Novel green and orange fluorescent labels and their uses

A fluorescent compound of the structure:

• 13
• where R2=—OH, —NH2, SO3— or SH(NH2)2,
• M is one of the alkali metals (Li, Na, K), ammonium (NH4), or pyridinium (Py),
• R1 is a spacer comprised of 2 to 20 carbon, oxygen or nitrogen atoms, with the carbon containing sequences chosen from alkyl, alkenyl, arylalkyl, or alkoxy groups, which can bear any of several substitutions along the carbon chain, including but not limited to amino, carbonyl, carboxyl, hydroxyl, sulfonyl, sulfonamide, oxyethylene, ethylene oxide, or hydroxyl moieties, and,
• R′=a succinimidyl ester, hydrazide, isothiocynato, isocyanato, maleimido, halo-acetamido, biotin or other homofunctional linker chemistry capable of covalently binding with a complementary reactive chemical entity on another molecule for the purpose of permanently linking the fluorophore to the second molecule.

A fluorescent compound of the structure

• 15
• R is chosen from any of alkyl, alkenyl, alkylaryl, aromatic, or alkoxy groups which can be substituted by various constituents including but not limited to amino, carbonyl, carboxyl, hydroxyl, sulfonyl, sulfonamide, oxyethylene, ethylene oxide, or hydroxyl moieties,
• R4 is chosen from —H, —CH3, alkyl, alkenyl, aromatic, or alkylaryl groups which can, in turn be substituted with any of several substitutions along the carbon chain, including but not limited to amino, carbonyl, carboxyl, hydroxyl, sulfonyl, sulfonamide, oxyethylene, ethylene oxide, or hydroxyl moieties,
• X is chosen from one of (CH2)n, aryl-(CH2)n, PEG, DEXTRAN, (CH2)n-CONH—(CH2)n, (CH2)N—CONH-Z, where n=1-15, Z=PEG, DEXTRAN or a polypeptide, which can bear any of several substitutions along the carbon chain, including but not limited to amino, carbonyl, carboxyl, hydroxyl, sulfonyl, sulfonamide, oxyethylene, ethylene oxide, or hydroxyl moieties, and,
• Y is chosen from one of —NH2, —NCS, —SH, —OH, —COOH, —COONHS, —NH2, -Maleimide, -hydrazide, CHO, biotinyl group, avidinyl group, NHCOCH2I or other monofunctional linker chemistry capable of covalently binding with a complementary reactive chemical entity on another molecule for the purpose of permanently linking the fluorophore to the second molecule.

A kit comprising the fluorescent of the general structure:

• 16
• where R2=—OH, —NH2, SO3— or SH(NH2)2,
• M is one of the alkali metals (Li, Na, K), ammonium (NH4), or pyridinium (Py),
• R1 is a spacer comprised of 2 to 20 carbon, oxygen or nitrogen atoms, with the carbon containing sequences chosen from alkyl, alkenyl, arylalkyl, or alkoxy groups, which can bear any of several substitutions
• along the carbon chain, including but not limited to amino, carbonyl, carboxyl, hydroxyl, sulfonyl, sulfonamide, oxyethylene, ethylene oxide, or hydroxyl moieties, and,
• R′=a terminal carboxyl, amino, sulfhydryl or biotinyl group.

A kit comprising a compound of the structure:

• 18
• where R2=—OH, —NH2, SO3— or SH(NH2)2,
• M is one of the alkali metals (Li, Na, K), ammonium (NH4), or pyridinium (Py),
• R1 is a spacer comprised of 2 to 20 carbon, oxygen or nitrogen atoms, with the carbon containing sequences chosen from alkyl, alkenyl, arylalkyl, or alkoxy groups, which can bear any of several substitutions along the carbon chain, including but not limited to amino, carbonyl, carboxyl, hydroxyl, sulfonyl, sulfonamide, oxyethylene, ethylene oxide, or hydroxyl moieties, and,
• R′=a succinimidyl ester, hydrazide, isothiocynato, isocyanato, maleimido, halo-acetamido, biotin or other homofunctional linker chemistry capable of covalently binding with a complementary reactive chemical entity on another molecule for the purpose of permanently linking the fluorophore to the second molecule.

