Fluorescent metal ion indicators with large stokes shifts

Abstract

The present invention provides fluorogenic compounds for the detection of target metal ions wherein the compounds exhibit a Stokes shift greater than 50 nm and the detectable signal is modulated by photoinduced electron transfer (PET). The present compounds consist of three functional elements, the ion sensing moiety (chelating moiety), the reporter moiety (fluorophore or fluorescent protein) and spacer or linker between the sensing and reporter moieties of the present compound that allows for PET upon binding of a metal ion and excitation by an appropriate wavelength.

1 . A compound for the detection of metal ions wherein a detectable response is modulated by photoinduced electron transfer (PET), wherein the compound comprises a metal chelating moiety and a fluorophore or a fluorescent protein that is covalently bonded to the metal chelating moiety by a linker —(CR 2 ) n NR′— or —(CR 2 ) n — wherein R and R′ are independently hydrogen, alkyl, or substituted alkyl; and n is 1-10. 
   
   
       2 . A compound for the detection of calcium ions wherein a detectable response is modulated by photoinduced electron transfer (PET), wherein the compound comprises a metal chelating moiety that is capable of binding calcium ions and a fluorophore that is covalently bonded to the metal chelating moiety by a linker —(CR 2 ) n NR′— or —(CR 2 ) n — wherein R and R′ are independently hydrogen, alkyl, or substituted alkyl; and n is 1-10. 
   
   
       3 . A compound of the formula: 
     
       
         
         
             
             
         
       
       wherein
 R 15  is hydrogen or C 1 -C 6  alkyl; 
 R 16  is hydrogen or C 1 -C 6  alkyl; 
 R 17  is hydrogen or C 1 -C 6  alkyl; 
 R 18  is hydrogen or C 1 -C 6  alkyl; and 
 R 13  is hydrogen, C 1 -C 6  alkyl, —CH 2 OCOCH 3  or a salt ion; 
 R 14  is hydrogen, C 1 -C 6  alkyl, —CH 2 OCOCH 3  or a salt ion; 
 
       R 1  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; 
       R 2  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; 
       R 3  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; 
       R 4  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; 
       R 5  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; 
       R 6  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; 
       R 7  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; 
       R 8  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; or
 a member independently selected from 
 R 1  in combination with R 2 ; 
 R 2  in combination with R 3 ; 
 R 3  in combination with R 4 ; 
 R 5  in combination with R 6 ; 
 R 6  in combination with R 7 ; and 
 R 7  in combination with R 8  
 together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl; 
 
 wherein R is hydrogen, alkyl or substituted alkyl; 
 R′ is hydrogen, alkyl, or substituted alkyl; 
 R″ is hydrogen, alkyl, substituted alkyl, fluorophore, carrier molecule, solid support or reactive group; and n is 1-10; and, 
 wherein R 19  is hydrogen, C 1 -C 6  alkyl, substituted alkyl, C 1 -C 6  carboxyalkyl (—(CH 2 ) 1-6 COOR 13 ), an alpha-acyloxyalkyl, a biologically compatible salt, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl; 
 R 20  is hydrogen, C 1 -C 6  alkyl, substituted alkyl, C 1 -C 6 -carboxyalkyl (—(CH 2 ) 1-6 COOR 13 ), an alpha-acyloxyalkyl, a biologically compatible salt, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl; 
 
       R 9  is hydrogen, a reactive group, a carrier molecule, a solid support, —(CR 2 ) n NR′R″, (CR 2 ) n NR′-fluorophore and C 1 -C 6  alkyl; 
       R 10  is hydrogen, a reactive group, a carrier molecule, a solid support, —(CR 2 ) n NR′R″, (CR 2 ) n NR′-fluorophore and C 1 -C 6  alkyl; 
       R 11  is hydrogen, a reactive group, a carrier molecule, a solid support, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore and C 1 -C 6  alkyl; 
       R 12  is hydrogen, a reactive group, a carrier molecule, a solid support, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore and C 1 -C 6  alkyl; or
 a member selected from 
 R 9  in combination with R 10 ; or 
 R 11  in combination with R 12  
 together with the atoms to which they are joined, form a ring which is a 5-, or 6-membered alicyclic ring, a substituted 5-, or 6-membered alicyclic ring, a 5-, or 6-membered heterocyclic ring, or a substituted 5-, or 6-membered heterocyclic ring; 
 
 
       p is 0, 1, 2 or 3; and 
       wherein at least one of R 1 -R 12  is —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n —fluorophore with the proviso that when at least one of R 1 -R 12  is —(CR 2 ) n -fluorophore that the fluorophore comprise a nitrogen atom that is covalently bonded to —(CR 2 ) n —. 
     
