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AMINOACRIDINE AND AMINOPYRENE DYES AND THEIR USE AS FLUORESCENT TAGS, IN PARTICULAR FOR CARBOHYDRATE ANALYSIS

20230040324 · 2023-02-09

    Inventors

    Cpc classification

    International classification

    Abstract

    The invention relates to novel fluorescent dyes with multiple negatively charged groups in their ionized form which are 9-aminoacridines or 1-aminopyrene shaving of one of the following general formulae A-E: or salts or protonated forms thereof, wherein the ionizable groups are typically selected from the following: OH, SH, COOH, SO.sub.3H, OSO.sub.3H, SO.sub.2NHCN, P(O)(OH).sub.2, P(O) (OH).sub.2. The invention further relates to the use of these dyes as fluorescent tags, in particular for reducing sugars and glycans, and to carbohydrate-dye conjugates comprising these dyes as well as to methods for preparing the same.

    ##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 formulae A, B, C, D, E, including salts or protonated forms thereof: ##STR00069## wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6 are independent from each other and selected from the group consisting of a) hydrogen; b) CH.sub.3, C.sub.2H.sub.5, a straight or branched C.sub.3-C.sub.6 alkyl, deuterated alkyl, or perfluoroalkyl group; c) straight or branched C.sub.2-C.sub.6 alkyl group that is substituted with 1 to 3 halogen atoms, or incorporates 1 to 3 heteroatoms selected from O, S, and N, or that is optionally substituted with solubilizing groups selected from OH, NH.sub.2, SH, and COOH and/or ionizable anionic residues selected from the group consisting of (alkyl) carboxy (CH.sub.2).sub.nCO.sub.2H, (alkyl) sulfonate (CH.sub.2).sub.nSO.sub.3H, (alkyl) sulfate (CH.sub.2).sub.nOSO.sub.3H, (alkyl) phosphate (CH.sub.2).sub.nOP(O)(OH).sub.2 and (alkyl) phosphonate (CH.sub.2).sub.nP(O)(OH).sub.2, wherein n is an integer ranging from 0 to 6, or analogs thereof wherein one or more of the CH.sub.2 groups are replaced by CF.sub.2 and/or CD.sub.2; d) (CH.sub.2).sub.nCOOR.sup.7, where n=1-6, and R.sup.7 is alkyl CH.sub.2CN, CH.sub.2CF.sub.3, benzyl, substituted benzyl, polyhaloalkyl, polyhalophenyl, 2- and 4-nitrophenyl, N-succinimidyl, sulfo-N-succinimidyl, 1-benzotriazolyl, 9-aza-1-benzotriazolyl or other forming an active ester of a carboxylic acid; e) a carbonate or carbamate derivative (CH.sub.2).sub.mOCOOR.sup.8 or (CH.sub.2).sub.mNHCOOR.sup.8, where m=1-6 and R.sup.8═C.sub.1-C.sub.4 alkyl, benzyl, 9-fluorenylmethyl, CH.sub.2CN, N-succinimidyl, sulfo-N-succinimidyl, 1-benzotriazolyl, phenyl, substituted phenyl group, 2-pyridyl, 4-pyridyl, or pyrimid-4-yl; f) (CH.sub.2).sub.mNR.sub.aR.sub.b, where m=1-6, with a straight or branched alkyl chain; R.sup.a, R.sup.b are independent from each other and represent hydrogen, C.sub.1-C.sub.4 alkyl and/or C.sub.1-C.sub.4 acyl groups, optionally substituted with a hydroxyalkyl group (CH.sub.2).sub.nOH, where n=1-6, with a straight or branched alkyl chain, a phosphorylated hydroxyalkyl group (CH.sub.2).sub.pOP(O)(OH).sub.2, where p=1-6, with a straight or branched alkyl chain; g) an alkyl azide (CH.sub.2).sub.qN.sub.3, where q=1-8, with a straight or branched alkyl chain; h) an alkylalkyne (CH.sub.2).sub.rC≡CR.sup.9, where r=1-8, with a straight or branched alkyl chain in (CH.sub.2).sub.r and R.sup.9=hydrogen, C.sub.1-C.sub.6 alkyl or (functionally) substituted a C.sub.1-C.sub.6 alkyl; i) (CH.sub.2).sub.sCONHR.sup.10, with s=1-8, R.sup.10═H, C.sub.1-C.sub.6 alkyl, (CH.sub.2).sub.qN.sub.3, (CH.sub.2).sub.rC≡CH or (CH.sub.2).sub.t—N-maleimido, (CH.sub.2).sub.u—NH—COCH.sub.2X with X═Br or I, where q, r, t, u indices are independent in the range 1-8, and with straight or branched alkyl chains in (CH.sub.2).sub.s, (CH.sub.2).sub.q, (CH.sub.2).sub.r, (CH.sub.2).sub.t, (CH.sub.2).sub.u and R.sup.10; j) a primary amino group (NH.sub.2) or secondary amino group (NHR.sup.11); R.sup.11═C.sub.1-C.sub.6 alkyl or (functionally) substituted C.sub.1-C.sub.6 alkyl; R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6 may contain a terminal alkyloxyamino group (CH.sub.2).sub.vONH.sub.2, where v=1-12 with a straight or branched alkyl chain that can include one or multiple (alkyl)oxy (CH.sub.2).sub.kO, (alkyl)amino (CH.sub.2).sub.lNH or (alkyl)amido (CH.sub.2).sub.wCONH groups in all possible combinations with indices k, l and w=0-12 (independent from each other; R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6 may contain a terminal acylhydrazino group (CH.sub.2).sub.xCONHNH.sub.2, where x=1-12, with a straight or branched alkyl chain that can include one or multiple (alkyl)oxy (CH.sub.2).sub.kO, (alkyl)amino (CH.sub.2).sub.lNH or (alkyl)amido (CH.sub.2).sub.wCONH groups in all possible combinations with indices k, l and w=0-12 independent from each other; one of the residues R.sup.1, R.sup.2, R.sup.3, R.sup.4 may represent C(O)R.sup.12 or COOR.sup.13, where R.sup.12 or R.sup.13═C.sub.1-C.sub.4 alkyl, optionally substituted with a hydroxyalkyl group (CH.sub.2).sub.yOH, where y=1-6, with a straight or branched alkyl chain, a phosphorylated hydroxyalkyl group (CH.sub.2).sub.mOP(O)(OH).sub.2, where m=1-6, with a straight or branched alkyl chain; one of the residues R.sup.5, R.sup.6 may represent CN; further, one of the residues R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6 may represent (CH.sub.2).sub.z—C.sub.6H.sub.4—NH.sub.2, COC.sub.6H.sub.4—NH.sub.2, CONHC.sub.6H.sub.4—NH.sub.2 or CSNHC.sub.6H.sub.4—NH.sub.2 with z=1-4, and C.sub.6H.sub.4 being a 1,2-, 1,3- or 1,4-phenylene, COC.sub.5H.sub.3N—NH.sub.2 or (CH.sub.2).sub.s—C.sub.5H.sub.3N—NH.sub.2, with z=1-4, and C.sub.5H.sub.3N being pyridin-2,4-diyl, pyridin-2,5-diyl, pyridin-2,6-diyl, or pyridin-3,5-diyl; additionally, R.sup.1-R.sup.2 (R.sup.3-R.sup.4, R.sup.5-R.sup.6) may form a four-, five, six-, or seven-membered heterocycle comprising the nitrogen atom; which heterocycle may be unsubstituted or substituted with a straight or branched (C.sub.2-C.sub.6) alkyl group, which is unsubstituted, substituted with 1 to 3 halogen atoms or incorporates 1 to 3 heteroatoms selected from O, S, and N, or which is optionally substituted with solubilizing groups selected from OH, NH.sub.2, SH, and COOH and/or ionizable anionic residues selected from (alkyl) carboxy (CH.sub.2).sub.nCO.sub.2H, (alkyl) sulfonate (CH.sub.2).sub.nSO.sub.3H, (alkyl) sulfate (CH.sub.2).sub.nOSO.sub.3H, (alkyl) phosphate (CH.sub.2).sub.nOP(O)(OH).sub.2 and (alkyl) phosphonate (CH.sub.2).sub.nP(O)(OH).sub.2, wherein n is an integer ranging from 0 to 6, or analogs thereof wherein one or more of the CH.sub.2 groups are replaced by CF.sub.2 and/or CD.sub.2; or R.sup.1-R.sup.2 (R.sup.3-R.sup.4, R.sup.5-R.sup.6) may form a four-, five, six-, or seven-membered cycle (comprising the nitrogen atom) with a hydroxyl group OH, or a phosphorylated hydroxyl group OP(O)(OH).sub.2 attached to one of the carbon atoms in this cycle, a primary amino group NH.sub.2, secondary amino group NHR.sup.a (where R.sup.a═C.sub.1-C.sub.6 alkyl of functionally substituted alkyl group), or the combination of such groups; with the proviso that in all compounds of Formula A 1, 2, 3, 4, 5 or 6 negatively charged groups are present under basic conditions, i.e. 7<pH<14, and these negatively charged groups represent at least partially deprotonated residues of ionizable groups selected from the following set: OH, SH, COOH, a sulfonic acid residue SO.sub.3H, a sulfate residue OSO.sub.3H, an N-cyanosulfonamide residue SO.sub.2NHCN, a phosphate group OP(O)(OH).sub.2, and a phosphonate group P(O)(OH).sub.2; ##STR00070## wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4 are independent from each other and may represent: a) hydrogen; b) CH.sub.3, C.sub.2H.sub.5, a straight or branched C.sub.3-C.sub.6 alkyl, deuterated alkyl, or perfluoroalkyl group; c) straight or branched C.sub.2-C.sub.6 alkyl group that is substituted with 1 to 3 halogen atoms, or incorporates 1 to 3 heteroatoms selected from O, S, and N, or which is optionally substituted with solubilizing groups selected from OH, NH.sub.2, SH, and COOH and/or ionizable anionic residues selected from (alkyl) carboxy (CH.sub.2).sub.nCO.sub.2H, (alkyl) sulfonate (CH.sub.2).sub.nSO.sub.3H, (alkyl) sulfate (CH.sub.2).sub.nOSO.sub.3H, (alkyl) phosphate (CH.sub.2).sub.nOP(O)(OH).sub.2 and (alkyl) phosphonate (CH.sub.2).sub.nP(O)(OH).sub.2, wherein n is an integer ranging from 0 to 6, or analogs thereof wherein one or more of the CH.sub.2 groups are replaced by CF.sub.2 and/or CD.sub.2 residues; d) (CH.sub.2).sub.nCOOR.sup.7, where n=1-6, and R.sup.7 is alkyl, CH.sub.2CN, CH.sub.2CF.sub.3, benzyl, substituted benzyl, polyhaloalkyl, polyhalophenyl, 2- and 4-nitrophenyl, N-succinimidyl, sulfo-N-succinimidyl, 1-benzotriazolyl, 9-aza-1-benzotriazolyl or other groups forming an active ester of a carboxylic acid; e) a carbonate or carbamate derivative (CH.sub.2).sub.mOCOOR.sup.8 or (CH.sub.2).sub.mNHCOOR.sup.8, where m=1-6 and R.sup.8═C.sub.1-C.sub.4 alkyl, benzyl, 9-fluorenylmethyl, CH.sub.2CN, N-succinimidyl, sulfo-N-succinimidyl, 1-benzotriazolyl, phenyl, substituted phenyl group, 2-pyridyl, 4-pyridyl, or pyrimid-4-yl; f) (CH.sub.2).sub.mNR.sup.aR.sup.b, where m=1-6, with a straight or branched alkyl chain; R.sup.a, R.sup.b are independent from each other and represent hydrogen, C.sub.1-C.sub.4 alkyl and/or C.sub.1-C.sub.4 acyl groups, optionally substituted with a hydroxyalkyl group (CH.sub.2).sub.nOH, where n=1-6, with a straight or branched alkyl chain, a phosphorylated hydroxyalkyl group (CH.sub.2).sub.pOP(O)(OH).sub.2, where p=1-6, with a straight or branched alkyl chain; g) an alkyl azide (CH.sub.2).sub.qN.sub.3, where q=1-8, with a straight or branched alkyl chain; h) an alkylalkyne (CH.sub.2).sub.r≡CR.sup.9, where r=1-8, with a straight or branched alkyl chain in (CH.sub.2).sub.r and R.sup.9=hydrogen, alkyl C.sub.1-C.sub.6 or (functionally) substituted alkyl C.sub.1-C.sub.6; i) (CH.sub.2).sub.sCONHR.sup.10, with s=1-8, R.sup.10═H, C.sub.1-C.sub.6 alkyl, (CH.sub.2).sub.qN.sub.3, (CH.sub.2).sub.rC≡CH or (CH.sub.2).sub.t—N-maleimido, (CH.sub.2).sub.u—NH—COCH.sub.2X (X ═Br or I), where q, r, t, u indices are independent in the range 1-8, and with straight or branched alkyl chains in (CH.sub.2).sub.s, (CH.sub.2).sub.q, (CH.sub.2).sub.r, (CH.sub.2).sub.t, (CH.sub.2).sub.u and R.sup.10; j) a primary amino group (NH.sub.2) or secondary amino group (NHR.sup.11); R.sup.11═C.sub.1-C.sub.6 alkyl (functionally) substituted a C.sub.1-C.sub.6 alkyl; R.sup.1, R.sup.2, R.sup.3, R.sup.4 may contain a terminal alkyloxyamino group (CH.sub.2).sub.vONH.sub.2, where v=1-12, with a straight or branched alkyl chain that can include one or multiple (alkyl)oxy (CH.sub.2).sub.kO, (alkyl)amino (CH.sub.2).sub.lNH or (alkyl)amido (CH.sub.2).sub.wCONH groups in all possible combinations with indices k, 1 and w=0-12 independent from each other; R.sup.1, R.sup.2, R.sup.3, R.sup.4 may contain a terminal acylhydrazino group (CH.sub.2).sub.xCONHNH.sub.2, where x=0-12, with a straight or branched alkyl chain that can include one or multiple (alkyl)oxy (CH.sub.2).sub.kO, (alkyl)amino (CH.sub.2).sub.lNH or (alkyl)amido (CH.sub.2)nCONH groups in all possible combinations with indices k, 1 and w=0-12 (independent from each other; one of the residues R.sup.1, R.sup.2, R.sup.3, R.sup.4 may represent C(O)R.sup.12 or COOR.sup.13, where R.sup.12 or R.sup.13═C.sub.1-C.sub.4 alkyl, optionally substituted with a hydroxyalkyl group (CH.sub.2).sub.yOH, where y=1-6, with a straight or branched alkyl chain, a phosphorylated hydroxyalkyl group (CH.sub.2).sub.mOP(O)(OH).sub.2, where m=1-6, with a straight or branched alkyl chain; further, one of the residues R.sup.1, R.sup.2, R.sup.3, R.sup.4 may represent (CH.sub.2).sub.z—C.sub.6H.sub.4—NH.sub.2, COC.sub.6H.sub.4—NH.sub.2, CONHC.sub.6H.sub.4—NH.sub.2 or CSNHC.sub.6H.sub.4—NH.sub.2 with z=1-4, and C.sub.6H.sub.4 being a 1,2-, 1,3- or 1,4-phenylene, COC.sub.5H.sub.3N—NH.sub.2 or (CH.sub.2).sub.s—C.sub.5H.sub.3N—NH.sub.2, with z=1-4, and C.sub.5H.sub.3N being pyridin-2,4-diyl, pyridin-2,5-diyl, pyridin-2,6-diyl, or pyridin-3,5-diyl; additionally, R.sup.1-R.sup.2 (R.sup.3-R.sup.4) may form a four-, five, six-, or seven-membered cycle (comprising the nitrogen atom); which heterocycle may be unsubstituted or substituted with a straight or branched C.sub.2-C.sub.6 alkyl group, which is unsubstituted, substituted with 1 to 3 halogen atoms or incorporates 1 to 3 heteroatoms selected from O, S, and N, or which is optionally substituted with solubilizing groups selected from OH, NH.sub.2, SH, and COOH) and/or ionizable anionic residues selected from (alkyl) carboxy (CH.sub.2).sub.nCO.sub.2H, (alkyl) sulfonate (CH.sub.2).sub.nSO.sub.3H, (alkyl) sulfate (CH.sub.2).sub.nOSO.sub.3H, (alkyl) phosphate (CH.sub.2)nOP(O)(OH).sub.2 and (alkyl) phosphonate (CH.sub.2).sub.nP(O)(OH).sub.2, wherein n is an integer ranging from 0 zero to 6, or analogs thereof wherein one or more of the CH.sub.2 groups are replaced by CF.sub.2 and/or CD.sub.2; or R.sup.1-R.sup.2 (R.sup.3-R.sup.4) may form a four-, five, six-, or seven-membered cycle (comprising the nitrogen atom) with a hydroxyl group OH, or a phosphorylated hydroxyl group OP(O)(OH).sub.2 attached to one of the carbon atoms in this cycle, a primary amino group NH.sub.2, secondary amino group NHR.sup.a (where R.sup.a═C.sub.1-C.sub.6 alkyl of functionally substituted alkyl group), or the combination of such groups; with the proviso that in all compounds of Formula A 1 2, 3, 4, 5 or 6 negatively charged groups are present under basic conditions, i.e. 7<pH<14, and these negatively charged groups represent at least partially deprotonated residues of ionizable groups selected from the following set: OH, SH, COOH, a sulfonic acid residue SO.sub.3H, a sulfate residue OSO.sub.3H, an N-cyanosulfonamide residue SO.sub.2NHCN, a phosphate group OP(O)(OH).sub.2, and a phosphonate group P(O)(OH).sub.2; the linker L (connecting the dye core with the group X) comprises at least one carbon atom and can be represented by alkyl, alkyloxy, CH.sub.2OCH.sub.2, CH.sub.2CH.sub.2O, CH.sub.2CH.sub.2OCH.sub.2, difluoromethyl (CF.sub.2), alkene or alkyne moieties in any combinations, at any occurrence, linear or branched, with the length ranging from C.sub.1 to C.sub.12, which allow further connection to a solubilizing and/or ionizable moieties X, or incorporation of heteroatoms selected from O, N, and S; into such (a) linker(s); in particular, the linker L may consist of alkanediyl (C.sub.2-C.sub.12), heterosubstituted alkanediyl containing heteroatoms selected from O, N and/or S in any combination, or alkyloxyalkyl (CH.sub.2).sub.nO(CH.sub.2).sub.m, alkylthiaalkyl (CH.sub.2).sub.nS(CH.sub.2).sub.m, or alkylazaalkyl (CH.sub.2).sub.nNR(CH.sub.2).sub.m, where n=1-6, m=1-6 (independent from each other) and R═H or (functionally substituted) alkyl (C.sub.1-C.sub.4); further on, the linker L can be represented by a divalent residue derived from dialkylamine, diethanolamine or N-alkylmonoethanolamine moieties such as —N(CH.sub.3)CH.sub.2CH.sub.2O- and N(CH.sub.2CH.sub.2O-).sub.2, difluoromethyl (CF.sub.2), alkene or alkyne moieties in any combinations, at any occurrence, linear or branched, with the number of C-atoms ranging from C.sub.1 to C.sub.12; additionally, the linker L may also include a carbonyl (>CO) moiety, the group CF.sub.2CO, one or several CD.sub.2 groups and/or terminal CD.sub.3 groups; wherein X denotes a solubilizing and/or ionizable anion-providing moiety selected from 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).sub.nOSO.sub.3H, alkyl phosphate (CH.sub.2).sub.nOP(O)(OH).sub.2 or phosphonate (CH.sub.2).sub.nP(O)(OH).sub.2, wherein n is an integer ranging from 0 to 12 or 1 to 12, or an analog thereof wherein one or more of the CH.sub.2 groups are replaced by CF.sub.2 and/or CD.sub.2; with the proviso that in all compounds of Formula B 1 2, 3, 4, 5 or 6 negatively charged groups are present under basic conditions, i.e. 7<pH<14, and these negatively charged groups represent at least partially deprotonated residues of ionizable groups selected from hydroxyl OH, thiol SH, carboxyl COOH, a sulfonic acid residue SO.sub.3H, a sulfate residue OSO.sub.3H, an N-cyanosulfonyl group SO.sub.2NHCN, a phosphate group OP(O)(OH).sub.2, and a phosphonate group P(O)(OH).sub.2; ##STR00071## wherein R.sup.1, R.sup.2 are independent from each other and selected from the group consisting of a) hydrogen; b) CH.sub.3, C.sub.2Hs, a straight or branched C.sub.3-C.sub.6, alkyl, deuterated alkyl, or perfluoroalkyl group; c) straight or branched C.sub.2-C.sub.6 alkyl group that is unsubstituted, substituted with 1 to 3 halogen atoms, or incorporates 1 to 3 heteroatoms selected from O, S, and N, or optionally substituted with solubilizing groups selected from OH, NH.sub.2, SH, and COOH and/or ionizable anionic residues selected from the group consisting of (alkyl) carboxy (CH.sub.2).sub.nCO.sub.2H, (alkyl) sulfonate (CH.sub.2).sub.nSO.sub.3H, (alkyl) sulfate (CH.sub.2).sub.nOSO.sub.3H, (alkyl) phosphate (CH.sub.2).sub.nOP(O)(OH).sub.2 and (alkyl) phosphonate (CH.sub.2).sub.nP(O)(OH).sub.2, wherein n is an integer ranging from 0 to 6, or analogs thereof wherein one or more of the CH.sub.2 groups are replaced by CF.sub.2 and/or CD.sub.2 residues; d) (CH.sub.2).sub.nCOOR.sup.7, where n=1-6, and R.sup.7 may be alkyl, CH.sub.2CN, CH.sub.2CF.sub.3, benzyl, substituted benzyl, CH.sub.2CF.sub.3, polyhaloalkyl, polyhalophenyl, 2- and 4-nitrophenyl, N-succinimidyl, sulfo-N-succinimidyl, 1-benzotriazolyl, 9-aza-1-benzotriazolyl or other groups forming an active ester of a carboxylic acid; e) a carbonate or carbamate derivative (CH.sub.2).sub.mOCOOR.sup.8 or (CH.sub.2).sub.mNHCOOR.sup.8, where m=1-6 and R.sup.8═C.sub.1-C alkyl, benzyl, 9-fluorenylmethyl, CH.sub.2CN, N-succinimidyl, sulfo-N-succinimidyl, 1-benzotriazolyl, phenyl, substituted phenyl group, 2-pyridyl, 4-pyridyl, or pyrimid-4-yl; f) (CH.sub.2).sub.mNR.sup.cR.sup.d, where m=1-6, with a straight or branched alkyl chain; R.sup.c, R.sup.d are independent from each other and represent hydrogen and/or C.sub.1-C.sub.4 alkyl groups, a hydroxyalkyl group (CH.sub.2).sub.mOH, where m=2-6, with a straight or branched alkyl chain, a phosphorylated hydroxyalkyl group (CH.sub.2).sub.mOP(O)(OH).sub.2, where m=2-6, with a straight or branched alkyl chain; g) an alkyl azide (CH.sub.2).sub.mN.sub.3, where m=1-8, with a straight or branched alkyl chain; h) an alkylalkyne (CH.sub.2)C≡CR.sup.9, where r=1-8, with a straight or branched alkyl chain in (CH.sub.2).sub.r and R.sup.9=hydrogen, alkyl C.sub.1-C.sub.6 or (functionally) substituted alkyl C.sub.1-C.sub.6. i) (CH.sub.2).sub.sCONHR.sup.10, with s=1-8, R.sup.10═H, C.sub.1-C.sub.6 alkyl, (CH.sub.2).sub.qN.sub.3, (CH.sub.2).sub.rC≡CH or (CH.sub.2).sub.rN-maleimido, (CH.sub.2).sub.n—NH—COCH.sub.2X with X═Br or I, where q, r, t, u indices are independent in the range 1-8, and with straight or branched alkyl chains in (CH.sub.2)s, (CH.sub.2).sub.q, (CH.sub.2).sub.r, (CH.sub.2)t, (CH.sub.2).sub.u and R.sup.10. j) a primary amino group (NH.sub.2) or secondary amino group (NHR.sup.11); R.sup.11=alkyl C.sub.1-C.sub.6 or (functionally) substituted alkyl C.sub.1-C.sub.6; R.sup.1, R.sup.2 may contain a terminal alkyloxyamino group (CH.sub.2).sub.vONH.sub.2, where v=1-12, with a straight or branched alkyl chain that can include one or multiple (alkyl)oxy (CH.sub.2).sub.kO, (alkyl)amino (CH.sub.2).sub.lNH or (alkyl)amido (CH.sub.2).sub.wCONH groups in all possible combinations with indices k, 1 and w=0-12 independent from each other); R.sup.1, R.sup.2 may contain a terminal acylhydrazino group (CH.sub.2).sub.xCONHNH.sub.2, where x=1-12, with a straight or branched alkyl chain that can include one or multiple (alkyl)oxy (CH.sub.2).sub.kO, (alkyl)amino (CH.sub.2).sub.lNH or (alkyl)amido (CH.sub.2).sub.wCONH groups in all possible combinations with indices k, 1 and w=0-12 independent from each other; one of the residues R.sup.1, R.sup.2 or both residues R.sup.1 and R.sup.2 may represent C(O)R.sup.12 or COOR.sup.13, where R.sup.12 or R.sup.13═C.sub.1-C.sub.4 alkyl, optionally substituted with a hydroxyalkyl group (CH.sub.2).sub.yOH, where y=1-6, with a straight or branched alkyl chain, a phosphorylated hydroxyalkyl group (CH.sub.2).sub.mOP(O)(OH).sub.2, where m=1-6, with a straight or branched alkyl chain; further, one of the residues R.sup.1, R.sup.2 may represent (CH.sub.2).sub.z—C.sub.6H.sub.4—NH.sub.2, COC.sub.6H.sub.4—NH.sub.2, CONHC.sub.6H.sub.4—NH.sub.2 or CSNHC.sub.6H.sub.4—NH.sub.2 with z=1-4, and C.sub.6H.sub.4 being a 1,2-, 1,3- or 1,4-phenylene, COC.sub.5H.sub.3N—NH.sub.2 or (CH.sub.2)S-C.sub.5H.sub.3N—NH.sub.2, with z=1-4, and C.sub.5H.sub.3N being pyridin-2,4-diyl, pyridin-2,5-diyl, pyridin-2,6-diyl, or pyridin-3,5-diyl; additionally, R.sup.1-R.sup.2 may form a four-, five, six-, or seven-membered cycle (comprising the nitrogen atom); which (hetero)cycle may be unsubstituted or substituted with a straight or branched C.sub.2-C.sub.6 alkyl group, which is unsubstituted, substituted with 1 to 3 halogen atoms or incorporates 1 to 3 heteroatoms selected from O, S, and N, or which is optionally substituted with solubilizing groups selected from OH, NH.sub.2, SH, and COOH and/or ionizable anionic residues, selected from (alkyl) carboxy (CH.sub.2).sub.nCO.sub.2H, (alkyl) sulfonate (CH.sub.2).sub.nSO.sub.3H, (alkyl) sulfate (CH.sub.2).sub.nOSO.sub.3H, (alkyl) phosphate (CH.sub.2).sub.nOP(O)(OH)2 and (alkyl) phosphonate (CH.sub.2).sub.nP(O)(OH).sub.2, wherein n is an integer ranging from 0 to 6, or analogs thereof wherein one or more of the CH.sub.2 groups are replaced by CF.sub.2 and/or CD.sub.2; or R.sup.1-R.sup.2 may form a four-, five, six-, or seven-membered cycle (comprising the nitrogen atom) decorated with a hydroxyl group OH, or a phosphorylated hydroxyl group OP(O)(OH).sub.2 attached to one of the carbon atoms in this cycle, a primary amino group NH.sub.2, secondary amino group NHR.sup.3 (where R.sup.a═C.sub.1-C.sub.6 alkyl of functionally substituted alkyl group), or the combination of such groups; with the proviso that in all compounds of Formulae C, D, E at least 3 negatively charged groups are present under basic conditions, i.e. 7<pH<14, and these negatively charged groups represent at least partially deprotonated residues of ionizable groups selected from the following set: OH, SH, COOH, a sulfonic acid residue SO.sub.3H, a sulfate residue OSO.sub.3H, an N-cyanosulfonamide residue SO.sub.2NHCN, a phosphate group OP(O)(OH).sub.2, and a phosphonate group P(O)(OH).sub.2; R.sup.14═H or primary phosphate group (P(O)(OH).sub.2).

