyl, pound of formula II : 30U N ( C , -C .) alkyl ) 2, N (( C . -C . ) alkynyl )2 , mercapto , ( C ,- C20 ) alkylthio , (Co -C30 ) aryl, (Co - C10 )arylthio , trifluoromethyl, II = O , heteroaryl, and heterocycle , and each substituent is optionally substituted with one to three R groups; B ' 35 R * isis H or ( C , - C . ) alkyl; R when Z or Z ' comprises a nitrogen moiety , a hydrogen of LKw N NO21 the Z or Z ' nitrogen moiety may be replaced by the group L , 2' ? A wherein L is an amino acid radical, a peptide radical having W2 A up to 20 amino acid moieties , or any other small molecule WIS W2 40 that is a substrate for a nonluciferase ; with the proviso that when L is an amino acid radical or a peptide radical, at least wherein one of W ' or a W ? is not H ; Z and Z ' are independently ORT, NHR ' , or NR ' R ' ; when Z is a hydroxyl group or a nitrogen moiety , H of the Z " is O , S , NH , NHR , or N = N ; hydroxyl or nitrogen moiety may be replaced by (HO ) 2P Q is carbonyl or CH2; 45 ( 0 ) - OCH , — , sulfo , - POZH , , or by a cephalosporanic wl is H , halo , ( C , -C . )alkyl , (C2 -C20 )alkenyl , hydroxyl, acid attached to the group Z via a carbon chain of one to or ( C , -Colalkoxy ; or about 12 carbon atoms ; with the proviso that the sulfo or the W and Z are both keto groups on ring A , and at least one - POZH2 group is attached to the hydroxyl oxygen via a of the dotted lines denoting optional double bonds in ring A (C .- C . )alkylene group ; is absent; when Z or Z ' is a hydroxyl group or a nitrogen moiety , or each W ? is independently H , halo , ( C , - C . )alkyl , ( C2 -C4 ) when 7 " _ R is a hydroxyl group one H of the hydroxyl or alkenyl, hydroxyl, or ( C , - C . ) alkoxy ; each of K1, K², K ’ , and K4 are independently CH , N , nitrogen moiety may be replaced by the group L ' - linker, N - oxide , or N - ( C , - C ) alkyl , and the dotted lines between wherein L ' is a group removable by an enzyme to free the K and K ?, and K and K4 , denote optional double bonds ; 55 linker , and linker is a carbon chain that can self -cleave , A ' and B ' are optional aromatic rings fused to ring A , only optionally interrupted by one or more nitrogen atoms, oxy one of which is present in the compound , so as to form a gen atoms, carbonyl groups, optionally substituted aromatic fused tricyclic system ; and rings, or peptide bonds, when B ' is present, the group Z is present, and linker is attached to L ' via an oxygen atom or an NH group when A ' is present, the group Z is absent; and 60 at one terminus of the linker and the other terminus of the Ris H , ( C , - C Jalkyl, ( C , -C20 ) alkenyl, ( C , -Co ) alkynyl, linker forms an ether, ester , or amide linkage with a group (C3 -C20 ) cycloalkyl, ( C , - C12 ) alkoxy, (Co -C30Jaryl , het- Z , Z ', or Z " — R , eroaryl, heterocycle , ( C , -C20 )alkylsulfoxy , (Co - C30 Jarylsul when Z is OR " , formula II is optionally a dimer connected foxy , heteroarylsulfoxy , ( C , -C20 Jalkoxycarbonyl, amino , at the two A rings via linker comprising a ( C2 - C 2 )alkyl NH (C7 - C . ) alkyl, N ( ( ( C / - C . ) alkyl) 2 , tri (C2 -C20 ) ammonium 65 diradical that is optionally interrupted by one to four O (C7 -C20 ) alkyl , heteroaryl( C / - C20 ) alkyl, heteroaryl having atoms, N atoms, or an optionally substituted aryl , heteroaryl, quaternary nitrogen , heteroarylcarbonyl having quaternary or heterocycle group to form a bridge between the dimer of US 10 ,077 ,244 B2 39 40 formula II , and the R ' group of each Z group connecting the or heterocycle group to form a bridge between the dimer of dimer of formula II is replaced by the bridge ; formula IIA , and the R ' group of each Z group connecting provided that a saccharide is not directly attached to K ; the dimer of formula II is replaced by the bridge ; A is an anion , present when a quaternary nitrogen is provided that a saccharide is not directly attached to K ? ; present; A is an anion , present when a quaternary nitrogen is or a suit thereof. present; In yet another embodiment, the invention provides a or a salt thereof. As illustrated by formulas II and IIA , the core structure of compound of formula IIA : rings A and B can also be a variety of ring systems. In 10 addition to benzo [ d ] thiazole , substituting a carbon atom of ring A of formula II with N , N -oxide , or N - alkyl allows IIA access to thiazolo 4 , 5 -b ]pyridine , thiazolo [ 4 , 5 - c ]pyridine , R thiazole [ 5 , 4 - c ]pyridine , and thiazolo [ 5 , 4 - b ]pyridine ring 2 - systems, and their corresponding N - oxides and N - alkyl 2F- AL BV 15 derivatives . U Formula II also illustrates that by substituting a second 2 carbon atom of ring A with N , N - oxide, or N -alkyl , the corresponding pyrazine , pyramidine , and pyridazine ring A analogs can be obtained for each of the above described ring wherein 20 systems. The substitution of a third carbon atom in ring A of Z is OR , NHR !, or NR 'R '; formula II provides access to the corresponding 1 , 2 ,4 Z " is O , S , NH , NHR , or N = N ; triazine derivatives . W is H , halo , hydroxyl, (C - C . )alkyl , or (C1 - C6 )alkoxy ; Formulas II and IIA include the various dihydro and R is H , ( C , -C20 )alkyl , (C2 - C20 )alkenyl , (C2 -C20 )alkynyl , tetrahydro derivatives of each of its ring systems. ( Cz- C20 ) cycloalkyl, ( C - C12 ) alkoxy , ( Co - C30 )aryl , het - 25 All ring systems described above can be substituted as eroaryl, heterocycle , ( C -C20 )alkylsulfoxy , (Co - C30 ) arylsul- illustrated in the formulas described herein , for example , foxy, heteroarylsulfoxy , (C1 - C20 )alkoxycarbonyl , amino formulas I and / or II . Each of the above - described individual NH (CZ - C ) alkyl, N (( CZ- C6) alkyl) 2 , tri (C2 -C20 ) ammonium ring systems and their derivatives, substituted as described (C2 -C20 Jalkyl heteroaryl (C1 - C20 ) alkyl, heteroaryl having in the formulas herein , are separate embodiments of the quaternary nitrogen , heteroarylcarbonyl having quaternary 30 invention . nitrogen , saccharide , or M * optionally when Z " is oxygen , Other deriviates and their use in luminogenic assays is wherein M is an alkali metal; described hereinbelow . Rl is (C6 -C30 ) aryl , heteroaryl, heterocycle , (C ,- C20 ) alky - The use of the luciferin derivatives described herein can Ithio , ( C , - C20 ) alkyl- S ( O ) - , (C1 - C20 ) alkyl- SO2 , S03( C1 - result in an assay which produces a measurable change in C20 ) alkyl, saccharide, ( C , -C20 ) alkylphosphate , ( C , -C20 ) 35 optical properties upon interaction with a nonluciferase alkylphosphonate , (Co - C30) arylthio , (Co -C30 )aryl - S (O ) - , molecule , which interaction may alter the structure of the (Co - C30 )aryl -SO2 , S03 (Co -C30 )aryl , (Co - C30Jarylphos luciferin derivative. As described herein , the product of a phate , (C . - C30 ) arylphosphonate , or R ' is (C1 -C20 )alkyl sub reaction between a luciferin derivative and a nonluciferase stituted by R2; enzyme or other molecule of interest need not be D - luciferin R2 is (C2 - C20 ) alkenyl, (C2 -C20 ) alkynyl , (Cz - C20 ) cy - 40 or aminoluciferin . For example , a luciferin derivative may cloalkyl, ( C , -C20 ) alkoxyl, ( C -C20 ) alkylcarbonyl, (C1 - C20 ) include a substrate that includes a reactive chemical group alkylcarboxyl , hydroxyl, — COOR ", SO3R ", ( C , -C20 ) (or a nonluciferase enzyme linked to luciferin or aminolu alkylthio , (C6 - C3 . ) arylthio , ( C , - C20 ) alkyl- S (O ) - , (C -C20 ) ciferin via a chemical linker . Transformation of the reactive alkyl- S02 — , nitro , amino , NH ( C , - C . ) alkyl , NH ( C , - C . ) chemical group of the derivative by the nonluciferase alkynyl, N ( C1 - C . ) alkyl) 2 orN ( ( C - C . ) alkynyl) 2 , 45 enzyme may yield a product that contains (retains ) a portion mercapto , saccharide, or trifluoromethyl; of the substrate , a portion of the chemical linker , the or when Z or Z ' is NR?R , R 'Rl together with the N to chemical linker, or a portion of the substrate and the chemi which they are attached forms a heteroaryl or heterocycle cal linker, and that product is a substrate for luciferase . Also group ; provided are luciferin derivatives which , after interaction wherein any alkyl, alkenyl, alkynyl , cycloalkyl, alkoxy, 50 with a nonluciferase enzyme or other molecule , may yield a amino , aryl, heteroaryl, or heterocycle group is optionally product that optionally undergoes one or more further reac substituted with 1 , 2 , 3 , 4 , or 5 substituents selected from tions, e . g . , B - elimination , to yield a suitable substrate for (C7 -C20 ) alkyl, (C2 -C20 ) alkenyl, (C2 - C20 Jalkynyl , (Cz - C20 ) luciferase . Luciferin derivatives in which the backbone of cycloalkyl, ( C , -C20 ) alkoxyl, (C ,- C20 ) alkylcarbonyl, (C ,- luciferin is further modified in its ring structure , e .g ., a C20) alkylcarboxyl, halo , hydroxyl, COOR , — SO R , 55 quinolyl or naphthyl luciferin , are provided , an well as SO3R * , ( C , -C20 ) alkyl- S ( O ) , ( C , - C20 )alkyl -SO2 advantageously providing modifications at the carboxy posi phosphate , ( C , -C20 ) alkylphosphate , (C2 -C20 ) alkylphospho - tion of the thiazole ring , to provide improved characteristics nate , nitro , amino , NH ( C - C . ) alkyl, NH (C7 - C . ) alkynyl, to the luciferin derivative . Derivatives with certain modifi N ( (Cz - C . ) alkyl) 2, N ( C1- C . ) alkynyl) 2 , mercapto , (C1 -C20 ) cations provide for or improve assays for certain nonlu alkylthio , (Co - C30 )aryl , (C6 - C30 Jarylthio , trifluoromethyl, 60 ciferase enzymes or molecules. For instance, a described = O , heteroaryl, and heterocycle , and each substituent is hereinbelow , a pH insensitive derivative of luciferin was optionally substituted with one to three R groups; identified that is useful in biological assays that may be run R * is H or ( C - C . ) alkyl ; at a pH other than physiological pH , i. e . , less than about pH when Z is OR , formula IIA is optionally a dimer con 7 .0 and greater than about pH 7 . 