Sulfonated 2(7)-aminoacridone and 1-aminopyrene dyes and their use as fluorescent tags, in particular for carbohydrate analysis

Abstract

Sulfonated 2(7)-aminoacridone and 1-aminopyrene dyes and their use as fluorescent tags, in particular for carbohydrate analysis The invention relates to fluorescent dyes with multiple negatively charged groups in their ionized form which are aminoacridone sulfonamides or 1-aminopyrenes having of one of the following general formulae A-D: Formula (A), Formula (B), Formula (C), Formula (D), wherein the ionizable groups X are typically selected from the following: SH, COOH, SO.sub.3H, OSO.sub.3H, OP(O)(OH).sub.2, OP(O)(OH)R.sup.a, P(O)(OH).sub.2, P(O)(OH)R.sup.a, where R.sup.aC.sub.1-C.sub.4alkyl or substituted C.sub.1-C.sub.4alkyl. The invention further relates to the use of these dyes as fluorescent tags, in particular for reducing sugars and glycans. ##STR00001##

Claims

1. A fluorescent dye having multiple ionizable and/or negatively charged groups which is selected from the group consisting of compounds of the following general Formula B or of salts thereof: ##STR00050## wherein R.sup.1 and/or R.sup.2 are independent from each other and may represent: H, deuterium (D), alkyl or deutero-substituted alkyl; R.sup.1-R.sup.2 may form a four-, five-, six-, or seven-membered non-aromatic carbocycle with an additional primary amino group NH.sub.2, secondary amino group NHR.sup.a, where R.sup.aC.sub.1-C.sub.6 alkyl, or hydroxyl group OH attached to one carbon atom of this carbocycle; or R.sup.1-R.sup.2 may form a four-, five-, six-, or seven-membered non-aromatic heterocycle with an additional heteroatom included in the heterocycle; a hydroxyalkyl group (CH.sub.2).sub.mOH, where m=1-12, with a straight or branched alkyl chain; one of R.sup.1 or R.sup.2 groups may be a carbonate or carbamate derivative (CH.sub.2).sub.mOCOOR.sup.4 or (CH.sub.2).sub.mNHCOOR.sup.4, where m=1-12 and R.sup.4=methyl, ethyl, 2-chloroethyl, N-succinimidyl, sulfo-N-succinimidyl, 1-oxybenzotriazolyl, a phenyl group or a substituted phenyl group; (CH.sub.2).sub.mNR.sup.aR.sup.b, where m=1-12, with a straight or branched alkyl chain; R.sup.a, R.sup.b are independent from each other and may be H, or optionally substituted C.sub.1-C.sub.4 alkyl group(s); one of R.sup.1 or R.sup.2 groups may be a primary amino group to form aryl hydrazines Ar-NR.sup.6NH.sub.2 where Ar is the entire pyrene residue in Formula B and R.sup.6H or alkyl; one of R.sup.1 or R.sup.2 groups may be a hydroxy group to form aryl hydroxylamines Ar-NR.sup.7OH where Ar is an entire pyrene residue in Formula B and R.sup.7H or alkyl; one of R.sup.1 or R.sup.2 groups may be an alkyloxyamino group (CH.sub.2).sub.nONH.sub.2 with n=1-12; one of R.sup.1 or R.sup.2 groups may be CO(CH.sub.2).sub.nCOOR, with n=1-5 and a straight or branched alkyl chain (CH.sub.2)n and with R.sup.8 selected from H, straight or branched C.sub.1-C.sub.6 alkyl, CH.sub.2CN, 2- and 4-nitrophenyl, 2,3,5,6-tetrafluorophenyl, pentachlorophenyl, pentafluoro-phenyl, N-succinimidyl, sulfo-N-succinimidyl, 1-oxybenzo-triazolyl; further, one of R.sup.1 or R.sup.2 may be (CH.sub.2).sub.nCONHR.sup.9, with n=1-5 and R.sup.9H, C.sub.1-C.sub.6 alkyl, (CH.sub.2).sub.mN.sub.3, (CH.sub.2).sub.mN-maleimido, (CH.sub.2).sub.mNHCOCH.sub.2X (XBr or I), where m=2-6 and with straight or branched alkyl chains in (CH.sub.2)n and R.sup.9; or one of R.sup.1 or R.sup.2 may represent CH.sub.2C.sub.6H.sub.4NH.sub.2, COC.sub.6H.sub.4NH.sub.2, CONHC.sub.6H.sub.4NH.sub.2 or CSNHC.sub.6H.sub.4NH.sub.2 with C.sub.6H.sub.4 being a 1,2-, 1,3- or 1,4-phenylene, COC.sub.5H3NNH.sub.2 or CH.sub.2C.sub.5H3NNH.sub.2 with C.sub.5H.sub.3N being pyridin-2,4-diyl, pyridin-2,5-diyl, pyridin-2,6-diyl, or pyridin-3,5-diyl; or one of R.sup.1 or R.sup.2 may be an alkyl azide (CH)N.sub.3 or alkine; wherein linker L may be divalent or polyvalent and comprises at least one carbon atom and may represent alkyl, heteroalkyl, perfluoroalkyl, alkyloxy, single or multiple difluoromethyl (CF.sub.2), and alkene or alkine moieties in any combinations, at any occurrence, linear or branched, with a length ranging from C.sub.1 to C.sub.12; the linker L may also include a carbonyl (CH.sub.2CO, CF.sub.2CO) moiety, also as a part of an amide group; or the linker L may also comprise or contain a residue of 1,3,5-triazine, thus providing two attachment points for group X; X denotes a solubilizing and/or ionizable anion-providing moiety, further, the anion-providing moiety is optionally linked through non-aromatic O, N and S-containing heterocycles, or, alternatively, one of the groups X optionally bears any of the moieties listed above for groups R.sup.1 and R.sup.2, also with any type of linkage listed for group L, and independently from other substituents; with the proviso that in all compounds represented by Formula B three or six negatively charged groups are present in the residues X of Formula B under basic conditions, and the negatively charged groups represent at least partially deprotonated residues of ionizable groups selected from the following: OH, SH, COOH, SO.sub.3H, OSO.sub.3H, OP(O)(OH).sub.2, OP(O)(OH)R.sup.a, where R.sup.aC.sub.1-C.sub.4 alkyl or substituted C.sub.1-C.sub.4 alkyl, P(O)(OH).sub.2, and P(O)(OH)R.sup.a, where R.sup.aC.sub.1-C.sub.4 alkyl or substituted C.sub.1-C.sub.4 alkyl.

2. The fluorescent dye according to claim 1, wherein X at each occurrence represents 1 to 4 groups SO.sub.3H attached to the respective linker group L at one or more sites.

3. The fluorescent dye according to claim 1 having Formula C and at least one alkyl sulfonyl group ##STR00051## or a salt thereof, wherein n=1-12; R.sup.1 and/or R.sup.2 are independent from each other and may represent: H, deuterium (D), alkyl or deutero-substituted alkyl; R.sup.1-R.sup.2 may form a four-, five, six-, or seven-membered non-aromatic carbocycle with an additional primary amino group NH.sub.2, secondary amino group NHR.sup.a, where R.sup.aC.sub.1-C.sub.6 alkyl, or hydroxyl group OH attached to one carbon atom of this carbocycle; or R.sup.1-R.sup.2 may form a four-, five-, six-, or seven-membered non-aromatic heterocycle with an additional heteroatom included in the heterocycle; a hydroxyalkyl group (CH.sub.2).sub.mOH, where m=2-12, with a straight or branched alkyl chain; one of R.sup.1 or R.sup.2 groups may be a carbonate or carbamate derivative (CH.sub.2).sub.mOCOOR.sup.4 or (CH.sub.2).sub.mNHCOOR.sup.4, where m=1-12 and R.sup.4=methyl, ethyl, 2-chloroethyl, N-succinimidyl, sulfo-N-succinimidyl, 1-oxybenzotriazolyl, a phenyl group or a substituted phenyl group; (CH.sub.2).sub.mNR.sup.aR.sup.b, where m=2-12, with a straight or branched alkyl chain; R.sup.a, R.sup.b are independent from each other and may be H, or optionally substituted C.sub.1-C.sub.4 alkyl group(s); one of R.sup.1 or R.sup.2 groups may be a primary amino group to form aryl hydrazines, as described for Formula B; one of R.sup.1 or R.sup.2 groups may be a hydroxy group to form aryl hydroxylamines, as described for Formula B; one of R.sup.1 or R.sup.2 groups may be an alkyloxyamino group (CH.sub.2).sub.nONH.sub.2 with n=1-12; one of R.sup.1 or R.sup.2 may be (CH.sub.2).sub.nCONHR.sup.6, with n=1-12, and R.sup.6H, C.sub.1-C.sub.6 alkyl, (CH.sub.2).sub.mN.sub.3, (CH.sub.2).sub.mN-maleimido, (CH.sub.2).sub.mNHCOCH.sub.2X (XBr or I), where m=2-6 and with straight or branched alkyl chains in (CH.sub.2)n and R.sup.6; or one of R.sup.1 or R.sup.2 may represent CH.sub.2C.sub.6H.sub.4NH.sub.2, COC.sub.6H.sub.4NH.sub.2, CONHC.sub.6H.sub.4NH.sub.2 or CSNHC.sub.6H.sub.4NH.sub.2 with C.sub.6H.sub.4 being a 1,2-, 1,3- or 1,4-phenylene, COC.sub.5H3NNH.sub.2 or CH.sub.2C.sub.5H.sub.3NNH.sub.2 with C.sub.5H.sub.3N being pyridin-2,4-diyl, pyridin-2,5-diyl, pyridin-2,6-diyl, or pyridin-3,5-diyl; the (CH.sub.2)nCH.sub.2 linker, with n=1-12, between the SO.sub.2 fragment and the residue X in Formula B may represent a straight-chain, branched or cyclic group having 2-6 carbon atoms; XSH, COOH, SO.sub.3H, OSO.sub.3H, OP(O)(OH).sub.2, OP(O)(OH)R.sup.a, where R.sup.a=optionally substituted C.sub.1-C.sub.4 alkyl, P(O)(OH).sub.2, P(O)(OH)R.sup.a, where R.sup.a=optionally substituted C.sub.1-C.sub.4 alkyl; with the proviso that in all compounds represented by Formula C three or six negatively charged groups are present in the residues X of Formula C under basic conditions, and the negatively charged groups represent at least partially deprotonated residues of ionizable groups selected from the following: OH, SH, COOH, SO.sub.3H, OSO.sub.3H, OP(O)(OH).sub.2, OP(O)(OH)R.sup.a, where R.sup.aC.sub.1-C.sub.4 alkyl or substituted C.sub.1-C.sub.4 alkyl, P(O)(OH).sub.2, and P(O)(OH)R.sup.a, where R.sup.aC.sub.1-C.sub.4 alkyl or substituted C.sub.1-C.sub.4 alkyl.

4. The fluorescent dye according to claim 2, wherein X at each occurrence is SO.sub.3H and n is 1-12.

5. The fluorescent dye according to claim 3 one of Formula B or C, wherein R.sup.1 and/or R.sup.2 in Formula B or C represents H, deuterium (D), alkyl or deutero-substituted alkyl, wherein one, several or all H atoms in the alkyl group may be replaced by deuterium atoms, 4,6-dihalo-1,3,5-triazinyl (C.sub.3N.sub.3X.sub.2) where X is halogen, 2-, 3- or 4-aminobenzoyl (COC.sub.6H.sub.4NH.sub.2), N-[(2-, N-[(3- or N-[(4-aminophenyl)ureido group (NHCONHC.sub.6H.sub.4NH.sub.2), N-[(2-, N-[(3- or N-[(4-aminophenyl)thioureido group (NHCSNHC.sub.6H.sub.4NH.sub.2) or linked carboxylic acid residues and their reactive esters of the general formulae (CH.sub.2).sub.m1COOR.sup.3, (CH.sub.2).sub.m1OCOOR.sup.3, (CH.sub.2).sub.n1COOR.sup.3 or (CO).sub.m1(CH.sub.2).sub.m2(CO).sub.n1(NH).sub.n2(CO).sub.n3(CH.sub.2).sub.n4COOR.sup.3 where the integers m1, m2 and n1, n2, n3, and n4 independently range from 1 to 12 and from 0 to 12, respectively, with the linked carboxylic acid residues and their respective esters being straight, branched, saturated, unsaturated, partially or completely deuterated, and/or included into a carbo- or heterocycle containing N, O or S, whereas R.sup.3 is H, D or a nucleophile-reactive leaving group.

6. The fluorescent dye according to claim 1 that has a hydroxylamine or a hydrazine moiety as an analyte-reactive group, which is connected directly with a dye core or where the hydroxylamine, hydrazine or sulfonyl hydrazide moieties are connected via a linker group.

7. The fluorescent dye according to claim 1 having one of the following formulae: ##STR00052## or a salt thereof.

8. The fluorescent dye according to claim 3 having Formula B or C which has a net charge z of 0, 1, 3, 6 or 12.

9. The fluorescent dye according to any one of claims 1, 6 or 8, which is a salt comprising negatively charged sulfonate and/or phosphate groups and counterions selected from inorganic or organic cations, and/or comprising a positively charged group or a charge-transfer complex formed at the nitrogen site N(R.sup.1)R.sup.2 in the dye of formula B or C with a counterion selected from anions of a strong mineral, organic or a Lewis acid.