    2. The fluorescent dye according to claim 1, wherein the analyte-reactive group at variable positions R.sup.1, R.sup.2 R.sup.3, R.sup.4, R.sup.5, R.sup.6 in Formula A or the analyte-reactive group at variable positions R.sup.1, R.sup.2, R.sup.3, R.sup.4 in Formula B may be represented by an aromatic or heterocyclic amine, carboxylic acid, ester of the carboxylic acid; or represented by alkyl azide (CH.sub.2).sub.nN.sub.3, alkyne, strained cyclic alkene, strained cyclic alkyne, amino (NH.sub.2), amino-oxyalkyl (CH.sub.2).sub.mONH.sub.2, maleimido or halogeno ketone function COCH.sub.2X with X selected from ═Cl, Br and I, as well as halogeno amide group NRCOCH.sub.2X, with R═H, C.sub.1-C.sub.6-alkyl, X═Cl, Br, I) connected either directly or indirectly via alkandiyl —(CH.sub.2).sub.p—, carbonyl (>CO), amido (—CONR.sup.a—), nitrogen (—NR.sup.b—), oxygen (—O—) or sulfur-containing linkers (—S—, >SO, >SO.sub.2)n R, R.sup.a, R.sup.b are independent from each other and represent H, C.sub.1-C.sub.6 alkyl group, or (functionally substituted) C.sub.1-C.sub.6 alkyl group; n, m, p=1-8 (independent from each other).

    3. The fluorescent dye according to claim 1, wherein the aryl amino groups (NR.sup.1R.sup.2 and/or NR.sup.3R.sup.4) in Formula A or B are connected to an analyte-reactive group via (poly)methylene, poly(ethylene glycol), carbonyl, nitrogen or sulfur-containing linear or branched linkers their combinations, or linked as a part of nitrogen-containing non-aromatic heterocycles or alternatively the aryl amino groups (NR.sup.1R.sup.2 and/or NR.sup.3R.sup.4) in Formula A or B are connected to an acyl hydrazine or alkyl hydrazine moiety indirectly, via linkers, thus comprising hydrazides (ZCONHNH.sub.2) or hydrazines (ZNHNH.sub.2), respectively, wherein Z denotes the dye residue of Formula A or B that includes aryl amino groups and linkers; R.sup.1 and R.sup.3 may be represented by: (CH.sub.2).sub.mCONR, CO(CH.sub.2).sub.nNR (R═H, low (substituted) alkyl), CO(CH.sub.2).sub.pS(CH.sub.2).sub.n, (CH.sub.2).sub.qS(CH.sub.2).sub.rCO, CO(CH.sub.2).sub.sSO.sub.2(CH.sub.2).sub.t, (CH.sub.2).sub.uSO.sub.2(CH.sub.2).sub.wCO and their combinations; m, n, p, q, r, s, t, u, w are independent integers ranging from 0 to 12; linkers may also be represented by non-aromatic O, N and S-containing heterocycles.

    4. The fluorescent dye according to claim 1 of one of general Formulae C-E wherein R.sup.1, R.sup.2 are independent from each other and selected from the group consisting of a) hydrogen; b) CH.sub.3, C.sub.2H.sub.5, a straight or branched C.sub.3-C.sub.6 group; c) aminooxyalkyl H.sub.2NO(CH.sub.2).sub.n, wherein n is an integer ranging from 2 to 6, or analogs thereof wherein one or more of the CH.sub.2 groups are replaced by CF.sub.2 and/or CD.sub.2 residues; d) (CH.sub.2).sub.mCOOR.sup.7, where m=1-6, and R.sub.7 may be H, CH.sub.2CN, CH.sub.2CF.sub.3, CF.sub.3, polyhaloalkyl, polyhalophenyl, e.g. tetra- or pentafluorophenyl, pentachlorophenyl, 2- and 4-nitrophenyl, N-succinimidyl, sulfo-N-succinimidyl, 1-benzotriazolyl, 9-aza-1-benzotriazolyl or other groups forming an active ester of a carboxylic acid; e) a carbonate or carbamate derivative (CH.sub.2).sub.mOCOOR.sup.8 or (CH.sub.2).sub.mNHCOOR.sup.8, where m=1-6 and R.sup.8═CH.sub.2CN, N-succinimidyl, sulfo-N-succinimidyl, 1-benzotriazolyl, phenyl, substituted phenyl group, e.g., 2- or 4-nitrophenyl, pentachlorophenyl, pentafluorophenyl, 2,3,5,6-tetrafluorophenyl, 2-pyridyl, 4-pyridyl, pyrimid-4-yl; f) an alkyl azide (CH.sub.2).sub.mN.sub.3, where m=1-8, with a straight or branched alkyl chain; and g) a terminal acylhydrazino group (CH.sub.2).sub.xCONHNH.sub.2, where x=1-12, with the proviso that in all compounds of Formulae C, D, E at least 3 negatively charged groups are present under basic conditions, i.e. 7<pH<14, and these negatively charged groups represent at least partially deprotonated residues of ionizable groups selected from the following set: OH, SH, COOH, a sulfonic acid residue SO.sub.3H, a sulfate residue OSO.sub.3H, an N-cyanosulfonamide residue SO.sub.2NHCN, a phosphate group OP(O)(OH).sub.2, and phosphonate group P(O)(OH).sub.2; R.sup.14═H or primary phosphate group (P(O)(OH).sub.2).

    5. The fluorescent dye according to claim 1 of Formulae A or B above which has a negative net charge q of −1, −3, −5, or higher; or of Formula C which has a net charge z of −3, or of Formulae D or E above which has a net charge z of 0, −3, or −6, in an aqueous medium at pH ranging from 7 to 13.

    6. The fluorescent dye according claim 1 of general Formula A above having one of the following formulae, including salts or protonated forms thereof: ##STR00072##

    7. The fluorescent dye according claim 1 of general Formula B having one of the following formulae, including salts or protonated forms thereof: ##STR00073##

    8. The fluorescent dye according claim 1 of one of general Formulae C-E having one of the following formulae, including salts or protonated forms thereof: ##STR00074##

    9. The fluorescent dye salt according to claim 1, comprising negatively charged N-cyanamidosulfonate 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 (with R.sup.1 and R.sup.2 as defined in claim 1 above) in the dye of formulae A-E with a counterion selected from anions of a strong mineral acid, an organic acid or a Lewis acid.