A kit comprising a fluorescent compound of the structure:

• 20
• R= is chosen from any of alkyl, alkenyl, alkylaryl, aromatic, or alkoxy groups which can be substituted by various constituents including but not limited to amino, carbonyl, carboxyl, hydroxyl, sulfonyl, sulfonamide, oxyethylene, ethylene oxide, or hydroxyl moieties,
• R4 is chosen from —H, —CH3, alkyl, alkenyl, aromatic, or alkylaryl groups which can, in turn be substituted with any of several substitutions along the carbon chain, including but not limited to amino, carbonyl, carboxyl, hydroxyl, sulfonyl, sulfonamide, oxyethylene, ethylene oxide, or hydroxyl moieties,
• X is chosen from one of (CH2)n, aryl-(CH2)n, PEG, DEXTRAN, (CH2)n—CONH—(CH2)n, (CH2)N—CONH-Z, where n=1-15, Z=PEG, DEXTRAN or a polypeptide, which can bear any of several substitutions along the carbon chain, including but not limited to amino, carbonyl, carboxyl, hydroxyl, sulfonyl, sulfonamide, oxyethylene, ethylene oxide, or hydroxyl moieties, and,
• Y is chosen from one of —NH2, —NCS, —SH, —OH, —COOH, —COONHS, —NH2, -Maleimide, -hydrazide, CHO, biotinyl group, avidinyl group, NHCOCH2I or other monofunctional linker chemistry capable of covalently binding with a complementary reactive chemical entity on another molecule for the purpose of permanently linking the fluorophore to the second molecule.

A method for detecting biomolecules comprising:

• a) providing a fluorescent compound of the general structure
• 21
• where R2=—OH, —NH2, SO3— or SH(NH2)2,
• M is one of the alkali metals (Li, Na, K), ammonium (NH4), or pyridinium (Py),
• R1 is a spacer comprised of 2 to 20 carbon, oxygen or nitrogen atoms, with the carbon containing sequences chosen from alkyl, alkenyl, arylalkyl, or alkoxy groups, which can bear any of several substitutions
• along the carbon chain, including but not limited to amino, carbonyl, carboxyl, hydroxyl, sulfonyl, sulfonamide, oxyethylene, ethylene oxide, or hydroxyl moieties, and,
• R′=a terminal carboxyl, amino, sulfhydryl or biotinyl group;
• and
• b) detecting the presence or absence of a fluorescence signal.

A method for detecting biomolecules comprising:

• a) providing a fluorescent compound of the general structure
• 22
• where R2=—OH, —NH2, SO3— or SH(NH2)2,
• M is one of the alkali metals (Li, Na, K), ammonium (NH4), or pyridinium (Py),
• R1 is a spacer comprised of 2 to 20 carbon, oxygen or nitrogen atoms, with the carbon containing sequences chosen from alkyl, alkenyl, arylalkyl, or alkoxy groups, which can bear any of several substitutions along the carbon chain, including but not limited to amino, carbonyl, carboxyl, hydroxyl, sulfonyl, sulfonamide, oxyethylene, ethylene oxide, or hydroxyl moieties, and,
• R′= a succinimidyl ester, hydrazide, isothiocynato, isocyanato, maleimido, halo-acetamido, biotin or other homofunctional linker chemistry capable of covalently binding with a complementary reactive chemical entity on another molecule for the purpose of permanently linking the fluorophore to the second molecule;
• and
• b) detecting the presence or absence of a fluorescence signal.

A method for detecting biomolecules comprising:

• a) providing a fluorescent compound of the general structure
• 23
• R= is chosen from any of alky, alkenyl, alkylaryl, aromatic, or alkoxy groups which can be substituted by various constituents including but not limited to amino, carbonyl, carboxyl, hydroxyl, sulfonyl, sulfonamide, oxyethylene, ethylene oxide, or hydroxyl moieties,
• R4 is chosen from —H, —CH3, alkyl, alkenyl, aromatic, or alkylaryl groups which can, in turn be substituted with any of several substitutions along the carbon chain, including but not limited to amino, carbonyl, carboxyl, hydroxyl, sulfonyl, sulfonamide, oxyethylene, ethylene oxide, or hydroxyl moieties,
• X is chosen from one of (CH2)n, aryl-(CH2)n, PEG, DEXTRAN, (CH2)n—CONH—(CH2)n, (CH2)N—CONH-Z, where n=1-15, Z=PEG, DEXTRAN or a polypeptide, which can bear any of several substitutions along the carbon chain, including but not limited to amino, carbonyl, carboxyl, hydroxyl, sulfonyl, sulfonamide, oxyethylene, ethylene oxide, or hydroxyl moieties, and,
• Y is chosen from one of —NH2, —NCS, —SH, —OH, —COOH, —COONHS, —NH2, -Maleimide, -hydrazide, CHO, biotinyl group, avidinyl group, NHCOCH2I or other monofunctional linker chemistry capable of covalently binding with a complementary reactive chemical entity on another molecule for the purpose of permanently linking the fluorophore to the second molecule;
• and
• b) detecting the presence or absence of a fluorescence signal.