10. The fluorescent dye according to any one of claims 1 6, or 8, wherein carbohydrate-reactive groups, are connected to the nitrogen site N(R.sup.1)R.sup.2 in the dye of formula B or C via all types of linkers listed for group L in formula B.

11. The fluorescent dye according to any one of claims 1, 6 or 8 and salts thereof for use in a reductive amination and in a condensation with reducing sugars.

12. The fluorescent dye according to any one of claims 1, 6 or 8 and salts thereof for use as a fluorescent label for amino acids, peptides, proteins, including primary and secondary antibodies, single-domain antibodies, docetaxel, avidin, streptavidin and their modifications, aptamers, nucleotides, nucleic acids, toxins, lipids, carbohydrates, including 2-deoxy-2-amino glucose and other 2-deoxy-2-aminoaminopyranosides, glycans, biotin, and other small molecules having molecular masses of less than 1500 Da.

13. The fluorescent dye according to claim 12 belongs to the following compound set: 13a, 13b, 16 and 18 ##STR00053## ##STR00054##

14. A method of using the fluorescent dye according to any one of claims 1, 6 or 8 and salts thereof as fluorescent reagents for conjugation to analytes, wherein the method comprises a conjugation step comprising forming at least one covalent chemical bond or at least one molecular complex with a chemical entity or substance.

15. Carbohydrate-dye conjugates comprising the fluorescent dye according to any one of claims 1, 6 or 8.

16. The carbohydrate-dye conjugates according to claim 15 wherein the fluorescent dye is selected from the compounds shown below: ##STR00055##

17. A kit or composition comprising one or more of the fluorescent dye of claims 1, 6 or 8.

18. A method for synthesizing fluorescent aminopyrene dyes with an absorbance maxima of 477-510 nm, a net charge of 0 or down to 6, and an emission maxima of 535-560 nm in water, said method comprising an exchange reaction of an aryl halogen to a substituted thioalkyl function, followed by oxidation to a sulfone and, optionally, by phosphorylation at the hydroxyl site, wherein the fluorescent aminopyrene dyes have the general formula B or of salts thereof: ##STR00056## wherein R.sup.1 and/or R.sup.2 are independent from each other and may represent: H, deuterium (D), alkyl or deutero-substituted alkyl; R.sup.1-R.sup.2 may form a four-, five-, six-, or seven-membered non-aromatic carbocycle with an additional primary amino group NH.sub.2, secondary amino group NHR.sup.a, where R.sup.1 C.sub.1-C.sub.6 alkyl, or hydroxyl group OH attached to one carbon atom of this carbocycle; or R.sup.1-R.sup.2 may form a four-, five-, six-, or seven-membered non-aromatic heterocycle with an additional heteroatom included in the heterocycle; a hydroxyalkyl group (CH.sub.2).sub.mOH, where m=1-12, with a straight or branched alkyl chain; one of R.sup.1 or R.sup.2 groups may be a carbonate or carbamate derivative (CH.sub.2).sub.mOCOOR.sup.4 or (CH.sub.2).sub.mNHCOOR.sup.4, where m=1-12 and R.sup.4=methyl, ethyl, 2-chloroethyl, 1V-succinimidyl, sulfo-N-succinimidyl, 1-oxybenzotriazolyl, a phenyl group or a substituted Phenyl group; (CH.sub.2).sub.mNR.sup.aR.sup.b, where m=1-12, with a straight or branched alkyl chain; R.sup.a, R.sup.b are independent from each other and may be H or optionally substituted C.sub.1-C.sub.4 alkyl group(s); one of R.sup.1 or R.sup.2 groups may be a primary amino group to form aryl hydrazines Ar-NR.sup.6NH.sub.2 where Ar is the entire pyrene residue in Formula B and R.sup.6H or alkyl; one of R.sup.1 or R.sup.2 groups may be a hydroxy group to form aryl hydroxylamines Ar-NR.sup.7OH where Ar is an entire pyrene residue in Formula B and R.sup.7H or alkyl; one of R.sup.1 or R.sup.2 groups may be an alkyloxyamino group (CH.sub.2).sub.nONH with n=1-12; one of R.sup.1 or R.sup.2 groups may be CO(CH.sub.2).sub.nCOOR.sup.8, with n=1-5 and a straight or branched alkyl chain (CH.sub.2).sub.n and with R.sup.8 selected from H, straight or branched C.sub.1-C.sub.6 alkyl, CH.sub.2CN, 2- and 4-nitrophenyl, 2,3,5,6-tetrafluorophenyl, pentachlorophenyl, pentafluoro-phenyl, N-succinimidyl, sulfo-N-succinimidyl, 1-oxybenzo-triazolyl; further, one of R.sup.1 or R.sup.2 may be (CH.sub.2).sub.nCONHR.sup.9, with n=1-5 and R.sup.9 H, C.sub.1-C.sub.6 alkyl, (CH.sub.2).sub.mN.sub.3, (CH.sub.2).sub.mN-maleimido, (CH.sub.2).sub.mNHCOCH.sub.2X (XBr or I), where m=2-6 and with straight or branched alkyl chains in (CH.sub.2)n and R.sup.9; or one of R.sup.1 or R.sup.2 may represent CH.sub.2-C.sub.6H.sub.4NH.sub.2, COC.sub.6H.sub.4NH.sub.2, CONHC.sub.6H.sub.4NH.sub.2 or CSNHC.sub.6H.sub.4NH.sub.2 with C.sub.6H.sub.4 being a 1,2-, 1,3- or 1,4-phenylene, COC.sub.5H.sub.3NNH.sub.2 or CH.sub.2-C.sub.4H.sub.3NNH.sub.2 with C.sub.5H.sub.3N being pyridin-2,4-diyl, pyridin-2,5-diyl, pyridin-2,6-diyl, or pyridin-3,5-diyl; or one of R.sup.1 or R.sup.2 may be an alkyl azide (CH)N.sub.3 or alkine; wherein linker L may be divalent or polyvalent and comprises at least one carbon atom and may represent alkyl, heteroalkyl, perfluoroalkyl, alkyloxy, single or multiple difluoromethyl (CF.sub.2), and alkene or alkine moieties in any combinations, at any occurrence, linear or branched, with a length ranging from C.sub.1 to C.sub.12; the linker L may also include a carbonyl (CH.sub.2CO, CF.sub.2CO) moiety, also as a tart of an amide group; or the linker L may also comprise or contain a residue of 1,3,5-triazine, thus providing two attachment points for group X; X denotes a solubilizing, and/or ionizable anion-providing moiety, further, the anion-providing moiety is optionally linked through non-aromatic O, N and S-containing heterocycles, or, alternatively, one of the groups X optionally bears any of the moieties listed above for groups R.sup.1 and R.sup.2, also with any type of linkage listed for group L, and independently from other substituents; with the proviso that in all compounds represented by Formula B three or six negatively charged groups are present in the residues X of Formula B under basic conditions, and the negatively charged groups represent at least partially deprotonated residues of ionizable groups selected from the following: OH, SH, COOH, SO.sub.3H, OSO.sub.3H, OP(O)(OH).sub.2, OP(O)(OH)R.sup.a, where R.sup.aC.sub.1-C.sub.4 alkyl or substituted C.sub.1-C.sub.4 alkyl, P(O)(OH).sub.2, and P(O)(OH)R.sup.a, where R.sup.aC.sub.1-C.sub.4 alkyl or substituted C.sub.1-C.sub.4 alkyl, or wherein the fluorescent aminopyrene dues have the general formula C: ##STR00057## or a salt thereof, wherein n=1-12; R.sup.1 and/or R.sup.2 are independent from each other and may represent; H, deuterium (D), alkyl or deutero-substituted alkyl; R.sup.1-R.sup.2 may form a four-, five, six-, or seven-membered non-aromatic carbocycle with an additional primary amino group NH.sub.2, secondary amino group NHR.sup.a, where R.sup.a C.sub.1-C.sub.6, alkyl, or hydroxyl group OH attached to one carbon atom of this carbocycle; or R.sup.1-R.sup.2 may form a four-, five-, six-, or seven-membered non-aromatic heterocycle with an additional heteroatom included in the heterocycle; a hydroxyalkyl group (CH.sub.2).sub.mOH, where m=2-12, with a straight or branched alkyl chain, one of R.sup.1 or R.sup.2 groups may be a carbonate or carbamate derivative (CH.sub.2).sub.mOCOOR.sup.4 or (CH.sub.2).sub.mNHCOOR.sup.4, where m=1-12 and R.sup.4=methyl, ethyl, 2-chloroethyl, N4-succinimidyl, sulfo-N-succinimidyl, 1-oxybenzotriazolyl, a phenyl group or a substituted phenyl group; (CH.sub.2).sub.mNR.sup.aR.sup.b, where in m=2-12, with a straight or branched alkyl chain, R.sup.a, R.sup.b are independent from each other and may be H, or optionally substituted C.sub.1-C.sub.4 alkyl group(s); one of R.sup.1 or R.sup.2 groups may be a primary amino group to form aryl hydrazines, as described for Formula B; one of R.sup.1 or R.sup.2 groups may be a hydroxy group to form aryl hydroxylamines, as described for Formula B; one of R.sup.1 or R.sup.2 groups may bean alkyloxyamino group (CH.sub.2).sub.nONH.sub.2 with n=1-12; one of R.sup.1 or R.sup.2 may be (CH.sub.2).sub.nCONHR.sup.6, with n=1-12, and R.sup.6H, C.sub.1-C.sub.6 alkyl, (CH.sub.2).sub.mN.sub.3, (CH.sub.2).sub.mN-maleimido, (CH.sub.2).sub.mNHCOCH.sub.2X (XBr or I), where m=2-6 and with straight or branched alkyl chains in (CH.sub.2)n and R.sup.6; or one of R.sup.1 or R.sup.2 may represent CH.sub.2-C.sub.6H.sub.4NH.sub.2, COC.sub.6H.sub.4NH.sub.2, CONHC.sub.6H.sub.4NH.sub.2 or CSNHC.sub.6H.sub.4NH.sub.2 with C.sub.6H.sub.4 being a 1,2, 1,3- or 1,4-phenylene, COC.sub.5H.sub.3NNH.sub.2 or CH.sub.2CH.sub.2NNH.sub.2 with C.sub.5H.sub.3N being pyridin-2,4-diyl, pyridin-2,5-diyl, pyridin-2,6-diyl, or pyridin-3,5-diyl; the (CH.sub.2)n-CH.sub.2 linker, with n=1-12, between the SO.sub.2 fragment and the residue X in Formula B may represent a straight-chain, branched or cyclic group having 2-6 carbon atoms, XSH, COOH, SO.sub.3H, OSO.sub.3H, OP(O)(OH).sub.2, OP(O)(OH)R.sup.a, where R.sup.a=optionally substituted C.sub.1-C.sub.4 alkyl, P(O)(OH).sub.2, P(O)(OH)R.sup.a, where R.sup.a=optionally substituted C.sub.1-C.sub.4 alkyl; with the proviso that in all compounds represented by Formula C three or six negatively charged groups are present in the residues X of Formula C under basic conditions, and the negatively charged groups represent at least partially deprotonated residues of ionizable groups selected from the following OH, SH, COOH, SO.sub.3H, OSO.sub.3H, OP(O)(OH).sub.2, OP(O)(OH)R.sup.a, where R.sup.a C.sub.1-C.sub.4 alkyl or substituted C.sub.1-C.sub.4 alkyl, P(O)(OH).sub.2, and P(O)(OH)R.sup.a, where R.sup.aC.sub.1-C.sub.4 alkyl or substituted C.sub.1-C.sub.4 alkyl.

19. A kit or composition comprising one or more of the carbohydrate-dye conjugates of claim 15.

20. The fluorescent dye according to claim 1, wherein X comprises a moiety selected from the group consisting of hydroxyalkyl (CH.sub.2).sub.nOH, thioalkyl ((CH.sub.2).sub.nSH), carboxy alkyl ((CH.sub.2).sub.nCO.sub.2H), alkyl sulfonate ((CH.sub.2).sub.nSO.sub.3H), alkyl sulfate ((CH.sub.2)OSO.sub.3H), alkyl phosphate ((CH.sub.2).sub.nOP(O)(OH).sub.2) and phosphonate ((CH.sub.2).sub.nP(O)(OH).sub.2), wherein n is an integer ranging from 0 to 12, or an analog thereof wherein one or more of the CH.sub.2 groups are replaced by CF.sub.2.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 Principal steps of separation-based glycan analysis.

(2) FIG. 2A shows actual normalized absorbance and fluorescence spectra of the new aminoacridone dyes: 6-H, 6-Me in TEAB buffer (pH 8-8.5)

(3) FIG. 2B shows actual normalized absorbance and fluorescence spectra of the new phosphorylated aminoppyrene dyes 8-H and 15 in TEAB buffer (pH 8-8.5) FIG. 3 is a scheme showing the information flow from the genome to the glycome.

(4) The following Examples illustrate the present invention in more detail.

GENERAL MATERIALS AND METHODS

(5) 1.1.1 Preparative Methods

(6) If not otherwise stated, reactions were carried out under an argon atmosphere. Solvents met Reag. PhEur purity standard (p.a.). Commercially available substances were used without further purification and purchased from Sigma Aldrich, Acros Organics, Alfa Aesar, ABCR, Merck Millipore, Carbolution Chemicals, etc.