    10. A fluorescent dye or dye salt according to claim 1, wherein carbohydrate-reactive groups selected from the group consisting of hydrazine —N(R)NH.sub.2, hydroxylamine —N(R)OH and aminooxy —ONH.sub.2 groups with R being H, (C.sub.1-C.sub.6) alkyl, heteroalkyl; n, m=1-6), alkenyl, or (per)fluoroalkyl (C.sub.1-C.sub.6) are connected to the nitrogen site N(R.sup.1)R.sup.2 in the dye of formulae A or B via all types of linkers (groups R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 or R.sup.6 in structures A and B), or linkers L in formula B.

    11. A compound according to claim 1 and salts thereof for use in the reductive amination and in the condensation with reducing sugars, which are monomeric, oligomeric or polymeric carbohydrates possessing an aldehyde group in a free form or as hemiacetal, including glycans.

    12. A compound according to claim 1 and salts thereof for use as a fluorescent label for natural products, including amino acids, peptides, proteins, including primary and secondary antibodies, single-domain antibodies, 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, and selected from docetaxel, cabazitaxel, larotaxel, aminophalloidin, jasplakinolide and their modifications.

    13. A method of using the compounds according to claim 1 and salts thereof as fluorescent reagents for conjugation to analytes, wherein the conjugation comprises formation of at least one covalent chemical bond or at least one molecular complex with a chemical entity or substance selected from the group consisting of an amine, carboxylic acid, aldehyde, alcohol, aromatic compound, heterocycle, dye, amino acid, peptide, protein, carbohydrate, nucleic acid, toxin and lipid, followed by fluorescence detection, which can be used alone or combined with any other detection method, including mass-spectrometric detection.

    14. A carbohydrate-dye conjugate comprising fluorescent dyes according to claim 1, wherein the carbohydrate moiety is a reducing glycans.

    15. The carbohydrate-dye conjugate according to claim 14 which is selected from the following structures: ##STR00075## ##STR00076## wherein the carbohydrate moiety is a reducing glycan.

    16. A kit or composition comprising one or more of the dyes of claim 1.

    17. A method for obtaining of dyes' conjugates with reducing sugars based on a two-step procedure: 1) formation of the (protonated) imine intermediate (Schiff's base) prepared from the fluorescent dye, in particular selected from 8-aminopyrene-1,3,6-trisulfonic acid (APTS), the salt thereof, or any dye according to claim 1, and an unlabeled mixture of reducing mono-, di- and oligosaccharides with incremental addition of monomeric units and stepwise increasing molecular masses, and 2) reduction of the said intermediate.

    18. The method for obtaining of dyes' conjugates with reducing sugars according to claim 17 (step 1), in which the formation of the protonated imine intermediate (Schiff's base) is facilitated by removal of water and high-boiling solvent from the reaction solution containing: a) APTS, the salt thereof, or the dye, b) a mixture of mixture of reducing mono-, di- and oligosaccharides with incremental addition of monomeric units and stepwise increasing molecular masses; and c) acid with pKa=2-6.

    19. The method for obtaining of dyes' conjugates with reducing sugars according to claim 17 (step 1), in which a dye, an individual reducing sugar, or mixture of reducing mono-, di- and oligosaccharides with incremental addition of monomeric units and stepwise increasing molecular masses are dissolved in water, combined with an organic acid dissolved in a suitable solvent, incubated at elevated temperature in a range of 25-70° C. for 0.5-2 h, followed by removal of solvents under reduced pressure.

    20. The method according to claim 17, in which a dye is mixed with maltooligosaccharides with incremental addition of monomeric units and stepwise increasing molecular masses, and malonic acid in DMSO, followed by incubation at 25-70° C. for 0.5-2 h, removal of the solvents under reduced pressure, addition of a solution of 2-picoline-borane complex, incubation at 25-70° C. for 1-48 h, and, finally, isolation of the products.

    21. A method of characterizing a sample containing one or more glycans, the method comprising: a) providing a sample containing one or more glycans conjugated with a fluorescent dye “A” as analytes of unknown structure and quantity or concentration and a known quantity of a fluorescent reference glycan standard, wherein the reference glycan standard is labeled with a dye (“B”) according to Formulae A-E of claim 1; b) contacting the sample with a dye “B” according to Formulae A-E conjugated with known reference glycans that is different from the fluorescent dye “A” conjugated with one or more unknown glycans used in step (a); c) separating the sample by electrophoresis or HPLC; d) selective/separate detection of the analytes and reference glycans due to different emission signatures of dyes “A” and “B” and e) quantifying at least one glycan in the sample relative to the reference glycan standard.

    22. A method for spectral calibration of a fluorescence detector which comprises injecting a solution of a dye mixture containing 2 or more fluorescent dyes according to claim 1 into an electrophoresis device followed by separation and detection of light emitted by each component of the dye mixture by a CCD camera, diodes array detector or similar device providing the spectral resolution and separate detection of the emitted light.

    23. A method for spectral calibration of a fluorescence detector which comprises injecting a solution containing 2 or more of the carbohydrate-dye conjugate according to claim 14 as a dye mixture into an electrophoresis device followed by separation and detection of light emitted by each component of the dye mixture by a CCD camera, diodes array detector or similar device providing the spectral resolution and separate detection of the emitted light.

    24. The carbohydrate-dye conjugate according to claim 14, wherein the glycans are selected from mannose, N-acetylglucosamine and N-acetylgalactosamine residues, galactose, fucose, glucose, maltose and/or their oligomers, as well as sialic acids in various possible combinations.

    25. The carbohydrate-dye conjugate according to claim 15, wherein the glycans are selected from mannose, N-acetylglucosamine and N-acetylgalactosamine residues, galactose, fucose, glucose, maltose and/or their oligomers, as well as sialic acids in various possible combinations.

    26. The method for spectral calibration of a fluorescence detector according to claim 22, wherein the detector is a detector for detection of laser induced fluorescence (LIF) including a detector used in capillary gel electrophoresis with detection by laser induced fluorescence (CGE-LIF).

    Description

    BRIEF DESCRIPTION OF THE FIGURES

    [0190] FIG. 1 shows the absorption and emission (λ.sub.exc=450 nm) spectra of compounds 13 and 13-Glc in HEPES (25 mM, pH 8).

    [0191] FIG. 2 shows gel electrophoresis results for several dyes and their conjugates (migration from “north” to “south”, pH 8.3). Lanes I, III and VII are empty. Reading from left to right, the following samples were loaded on each lane (from bottom to top): lane II, APTS, and its conjugates with glucose, maltotriose, maltoheptaose; lane IV, VBDP, and its conjugates with glucose, maltotriose, maltoheptaose; lane V, compound 12, and its conjugates with glucose, maltotriose, maltoheptaose; lane VI, compound 13, and its conjugates with glucose, maltotriose, maltoheptaose. Bands were detected by emission (excitation at 365 nm).

    [0192] FIG. 3 shows gel electrophoresis results (migration from “north” to “south”, pH 8) observed using an Amersham Imager 600 (excitation at 460 nm, Cy2/Cy3 filter overlay) for various labelled oligosaccharides. The following samples were loaded on each lane (from bottom to top): lane I, 2 nmol of APTS-G; lane II, 2 nmol of APTS-G7; lane III, 5 nmol of APTS labelled dextran ladder; lane IV, 40 nmol of dextran ladder labelled with compound 13; lane V, 10 nmol of 13-G7; lane VI, 10 nmol of 13-G.

    [0193] FIG. 4 shows gel electrophoresis results (migration from “north” to “south”, pH 8.3) for various dyes and labelled oligosaccharides. Right (reference) lane: free dye 40 (FIG. 20). Middle lane: APTS-conjugates prepared from the “dextran ladder” (partially hydrolyzed dextran) and APTS dye (FIG. 10); from the bottom up: glucose (G), maltose (G.sub.2), maltotriose (G.sub.3), etc. up to G.sub.14. Left lane: dye 40 (lowest band) and its conjugates obtained from the same “dextran ladder” (middle lane). Bands were detected by emission.

    [0194] FIG. 5 is a bar chart showing increasing information content by passing from genome (whole DNA) over the transcriptome (all RNAs) and the proteome (all proteins) to the glycome (all glycans). [J. E. Turnbull, R. A. Field, Nat. Chem. Biol. 2007, 3, 74-77.]

    [0195] FIG. 6 shows reductive amination of mono and oligosaccharides. If R.sup.3 incorporates the residue of a fluorescent dye, the final product—carbohydrate derivative R.sup.2CH.sub.2NHR.sup.3—is also fluorescent.

    [0196] FIG. 7 shows the structure and spectral properties of APTS and its N-alkylated derivatives in aqueous buffers (according to Z. Sharrett, S. Gamsey, L. Hirayama, B. Vilozny, J. T. Suri, R. A. Wessling, B. Singaram, Org. Biomol. Chem. 2009, 7, 1461-1470).

    [0197] FIG. 8 shows UV-Vis spectra of free APTS (spectrum A) and APTS-maltohexaose (spectrum B). Both were measured as 15 μM solutions in 50 mM phosphate buffer at pH 7.0. The vertical dashed line at 488 nm marks the main wavelength of the argon-ion laser. Adapted from R. A. Evangelista, M.-S. Liu, F.-T. A. Chen, Anal. Chem. 1995, 67, 2239-2245. Copyright (1995) American Chemical Society.

    [0198] FIG. 9 shows model 7,9-diaminoacridines (6-11) with electron-acceptor cyano, alkylsulfonyl and sulfonamido groups at C-2.

    [0199] FIG. 10 show negatively charged 7,9-diaminoacridines (12, 13) with primary phosphate groups and reference compounds (APTS and acridone dye VBDP).

    [0200] FIG. 11 shows a synthesis of 2-bromo-7-nitro-9-phenoxyacridine (18).

    [0201] FIG. 12 shows synthesis of 2-amino-7-cyano-9-(6-hydroxyhexyl)aminoacridine (6).

    [0202] FIG. 13 shows synthesis of 7-amino-9-(3-hydroxypropyl)amino-2-[(3-hydroxypropyl)sulfonyl]-acridine 7.

    [0203] FIG. 14 shows synthesis of acridine-7-sulfonamides 8 and 9 with two amino groups in 2- and 9-positions.

    [0204] FIG. 15 shows synthesis of acridine-7-sulfonamides 10 and 11.

    [0205] FIG. 16 shows glucose conjugates prepared from dyes 6-7 and 9-11 via reductive amination.

    [0206] FIG. 17 shows synthesis of aminoacridine dye 12 decorated with two primary phosphate groups.

    [0207] FIG. 18 shows synthesis of aminoacridine-triphosphate (13) and acridone-diphosphate (39) dyes.

    [0208] FIG. 19 shows glycoconjugates prepared via reductive amination from the negatively charged amino-dyes and glucose oligomers.

    [0209] FIG. 20 shows conversion of APTS dye (trisodium salt) into 8-amino-1,3,6-tri[(cyanamido)sulfonyl]pyrene (40).

    [0210] FIG. 21 shows triple O-phosphorylated 1,6,8-tris[(3-hydroxy-trans cis-cyclobutyl)sulfonyl]-pyrene-3-amines 44: a) trans- or cis-3-mercaptocyclobutanol (see scheme), DIPEA, Pd.sub.2(dba).sub.3, XantPhos, DMF, 100° C., overnight; b) aq. H.sub.2O.sub.2, Na.sub.2WO.sub.4, AcOH, r. t., overnight; c) aq. NaOH, MeOH; d) POCl.sub.3, (MeO).sub.3PO, r. t., then aq. Et.sub.3N*H.sub.2CO.sub.3 buffer (pH 8-9); e) aq. Na2CO3.

    [0211] FIG. 22 shows synthesis of tris-O-phosphorylated 1-aminopyrene 52: a) di-tert-butyl N,N-diisopropylphosphoramidite, 1H-tetrazole, DMF, r. t., 1 h, then 30% H.sub.2O.sub.2, 0° C..fwdarw.r. t., 6 h; b) ClSO.sub.3H, 65° C., 3 h; c) H.sub.2, Pd/C, MeOH, r. t., overnight; d) Et.sub.3N, MeCN, r. t., 10 min; e) 3-(tert-butyldimethylsilyloxy)azetidine, Et.sub.3N, MeCN, r. t., 1 h; then 50-55% aq. HF, MeCN, 0° C..fwdarw.r. t., overnight; f) CF.sub.3CO.sub.2H, DCM, r. t., 1 h, then 1 M aq. Et.sub.3N*H.sub.2CO.sub.3 (pH 8-9), overnight.

    [0212] FIGS. 23, 24, 25, 26 and 27 show absorption and emission spectra.

    [0213] The following Examples illustrate the present invention in more detail.

    [0214] General Materials and Methods

    [0215] All commercially available chemicals (Acros, Aldrich, Fluka, Merck, Alfa Aesar, TCI) were used as received. The solvents had the purity “pro analysis”. For photophysical measurements solvents of spectrophotometric grade were used. Anhydrous solvents were stored over molecular sieves. Deuterated solvents were purchased from Deutero GmbH (CDCl.sub.3, CD.sub.3OD, D.sub.2O, DMF-d.sub.7, DMSO-d.sub.6, TFA-d). Reactions at “0° C.” were carried out using an ice-bath.

    [0216] TLC. Normal phase TLC was performed on silica gel 60 F.sub.254 (Merck Millipore). Compounds were detected by exposing TLC plates to UV-light (254 or 366 nm).

    [0217] Analytical HPLC. Analytical HPLC was performed on a Knauer Azura liquid chromatography system with a binary P 6.1 L pump (Article No. EPH35, Knauer), UV diode array detector DAD 6.1 L (Article No. ADC11, Knauer), an injection valve with a 20 μL loop and two electrical switching valves V 2.1 S with 6-port multiposition valve head (Article No. EWA10, Knauer). The UV detection was carried out with double wavelength pattern at wavelength channel 1 of 254 nm and wavelength channel 2 of 350 nm. The column temperature was not standardized, but remained at around 25° C.

    [0218] Analytical columns: Knauer Eurospher II 100-5 C18, 5 m, 150×4 mm (if not stated otherwise), or Knauer Eurospher II 100-5 C18A, 5 m, 150×4 mm, or Knauer Eurospher II 100-10 C18A, m, 150×4 mm, or Interchim Uptisphere Strategy C18-HQ, 10 μm, 250×4.6 mm.

    [0219] Flow rate: 1.2 mL/min.

    [0220] Phase A: water+0.1% v/v trifluoroacetic acid (TFA).

    [0221] Phase B: MeCN+0.1% v/v TFA.

    [0222] Method 20-100: 20B (3 min); 20-100B (12 min).

    [0223] Method 5-50: 5B (3 min); 5-50B (12 min); 50-100B (3 min).

    [0224] For isolation and purification of phosphorylated dyes, acetonitrile (phase B)—aqueous systems containing 0.05 M of TEAB buffer (phase A, pH=8; self-prepared from 1 M aq. Et.sub.3N and CO.sub.2 gas obtained by evaporation of solid CO.sub.2).

    [0225] Method TEAB-0-25: OB (3 min); 0-25B (12 min); 25-100B (3 min).

    [0226] Method TEAB-0-40: OB (3 min); 0-40B (12 min); 40-100B (3 min).

    [0227] FC. Flash chromatography was performed on an automated Biotage Isolera One or Interchim puriFlash™ flash purification system using the cartridges and solvent gradients indicated in the text.

    [0228] MS. Mass spectra with ESI Ion source were recorded by J. Bienert (Chemical Facility, Max Planck Institute for Biophysical Chemistry, Göttingen) using a Varian 500 MS (Agilent). High resolution mass spectra were obtained on a Bruker maXis (ESI-QTOF-HRMS) or Bruker Autoflex Speed (MALDI-TOF HRMS) spectrometer by the team of Dr. H. Frauendorf (Facility of Mass Spectrometry, Georg-August-Universitat Gottingen).

    [0229] NMR. NMR spectra were recorded on an Agilent 400MR DD2 spectrometer. All spectra are referenced to tetrametylsilane as an internal standard (6=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 J are given in Hz. For .sup.13C-signals, which were revealed by indirect detection by HSQC, only resonances of the carbon atoms linked to H-atoms were recorded.

    [0230] UV-Vis and fluorescence. Absorption spectra was recorded on a double-beam UV-Vis spectrophotometer (Varian, series 4000). Measurements were performed in 1-cm quartz cells or UV-Vis disposable cuvettes (BRAND semi-micro), and in air-equilibrated solutions at ambient temperature (24-25° C.). 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). Emission and UV-Vis scan spectra were recorded using following parameters: average time 0.1 s; data interval 1 nm; scan rate 600 nm/min; with base line correction.

    [0231] Gel electrophoresis. The gel contained 50 mL of 20% of acrylamide, which was made up in the standard TBE buffer (89 mM Tris, 89 mM borate, 2 mM EDTA) containing 7 M urea. Polymerization was catalyzed by the addition of 162.5 μL of a ammoniumpersulfate (APS, 25 wt. % solution in water) and 43.8 μL of N,N,N′,N′-tetramethylethyleendiamine (TEMED). The gels were of the 8- or 17-well format (width 20 cm) with 30 cm well-to-read length and 0.75 mm thickness. The running buffer was 89 mM Tris-borate pH 8.3 containing 2 mM EDTA. Electrophoresis was performed at a constant power of 35 W (Consort EV3330) at ambient temperature with forced air cooling, the front glass plate was equipped with an external aluminium plate. After pre-running the gel for 30 min, the wells were thoroughly rinsed with the TBE buffer, and appropriate volumes (30-50 μL, ca. 50% formamide) of the samples were loaded. Usually one gel lane was skipped between each two samples, to avoid cross-contamination and to ease the lane tracking process. The electrophoresis voltage during separation was 1700-2200 V and the analysis was run until APTS reached the bottom of the gel (1.5-2 h). The fluorescence of APTS- and acridine derivatized carbohydrates was readily resolved in a UV viewing cabinet (254/365 nm) equipped with a digital camera or using Amersham Imager 600.