   
   
       4 . The compound according to  claim 3 , wherein R 15 , R 16 , R 17  and R 18  are each hydrogen. 
   
   
       5 . The compound according to  claim 3 , wherein R 13  and R 14  are independently hydrogen or a salt ion. 
   
   
       6 . The compound according to  claim 3 , wherein R 13  and R 14  are independently CH 2 OCOCH 3 . 
   
   
       7 . The compound according to  claim 3 , wherein exactly one of R 1 -R 8  is —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore. 
   
   
       8 . The compound according to  claim 3 , wherein exactly one of R 2 , R 3 , R 6  or R 7  is (CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore. 
   
   
       9 . The compound according to  claim 3 , wherein R and R′ are each hydrogen. 
   
   
       10 . The compound according to  claim 3 , wherein the fluorophore is dansyl, xanthene, cyanine, borapolyazaindacene, pyrene, naphthalene, coumarin, oxazine or derivatives thereof. 
   
   
       11 . The compound according to  claim 10 , wherein the xanthene is fluorescein or derivatives thereof, rhodamine or derivatives thereof, rhodol or derivatives thereof, or rosamine or derivatives thereof. 
   
   
       12 . The compound according to  claim 11 , wherein the xanthene is substituted by at least one fluorine. 
   
   
       13 . The compound according to  claim 3 , wherein the reactive group, solid support and carrier molecule comprise a linker that is a single covalent bond, or a covalent linkage that is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1-20 nonhydrogen atoms selected from the group consisting of C, N, P, O and S; and are composed of any combination of ether, thioether, amine, ester, carboxamide, sulfonamide, hydrazide bonds and aromatic or heteroaromatic bonds. 
   
   
       14 . The compound according to  claim 3 , wherein the reactive group is selected from the group consisting of carboxylic acid, succinimidyl ester of a carboxylic acid, hydrazide, amine and a maleimide. 
   
   
       15 . The compound according to  claim 3 , wherein the carrier molecule is selected from the group consisting of an amino acid, a peptide, a protein, a polysaccharide, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a hapten, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a polymeric microparticle, a biological cell or a virus. 
   
   
       16 . The compound according to  claim 3 , wherein the solid support is selected from the group consisting of a microfluidic chip, a silicon chip, a microscope slide, a microplate well, silica gels, polymeric membranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels, polysaccharides, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, and superparamagnetic bead. 
   
   
       17 . The compound according to  claim 3 , having the formula: 
     
       
         
         
             
             
         
       
       wherein
 R 13  is hydrogen, C 1 -C 6  alkyl, —CH 2 OCOCH 3  or a salt ion; 
 R 14  is hydrogen, C 1 -C 6  alkyl, —CH 2 OCOCH 3  or a salt ion; 
 
       R 1  is hydrogen, halogen, or C 1  to C 10  alkyl (CH 2 ), carboxyl, sulfonyl; 
       R 2  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), carboxyl, sulfonyl; 
       R 4  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), carboxyl, sulfonyl; 
       R 5  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), carboxyl, sulfonyl; 
       R 6  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), carboxyl, sulfonyl; 
       R 7  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), carboxyl, sulfonyl; 
       R 8  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), carboxyl, sulfonyl; 
       R′ is hydrogen, alkyl, or substituted alkyl; 
       R″ is a fluorophore, carrier and n is 1-10. 
     
   
   
       18 . The compound according to  claim 3 , wherein the compound is selected from the group consisting of Compound 5, 6, 9, 10, 13, 14 and 15. 
   