(7) 1.1.2 Chromatographic Methods

(8) 1.1.2.1 Thin Layer Chromatography (TLC)

(9) Normal phase TLC was performed on silica gel 60 F.sub.254 (Merck Millipore). For TLC on reversed phase silica gel 60 RP-18 F.sub.254 (Merck Millipore) was used. Preparative TLC was performed on HPTLC Silica gel 60 F.sub.254 with concentrating zone 102.5 cm (Merck Millipore). Compounds were detected by exposing TLC plates to UV-light (254 or 366 nm). Amines were detected by using ninhydrin solution (0.5% in EtOH).

(10) 1.1.2.2 Column Chromatography

(11) Silica gel 60 with a particle size of 40-63 m was purchased from Merck Millipore. Reversed phase column chromatography was performed on POLYGOPREE 60-50 C.sub.18 (Macherey Nagel). Deactivated silica gel 60 was purchased from MP Biomedical. Routine separation was performed with an automated Isolera One system (Biotage GmbH) with commercially available cartridges.

(12) 1.1.3 Analytical Instruments

(13) 1.1.3.1 Absorption Spectroscopy

(14) Absorption spectra were recorded with a double-beam UV-vis spectrophotometer (Varian, series 4000). For the determination of the absorption spectra, quartz cells with a 1 cm path length were used. Emission spectra and fluorescence quantum yield were obtained on a Quantaurus-QY Absolute PL quantum yield spectrometer C11347 (Quantaurus QY) or on a Cary Eclipse fluorescence spectrometer (Varian).

(15) 1.1.3.2 Nuclear Magnetic Resonance (NMR)

(16) NMR spectra were recorded on an Agilent 400MR DD2 spectrometer. All spectra are referenced to tetrametylsilane as an internal standard (=0.00 ppm) using the signals of the residual protons of CHCl.sub.3 (7.26 ppm) in CDCl.sub.3, CHD.sub.2OD (3.31 ppm) in CD.sub.3OD, CHD.sub.2COCD.sub.3 (2.05 ppm) in (CD.sub.3).sub.2CO or DMSO-d.sub.5 in DMSO-d.sub.6. Multiplicities of the signals are described as follows: s=singlet, br. s=broad singlet, d=doublet, t=triplet, q=quartet, m=multiplet. Coupling constants .sup.nJ.sub.x,y are given in Hz, where n is the number of bonds between the coupled nuclei x and y. For .sup.13C-signals, which were revealed by indirect detection by HSQC, only resonances of the carbon atoms linked to H-atoms were recorded.

(17) 1.1.3.3 Mass-Spectrometry (MS)

(18) Low resolution mass spectra (ESI-MS) with electro-spray ionization (ESI) were obtained on a Varian 500-MS spectrometer. High resolution mass spectra (ESI-HRMS) were obtained on a Bruker micro TOF (ESI-TOF-MS) spectrometer.

(19) 1.1.3.4 High-Performance Liquid Chromatography (HPLC)

(20) HPLC system (Knauer): Smartline pump 1000 (2) with 10 mL pump-head, UV detector 2500, column thermostat 4000, mixing chamber, injection valve with a 20 or 50 L loop for the analytical and 500 L loop for preparative columns; 6-port-3-channel switching valve; analytical column: Eurospher-100 C18 5 m (if not stated otherwise), or Kinetex C18 100, 5 m, 2504.6 mm, flow 1.2 mL/min; preparative column: Kinetex C18 100, 5 m, 25021 mm, flow 10 mL min/mL, solvent A: water+0.1% v/v trifluoroacetic acid (TFA); solvent B: MeCN+0.1% v/v TFA (if not stated otherwise.) For isolation and purification of phosphorylated dyes on preparative scales, acetonitrile-water systems containing 0.05-0.1 M of Et.sub.3N*H.sub.2CO.sub.3 buffer (pH=8-8.9; Sigma-Aldrich, or self-prepared from 1 M aq. Et.sub.3N and CO.sub.2 gas obtained by evaporation of solid CO.sub.2).

Example 1

(21) Synthesis of Fluorescent Acridone Dyes and their Precursors

2-Nitroacridine-9 (10H)-one (1-H)

(22) ##STR00022##

(23) The reaction between 2-chloro-5-nitrobenzoic acid and aniline in the presence of K.sub.2CO.sub.3 and catalytic amounts of Cu(NO.sub.2).sub.2 (185 C., 2-4 h) afforded 4-nitro-N-phenylanthranilic acid according to the procedure described in WO 2007/049057 (3 May 2007) by R. Ramage, B. Maltman, G. Cotton, S. C. M. Couturier, and R. A. S. McMordie; see also: US2012/0015373 (19 Jan. 2012) by J. A. Smith and R. M. West. 4-Nitro-N-phenylanthranilic acid was cyclized to 2-nitroacridine-9 (10H)-one (1-H)(POCl.sub.3, reflux, 3 h). .sup.1H NMR (400 MHz, DMSO-d.sub.6) =12.4 (s, 1H, NH), 8.96 (d, J=2.8, 1H, H-1), 8.46 (dd, J=9.2, 2.7 Hz, 1H, H-3), 8.23 (dd, J=8.1, 1.6 Hz, 1H, H-8), 7.81 (ddd, J=8.5, 7.0 and 1.6 Hz, 1H, H-6/7), 7.66 (d, J=9.1 Hz, 1H, H-4), 7.58 (dm, 1H, H-5), 7.37 (ddd, J=8.1, 7.0 and 1.1 Hz, 1H, H-7/6) ppm.

2-Nitro-9-oxo-9,10-dihydroacridine-7-sulfonic acid (2-H)

(24) ##STR00023##

(25) Compound 1-H was dried and sulfonated with a 20% oleum (100 C., 1.5 h) to afford the title compound 2-H (US2012/0015373). For example, to 530 mg (2.21 mmol) of 2-nitro-10H-acridine-9-one (1-H), 20% SO.sub.3 in H.sub.2SO.sub.4 (25 mL) was added dropwise and the reaction mixture was stirred at 100 C. for 90 minutes. Then the reaction mixture was very carefully and slowly poured onto ice (>30 g), and 4 mL of conc. aq. HCl was added. The brown precipitate was separated by centrifugation, washed with 4 M aq. HCl (25 mL) and lyophilized. Yield463 mg (65%) of brown solid. HPLC: t.sub.R=5.6 min; H.sub.2O/ACN (+0.1% TFA): 80/20.fwdarw.50/50 in 25 min, detection at 254 nm. .sup.1H NMR (400 MHz, D.sub.2O) =7.61 (dd, J=8.0 and 2.6 Hz, 1H, H-3), 7.59 (br. s, 1H, H-1), 7.57 (d, J=2.6 Hz, 1H, H-8), 7.33 (dd, J=8.6 and 2.1 Hz, H-6), 6.53 (dd, J=8.8 and 1.0 Hz, 1H, H-4), 6.47 (d, J=8.6 Hz, 1H, H-5) ppm. ESI-MS (C.sub.13H.sub.8N.sub.2O.sub.6S, M=320), negative mode: m/z (rel. int., %)=319 (100) [MH].sup..

N,N-(2-Hydroxyethyl)-2-nitro-9-oxo-9,10-dihydroacridine-7-sulfonamide (4-H)

(26) ##STR00024##

(27) Compound 2-H (55 mg, 0.17 mmol) was cooled down to 0 C. and chlorosulfonic acid (1.7 mL, d=1.75 g/mL, 3.0 g, 26 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 6 h and poured onto ice (7-10 g). The beige precipitate was separated by centrifugation, washed with ice-water (25 mL) and dried in vacuo. The obtained sulfonyl chloride was added to a solution of N-methyl ethanolamine (86 mg, 0.80 mmol) in MeCN/H.sub.2O (1:1) at 0 C. The reaction mixture was warmed-up to room temperature and stirred at 50 C. overnight. The precipitate dissolved gradually. Yield33 mg (48%) of a yellow solid which was obtained upon concentration of the reaction mixture in vacuo (in the course of evaporation of acetonitrile). HPLC: t.sub.R=16.7 min H.sub.2O/CAN (+0.1% TFA): 80/20.fwdarw.50/50 in 25 min, 254 nm. .sup.1H NMR (400 MHz, DMSO-d.sub.6) =12.7 (br. s, 1H, NH), 8.95 (d, J=2.6 Hz, 1H, H-1), 8.55 (d, J=2.2 Hz, 1H, H-8), 8.51 (dd, J=9.2, 2.7 Hz, 1H, H-6), 8.13 (dd, J=8.8, 2.2 Hz, 1H, H-3), 7.72 (t, J=8.9 Hz, 2H, H-4,5), 4.83 (t, J=5.5 Hz, 2H, OH), 3.51 (q, J=6.3 Hz, 4H, CH.sub.2), 3.18 (t, J=6.4 Hz, 4H, CH.sub.2) ppm. C.sub.17H.sub.17N.sub.3O.sub.7S, M=407 g/mol); ESI-MS, negative mode: m/z (rel. int., %)=406 (100) [MH].sup..

N,N-(2-Hydroxyethyl)-2-nitro-9-oxo-9,10-dihydroacridine-7-sulfonamide O,O-diphosphate (5-H)

(28) ##STR00025##

(29) Compound 4-H (50 mg, 0.12 mmol) was dissolved in 10 mL MeCN, and this solution was added under Ar to 5 mL of freshly distilled POCl.sub.3. The suspension was stirred at 30 C. for 2 h. The solvent and the excess of POCl.sub.3 were removed in vacuo, the residue was dissolved in aqueous TEAB buffer (1 M), and the title compound was isolated on RP-18 using MeCN0.05 M aq. TEAB buffer (1:3) as an eluent. Yield27 mg (40%) of a yellow solid (after lyophilization). HPLC: t.sub.R=3.5 min; H.sub.2O/ACN (+0.1% TFA): 70/30.fwdarw.0/100 in 25 min, 254 nm. HPLC: t.sub.R=9.2 min, H.sub.2O/ACN (+0.1% TFA): 80/20.fwdarw.50/50 in 25 min, 254 nm. .sup.1H NMR (400 MHz, D.sub.2O) =8.72 (d, J=2.6 Hz, 1H, H-1), 8.35 (d, J=2.2 Hz, 1H, H-8), 8.24 (dd, J=9.3, 2.6 Hz, 1H, H-6), 7.95 (dd, J=8.9, 2.2 Hz, 1H, H-3), 7.47 (d, J=8.9 Hz, 1H, H-4/5), 7.39 (d, J=9.2 Hz, 1H, H-5/4), 3.84-3.78 (m, 4H, CH.sub.2), 3.37 (t, J=5.9 Hz, 4H, CH.sub.2). .sup.31P NMR (81 MHz, D.sub.2O): =1.0 ppm. C.sub.17H.sub.19N.sub.3O.sub.13P.sub.2S, M=567 g/mol). ESI-MS, negative mode: m/z (rel. int., %)=566 (100) [MH].sup..

2-Amino-N,N-(2-hydroxyethyl)-9-oxo-9,10-dihydroacridine-7-sulfonamide O,O-diphosphate (6-H)

(30) ##STR00026##

(31) Pd/C (the oxidized form (VWR International, 10% Pd, 7 mg) was mixed under argon with propanol-2 (attention: Pd/C causes ignition when gets dry in air during the reaction workup, especially filtration!), water (1 mL) was added; the mixture was flushed with hydrogen, and stirred for 30 min. Compound 5-H (27 mg, 0.48 mmol) was dissolved in propanol-2 (0.5 mL), and this solution was added to the suspension of the pre-reduced catalyst. The reaction mixture was stirred overnight in the atmosphere of hydrogen. HPLC control indicated nearly full conversion to a new substance with yellow fluorescence and t.sub.R=2.9 min (HPLC area 92%), 8.8 min (7.5%; starting material) H.sub.2O/ACN (+0.1% TFA): 80/20.fwdarw.50/50 in 25 min, 254 nm. The reaction mixture was flushed with argon, the catalyst was separated by centrifugation, washed with aqueous propanol-2, and the combined supernatant was concentrated in vacuo. The title compound was isolated by preparative HPLC (see below). C.sub.17H.sub.21N.sub.3O.sub.11P.sub.2S (exact mass 537.0372), ESI-MS, negative mode: m/z (rel. int., %)=536 (100) [MH].sup.. ESI-HRMS: 536.0278 (found [MH].sup.), calculated: 536,0299. Final purification (for reductive amination of sugars) was achieved by preparative HPLC using an aqueous 0.05 M TEAB buffer (pH 8) and Kinetex column (5 m C18 100, 25010 mm), ACN/H.sub.2O: 5/95-30/70 in 20 min, 4 mL/min; t.sub.R10-11 min (strongly depends on concentration of the analyte). Purity control: 0.05 M TEAB aqueous buffer (pH 8) and Kinetex column (5 m C18 100, 2504.6 mm), ACN/H.sub.2O: 5/95-50/50 in 20 min, 1.2 mL/min; t.sub.R=8.1 min. .sup.1H NMR (400 MHz, D.sub.2O) =8.35 (d, J=2.2 Hz, 1H, H-8), 7.82 (dd, J=9.3 and 2.6 Hz, 1H, H-6), 7.29 (d, J=9.3, 1H, H-5), 7.12 (d, J=2.2 Hz, 1H, H-1), 7.04 (dd, J=2.4 and 8.9 Hz, 1H, H-3), 7.01 (d, J=8.9 Hz, 1H, H-4), 3.87 (q, J=6.0, 4H, (OH).sub.2POCH.sub.2), 3.40 (t, J=5.9 Hz, 4H, NCH.sub.2), 3.05 (q, 16H, CH.sub.2N in Et.sub.3N), 1.13 (t, 23H, CH.sub.3 in Et.sub.3N) ppm. .sub.max (absorption, H.sub.2O (, M.sup.1 cm.sup.1)=217 (13500), 260 (26000), 295 (28000), 420 (3700) nm; .sub.max (emission, H.sub.2O)=485 nm (excitation at 405 nm), 586 nm (excitation at 300 nm, 420 nm or 470 nm); fluorescence lifetimes22.3 ns (excitation at 405 nm, emission detected at 485 nm) and 3.7 ns (excitation at 470 nm, emission detected at 585 nm).