    Example 1

    Synthesis of Fluorescent Acridone Dyes and their Precursors

    [0232] ##STR00014##

    [0233] 2-Nitroacridone was synthesized according to the literature (P. Graves and J. A. Smith. “Fluorogenic peptides and their method of production.” US 2011/0039289 A1, Mar. 14, 2008). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=12.33 (s, 1H), 8.95 (d, J=2.7 Hz, 1H), 8.45 (dd, J=9.4, 2.7 Hz, 1H), 8.22 (dd, J=8.2, 1.2 Hz, 1H), 7.80 (ddd, J=8.2, 7.0, 1.6 Hz, 1H), 7.65 (d, J=9.4 Hz, 1H), 7.58 (d, J=8.2 Hz, 1H), 7.36 (ddd, J=8.2, 7.0, 0.8 Hz, 1H) ppm.

    ##STR00015##

    [0234] 2-Bromo-7-nitroacridone was synthesized according to the literature (A. Kliegl, L. Schaible, Chem. Ber. 1957, 90, 60-65.). 1H NMR (400 MHz, DMSO-d.sub.6): δ=12.47 (s, 1H), 8.92 (d, J=2.7 Hz, 1H), 8.46 (dd, J=9.2, 2.7 Hz, 1H), 8.27 (d, J=2.3 Hz, 1H), 7.93 (dd, J=8.6, 2.3 Hz, 1H), 7.65 (d, J=9.2 Hz, 1H), 7.54 (d, J=8.6 Hz, 1H) ppm.

    ##STR00016##

    [0235] 2-Bromo-9-chloro-7-nitroacridine was synthesized according to the literature (K. Singh, G. Singh, Indian J. Pharm. 1952, 14, 47-49.). TLC (SiO.sub.2): R.sub.f=0.4 (DCM/MeOH 20:0.05). 1H NMR (400 MHz, CDCl.sub.3): δ=9.41 (d, J=2.7 Hz, 1H), 8.64 (d, J=2.0 Hz, 1H), 8.53 (dd, J=9.4, 2.7 Hz, 1H), 8.33 (d, J=9.4 Hz, 1H), 8.13 (d, J=9.0 Hz, 1H), 7.96 (dd, J=9.0, 2.0 Hz, 1H) ppm.

    ##STR00017##

    [0236] A mixture of 7-bromo-9-chloro-2-nitroacridine (76 mg, 225 μmol) and phenol (1 g) was stirred at 100° C. for 1.5 hours under Ar. The reaction mixture was allowed to cool down to rt, diluted with DCM and washed with 0.1 M NaOH to remove phenol. The organic phase was further washed with brine (until pH became neutral), dried (Na.sub.2SO.sub.4), and the solvent was removed in vacuo to give a yellow solid (85 mg, 95%). TLC (SiO.sub.2): R.sub.f=0.35 (DCM/MeOH 20:0.05). .sup.1H NMR (400 MHz, CDCl.sub.3): δ=9.05 (d, J=2.0 Hz, 1H), 8.50 (dd, J=9.4, 2.3 Hz, 1H), 8.34 (dd, J=9.4, 0.8 Hz, 1H), 8.27 (d, J=1.6 Hz, 1H), 8.15 (d, J=9.4 Hz, 1H), 7.92 (dd, J=9.4, 2.3 Hz, 1H), 7.32-7.38 (m, 2H), 7.12-7.19 (m, 1H), 6.86-6.91 (m, 2H) ppm. .sup.11C NMR (101 MHz, CDCl.sub.3): δ=159.2, 157.3, 151.0, 150.9, 145.2, 136.3, 131.9, 131.7, 130.4, 124.9, 123.8, 123.7, 121.6, 121.6, 120.8, 118.9, 115.7 ppm. HRMS: m/z 395.0022 ([M+H].sup.+) calculated for C.sub.19H.sub.12BrN.sub.2O.sub.3+: 395.0026 (Δ1.0 ppm).

    ##STR00018##

    [0237] A mixture of 7-bromo-2-nitro-9-phenoxyacridine (85 mg, 215 μmol) and 6-amino-1-hexanol (50 mg, 430 μmol) in dry DMF (5 mL) was stirred at rt for 1 hour under Ar. The reaction mixture was diluted with DCM (50 mL) and washed with 0.1 M NaOH to remove phenol. The organic phase was further washed with brine (until pH became neutral), dried (Na.sub.2SO.sub.4), and the solvent was removed in vacuo to give a red-orange powder (85 mg, 95%). TLC (SiO.sub.2): R.sub.f=0.19 (DCM/MeOH 20:1), R.sub.f=0.39 (DCM/MeOH 20:2). HPLC (20-100, Knauer Eurospher II 100-10 C18A): t.sub.R=8.7 min. ESI-MS: m/z 418.2 [M+H].sup.+. .sup.1H NMR (400 MHz, MeOD-d.sub.3 with TFA-d): δ=9.22-9.72 (m, 1H), 8.60-8.84 (m, 1H), 8.71 (dd, J=9.4, 2.3 Hz, 1H), 8.15 (dd, J=9.0, 2.0 Hz, 1H), 7.91 (d, J=9.4 Hz, 1H), 7.77 (d, J=9.0 Hz, 1H), 4.14-4.20 (m, 2H), 3.56-3.61 (m, 2H), 1.99-2.18 (m, 2H), 1.57-1.67 (m, 4H), 1.47-1.57 (m, 2H) ppm.

    ##STR00019##

    [0238] Compound 19 (127 mg, 304 μmol), Zn(CN).sub.2 (71 mg, 608 μmol) and Pd(PPh.sub.3).sub.4 (35 mg, 30 μmol) were placed in a heat dried Schlenk tube filled with Ar, which was evacuated-backfilled with Ar (3×). DMF (2.5 mL) was added and the reaction mixture was immediately heated up to 100° C. After 30 min of stirring at the same temperature, the reaction mixture was allowed to cool down to rt, diluted with DCM, washed with brine, dried (Na.sub.2SO.sub.4) and concentrated in vacuo. The residue was purified by FC (SiO.sub.2, RediSep Rf 24 g cartridge, dry-load, DCM/MeOH with 0.5-5% MeOH gradient) to give a red solid (96 mg, 87%). TLC (SiO.sub.2): R.sub.f=0.42 (DCM/MeOH 10:1). HPLC (20-100, Knauer Eurospher II 100-10 C18A): t.sub.R=7.0 min. ESI-MS: m/z 365.2 [M+H].sup.+. .sup.1H NMR (400 MHz, MeOD-d.sub.3 with TFA-d): δ=9.26-9.72 (m, 1H), 8.81-9.08 (m, 1H), 8.75 (dd, J=9.4, 2.0 Hz, 1H), 8.23 (dd, J=9.0, 1.6 Hz, 1H), 7.85-7.98 (m, 2H), 4.16-4.26 (m, 2H), 3.54-3.63 (m, 2H), 1.98-2.22 (m, 2H), 1.57-1.72 (m, 4H), 1.48-1.56 (m, 2H) ppm.

    ##STR00020##

    [0239] Compound 20 (64 mg, 176 μmol) and SnCl.sub.2×2 H.sub.2O (199 mg, 880 μmol) were placed in a Schlenk flask, which was evacuated-backfilled with Ar. Absolute EtOH (0.5 mL) was added and the reaction mixture was immediately heated up to 70° C. After 15 min of stirring at the same temperature, the reaction mixture was allowed to cool down to rt, diluted with 18 mL of 0.1% aq. TFA and purified by FC (C18, 30C18AQ-F0025 cartridge, H.sub.2O/ACN (with 0.1% TFA) with 0-50% ACN gradient). Good fractions were pooled, concentrated in vacuo, and the excess of TFA was removed by filtration through C18 column using H.sub.2O-ACN as eluents. The resulting orange filtrate was diluted with H.sub.2O and lyophilized to give a red-orange powder (48 mg, 48%, TFA salt). HPLC (Knauer Eurospher II 100-10 C18A): t.sub.R=4.8 min (20-100); t.sub.R=10.7 min (5-50). ESI-MS: m/z 335.4 [M+H].sup.+. HRMS: m/z 335.1867 ([M+H].sup.+) calculated for C.sub.20H.sub.23N.sub.4O.sup.+: 335.1866 (Δ0.3 ppm). .sup.1H NMR (400 MHz, D.sub.2O): δ=8.29 (s, 1H), 7.82 (br d, J=8.6 Hz, 1H), 7.47 (d, J=9.0 Hz, 1H), 7.32-7.42 (m, 2H), 7.10 (s, 1H), 3.81 (br dd, J=7.4, 7.0 Hz, 2H), 3.56 (dd, J=6.7, 6.3 Hz, 2H), 1.69-1.89 (m, 2H), 1.46-1.60 (m, 2H), 1.28-1.45 (m, 4H) ppm. The following signals were visible in the .sup.13C NMR (100 MHz, D.sub.2O/ACN-d.sub.3, HSQC) spectra of 6: 6=134.3 (CH), 124.8, 120.1 (CH), 119.7 (CH), 118.7, 61.6 (CH.sub.2), 48.9 (CH.sub.2), 31.3 (CH.sub.2), 29.0 (CH.sub.2), 25.8 (CH.sub.2), 24.8 (CH.sub.2) ppm. .sup.19F NMR (376 MHz, D.sub.2O): δ=−75.53 ppm.

    ##STR00021##

    [0240] A mixture of 7-bromo-2-nitro-9-phenoxyacridine (500 mg, 1.27 mmol) and 3-amino-1-propanol (191 mg, 2.54 mmol) in dry DMF (15 mL) was stirred at rt for 1 hour under Ar. The reaction mixture was diluted with DCM-MeOH (10:1, 500 mL) and washed with 0.1 M NaOH to remove phenol. MeOH was added to the organic phase whenever precipitates were formed. The organic phase was further washed with brine (until pH became neutral), dried (Na.sub.2SO.sub.4), and the solvent was removed in vacuo to give a red powder (440 mg, 92%). TLC (SiO.sub.2): R.sub.f=0.18 (DCM/MeOH 20:1); R.sub.f=0.46 (DCM/MeOH 10:1); R.sub.f=0.55 (DCM/MeOH/H.sub.2O 90:10:1). HPLC (20-100): t.sub.R=7.2 min; λ.sub.max=442, 423, 363, 313, 271, 251 nm. ESI-MS: m/z 376.0 [M+H].sup.+. HRMS: m/z 376.0291 ([M+H].sup.+) calculated for C.sub.16H.sub.15BrN.sub.3O.sub.3: 376.0291 (Δ0.0 ppm). .sup.1H NMR (400 MHz, MeOD-d.sub.3 with TFA-d): δ=9.45 (d, J=2.0 Hz, 1H), 8.64-8.75 (m, 2H), 8.13 (dd, J=9.0, 2.0 Hz, 1H), 7.92 (d, J=9.0 Hz, 1H), 7.78 (d, J=9.0 Hz, 1H), 4.39 (t, J=6.6 Hz, 2H), 3.91 (t, J=5.9, 5.5 Hz, 2H), 2.23 (quin., J=6.6, 5.9, 5.5 Hz, 2H) ppm.

    ##STR00022##

    [0241] Compound 21 (50 mg, 133 μmol), Pd.sub.2(dba).sub.3 (7.4 mg, 8 μmol) and Xantphos (8.9 mg, 15 μmol) were placed in a heat dried Schlenk tube filled with Ar, which was evacuated-backfilled with Ar (3×). DMF (1.3 mL), DIPEA (46 μL, 266 μmol) and 3-mercapto-1-propanol (15 mg, 160 μmol) were added and the reaction mixture was immediately heated up to 100° C. After 1 h of stirring at the same temperature, the reaction mixture was allowed to cool down to rt, diluted with DCM-MeOH (10:1), washed with 10% aq. Na.sub.2SO.sub.3— aq. sat. NaHCO.sub.3 (1:1) and brine, dried (Na.sub.2SO.sub.4) and concentrated in vacuo. The residue was purified by FC (SiO.sub.2, RediSep R.sub.f 24 g cartridge, dry-load, DCM/MeOH with 0-15% MeOH gradient) to give a dark-red solid (46 mg, 89%). TLC (SiO.sub.2): R.sub.f=0.24 (DCM/MeOH 10:1); R.sub.f=0.3 (DCM/MeOH/H.sub.2O 90:10:1). HPLC: t.sub.R=7.0 min (20-100); t.sub.R=12.1 min (5-50); λ.sub.max=442, 345, 273 nm. ESI-MS: m/z 388.0 [M+H].sup.+. HRMS: m/z 388.1327 ([M+H].sup.+) calculated for C.sub.19H.sub.22N.sub.3O.sub.4S.sup.+: 388.1326 (Δ0.3 ppm). .sup.1H NMR (400 MHz, CDCl.sub.3/MeOD-d.sub.3 7:3): δ=9.29 (d, J=2.3 Hz, 1H), 8.26 (dd, J=9.4, 2.3 Hz, 1H), 7.99 (d, J=2.0 Hz, 1H), 7.80 (d, J=9.4 Hz, 1H), 7.76 (d, J=9.0 Hz, 1H), 7.61 (dd, J=9.0, 2.0 Hz, 1H), 4.19 (t, J=6.3, 5.9 Hz, 2H), 3.93 (t, J=5.5, 5.1 Hz, 2H), 3.69 (t, J=5.9 Hz, 2H), 3.10 (t, J=7.4, 7.0 Hz, 2H), 2.04-2.11 (m, 2H), 1.82-1.97 (m, 2H) ppm.

    ##STR00023##

    [0242] Compound 22 (110 mg, 284 μmol) was suspended in H.sub.2O (6 mL). Oxone (166 mg, 270 μmol) was added in small portions. The suspension changed from red to yellow color. After 30 min of stirring at rt, the reaction mixture was diluted with 10% aq. Na.sub.2SO.sub.3— aq. sat. NaHCO.sub.3 (1:1), and the product was extracted into EtOAc (containing small amounts of MeOH). The organic phase was washed with brine, dried (Na.sub.2SO.sub.4) and concentrated in vacuo. The residue was purified by FC (SiO.sub.2, RediSep R.sub.f 24 g cartridge, DCM/MeOH/H.sub.2O 90:10:1) to give a light-orange solid (60 mg, 50%). TLC (SiO.sub.2): R.sub.f=0.3 (DCM/MeOH/H.sub.2O 90:10:1); R.sub.f=0.1 (DCM/MeOH 20:1). HPLC: t.sub.R=10.3 min (5-50); λ.sub.max=426, 408, 357, 303, 266 nm. ESI-MS: m/z 420.2 [M+H].sup.+. HRMS: m/z 420.1227 ([M+H].sup.+) calculated for C.sub.19H.sub.22N.sub.3O.sub.6S.sup.+: 420.1224 (Δ0.7 ppm). .sup.1H NMR (400 MHz, MeOD-d.sub.3 with TFA-d): δ=9.39-9.57 (m, 1H), 9.04 (d, J=1.6 Hz, 1H), 8.76 (dd, J=9.4, 1.6 Hz, 1H), 8.43 (dd, J=9.0, 1.6 Hz, 1H), 7.99 (d, J=9.0 Hz, 1H), 7.95 (d, J=9.4 Hz, 1H), 4.33-4.53 (m, 2H), 3.86-3.99 (m, 2H), 3.63 (t, J=6.3 Hz, 2H), 3.39-3.47 (m, 2H), 2.26 (quin., J=6.7, 6.3, 5.9, 5.5 Hz, 2H), 1.88-1.98 (m, 2H) ppm.

    ##STR00024##

    [0243] Compound 23 (15 mg, 36 μmol) was dissolved in MeOH (10 mL) and AcOH (10 μL) under Ar. A 10% Pd/C catalyst (7 mg) was added, and hydrogen atmosphere was applied. After 1 hour of stirring at rt, orange fluorescent solution was filtered through a pad of Celite, and concentrated in vacuo. The residue was dissolved in 10 mL of H.sub.2O and purified by FC (C18, 30C18AQ-F0025 cartridge, ACN/10 mM TEAAc pH 7.0) to give a red solid (3.4 mg, 22%, AcOH salt). HPLC: t.sub.R=9.0 min (5-50); λ.sub.max=428, 408, 301, 273, 227 nm. HRMS: m/z 390.1488 ([M+H].sup.+) calculated for C.sub.19H.sub.24N.sub.3O.sub.4S.sup.+: 390.1482 (Δ1.5 ppm). .sup.1H NMR (400 MHz, D.sub.2O/MeCN-d.sub.3 6:1): δ=8.69 (d, J=2.0 Hz, 1H), 8.07 (dd, J=9.0, 1.6 Hz, 1H), 7.78 (d, J=9.0 Hz, 1H), 7.56 (d, J=9.0 Hz, 1H), 7.45 (dd, J=9.0, 2.0 Hz, 1H), 7.24 (d, J=1.6 Hz, 1H), 4.09 (t, J=7.0 Hz, 2H), 3.91 (t, J=5.9 Hz, 2H), 3.73 (t, J=6.3 Hz, 2H), 3.51-3.63 (m, 2H), 2.17 (quin, J=6.3 Hz, 2H), 2.00-2.07 (m, 2H), 1.99 (s, AcOH) ppm.