8 . Thus, bioluminescent nected at the two A rings via linker comprising a (C1 - C12 ) 65 methods that employ a luciferin derivative of the invention alkyl diradical that is optionally interrupted by one to four o may be used to detect one or more molecules, e . g . , an atoms, N atoms, or an optionally substituted aryl , heteroaryl, enzyme, a cofactor for an enzymatic reaction such as ATP, US 10 ,077 , 244 B2 42 an enzyme substrate , an enzyme inhibitor, an enzyme acti or - POZH2, or by cephalosporanic acid attached to the vator, or OH radicals , or one or more conditions , e . g ., redox group Z via a carbon chain of one or more carbon atoms; conditions . with the proviso thatwhen ring B is a thiazole ring, the sulfo In one embodiment, the invention provides a compound or the - POZH2 group is attached to the hydroxyl oxygen via of formula ( V ) : 5 a lower alkylene chain ; and 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 the linker is a carbon chain that may optimally self -cleave , to 10 _ optionally interrupted by one or more nitrogen atoms, oxy {;_ gen atoms, carbonyl groups, ( substituted ) aromatic rings, or ? ' ? ' ? ? --- 21R peptide bonds, and -- K linker is attached to L ' via an oxygen atom or an NH group ? K41w ( W ) n 15 at one terminus of the linker and the other terminus of the linker forms an ether, ester , or amide linkage with the group Z ; and wherein when Z is hydroxyl , formula V includes a luciferin dimer Y is N , N -oxide , N - loweralkyl, or CH ; connected at the two A rings via an OCH O — bridge ; and X is S , CH = CH , or N = C , 20 wherein Z and Z ' are H , OR , NHR , of NRR , A ' and B ' are optional aromatic rings fused to ring A , only Z " is O , S , NH , NHR , or N = N , one of which may be present at a time, so as to form a fused each Wis independently H ,halo , C1- 6 - alkyl, C2- 20alkenyl , tricyclic system ; and hydroxyl, or when B ' is present, the group Z is present, and C1- 6alkoxy ; or 25 when A ' is present, the group Z is absent, and W and Z on ring A are both keto groups ; wherein each of K ' , K², K3, and K4 are independently CH , one carbon of ring A may be replaced by an N - oxide N , N - oxide , or N - loweralkyl; moiety ; R is H , C1- 20alkyl, substituted C1- 20alkyl, C2- 20alkenyl, the dotted line in ring B is an optional double bond ; substituted C220alkenyl, halogenated C2- 20alkenyl, substi - 30 if X is N = C , ring C is attached at the carbon atom of the tuted halogenated C2- 20alkenyl , C3- 20alkynyl , substituted N = C moiety , and C3- 20alkynyl, C2- 20alkeny1C1 - 20alkyl, substituted C2- 20alk - A is an anion , present when a quaternary nitrogen is eny1C1- 20alkyl, C3 - 20alkyny1C2- 2oalkenyl, substituted present; or a salt thereof, with the proviso that W is not C1- 20alkyny1C2- 20alkenyl, C3- 20cycloalkyl, substituted hydrogen when the compound to which W is attached is C3 - 20cycloalkyl, C6- 30aryl, heteroaryl , C6 - 30ary1C1- 20alkyl, 35 luciferin , luciferin methyl ester, or aminoluciferin or when substituted Co - soaryl, substituted heteroaryl, substituted rings A and B form a naphthalene or quinoline ring system . Cozgary1C , zoalkyl, alkylsulfoxyC , zoalkyl, C , alkoxy . Further derivatives of luciferin or aminoluciferin have the carbonyl, Co. zgary1C , zoalkoxycarbonyl, Co -znarylthio general formula V1. C1- 20alkyl, hydroxyC1- 20alkyl, triC - 20 ammoniumC - 2oalkyl, heteroary1C1- 20alkyl, substituted heteroary1C1. * 20alkyl, heteroaryl having quaternary nitrogen , heteroaryl carbonyl having quaternary nitrogen , and N -methyl 7 RO tetrahydropyridinyl; and M * when Z " is oxygen , wherein M is an alkali metal; wherein the alkyl, cycloalkyl , alkenyl, 45 and / or alkynyl groups may be optionally substituted by one RI more C1- zoalkyl, halo , hydroxyl, acetyl, amino , lower alky lamino , lower alkynylamino , imidazolinylmethylamino , di wherein lower lower alkylamino , di- lower alkynylamino , piperidino , Rrepresents hydrogen , hydroxyl, amino , C1- 20alkoxy , sub pyrrolidine, azetidino , aziridino , 50 stituted C1- 20alkoxy , C2- 2oalkenyloxy, substituted C2- 20alk di- imidazolinylmethylamino , mercapto , C1- 20alkylthio , enyloxy, halogenated C1- 20alkoxy , substituted halogenated C6- 30arylthio , or trifluoromethyl groups, substituted C1-20alkoxy , C3- 20alkynyloxy , substituted C3 - 20alkynyloxy , C6- 30ary1C1 - 20 alkyl carbonyl ; and each group R is defined C3- 20cycloalkoxy, substituted C3 - 20 cycloalkoxy, C3- 20cy independently if more than one is present; cloalkylamino , substituted C3- 20cycloalkylamino , W is ( C = O ), or (CH2 ) n; 55 C1- 20alkylamino substituted C1- 20alkylamino , diC1- 20alky n is 0 , 1 , or 2 ; lamino , substituted diC1- 20alkylamino , C2- 20alkenylamino , and wherein substituted C2- 20 alkenylamino, diC2- 20alkenylamino , substi when Z or Z " is amino , one or both of the hydrogens may tuted diC2- 20alkenylamino , C2- 20alkeny1C1- 20alkylamino , be replaced by C1- 2oalkyl, or the group L , wherein substituted C2- 20alkeny1C1- 20alkylamino , C3- 20alky L is an amino acid radical, a peptide radical having up to 60 nylamino , substituted C3- 20alkynylamino , diC3- 2oalky 20 amino acid moieties, or may be any other small molecule nylamino, substituted diC3 -20alkynylamino , C3- 20 alkynyl that is a substrate for a nonluciferase ; with the proviso that C2- 20alkenylamino , when L is an amino acid radical or a peptide radical , W is not or substituted C3 - 2oalkyny1C2- 20 alkenylamino ; H ; R “ represents CH2OH ; CORll wherein Rll represents H , and wherein 65 OH , C1- 20alkoxide, C2- 20alkenyl, or NR12R13 wherein R12 when Z is hydroxyl or amino , H may be replaced by and R13 are independently H or C1- 20alkyl; or — OM (HO )2P ( O ) _ OCH2 – , sulfo , wherein M * is an alkali metal; or a salt; and US 10 ,077 ,244 B2 43 44 R ? represents H , C1- galkyl , C2- 20alkenyl , halogen , or - POZH2, or by a cephalosporanic acid attached to the C1- alkoxide, group Z via a carbon chain of one or more carbon atoms; and with the proviso that when Rl is OH or NH ,, R7 is not H , when Z is hydroxyl or amino or when Z " - R is hydroxyl, R® is not COR " wherein Rll is OH or OMe formula VI does H may be replaced by the group L' -linker , wherein L ' is a not include luciferin , luciferin methyl ester , and aminolu - 5 group removable by an enzyme to free the linker, and linker ciferin . is carbon chain that can self - cleave , optionally interrupted Further derivatives of luciferin or aminoluciferin have the by one or more nitrogen atoms, oxygen atoms , carbonyl general formula VII ; groups , (substituted )aromatic rings , or peptide bonds, and linker is attached to L ' via an oxygen atom or an NH group 10 at one terminus of the linker and the other terminus of the VII linker forms an ether, ester, or amide linkage with the group Z ; and when Z is hydroxyl, formula VII includes a luciferin B' KVY 2 - R 15 dimer connected at the two A rings via an OCH2O Z - A A | B ) bridge ; and wherein Z * * (Win W ) n A A ' and B ' are optional aromatic rings fused to ring A , only one of which may be present at a time, so as to form a fused 20 tricyclic system ; and wherein Y is N -oxide , N - loweralkyl, or CH ; when B ' is present, the group Z is present, and X is S or CH = CH ; or when A ' is present, the group Z is absent ; and Y is N and X is N = C or CH = CH ; wherein Z and Z are H , OR , NHR , or NRR ; or one carbon of ring A may be replaced by an N -oxide Z is a cyclic dietherified dihydroxyborane group attached to 25 moiety ; ring A via the boron atom ; the dotted line in ring B is an optional double bond ; Z " is O , S , NH , NHR , or N = N ; if X is N = C , ring C can optionally be attached at the each W is independently H , halo , C1- galkyl , C2- 20alkenyl, carbon atom of the N = C moiety ; and hydroxyl, or C1- galkoxy ; A is an anion , present when a quaternary nitrogen is each of K ', K², Kº, and K4 are independently CH , N , present; or a salt thereof; with the proviso that W is not N - oxide, or N - loweralkyl; hydrogen when rings A and B form a naphthalene ring R is H , C1- 20 alkyl, substituted C1- 20alkyl, C2- 20 alkenyl, system . substituted C2- 20 alkenyl , halogenated C2- 20alkenyl, substi Other derivatives include a compound of formula VIII: tuted halogenated C2- 20alkenyl , C2- 20alkeny1C1- 30 alkyl, 35 substituted C2- 20alkeny1C1 - 20alkyl , C3- 20alkynyl, substituted C3- 20alkynyl , C3- 20alkyny1C2- 20alkenyl, substituted VIII C3- 20alkyny1C2- 20alkenyl, C3- 20cycloalkyl , substituted C3- 20cycloalkyl, C6- 30aryl, heteroaryl, C6- 30arylC1- 20alkyl, substituted C6- 30aryl substituted heteroaryl, substituted 40 C6- 30ary1C1- 20alkyl, C1- 20alkoxycarbonyl, C6- 10aryl C1- 20alkoxycarbonyl, C6- 30 arylthioC1- 20alkyl , hydroxy C1- 20alkyl, triC1- 20ammoniumC1- 20 alkyl, heteroaryl RI C1- 2oalkyl, substituted heteroary1C1- 20alkyl, heteroaryl hav koko ing quaternary nitrogen , heteroarylcarbonyl having quater - 45 nary nitrogen , N -methyl - tetrahydropyridinyl , pentafluoro phenylsulphonyl, and M + when Z " is oxygen , wherein M is COOR2 an alkali metal ; wherein the alkyl, cycloalkyl , alkenyl, X and / or alkynyl groups may be optionally substituted by one hin more C1- 20alkyl, halo , hydroxyl , acetyl, amino , lower alky - 50 lamino , lower alkynylamino , imidazolinylmethylamino , di lower alkylamino , di- lower alkynylamino , piperidino , pyr wherein rolidino , azetidino , aziridino , di- imidazolinylmethylamino , n = 0 or 1 and when mercapto , C1- 20alkylthio , n + 0 , then X = S , and is a single bond ; or when C6 -30 arylthio , or trifluoromethyl groups; and each group 55 n = 1 , then X = CH , and a double bond ; R is defined independently if more than one is present; Ri = H , F , or OH : Q is ( C = O ) , or (CH2 ) n , R2= H ,methyl , ethyl, propyl, butyl, benzyl, hydroxyethyl, n is 0 , 1 , or 2 ; or an ester of hydroxyethyl; and wherein Rz and R4 are independently H , methyl, ethyl , propyl, when Z is amino , one or both of the hydrogens may be 60 allyl , imidazolinylmethyl, or replaced by C1-20alkyl , or the group L , wherein Rz and R4 together with the nitrogen atom to which they L is an amino acid radical, a peptide radical having up to are attached form a piperidino , pyrrolidino , azetidino, or 20 amino acid moieties , or may be any other small molecule aziridino ring ; that is a substrate for a nonluciferase ; R , and Ro are independently H or methyl; and wherein 65 R , is H or methyl; when Z is hydroxyl or amino , H may be replaced by Rg is H , methyl, hydroxyl, or acetyl ; and (HO )2P ( O ) _ OCH — , sulfo or R , is H or methyl, US 10 ,077 ,244 B2 45 46 Compounds of formula VIII may be useful as MAO Co - 3oary1C1- 20alkoxyl, heterocycle C1- 20alkyl, substituted substrates Co- 30ary1C1- 20alkoxyl, C6- 