10-Methyl-2-nitroacridin-9 (10H)-one (1-Me)

(32) ##STR00027##

(33) NaH (600 mg of a 60% suspension in mineral oil; 360 mg, 15 mmol) was washed with dry pentane (315 mL) under argon atmosphere, suspended in dry DMSO, and then 2-nitroacridine-9 (10H)-one (1-H, 2.5 g, 10.4 mmol) was added to this suspension in small portions. The organic substance dissolves gradually, and the solution turned magenta (violet). It was warmed-up to 50-60 C., stirred for 30 min, cooled to room temperature, and then MeI (4 mL, 64 mmol) was added to the reaction mixture. In the course of stirring overnight at room temperature, the suspension gradually turned yellow. Then the reaction mixture was poured onto ice with water (75 mL). The precipitate was collected by filtration, washed with water, and recrystallized from AcOH (200 mL). Yield2.1 g (80%) of a yellow solid. HPLC: t.sub.R=13.9 min A/B: 70/30.fwdarw.0/100 in 25 min, 254 nm. C.sub.14H.sub.10N.sub.2O.sub.3, M=254 g/mol). ESI-MS, positive mode: m/z (rel. int., %)=277.0579 (100) [found M+Na].sup.+; 277.0584 (calculated). .sup.1H NMR (400 MHz, DMSO-d.sub.6) =9.01 (d, J=2.9, 1H, H-1), 8.50 (dd, J=9.6, 2.9 Hz, 1H, H-3), 8.32 (ddd, J=7.9, 1.6, 0.7 Hz, 1H, H-8), 8.02 (d, J=9.6 Hz, 1H, H-4), 7.95-7.85 (m, 2H, H-6/7), 7.44 (ddd, J=8.0, 6.2, 1.7 Hz, 1H, H-5), 3.98 (s, 3H, CH.sub.3) ppm.

10-Methyl-2-nitro-9-oxo-9,10-dihydroacridine-7-sulfonic acid (2-Me)

(34) ##STR00028##

(35) To compound 1-Me (400 mg, 1.57 mmol), 20% SO.sub.3 in H.sub.2SO.sub.4 (7.2 mL, 16.8 mmol) was added dropwise, and the reaction mixture was stirred at 100 C. for 90 min. Then the reaction mixture was poured very carefully and slowly onto ice (>30 g), and 4 mL of conc. HCl was added. The brown precipitate was separated by centrifugation, washed with cold 4 M HCl (25 mL) and lyophilized. Yield376 mg (72%) of the title compound as a brown solid. HPLC: t.sub.R=7.9 min A/B: 80/20.fwdarw.50/50 in 25 min, 254 nm. .sup.1H NMR (400 MHz, DMSO-d.sub.6): =9.03 (d, J=2.9 Hz, 1H, H-1), 8.55 (d, J=2.1 Hz, 1H, H-8), 8.52 (dd, J=9.5, 2.9 Hz, 1H, H-3), 8.08-8.02 (m, 2H, H-4, H-6), 7.90 (d, J=9.1 Hz, 1H, H-5), 4.00 (s, 3H, CH.sub.3) ppm. C.sub.14H.sub.10N.sub.2O.sub.6S, M=334 g/mol. ESI-MS, negative mode: m/z (rel. int., %)=333 (100) [MH].sup.. .sup.1H NMR (100 MHz, DMSO-d.sub.6): =176.7 (CO), 146.2, 143.3, 142.6, 141.2, 132.8, 128.1, 123.8, 123.3, 121.4, 121.0, 118.8, 117.3, 35.3 (N-Me) ppm.

N,N-(2-Hydroxyethyl)-10-methyl-2-nitro-9-oxo-9,10-dihydroacridine-7-sulfonamide (4-H)

(36) ##STR00029##

(37) To compound 2-Me (62 mg, 0.19 mmol), chlorosulfonic acid (3.5 mL, 53 mmol) was added dropwise at 0 C. The reaction mixture was stirred at 50 C. for 30 min and at room temperature overnight. Then the reaction mixture was dropwise poured into ice (10 g), and the beige precipitate separated by centrifugation. It was washed with ice-water (210 mL) and lyophilized. The obtained sulfonyl chloride was added to the solution of diethanolamine (185 mg, 1.76 mmol) in THF (2 mL) at 0 C. The reaction mixture was stirred at room temperature overnight. The precipitate was separated by centrifugation, washed with ice water and lyophilized. Yield57 mg (70%) of the title product as a yellow solid. HPLC: t.sub.R=16.9 min A/B: 80/20.fwdarw.50/50 in 25 min, 254 nm. ESI-MS, positive mode: m/z (rel. int., %)=444 (100) [M+Na].sup.+. C.sub.18H.sub.19N.sub.3O.sub.7S, M=421 g/mol. ESI-MS, positive mode: m/z=422.1005 [found M+H].sup.+; 422.1016 (calculated); 444.0829 [found M+Na].sup.+; 444.0836 (calculated). .sup.1H NMR (400 MHz, DMSO-d.sub.6): =8.99 (d, J=2.8 Hz, 1H, H-1), 8.61 (d, J=1.6 Hz, 1H, H-8), 8.56 (dd, J=9.5 and 2.9 Hz, 1H, H-3), 8.19 (dd, J=9.1, 2.4 Hz, 1H, H-6), 8.10 (d, J=9.4 Hz, 2H, H-4,5), 4.82 (t, J=5.5 Hz, 2H, OH), 4.02 (s, 3H, NMe), 3.52 (q, J=6.0 Hz, 4H, OCH.sub.2), 3.20 (t, J=6.3 Hz, 4H, NCH.sub.2) ppm.

N,N-(2-Hydroxyethyl)-9,10-dihydro-9-oxo-10-methyl-2-nitroacridine-7-sulfonamide O,O-diphosphate (5-Me)

(38) ##STR00030##

(39) To a solution of 4-Me (31 mg, 73 mol) in trimethyl phosphate (0.5 mL) POCl.sub.3 (0.2 mL) was added under Ar at 0 C. The suspension was stirred at room temperature for 2 hours. Then the solvent and the excess of POCl.sub.3 were removed in vacuo the residue was diluted with an excess of aq. Et.sub.3NH.sub.2CO.sub.3 buffer (pH 7.5) and kept at 4 C. overnight. The title compound was isolated on RP-18 using MeCN0.1 M aq. Et.sub.3NH.sub.2CO.sub.3 buffer (1:5) as an eluent, lyophilized and used in the final reduction step (see below). C.sub.18H.sub.21N.sub.3O.sub.13P.sub.2S, M=581 g/mol. ESI-MS, negative mode: m/z (rel. int., %)=580.0191 (100) [found MH].sup.; 580.0198 (calculated).

2-Amino-N,N-(2-hydroxyethyl)-9,10-dihydro-9-oxo-10-methylacridine-7-sulfonamide O,O-diphosphate (6-Me)

(40) ##STR00031##

(41) Pd/C(VWR International, the oxidized form, 10% Pd) 7 mg of was mixed under argon with propanol-2, water (1 mL) was added; the mixture was flushed with hydrogen, and stirred for 30 min. Compound 5-Me (27 mg) was dissolved in propanol-2 (0.5 mL), and this solution was added to the suspension of the pre-reduced catalyst. The reaction mixture was stirred overnight in the atmosphere of hydrogen. HPLC control indicated nearly full conversion to a new substance with yellow fluorescence and t.sub.R=16.1 min (HPLC area 95%), 19.2 min (5%; starting material?) H.sub.2O/ACN (+0.05 M Et.sub.3N*H.sub.2CO.sub.3 buffer in aqueous phase): 99/1.fwdarw.80/20 in 20 min, dioden array detector, Kinetex column 5 m C18 100, 4.6250 mm, 20 C., 1.2 mL/min. The reaction mixture was flushed with argon, the catalyst was separated by centrifugation, washed with aqueous propanol-2, and the combined supernatant was concentrated in vacuo. The title compound was isolated by preparative HPLC using an aqueous 0.05 M TEAB buffer (pH 8) and Kinetex column (5 m C18 100, 25010 mm), ACN/H.sub.2O: 5/95-30/70 in 20 min, 4 mL/min; t.sub.R11.4 min. C.sub.18H.sub.23N.sub.3O.sub.11P.sub.2S (exact mass 551.0529). ESI-MS, negative mode: m/z (rel. int., %)=550 (100) [MH].sup.. ESI-HRMS: 550.0441 (found [MH].sup.), calculated: 550,0456. .sup.1H NMR (400 MHz, D.sub.2O): =8.29 (d, J=2.4 Hz, 1H, H-8), 7.84 (dd, J=9.3 and 2.4 Hz, 1H, H-6), 7.43 (d, J=9.3, 1H, H-5), 7.12 (d, J=9.3 Hz, 1H, H-3), 7.03 (d, J=2.8 Hz, 1H, H-1), 6.97 (dd, J=9.2 and 2.8 Hz, 1H, H-4), 3.85 (q, J=6.1, 4H, POCH.sub.2), 3.49 (s, 3H, NMe), 3.37 (t, J=5.9 Hz, 4H, NCH.sub.2), 3.04 (q, 14H, CH.sub.2N in Et.sub.3N), 1.13 (t, 20H, CH.sub.3 in Et.sub.3N) ppm. .sup.31P NMR (162 MHz, CDCl.sub.3): =1.1 ppm. .sub.max (0.05 M aq Et.sub.3N*H.sub.2CO.sub.3 buffer, pH 8, c, M.sup.1 cm.sup.1)=219 (10 300), 263 (18 600), 299 (18 500), 430 (2900) nm; .sub.max (emission, 0.05 M aq Et.sub.3N*H.sub.2CO.sub.3 buffer, pH 8)=485 nm and 585 nm (two maxima in ca. 1:2 ratio, independent from excitation wavelength which may be at 300 nm, 420 nm or 470 nm); fluorescence quantum yield: 5-6% (absolute value).

Example 2

(42) Synthesis of Fluorescent 1-Aminopyrene Dyes and their Precursors

(43) 3-Aminopyrene-1,6,8-trisulfonic acid trisodium salt (APTS) was prepared from 1-aminopyrene and a 20% oleum in the presence of Na.sub.2SO.sub.4 as described by Z. Sharrett, S. Gamsey, L. Hirayama, B. Vilozny, J. T. Suri, R. A. Wessling, B Singaram, Org. Biomol. Chem. 2009, 7, 1461-1470.

3-Amino-N,N,N-tris(2-hydroxyethyl)-N,N,N-trimethylpyrene-1,6,8-trisulfonamide (7-H)

(44) ##STR00032##

(45) 446 mg (0.98 mmol) of APTS (trisodium salt) was cooled down to 0 C. in an ice bath, and then chlorosulfonic acid (7.5 mL, 0.11 mol) was added dropwise with stirring. The reaction mixture was stirred at 65 C. for 3 h. After cooling down to room temperature, the reaction mixture was transferred onto crushed ice. The red precipitate of trisulfonyl chloride was isolated by centrifugation, washed with ice-water (210 mL) and added to a solution of N-methylethanolamine (1.0 g, 13 mmol) in aqueous acetonitrile (1:1, 25 mL) at 0 C. The reaction mixture was vigorously stirred at room temperature, until it became homogeneous, and then lyophilized. The title compound was isolated by chromatography on SiO.sub.2 (100 g) with CHCl.sub.3/MeOH/H.sub.2O (80:18:2) mixture as an eluent. Yield252 mg (41%) of a brown-orange solid obtained after the second chromatographic purification. HPLC: t.sub.R=15.8 min, ACN/H.sub.2O: 20/80-50/50 in 25 min, 1.2 mL/min, 254 nm. C.sub.25H.sub.32N.sub.4O.sub.9S.sub.3 (exact mass 628.1331); ESI-HRMS: 651.1212 (found [M+Na].sup.+), calculated: 651,1224. .sup.1H NMR (400 MHz, CD.sub.3OD) =9.19 (d, J=9.8 Hz, 1H), 9.06 (s, 1H), 8.99 (d, J=9.6 Hz, 1H), 8.82 (d, J=9.8 Hz, 1H), 8.74 (d, J=9.7 Hz, 1H), 8.18 (s, 1H), 3.69 (m, 6H, CH.sub.2), 3.42 (m, 6H, CH.sub.2), 3.04 (s, 3H, NCH.sub.3), 3.01 (s, 6H, 2NCH.sub.3) ppm; .sub.max (absorption)=477 nm (=22 400 M.sup.1 cm.sup.1, MeOH), max (emission)=535 nm (MeOH, excitation at 470 nm); fluorescence lifetime 5.6 ns (MeOH); fluorescence quantum yield (0.96; absolute value in MeOH).