   
       19 . A composition comprising:
 a) a compound having the formula:   
     
       
         
         
             
             
         
       
       wherein
 R 15  is hydrogen or C 1 -C 6  alkyl; 
 R 16  is hydrogen or C 1 -C 6  alkyl; 
 R 17  is hydrogen or C 1 -C 6  alkyl; 
 R 18  is hydrogen or C 1 -C 6  alkyl; and 
 R 13  is hydrogen, C 1 -C 6  alkyl, —CH 2 OCOCH 3  or a salt ion; 
 R 14  is hydrogen, C 1 -C 6  alkyl, —CH 2 OCOCH 3  or a salt ion; 
 
       R 1  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; 
       R 2  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; 
       R 3  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; 
       R 4  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; 
       R 5  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; 
       R 6  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; 
       R 7  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; 
       R 8  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; or
 a member independently selected from 
 R 1  in combination with R 2 ; 
 R 2  in combination with R 3 ; 
 R 3  in combination with R 4 ; 
 R 5  in combination with R 6 ; 
 R 6  in combination with R 7 ; and 
 R 7  in combination with R 8  
 together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl; 
 
 wherein R is hydrogen, alkyl or substituted alkyl; 
 R′ is hydrogen, alkyl, or substituted alkyl; 
 R″ is hydrogen, alkyl, substituted alkyl, fluorophore, carrier molecule, solid support or reactive group; and n is 1-10; and, 
 wherein R 19  is hydrogen, C 1 -C 6  alkyl, substituted alkyl, C 1 -C 6  carboxyalkyl (—(CH 2 ) 1-6 COOR 13 ), an alpha-acyloxyalkyl, a biologically compatible salt, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl; 
 R 20  is hydrogen, C 1 -C 6  alkyl, substituted alkyl, C 1 -C 6 -carboxyalkyl (—(CH 2 ) 1-6 COOR 13 ), an alpha-acyloxyalkyl, a biologically compatible salt, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl; 
 
       R 9  is hydrogen, a reactive group, a carrier molecule, a solid support, —(CR 2 ) n NR′R″, (CR 2 ) n NR′-fluorophore and C 1 -C 6  alkyl; 
       R 10  is hydrogen, a reactive group, a carrier molecule, a solid support, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore and C 1 -C 6  alkyl; 
       R″ is hydrogen, a reactive group, a carrier molecule, a solid support, —(CR 2 ) n NR′R″, (CR 2 ) n NR′-fluorophore and C 1 -C 6  alkyl; 
       R 12  is hydrogen, a reactive group, a carrier molecule, a solid support, —(CR 2 ) n NR′R″, (CR 2 ) n NR′-fluorophore and C 1 -C 6  alkyl; or
 a member selected from 
 R 9  in combination with R 10 ; or 
 R 11  in combination with R 12  
 together with the atoms to which they are joined, form a ring which is a 5-, or 6-membered alicyclic ring, a substituted 5-, or 6-membered alicyclic ring, a 5-, or 6-membered heterocyclic ring, or a substituted 5-, or 6-membered heterocyclic ring; 
 
 
       p is 0, 1, 2 or 3; and 
       wherein at least one of R 1 -R 12  is —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore with the proviso that when at least one of R 1 -R 12  is —(CR 2 ) n -fluorophore that the fluorophore comprise a nitrogen atom that is covalently bonded to —(CR 2 ) n —; and
 b) a metal ion that is capable of being chelated by the compound. 
 
     
   
   
       20 . A composition according to  claim 19 , wherein the metal ion is selected from the group consisting of Ca 2+ , Zn 2+ , Mg 2+ , Ga 3+ , Tb +3 , La 3+ , Pb 2+ , Hg 2+ , Cd 2+ , Cu 2+ , Ni 2+ , Co 2+ , Fe 2+ , Mn 2+ , Ba 2+ , and Sr 2+ . 
   
   
       21 . A method for binding a target metal ion in a sample, comprising steps of:
 a. contacting the sample with a metal ion bind compound to form a contacted sample, wherein the metal ion bind compound has the formaula   
     
       
         
         
             
             
         
       
       wherein
 R 15  is hydrogen or C 1 -C 6  alkyl; 
 R 16  is hydrogen or C 1 -C 6  alkyl; 
 R 17  is hydrogen or C 1 -C 6  alkyl; 
 R 18  is hydrogen or C 1 -C 6  alkyl; and 
 R 13  is hydrogen, C 1 -C 6  alkyl, —CH 2 OCOCH 3  or a salt ion; 
 R 14  is hydrogen, C 1 -C 6  alkyl, —CH 2 OCOCH 3  or a salt ion; 
 