30ary1C1 - 20alkyl carbonyl , substi Yet other derivatives include a compound of formula IX : tuted C6- 30ary1C1- 20alkyl carbonyl or additional unsubsti tuted R groups ; and wherein each group R is defined 5 independently if more than one is present ; IX wherein heterocycle C1- 20alkyl is optionally substituted N N . RoRO with one or more , e. g ., 1 , 2 , 3 , 4 , or 5 , R groups ; Rl is hydrogen or Cl- alkyl ; RI 10 Q is CEO ) or CH2, 10 n is 0 , 1 , or 2 ; and wherein wherein Rl is H , OR , NH - C (O ) O -benzyl , or NHO when Z or Z " is amino , one or both of the hydrogens of iso - butyl ; the amino group may be replaced by C1- 20alkyl, or the group R is lower alkyl, benzyl, 2 , 4 , 6 - trimethylbenzyl, phe 15 L , wherein nylpiperazinobenzyl, 6 - trifluoromethylbenzyl, or 3 - picoli L is an amino acid radical , a peptide radical having up to nyl; 20 amino acid moieties, or any other small molecule that is R™ is a carboxyl group esterified by lower alkyl, 3 - picoli1 . a substrate for a nonluciferase ; with the proviso that when L nyl, ethylene glycol when Ris H or OR , or Rºis carboxyl is an amino acid radical or a peptide radical, W is not H ; when Rl is NH - C ( O ) - O -benzyl or NH – O - iso - butyl or 20 a when R is OR wherein R is 2 ,4 ,6 - trimethylbenzyl, phe when Z is hydroxy ; or amino , H of the hydroxyl or amino nylpiperazinobenzyl, o - trifluoromethylbenzyl. Such deriva may be replaced by (HO ) 2P (O ) _ OCH — , sulfo , tives may be useful as P450 substrates . - POZH , , or by a cephalosporanic acid attached to the Also provided is a compound of formula X : group Z via a carbon chain of one or more carbon atoms; with the proviso that when ring B is a thiazole ring , the sulfo or the - POZH , group is attached to the hydroxyl oxygen via a loweralkylene chain ; and B ' when Z or Z ' is hydroxyl or amino or when Z " - R is 20 hydroxyl , one H of the hydroxyl or amino may be replaced by the group L ' - linker, wherein L ' is a group removable by ZT A | A an enzyme to free the linker , and linker is carbon chain that can self - cleave , optionally interrupted by one or more nitro W (Win W ) n A gen atoms, oxygen atoms, carbonyl groups , ( substituted ) 35 aromatic rings, or peptide bonds, and wherein linker is attached to L ' via an oxygen atom or an NH group Y is N , N - oxide, N - loweralkyl, or CH ; at one terminus of the linker and the other terminus of the X is S , CH = CH , or N = C , linker forms an ether , ester , or amide linkage with the group Z and Z ' are independently H , OR , NHR , or NRR . Z , Z' , or Z " — R ; and Z " is O , S , NH , NHR , or N = N ; 40 when Z is OR , formula X can optionally be a dimer each W is independently H , halo , Cl- alkyl , C2- 20alkenyl, connected at the two A rings via a CH , or CH2- C6H4 hydroxyl, or C1-talkoxy ; or CH , bridge , and the R group of each Z group connecting the W and Z are both keto groups on ring A , and the dotted dimer of formula X is replaced by the bridge ; and lines in ring A are absent; wherein each of K , K™, K , and K * are independently CH , N , 45 A ' and B ' are optional aromatic rings fused to ring A , only N -oxide , or N - loweralkyl, and the dotted lines between K one of which may be present at a time, so as to form a fused and K ? , and K and K4 denote optional double bonds ; tricyclic system ; and R is H , amino , C1- 20alkyl , C2- 20alkenyl, halogenated C2- 20alkenyl , C3- 20alkynyl, C2- 20alkeny1C1 - 20alkyl, when B ' is present, the group Z is present, and C3- 20alkynylC2- 20 alkenyl, C3- 20cycloalkyl, C6- 30aryl, het - 50 when A ' is present, the group Z is absent ; and eroaryl, C6-30 ary1C1- 20alkyl, C1-12alkoxy , C1- 20alkylsulfoxy , wherein C6 -30arylsulfoxyC1 - 20alkyl, C1- 20alkylsulfoxyC1- 20alkyl, one carbon of ring A may be replaced by an N -oxide C1- 20alkoxycarbonyl , C6- 30 ary1C1- 20alkoxycarbonyl , moiety ; C6- 30arylthioC1 - 20alkyl, hydroxyC1- 20alkyl, triC3- 20ammo the dotted line in ring B is an optional double bond : niumC1- 20alkyl, heteroarylsulfoxy, heteroarylC1- 20alkyl , 55 if X is N — C . ring C can optionally be attached at the heteroaryl having quarternary nitrogen , heteroarylcarbonyl having quaternary nitrogen , N -methyl - tetrahydropyridinyl; carbon atom of the N = C moiety ; and or M * and Z " is oxygen , wherein M is an alkali metal; A is an anion , present when a quaternary nitrogen is wherein any alkyl, alkenyl , alkynyl, cycloalkyl, alkoxy, present; or a salt thereof, with the proviso that W is not aryl, or heteroaryl groups of R can be optionally substitutedin 60 hydrogen when the compound to which W is attached is by one or more , e . g . , 1 , 2 , 3 , 4 , or 5 , C1- 20alkyl, halo , luciferin , luciferin methyl ester , or aminoluciferin or when hydroxyl, acetyl, COOR ', - SO3R ' , amino , nitro , lower rings A and B form a naphthalene or quinoline ring system , alkylamino , lower alkynylamino , imidazolinylmethylamino , with the proviso that - Q -Z " - R is not - C (O ) - NH di- lower alkylamino , di - lower alkynylamino , piperidino , NH2 when a ring A substituent is OH . pyrrolidino , azetidino , aziridino , di- imidazolinylmethyl- 65 In one embodiment, the W group attached to ring C is amino , mercapto , C1- 20alkylthio , C6- 30arylthio , trifluorom - absent ( i. e . , the value of “ n ” is 0 ) . In another embodiment, ethyl, C1- 20alkylcarboxyl, C6- 30aryl , substituted C6- 30aryl, the W group attached to ring C is H or F . US 10 , 077 , 244 B2 47 48 Further provided is a compound of formula XI: 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 nitrogen XI atoms, oxygen atoms, carbonyl groups , ( substituted ) aro 5 matic rings, or peptide bonds , and linker is attached to L ' via an oxygen atom or an NH group -R at one terminus of the linker and the other terminus of the ZA À A linker forms an ether , ester, or amide linkage with the group Z , Z ', or Z " _ R ; and ?si== 10 ? WW ( W ) n when Z is hydroxyl, formula XI includes a luciferin dimer connected at the two A rings via an OCH , O bridge, and wherein wherein A ' and B ' are optional aromatic rings fused to ring A , only Y is N -oxide , N - loweralkyl, or CH ; 15 one of which may be present at a time, so as to form a fused X is S or CH = CH ; or tricyclic system ; and Y is N and X is N = C or CH = CH ; when B ' is present, the group Z is present, and Z and Z are H , OR , NHR , or NRR ; or when A ' is present, the group Z is absent; and Z is a cyclic dietherified dihydroxyborane group attached wherein to ring A via the boron atom ; 20 the dotted line in ring B is an optional double bond ; Z " is O , S , NH , NHR , or N = N ; if X is N = C , ring C can optionally be attached at the each W is independently H , halo , C1- galkyl , C2 -20alkenyl , carbon atom of the N - C moiety ; and hydroxyl, or Cl- balkoxy ; A is an anion , present when a quaternary nitrogen is each of K , K , Kº , and K * are independently CH , N , present; or a salt thereof; with the proviso that W is not N -oxide , or N - loweralkyl, and the dotted lines between K ' 25 hydrogen when rings A and B form a naphthalene ring and K ? , and K and K4, denote optional double bonds ; system . In one embodiment , the W group attached to ring C R is H , C1- 20 alkyl, C2- 20alkenyl, halogenated C2- 20alk - is absent ( i. e ., the value of “ n ” is 0 ) . In another embodiment enyl, C3 -20alkynyl , C2- 20alkeny1C1- 20alkyl , C3- 20alkynyl the W group attached to ring C is H or F . C2- 20alkenyl , C3- 20cycloalkyl, C6- 30aryl , heteroaryl, Also provided is a compound of formula XII: C6- 30ary1C1- 20alkyl, C1- 20alkylsulfoxy , C6- 30 arylsulfoxy, 30 C6 -30arylsulfoxyC1 - zoalkyl , C1- 20alkylsulfoxyC1- 20alkyl, C1 - 20alkoxycarbonyl, C6 - 30ary1C1- 20alkoxycarbonyl, XII C6- 30arylthioC1- 20alkyl , hydroxyC1- 20alkyl, triC1- 20ammo niumC1- 2oalkyl, heteroaryl - sulfoxy, heteroarylC1- 20alkyl, do heteroaryl having quaternary nitrogen , heteroarylcarbonyl 35 i N having quaternary nitrogen , N -methyl - tetrahydropyridinyl; ? or M * when Z " is oxygen , wherein M is an alkali metal; ZA wherein the alkyl, alkenyl , alkynyl , cycloalkyl , aryl, and k ' w 1 heteroaryl groups of R can be optionally substituted by one ? (W )n or more , e. g. , 1, 2 , 3, 4 , or 5 , C1- 20alkyl, halo , hydroxyl, 40 acetyl, COOR ', SO3R ', amino , nitro , lower alky wherein lamino , lower alkynylamino , imidazolinylmethylamino , di Y is N , N -oxide , N - loweralkyl, or CH ; lower alkylamino , di- lower , alkynylamino , piperidino, pyr X is S , CH = CH , or NEC , rolidino , azetidino , aziridino , di- imidazolinylmethyl- amino , Z and Z are independently H , OR , NHR , or NRR ; mercapto , C1- 20alkylthio , C6- 30arylthio , trifluoromethyl, 45 Z " is O , S , NH , NHR , or N = N ; substituted C6- 3oaryl , C1- 20alkylcarboxyl , substituted each W is independently H , halo , C1- talkyl , C2- 20alkenyl , C6 -3oaryl , substituted Co- 30ary1C1- 20alkoxyl, substituted hydroxyl , or C - alkoxy ; or C6- 3oary1C1 - 20alkyl carbonyl or additional unsubstituted R W and Z are both keto groups on ring A , and the dotted group ; and each group R is defined independently if more lines in ring A are absent; than one is present; 50 each of K1, K², K ’, and K4 are independently CH , N , R is hydrogen or C1- galkyl ; N -oxide , or N -loweralkyl , and the dotted lines between K Q is C ( O ) or CH2; and K ? , and K and K4, denote optional double bonds; each n is independently 0 , 1 , or 2 ; R is H , C1- 2oalkyl, C2- 20alkenyl, halogenated C2- 20 alk and wherein enyl, C3 - 20alkynyl, C2- 20alkeny1C1- 2oalkyl, C3- 20alkynyl when Z is amino , both of the hydrogens of the amino 55 C2- 20alkenyl, C3- 20cycloalkyl, C6- 30aryl , heteroaryl , hetero group may be replaced by C1- 20alkyl , or the group L , cyclic , substituted heterocyclic , C6 -30ary1C1 - 20alkyl , wherein C1- 20alkylsulfoxy, C6- 30arylsulfoxy, C6- 30arylsulfoxy L is an amino acid radical, a peptide radical having up to C1-20alkyl , C1- 20alkylsulfoxyC1- 20alkyl, C1- 20alkoxycarbo 20 amino acid moieties , ormay be any other small molecule nyl, C6- 30ary1C1- zoalkoxycarbonyl , C6- 3oarylthioC1- 20alkyl , that is a substrate for a nonluciferase ; 60 hydroxyC1- 20alkyl , triC1- 2oammoniumC1- 2oalkyl, heteroar and wherein ylsulfoxy, heteroary1C1- 20alkyl, heteroaryl having quater when Z or Z ' is hydroxyl or amino , H of the hydroxyl or nary nitrogen , heteroarylcarbonyl having quaternary