    ##STR00025##

    [0244] A mixture of 2-bromo-7-nitro-9-phenoxyacridine (100 mg, 253 μmol) and 25% aq. NH.sub.3 (75 μL, 1 mmol) in DMF (5 mL) was stirred at rt for 1 hour. The reaction mixture was diluted with DCM-MeOH (10:1) and washed with brine (until pH became neutral, MeOH was added to the organic phase whenever precipitates were formed), dried (Na.sub.2SO.sub.4) and concentrated in vacuo. The red residue was purified by FC (SiO.sub.2, RediSep R.sub.f 24 g cartridge, dry-load, DCM—DCM/MeOH/H.sub.2O 90:10:1) to give a red solid (67 mg, 83%). HPLC: t.sub.R=7.2 min (20-100); λ.sub.max=435, 415, 357, 311, 265, 250 nm. ESI-MS: m/z 318.1 [M+H].sup.+. .sup.1H NMR (400 MHz, MeOD-d.sub.3 with TFA-d): δ=9.64 (d, J=2.3 Hz, 1H), 8.82 (d, J=2.0 Hz, 1H), 8.72 (dd, J=9.4, 2.3 Hz, 1H), 8.17 (dd, J=9.0, 2.0 Hz, 1H), 7.96 (d, J=9.4 Hz, 1H), 7.81 (d, J=9.0 Hz, 1H) ppm. .sup.3C NMR (101 MHz, MeOD-d.sub.3 with TFA-d): δ=159.4, 143.5, 142.2, 139.6, 138.8, 129.0, 126.8, 122.1, 120.8, 120.2, 118.2, 113.7, 111.0 ppm.

    ##STR00026##

    [0245] Compound 24 (24 mg, 75 μmol), Pd.sub.2(dba).sub.3 (4 mg, 4 μmol) and Xantphos (5 mg, 8 μmol) were placed in a heat dried Schlenk tube filled with Ar, which was evacuated-backfilled with Ar (3×). DMF (1 mL), DIPEA (26 μL, 150 μmol) and p-methoxybenzylmercaptane (15 mg, 90 μmol) were added and the reaction mixture was immediately heated up to 100° C. After 2 h of stirring at the same temperature, the reaction mixture was allowed to cool down to rt, diluted with DCM-MeOH (10:1), washed with 10% aq. Na.sub.2SO.sub.3— aq. sat. NaHCO.sub.3 (1:1) and brine, dried (Na.sub.2SO.sub.4) and concentrated in vacuo. The residue was purified by FC (SiO.sub.2, RediSep R.sub.f 24 g cartridge, dry-load, DCM—DCM/MeOH/H.sub.2O 90:10:1) to give a red solid (23 mg, 79%). HPLC: t.sub.R=9.5 min (20-100); λ.sub.max=434, 341, 273 nm. ESI-MS: m/z 392.3 [M+H].sup.+. .sup.1H NMR (400 MHz, MeOD-d.sub.3 with TFA-d): δ=9.66 (d, J=2.3 Hz, 1H), 8.71 (dd, J=9.4, 2.3 Hz, 1H), 8.46 (d, J=2.0 Hz, 1H), 8.01 (dd, J=9.0, 2.0 Hz, 1H), 7.93 (d, J=9.4 Hz, 1H), 7.78 (d, J=9.0 Hz, 1H), 7.24-7.30 (m, 2H), 6.79-6.85 (m, 2H), 4.32 (s, 2H), 3.74 (s, 3H) ppm.

    ##STR00027##

    [0246] Compound 21 (290 mg, 0.77 mmol), Pd.sub.2(dba).sub.3 (37 mg, 0.04 mmol) and Xantphos (46 mg, 0.08 mmol) were placed in a heat dried Schlenk tube filled with Ar, which was evacuated-backfilled with Ar (3×). DMF (7.7 mL), DIPEA (268 μL, 1.54 mmol) and PMBSH (142 μg, 0.92 mmol) were added and the reaction mixture was immediately heated up to 100° C. After 2 h of stirring at the same temperature, the reaction mixture was allowed to cool down to rt, diluted with DCM-MeOH (10:1), washed with 10% aq. Na.sub.2SO.sub.3— aq. sat. NaHCO.sub.3 (1:1) and brine, dried (Na.sub.2SO.sub.4) and concentrated in vacuo. The residue was purified by FC (SiO.sub.2, RediSep R.sub.f 24 g cartridge, dry-load, DCM—DCM/MeOH/H.sub.2O 90:10:1) to give a red solid (325 mg, 94%). TLC (SiO.sub.2): R.sub.f=0.35 (DCM/MeOH/H.sub.2O 90:10:1). HPLC: t.sub.R=9.6 min (20-100); λ.sub.max=441, 348, 274 nm. ESI-MS: m/z 450.3 [M+H].sup.+. HRMS: m/z 450.1491 ([M+H].sup.+) calculated for C.sub.24H.sub.24N.sub.3O.sub.4S.sup.+: 450.1482 (Δ2.0 ppm). .sup.1H NMR (400 MHz, MeOD-d.sub.3 with TFA-d): δ=9.43 (d, J=2.0 Hz, 1H), 8.67 (dd, J=9.4, 2.3 Hz, 1H), 8.24 (br s, 1H), 7.92-8.05 (m, 1H), 7.88 (d, J=9.4 Hz, 1H), 7.75 (d, J=9.0 Hz, 1H), 7.26 (d, J=8.6 Hz, 2H), 6.76-6.88 (m, 2H), 4.28 (s, 2H), 4.00-4.63 (m, 2H), 3.82-3.99 (m, 2H), 3.74 (s, 3H), 1.93-2.31 (m, 2H) ppm.

    ##STR00028##

    [0247] Compound 29 was synthesized from a corresponding sulfide 25 through a sulfonyl chloride intermediate 27 generated as described in Y-M. Pu, A. Christesen, Y.-Y. Ku, Tetrahedron Lett. 2010, 51, 418-421. Compound 25 (10 mg, 25 μmol) was suspended in the mixture of MeCN (500 μL), AcOH (20 μL) and H.sub.2O (12 μL) at 0° C., further 100 μL of DCM was added. DCDMH (10 mg, 50 μmol) was added in portions. The color changed to yellow. The formation of the sulfonyl chloride intermediate was monitored by HPLC (t.sub.R=7.3 min; λ.sub.max=416 sh., 401, 357, 300, 261 nm; method 20-100). After 40 min of stirring at 0° C., the educt was fully converted. Mixture of DEA (10 mg, 100 μmol), DIPEA (100 μL) and ACN (100 μL) was added, and the reaction mixture was allowed to warm to rt. The reaction mixture became a clear orange solution. After 15 min of stirring at rt, the reaction mixture was concentrated in vacuo. The residue was dissolved in 10 mL of 0.1% aq. TFA and purified by FC (C18, 30C18AQ-F0012 cartridge, ACN—0.1% aq. TFA). Good fractions were pooled, basified with sat. NaHCO.sub.3, and the product was extracted into EtOAc-MeOH (10:1). The combined organic phase was washed with brine, dried (Na.sub.2SO.sub.4) and concentrated in vacuo to give a light orange solid (1 mg, 10%). TLC (SiO.sub.2): R.sub.f=0.2 (DCM/MeOH/H.sub.2O 90:10:1). HPLC: t.sub.R=10.3 min (5-50); λ.sub.max=424, 404, 356, 305, 260 nm. ESI-MS: m/z 407.3 [M+H].sup.+.

    ##STR00029##

    [0248] Compound 30 was synthesized from a corresponding sulfide 26 through a sulfonyl chloride intermediate 28 generated as described in Y-M. Pu, A. Christesen, Y.-Y. Ku, Tetrahedron Lett. 2010, 51, 418-421. Compound 26 (61 mg, 136 μmol) was suspended in 3 mL of MeCN/AcOH/H.sub.2O (20:0.75:0.5) at 0° C. DCDMH (75 mg, 381 μmol) was added portionwise. The color of suspension changed to yellow. The reaction mixture was stirred at 0° C. for 30 min, then 30 min at rt. The formation of the sulfonyl chloride intermediate was assessed by HPLC (t.sub.R=7.7 min, λ.sub.max=423, 406, 360, 302, 265 nm, method 20-100), the educt was fully converted. The solvent was removed in vacuo (35° C., <20 mbar). DEA (75 mg) in ACN (15 mL) was added. The color changed immediately to orange-brown. After 1 h of stirring at rt, the reaction mixture was diluted with EtOAc-MeOH (10:1), washed with sat. NaHCO.sub.3 and brine, dried (Na.sub.2SO.sub.4) and concentrated in vacuo. The residue was purified by FC (SiO.sub.2, RediSep Rf 24 g cartridge, dry-load, DCM—DCM/MeOH/H.sub.2O 90:10:1) to give a light orange solid (52 mg, 82%). TLC (SiO.sub.2): R.sub.f=0.2 (DCM/MeOH/H.sub.2O 90:10:1). HPLC: t.sub.R=10.5 min (5-50); λ.sub.max=428, 410, 360, 307, 267 nm. HRMS: m/z 465.1441 ([M+H].sup.+) calculated for C.sub.20H.sub.25N40.sub.7S.sup.+: 465.1438 (Δ0.6 ppm). .sup.1H NMR (400 MHz, MeOD-d.sub.3 with TFA-d): δ=9.33-9.63 (m, 1H), 8.95 (s, 1H), 8.73 (br dd, J=9.0, 0.8 Hz, 1H), 8.35 (br d, J=9.0 Hz, 1H), 7.95 (d, J=9.0 Hz, 2H), 4.31-4.55 (m, 2H), 3.83-4.04 (m, 2H), 3.74 (t, J=5.9, 5.5 Hz, 4H), 3.42 (t, J=5.5 Hz, 4H), 2.25 (quin, J=6.7, 6.3, 5.9, 5.5 Hz, 2H) ppm.

    ##STR00030##

    [0249] Compound 29 (1 mg, 2.5 μmol) was dissolved in MeOH (1 mL), containing AcOH (10 μL), under Ar. A 10% Pd/C catalyst (2 mg) was added, and hydrogen atmosphere was applied. After 1 hour of stirring at rt, orange fluorescent solution was filtered through a pad of Celite and concentrated in vacuo. The residue was dissolved in 10 mL of H.sub.2O (with 0.1% TFA) and purified by FC (C18, 15C18HP-F0012 cartridge, ACN—0.1% aq. TFA). Good fractions were pooled and lyophilized to give a red solid (0.8 mg, TFA salt). HPLC: t.sub.R=9.0 min (5-50); λ.sub.max=453 sh., 422, 401, 302 sh., 270 nm. ESI-MS: m/z 377.3 [M+H].sup.+.

    ##STR00031##

    [0250] Compound 30 (5 mg, 11 μmol) was dissolved in MeOH (10 mL), containing AcOH (10 μL), under Ar. A 10% Pd/C catalyst (1 mg) was added, and hydrogen atmosphere was applied. After 1 hour of stirring at rt, orange fluorescent solution was filtered through a pad of Celite and concentrated in vacuo. The residue was dissolved in 10 mL of H.sub.2O (with 0.1% TFA) and purified by FC (C18, 15C18HP-F0012 cartridge, ACN—0.1% aq. TFA). Good fractions were pooled and lyophilized (2×) to give a red solid (4.94 mg, 84%, TFA salt). HPLC: t.sub.R=9.0 min (5-50); λ.sub.max=430, 410, 272 nm. HRMS: m/z 435.1693 ([M+H].sup.+) calculated for C.sub.20H.sub.27N.sub.4O.sub.5S.sup.+: 435.1697 (Δ0.9 ppm). .sup.1H NMR (400 MHz, D.sub.2O): δ=8.35 (d, J=2.0 Hz, 1H), 7.95 (dd, J=9.0, 2.0 Hz, 1H), 7.48 (d, J=9.0 Hz, 1H), 7.25-7.38 (m, 2H), 7.07 (br s, 1H), 3.97 (t, J=7.0, 6.7 Hz, 2H), 3.81 (t, J=5.9, 5.5 Hz, 2H), 3.70 (t, J=5.9, 5.5 Hz, 4H), 3.37 (t, J=5.9, 5.5 Hz, 4H), 2.04 (quin, J=6.7, 6.3, 5.9 Hz, 2H) ppm. .sup.19F NMR (376 MHz, D.sub.2O): δ=−75.53 ppm.

    [0251] Reaction of sulfide 26 with 3-hydroxyazetidine through a sulfonyl chloride intermediate.

    [0252] Compound 26 (22 mg, 50 μmol) was suspended in 1 mL of MeCN/AcOH/H.sub.2O (20:0.75:0.5) at 0° C. DCDMH (25 mg, 125 μmol) was added portionwise. The color of suspension changed to yellow. The reaction mixture was stirred at 0° C. for 30 min, then 30 min at rt. The formation of the sulfonyl chloride intermediate 26-SO.sub.2Cl was assessed by HPLC (t.sub.R=7.5 min, λ.sub.max=423, 406, 360, 302, 265 nm, method 20-100), the educt was fully converted. The solvent was removed in vacuo (35° C., <20 mbar). MeCN (10 mL), 3-hydroxyazetidine hydrochloride (50 mg, 456 μmol) and DIPEA (15 mL) were added. The color changed immediately to orange-brown. After 1 h of stirring at rt, the reaction mixture was stored at −20° C. overnight. The reaction mixture was diluted with EtOAc-MeOH (10:1), washed with sat. NaHCO.sub.3 and brine, dried (Na.sub.2SO.sub.4) and concentrated in vacuo. The residue was purified by FC (SiO.sub.2, SNAP Ultra 10 g cartridge, dry-load, DCM—DCM/MeOH/H.sub.2O 90:10:1) to give the desired product 31 (3 mg, 14%) as a brown solid and side-product 32 (6 mg, 28%) as a light orange solid.

    ##STR00032##

    [0253] HPLC: t.sub.R=11.0 min (5-50); λ.sub.max=428, 410, 359, 305, 267 nm. ESI-MS: m/z 433.2 [M+H].sup.+. HRMS: m/z 433.1177 ([M+H].sup.+) calculated for C.sub.19H.sub.21N.sub.4O.sub.6S.sup.+: 433.1176 (Δ0.2 ppm). .sup.1H NMR (400 MHz, MeOD-d.sub.3 with TFA-d): δ=9.48 (br s, 1H), 8.94 (d, J=1.6 Hz, 1H), 8.76 (dd, J=9.4, 2.0 Hz, 1H), 8.35 (dd, J=9.0, 1.6 Hz, 1H), 8.02 (d, J=9.0 Hz, 1H), 7.96 (d, J=9.4 Hz, 1H), 4.43 (quin, J=6.3, 5.9, 5.5 Hz, 1H), 4.31-4.55 (m, 2H), 4.02-4.15 (m, 2H), 3.82-4.01 (m, 2H), 3.59 (dd, J=8.6, 5.5 Hz, 2H), 2.26 (quin, J=6.3, 5.9, 5.5 Hz, 2H) ppm.

    ##STR00033##

    [0254] HPLC: t.sub.R=10.5 min (5-50); λ.sub.max=425, 410, 361, 305, 269 nm. ESI-MS: m/z 431.3 [M+H].sup.+. HRMS: m/z 431.1022 ([M+H].sup.+) calculated for C.sub.19H.sub.19N.sub.4O.sub.6S.sup.−: 431.1020 (Δ0.5 ppm). .sup.1H NMR (400 MHz, MeOD-d.sub.3 with TFA-d): δ=9.00 (d, J=2.3 Hz, 1H), 8.68 (dd, J=9.4, 2.3 Hz, 1H), 8.51 (d, J=2.0 Hz, 1H), 8.25 (dd, J=9.0, 2.0 Hz, 1H), 7.95 (d, J=9.0 Hz, 1H), 7.91 (d, J=9.4 Hz, 1H), 5.27-5.38 (m, 2H), 5.15-5.26 (m, 2H), 4.88 (dddd, J=6.6, 4.4 Hz, 1H), 4.45 (dddd, J=6.6, 5.5 Hz, 1H), 4.02-4.12 (m, 2H), 3.54-3.63 (m, 2H) ppm.

    ##STR00034##

    [0255] Compound 31 (3 mg, 7 μmol) was dissolved in MeOH (10 mL), containing AcOH (10 μL), under Ar. A 10% Pd/C catalyst (1 mg) was added, and hydrogen atmosphere was applied. After 1 hour of stirring at rt, orange fluorescent solution was filtered through a pad of Celite and concentrated in vacuo. The residue was dissolved in 10 mL of H.sub.2O (with 0.1% TFA) and purified by FC (C18, 15C18HP-F0012 cartridge, ACN—0.1% aq. TFA). Good fractions were pooled and lyophilized to give a red powder (2.26 mg, 63%, TFA salt). HPLC: t.sub.R=9.7 min (5-50); λ.sub.max=431, 410, 303, 273 nm. HRMS: m/z 403.1439 ([M+H].sup.+) calculated for C.sub.19H.sub.23N.sub.4O.sub.4S.sup.+: 403.1435 (Δ1.0 ppm). .sup.1H NMR (400 MHz, D.sub.2O): δ=8.45 (d, J=1.6 Hz, 1H), 8.00 (dd, J=9.0, 1.6 Hz, 1H), 7.64 (d, J=9.4 Hz, 1H), 7.37-7.48 (m, 2H), 7.25 (br s, 1H), 4.45 (quin, J=6.7, 6.3, 5.5, 5.1 Hz, 1H), 4.01-4.13 (m, 4H), 3.82 (t, J=5.9, 5.5 Hz, 2H), 3.60 (d, J=5.1 Hz, 1H), 3.57 (d, J=5.1 Hz, 1H), 2.07 (quin, J=6.7, 6.3, 5.9 Hz, 2H) ppm.