3-Amino-N,N,N-tris(2-hydroxyethyl)-N,N,N-trimethyl-pyrene-1,6,8-trisulfonamide O,O,O-triphosphate tris(triethylammonium salt) (8-H)

(46) ##STR00033##

(47) Compound 7-H (40 mg, 64 mol) was dissolved in (MeO).sub.3PO (5 mL) and this solution was added to freshly distilled POCl.sub.3 (0.2 mL) with stirring at room temperature. A weak exothermic reaction was observed, and the solution turned orange-brown. The reaction mixture was stirred several hours at room temperature. All volatile materials (excess of POCl.sub.3 and most of trimethyl phosphate) were removed in vacuum (first using rotary evaporator and thenoil pump; 0.5 mbar, 60 C., cold trap cooled with dry ice for collecting trimethyl phosphate and POCl.sub.3). The residue was treated and stirred with 1 M Et.sub.3N*H.sub.2CO.sub.3 buffer (TEAB; initial pH=8), and the pH was controlled. Phosphorylation of the amino group also takes place, but this phosphate group is readily cleaved. Fresh portions of the TEAB buffer were added when the solution became acidic, until the pH stabilized at about 5-7. TLC control on regular SiO.sub.2 was possible in iPrOH/H.sub.2O/25% aq. NH.sub.3 (10/5/1); R.sub.f 0.3 (yellow spot with green fluorescence). The reaction mixture was lyophilized, and the title compound was isolated by chromatography on regular SiO.sub.2 using iPrOH/H.sub.2O/25% aq. NH.sub.3 (10/5/1) mixture as an eluent. HPLC control indicated homogeneous fractions containing the substance with t.sub.R=10.2 min (ACN/H.sub.2O (+0.1% TFA): 20/80-50/50 in 25 min, 1.2 mL/min, 254 nm). Lyophilization afforded 60 mg of the red-orange foam. C.sub.25H.sub.35N.sub.4O.sub.18P.sub.3S.sub.3 (exact mass 868.0321)*NH.sub.3 ESI-MS, negative mode: m/z (rel. int., %)=867 (100) [MH].sup., 889 (60) [M2H+Na].sup..

(48) Final purification was achieved by preparative HPLC using a 0.1 M TEAB buffer and a preparative Kintex column (e.g., Kinetex, 5 m C18 100, 2504.6 mm, ACN/H.sub.2O: 10/90-30/70 in 20 min, 1.2 mL/min; t.sub.R=10.8 min). Freeze-drying of the eluate gave the title compound as yellow foam. .sup.1H NMR (400 MHz, CD.sub.3OD) =9.14 (d, J=9.7 Hz, 1H), 9.03 (s, 1H), 8.96 (d, J=9.7 Hz, 1H), 8.77 (d, J=9.8 Hz, 1H), 8.67 (d, J=9.7 Hz, 1H), 8.17 (s, 1H), 3.94-4.06 (m, 6H, CH.sub.2), 3.49-3.59 (m, 6H, CH.sub.2), 3.08 (s, 3H, NCH.sub.3), 3.04 (q, J=7.3 Hz, 18H, CH.sub.2N in Et.sub.3N), 3.02 (s, 3H, NCH.sub.3), 1.22 (t, J=7.3 Hz, 27H, CH.sub.3CH.sub.2 in Et.sub.3N) ppm; .sub.max (absorption)=205, 237, 305 and 471 nm (0.05 M aq Et.sub.3N*H.sub.2CO.sub.3 buffer, pH 8), .sub.max (emission)=544 (0.05 M aq Et.sub.3N*H.sub.2CO.sub.3 buffer, pH 8); fluorescence lifetime 5.9 ns (in H.sub.2O; excitation at 470 nm); fluorescence quantum yield: 0.77 (H.sub.2O, standard: Coumarin 153 with emission efficiency of 0.54 in ethanol, .sub.excit=400 nm), 0.88 (absolute value in 0.05 M aq Et.sub.3N*H.sub.2CO.sub.3 buffer, pH 8; excitation at 460 nm).

3-(Trifluoroacetyl)amino-N,N,N-tris(2-hydroxyethyl)-N,N,N-trimethylpyrene-1,6,8-trisulfonamide (9-H)

(49) ##STR00034##

(50) Compound 7-H (179 mg, 0.28 mmol) was suspended in 10 mL of DCM, and a 10% solution of trifluoroacetic anhydride in DCM (d=1.33 g/mL, 0.9 mL, 0.57 mmol) followed by Et.sub.3N (126 L, d=0.73 g/mL, 0.91 mmol) was added at room temperature. The reaction mixture was stirred for 30 min. All volatile materials were evaporated under reduced pressure, the residue was dissolved in methanol (50 mL) and NaHCO.sub.3 (50 mg) was added. After stirring at room temperature for 30 min, the reaction mixture was neutralized with acetic acid, and all volatile materials were removed in vacuo. These operations remove trifluoroacetate groups from hydroxyl groups. The title compound was isolated by chromatography regular silica gel (50 g) using CH.sub.2Cl.sub.2/aceton (2:1) as an eluent. Yield165 mg (82%) of a yellow solid. HPLC: t.sub.R=10.8 min, ACN/H.sub.2O: 20/80-100/0 in 25 min, 254 nm. C.sub.27H.sub.31F.sub.3N.sub.4O.sub.10S.sub.3, (exact mass 724.1154). ESI-MS, negative mode: m/z (rel. int., %)=723 (100) [MH].sup.. .sup.1H NMR (400 MHz, acetone-d.sub.6) =11.2 (br. s, 1H, NH), 9.49 (d, J=10.0 Hz, 1H), 9.38 (dd, J=9.7 and 1.9 Hz, 2H), 9.26 (s, 1H), 9.02 (s, 1H), 8.76 (d, J=9.7 Hz, 1H), 3.83-3.90 (m, 3H, OH), 3.71 (m, 6H, OCH.sub.2), 3.43 (m, 6H, NCH.sub.2), 3.08/3.06/3.06 (3s, 9H, NMe) ppm. .sup.19F NMR (376 MHz, acetone-d.sub.6) =75.7 ppm

3-[N-methyl-N-(trifluoroacetyl)]amino-N,N,N-tris(2-hydrox-yethyl)-N,N,N-trimethylpyrene-1,6,8-trisulfonamide (9-Me)

(51) ##STR00035##

(52) To a solution of compound 7-H (120 mg, 0.17 mmol) in DMF (2 mL), CsCO.sub.3 (42 mg, 0.13 mmol) and CH.sub.3I (960 mg, 6.7 mmol) were added under argon. The reaction mixture was stirred at 70 C. for 40 min, and the solvent was evaporated under reduced pressure. The title compound was isolated by chromatography on regular SiO.sub.2 (50 g) using a 15:1 mixture of DCM and methanol as an eluent; yield110 mg (88%) of a yellow solid. Due to the presence of the secondary amide group, two rotamers (two sets of signals) were detected in .sup.1H NMR spectrum of this substance, and therefore it was difficult to interpret. HPLC: t.sub.R=12.2 min, ACN/H.sub.2O: 20/80-100/0 in 25 min, 254 nm. C.sub.28H.sub.33F.sub.3N.sub.4O.sub.10S.sub.3 (exact mass 738.1311). ESI-MS, positive mode: m/z (rel. int., %)=739 (100) [M+H].sup.+, 761 (35) [M+Na].sup.+.

3-Methylamino-N,N,N-tris(2-hydroxyethyl)-N,N,N-trimethylpyrene-1,6,8-trisulfonamide (7-Me)

(53) ##STR00036##

(54) The title compound was obtained from the corresponding N-methyl-N-trifluoroacetamide (9-Me; see above) upon treatment with Na.sub.2CO.sub.3, which was taken in excess as a saturated solution, in aqueous methanol (ca. 1:1) so that reaction mixture remained homogeneous. The product was isolated as an orange solid (20 mg) by chromatography on SiO.sub.2 using a 15:1 mixture of DCM and methanol as an eluent.

(55) .sup.1H NMR (400 MHz, CD.sub.3OD) =9.15 (d, J=9.8 Hz, 1H), 9.06 (s, 1H), 8.98 (d, J=9.6 Hz, 1H), 8.77 (d, J=9.8 Hz, 1H), 8.63 (d, J=9.7 Hz, 1H), 7.93 (s, 1H), 3.77-3.63 (m, 6H, CH.sub.2), 3.40 (m, 6H, CH.sub.2), 3.21 (s, 3H, NCH.sub.3), 3.00 (s, 3H, NCH.sub.3), 2.99 (s, 6H, 2NCH.sub.3) ppm; C.sub.26H.sub.34N.sub.4O.sub.9S.sub.3 (exact mass 642.1488). HRMS (ESI): 665.1353 (found [M+Na].sup.+), calculated: 665.1380. .sub.max(absorption)=493 nm (=23000 M.sup.1 cm.sup.1, MeOH), .sub.max (emission)=549 nm (MeOH); fluorescence lifetime 5.9 ns (MeOH), fluorescence quantum yield: 0.97 (absolute value in MeOH); 0.83 (relative value obtained in MeOH using Rhodamine 6G as a standard (QY=0.94 in ethanol), excitation at 480 nm).

3-Methylamino-N,N,N-tris(2-hydroxyethyl)-N,N,N-trimethylpyrene-1,6,8-trisulfonamide O,O,O-triphosphate (8-Me) as tris(triethylammonium salt)

(56) ##STR00037##

(57) To a solution of POCl.sub.3 (74 mg, 480 mol) in 0.1 mL of (MeO).sub.3PO, the solution of compound 7-Me (40 mg, 54 mol) in 0.5 mL of (MeO).sub.3PO was added dropwise at 0 C. Then the reaction mixture was stirred for 1.5 h at room temperature. All volatile materials (excess of POCl.sub.3 and most of trimethyl phosphate) were removed in vacuum (first, using rotary evaporator and then an oil pump; 0.5 mbar, 60 C., cold trap cooled with dry ice for collecting trimethyl phosphate). The residue was treated and stirred with 1 M Et.sub.3N*H.sub.2CO.sub.3 buffer (TEAB; initial pH=8) and pH-value was controlled. Fresh portions of the TEAB buffer were added when the solution became acidic, until the pH-value stabilized at about 5-7. Then the reaction mixture was loaded on RP-18 (ca. 30 g) and the title compound was eluted using 1:4 mixture of MeCN and aqueous 0.1 M Et.sub.3N*H.sub.2CO.sub.3 buffer (pH 7.5). Yield63%, a yellow solid. HPLC: t.sub.R=6.9 min H.sub.2O/ACN (+0.1% TFA): 80/20.fwdarw.0/100 in 25 min, 254 nm. C.sub.26H.sub.37N.sub.4O.sub.18P.sub.3S.sub.3 (exact mass 882,0478). ESI-MS, negative mode: m/z (rel. int., %)=881 (100) [MH].sup., 440 (15) [M2H].sup.2. .sub.max (absorption)=502 nm (H.sub.2O), .sub.max (emission)=563 nm (H.sub.2O); fluorescence quantum yield 0.85 (H.sub.2O, standard: Rhodamine 6G with emission efficiency of 0.94 in ethanol, .sub.excit=500 nm). This substance was found to be unreactive in the reductive amination of sugars.

2,2,2-Trifluoro-N-(pyren-1-yl) acetamide (11)

(58) ##STR00038##

(59) 1-Aminopyrene (10)(1.00 g, 4.60 mmol) was dissolved in dry DCM (30 mL) under stirring and by using an ultrasonic bath. Trifluoroacetic anhydride (d=1.51, 772 L, 1.17 g, 5.55 mmol, 1.21 eq.) was added dropwise over a period of 10 min. The obtained suspension was stirred for 30 min at r.t. The precipitate was removed by filtration and washed with cyclohexane (315 mL) and dried in vacuo. A white precipitate observed in the filtrate and was also filtered off. The combined precipitates (a light grey powder) represented the title compound (1.13 g, 3.61 mmol, 79%). .sup.1H NMR (400 MHz, aceton-d.sub.6): =8.11 (t, .sup.3J.sub.H,H=7.6 Hz, 1H, 7-H), 8.20 (d, .sup.3J.sub.H,H=9.1 Hz, 1 H), 8.22 (d, .sup.3J.sub.H,H=8.4 Hz, 1H, 2-H), 8.23 (d, .sup.3J.sub.H,H=9.3 Hz, 1H), 8.23 (d, .sup.3J.sub.H,H=9.1 Hz, 1H), 8.34 (d, .sup.3J.sub.H,H=7.6 Hz, 2H, 6-H, 8-H), 8.35 (d, .sup.3J.sub.H,H=8.4 Hz, 1H, 3-H), 8.82 (d, .sup.3J.sub.H,H=9.3 Hz, 1H), 10.78 (br. s, 1H, NH) ppm. 13C NMR (101 MHz, aceton-d.sub.6): (ppm)=122.5 (CH), 125.2 (C.sub.q), 125.4 (CH), 125.9 (C.sub.q), 126.0 (CH), 126.7 (CH), 126.9 (CH), 127.0 (C.sub.q) 127.6 (CH), 128.2 (CH), 128.8 (CH), 129.1 (C.sub.q) 129.2 (CH), 131.7 (C.sub.q), 131.8 (C.sub.q), 132.2 (C.sub.q) ppm. CF.sub.3 and CO signals were not detected due to low intensities. .sup.19F NMR (376 MHz, aceton-d.sub.6): (ppm)=75.7 (s, CF.sub.3) ppm. C.sub.18H.sub.10F.sub.3NO (313.0714) HR-MS (ESI): found 314.0783 [M+H].sup.+; calcd. 314.0787.