       R 1  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; 
       R 2  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; 
       R 3  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; 
       R 4  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; 
       R 5  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; 
       R 6  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; 
       R 7  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; 
       R 8  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; or
 a member independently selected from 
 R 1  in combination with R 2 ; 
 R 2  in combination with R 3 ; 
 R 3  in combination with R 4 ; 
 R 5  in combination with R 6 ; 
 R 6  in combination with R 7 ; and 
 R 7  in combination with R 8  
 together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl; 
 
 wherein R is hydrogen, alkyl or substituted alkyl; 
 R′ is hydrogen, alkyl, or substituted alkyl; 
 R″ is hydrogen, alkyl, substituted alkyl, fluorophore, carrier molecule, solid support or reactive group; and n is 1-10; and, 
 wherein R 19  is hydrogen, C 1 -C 6  alkyl, substituted alkyl, C 1 -C 6  carboxyalkyl (—(CH 2 ) 1-6 COOR 13 ), an alpha-acyloxyalkyl, a biologically compatible salt, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl; 
 R 20  is hydrogen, C 1 -C 6  alkyl, substituted alkyl, C 1 -C 6 -carboxyalkyl (—(CH 2 ) 1-6 COOR 13 ), an alpha-acyloxyalkyl, a biologically compatible salt, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl; 
 
       R 9  is hydrogen, a reactive group, a carrier molecule, a solid support, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore and C 1 -C 6  alkyl; 
       R 10  is hydrogen, a reactive group, a carrier molecule, a solid support, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore and C 1 -C 6  alkyl; 
       R 11  is hydrogen, a reactive group, a carrier molecule, a solid support, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore and C 1 -C 6  alkyl; 
       R 12  is hydrogen, a reactive group, a carrier molecule, a solid support, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore and C 1 -C 6  alkyl; or
 a member selected from 
 R 9  in combination with R 10 ; or 
 R 11  in combination with R 12  
 together with the atoms to which they are joined, form a ring which is a 5-, or 6-membered alicyclic ring, a substituted 5-, or 6-membered alicyclic ring, a 5-, or 6-membered heterocyclic ring, or a substituted 5-, or 6-membered heterocyclic ring; 
 
 
       p is 0, 1, 2 or 3; and 
       wherein at least one of R 1 -R 12  is —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore with the proviso that when at least one of R 1 -R 12  is —(CR 2 ) n -fluorophore that the fluorophore comprise a nitrogen atom that is covalently bonded to —(CR 2 ) n —; and,
 b) incubating the contacted sample for sufficient time to allow the metal ion binding compound to chelate the target metal ion whereby the metal ion is bound. 
 
     
   
   
       22 . The method according to  claim 21 , wherein the sample comprises live cells, intracellular fluids, extracellular fluids, biological fluids, biological fermentation media, environmental sample, industrial samples, proteins, peptides, buffer solutions or biological fluids or chemical reactors. 
   
   
       23 . The method according to  claim 21 , wherein the sample comprises blood cells, immune cells, cultured cells, muscle tissue, neurons, extracellular vesicles; vascular tissue, blood fluids, saliva, urine; water, soil, waste water, sea water; pharmaceuticals, foodstuffs or beverages. 
   
   
       24 . The method according to  claim 21 , wherein the method further comprises detecting a target metal ion wherein the sample is illuminated with an appropriate wavelength whereby the target metal ion is detected. 
   
   
       25 . The method according to  claim 21 , wherein the metal ion is Ca 2+ , Zn 2+ , Mg 2+ , Ga 3+ , Tb 3+ , La 3+ , Pb 2+ , Hg 2+ , Cd 2+ , Cu 2+ , Ni 2+ , Co 2+ , Fe 2+ , Mn 2+ , Ba 2+ , or Sr 2+ . 
   