    ##STR00035##

    [0256] Compound 32 (6 mg, 14 μmol) was dissolved in MeOH (10 mL), containing AcOH (10 μL), under Ar. A 10% Pd/C catalyst (1 mg) was added, and hydrogen atmosphere was applied. After 1 hour of stirring at rt, orange fluorescent solution was filtered through a pad of Celite and concentrated in vacuo. The residue was dissolved in 10 mL of H.sub.2O (with 0.1% TFA) and purified by FC (C18, 15C18HP-F0012 cartridge, ACN—0.1% aq. TFA). Good fractions were pooled and lyophilized to give a red powder (6 mg, 86%, TFA salt). HPLC: t.sub.R=8.6 min (5-50); λ.sub.max=430, 410, 277 nm. HRMS: m/z 401.1281 ([M+H].sup.+) calculated for Cl.sub.9H.sub.21N.sub.4O.sub.4S.sup.+: 401.1278 (Δ0.7 ppm). 1H NMR (400 MHz, MeOD-d.sub.3): δ=8.47 (d, J=1.6 Hz, 1H), 8.08 (dd, J=9.0, 1.6 Hz, 1H), 7.85 (d, J=9.0 Hz, 1H), 7.64 (d, J=9.0 Hz, 1H), 7.47 (dd, J=9.0, 2.0 Hz, 1H), 7.32 (d, J=2.0 Hz, 1H), 5.17-5.43 (m, 2H), 4.98-5.16 (m, 2H), 4.83-4.91 (m, 1H), 4.45 (dq, J=6.3, 5.9 Hz, 1H), 4.06 (t, J=7.8 Hz, 2H), 3.57 (dd, J=8.6, 5.5 Hz, 2H) ppm. .sup.13C NMR (101 MHz, MeOD-d.sub.3, HSQC): δ=158.4, 146.0, 142.4, 134.5, 132.4 (CH), 129.7 (CH), 127.9 (CH), 127.8, 120.5 (2×CH), 115.5, 111.3, 107.3 (CH), 72.6 (2×CH.sub.2), 63.4 (CH), 61.6 (2×CH.sub.2), 60.8 (CH) ppm. .sup.19F NMR (376 MHz, MeOD-d.sub.3): δ=−76.71 ppm.

    Example 2

    Synthesis of Aminoacridine Dye-Glucose Conjugates

    [0257] ##STR00036##

    [0258] Compound 6 (20 mg, 36 μmol), glucose (32 mg, 178 μmol) and AcOH (4 mg, 71 μmol) were mixed in 1 mL of water. NaBH.sub.3CN (22 mg, 356 μmol) in 0.5 mL of MeOH was added. The reaction mixture was heated to 60° C. The reaction progress was monitored by HPLC. After 5 h of stirring at 60° C., the reaction mixture was cooled to rt, diluted with 10 mL of 0.1% aq. TFA and purified by FC (C18, 30C18AQ-F0025 cartridge, ACN—0.1% aq. TFA). The good fractions were pooled and lyophilized to give the title product 6-Glc (10 mg, TFA salt) as a red-purple solid. HPLC: t.sub.R=10.9 min (5-50). ESI-MS: m/z 499.4 [M+H].sup.+. HRMS: m/z 499.2553 ([M+H].sup.+) calculated for C.sub.26H.sub.34N.sub.4O.sub.6.sup.+: 499.2551 (Δ0.4 ppm). .sup.1H NMR (400 MHz, D.sub.2O): 6=8.12 (s, 1H), 7.76 (dd, J=9.0, 1.6 Hz, 1H), 7.39 (d, J=9.0 Hz, 1H), 7.30 (dd, J=9.0, 2.0 Hz, 1H), 7.25 (d, J=9.0 Hz, 1H), 6.62 (d, J=2.0 Hz, 1H), 4.06 (ddtd, J=5.1, 4.3, 3.9, 0.9 Hz, 1H), 3.91 (dd, J=5.3, 2.2 Hz, 1H), 3.86 (dd, J=15.7, 2.7 Hz, 1H), 3.80-3.89 (m, 1H), 3.66-3.78 (m, 4H), 3.57 (t, J=6.7 Hz, 2H), 3.33 (dd, J=13.3, 3.9 Hz, 1H), 3.18 (dd, J=13.3, 8.2 Hz, 1H), 1.76 (quin, J=7.0 Hz, 2H), 1.54 (quin, J=6.7 Hz, 2H), 1.30-1.45 (m, 4H) ppm. 19F NMR (376 MHz, D.sub.2O): δ=−75.50 ppm.

    [0259] General method for labelling of glucose with model compounds (7, 9-11). 1.5 mL Brand® micro tube with screw cap was charged with dye (1 equiv., 0.1 M solution in water), glucose (1 equiv., 0.1 M solution in water), malonic acid (10 equiv., 1 M solution in DMSO) and 2-picoline-borane complex (10 equiv., 1 M solution in DMSO). After vortexing for 10 s, the reaction mixture was incubated in an Eppendorf ThermoMixer® with shaking (400-600 rpm) at 40° C. for 18 h. The reaction mixture was cooled to rt, diluted with 10 mL of 0.1% aq. TFA and purified by FC (C18, 15C18HP-F0012 cartridge, ACN—0.1% aq. TFA). The good fractions were pooled and lyophilized.

    ##STR00037##

    [0260] The title compound 7-Glc (0.8 μmol, TFA salt) was obtained as an orange-red solid from dye 7 (1.3 μmol) according to the general method for labelling of glucose. HPLC: t.sub.R=9.1 min (5-50); λ.sub.max=482, 319, 275 nm. ESI-MS: m/z 554.3 [M+H].sup.+. HRMS: m/z 554.2160 ([M+H].sup.+) calculated for C.sub.25H.sub.36N.sub.3O.sub.9S.sup.+: 554.2167 (Δ1.3 ppm). 1H NMR (400 MHz, D.sub.2O): δ=8.63 (d, J=1.0 Hz, 1H), 8.06 (d, J=9.0 Hz, 1H), 7.69 (dd, J=9.0, 0.4 Hz, 1H), 7.46 (d, J=9.0 Hz, 1H), 7.38-7.44 (m, 1H), 6.91 (s, 1H), 4.03-4.20 (m, 3H), 3.91 (dd, J=5.5, 2.2 Hz, 1H), 3.78-3.88 (m, 4H), 3.75 (dd, J=8.2, 2.2 Hz, 1H), 3.64-3.71 (m, 1H), 3.62 (t, J=6.3 Hz, 2H), 3.46-3.53 (m, 2H), 3.43 (dd, J=13.3, 3.5 Hz, 1H), 3.26 (dd, J=13.3, 8.2 Hz, 1H), 2.04-2.17 (m, 2H), 1.85-1.97 (m, 2H) ppm. .sup.19F NMR (376 MHz, D.sub.2O): δ=−75.53 ppm.

    ##STR00038##

    [0261] The title compound 9-Glc (0.7 μmol, TFA salt) was obtained as an orange-red solid from dye 9 (1.6 μmol) according to the general method for labelling of glucose. HPLC: t.sub.R=9.1 min (5-50); λ.sub.max=480, 319, 276 nm. ESI-MS: m/z 599.3 [M+H].sup.+. HRMS: m/z 599.2381 ([M+H].sup.+) calculated for C.sub.26H.sub.39N.sub.4O.sub.10S.sup.+: 599.2381 (Δ0.0 ppm). .sup.1H NMR (400 MHz, D.sub.2O): δ=8.53 (s, 1H), 8.01 (dd, J=9.0, 1.0 Hz, 1H), 7.61 (d, J=9.0 Hz, 1H), 7.36-7.47 (m, 2H), 6.88 (s, 1H), 4.01-4.17 (m, 3H), 3.93 (dd, J=5.5, 2.0 Hz, 1H), 3.80-3.89 (m, 4H), 3.75-3.80 (m, 1H), 3.62-3.75 (m, 5H), 3.33-3.48 (m, 5H), 3.27 (dd, J=13.1, 8.4 Hz, 1H), 2.04-2.17 (m, 2H) ppm. .sup.19F NMR (376 MHz, D.sub.2O): δ=−75.52 ppm.

    ##STR00039##

    [0262] The title compound 10-Glc (0.6 μmol, TFA salt) was obtained as an orange-red solid from dye 10 (1.5 μmol) according to the general method for labelling of glucose. HPLC: t.sub.R=9.6 min (5-50); λ.sub.max=482, 320, 276 nm. ESI-MS: m/z 567.3 [M+H].sup.+. HRMS: m/z 567.2120 ([M+H].sup.+) calculated for C.sub.25H.sub.35N.sub.4O.sub.9S.sup.+: 567.2119 (Δ0.2 ppm). 1H NMR (400 MHz, D.sub.2O): δ=8.53 (s, 1H), 7.95-8.12 (m, 1H), 7.60-7.82 (m, 1H), 7.33-7.57 (m, 2H), 6.82-7.00 (m, 1H), 4.39-4.56 (m, 1H), 4.00-4.25 (m, 5H), 3.90-3.97 (m, 1H), 3.80-3.89 (m, 4H), 3.74-3.80 (m, 1H), 3.66-3.74 (m, 1H), 3.56-3.65 (m, 2H), 3.37-3.51 (m, 1H), 3.20-3.32 (m, 1H), 2.07-2.18 (m, 2H) ppm. .sup.19F NMR (376 MHz, D.sub.2O): δ=−75.52 ppm.

    ##STR00040##

    [0263] The title compound 11-Glc (1.3 μmol, AcOH salt) was obtained as a red solid from dye 11 (5.1 μmol) according to the general method for labelling of glucose. The material was further purified by FC (C18, 15C18HP-F0012 cartridge, ACN—0.1% aq. AcOH). The product appeared to be unstable in the presence of TFA (by HPLC). HPLC: t.sub.R=8.8 min (5-50); λ.sub.max=485, 431, 320, 277 nm. HRMS: m/z 565.1959 ([M+H].sup.+) calculated for C.sub.25H.sub.33N.sub.4O.sub.9S.sup.+: 565.1963 (Δ0.7 ppm). .sup.1H NMR (400 MHz, D.sub.2O): δ=8.12 (d, J=1.2 Hz, 1H), 7.96 (dd, J=9.0, 1.2 Hz, 1H), 7.54 (br d, J=9.0 Hz, 1H), 7.37-7.48 (m, 2H), 6.87 (s, 1H), 5.04-5.25 (m, 2H), 4.87-5.01 (m, 2H), 4.78-4.82 (m, 1H), 4.47 (dt, J=11.7, 5.9 Hz, 1H), 4.00-4.16 (m, 3H), 3.91 (dd, J=5.5, 2.3 Hz, 1H), 3.78-3.86 (m, 2H), 3.75 (dd, J=8.0, 2.2 Hz, 1H), 3.64-3.71 (m, 1H), 3.61 (dd, J=9.2, 5.3 Hz, 2H), 3.47 (dd, J=13.5, 4.1 Hz, 1H), 3.29 (dd, J=13.5, 8.0 Hz, 1H), 1.88 (s, 2H, AcOH) ppm.

    Example 3

    Synthesis of Phosphorylated Acridines (and Acridone)

    [0264] ##STR00041##

    [0265] Compound 22 (330 mg, 0.79 mmol) and 1H-terazole (330 mg, 4.72 mmol) were placed in Ar-filled flask, which was evacuated-backfilled with Ar (3×). DMF (8 mL) and (t-BuO).sub.2PNi-Pr.sub.2 (994 μL, 3.15 mmol) were added, and the reaction mixture was stirred 1 h at rt. Formation of the phosphite intermediate was monitored by HPLC (t.sub.R=14.3 min, 5-50). Then 50% aq. H.sub.2O.sub.2 (358 μL, 7.3 mmol) was added in one portion. After 30 min of stirring at rt, the reaction mixture was diluted with DCM (200 mL), washed with 10% aq. Na.sub.2SO.sub.3— aq. sat. NaHCO.sub.3 (100 mL, 1:1) and brine, dried (Na.sub.2SO.sub.4) and concentrated in vacuo. The residue was purified by FC (SiO.sub.2, 15SIHP-F0040 cartridge, DCM/MeOH with 2-5% MeOH gradient) to give a light orange solid (260 mg, 41%). TLC (SiO.sub.2): R.sub.f=0.3 (DCM/MeOH 20:1). HPLC: t.sub.R=10.9 min (20-100); t.sub.R=17.6 min (5-50); λ.sub.max=427, 410, 357, 303, 267 nm. ESI-MS: m/z 804.5 [M+H].sup.+.

    [0266] HRMS: m/z 804.3056 ([M+H].sup.+) calculated for C.sub.35H.sub.56N.sub.3O.sub.12P.sub.2S.sup.+: 804.3054 (Δ0.2 ppm). .sup.1H NMR (400 MHz, MeOD-d.sub.3 with TFA-d): δ=9.29-9.75 (m, 1H), 8.89-9.28 (m, 1H), 8.75 (dd, J=9.4, 2.3 Hz, 1H), 8.42 (dd, J=9.0, 1.6 Hz, 1H), 8.00 (d, J=9.0 Hz, 1H), 7.95 (d, J=9.4 Hz, 1H), 4.36 (t, J=6.7 Hz, 2H), 4.19 (q, J=6.7, 6.3, 5.9 Hz, 2H), 4.06 (q, J=7.4, 6.3 Hz, 2H), 3.42-3.52 (m, 2H), 2.44 (quin, J=6.3 Hz, 2H), 2.04-2.17 (m, 2H), 1.44 (s, 9H), 1.44 (s, 9H), 1.42 (s, 18H) ppm. .sup.31P NMR (162 MHz, CDCl.sub.3): δ=−7.99 (br s, 1P), −10.13 (s, 1P) ppm.

    ##STR00042##

    [0267] Compound 33 (50 mg, 62 μmol) was dissolved in MeOH (4 mL), containing AcOH (20 μL), under Ar. A 10% Pd/C catalyst (5 mg) was added, and hydrogen atmosphere was applied. After 1 h of stirring at rt, orange fluorescent solution was filtered through a pad of Celite and concentrated in vacuo. The residue was dissolved in 10 mL of ACN/0.1% aq. TFA (1:5) and purified by FC (C18, 30C18AQ-F0025 cartridge, ACN—0.1% aq. TFA). Good fractions were pooled and lyophilized to give a red powder (42 mg, 76%, TFA salt). HPLC: t.sub.R=10.3 min (20-100). ESI-MS: m/z 774.6 [M+H].sup.+. HRMS: m/z 774.3315 ([M+H].sup.+) calculated for C.sub.35H.sub.58N.sub.3O.sub.10P.sub.2S.sup.+: 774.3313 (Δ0.3 ppm). .sup.1H NMR (400 MHz, MeOD-d.sub.3): δ=9.02 (d, J=1.6 Hz, 1H), 8.21 (dd, J=9.0, 1.6 Hz, 1H), 7.91 (d, J=9.0 Hz, 1H), 7.69 (dd, J=7.4, 2.3 Hz, 1H), 7.53 (s, 1H), 7.54 (dd, J=7.4, 2.3 Hz, 1H), 4.32 (t, J=7.0, 6.7 Hz, 2H), 4.20 (q, J=6.3, 5.9 Hz, 2H), 4.06 (q, J=7.8, 6.3, 5.9 Hz, 2H), 3.45 (m, J=7.8, 7.4, 2.3, 2.0 Hz, 2H), 2.38 (quin, J=6.3, 5.9 Hz, 2H), 2.05-2.16 (m, 2H), 1.45 (s, 18H), 1.44 (s, 18H) ppm. .sup.31P NMR (162 MHz, MeOD-D.sub.3): δ=−10.61 (s, 1P), −10.62 (s, 1P) ppm.

    ##STR00043##

    [0268] Compound 34 (43 mg, 48 μmol) was stirred in 5% TFA in DCM (4 mL) for 4 h at rt under Ar. The reaction mixture was then concentrated in vacuo (without heating bath). The residue was dissolved in 10 mL of TEAAc buffer (1.0 M, pH 7) and purified by FC (C18, 30C18AQ-F0025, ACN—10 mM TEAAc pH 7). Good fractions were pooled and lyophilized to give a red powder (20 mg, 65%, TEA salt). HPLC (Knauer Eurospher II 100-5 C18A): t.sub.R=9.3 min (TEAB-O-25); λ.sub.max=455, 303, 273 nm. ESI-MS: m/z 548.2 [M−H].sup.−. HRMS: m/z 550.0805 ([M+H].sup.+) calculated for C.sub.19H.sub.26N.sub.3O.sub.10P.sub.2S.sup.+: 550.0809 (Δ0.7 ppm). 1H NMR (400 MHz, D.sub.2O): δ=8.35 (s, 1H), 7.94 (dd, J=9.0, 1.6 Hz, 1H), 7.49 (d, J=9.0 Hz, 1H), 6.94-7.13 (m, 2H), 6.83 (s, 1H), 4.06-4.22 (m, 2H), 3.85-3.98 (m, 4H), 3.46-3.59 (m, 2H), 3.18 (q, J=7.2 Hz, 6H, TEA), 2.09-2.20 (m, 2H), 1.98-2.08 (m, 2H), 1.26 (t, J=7.2 Hz, 9H, TEA) ppm. .sup.31P NMR (162 MHz, D.sub.2O): δ=0.35 (s, 1P), 0.25 (s, 1P) ppm.