2,2,2-Trifluoro-N-[3,6,8-tris(bromo)pyren-1-yl]-acetamide (12)

(60) ##STR00039##

(61) N-Trifluoroacetyl-1-aminopyrene 11 (870 mg, 2.76 mmol) was suspended in nitrobenzene (30 mL) and stirred at 50 C. for 30 min. Bromine (500 L, 19.5 mmol, 7.07 eq.), dissolved in nitrobenzene (5 mL), was added to the solution of pyrene. The reaction mixture was stirred in a closed vessel at 80 C. for 30 min. Afterwards, the mixture was allowed to reach rt. After diluting with cyclohexane (20 mL), the precipitate was removed by filtration, washed with cyclohexane (225 mL) and dried in vacuo. The product was obtained as a light yellow powder (1224 mg, 2.26 mmol, 81%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): =8.39 (d, .sup.3J.sub.H,H=9.7 Hz, 1H, 10-H), 8.47 (d, .sup.3J.sub.H,H=9.7 Hz, 1H, 9-H), 8.51 (d, .sup.3J.sub.H,H=9.3 Hz, 1H, 5-H), 8.59 (d, .sup.3J.sub.H,H=9.3 Hz, 1H, 4-H), 8.66 (s, 1H, 2-H), 8.80 (s, 1H, 7-H) ppm. .sup.19F NMR (376 MHz, DMSO-d.sub.6): =73.57 (s, CF.sub.3). MS (ESI): m/z (negative mode, %)=548 (100) [M].sup.. HR-MS (ESI): calcd. for C.sub.18H.sub.7NOF.sub.3.sup.79Br.sub.2.sup.80Br ([MH].sup.) 547.7937, found 547.7930.

2,2,2-Trifluoro-N-[3,6,8-tris[(3-hydroxypropyl) sulfanyl]pyrene-1-yl]-acetamide (14)

(62) ##STR00040##

(63) K.sub.2CO.sub.3 (221.2 mg, 400 mol, 0.53 eq.) and NEt.sub.3 (40 L) were suspended in dry DMF (4 mL) and flushed with argon for 15 min. 3-Mercapto-1-propanol (d=1.067 mg/mL, 272 L, 290 mg, 3160 mol, 4.35 eq.), brominated pyrene derivative 12 (400 mg, 757 mol) and dry DMF (40 mL) were added, and a gentle argon stream was bubbled through the solution for 20 min. Afterwards, Pd.sub.2(dba).sub.3 (197.6 mg, 216 mol, 0.30 eq.) and Xantphos (140.4 mg, 242.8 mol, 0.33 eq.) were added. The mixture was stirred at 85 C. under argon for 18 h. The solvents were removed in vacuo, the residue was dissolved in MeOH, applied to Celite and submitted to flash chromatography (SNAP Ultra 100 g cartridge, DCM/MeOH with 2-18% MeOH-gradient over 15 CV) to provide the title compound 14 (298 mg, 511 mol, 70%) as a pale-yellow solid. This procedure has been repeated several times on various scales and reliably gave 50-70% yields of the title product. .sup.1H NMR (400 MHz, DMSO-d.sub.6): =1.80 (m, 6H, CH.sub.2CH.sub.2CH.sub.2), 3.28 (m, 6H, SCH.sub.2CH.sub.2), 3.55 (m, 6H, HOCH.sub.2CH.sub.2), 4.63 (m, 3H, OH), 8.06 (d, .sup.3J.sub.H,H=9.47 Hz, 1H, 9-H), 8.17 (br. s, 2H, 2-H, 7-H), 8.50 (d, .sup.3J.sub.H,H=9.47 Hz, 1H, 9-H), 8.53 (d, .sup.3J.sub.H,H=9.50 Hz, 1H, 5-H), 8.53 (d, .sup.3J.sub.H,H=9.50 Hz, 1H, 4-H), 11.85 (s, NH, 1H) ppm. 13C NMR (101 MHz, DMSO-d.sub.6): =29.9 (CH.sub.2), 30.0 (CH.sub.2), 31.7 (CH.sub.2), 31.9 (CH.sub.2), 32.0 (CH.sub.2), 59.2 (CH.sub.2), 59.2 (CH.sub.2), 59.2 (CH.sub.2), 116.2 (d, .sup.1J.sub.F,C=289.3 Hz), 121.8 (CH), 123.2 (CH), 123.7 (CH), 124.2 (CH), 124.4 (C.sub.q), 124.6 (C.sub.q), 124.7 (C.sub.q), 125.5 (CH), 127.2 (CH), 127.4 (C.sub.q), 127.6 (C.sub.q), 128.6 (2C.sub.q), 132.3 (C.sub.q), 132.8 (C.sub.q), 132.9 (C.sub.q), 156.2 (d, .sup.2J.sub.C,F=36.7 Hz) ppm.

(64) .sup.19F NMR (376 MHz, DMSO-d.sub.6): =73.15 (s, CF.sub.3) ppm. HPLC: t.sub.R=8.3 min (MeCN/H.sub.2O 50:50.fwdarw.100:0+0.1% TFA in 25 min detected at 254 nm). HR-MS (ESI) Calcd. for C.sub.27H.sub.28NOF.sub.3S.sub.3 ([MH].sup.) 582.1060, found 582.1049.

2,2,2-Trifluoro-N-[3,6,8-tris[(3-hydroxypropyl) sulfonyl]pyren-1-yl]-acetamide (13a)

(65) ##STR00041##

(66) Triol 14 (135 mg, 232 mol) was suspended in a mixture of AcOH (20 mL) and H.sub.2O (1 mL). Afterwards, sodium tungstate dihydrate (19.1 mg, 57.9 mol, 0.25 eq.) was added, and the solution was cooled with an ice-bath until the mixture became viscous. Then aq. H.sub.2O.sub.2 solution (85-90%, d=1.45, 5.00 mL, 189 mmol, 82 eq.) was added over a period of 10 min. The solution was stirred for 30 min in the ice-bath. After removing the ice-bath, the mixture was stirred for 2 at rt, until the reaction was complete (TLC). The solvents were removed by freeze-drying, and the residue was dissolved in MeCN/H.sub.2O. Celite was added and, after removing all solvents in vacuo, the sample was submitted to flash chromatography (SNAP Ultra 25 g cartridge, ACN/MeOH+5% DCM with MeOH 1-10%-gradient over 20 CV). The isolated compound 13a was obtained as a yellow solid (113 mg, 116 mol, 50%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): )=1.78 (m, 6H, CH.sub.2CH.sub.2CH.sub.2), 3.41 (m, 6H, HOCH.sub.2CH.sub.2), 3.73 (m, 6H, SCH.sub.2CH.sub.2), 4.59 (m, 3H, OH), 8.83 (d, .sup.3J.sub.H,H=9.46 Hz, 1H, 10-H), 9.02 (s, 1H, 2-H), 9.26 (s, 1H, 7-H), 9.35 (m, 2H, 4-H, 4-H), 9.47 (d, .sup.3J.sub.H,H=9.46 Hz, 1H, 9-H), 12.35 (s, NH, 1H) ppm. 13C NMR (101 MHz, DMSO-d.sub.6): =25.7 (CH.sub.2), 25.9 (CH.sub.2), 53.2 (CH.sub.2), 53.3 (CH.sub.2), 53.5 (CH.sub.2), 58.4 (CH.sub.2), 58.4 (CH.sub.2), 115.91 (d, .sup.1J.sub.F,C=289.0 Hz), 124.3 (CH), 124.8 (CH), 125.0, 125.3, 127.3, 127.9, 128.0, 128.1, 129.5 (CH), 131.1 (CH), 132.1 (CH), 132.2, 132.3, 134.5 (CH), 156.5 (t, 2J.sub.C,F=38.2 Hz) ppm. .sup.19F NMR (376 MHz, DMSO-d.sub.6): =73.46 (s, CF.sub.3) ppm. TLC (SiO.sub.2): R.sub.f=0.68 (ACN:DCM:H.sub.2O=10:1:1). HR-MS (ESI): Calcd. for C.sub.27H.sub.28NO.sub.10F.sub.3S.sub.3 ([MH].sup.) 678.0755, found 678.0756.

3,6,8-Tris[(3-hydroxypropyl)sulfonyl]-pyrene-1-amine (13b)

(67) ##STR00042##

(68) Protected pyrene derivative 13a (20 mg, 29 mol) was suspended in MeOH (6 mL). Diluted aq. Na.sub.2CO.sub.3 (750 L of the sat. aq. Na.sub.2CO.sub.3-solution mixed with 5 mL H.sub.2O) was added, and the solution was stirred for 30 min at rt, followed by 30 min at 50 C. and 90 min at rt. Celite and MeOH were added and the solvents were removed under reduced pressure. The crude product was purified by fcc (SNAP Ultra 10 g cartridge, DCM/MeOH with 2-20% MeOH-gradient over 12 CV), and the title product 13b was isolated as an orange solid (14 mg, 24 mol, 81%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): (ppm)=1.73 (m, 6H, CH.sub.2CH.sub.2CH.sub.2), 3.45 (m, 6H, OCH.sub.2CH.sub.2), 3.51 (m, 6H, SCH.sub.2CH.sub.2), 4.60 (m, 3H, OH), 8.25 (s, 1H, 2-H), 7.83 (br. s, 2H, NH.sub.2), 8.77 (d, .sup.3J.sub.H,H=9.56 Hz, 1H), 8.87 (d, .sup.3J.sub.H,H=9.66 Hz, 1H), 8.93 (d, .sup.3J.sub.H,H=9.66 Hz, 1H), 9.02 (s, 1H), 9.15 (d, .sup.3J.sub.H,H=9.56 Hz, 1H) ppm. 13C NMR (101 MHz, DMSO-d.sub.6): =25.9, 26.0, 26.1, 52.3, 52.5, 53.4, 58.5, 58.5, 58.6, 115.8, 115.9, 116.9, 118.5, 121.0, 126.1, 126.4, 127.3, 127.9, 128.0, 128.8, 130.0, 132.8, 134.1, 136.9, 148.5 ppm. HPLC: t.sub.R=13.1 min (MeCN/H.sub.2O 10:90.fwdarw.100:0+0.1% TFA in 25 min detected at 225 nm). TLC (SiO.sub.2) R.sub.f=0.38 (DCM:MeOH=9:2). HR-MS (ESI): Calcd. for C.sub.25H.sub.29NO.sub.9S.sub.3 ([MH].sup.) 582.0932, found 582.0927.UV-VIS (In MeOH): =21000 M.sup.1 cm.sup.1, .sub.max (absorption)=486 nm, .sub.max (florescence)=534 nm, QY=0.80.

Tri-O-phosphorylated 3,6,8-tris[(3-hydroxypropyl)sulfonyl]-pyrene-1-amine (15)

(69) ##STR00043##

(70) A solution of the deprotected pyrene derivative 13b (2.00 mg, 3.43 mol) in trimethylphosphate (0.2 mL) was added dropwise to freshly distilled and ice-cooled POCl.sub.3 (0.25 mL, 2.69 mmol) under argon atmosphere. The mixture was stirred at 0 C. for 30 min and 4 h at room temperature. All volatile components were distilled in vacuo (0.7 mbar) into a flask cooled in a dry ice/acetone bath (first at room temperature, later heating until 60 C.) and the residue was further dried by lyophilization (0.02 mbar). An aqueous Et.sub.3NH.sub.2CO.sub.3 buffer (1 M, ca. 6-8 mL) was added to the residue until the pH is adjusted to 8 (gas evolution). The sample was concentrated to a volume of ca 1 mL by freeze-drying and purified by preparative HPLC (Kinetex 5 m EVO C18 100 A 25021 mm column, MeCN/water+0.05 M TEAB, with MeCN-gradient 10-30% over 20 min, peak at 9.1 min).

(71) .sup.1H NMR (400 MHz, CD.sub.3OD): =1.24 (t, 36H, J=7 Hz, 12 CH.sub.3, 3 Et.sub.3N) 2.04 (m, 6H, CH.sub.2CH.sub.2CH.sub.2), 3.58-3.72 (m, 6H, ArSO.sub.2CH.sub.2CH.sub.2), 3.07 (q, J=7 Hz, 24H, CH.sub.2CH.sub.3, 3 Et.sub.3N), 3.88-3.93 (m, 6H, CH.sub.2CH.sub.2SO.sub.3), 8.17 (s, 1H), 8.73 (d, J=9.5 Hz, 1H), 8.73 (d, J=9.5 Hz, 1H), 8.86 (d, J=9.5 Hz, 1H), 9.18 (s, 1H) 9.21 (d, J=9.5 Hz, 1H), ppm.