nitro amino may be replaced by (HO ) P (O ) - OCH — , sulfo , gen , N -methyl - tetrahydropyridinyl ; or M + when Z " is - POZH2, or by a cephalosporanic acid attached to the group oxygen , wherein M is an alkali metal ; Z via a carbon chain of one or more carbon atoms; and 65 wherein the alkyl, alkenyl, alkynyl , cycloalkyl, aryl, and when Z is hydroxyl or amino or when Z " / R is hydroxyl, heteroaryl groups of R can be optionally substituted by one one H of the hydroxyl or amino may be replaced by the or more , e . g . , 1 , 2 , 3 , 4 , or 5 , C1- 20alkyl, halo , hydroxyl , US 10 ,077 , 244 B2 49 50 acetyl, — COOR , SO3R ', amino , nitro , lower alky - luciferin , luciferin methyl ester , or aminoluciferin or when lamino , lower alkynylamino , imidazolinylmethylamino , di- rings A and B form a naphthalene or quinoline ring system . lower alkylamino , di- lower alkynylamino , piperidino , pyr - In one embodiment, the W group attached to ring C is rolidino , azetidino , aziridino , di- imidazolinylmethyl - amino , absent (i . e. , the value of “ n ” is 0 ) . In another embodiment, mercapto , C1- 20alkylthio , C6- 3oarylthio , trifluoromethyl, 5 the W group attached to ring C is H or F . C1- 20alkylcarboxyl, C6- 30aryl , substituted C6- 30 aryl, C6- 30ary1C1- 20alkoxyl, substituted C6- 3oary1C1 - 20alkoxyl, IV . Exemplary Luciferin Derivatives Co- 30ary1C1- 20alkyl carbonyl, substituted C6- 30aryl C1- 2oalkyl carbonyl or additional unsubstituted R groups ; Derivatives having a six membered B ring include : and wherein each group R is defined independently if moreNore 10" than one is present; wherein substituted aryl groups are substituted with one compound of formula (XIX ) or alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclic , LJX HN substituted heterocyclic , and heteroaryl groups of R can be 15 optionally substituted by one or more C1- 20alkyl, O Co- Cloaryl , halo , hydroxyl, acetyl, — COOR ' , amino , nitro , lower alkylamino , lower alkynylamino , imidazolinylmeth ylamino , di- lower alkylamino , di- lower alkynylamino , pip Y - R eridino , pyrrolidino , azetidino , aziridino , di- imidazolinylm - 20 ethylamino , mercapto , Cl- 20alkylthio , C6- 30arylthio , or wherein L may be any structure for an enzyme , such as a trifluoromethyl groups ; peptide sequence or small molecules : X is O , NH , or a R ! is hydrogen or C1- alkyl; self- cleavable linker ; Z is N (aminoquinolinyl luciferin ) or Q is CCO) or CH2; CH (naphthyl luciferin ), Y is 0 ( ester ), NH ( amide ), NH n is 0 , 1, or 2 ; 25 NH (hydrazide ) , or S (thioester ); and R may be alkyl, and wherein aromatic , a peptide , an oligonucleotide , or a self -cleavable when Z or Z " is amino, one or both of the hydrogens of linkerlink attached to a substrate for an enzyme. the amino group may be replaced by C1- 20alkyl, or the suupgroup L , wherein In one embodiment, a derivative may be employed to L is an amino acid radical, a peptide radical having up to 30 detect N -dealkylation , e .g ., by cytochrome (CYP ) P450 20 amino acid moieties, or any other small molecule that is isozymes . In the case of an ester ( R — alkyl chain ) , an a substrate for a nonluciferase ; with the proviso that when L esterase may be added after the dealkylation reaction is is an amino acid radical or a peptide radical, W is not H ; commenced , or at the time the dealkylation reaction is and wherein initiated , and before a luciferase -mediated reaction is initi when Z is hydroxyl or amino , H may be replaced by 35 ated . (HO ) P ( O ) OCH — , sulfo , or - POZH2, or by a cepha losporanic acid attached to the group Z via a carbon chain of one or more carbon atoms; with the proviso that when ring B is a thiazole ring , the sulfo or the - POZH2 group is ? attached in the hydroxyl oxygen via a lower alkyl chain ; and 40 1) CYP N - Dealkylation when Z or Z ' is hydroxyl or amino or when Z " - R is 2 ) Esterase hydroxyl, one H may be replaced by the group L ' - linker, SOR- R wherein L ' is a group removable by an enzyme to free the linker, and linker is carbon chain that can self -cleave , compound of formula XX optionally interrupted by one or more nitrogen atoms, oxy - 45 H2N gen atoms, carbonyl groups , (substituted ) aromatic rings , or peptide bonds , and linker is attached to L ' via an oxygen atom or an NH group at one terminus of the linker and the other terminus of the OH linker forms an ether, ester, or amide linkage with the group 50 Z , Z ' , or Z " - R ; and compound of formula XXI when Z is hydroxyl, formula XII includes a luciferin L = alkyl dimer connected at the two A rings via an _ OCH , 0 Z = N or CH bridge ; and R = H or alkyl wherein 55 A ' and B ' are optional aromatic rings fused to ring A , only Although light output with 6 -aminoquinolinyl luciferin one of which may be present at a time, so as to form a fused ( formula V ) is pH sensitive , reactions including control tricyclic system ; and reactions may be conducted at the same pH . Alternatively , when B ' is present, the group Z is present, and 6 -aminoquinolinyl luciferin may be modified so as to alter when A ' is present, the group Z is absent ; and 60 electron density distribution of the aromatic ring, for wherein example , with halogenation . the dotted line in ring B is an optional double bond ; Derivatives of the invention include fluorinated luciferin , if X is N = C , ring C can optionally be attached at the quinolinyl luciferin , aminoquinolinyl luciferin , and naphthyl carbon atom of the NFC moiety ; and luciferin . For instance , the optical properties of fluorinated A is an anion , present when a quaternary nitrogen is 65 derivatives of aminoquinolinyl luciferin may be altered due present; or a salt thereof , with the proviso that W is not to the electron withdrawing power of fluorine . Fluorinated hydrogen when the compound to which W is attached is derivatives include compound of formula XXII and XXIII : 51 US 10 ,077 , 244 B2 XXII HO 54' HX 6 w N LCOOH F81 OOH "; 10 wherein X = O or NH ; Y = N or CH ; and F may be at 3' , 4 , N 5 ' , 7 ', or 8 -O er NIHY= N ex Cits and may be a3 , 40 ; " W i coDOH XXIII 15 HN HX6 COOH 55 ' N DOH 20 rodinyF 4' 3' ovoce wherein X = O or NH ; and F may be at 4 ', 5 ', or 7 ' . wherein OH is at positions 6 ' and/ or 8 ' An exemplary fluorinated derivative is 5 - fluoro - 6 -amino - 25 Derivatives of the invention may be employed as sensors luciferin ( FIG . 3 ) . for reactive oxygen species (ROS ) or a redox sensor for In one embodiment, the invention provides for quinolinyl horseradish peroxidase (HRP ) and other enzymes. Those derivatives ( compounds of formula XXIV - XXVI ): derivatives include compounds of formula XXVII -XXXII : tron OH 1 ) H2O2 tendentecompound of formula XXVII - N OH 2 ) Luciferase Light HN aminoluciferinxph . .. HX OH 1 ) AROS O axshcompound of formula XXVIII US 10 ,077 , 244 B2 5454 -continued N N , OH 2 ) Luciferase Light HO ss- luciferin ( H .NH2 ???? 1 ) H202 compound of formula XXIX 0H 2 ) Luciferase?? Light HO ?????luciferin ??? F 0 - OH F 1 ) H202 - ?? ?????compound of formula XXX 0H 2 ) Luciferase Light HO luciferin 7 OH 1 OH radical 0H 2 ) Luciferase Light HO ???compound of formula XXXI ?? luciferin??? ????? H ( 2 OH 1 ) Reduction 0H 2 ) Luciferase Light H ( ?????compound of formula XXXII ????5 'hydroxyluciferin ? US 10 , 077 ,244 B2 55 56 Exemplary bioluminogenic MAO substrates include : compound of formula XXXV compound of formula XXXIII COOH 5 5 R2 COOR 10 x = HO X compound of formula XXXVI COOH wherein n = 0 and X = S or n = 1 and X = CH ; ?15 wherein R = H , F , or OH ; wherein R2 , CH3, CH2CH3, CH2CH2CH3, CH2CH (CH3 ) 5 2 , CH Ar, CH2CH2OH , or any ester ; - S wherein Rz= H , CH3, CH2CH3, CH2CH2CH3, H2C HO 6 1 C = CH , — H , C 20 Other A ring modifications include a stable substrate for phosphatase which may also be a luciferase substrate (com of pound of formula XXXVII ) : or Rz = ( CH2) mR4 : R ; = R2 = CH , and n + 0 , 1 , 2 , or 3 ; compound of formula XXXVII wherein R , and R . are independently H or CH3; COOH wherein R = H or CH3; 30 HO . wherein R = H , CH3, OH , or COCHZ. HO Exemplary bioluminogenic FMO substrates include formula XXXIV " Lace The C ring of luciferin may be modified to include a 35 substrate for acetylcholinesterase ( ACh ) , which optionally has a modified A ring or a modified C and A ring . ACh has a very high turnover number (25 ,000 s ) and has attained kinetic perfection as indicated by its kcal/ Km value of 2x108 N COOR2 40 M - 's - 1 . Two types of derivatives which are substrates for ACh include ( compounds of formula XXXIX -XXXX ) : R2 - S xBn S compound of formula XXXVIII wherein n = 0 and X = S or wherein n = 1 and X = CH ; 4545 wherein R , = H , F, or OH ; F wherein Rz = H , CH2, CH2CH3, CH CH CH3, CH CH . CH ( CH3) 2 , CH Ar, CH2CH2OH , or any ester; wherein Rz = H , CH3, CH2CH3, CH2CH2CH3, any alkyl 50 HX chain , est or com wherein X = O or NH R, 55 compounddof of formulaformula XXXXIX R2 60 wherein R ,' , R2' , H , F , CI, Br, CN , NO2, CH3, OCH3, NH2, or N (CH3 ) 2 ; HX wind wherein Rs and Ro are independently H or CHz. The A ring of D - luciferin may also be modified to include 65 anotherCara ring structure memasane( compound of formulas. co . XXXV me . wherein X = O or NH , and XXXVI) : Y = N or CH US 10 , 077 ,244 B2 57 58 - continued ChE - Ser - OH compound of formula XXXXVI ?? Qurangka personen 5 ?? compound of formula XXXX wherein X = 0 or NH O 2 ) Hydrolysis N N Ser- ACHE 3 ) Luciferase 10 e n XH?? is Desde compound of formula XXXXVII compound of formula XXXXI Light 15 Derivatives useful to detect B - lactamase -mediated reac tions include quinolinyl luciferin , aminoquinolinyl luciferin and naphthyl luciferin derivatives, optionally also modified 20 with a halogen , e .g ., a A ring modification that replaces a H linked to a C ring atom with a F . COOH beta - lactamase + OH compound of formula XXXXII OH LE HZ - 70 COOH compound of formula XXXXIII 40 mercado wherein R = different group , X = N or CH , Y = S or -continued CH = CH , andthe sonxen en el compound of formula XXXXIII Z = 0 or NH Derivatives which include esters of luciferin ( a C ring 45 modification ) optionally with a A ring modification or a B ring modification , or a A and a C ring modification , include compound of formulas XXXXIV - LVIII : 50 coding compound of formula XXXXIV compound of formula XXXXIX 55 compound of formula L compound of formula XXXXV 60 OH N com 65 HO cabing US 10 , 077, 244 B2 59 60 -continued - continued compound of formula LI 0H HO S ' s L -luciferin methyl ester compound of formula LII 1 ) To prepare esters for a derivative of luciferin that are substrates for enzymes including P450 , MAO and GST, a cyclization procedure , may be employed with TCEP to 15 reduce ethylene glycol ester D -cysteine before adding to a cyano - containing molecule , is utilized . For instance , to a solution of D - cysteine ester neutralized with potassium bicarbonate , add TECP . After a few minutes , add the solu tion to the appropriate nitrile dissolved in methanol. Also , compound of formula LIII 20 methyl ester luciferin derivatives are made by direct cycl ization of benzothiole derivatives with methyl ester D -cyteine or methylation of carboxylic acids of luciferin with diazomethane . 