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US 2012/0009683 A1Published application
Fluorescent Metal Ion Indicators with Large Stokes Shifts
AssigneeLife Technologies Corporation
InventorsKyle Richard GEE, Vladimir MARTIN
Life of this patent
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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.
Claims24
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 —(CR2)nNR′— or —(CR2)n- wherein R and R′ are independently hydrogen, alkyl, or substituted alkyl;
- and n is 1-10.
(canceled)
A compound of the formula:
- wherein
- R15 is hydrogen or C1-C6 alkyl;
- R16 is hydrogen or C1-C6 alkyl;
- R17 is hydrogen or C1-C6 alkyl;
- R18 is hydrogen or C1-C6 alkyl;
- and
- R13 is hydrogen, C1-C6 alkyl, —CH2OCOCH3 or a salt ion;
- R14 is hydrogen, C1-C6 alkyl, —CH2OCOCH3 or a salt ion;
- R1 is hydrogen, halogen, C1 to C10 alkyl (CH2), methoxy (—OCH3), hydroxyl (—OH), C2-C6 alkoxy (—OCH2), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR19R20), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR2)nNR′R″, —(CR2)nNR′-fluorophore or —(CR2)n-fluorophore;
- R2 is hydrogen, halogen, C1 to C10 alkyl (CH2), methoxy (—OCH3), hydroxyl (—OH), C2-C6 alkoxy (—OCH2), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR19R20), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR2)nNR′R″, —(CR2)nNR′-fluorophore or —(CR2)n-fluorophore;
- R3 is hydrogen, halogen, C1 to C10 alkyl (CH2), methoxy (—OCH3), hydroxyl (—OH), C2-C6 alkoxy (—OCH2), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR19R20), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR2)nNR′R″, —(CR2)nNR′-fluorophore or —(CR2)n-fluorophore;
- R4 is hydrogen, halogen, C1 to C10 alkyl (CH2), methoxy (—OCH3), hydroxyl (—OH), C2-C6 alkoxy (—OCH2), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR19R20), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR2)nNR′R″, —(CR2)nNR′-fluorophore or —(CR2)n-fluorophore;
- R5 is hydrogen, halogen, C1 to C10 alkyl (CH2), methoxy (—OCH3), hydroxyl (—OH), C2-C6 alkoxy (—OCH2), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR19R20), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR2)nNR′R″, —(CR2)nNR′-fluorophore or —(CR2)n-fluorophore;
- R6 is hydrogen, halogen, C1 to C10 alkyl (CH2), methoxy (—OCH3), hydroxyl (—OH), C2-C6 alkoxy (—OCH2), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR19R20), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR2)nNR′R″, —(CR2)nNR′-fluorophore or —(CR2)n-fluorophore;
- R7 is hydrogen, halogen, C1 to C10 alkyl (CH2), methoxy (—OCH3), hydroxyl (—OH), C2-C6 alkoxy (—OCH2), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR19R20), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR2)nNR′R″, —(CR2)nNR′-fluorophore or —(CR2)n-fluorophore;
- R8 is hydrogen, halogen, C1 to C10 alkyl (CH2), methoxy (—OCH3), hydroxyl (—OH), C2-C6 alkoxy (—OCH2), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR19R20), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR2)nNR′R″, —(CR2)nNR′-fluorophore or —(CR2)n-fluorophore;
- or
- a member independently selected from
- R1 in combination with R2;
- R2 in combination with R3;
- R3 in combination with R4;
- R5 in combination with R6;
- R6 in combination with R7;
- and
- R7 in combination with R8
- 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 R19 is hydrogen, C1-C6 alkyl, substituted alkyl, C1-C6 carboxyalkyl (—(CH2)1-6COOR13), an alpha-acyloxyalkyl, a biologically compatible salt, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl;
- R20 is hydrogen, C1-C6 alkyl, substituted alkyl, C1-C6 carboxyalkyl (—(CH2)1-6COOR13), an alpha-acyloxyalkyl, a biologically compatible salt, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl;
- R9 is hydrogen, a reactive group, a carrier molecule, a solid support, —(CR2)nNR′R″, —(CR2)nNR′-fluorophore and C1-C6 alkyl;
- R10 is hydrogen, a reactive group, a carrier molecule, a solid support, —(CR2)nNR′R″, —(CR2)nNR′-fluorophore and C1-C6 alkyl;
- R11 is hydrogen, a reactive group, a carrier molecule, a solid support, —(CR2)nNR′R″, —(CR2)nNR′-fluorophore and C1-C6 alkyl;
- R12 is hydrogen, a reactive group, a carrier molecule, a solid support, —(CR2)nNR′R″, —(CR2)nNR′-fluorophore and C1-C6 alkyl;
- or
- a member selected from
- R9 in combination with R10;
- or
- R11 in combination with R12
- 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 R1-R12 is —(CR2)nNR′R″, —(CR2)nNR′-fluorophore or —(CR2)n-fluorophore with the proviso that when at least one of R1-R12 is —(CR2)n-fluorophore that the fluorophore comprise a nitrogen atom that is covalently bonded to —(CR2)n—.