(72) .sup.13C NMR (101 MHz, CD.sub.3OD): =9.2 (CH.sub.3), 21.6 (CH.sub.2), 22.1 (CH.sub.2), 25.8 (CH.sub.2), 47.4 (CH.sub.2), 54.1 (CH.sub.2), 55.3 (CH.sub.2), 56.5 (CH.sub.2), 63.8 (CH.sub.2), 71.0 (CH.sub.2), 71.6 (CH.sub.2), 117.8 (C), 118.3 (CH), 120.2 (CH), 122.9 (CH), 127.7 (C), 128.2 (C), 128.7 (CH), 129.3 (C), 129.8 (C), 130.2 (CH), 131.8 (CH), 134.8 (C), 136.0 (C), 138.3 (C), 149.8 (C), ppm. .sup.31P NMR (161.9 MHz, CD.sub.3OD): =0.97 ppm (s, OP(O)(OH) 2))

(73) HPLC: t.sub.R=5.48 min (MeCN/H.sub.2O 10:90.fwdarw.100:0+0.05 M TEAB in 20 min detected at 254 nm). TLC (RPSiO.sub.2 C18): R.sub.f=0.7-0.8 (MeCN:H.sub.2O+0.05 M TEAB=9:2). MS (ESI): Calcd. for C.sub.25H.sub.32NO.sub.18P.sub.3S.sub.3 (822.9994): [MH].sup. 821.9922. UV-VIS (0.05 M aq. Et.sub.3N*H.sub.2CO.sub.3 (TEAB) buffer, pH 8-8.5): .sub.max (absorption)=203, 236, 305, 477 nm; .sub.max (emission)=542 nm, =19600 M.sup.1 cm.sup.1, QY.sub.fl.=92% (absolute value in a TEAB buffer, pH 8-8.5, as measured by Hamamatsu apparatus C11347-12 with an integration sphere) and 74-76%, respectively, as measured with a Fluorolog-3 spectrometer with an R2658 PMT, relative to an alkaline solution of fluorescein as a standard, whose QY.sub.fl.=90% in a 0.1M NaOH under excitation at 496 nm).

N-[3,6,8-Tris[(3-hydroxypropyl) sulfonyl]pyren-1-yl]glycine (16)

(74) ##STR00044##

(75) The deprotected amino pyrene 13b (19 mg, 34 mol) was suspended in MeOH (11 mL). Glyoxylic acid monohydrate (12.6 mg, 136 mol, 4.0 eq.) was added, and the mixture was acidified by addition of conc. HCl (12 L, 140 mol, 4.3 eq.). After 1 h stirring at rt, NaBH.sub.3CN (17 mg, 270 mol, 8.0 eq.) was added. After 3 h, another portion of glyoxylic acid monohydrate (11 mg, 120 mol, 3.5 eq.) was added. After stirring overnight, a second portion of NaBH.sub.3CN (29 mg, 460 mol, 14 eq.) and conc. HCl (20 L, 240 mol, 7.1 eq.) were added. After stirring for one more day at rt, a third portion of glyoxylic acid monohydrate (91 mg, 990 mol, 29 eq.), NaBH.sub.3CN (27 mg, 430 mol, 12.6 eq.) and conc. HCl (20 L, 240 mol, 7.1 eq.) were added, and the reaction was stirred for one night. The solvent was removed in vacuo, and the crude product was purified by RP-column chromatography with H.sub.2O/ACN+0.2% TFA with 9-25%-ACN gradient. The product was isolated as a red solid (8.3 mg, 13 mol, 38%).

(76) .sup.1H NMR (400 MHz, DMSO-d.sub.6): =1.74 (m, 6H, CH.sub.2CH.sub.2CH.sub.2), 3.38 (m, 6H, SCH.sub.2CH.sub.2), 3.63 (m, 6H, HOCH.sub.2CH.sub.2), 4.26 (br. s, 2H, CH.sub.2COOH), 4.59 (m, 3H, OH), 7.89 (br. s, 1H, NH) 8.45 (m, 2H, 4-H, 5-H), 8.81 (d, .sup.3J.sub.H,H=9.46 Hz, 1H, 10-H), 8.89 (s, 1H, 2-H), 9.06 (s, 1H, 7-H), 9.21 (d, .sup.3J.sub.H,H=9.46 Hz, 1H, 9-H) ppm. TLC (SiO.sub.2) R.sub.f=0.22 (ACN:H.sub.2O:DCM+formic acid=20:2:1).

(77) HPLC: t.sub.R=4.4 min (MeCN/H.sub.2O 30:70.fwdarw.100:0+0.1% TFA in 25 min detected at 254 nm). HPLC: t.sub.R=9.5 min (MeCN/H.sub.2O 10:90.fwdarw.100:0+0.1% TFA in 25 min detected at 465 nm). MS (ESI): m/z (negative mode, %)=641 (100) [M].sup.. HR-MS (ESI): Calcd. for C.sub.27H.sub.31NO.sub.11S.sub.3 ([M+Na].sup.+) 664.0951, found 664.0925. UV-VIS (In MeOH): =18000 M.sup.1 cm.sup.1, .sub.max. (absorption)=499 nm, .sub.max. (fluorescence)=553 nm, QY=0.71.

3,6,8-Tris[(3-hydroxypropyl) sulfonyl]pyrene-1-(methylamine) (18)

(78) ##STR00045##

(79) Compound 14 (75 mg, 0.13 mmol) was suspended in dry DMF (0.1 mL) under argon, Cs.sub.2CO.sub.3 (55 mg, 0.17 mmol) was added followed by MeI (0.15 mL). The reaction mixture was stirred for 1 h in a screw-cap tube at 50 C. HPLC indicated that the reaction was complete. HPLC: starting material t.sub.R=13.7 min (MeCN/H.sub.2O 30:70.fwdarw.100:0+0.1% TFA in 25 min detected at 254 nm). HPLC: product t.sub.R=15.8 min (MeCN/H.sub.2O 30:70.fwdarw.100:0+0.1% TFA in 25 min detected at 254 nm). DMF was removed under vacuum, and the residue was taken up in dichloromethane-water mixture. The organic layer was separated, washed with brine, dried over sodium sulfate, filtered and evaporated in vacuo. Compound 17 (56 mg, 78%, the N-methylated product, see the general scheme of the synthesis for structure) was isolated by chromatography on SiO.sub.2 (25 g) in the course of elution with 0-5% MeOH in ethyl acetate. To oxidize it into the sulfone, compound 17 was dissolved in acetic acid (5 mL), water was added (3 mL), followed by Na.sub.2WO.sub.4*2H.sub.2O (12 mg, catalyst), and the mixture was cooled to 0 C. Hydrogen peroxide (1.5 mL of a ca. 80% solution) was added at 0 C. The reaction mixture turned dark, and then the dark color disappeared. HPLC indicated complete conversion to a new substance with t.sub.R=8.48 min (MeCN/H.sub.2O 30:70.fwdarw.100:0+0.1% TFA in 25 min detected at 254 nm). All volatile materials were removed in vacuo, and the dry residue was dissolved in aqueous acetonitrile. The cleavage of the CF.sub.3CO group (deprotection) in the intermediate compound was performed as follows: a saturated aqueous solution of Na.sub.2CO.sub.3 (ca. 20 wt.-%, 1.5 mL) was added, and the solution was stirred at room temperature. The ratio acetonitrile-water has to be chosen in such a way, that the addition of 1.5 mL of saturated aqueous Na.sub.2CO.sub.3 solution will provide a homogeneous reaction mixture. The reaction mixture turns to be bright orange in several minutes; it was stirred overnight at room temperature. HPLC indicated complete conversion to a new substance (title compound) with t.sub.R=6.4 min (MeCN/H.sub.2O 30:70-100:0+0.1% TFA in 25 min detected at 254 nm). Sodium carbonate was neutralized by addition of glacial AcOH, and the frozen reaction mixture was lyophilized. The solid residue was dissolved in hot aqueous acetonitrile and applied onto a column with RPSiO.sub.2 (C18, 50 g). Elution with acetonitrile-water mixture (1/3-1/2, +1% AcOH) resulted first in a green-yellow band of impurity, followed by an orange zone of the title compound (17). Lyophilization of the orange solution afforded 48 mg (80%) of the title compound as an orange-red powder (slightly soluble in methanol and water). HPLC: t.sub.R=6.0 min (MeCN/H.sub.2O 30:70.fwdarw.100:0+0.1% TFA in 20 min detected at 254 nm; Kinetex column). .sup.1H NMR (400 MHz, CD.sub.3OD+DMSO-d.sub.6, ref. 3.30 ppm for CHD.sub.2OD): =1.92 (m, 6H, CH.sub.2CH.sub.2CH.sub.2), 3.28 (s, 3H, MeNH), 3.57 (q, 6H, .sup.3J.sub.H,H=6.1 Hz, OCH.sub.2CH.sub.2), 3.68 (m, 6H, SO.sub.2CH.sub.2CH.sub.2), 8.12 (s, 1H, 2-H), 8.89 (d, .sup.3J.sub.H,H=9.6 Hz, 1H), 8.93 (d, .sup.3J.sub.H,H=9.6 Hz, 1H), 9.10 (d, .sup.3J.sub.H,H=9.6 Hz, 1H), 9.22 (s, 1H), 9.32 (d, .sup.3J.sub.H,H=9.7 Hz, 1H) ppm. HR-MS (ESI): C.sub.26H.sub.31NO.sub.9S.sub.3 (597.1116); [MH].sup. found 596,1078, calc. 596.1088. UV-VIS (MeOH): =23400 M.sup.1 cm.sup.1, .sub.max. (absorption)=502 nm, .sub.max. (fluorescence)=550 nm, QY=0.88. UV-VIS (H.sub.2O): =19500 M.sup.1 cm.sup.1, .sub.max. (absorption)=509 nm, .sub.max. (fluorescence)=563 nm, QY=0.88.

(80) Some Additional Selected Fluorescent Dyes with Large Negative Net Charges and Favorable Spectral Properties (See Also Scheme 12 and Table 2).

(81) ##STR00046##

(82) The red-emitting rhodamine dye with multiple ionizable groups of structure 20 was obtained by phosphorylation of the corresponding hydroxyl-substituted rhodamine precursor and isolated analogously to compound 19 (another phosphorylated rhodamine dye, see Schemes 6 and 11 above) previously described by K. Kolmakov, C. A. Wurm, R. Hennig, E. Rapp, S. Jakobs, V. N. Belov and S. W. Hell in Chem. Eur. J. 2012, 18, 12986-12998 (see compound 7-H therein for the properties and the phosphorylation details). The hydroxyl-substituted precursor for compound 20 was synthesized according to K. Kolmakov, C. A. Wurm, D. N. H. Meineke, F. Gttfert, V. P. Boyarskiy, V. N. Belov and S. W. Hell (Chem. Eur. Journal, 2013, 20, 146-157; see compound 14-Et therein). The phosphorylation was followed by saponification of the ethyl ester group via a routine procedure, as described.

(83) Purity and identity of compound 20 was confirmed by the following analytical data: .sup.1H NMR (400 MHz, DMSO-d.sub.6): =1.23 (s, 6H, CH.sub.3), 1.28 (s, 6H, CH.sub.3), 2.62 (s, 6H, NCH.sub.3), 4.21 (m, 4H, 2CH.sub.2), 5.70 (s, 2H), 6.76 (s, 2H), 7.16-7.30 (br. m, 4H), 8.55 (m, 1H), 8.36 (m, 1H) ppm. .sup.13C NMR (101 MHz, DMSO-d.sub.6): =29.1 (CH.sub.3), 34.2 (CH.sub.3), 95.8 (CH.sub.2), 118.2 (CH), 121.7 (C) 122.6 (C), 125.5 (CH), 127.3 (CH), 127.4 (CH), 128.0 (CH), 129.8 (CH), 133.9 (C), 136.8 (C), 155.0 (CO), 157.0 (CO) ppm.

(84) .sup.1H NMR (400 MHz, CD.sub.3OD, 20 as a Et.sub.3N-salt): =1.12 (t, J=7 Hz, 9H, CH.sub.3CH.sub.2), 1.25 (t, J=7 Hz, 27H, CH.sub.3CH.sub.2), 1.52 (s, 6H, CH.sub.3), 1.53 (s, 6H, CH.sub.3), 3.11, 3.31 (m, 24H, CH.sub.3CH.sub.2), 3.18 (s, 6H, NCH.sub.3), 3.61 (m, 2H, CH.sub.2), 4.45 (m, 2H, CH.sub.2), 6.03 (s, 2H), 6.8 (s, 2H), 6.9 (s, 2H), 7.28 (d, J=8 Hz, 1H), 8.16 (d, J=8 Hz, 1H), 8.66 (m, 1H) ppm. .sup.31P NMR (161.9 MHz): =0.2 (DMSO-d.sub.6) and 0.63 (CD.sub.3OD) ppm (s, OP(O)(OH).sub.2)).

(85) HPLC: t.sub.R=3.9 min (Kinetex EVO C-18 column, with 0.02 M aq. Et.sub.3N (A) and 3% MeCN (B), isocratic flow 0.5 mL/min, detection at 254 nm). TLC: R.sub.f=0.25 (silica gel plates, MeCN/H.sub.2O 5:1+0.2% Et.sub.3N).

(86) HR-MS (ESI): calc. for C.sub.35H.sub.35N.sub.2O.sub.13P.sub.2.sup. ([MH].sup.) 753.1614, found 753.1672. A red-violet highly water-soluble dye with a very intense red fluorescence: UV-VIS (PBS buffer, pH=7.4) .sub.max. abs.=582 nm, .sub.max. fl.=609 nm, =120000 M.sup.1 cm.sup.1, QY.sub.fl.=0.76% (abs. value in TEAB buffer, excitation at 540 nm).