25 ??compound of formula LIV. 0H 3 ) 27 HO luciferin 35 formula LIX ?? ????H3N . OH compound of formula LV 40) ?? NIO 45 H , N "OH compound of formula LVII formula LX ? 56 ) HS . 111111 CH3 55 Derivatives of luciferin with A , B and/ or C ring modifi cations include: ( compounds of formula LXI- LXX below ) ????compound of formula LVIII 6 ) OCH , COH ????65 H , CO US 10 ,077 , 244 B2 61 62 - continued -continued COOH N COOH Me0 " NMe2 ???X = 0 orS 10 COOH ??? COOH Me0 HO COOH COOH 20 HC HO) 25 COOH NMe2 - ( H 30 COOH Bn0 . 35 COOH ?? 40 Yet further derivatives include compounds of formula LXXI, LXXII and LXXIII (below ) H2 20 HO0C COOH COOR2 ? ? US 10 , 077 ,244 B2 63 64 wherein R?H , F , or OH ; wherein Rs and Rg are independently H or CHz. LXXVIII In one embodiment, the derivative is a GST substrate ( formula LXXIV -LXXV ) 5 HN N COOR2 Rs LXXIV xen 's 10 Re LP R3 N COOR2 RzZ GST /GSH x =tln ' s wherein n = 0 and X = S or n = 1 and X = CH ; á 15 wherein Ri = H , F , or OH ; wherein Rz = H , CH3, CH2CH3, CH2CH2CH3, CH CH (CH3 ) 2, CH2, Ar, CH ,CH OH , or any ester ; wherein Rz, Rz', R4 independently are NO2, CF3, or H . 20 ?? COOR LXXIX + R4 R3 = Rz' x = + An 25 HN ACOOR @ N R32 = 30 Xin =V n = 0 , X = S n = 1 , X = CH 35 wherein n = 0 and X = S or n = 1 and X = CH ; Y = 0 , - 0802 — , - OP ( O )OR wherein Ri= H , F , or OH ; ( R = any alkyl or aromatic ester ) = H , F , OH wherein Rz = H , CH2CH3, CH2CH2CH3, CH2CH (CH3 ) 2, R2 = H , CH3, CH2CH3, CH2CH2CH3, CH2CH (CH3 ) 2 , CH Ar, CH2CH2OH , or any ester R3 = R3' , R4 = NO2, CF3, H CH Ar, CH2CH2OH , or any ester ; R , R6 = H , CH3 40 wherein R3, R3', R4 are independently NO2, CF3, or H Z = CH , N Exemplary phosphatase and sulfatase substrates include formulas LXXX -LXXXIII : wherein n = 0 and X = S or n = 1 and X = CH ; wherein R = H , F , or OH ; 45 HO OH wherein Rz = H , CH3, CH2CH3, CH2CH2CH3, CH CH O = P (CH3 ) 2, CH Ar; CH2CH2OH , or any ester ; COOR2 wherein R3, R3' , R4 independently = NO2, CF3, or H . 50 - Xin LXXVLXXV HOO 0 = S COOR2 55 ?žeCOOR2 Xin X + An Zain R3Rz' de6060 Ere wherein n = 0 and X = S or n = 1 and X = CH ; wherein R = H , F, or OH ; COORZ wherein Rp = H , CH? , CH?CH , CH?CH , CH, CH?CH , O P Ž (CH3 ) 2, CH Ar, CH2CH2OH , or any ester ; HO OH wherein R3, Rz' or R4 independently = NO2, CF3 , or H . US 10 ,077 , 244 B2 65 66 - continued - continued LXXXIX COOR ? COOR2 H0 0 ? Cl - ( X - Fn s wherein n = 0 and X - S or n = 1 and X - CH ; 10) wherein R - H , D , or OH ; wherein R - H , CH , CH ,CH , CH ,CH ,CH , CHCH wherein n = 0 and X - S or n = 1 and X - CH ; ( CH ) , CHAr, CH , CH2OH , or any ester. wherein R = H , F , or OH ; wherein R , H , CH , CH , CH , CH , CH , CH , CH , CH LXXXIV (CH ) , CH2Ar, CHCH , OH , or any ester ; HO | OH whereinR H , F , Cl, Br, CN , NO , CH , OCH , NH ) , or 0 = p 20 N (CH , )2 ; or HN COOR2 wherein Rs and Rg are independently H or CH3. ? Derivatives for use in redox or dealkylase bioluminescent sr ? assays include compounds of formulas LXXXX -LXXXXI : 25 LXXXV LXXXX HO 0s N , COOR 30 ??? ???? 35 LXXXVI LXXXXI COOR2 OR 4 { ?? N HOOH ?? LXXXVII A5 COOR Specific derivatives useful as reduction sensors or a ? dealkylase sensor include compounds of formula HO 0 ? 30 LXXXXII/ -LXXXXVI : Other derivatives of the invention include compounds of for Redox sensor; Z = 0 formula LXXXVIII -LXXXIX : for dealkylase ; 5555 ? 7 = Me LXXXVIII LXXXXII COOR, 60 ? OH 65 Y = OH ,NH2 US 10 , 077 ,244 B2 67 68 - continued - continued N OH 5 LXXXXIII 10 Y ' LXXXXVII OH OH 15 Y = OH , NH2 N OH 20 Y = OH , NH2 2 * LXXXXIV Other examples of redox sensors are shown in FIGS . 4 and 25 6 . Other esterase substrates are shown in FIGS. 8 - 10 , 22 - 23 , OH 26 , 37B , 40 and 42 . V . Derivatives of Fluorophores 30 CroiseIn one embodiment, derivatives of a fluorophore of the invention have the following structure L - F , where F is a OH fluorophore and L is a substrate linked via O to the fluoro 36 phore . In one embodiment, fluorescent substrates according to the present invention are benzopyran derivatives having NZ the general formula XIII : LXXXXV XIII 40 24 W3 ?? 21 - 0 W2 yol AS Y = OH , NH wherein xona the dashed ring is an optionally present benzo ring ; 50 the dashed bond in the B ring is present only when the OH dashed ring is absent; X ' is CH when the dashed ring is absent; X ' is NH when the dashed ring is present; X ' is a carbon atom when the dashed ring is present and X ' LXXXVI 55 forms part of a spiro ring system which is a y -butyrolactone ring having an optionally substituted benzo ring fused at the avoin a and ß carbons of the lactone ring and attached to X ' at the y carbon ; Wl, W3, and W4 are independently H , halo , carboxyl, 60 carboxy ester , loweralkyl, hydroxyloweralkyl, C6 -20aryl , or substituted C6- 20aryl ; W2 is hydroxyl, loweralkoxy, or " ?? amino, wherein one or both amino hydrogens may be replaced by lower alkyl; z is a lower alkylene chain terminated by an amino group , 65 a loweralkylamino group , a diloweralkylamino group , a Y = OH , NH2 thiol group , or a lower alkylthio group ; and W2 is alkoxy, or DusliOCH (R4 ) CH ( R ) CH (R2 ) N ( R3R4 ) ; and US 10 ,077 , 244 B2 69 70 Z ? is a keto group present only when the benzo ring is -continued absent. CII Exemplary fluorogenic MAO substrates include formulas LXXXXVIII- C : 5 LXXXXVIII Ray R7 R3N 12 CIII LXXXXIX 25 DLANRO 30 R1, R2, R2 = H , F , C1, Br, I, COOH , COOR (any ester ) H?N R3, R = H , CH3, CH2CH3, CH2CH2CH2, - H ,CECH , 35 0 or Rz( C2) , R _ R = RACH , , n = 0 , 1 , 2 , 3 , R = H , CH2 R = H , CH3, OH , COCHZ R , = H , CHZ 45 R = Alkoxyl ( any ) , or symetric chain OCH (R7 )CH (R8 ) CH (R9 ) N (R3R4 ) Meo See FIGS. 5 and 11 for exemplary fluorogenic substrates for redox and MAO reactions . - NH2 Any fluorophore may be employed to prepare a fluoro 50 genic substrate, fluorophores including but not limited to fluorescein , Texas Red , DAPI, PI, acridine orange , Alexa Fluorogenic FMO substrates include formulas CI- CIII : fluors , e . g ., Alexa 350 , Alexa 405 or Alexa 488 , cyanine dyes , e . g . , Cy3 , coumarin , ethidium bromide , fluorescein , BODIPY, a rhodol, Rox , 5 - carboxyfluorescein , 6 -carboxy fluorescein , and anthracene , 2 -amino -4 -methoxynaphtha CI 55 lene , a phenalenone , an acridone , fluorinated xanthene derivatives, a -naphtol , B - napthol, 1 -hydroxypyrene , cou marins , e . g ., 7 -amino -4 -methylcoumarin (AMC ) or 7 -amino - 4 - trifluoromethylcoumarin (AFC ) , rhodamines , e . g ., tetramethylrhodamine , rhodamine - 110 , or carboxyrho 60 damine , cresyl violet , or resorufin , as wells as fluorophores Rom disclosed in U . S . Pat. No . 6 , 420 , 130 , the disclosure of which R7 is incorporated by reference herein . VI. Linkers 65 A linker strategy may be employed for either the A or C ring modified luciferins, or with fluorophore containing US 10 ,077 ,244 B2 72 derivatives, to introduce a substrate for an enzyme of interest wherein R is as defined for any one of formulas I - III or such as a deocetylase , deformylase, demethylase or other enzyme that can remove the L group of formula I ( a substrate V -XII , which is a group removable by an enzyme, e .g ., an for that enzyme ) to free the linker, yielding a substrate of enzyme that is being assayed ; the quinine methide linker luciferase, or a prosubstrate , where the remaining linker may 5 replaces a hydrogen atom of one of the groups Z , Z " , or optionally be removed by a nonenzymatic reaction . Linkers can be alkyl or alkoxy chains, such as ( C . - C . ) Z " - R ; and ' leaving group ' is the remainder of the structure alkyl or (C . - C . Jalkoxy groups. The chain can have one or of formula I - III or V - XII . See Greenwald et al. , J. Med . more election withdrawing group substituents R , such is an Chem . 42 :3657 ( 1999 ) and Greenwald et al. , Bioconjugate aldehyde , acetyl, sulfoxide , sulfone , nitro , cyano group , or a combination thereof. Other linkers include trimethyl lock , 10 Chem ., 14 : 395 ( 2003 ) for the use of quinine methide linkers . quinine methide and diketopiperazine linkers , and their diketopiperazine linker can be illustrated as follows derivatives . A trimethyl lock linker can be illustrated as follows: 1515 CIV leaving group CVI 20 R RP N x leaving group © RR 25 wherein R is as defined for any one of formulas I- III or V - XII , which is a group removable by an enzyme, e . g ., an enzyme that is being assayed ; the trimethyl lock linker replaces a hydrogen atom of one of the groups Z , Z " , or Z " — R , and ‘ leaving group ' is the remainder of the structure 30 of formula I - III or V - XII . See Wang et al ., J . Org . Chem ., 62 : 1363 ( 1997 ) and Chandran et al, J. Am . Chem . Soc. wherein R is as defined for any one of formulas I- III or V -XII 127 : 1652 ( 2005 ) for the use of trimethyl lock linkers . which is a group removable by an enzyme, e. g ., an enzyme A quinine methide linker can be illustrated as follows: that is being assayed ; each R ' of the diketopiperazine linker 35 is independently H or an alkyl chain optionally interrupted leaving group by O , S , or NH , preferably a methyl group , the diketopip erazine linker replaces a hydrogen atom of one of the groups 40 Z , Z' , or Z " _ R ; and "leaving group ' is the remainder of structure of formula I - III or V - XII . See Wei et al, Bioorg. Med . Chem . Lett. , 10 : 1073 ( 2000 ) for the use of diketopip K erazine linkers . Other linker containing derivatives include: compound of formula CVII H2N COOH NH2 wytingumoX = 0 or NH compound of formula CVIII COOH ????????? HON ???X = O or NH US 10 , 077 ,244 B2 73 74 - continued compound of formula CIX COOH N compound of formula CX econdaronNH COOH B - elimination of a product of a bioluminogenic or fluo - Any fluorogenic chromophore or luminophore rogenic reaction with a substrate having a group R that in the To detect FMO , the derivative may undergo the following product is an electron withdrawing group R , such as alde - reaction : hyde , acetyl , sulfoxide , sulfone , nitro , or cyano , may yield a 30 substrate for luciferase or a fluorophore . On Enzyme X FMO B -elimination - OH presin O + 40. O B - elimination O SEFO )n R n = 1 , 2 Any fluorogenic chromophore or luminophore For instance , to detectMAO , the derivative may undergo - OH + the following reaction . Oon ^ (O )n 50 n = 1 , 2 Ri MAO A / B 55 Any fluorogenic chromophore or luminophore Specific embodiments of reactions and derivatives having formulas CXI- CXV which employ a linker include : - OH + - H 60 L - Linker voor h COOH 1 ) Enzyme cleavage compound of formula CXIet US 10 , 077 ,244 B2 75 76 - continued75 - continued HZ Linker 2 ) self- elimination 2 ) self -elimination COOH 5 - O - Linker HZ . other COOH 10 HZ wherein L = Me for demethylase, Ac for deacetylase, or CHO - N for deformylase , 15 COOH X = S or CH = CH , Y?N or CH , and Z = O or NH ; HZ . 