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

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:

• wherein R1 and/or R2 are independent from each other and may represent:
• H, deuterium (D), alkyl or deutero-substituted alkyl;
• R1-R2 may form a four-, five-, six-, or seven-membered non-aromatic carbocycle with an additional primary amino group NH2, secondary amino group NHRa, where R1 ═C1-C6 alkyl, or hydroxyl group OH attached to one carbon atom of this carbocycle;
• or
• R1-R2 may form a four-, five-, six-, or seven-membered non-aromatic heterocycle with an additional heteroatom included in the heterocycle;
• a hydroxyalkyl group (CH2)mOH, where m=1-12, with a straight or branched alkyl chain;
• one of R1 or R2 groups may be a carbonate or carbamate derivative (CH2)mOCOOR4 or (CH2)mNHCOOR4, where m=1-12 and R4=methyl, ethyl, 2-chloroethyl, 1V-succinimidyl,
• sulfo-N-succinimidyl, 1-oxybenzotriazolyl, a phenyl group or a substituted Phenyl group;
• (CH2)mNRaRb, where m=1-12, with a straight or branched alkyl chain;
• Ra, Rb are independent from each other and may be H or optionally substituted C1-C4 alkyl group(s);
• one of R1 or R2 groups may be a primary amino group to form aryl hydrazines Ar-NR6NH2 where Ar is the entire pyrene residue in Formula B and R6═H or alkyl;
• one of R1 or R2 groups may be a hydroxy group to form aryl hydroxylamines Ar-NR7OH where Ar is an entire pyrene residue in Formula B and R7═H or alkyl;
• one of R1 or R2 groups may be an alkyloxyamino group (CH2)nONH with n=1-12;
• one of R1 or R2 groups may be CO(CH2)nCOOR8, with n=1-5 and a straight or branched alkyl chain (CH2)n and with R8 selected from H, straight or branched C1-C6 alkyl, CH2CN, 2- and 4-nitrophenyl, 2,3,5,6-tetrafluorophenyl, pentachlorophenyl, pentafluoro-phenyl, N-succinimidyl, sulfo-N-succinimidyl, 1-oxybenzo-triazolyl;
• further, one of R1 or R2 may be (CH2)nCONHR9, with n=1-5 and R9 ═H, C1-C6 alkyl, (CH2)mN3, (CH2)m—N-maleimido, (CH2)mNHCOCH2X (X═Br or I), where m=2-6 and with straight or branched alkyl chains in (CH2)n and R9;
• or
• one of R1 or R2 may represent CH2-C6H4—NH2, COC6H4—NH2, CONHC6H4—NH2 or CSNHC6H4—NH2 with C6H4 being a 1,2-, 1,3- or 1,4-phenylene, COC5H3N—NH2 or CH2-C4H3N—NH2 with C5H3N being pyridin-2,4-diyl, pyridin-2,5-diyl, pyridin-2,6-diyl, or pyridin-3,5-diyl;
• or one of R1 or R2 may be an alkyl azide (CH)N3 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 (CF2), and alkene or alkine moieties in any combinations, at any occurrence, linear or branched, with a length ranging from C1 to C12;
• the linker L may also include a carbonyl (CH2CO, CF2CO) 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 R1 and R2, 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, SO3H, OSO3H, OP(O)(OH)2, OP(O)(OH)Ra, where Ra═C1-C4 alkyl or substituted C1-C4 alkyl, P(O)(OH)2, and P(O)(OH)Ra, where Ra═C1-C4 alkyl or substituted C1-C4 alkyl,
• or wherein the fluorescent aminopyrene dues have the general formula C:
• or a salt thereof,
• wherein
• n=1-12;
• R1 and/or R2 are independent from each other and may represent;
• H, deuterium (D), alkyl or deutero-substituted alkyl;
• R1-R2 may form a four-, five, six-, or seven-membered non-aromatic carbocycle with an additional primary amino group NH2, secondary amino group NHRa, where Ra ═C1-C6, alkyl, or hydroxyl group OH attached to one carbon atom of this carbocycle;
• or
• R1-R2 may form a four-, five-, six-, or seven-membered non-aromatic heterocycle with an additional heteroatom included in the heterocycle;
• a hydroxyalkyl group (CH2)mOH, where m=2-12, with a straight or branched alkyl chain,
• one of R1 or R2 groups may be a carbonate or carbamate derivative (CH2)mOCOOR4 or (CH2)mNHCOOR4, where m=1-12 and R4=methyl, ethyl, 2-chloroethyl, N4-succinimidyl, sulfo-N-succinimidyl, 1-oxybenzotriazolyl, a phenyl group or a substituted phenyl group;
• (CH2)mNRaRb, where in m=2-12, with a straight or branched alkyl chain, Ra, Rb are independent from each other and may be H, or optionally substituted C1-C4 alkyl group(s);
• one of R1 or R2 groups may be a primary amino group to form aryl hydrazines, as described for Formula B;
• one of R1 or R2 groups may be a hydroxy group to form aryl hydroxylamines, as described for Formula B;
• one of R1 or R2 groups may bean alkyloxyamino group (CH2)nONH2 with n=1-12;
• one of R1 or R2 may be (CH2)nCONHR6, with n=1-12, and R6═H, C1-C6 alkyl, (CH2)mN3, (CH2)m—N-maleimido, (CH2)m—NHCOCH2X (X═Br or I), where m=2-6 and with straight or branched alkyl chains in (CH2)n and R6;
• or
• one of R1 or R2 may represent CH2-C6H4—NH2, COC6H4—NH2, CONHC6H4—NH2 or CSNHC6H4—NH2 with C6H4 being a 1,2, 1,3- or 1,4-phenylene, COC5H3N—NH2 or CH2—CH2N—NH2 with C5H3N being pyridin-2,4-diyl, pyridin-2,5-diyl, pyridin-2,6-diyl, or pyridin-3,5-diyl;
• the (CH2)n-CH2 linker, with n=1-12, between the SO2 fragment and the residue X in Formula B may represent a straight-chain, branched or cyclic group having 2-6 carbon atoms,
• X═SH, COOH, SO3H, OSO3H, OP(O)(OH)2, OP(O)(OH)Ra, where Ra=optionally substituted C1-C4 alkyl, P(O)(OH)2, P(O)(OH)Ra, where Ra=optionally substituted C1-C4 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, SO3H, OSO3H, OP(O)(OH)2, OP(O)(OH)Ra, where Ra ═C1-C4 alkyl or substituted C1-C4 alkyl, P(O)(OH)2, and P(O)(OH)Ra, where Ra═C1-C4 alkyl or substituted C1-C4 alkyl.