   
       26 . A kit for binding a metal ion in a sample, comprising at least one metal binding compound according to the formula: 
     
       
         
         
             
             
         
       
       wherein
 R 15  is hydrogen or C 1 -C 6  alkyl; 
 R 16  is hydrogen or C 1 -C 6  alkyl; 
 R 17  is hydrogen or C 1 -C 6  alkyl; 
 R 18  is hydrogen or C 1 -C 6  alkyl; and 
 R 13  is hydrogen, C 1 -C 6  alkyl, —CH 2 OCOCH 3  or a salt ion; 
 R 14  is hydrogen, C 1 -C 6  alkyl, —CH 2 OCOCH 3  or a salt ion; 
 
       R 1  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; 
       R 2  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; 
       R 3  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; 
       R 4  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; 
       R 5  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; 
       R 6  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; 
       R 7  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; 
       R 8  is hydrogen, halogen, C 1  to C 10  alkyl (CH 2 ), methoxy (—OCH 3 ), hydroxyl (—OH), C 2 -C 6  alkoxy (—OCH 2 ), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR 19 R 20 ), carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore; or
 a member independently selected from 
 R 1  in combination with R 2 ; 
 R 2  in combination with R 3 ; 
 R 3  in combination with R 4 ; 
 R 5  in combination with R 6 ; 
 R 6  in combination with R 7 ; and 
 R 7  in combination with R 8  
 together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl; 
 
 wherein R is hydrogen, alkyl or substituted alkyl; 
 R′ is hydrogen, alkyl, or substituted alkyl; 
 R″ is hydrogen, alkyl, substituted alkyl, fluorophore, carrier molecule, solid support or reactive group; and n is 1-10; and, 
 wherein R 19  is hydrogen, C 1 -C 6  alkyl, substituted alkyl, C 1 -C 6  carboxyalkyl (—(CH 2 ) 1-6 COOR 13 ), an alpha-acyloxyalkyl, a biologically compatible salt, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl; 
 R 20  is hydrogen, C 1 -C 6  alkyl, substituted alkyl, C 1 -C 6 -carboxyalkyl (—(CH 2 ) 1-6 COOR 13 ), an alpha-acyloxyalkyl, a biologically compatible salt, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl; 
 
       R 9  is hydrogen, a reactive group, a carrier molecule, a solid support, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore and C 1 -C 6  alkyl; 
       R 10  is hydrogen, a reactive group, a carrier molecule, a solid support, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore and C 1 -C 6  alkyl; 
       R 11  is hydrogen, a reactive group, a carrier molecule, a solid support, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore and C 1 -C 6  alkyl; 
       R 12  is hydrogen, a reactive group, a carrier molecule, a solid support, —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore and C 1 -C 6  alkyl; or
 a member selected from 
 R 9  in combination with R 10 ; or 
 R 11  in combination with R 12  
 together with the atoms to which they are joined, form a ring which is a 5-, or 6-membered alicyclic ring, a substituted 5-, or 6-membered alicyclic ring, a 5-, or 6-membered heterocyclic ring, or a substituted 5-, or 6-membered heterocyclic ring; 
 
 
       p is 0, 1, 2 or 3; and 
       wherein at least one of R 1 -R 12  is —(CR 2 ) n NR′R″, —(CR 2 ) n NR′-fluorophore or —(CR 2 ) n -fluorophore with the proviso that when at least one of R 1 -R 12  is —(CR 2 ) n -fluorophore that the fluorophore comprise a nitrogen atom that is covalently bonded to —(CR 2 ) n —; 
       and instructions for use thereof. 
     
   
   
       27 . The kit according to  claim 26 , wherein the kit independently further comprises one or more components selected from the group consisting of a calibration standard of a metal ion, an ionophore, a metal ion indicator other than for calcium ions, a detectable signal standard, an aqueous buffer solution, an antibody or fragment thereof, a reference dye standard and an organic solvent. 
   
   
       28 . A metal ion chelating compound comprising a chelating moiety and a reporter moiety wherein when the compound binds a metal ion capable of being bound by the chelating moiety and illuminated with an appropriate wavelength, wherein the compound has a Stokes shift greater than about 50 nm. 
   
   
       29 . The compound according to  claim 28 , wherein the Stokes shift is greater than about 100 nm. 
   
   
       30 . The compound according to  claim 28 , wherein the Stokes shift is greater than about 150 nm. 
   
   
       31 . The compound according to  claim 28 , wherein the Stokes shift is greater than about 200 nm. 
   
   
       32 . The compound according to  claim 28 , wherein the Stokes shift is greater than about 250 nm.