20 1 ) Enzyme cleavage T i O -Linker - R holatorenEnorme clave wherein R = Me for demethylase , Ac for deacetylase, or CHO compound of formula CXII for deformylase, X = S or CH = CH , Y?N or CH ,and Z = 0 " Cthem or NH 1 ) Enzyme cleavage COOH mothycompound of formula CXIII H2N elf- elimination COOH HO . COOH 50 wherein L =Me for demethylase , Ac for deacetylase , or CHO for deformylase , X = S or CH = CH , and Y = N or CH ; 1 ) Enzyme cleavage COOH compound of formula CXIV US 10 ,077 , 244 B2 77 -continued HZ COOH 2) selfself - eliminationelimination S HN COOH wherein L =Me for demethylase, Ac for deacetylase, or CHO for Quretanodeformylase, X = S or CH = CH , Y?N or CH , and Z = 0 or NH ; or 1 ) Enzyme cleavage wy .COOH compound of formula CXV za HZ HN 1 ) Self - eliminaion COOH COOH 40 wherein L = Me for demethylase, Ac for deacetylase, or CHO for deformylase, X = S or CH = CH , Y?N or CH , and Z = O XIV or NH . 4545 VII . Agents Useful to Stabilize Light Production in R1 R2 Luciferase -Mediated Reactions wherein X is S or Se ; R , and R2 are each independently Agents useful to stabilize light production in a luciferase hydrogen , (C1 - C20 ) alkyl, (C3 -Cg ) cycloalkyl, ( C , - C20 ) mediated reaction include organic compounds ( i. e . , com 50 alkoxy, (C2 - C20 )alkenyl , (C2 -C20 )alkynyl , aryl , heteroaryl, pounds that comprise one or more carbon atoms) . Such an or NR ,Rb ; or R , and R , together with the carbon to which agent may be added prior to , at the initiation of and / or during they are attached form a 5 , 6 , 7 , or 8 membered saturated or a nonluciferase enzyme-mediated reaction or a luciferase unsaturated ring comprising carbon and optionally compris mediated reaction . Suitable organic compounds can com - 55 ing 1 , 2 , or 3 heteroatoms selected from oxy ( 0 - ), thio prise a carbon - sulfur bond or a carbon - selenium bond , for ( 5 ) , or nitrogen ( NR ) , wherein said ring is option example suitable organic compounds can comprise a car ally substituted with 1 , 2 , or 3 halo , hydroxy, oxo , thioxo , bon -sulfur double bond ( C = S ), a carbon selenium double carboxy, ( C , -C20 )alkyl , (Cz - C3 ) cycloalkyl, ( C , -C20 )alkoxy , bond (C = Se ), a carbon - sulfur single bond ( C — S ), or car (C2 - C20 )alkanoyl , (C1 - C20 )alkoxycarbonyl , (C2 - C20 ) alk bon - selenium single bond ( C — Se ) . Suitable organic com - 60 enyl, (C2 - C20 ) alkynyl, aryl, or heteroaryl; and Ra, R , and R . pounds can also comprise a carbon bound mercapto group are each independently hydrogen , (C2 - C20 ) alkyl , ( Cz -C3 ) ( C — SH ) or a sulfur atom bound to two carbon atomsMUS cycloalkyl , (C2 -C20 ) alkenyl, (C1 - C20 ) alkanoyl, ( C , - C20 ) ( C - S - C ) . In one embodiment , compounds are lipophilic alkoxycarbonyl, (C2 - C20 ) alkynyl , aryl, heteroaryl ; wherein in nature . any ( C1- C20 ) alkyl, (Cz -C3 ) cycloalkyl , (C1 - C20 )alkoxy , (C2 Suitable compounds that comprise a carbon sulfur double 65 C20 )alkenyl ( C , -C20 )alkanoyl , ( C ,- C20) alkoxycarbonyl, or bond or a carbon selenium double bond include for example (C2 - C20 ) alkynyl or R1, R2, Ra, Rb, and R , is optionally compounds of formula (XIV ): substituted with one or more ( e . g . 1 , 2 , 3 , or 4 ) halo , US 10 ,077 , 244 B2 79 80 hydroxy, mercapto , oxo , thioxo , carboxy , ( C , -C20 )alkanoyl , Specifically , (C2 -C20 ) alkyl can be methyl, ethyl, propyl, ( C - C20 ) alkoxycarbonyl, aryl , or heteroaryl, and wherein isopropyl, butyl, iso -butyl , sec -butyl , pentyl, 3 - pentyl, or any aryl or heteroaryl is optionally substituted with one or hexyl ; (C3 - C )cycloalkyl can be cyclopropyl, cyclobutyl, more ( 1 , 2 , 3 , or 4 ) halo , hydroxy, mercapto , carboxy, cyano , cyclopentyl, or cyclohexyl, ( C - C20 Jalkoxy can be methoxy, nitro , trifluoromethyl, trifluoromethoxy, ( C , -C20 ) alkanoyl, 5 ethoxy, propoxy, isopropoxy , butoxy, iso -butoxy , sec -bu ( C , -C20 ) alkanoyloxy , sulfo or ( C7 - C20 ) alkoxycarbonyl; or a toxy, pentoxy, 3 -pentoxy , or hexyloxy ; (C2 - C20 )alkenyl can salt thereof. be vinyl, allyl, 1 -propenyl , 2 -propenyl , 1 -butenyl , 2 -butenyl , Suitable compounds that comprise a mercapto group 3 - butenyl, 1 -pentenyl , 2 -pentenyl , 3 -pentenyl , 4 -pentenyl , include for example compounds or the formula R SH 1 -hexenyl , 2 -hexenyl 3 -hexenyl , 4 -hexenyl , or 5 -hexenyl ; wherein ; R2 is (C7 -C20 ) alkyl, (C2 -C3 ) cycloalkyl, (C2 - C20 ) 10 (C2 - C20 )alkynyl can be ethynyl, 1 -propynyl , 2 -propynyl , alkenyl, ( C2 - C20 )alkynyl , aryl, heteroaryl; wherein any (C , 1 -butynyl , 2 -butynyl , 3 -butynyl , 1 -pentynyl , 2 -pentynyl , C20 )alkyl , (Cz - C )cycloalkyl , (C2 - C20 ) alkenyl, or (C2 - C20 ) 3 -pentynyl , 4 -pentynyl , 1- hexynyl , 2 -hexynyl , 3 -hexynyl , alkynyl or Rz is optionally substituted with one or more ( e . g . 4 - hexynyl, or 5 -hexynyl ; ( C , -C20 ) alkanoyl can be acetyl , 1 , 2 , 3 , or 4 ) halo , hydroxy, mercapto oxo , thioxo , carboxy , propanoyl or butanoyl; ( C1 -C20 ) alkoxycarbonyl can be ( C1- C20 ) alkanoyl, ( C1 -C20 ) alkoxycarbonyl, aryl, heteroaryl, 15 methoxycarbonyl, ethoxycarbonyl, ethoxycarbonyl, or NR , R .; wherein R and R are each independently hydro propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl , gen , ( C , -C20 ) alkyl, (Cz - C3) cycloalkyl, (C2 - C20 ) alkenyl, pentoxycarbonyl, or hexyloxycarbonyl; ( C2- C20 ) alkanoy (C2 -C20 ) alkynyl , (C1 - C20 )alkanoyl , (C1 - C20 ) alkoxycarbo - loxy can be acetoxy , propanoyloxy, butanoyloxy, isobu nyl aryl, or heteroaryl; and wherein any aryl or heteroaryl; tanoyloxy, pentanoyloxy, or hexanoyloxy ; aryl can be phe and wherein any aryl or heteroaryl is optionally substituted 20 nyl, indenyl, or naphthyl; and heteroaryl can be furyl, with one ormore ( 1 , 2 , 3 , or 4 ) halo , mercapto , hydroxy , oxo , imidazolyl, triazolyl, triazinyl, oxazoyl , isoxazoyl, thiazolyl, carboxy, cyano , nitro , trifluoromethyl, trifluoromethoxy, isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl , ( C , - C20 ) alkanoyl, ( C , -C20 ) alkanoyloxy, sulfo or ( C -C20 ) pyridyl, (or its N -oxide ) thienyl, pyrimidinyl (or its N -ox alkoxycarbonyl; or a salt thereof. ide ), indolyl, isoquinolyl ( or its N - oxide ) or quinolyl ( or its Other suitable compounds include for example com - 25 N -oxide ) . pounds of the formula RANCS wherein : R4 is ( C / - C20 ) alkyl, Specifically , R , and R2 can each independently be hydro ( C3 - C8) cycloalkyl, (C2 - C20 ) alkenyl, (C2 - C20) alkynyl , aryl, gen , (C , - C20 )alkyl , (C3 -C ) cycloalkyl, (C2 -C20 ) alkenyl, or heteroaryl; wherein any ( C1 -C20 ) alkyl, ( C3- C ) cy - (C2 - C20 ) alkynyl, aryl, heteroaryl, or NRRs ; wherein R , and cloalkyl, (C2 - C20) alkenyl , or (C2 - C20) alkenyl or Rz is R , are each independently hydrogen , (C ,- C20 )alkyl , (C3 -C3 ) optionally substituted with one or more ( e . g . 1 , 2 , 3 , or 4 ) 30 cycloalkyl, (C2 - C20 ) alkenyl, (C1 - C20 ) alkanoyl, (C1 - C20 ) halo , hydroxy, mercapto oxo , thioxo , carboxy, ( C , -C20 ) alkoxycarbonyl, (C2 - C20 ) alkynyl, aryl, or heteroaryl; alkanoyl, (C -C20 )alkoxycarbonyl , aryl, heteroaryl, or wherein any (C1 - C20 )alkyl , (C3 -C2 ) cycloalkyl, (C ,- C20 ) NRR ; wherein R and R , are each independently hydrogen , alkoxy, (C2 - C20 ) alkenyl (C7 -C20 )alkanoyl , ( C , -C20 ) alkoxy ( C - C20 ) alkyl, ( Cz- C3) cycloalkyl , (C2 - C20 ) alkenyl , (C2 carbonyl, or (C2 -C20 ) alkynyl or R1, R2, Ra, and Ry is C20) alkynyl , C - C20 ) alkanoyl, (C2 -C20 ) alkoxycarbonyl 35 optionally substituted with 1 or 2 halo , hydroxy, mercapto , aryl , or heteroaryl; and wherein any aryl or heteroaryl is oxo , thioxo , carboxy, (C2 - C20Jalkanoyl , (C2 - C2 . Jalkoxycar optionally substituted with one or more ( 1, 2 , 3 , or 4 ) halo bonyl, aryl , or heteroaryl; and wherein any aryl or heteroaryl mercapto , hydroxy, oxo , carboxy, cyano , nitro , trifluorom is optionally substituted with one or more halo , hydroxy , ethyl , trifluoromethoxy (C1 - C20 Jalkanoyl, (C1 -C20 ) alkanoy mercapto , carboxy, cyano , nitro , trifluoromethyl, trifluo loxy, sulfo or ( C , -C20 ) alkoxycarbonyl ; or a salt thereof. 