    [0269] Reaction of 30 with di-tert-butyl N,N-diisopropylphosphoramidite with H.sub.2O.sub.2 as oxidant.

    [0270] Compound 30 (57 mg, 0.12 mmol) and 1H-terazole (78 mg, 1.11 mmol) were placed in Ar-filled flask, which was evacuated-backfilled with Ar (3×). DMF (1.25 mL) and (t-BuO).sub.2PNi-Pr.sub.2 (233 μL, 0.74 mmol) were added, and the reaction mixture was stirred 1 h at rt. Formation of the phosphite intermediate was monitored by HPLC (t.sub.R=9.2 min, 20-100). Then 50% aq. H.sub.2O.sub.2 (278 μL, 4.9 mmol) was added in one portion. After 30 min of stirring at rt, the reaction mixture was diluted with DCM (200 mL), washed with 10% aq. Na.sub.2SO.sub.3— aq. sat. NaHCO.sub.3 (100 mL, 1:1) and brine, dried (Na.sub.2SO.sub.4) and concentrated in vacuo. The residue was purified by FC (SiO.sub.2, 30SIHP-F0025 cartridge, DCM/MeOH with 1-5% MeOH gradient) to give the desired product 35 (58 mg, 47%) as an orange oil and a side-product 36 (12 mg, 13%) as a yellow powder.

    ##STR00044##

    [0271] HPLC: t.sub.R=12.3 min (20-100); λ.sub.max=429, 412, 358, 307, 268 nm. HRMS: m/z 1041.4168 ([M+H].sup.+) calculated for C.sub.44H.sub.76N.sub.4O.sub.16P.sub.3S.sup.+: 1041.4184 (Δ1.5 ppm). .sup.1H NMR (400 MHz, MeOD-d.sub.3 with TFA-d): δ=9.29-9.80 (m, 1H), 8.83-9.23 (m, 1H), 8.76 (dd, J=9.4, 2.3 Hz, 1H), 8.40 (dd, J=9.0, 2.0 Hz, 1H), 7.97 (d, J=9.0 Hz, 1H), 7.96 (d, J=9.4 Hz, 1H), 4.37 (t, J=7.0, 6.7 Hz, 2H), 4.21 (q, J=6.7, 5.9 Hz, 2H), 4.13 (q, J=6.3, 5.9, 5.5 Hz, 4H), 3.66 (t, J=5.5 Hz, 4H), 2.47 (quin, J=6.3, 5.9 Hz, 2H), 1.46 (s, 36H), 1.45 (s, 18H) ppm. .sup.31P NMR (162 MHz, MeOD-d.sub.3 with TFA-d): δ=−10.23 (s, 1P), −10.90 (s, 2P) ppm.

    ##STR00045##

    [0272] HPLC: t.sub.R=12.6 min (20-100); λ.sub.max=354, 298, 249 nm. ESI-MS: m/z 790.4 [M−H].sup.−. HRMS: m/z 814.2515 ([M+Na].sup.+) calculated for C.sub.33H.sub.51N.sub.3NaO.sub.13P.sub.2S.sup.+: 814.2510 (Δ0.6 ppm). 1H NMR (400 MHz, CDCl.sub.3): δ=12.05 (s, 1H), 9.08 (d, J=2.3 Hz, 1H), 8.82 (d, J=2.3 Hz, 1H), 8.29 (dd, J=9.0, 2.7 Hz, 1H), 7.98 (dd, J=8.6, 2.3 Hz, 1H), 7.57 (d, J=9.0 Hz, 1H), 7.43 (d, J=9.0 Hz, 1H), 4.19 (q, J=7.4, 6.3, 5.9 Hz, 4H), 3.64 (t, J=6.3, 5.9 Hz, 4H), 1.51 (s, 36H) ppm. .sup.13C NMR (101 MHz, CDCl.sub.3, HSQC): δ=176.6, 144.4, 143.2, 142.0, 133.2, 131.3 (CH), 127.6 (CH), 127.2 (CH), 123.9 (CH), 120.8, 120.4, 119.0 (CH), 118.4 (CH), 83.4 (d, 4×C, J.sub.CP=7.6 Hz), 65.3 (d, 2×CH.sub.2, J.sub.CP=6.9 Hz), 48.9 (d, CH.sub.2, J.sub.CP=8.4 Hz), 29.9 (d, 12×CH.sub.3, J.sub.CP=3.8 Hz) ppm. .sup.31P NMR (162 MHz, CDCl.sub.3): δ=−11.19 (s, 2P) ppm.

    ##STR00046##

    [0273] Compound 35 (58 mg, 56 μmol) was dissolved in MeOH (5 mL), containing AcOH (100 μL), under Ar. A 10% Pd/C catalyst (5 mg) was added, and hydrogen atmosphere was applied. After 1 h of stirring at rt, orange fluorescent solution was filtered through a pad of Celite and concentrated in vacuo. The residue was dissolved in 10 mL of ACN/0.1% aq. TFA (1:5) and purified by FC (C18, 30C18AQ-F0025 cartridge, ACN—0.1% aq. TFA). Good fractions were pooled and lyophilized to give a red powder (27 mg, 43%, TFA salt). HPLC: t.sub.R=11.8 min (20-100); λ.sub.max=475, 312, 275 nm. HRMS: m/z 1011.4449 ([M+H].sup.+) calculated for C.sub.44H.sub.78N.sub.4O.sub.14P.sub.3S.sup.+: 1011.4443 (A 0.6 ppm). .sup.1H NMR (400 MHz, MeOD-d.sub.3): δ=8.95 (d, J=2.0 Hz, 1H), 8.17 (dd, J=9.0, 2.0 Hz, 1H), 7.86 (d, J=9.0 Hz, 1H), 7.66 (dd, J=8.2, 1.6 Hz, 1H), 7.51 (s, 1H), 7.49 (dd, J=9.0, 2.3 Hz, 1H), 4.30 (t, J=7.0, 6.7 Hz, 2H), 4.18 (q, J=6.3, 5.9 Hz, 2H), 4.10 (q, J=6.7, 5.9 Hz, 4H), 3.62 (t, J=5.9 Hz, 4H), 2.35 (quin, J=6.3, 5.9 Hz, 2H), 1.44 (s, 36H), 1.42 (s, 18H) ppm. .sup.31P NMR (162 MHz, CDCl.sub.3): δ=−10.02 (s, 2P), −10.86 (s, 1P) ppm.

    ##STR00047##

    [0274] Compound 36 (12 mg, 15 μmol) was dissolved in MeOH (1 mL), containing AcOH (25 μL), under Ar. A 10% Pd/C catalyst (1 mg) was added, and hydrogen atmosphere was applied. After 1 h of stirring at rt, the reaction mixture was filtered through a pad of Celite and concentrated in vacuo. The residue was dissolved in 10 mL of ACN/0.1% aq. TFA (1:5) and purified by FC (C18, 30C18AQ-F0025 cartridge, ACN—0.1% aq. TFA). Good fractions were pooled and lyophilized to give a light yellow powder (6.3 mg, 77%, TFA salt). HPLC: t.sub.R=9.6 min (20-100); λ.sub.max=397, 380, 318, 306, 280, 256 nm. ESI-MS: m/z 762.4 [M+H].sup.+. HRMS: m/z 762.2945 ([M+H].sup.+) calculated for C.sub.33H.sub.54N.sub.3O.sub.11P.sub.2S.sup.+: 762.2949 (Δ0.5 ppm). .sup.1H NMR (400 MHz, MeOD-d.sub.3): δ=8.78 (d, J=2.0 Hz, 1H), 8.07-8.13 (m, 2H), 7.66 (d, J=8.6 Hz, 1H), 7.62 (d, J=1.6 Hz, 2H), 4.09 (q, J=7.0, 6.3, 5.9 Hz, 4H), 3.56 (t, J=5.9 Hz, 4H), 1.45 (s, 36H) ppm. .sup.13C NMR (100 MHz, MeOD-d.sub.3, HSQC): δ=177.2, 143.1, 138.6, 132.1, 131.2, 130.9 (CH), 127.5 (CH), 126.8 (CH), 121.6, 119.3 (CH), 119.2, 118.8 (CH), 116.2 (CH), 83.3 (d, 2×C, J.sub.CP=7.6 Hz), 65.3 (d, 2×CH.sub.2, J.sub.CP=6.9 Hz), 48.7 (d, 2×CH.sub.2, J.sub.CP=8.4 Hz), 28.7 (d, 12×CH.sub.3, J.sub.CP=3.8 Hz) ppm. .sup.31P NMR (162 MHz, MeOD-d.sub.3): δ=−10.79 (s) ppm.

    ##STR00048##

    [0275] Compound 37 (11 mg, 10 μmol) was stirred in 5% TFA in DCM (0.75 mL) for 2 h at rt under Ar. The reaction mixture was then cooled to 0° C., and 5 mL of TEAAc buffer (1.0 M, pH 7) was added. The resulting emulsion was concentrated in vacuo (without heating bath) to obtain clear aqueous solution, which was purified by FC (C18, 30C18AQ-F0025, ACN—10 mM TEAAc pH 7). Good fractions were pooled and lyophilized (3×) to give a dark red solid (8 mg, 72%, TEA salt). HPLC (Knauer Eurospher II 100-5 C18A): t.sub.R=8.7 min (TEAB-0-25); t.sub.R=7.0 min (TEAB-0-40); λ.sub.max=458, 305, 273 nm. ESI-MS: m/z 673.2 [M−H].sup.−. HRMS: m/z 673.0532 ([M−H].sup.−) calculated for C.sub.20H.sub.28N.sub.4O.sub.14P.sub.3S.sup.−: 673.0541 (Δ1.3 ppm). 1H NMR (400 MHz, D.sub.2O): 6=8.46 (s, 1H), 8.05 (d, J=9.0 Hz, 1H), 7.63 (d, J=9.4 Hz, 1H), 7.30 (d, J=8.6 Hz, 1H), 7.25 (d, J=9.0 Hz, 1H), 7.16 (s, 1H), 4.08-4.20 (m, 2H), 4.04 (t, J=6.3, 5.9 Hz, 2H), 3.98 (q, J=5.9, 5.5, 5.1 Hz, 4H), 3.58 (t, J=5.5, 5.1 Hz, 4H), 3.19 (q, J=7.4 Hz, 26H, TEA), 2.12-2.26 (m, 2H), 1.26 (t, J=7.4 Hz, 38H, TEA) ppm. .sup.31P NMR (162 MHz, D.sub.2O): δ=1.32 (s, 1P), 0.72 (s, 2P) ppm.

    ##STR00049##

    [0276] Compound 38 (6.3 mg, 7 μmol) was stirred in 5% TFA in DCM (0.75 mL) for 2 h at rt under Ar. The reaction mixture was then cooled to 0° C., and 5 mL of TEAAc buffer (1.0 M, pH 7) was added. The resulting emulsion was concentrated in vacuo (without heating bath) to obtain clear aqueous solution, which was purified by FC (C18, 30C18AQ-F0025, ACN—10 mM TEAAc pH 7). Good fractions were pooled and lyophilized (3×) to give a yellow-brown solid (4 mg, 79%, TEA salt). HPLC (Knauer Eurospher II 100-5 C18A): t.sub.R=8.6 min (TEAB-0-40); λ.sub.max=425, 297, 260 nm. ESI-MS: m/z 536.1 [M−H].sup.−. HRMS: m/z 536.0298 ([M−H].sup.−) calculated for C.sub.17H.sub.20N.sub.3O.sub.11P2S 536.0299 (Δ0.2 ppm). 1H NMR (400 MHz, D.sub.2O): δ=8.32 (d, J=2.0 Hz, 1H), 7.84 (dd, J=9.0, 1.2 Hz, 1H), 7.27 (d, J=9.0 Hz, 1H), 7.23 (s, 1H), 7.18 (dd, J=8.6, 0.8 Hz, 1H), 7.06 (d, J=9.0 Hz, 1H), 4.00 (q, J=5.9, 5.5 Hz, 4H), 3.51 (t, J=5.5, 5.1 Hz, 4H), 3.17 (q, J=7.4 Hz, TEA), 1.25 (t, J=7.4 Hz, TEA) ppm. .sup.13C NMR (101 MHz, D.sub.2O, HSQC): 6=177.5, 141.7, 139.1, 135.2, 130.0 (CH), 129.6, 126.8 (CH), 126.6 (CH), 120.6, 119.1 (CH), 119.0 (CH), 117.4, 110.1 (CH), 63.5 (d, 2×CH.sub.2, J.sub.CP=4.6 Hz), 49.1 (d, 2×CH.sub.2, J.sub.CP=6.9 Hz), 46.7 (CH.sub.2, TEA), 8.3 (CH.sub.3, TEA) ppm. .sup.31P NMR (162 MHz, D.sub.2O): δ=0.28 (s) ppm.

    Example 4

    Reductive Amination of Carbohydrates with Negatively Charged Amino-Dyes

    [0277] General method for labelling of carbohydrates with negatively charged amino-dyes for electrophoresis. 1.5 mL Brand® micro tube with a screw cap was charged with dye (1 equiv., 0.1 M solution in water), carbohydrate (1 equiv., 0.1 M solution in water), malonic acid (10 equiv., 1 M solution in DMSO) and 2-picoline-borane complex (10 equiv., 1 M solution in DMSO). After vortexing for 10 s, the reaction mixture was incubated in an Eppendorf ThermoMixer® with shaking (400-600 rpm) at 40° C. for 18 h. The reaction mixture was cooled to rt, diluted with 5 mL of TEAB buffer (1.0 M, pH 8) and purified by flash chromatography (RP C18, 15C18AQ-F0025 cartridge, ACN—20 mM TEAB, pH 8, 0-5% ACN, 10 column volumes). The appropriate fractions were pooled and lyophilized.

    [0278] Removal of buffer at reduced pressure and prolonged drying gave the conjugation products of dye 13 as red powders. These compounds did not dissolve any more in water, methanol, ethanol, DMSO, or DMF even on heating.

    [0279] General method for labelling of dextran ladder with negatively charged amino-dyes for electrophoresis. 1.5 mL Brand® micro tube with a screw cap was charged with a dextran ladder (0.4 mg, maltodextrin oligosaccharides—DP2 to DP15, Carbosynth), dye (5 μL, 0.1 M solution in water or DMSO), malonic acid (5 μL, 1 M solution in DMSO) and 2-picoline-borane complex (5 μL, 1 M solution in DMSO). After vortexing for 10 s, the reaction mixture was incubated in an Eppendorf ThermoMixer® with shaking (400-600 rpm) at 40° C. for 18 h. The reaction mixture was cooled to rt, diluted with 5 mL of TEAB buffer (1.0 M, pH 8) and purified by flash chromatography (RP C18, 15C18AQ-F0025 cartridge, ACN—20 mM TEAB, pH 8, 0-5% ACN, 10 column volumes). The appropriate fractions were pooled and concentrated in vacuo (rotary evaporator, then speedvac).

    ##STR00050##

    [0280] HPLC (Knauer Eurospher II 100-5 C18A): t.sub.R=9.5 min (TEAB-0-25); λ.sub.max=469, 317, 276 nm. ESI-MS: m/z 712.4 [M−H].sup.−. HRMS: m/z 712.1339 ([M−H].sup.−) calculated for C.sub.25H.sub.36N.sub.3O.sub.15P.sub.2S.sup.−: 712.1348 (Δ1.3 ppm). .sup.1H NMR (400 MHz, D.sub.2O): δ=8.60 (s, 1H), 8.04 (d, J=8.6 Hz, 1H), 7.69 (d, J=9.4 Hz, 1H), 7.39 (d, J=9.0 Hz, 1H), 7.16-7.35 (m, 1H), 6.91 (s, 1H), 4.06-4.17 (m, 4H), 4.04 (quin, J=5.5, 4.3, 3.5 Hz, 1H), 3.86-3.96 (m, 3H), 3.78-3.86 (m, 2H), 3.74-3.79 (m, 1H), 3.64-3.73 (m, 1H), 3.49-3.61 (m, 2H), 3.40 (dd, J=13.5, 3.7 Hz, 1H), 3.23 (dd, J=13.7, 8.2 Hz, 1H), 3.18 (q, J=7.4 Hz, TEA), 2.17-2.29 (m, 2H), 1.97-2.08 (m, 2H), 1.25 (t, J=7.4 Hz, TEA) ppm. .sup.31P NMR (162 MHz, D.sub.2O): δ=0.69 (s, 1P), 0.33 (s, 1P) ppm.

    ##STR00051##

    [0281] HPLC (Knauer Eurospher II 100-5 C18A): t.sub.R=8.8 min (TEAB-0-25). ESI-MS: m/z 1036.6 [M−H].sup.−.

    ##STR00052##

    [0282] HPLC (Knauer Eurospher II 100-5 C18A): t.sub.R=8.8 min (TEAB-0-25). ESI-MS: m/z 1685.6 [M−H].sup.−.