43 more elements
A composition comprising:
- a) a compound having the formula:
- wherein
- R15 is hydrogen or C1-C6 alkyl;
- R16 is hydrogen or C1-C6 alkyl;
- R17 is hydrogen or C1-C6 alkyl;
- R18 is hydrogen or C1-C6 alkyl;
- and
- R13 is hydrogen, C1-C6 alkyl, —CH2OCOCH3 or a salt ion;
- R14 is hydrogen, C1-C6 alkyl, —CH2OCOCH3 or a salt ion;
- R1 is hydrogen, halogen, C1 to C10 alkyl (CH2), methoxy (—OCH3), hydroxyl (—OH), C2-C6 alkoxy (—OCH2), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR19R20), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR2)nNR′R″, —(CR2)nNR′-fluorophore or —(CR2)n-fluorophore;
- R2 is hydrogen, halogen, C1 to C10 alkyl (CH2), methoxy (—OCH3), hydroxyl (—OH), C2-C6 alkoxy (—OCH2), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR19R20), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR2)nNR′R″, —(CR2)nNR′-fluorophore or —(CR2)n-fluorophore;
- R3 is hydrogen, halogen, C1 to C10 alkyl (CH2), methoxy (—OCH3), hydroxyl (—OH), C2-C6 alkoxy (—OCH2), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR19R20), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR2)nNR′R″, —(CR2)nNR′-fluorophore or —(CR2)n-fluorophore;
- R4 is hydrogen, halogen, C1 to C10 alkyl (CH2), methoxy (—OCH3), hydroxyl (—OH), C2-C6 alkoxy (—OCH2), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR19R20), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR2)nNR′R″, —(CR2)nNR′-fluorophore or —(CR2)n-fluorophore;
- R5 is hydrogen, halogen, C1 to C10 alkyl (CH2), methoxy (—OCH3), hydroxyl (—OH), C2-C6 alkoxy (—OCH2), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR19R20), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR2)nNR′R″, —(CR2)nNR′-fluorophore or —(CR2)n-fluorophore;
- R6 is hydrogen, halogen, C1 to C10 alkyl (CH2), methoxy (—OCH3), hydroxyl (—OH), C2-C6 alkoxy (—OCH2), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR19R20), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR2)nNR′R″, —(CR2)nNR′-fluorophore or —(CR2)n-fluorophore;
- R7 is hydrogen, halogen, C1 to C10 alkyl (CH2), methoxy (—OCH3), hydroxyl (—OH), C2-C6 alkoxy (—OCH2), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR19R20), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR2)nNR′R″, —(CR2)nNR′-fluorophore or —(CR2)n-fluorophore;
- R8 is hydrogen, halogen, C1 to C10 alkyl (CH2), methoxy (—OCH3), hydroxyl (—OH), C2-C6 alkoxy (—OCH2), alicyclic, heteroalicyclic, aryl, heteroaryl, amino (—NR19R20), aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfonyl, reactive group, carrier molecule, solid support, reporter molecule, —(CR2)nNR′R″, —(CR2)nNR′-fluorophore or —(CR2)n-fluorophore;
- or
- a member independently selected from
- R1 in combination with R2;
- R2 in combination with R3;
- R3 in combination with R4;
- R5 in combination with R6;
- R6 in combination with R7;
- and
- R7 in combination with R8
- 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 R19 is hydrogen, C1-C6 alkyl, substituted alkyl, C1-C6 carboxyalkyl (—(CH2)1-6COOR13), an alpha-acyloxyalkyl, a biologically compatible salt, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl;
- R20 is hydrogen, C1-C6 alkyl, substituted alkyl, C1-C6 carboxyalkyl (—(CH2)1-6COOR13), an alpha-acyloxyalkyl, a biologically compatible salt, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl;
- R9 is hydrogen, a reactive group, a carrier molecule, a solid support, —(CR2)nNR′R″, —(CR2)nNR′-fluorophore and C1-C6 alkyl;
- R10 is hydrogen, a reactive group, a carrier molecule, a solid support, —(CR2)nNR′R″, —(CR2)nNR′-fluorophore and C1-C6 alkyl;
- R11 is hydrogen, a reactive group, a carrier molecule, a solid support, —(CR2)nNR′R″, —(CR2)nNR′-fluorophore and C1-C6 alkyl;
- R12 is hydrogen, a reactive group, a carrier molecule, a solid support, —(CR2)nNR′R″, —(CR2)nNR′-fluorophore and C1-C6 alkyl;
- or
- a member selected from
- R9 in combination with R10;
- or
- R11 in combination with R12
- 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 R1-R12 is —(CR2)nNR′R″, —(CR2)nNR′-fluorophore or —(CR2)n-fluorophore with the proviso that when at least one of R1-R12 is —(CR2)n-fluorophore that the fluorophore comprise a nitrogen atom that is covalently bonded to —(CR2)n—;
- and
- b) a metal ion that is capable of being chelated by the compound.
46 more elements
-32. (canceled)
Dependencies are inferred from the claim text.