40 romethoxy, ( C , -C20 ) alkanoyl , ( C - C20 ) alkanoyloxy , sulfo or Other suitable compounds that comprise a carbon - sele - ( C , - C20 ) alkoxycarbonyl. nium single bond or a carbon sulfur single bond include Specifically , R , and R , can each independently be hydro compounds of formula Rs - X - R , wherein : gen , (C -C10 )alkyl , (C2 - C10 ) alkenyl, (C2 -C10 )alkynyl , aryl , X is - S — or — Se = ; or NR , R , Rs is ( C - C20 ) alkyl, (C3 - C3) cycloalkyl, (C2 -C20 ) alkenyl, 45 Specifically , R , and R , together with the carbon to which ( C2- C20 ) alkynyl, aryl, or heteroaryl, and Ro is hydrogen , they are attached can form a 5 or 6 membered saturated or ( C , -C20 ) alkyl, ( C3 -C2 ) cycloalkyl, (C2 - C20 ) alkenyl, (C20 undunsaturated ring comprising carbon and optionally compris C20 ) alkynyl, aryl, or heteroaryl; ing 1 or 2 heteroatoms selected from oxy ( 0 ) , thio or R , and Rg together with X form a heteroaryl; GS — ) , or nitrogen ( NR ) , wherein said ring is option wherein any (C , -C20 )alkyl , (C3 - C )cycloalkyl , (C2 -C20 ) 50 ally substituted with 1 , 2 , or 3 halo , hydroxy , oxo , thioxo , alkenyl, or (C2 - C20) alkynyl of R , or R is optionally sub - carboxy, (C ,- C20 ) alkyl, (C3 - C ) cycloalkyl , (C2 - C20 )alkoxy , stituted with one or more (e .g ., 1, 2 , 3, or 4) halo , hydroxy, (C1 -C20 ) alkanoyl, (C1 - C20 )alkoxycarbonyl , (C2 - C20 ) alk mercapto oxo , thioxo , carboxy, (C1 -C20 )alkanoyl , (C , - C20 ) enyl, (C2 -C20 )alkynyl , aryl, or heteroaryl; wherein R , is alkoxycarbonyl , aryl, heteroaryl, or NR , Rmi hydrogen , ( C , -C20 )alkyl , (C3 -Cg ) cycloalkyl, (C2 -C20 ) alk wherein Rz and Rm are each independently hydrogen , 55 enyl , (C1 - C20 ) alkanoyl, (C1 -C20 )alkoxycarbonyl , (C2 - C20 ) ( C2- C20 ) alkyl , (Cz - C3) cycloalkyl , (C2 - C20 ) alkenyl , (C2 alkynyl , aryl, heteroaryl; wherein any (CZ - C20 ) alkyl, (Cz C20) alkynyl, (C , - C20) alkanoyl , (C1 - C20 ) alkoxycarbonyl C20 )cycloalkyl , (C1 -C20 ) alkoxy, (C2 - C20 ) alkenyl (C -C20 ) aryl, or heteroaryl; and alkanoyl, ( C , - C20 )alkoxycarbonyl , or (C2 - C20 )alkynyl or wherein any aryl or heteroaryl is optionally substituted R1, R2, and R , is optionally substituted with one or more with one ormore ( 1 , 2 , 3 , or 4 ) halo , mercapto , hydroxy , oxo , 60 halo , hydroxy , mercapto , oxo , thioxo , carboxy , (C1 - C20 ) carboxy , cyano , nitro , trifluoromethyl, trifluoromethoxy, alkanoyl, ( C , - C20 )alkoxycarbonyl , aryl, or heteroaryl; and (C1 - C20 ) alkanoyl , (C1 - C20 ) alkanoyloxy, sulfo or (C , -C20 ) wherein any aryl or heteroaryl is optionally substituted with alkoxycarbonyl; or salt thereof. one or more halo , hydroxy , mercapto , carboxy, cyano , nitro , Specific and preferred values listed below for radicals , trifluoromethyl, trifluoromethoxy, ( C , -C20 ) alkanoyl , (C1 substituents , and ranges, are for illustration only ; they do not 65 C20 )alkanoyloxy , sulfo or (C1 -C20 )alkoxycarbonyl . exclude other defined values or other values within defined Specifically , R , and R2 can each independently by NR , R ; ranges for the radicals and substituents . wherein R , and R , are each independently hydrogen , ( C , US 10 ,077 ,244 B2 81 82 C20Jalkyl, ( Cz- Cg) cycloalkyl, (C2 - C20 )alkenyl , ( C , -C20 )al Specifically , the compound can be present in a lumines kanoyl, ( C , - C20 ) alkoxycarbonyl, (C2 - C20 ) alkynyl, aryl, het cence reaction at a concentration of at least about 0 .01 uM , eroaryl; wherein any (C1 -C20 )alkyl , (Cz - Co )cycloalkyl , (C2 or at a concentration of at least about 0 . 1 uM , e . g . , at least C20 ) alkenyl (C2 -C20 ) alkanoyl , (C1 - C20 ) alkoxycarbonyl, or about 0 . 1 mM . More specifically , the compound can be (C2 - C Jalkynyl is optionally substituted with one or more 5 present in the luminescence reaction at a concentration in the halo , hydroxy , mercapto , oxo , thioxo , carboxy , aryl, or range from about 0 . 1 uM to about 500 mM ( inclusive ) , or in heteroaryl ; and wherein any aryl or heteroaryl is optionally the range from about 0 . 001 uM , 0 .01 uM , or 0 . 1 uM to about substituted with one or more halo , hydroxy, mercapto , 250 mM ( inclusive ) . Preferably , the compound is present at carboxy , cyano , nitro , trifluoromethyl, trifluoromethyl, trif- a concentration in the range from about 0 .001 uM , 0 .01 uM , luoromethoxy , ( C , - C20 ) alkanoyl, ( C - C20 )alkanoyloxy , 10 0 . 1 uM , 1 uM or 10 uM to about 100 mM ( inclusive ) . sulfo or (C , -C20 )alkoxycarbonyl . Specifically , the assay can be performed in the presence of Specifically , R , and R2 can each independently be amino , purified enzymes, cell lys ( C2- C20 ) alkyl, R1(C1 and- C20 Rz Jalkylamino can each ,independently allylamino, 2 -behydroxy amino , purified enzymes , cell lystates or whole cells . ethylamino , phenylamino , or 4 -thiazoylamino . Specifically , the assay can be carried out in a solvent Specifically , R , and R2 can each independently be amino , is15 ;comprising at least about 10 % water . More specifically , the methyl, allylamino , 2 -hydroxyethylamino , phenylamino , or invention can be carried out in a solvent comprising at least 4 - thiazoylamino . about 25 % water, or at least about 40 % water . A specific value for Rz is (C , -C20 ) alkyl optionally sub stituted with one or more halo , mercapto oxo , thioxo , VII. Kits carboxy, (C1 - C20 ) alkanoyl, (C1 -C20 ) alkoxycarbonyl, aryl, 20 heteroaryl, or NR RE The present invention also provides kits for detecting the A specific value for Rz is 2 - aminoethyl, 2 - amino - 2 - car - presence or activity of one or more molecules which are boxyethyl , or 2 - acylamino - 2 -carboxyethyl . reagents for a reaction , enhance or inhibit a reaction , or for A specific value for R4 is aryl, optionally substituted with detecting a condition , in a sample such as a sample including one or more halo , mercapto , hydroxy , oxo , carboxy , cyano , 25 intact cells , a cell lysate , e . g ., a lysate which is at least nitro , trifluoromethyl, trifluoromethoxy , (C1 - C20 Jalkanoyl, partially purified , or a cellular supernatant. In one embodi ( C2 - C20 )alkanoyloxy , sulfo or (C , -C20 )alkoxycarbonyl , ment, the kit includes a derivative of the invention . For kits Specifically , R? is ( C . - C , Jalkyl, (Cz - C . )cycloalkyl , ( C , of the invention that include two or more of the following, C10 ) alkenyl, (C2 - C1oalkynyl, aryl, or heteroaryl; and Ro is a derivative of luciferin or aminoluciferin , a derivative of a hydrogen , ( C , -C10 )alkyl , ( C3 -C .) cycloalkyl, (C2 - C10 )alk - 30 fluorophore , another substrate , enzyme, or reaction mixture , enyl , ( C2- C10 ) alkynyl, aryl, or heteroaryl. each can be contained in a separate container , some com Specifically , R , and R . together with X form a heteroaryl. ponents may be combined in some containers , or they can be In one embodiment, the organic compound is not a contained in a single container . The kit can optionally polypeptide, or protein with one or more mercapto ( C — SH ) comprise a buffer solution suitable for use in an assay, and groups . 35 the derivative or enzyme, and the buffer solution can option In one embodiment, the organic compound is not a ally be contained in a single container Additionally , the compound that includes one or more mercapto ( C — SH ) derivative and the buffer solution can optionally be con groups. tained in a single container. The kits can also optionally Preferred compounds are coenzyme A and DTT. include a quenching agent for a nonbioluminescent reaction . The compounds described hereinabove are available from 40 commercial sources or can be prepared from commercially available starting materials using procedures that are known IX . Exemplary Synthesis in the field of synthetic chemistry . For example , see Jerry March , Advanced Organic Chemistry, 4th ed . Wiley -Inter - A . Experimental Procedures for Luciferin Modifications science . John Wiley and Sons, New York , 1992 . 45 Included below are experimentalprocedures for syntheses In cases where compounds are sufficiently basic or acidic of various luciferin derivatives : to form stable salts , use of the compounds as salts in the methods of the invention may be appropriate . Examples of I. General procedures for synthesizing esters of luciferins suitable salts include organic acid addition salts , for II . D - cysteine or D - cystine esters example , tosylate, methanesulfonate , acetate, citrate , 50 III . Esters of luciferin methyl ether or luciferin - H malonate , tartarate , succinate , benzoate, ascorbate , a - keto glutarate , and a - glycerophosphate salts . Suitable inorganic IV . Quinolinyl luciferin derivatives salts may also be formed , including hydrochloride , sulfate , V . Miscellaneous nitrate , bicarbonate , and carbonate salts . I. General Procedures for Synthesizing Luciferin Esters Salts can be obtained using standard procedures well 55 ThereThe are two ways of synthesizing esters of D - luciferin known in the art, for example by reacting a sufficiently basic and derivatives , including luciferin - H and quinolinyl compound with a suitable acid . Alkali metal ( for example , luciferin : a ) by direct cyclization of the corresponding cyano sodium , potassium or lithium ) or alkaline earth metal ( for group with D -cysteine or D - cysteine esters , and b ) by example calcium ) salts can also be used . CsF -promoted esterification with halogenated organic com When used in accord with the methods of the invention , 60 the compounds described herein can be present in a lumi pounds, such as 3 - iodomethylpyridine hydriodide (picolinyl nescence reaction at any effective concentration . The opti iodide) or 3 -iodopropanol . mum concentration of a given compound will depend on the A . Direct Cyclization luminescent reagent( s ) employed , and on the specific con Under this category , two D - cyteine esters are used , one is ditions under which a given assay is carried out. However , 65 D -cyteine methyl ester and the other is D - cystine 2 -hydroxy suitable concentrations can be determined using standard ethyl ester. The latter requires a reducing agent before techniques that are available in the art . cyclization . US 10 , 077 ,244 B2 83 84 i) D -Cyteine Methyl Ester - continued H3N OH IN- CN + 1 ) TCEP , H2O formula CXVI ý 2 ) MeOH , K2CO3 , pH 7 HEN OH © o H3N MeOH , K2CO3, pH 7 der wenigen, formula CXIX o HS formula CXX R = OH , OCH3, H X = S , CH = CH 25 formula CXVII The procedure of cyclization with D - cystine 2 - hydroxyethyl R = OH , OCH3, H ester is very similar to D - cyteine methyl ester except 1 X = S , CH = CH equivalent of tris ( 2 - carboxyethyl) phosphine hydroxychlo 30 ride ( TCEP ) was added before pH adjustment . A derivative of 2 - cyanobenzothiazole or 2 -cyanoquino - B . CsF -Promoted Esterification line ( 1 equivalent ) was dissolved in methanol and was degassed with a gentle stream of nitrogen for 10 minutes . Meanwhile, D - cyteine methyl ester ( 1 . 