3,6,8-Tris[(3-sulfopropyl)sulfonyl]-pyrene-1-amine (23a)

(87) The synthesis of a sulfoalkylsulfonyl-substituted 1-aminopyrene derivative 23a and of its homolog 23b involved halogen exchange in the tribromo-substituted precursor 12 (see Scheme 11 above) with synthones containing SH and SO.sub.3H functions and was followed by oxidation to the sulfone.

2,2,2-Trifluoro-N-[3,6,8-tris[(3-sulfopropyl) sulfanyl]pyrene-1-yl]-acetamide (21a)

(88) ##STR00047##

(89) 2,2,2-trifluoro-N-[3,6,8-tris(bromo)pyren-1-yl]-acetamide (tribromo-substituted precursor 12, 180 mg, 0.33 mmol; for preparation and properties see above) and sodium 3-mercapto-1-propanesulfonate (600 mg, 3.37 mmol, 10 equiv) was reacted in dry DMF (20 mL) at 90 C. upon stirring under an argon atmosphere in presence of triethylamine (0.6 mL) as a base with Pd.sub.2(dba).sub.3 (20 mg, 0.02 mmol) and xantphos reagent (30 mg, 0.05 mmol) as catalysts. As the reaction completed (3-5 h), the crude product (Na salt of 21a) was first purified over reversed-phase silica gel (C-18) and H.sub.2OMeCN (gradient 0-70%) as mobile phase. The homogeneous fractions were combined, filtered (syringe filters 0.45 m), concentrated and freeze-dried. The additional purification of the crude product (340 mg, containing inorganic salts) was performed by repeated preparative HPLC using Kinetex Gemini NX C-18 solid phase (5 m) with 50 mmol TEAB buffer (A) and MeCN (B) under gradient (0-20% B) conditions. The pure fractions were combined, concentrated at t40 C. and freeze-dried to furnish 70-80% of the yellow crystalline water-soluble solid as a triethylammonium salt of compound 21a.

(90) Analytical data: .sup.1H NMR (400 MHz, DMSO-d.sub.6): =1.08 (m, 27H, CH.sub.3CH.sub.2) 1.98 (m, 6H, CH.sub.2CH.sub.2CH.sub.2), 2.66 (m, 6H, SCH.sub.2CH.sub.2), 2.88 (m, 18H, CH.sub.2CH.sub.3), 3.39 (m, 6H, O.sub.3SCH.sub.2CH.sub.2), 8.13 (d, J=9.5 Hz, 1H), 8.15 (d, J=9.5 Hz, 1H), 8.26 (br. s, 1H), 8.45 (d, J=9.5 Hz, 1H), 8.48 (d, J=9.5 Hz, 1H), 8.52 (s, 2H) ppm. .sup.1H NMR (400 MHz, CD.sub.3OD): =1.21 (t, J=7 Hz, 27H, CH.sub.3CH.sub.2) 2.15 (m, 6H, CH.sub.2CH.sub.2CH.sub.2), 2.99 (m, 6H, SCH.sub.2CH.sub.2), 3.10 (q, J=7 Hz, 18H, CH.sub.2CH.sub.3), 3.38 (m, 6H, O.sub.3SCH.sub.2CH.sub.2), 8.06 (d, J=9.5 Hz, 1H), 8.22 (s, 1H), 8.31 (s, 1H), 8.43 (d, J=9.5 Hz, 1H), 8.51 (br. s, 1H, NH), 8.63-8.70 (m, 2H) ppm.

(91) .sup.13C NMR (101 MHz, DMSO-d.sub.6): =9.4 (CH.sub.3), 22.5 (CH.sub.2), 25.0 (CH.sub.2), 25.1 (CH.sub.2), 32.3 (CH.sub.2), 32.4 (CH.sub.2), 45.7 (CH.sub.2), 50.0 (CH.sub.2), 115.3 (CH), 118.2 (CH), 122.3 (CH), 123.1 (CH), 123.4 (CH), 124.3 (C), 124.8 (CH), 124.9 (C), 125.5 (CH), 127.2 (CH), 127.3 (C), 127.7 (C), 127.9 (C), 132.0 (C), 132.1 (C), 156.3 (C), 156.6 (C), 171.0 (CO) ppm. .sup.19F NMR (376 MHz, DMSO-d.sub.6): =73.20 (s, CF.sub.3) ppm.

(92) HPLC: t.sub.R=8.2 min (Kinetex column EVO C-18 100, 5 m, 4.6250 mm), 0.05 M aq. TEAB (A) and 25% MeCN (B), isocratic flow 0.5 mL/min, detection at 254 nm). TLC: R.sub.f=0.6 (silica gel plates, MeCN/H.sub.2O 7:1+0.2% Et.sub.3N, also seen by its intense blue fluorescence under illumination with UV-light at 365 nm). HR-MS (ESI): calc. for C.sub.27H.sub.27F.sub.3NO.sub.10S.sub.6.sup. ([MH].sup.) 773.9911, found 773.9845.

(93) UV-VIS (PBS buffer, pH=7.4) .sub.max. (absorption)=428 nm. The synthesis is based on the general recipe for palladium-catalized cross-coupling of thiols and aryl bromides described by C. Mispeleare-Canivet and co-workers, Tetrahedron 2005, 61, 5253-5259.

2,2,2-Trifluoro-N-[3,6,8-tris[(3-sulfopropyl) sulfonyl]pyrene-1-yl]-acetamide (22a)

(94) ##STR00048##

(95) The thioether 21a (as a triethylammonium salt) was oxidized to the sulfone as follows: 50 mg (0,046 mmol, as a Et.sub.3N salt with M=1077) of the substrate was dissolved in a mixture of HOAc (4 mL) and water (0.4 mL). Upon cooling to +5 C. 1 ml of 50 wt. % H.sub.2O.sub.2 and 0.5 mL of 1 wt. % aq. solution of Na.sub.2WO.sub.42H.sub.2O (a catalyst) was added. The reaction mixture was kept for 30 min at this temperature, left overnight at RT, diluted with water (20 mL) and freeze-dried. The product was purified by means of preparative HPLC using Kinetex Gemini NX C-18 solid phase (5 m) with 0.5 vol. % aqueous HOAc (A) and MeCN (B) under gradient (0-50% B) conditions. The pure fractions were pooled, concentrated at t40 C. and freeze-dried to furnish 28 mg (52%) of a red crystalline solid as a triethylammonium salt (M.W.=1173), which has an intense yellow-orange fluorescence in water.

(96) Analytical data for compound 22a: .sup.1H NMR (400 MHz, CD.sub.3OD): =1.27 (m, 27H, CH.sub.3CH.sub.2) 2.24 (m, 6H, CH.sub.2CH.sub.2CH.sub.2), 2.97 (m, 6H, SO.sub.2CH.sub.2CH.sub.2), 3.19 (m, 18H, CH.sub.2CH.sub.3), 3.84 (m, 6H, CH.sub.2CH.sub.2SO.sub.3), 8.73 (d, J=9 Hz, 1H), 9.03 (s, 1H), 9.39-9.41 (m, 2H), 9.02 (s, 1H), 9.52 (d, J=9 Hz, 1H) ppm. .sup.13C NMR (101 MHz, CD.sub.3OD): =8.9 (CH.sub.3), 20.1 (CH.sub.2), 47.9 (CH.sub.2), 50.2 (CH.sub.2), 55.7 (CH.sub.2), 56.2 (CH.sub.2), 56.6 (CH.sub.2), 126.4 (C), 126.6 (CH), 127.0 (C), 128.0 (CH), 129.6 (CH), 130.0 (C), 131.7 (C), 133.5 (C), 134.1 (C), 134.4 (C), 134.9 (C), 136.4 (C), 158.8 (CO), ppm. .sup.19F NMR (376 MHz, CD.sub.3OD): =76.50 (s, CF.sub.3) ppm.

(97) HPLC: t.sub.R=5.2 min (Kinetex column EVO C-18 100, with 0.05 M aq. TEAB (A) and 25% MeCN (B), isocratic flow 0.5 mL/min, detection at 254 nm). TLC: R.sub.f=0.5 (silica gel plates, MeCN/H.sub.2O 7:1+0.2% Et.sub.3N, seen by its intense orange fluorescence under illumination with UV-light at 365 nm). HR-MS (ESI): calc. for C.sub.27H.sub.27F.sub.3NO.sub.16S.sub.6.sup. ([MH].sup.) 869.9606, found 869.9536. UV-VIS (PBS buffer, pH=7.4) .sub.max abs.=476 nm.

3,6,8-Tris[(3-sulfopropyl)sulfonyl]-pyrene-1-amine (23a)

(98) ##STR00049##

(99) The protective COCF.sub.3 group in compound 22a was cleaved by a conventional method under basic conditions at RT (see T. W Greene, P. G. M. Wuts Protective groups in organic synthesis Third Edition 1999, p. 557). The reaction progress was monitored by TLC, the reaction mixture neutralized to pH 7-8 with HOAc and concentrated at t40 C. The residue was subjected to preparative HPLC using Gemini NX C-18 solid phase (5 m) with 0.3 vol. % aqueous Et.sub.3N (A) and 10% MeCN (B) under isocratic mode. The pure fractions were concentrated and freeze-dried to furnish 22 mg (79%) of compound 23a as a triethylammonium salt. Properties: red crystalline solid, well-soluble in water to give solutions with a very intense yellow-greenish fluorescence.

(100) Analytical data: .sup.1H NMR (400 MHz, CD.sub.3OD): =1.27 (t, 27H, J=7 Hz, CH.sub.3CH.sub.2, 3 Et.sub.3N) 2.13-2.29 (m, 6H, CH.sub.2CH.sub.2CH.sub.2), 2.88-3.17 (m, 6H, ArSO.sub.2CH.sub.2CH.sub.2), 3.16 (q, J=7 Hz, 18H, CH.sub.2CH.sub.3, 3 Et.sub.3N), 3.69-3.84 (m, 6H, CH.sub.2CH.sub.2SO.sub.3), 8.13 (s, 1H), 8.66 (d, J=9 Hz, 1H), 8.83 (d, J=9 Hz, 1H), 8.93 (d, J=9 Hz, 1H), 9.17 (d, J=9 Hz, 1H), 9.20 (s, 1H) ppm. .sup.13C NMR (101 MHz, CD.sub.3OD) =9.2 (CH.sub.3), 20.0 (CH.sub.2), 20.1 (CH.sub.2), 20.2 (CH.sub.2), 47.9 (CH.sub.2), 50.4 (CH.sub.2), 55.4 (CH.sub.2), 55.7 (CH.sub.2), 56.5 (CH.sub.2), 60.1 (CH.sub.2), 117.7 (C), 118.3 (CH), 120.3 (CH), 123.0 (CH), 127.7 (C), 128.1 (C), 128.6 (CH), 129.4 (C), 129.9 (C), 130.2 (CH), 131.7 (CH), 134.9 (C), 136.0 (C), 138.1 (C), 149.6 (C), ppm. HPLC: t.sub.R=4.7 m n (Kinetex column EVO C-18 100, with 0.05 M aa. TEAB (A) and 25% MeCN (B), isocratic, 0.5 mL/min, detection at 254 nm). TLC: R.sub.f=0.4 (silica gel plates, MeCN/H.sub.2O 7:1+0.2% Et.sub.3N, seen by its intense green fluorescence under illumination with UV-light at 365 nm). HR-MS (ESI) negative mode: calc. for C.sub.25H.sub.28NO.sub.15S.sub.6.sup. ([MH].sup.) 774.9873, found 774.9804; positive mode: calc. for C.sub.25H.sub.33N.sub.2O.sub.15S.sub.6+([M+NH.sub.4].sup.+) 793, 0205, found 793.0146. UV-VIS (TEAB buffer, pH=8.5): =21 000 M.sup.1 cm.sup.1, .sub.max. abs.=486 nm, .sub.max. fl.=542 nm., .sub.fl=86% (in TEAB buffer, relative to fluorescein as a reference dye with .sub.fl=0.9 in 0.1 M NaOH under excitation at 496 nm).

(101) Using the commercially available sodium 2-mercapto-ethanesulfonate as a thiol-containing reagent with a shorter alkyl chain, the dye 23b (a homolog of dye 23a depicted above, which also belongs to the general Formula C with each XSO.sub.3H and n=1) was obtained in the exactly same fashion and with a high yield from bromide 12. Its spectral and photophysical properties (, .sub.max and .sub.fl, see also Table 2) proved to be fully identical to those of the dye 23a.

Example 3

(102) Reductive Amination of Glycans

(103) For reductive amination of glycans using the compounds of the present invention, the prior art protocol for fluorescent labeling of N-glycans with 8-aminopyrene-1,3,6-trisulfonic acid trisodium salt (APTS) and a borane-based reducing agent (Bigge J C, Patel T P, Bruce J A, Goulding P N, Charles S M, Parekh R B, Analytical Biochemistry 1995, 230, 229-238; Ruhaak L R, Hennig R, Huhn C, Borowiak M, Dolhain RJEM, Deelder A M, Rapp E, Wuhrer M, Journal of Proteome Research 2010, 9, 6655-6664) or a modified version thereof may be followed.