5 equivalents ) was dissolved in water and its pH was adjusted to about 7 . This solution was also degassed with a gentle stream of nitrogen - OH + for 5 minutes. Then , the aqueous solution was added to above organic solution dropwise and the resultant solution R was stirred under nitrogen for 30 minutes to a few hours 10 formula CXXI depending on substrates . The reaction was monitored by TLC with methanol in dichloromethane . After the comple tion of the reaction , the mixture was filtered and was purified DMF by HPLC . Typical HPLC conditions are the following . 1I - R CsFCsF Column : Dynamax 60 Å , 1 inch , 250 mm , reversed phase 45 C - 18 column Mobile phase A : water or 100 mM ammonium acetate, pH 7 , or 0 . 1 % TFA in water Mobile phase B : acetonitrile 50 OR Flow rate : 20 mL/ min Detection wavelength : 300 nm formula CXXII Gradient : from 100 % A to 100 % B , 30 minutes, and keep R ' = alkyl , aromatic 100 % B for 30 minutes . 55 R = OMe, OH , H The purified compound was then characterized by NMR , X = S , CH = CH MS, UV - Vis , and HPLC . ii) D - Cystine 2 - Hydroxyethyl Ester To a solution luciferin or a derivative (1 equivalent) in DMF so was added cesium fluoride ( 1 . 5 equivalents ), followed by 3 - iodomethyl pyridine hydriodide or 3 - iodopropanol ( 1 . 5 equivalents ) . After the resultantmixture was stirred at room - CN + temperature for a few hours ,monitored by TLC with metha nol in dichloromethane , the solution was concentrated down 65 and water was added . The mixture was then filtered through formula CXIII a 0 . 2 um filter and was subjected to HPLC purification using similar condition described above . US 10 , 077 , 244 B2 85 86 II . D - Cysteine or D - Cystine Esters put on ice briefly and then filtered under vacuum , washed 1 . Synthesis of 2 -Hydroxyethyl Ester of D -Cystine with cold isopropyl alcohol, and pumped by maintaining the suction of the filtrate cake . ' H NMR (CD3OD ) : 4 . 35 ppm (t , 1H ) , 3 . 85 ppm ( s , 3 ) , HS 5 3 .10 ppm ( d , 2H ) H20 III . Luciferin Methyl Ether Esters HCI ( g) 3 . Synthesis of 2 -Hydroxyethyl Ester of Luciferin Methyl LOH + HO OH HN Ether 0 a. By 2- hydroxyethyl ester of D -cystine 10 formula CXXIII - CN + OZ OH 15 formula CXXVII - H3N . OH 20 “ OH?? 1 ) TCEP , H2O - 2 ) MeOH , K2CO3, pH 7 formula CXXIV 25 To anhydrous ethylene glycol solution ( 10 mL ) was OH charged D -cysteine hydrochloride monohydrate ( 1 g ). A gentle stream of hydrochloride gas was introduced to bubble OH the solution for 1 hour. The solution was then let stand over 30 night. Cold isopropyl alcohol ( 12 . 5 mL ) was added slowly and the resultantmixture was put at - 20° C . for 1 hour. The white precipitates were then filtered , washed with cold isopropyl alcohol, and pumped by maintaining the suction of the filtrate cake . White crystals (595 mg) were obtained 35 formula CXXVII (yield : 52 % ). ' H NMR (DMSO -d6 ) : 8 .6 -88 ppm (broad , 6H ), 4 .35 ppm 2 -cyano -6 -methoxybenzothiazole ( 100 mg, 0 .526 mmol) ( broad singlet , 2H ) , 4 . 20 ppm ( m , 4H ) , 3 . 63 ppm ( m , 4H ) , was dissolved in methanol ( 20 mL ) and the resultant solu 3 .05 ppm (broad singlet , 4H ) tion was degassed with a gentle stream of nitrogen for 10 ES + 330 . 53 ( M . W . 330 . 44 ) 40 minutes . 2 . Synthesis of Methyl Ester of D - Cysteine To a solution of 2 -hydroxyethyl ester of D - cysteine ( 87 mg, 0 . 263 mmol) in water ( 10 mL ) was added tris ( 2 carboxyethyl phosphine hydrochloride ( TCEP ) ( 77 mg, HS . H2OH2O 0 . 263 mmol) . The pH of this solution was adjusted to about + HO - CH2 HCl( g ) 45 7 with 1 M potassium carbonate . The resultant solution was OH + added to aforementioned solution dropwise and the mixture H?N was stirred for 30 minutes. The precipitate were filtered out through a 0 .2 um filler and the filtrate was purified by formula CXXV semi-preparative HPLC ( see general procedure : Mobile HS 50 phase A : water ). 102 mg of purr compound was obtained ( Yield : 52 % ). ' H NMR ( CD3CN ) : 8 . 0 ppm ( d , 1H ), 7 .55 ppm ( d , 1H ) , 7 .20 ppm (dd , 1H ), 5 .42 ppm (t , 1H ), 4 . 25 ppm ( m , 2H ), 3 .75 ppm (m , 4H ) 55 MS: ES + 338 .58 (M . W . 338 .40 ) formula CXXVI Extinction coefficient: 16 ,630 ( at 325 nm in acetonitrile ) b . By Ethylene Glycol To a methanol solution ( 100 mL ) was charged D - cysteine hydrochloride monohydrate ( 6 g ) . A gentle stream of hydro - 60 chloride gas was introduced to bubble the solution for 105 Ethylene glycol minutes. The solution was then stirred over night. Then the OH HCI ( g ) solvent was then removed under vacuum . The white residue was dissolved in hot methanol ( 25 mL ) and 25 mL of o n einemder s isopropyl alcohol was added . The mixture was put on a 65 v rotavap to remove some of the methanol until white pre formula CXXIX cipitates crashed out of the solution . The precipitates were US 10 , 077 ,244 B2 87 88 - continued - continued DMF CsF OH 5 formula CXXXV formula CXXX To a solution of luciferin methyl ether (100 mg, 0 .34 mmol) 10 in anhydrous ethylene glycol (4 mL ) in a 5 ml. Schlenk flask ss was introduced a gentle stream of hydrochloride gas . After formula CXXXVI 5 minutes of bubbling , the flask was sealed and put into an oil bath of 62° C . for 3 hours . The HCl bubbling was repeated for another 5 minutes and put back into the oil bath 15 To a solution of luciferin methyl ether ( 215 mg, 0 . 73 mmol ) for another 1 hour. After the solution was cooled to room in DMF ( 35 mL ) was added cesium fluoride ( 170 mg, 1 . 1 mmol) , followed by 3 - iodomethyl pyridine hydroiodide temperature, water (6 mL ) was added and the resultant (380 mg, 1 . 1 mmol) . After the resultant mixture was stirred mixture was filtered and purified with semi- preparative at room temperature for 3 hours and twenty minutes , the HPLC ( see general procedure : Mobile phase A : water) . 20 solution was concentrated down to about 10 mL and 6 mL 4 . Synthesis of 2 -Hydroxy Ethyl Ester of Luciferin - H of water was added . The mixture was filtered through a 0 . 2 um filter and was subjected to HPLC purification ( see general procedure : Mobile phase A : 0 . 1 % TFA in water ). 57 mg of pure compound was obtained . - CN 25 H NMR (CD2CN ) : 8 .62 ppm ( s , 1H ), 8 . 54 ppm ( d , 1H ) , 7 . 96 ppm ( d , 1H ) , 7 .84 ppm ( d , 1H ) , 7 . 56 ppm ( d , 1H ) , 7 .41 ppm ( m , 1H ), 7 .20 ppm (dd , 1H ) formula CXXXI MS: ES + 385 .77 ( M . W . 385 . 46 ) 6 . Synthesis of m - Picolinyl Ester of Luciferin H3N OH 30 1 ) TCEP , H2O - 2 ) MeOH , K2CO3 , pH 7 OH + u HO H?N OH formula CXXXVI DMF CsF formula CXXXII OH B formula CXXXVIII # s formula CXXXIII co HO 's This compound was synthesized in a similar way to proce - 50 formula CXXXIX dure described in section 3a . 'H NMR ( CD3CN ) : 8 .15 ppm (m , 2H ), 7 .60 ppm ( m , 2H ), To a solution of luciferin mono potassium salt (51 mg, 0 . 16 5 .45 ppm (t , 1H ), 4 .25 ppm ( m , 2H ), 3 .75 ppm ( m , 4H ), 1. 95 mmol) in DMF ( 10 mL ) was added cesium fluoride ( 24 mg. ppm ( t, 1H ) 0 . 16 mmol) , followed by 3 -bromomethyl pyridine hydro MS : ES + 308 .58 (M . W . 308 .38 ) 55 bromide (44 mg, 0 . 17 mmol) . After 16 hours of reaction at Extinction coefficient 14 ,050 (at 294 nm in acetonitrile ) room temperature , the solvent was stripped away under 5 . Synthesis of m - picolinyl Ester of Luciferin Methyl Ether vacuum . The residue was then dissolved in 10 mL of 60 % DMF in water. The resultant solution was filtered through on 0 . 2 um filter and was subjected to HPLC purification (see 60 general procedure : Mobile phase A : water ) . 8 . 6 mg of pure compound was obtained . + ' H NMR ( CD3CN -D20 ) : 8 . 58 ppm ( s , 1H ), 8 . 50 ppm ( d , OH 1H ) , 7 . 90 ppm ( d , 1H , 7 . 85 ppm ( dd , 1H ) , 7 .42 ppm ( dd , 1H ), 7 .40 ppm ( s , 1H ), 7 . 10 ppm ( dd , 1H ) , 5 . 45 ppm ( t, 1H ) , 65 5 .25 ppm ( s , 2H ) , 3 .75 ppm ( m , 2H ) formula CXXXIV MS: ES +: 371. 65 ( M . W . 371 ) van UV - Vis : 263 nm and 334 nm US 10 ,077 , 244 B2 89 90 7 . Synthesis of m - picolinyl Ester of Luciferin - H IV . Quinolinyl Luciferin Derivatives 9 . Synthesis of 2 -Hydroxy Ester of Quinolinyl Luciferin -H 5 OH + + N CN formula CXXXXVI formula CXXXX 1010 DMF CsF H3N OH formula CXXXXI formula CXXX 1 ) TCEP , H2O Dve2 ) MeOH , K2CO3os,, pHHP 7 , HN OH N 20 formula CXXXXII formula CXXXXVII OH This compound was synthesized and purified in a similar fashion to the synthesis of m - picolinyl ester of luciferin 25 methyl ether using luciferin - H as starting material. 13 mg of pure compound was obtained . ' H NMR (CD3CN -D20 ): 8 .60 ppm (s , 1H ), 8. 52 ppm (d , formula CXXXXVIII 1H ), 8. 10 ppm ( m , 2H ) , 7 .85 ppm (m , 1H ), 7 .59 ppm (m , 30 2H ), 7 . 42 ppm ( m , 1H ), 5 . 50 ppm ( t, 1H ) , 5 . 28 ppm ( s , 2H ) , A solution of 2 - cyanoquinoline (50 mg, 0 . 32 mmol) in 3 . 79 ppm ( m , 2H ) 1, 4 -dioxane ( 5 mL ) was degassed for 5 minutes by passing MS : ES + 3 .55 .54 and 711. 36 (dimer ) (M .W . 355 .43 ) through a gentle stream of nitrogen gas . 8 . Synthesis of 3 -Hydroxypropyl Ester of Luciferin Methyl 35 Cystine 2 -hydroxyethyl ester hydrochloride (53 mg, 0. 16 Ether mmol) in water ( 5 mL ) was treated with tris ( 2 -carboxyethyl ) phosphine hydrochloride (46 mg, 0 . 16 mmol) . The pH of this solution was adjusted to about 7 with 1 M potassium carbonate . It was then added dropwise to the solution of 40 2 -cyanoquinoline . The resultant solution was stirred at room OH + temperature for 7 . 5 hours and was purified in a similar way to m -picolinyl ester of luciferin ( see general procedure : Mobile phase A :water ). 13 mg of pure compound was obtained (yield . 26 . 7 % ) formula CXXXXIII H NMR ( CD3 - OD - D20 ) : 8 . 40 ppm ( d , 1H ) , 8 . 15 ppm DMF ( d , 1H ), 8 . 10 ppm ( d , 1H ) , 7 . 95 ppm ( d , 1H ) , 7 .82 ppm ( m , OH CsF 1H ), 7 .68 ppm (m , 1H ), 5 .53 ppm (t , 1H ), 4 .25 ppm (m , 2H ), 3 .75 ppm ( m , 2H ) , 3 .65 pppm ( m , 2H ) formula CXXXIV MS: EST: 302 .35 , 324 .47 ( M . W . 302 .35 ) 50 UV - Vis : 247 nm and 295 nm . Extinction coefficient: 7 ,500 ( at 288 nm in acetonitrile ) OH?? 10 . Synthesis of Quinolinyl Luciferin - H formula CXXXXV SS55. Bz - Cl, KCN This compound was synthesized in a similar way to m - pi ON 2008. colinyl ester of luciferin methyl ether using luciferin methyl ether and 3 - iodopropanol as starting materials . IH NMR (CD3OD ) : 7 .95 ppm ( d , 1H ), 7. 55 ppm (d , 1H ) formula CXXXXIX 7 . 18 ppm (dd , 1H ), 5. 41 ppm ( t, 1H ) , 4 .35 ppm (t , 2H ), 3. 92 ppm ( s , 3H ) , 3 .75 ppm ( dd , 2H ) , 3 .65 ppm (t , 2H ) , 1 .92 ppm D - Cys ( m , 2H ) 65 SN MS: EST: 353 .70 (M .W . 352 .43 ) formula CL UV - Vis: 330 nm and 264 nm .