    ##STR00053##

    [0283] HPLC (Knauer Eurospher II 100-5 C18A): t.sub.R=8.8 min (TEAB-0-25); λ.sub.max=473, 318, 276 nm. ESI-MS: m/z 836.9 [M−H].sup.−. HRMS: m/z 418.0568 ([M−2H].sup.2−) calculated for C.sub.26H.sub.39N.sub.4O.sub.19P.sub.3S.sup.2: 418.0577 (z 2.2 ppm).

    ##STR00054##

    [0284] HPLC (Knauer Eurospher II 100-5 C18A): t.sub.R=9.0 min (TEAB-0-25). ESI-MS: m/z 1161.1 [M−H].sup.−.

    ##STR00055##

    [0285] HPLC (Knauer Eurospher II 100-5 C18A): t.sub.R=8.8 min (TEAB-0-25). ESI-MS: m/z 904.1 [M−2H].sup.2−.

    ##STR00056##

    [0286] HPLC (Knauer Eurospher 11100-5 C18A): t.sub.R=10.6 min (TEAB-0-25); λ.sub.max=439, 306, 254 nm. ESI-MS: m/z 714.2 [M−H].sup.−. HRMS: m/z 356.5533 ([M−2H].sup.2−) calculated for C.sub.24H.sub.33N.sub.3O.sub.16P.sub.2S.sup.2−: 356.5534 (Δ0.3 ppm).

    ##STR00057##

    [0287] HPLC (Knauer Eurospher II 100-5 C18A): t.sub.R=10.2 min (TEAB-0-25). ESI-MS: m/z 1038.4 [M−H].sup.−.

    ##STR00058##

    [0288] HPLC (Knauer Eurospher II 100-5 C18A): t.sub.R=10.3 min (TEAB-0-25). ESI-MS: m/z 1686.6 [M−H].sup.−.

    ##STR00059##

    [0289] a) Anhydrous trisodium salt of 8-aminopyrene-1,3,6-trisulfonic acid (52 mg, 0.10 mmol) was introduced into a 10 mL flask, cooled down to 0° C. (ice bath), and chlorosulfonic acid (356 mg, 204 μL, 3 mmol) was added dropwise with stirring. The reaction mixture was stirred at r. t. for 4 h. After cooling down to 0° C., the reaction mixture was carefully transferred onto crushed ice (50 g). The red precipitate of trisulfonyl chloride was isolated by centrifugation and washed with ice water (3×50 mL; with centrifugation). The crude compound was lyophilized to afford 42 mg of 1,3,6-tri(chlorosulfonyl)-8-aminopyrene (49) (82% yield). The flask was purged with Ar and kept in the freezer (−20° C.).

    [0290] b) To a stirred and ice-cooled solution of cyanamide (50 mg, 1.2 mmol) and triethylamine (122 mg, 176 μL, 1.2 mmol) in aqueous MeCN (1:1, 1.5 mL), the solid sulfonylchloride (21 mg, 0.04 mmol) was added in portions. The reaction mixture turned orange. When the addition was complete, the reaction mixture was stirred for 10 min, diluted with aq. TEAB (Et3N*H.sub.2CO.sub.3, 2 mL, pH=8), frozen and lyophilized. Analytical HPLC: Kinetex, 5 μm C18 100, 250 mm, 4.6 mm, ACN/0.05 M TEAB: 5/95-60/40 in 20 min, 1.2 mL/min; t.sub.R=9.4 min. The title compound was isolated by preparative IPLC with UV-VIS detection (MeCN/TEAB 0.05 M in water, 5:95.fwdarw.30:70 in 20 min detected at 500 nm)

    [0291] .sup.1H NMR (400 MHz, D.sub.2O) δ 9.14 (s, 1H), 8.84 (d, J=9.7 Hz, 1H), 8.57 (d, J=9.6 Hz, 1H), 8.52 (d, J=9.7 Hz, 1H), 8.03 (d, J=9.6 Hz, 1H), 7.99 (s, 1H), 3.20 (d, J=7.2 Hz, 1H), 2.82 (q, J=7.3 Hz, 18H, CH.sub.2 in Et.sub.3N), 0.99 (t, J=7.3 Hz, 27H, CH.sub.3 in Et.sub.3N). .sup.13C NMR (101 MHz, D.sub.2O) δ 144.8, 138.2, 131.3, 131.1, 130.8, 130.4, 128.2, 126.4, 125.5, 124.8, 124.7, 122.5, 120.1, 116.6, 116.0, 115.4, 46.4, 8.0.

    [0292] HR-MS: C.sub.19H.sub.11N.sub.7O.sub.6S.sub.3 found 527.9850 [M−H].sup.−; calculated 527.9860.

    [0293] λ.sub.max (absorption)=454 nm (H.sub.2O), F=23 900 M.sup.−1 cm.sup.−1, λ.sub.max (emission)=531 nm (excitation at 440 nm); Stocks shift 75 nm, fluorescence lifetime 5.6 ns (H.sub.2O; excitation at 440 nm); fluorescence quantum yield: 0.93 (H.sub.2O). See FIG. 23.

    ##STR00060##

    [0294] A 1.5 mL Eppendorf vial was charged with dye 40 (100 μL of 0.02 M solution in water), glucose (5 equiv., 10 μmol, 2 mg), and malonic acid (10 equiv., 20 μL of 1 M solution in DMSO). The closed vial shaken at 40° C. for 1 h (Eppendorf ThermoMixer®), and then the solvents (water and DMSO) were removed by lyophilization (p<0.2 mbar). A solution of 2-picoline-borane complex (10 equiv., 20 μL of 1 M solution in DMSO) was added, and the sample was shaken at 40° C. for 16 hours (Eppendorf ThermoMixer®). The product was isolated by preparative HPLC with UV-VIS detection (MeCN/TEAB 0.05 M in water, 5:95.fwdarw.30:70 in 20 min, detected at 500 nm). The product was characterized by ESI-HRMS, UV-Vis and fluorescence spectroscopy.

    [0295] HR-MS: C.sub.25H.sub.23N.sub.7O.sub.11S.sub.3 found 692.9850 [M−H].sup.−; calculated 692.9860.

    [0296] λ.sub.max (absorption)=483 nm (H.sub.2O), λ.sub.max (emission)=544 nm (excitation at 460 nm); Stocks shift 61 nm, fluorescence lifetime 5.34 ns (H.sub.2O; excitation at 440 nm); fluorescence quantum yield: 0.92 (H.sub.2O). See FIG. 24.

    ##STR00061##

    [0297] N-enzyloxycarbonylazetidini-3-ol was synthesized according to the literature (T. A. Davis, M. W. Danneman, J. N. Johnston. Chem. Comm. 2012, 48, 5578-5580).

    [0298] .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 7.47-7.24 (m, 5H), 5.06 (d, J=0.7 Hz, 2H), 4.62-4.53 (m, 1H), 4.15 (s, 2H), 3.84-3.68 (m, 2H).

    ##STR00062##

    [0299] 1H-Tetrazole (840 mg, 12 mmol) and di-t-butyl N,N-diisopropylphosphoramidite (2.8 g, 3.18 mL, 9.6 mmol) were added to a solution of N-benzyloxycarbonylazetidin-3-ol (1.0 g, 4.8 mmol) in DMF (8 mL) under Ar, and the mixture was stirred at r.t. for 1 h. The course of the reaction was monitored by HPLC. After the starting compound disappeared, the mixture was cooled to 0° C., and H.sub.2O.sub.2 (70% aqueous solution, 1.5 mL) was added. After 15 min the cooling bath was removed, the reaction mixture analyzed by HPLC, and then aqueous Na.sub.2SO.sub.3 (10%, 30 mL) was added (with cooling of the reaction mixture in an ice bath). After 30 min, the reaction mixture was extracted with EtOAc (30 mL×3). The combined solutions were washed with brine, dried over MgSO.sub.4, and evaporated. The residue was submitted to flash chromatography (SNAP Ultra cartridge with 50 g SiO.sub.2, hexane/EtOAc with 20-65% EtOAc-gradient over 15 CV) to provide a white solid (1.2 g, 62% yield).

    [0300] HR-MS: C.sub.19H.sub.30NO.sub.6P found 400.1882 [M+H].sup.+, calculated 400.1884; found 422.1703 [M+Na].sup.+, calculated 422.1703.

    [0301] .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 7.41-7.28 (m, 5H), 5.08 (s, 2H), 5.06-4.98 (m, 1H), 4.33-4.23 (m, 2H), 4.06-3.97 (m, 2H), 1.47 (d, J=0.7 Hz, 18H).

    [0302] .sup.13C NMR (101 MHz, Acetone-d.sub.6) δ 138.0, 129.2, 128.7, 128.6, 83.2, 83.1, 66.9, 66.0, 65.9, 30.0. .sup.31P NMR (162 MHz, Acetone-d.sub.6) δ−10.93.

    ##STR00063##

    [0303] A solution of N-benzyloxycarbonylazetidin-3-yl di(t-butyl)phosphate (46) (640 mg, 1.6 mmol) in MeOH (10 mL) was added to a Schlenk-flask charged with Pd/C (10% Pd in an oxidized form, 160 mg) in THE (3 mL), which was pre-reduced with H.sub.2. The reaction mixture was stirred overnight at r.t. under hydrogen. The reaction mixture was flushed with argon, transferred into centrifuge tubes, the catalyst removed, and washed with THF. The supernatant was evaporated to give a colorless oil (390 mg, 92% yield).

    [0304] HR-MS: C.sub.11H.sub.24NO.sub.4P, found 266.1504 [M+H].sup.+, calculated 266.1516; found 288.1346 [M+Na].sup.+, calculated 288.1335.

    [0305] .sup.1H NMR (400 MHz, Acetonitrile-d.sub.3) δ 5.03-4.94 (m, 1H), 4.26-4.18 (m, 2H), 4.11-4.01 (m, 2H), 1.41 (d, J=0.7 Hz, 18H).

    [0306] .sup.13C NMR (101 MHz, Acetonitrile-d.sub.3) δ 85.6, 85.5, 67.2, 65.2, 55.3, 54.6, 30.0, 29.9, 1.9, 1.7.

    [0307] .sup.31P NMR (162 MHz, Acetonitrile-d.sub.3) δ −12.02.

    ##STR00064##

    [0308] A solid sulfonyl chloride 49 (0.02 mmol, 11 mg) was added in portions to a stirred and cooled (0° C.) solution of azetidin-3-yl di(t-butyl)phosphate 48 (0.2 mmol, 53 mg) and Et.sub.3N (52 μL, 0.4 mmol) in MeCN (2 mL). After stirring for 10 min, the reaction mixture was analyzed by TLC (DCM/MeOH, 10/1), and a spot with R.sub.f=0.5 was detected. The reaction mixture was lyophilized, and the residue was submitted to flash chromatography (SNAP Ultra cartridge with 10 g SiO.sub.2, gradient of DCM/MeOH with 2-15% MeOH over 15 CV) to provide an orange solid (2.0 mg, 8% yield).

    [0309] HR-MS: C.sub.49H.sub.77N.sub.4O.sub.18P.sub.3S.sub.3, found 1199.3645 [M+H].sup.+, calculated 1199.3681; found 1221.3505 [M+Na].sup.+, calculated 1221.3500

    [0310] .sup.1H NMR (400 MHz, Acetonitrile-d.sub.3) δ 9.24 (d, J=9.8, 0.6 Hz, 1H), 9.09 (d, J=0.6 Hz, 1H), 9.05 (d, J=9.7, 0.6 Hz, 1H), 8.89 (d, J=9.8, 0.6 Hz, 1H), 8.60 (d, J=9.7 Hz, 1H), 8.24 (d, J=0.6 Hz, 1H), 6.18 (s, 2H), 4.84-4.72 (m, 3H), 4.26-4.10 (m, 6H), 4.01-3.84 (m, 6H), 1.29 (s, 18H), 1.29 (s, 18H), 1.24 (s, 18H).

    [0311] .sup.31P NMR (162 MHz, Acetonitrile-d.sub.3) 6-11.63 (“d”, J=4.3 Hz), −11.75.

    Example 5

    Synthesis of Fluorescent 1-Aminopyrene Dyes and their Precursors

    [0312] ##STR00065##

    [0313] A solution of sulfonyl chloride 49 (0.2 mmol, 110 mg) in 3.0 mL of ACN was added dropwise to a stirred and cooled (0° C.) solution of 3-(tet-butyldimethylsilyloxy)azetidine (1.35 mmol, 253 mg) and Et.sub.3N (176 μL, 1.35 mmol) in MeCN (1 mL). After stirring for 1 h, the reaction mixture was analyzed by TLC (DCM/MeOH, 10/1), and a spot with R.sub.f=0.98 was detected. The reaction mixture was lyophilized, the residue submitted to flash chromatography (SNAP Ultra cartridge with 25 g SiO.sub.2, gradient of DCM/MeOH with 1-10% MeOH over 15 CV) to provide an orange solid (48 mg, 25% yield).

    [0314] HR-MS: C.sub.43H.sub.68N.sub.4O.sub.9S.sub.3Si.sub.3, found 965.3522 [M+H].sup.+, calculated 965.3529; found 987.3341 [M+Na].sup.+, calculated 987.3348

    [0315] .sup.1H NMR (400 MHz, Acetonitrile-d.sub.3) δ 9.24 (d, J=9.8 Hz, 1H), 9.08 (s, 1H), 9.06 (d, J=9.6 Hz, 1H), 8.90 (d, J=9.7 Hz, 1H), 8.57 (d, J=9.7 Hz, 1H), 8.22 (s, 1H), 6.11 (s, 2H), 4.55-4.43 (m, 3H), 4.11-4.01 (m, 6H), 3.75-3.62 (m, 6H), 0.67 (d, J=0.7 Hz, 18H), 0.61 (s, 10H), −0.09 (d, J=0.5 Hz, 12H), −0.13 (s, 6H).

    [0316] .sup.13C NMR (126 MHz, Acetonitrile-d.sub.3) δ 147.7, 135.4, 134.4, 131.9, 130.3, 128.0, 127.3, 127.2, 127.1, 126.6, 124.2, 121.4, 119.5, 61.5, 61.4, 61.3, 61.2, 25.8, 25.7, 18.3, −5.0, −5.05, −5.1.

    ##STR00066##

    [0317] The reaction was carried out in a plastic test-tube (with a screw-cap) used for centrifugation. To a stirred solution of TBDMS-pyrene (44 mg, 0.45 mmol) in MeCN, 50-55% aq. HF (74 μL, 2.33 mmol) was added at 0° C. After stirring for 4 h, an additional amount of HF was added (74 μL, 2.33 mmol), and the reaction mixture was stirred overnight at r.t. The reaction mixture was analyzed by TLC (DCM/MeOH, 10/1), a new spot with R.sub.f=0.25 was detected. The reaction was “quenched” by addition of an aqueous sodium bicarbonate solution (5%, 10 mL) and brine. The product was extracted with DCM/iPrOH (1:1; 3×100 mL). The combined organic solutions were dried over MgSO.sub.4 and concentrated in vacuo. The residue was submitted to flash chromatography (SNAP Ultra cartridge with 25 g SiO.sub.2, gradient of DCM/MeOH with 2-20% MeOH over 15 CV) to provide an orange solid (27 mg, 96%).

    [0318] HR-MS: C.sub.25H.sub.26N.sub.4O.sub.9S.sub.3, found 623.0913 [M+H].sup.+, calculated 623.0935; found 621.0773 [M−H].sup.−, calculated 624.0789.

    [0319] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.06 (d, J=9.7 Hz, 1H), 8.91 (s, 1H), 8.87 (d, J=9.7 Hz, 1H), 8.83 (d, J=9.7 Hz, 1H), 8.70 (d, J=9.8 Hz, 1H), 8.16 (s, 1H), 7.70 (s, 2H), 5.72-5.59 (m, 3H), 4.38-4.24 (m, 3H), 4.07-3.91 (m, 6H), 3.65-3.45 (m, 6H).

    [0320] .sup.13C NMR (101 MHz, DMSO-d.sub.6) δ 148.2, 134.3, 133.3, 133.1, 130.6, 129.1, 127.1, 126.6, 125.9, 125.0, 124.0, 121.9, 119.1, 117.1, 116.7, 115.8, 60.2, 60.0, 58.6, 58.5, 40.4.

    [0321] λ.sub.max (absorption)=483 nm (ε=19000 M.sup.−1 cm.sup.−1, MeOH), λ.sub.max (emission)=534 nm (MeOH; excitation at 450 nm); Stocks shift 51 nm, fluorescence lifetime 5.6 ns (MeOH), fluorescence quantum yield: 0.77 (absolute value in MeOH). See FIG. 25.

    ##STR00067##

    [0322] Ester 50 (2.0 mg, 1.67 μmol) was dissolved in dichloromethane (0.5 mL), the solution cooled to +5° C. in an ice bath, and then a solution of TFA in DCM (5% v/v, 0.5 mL) was added slowly with stirring. The reaction mixture was allowed to warm-up to r. t. and stirred for 2 h. The volatile materials were removed in vacuo. The residue was treated with 1 M aq. Et.sub.3N*H.sub.2CO.sub.3 buffer (TEAB; pH=8-9) and stirred, until the pH stabilized at 8-9. The title compound was isolated by preparative HPLC (Kinetex 5 μm EVO C18 100A 250×21 mm column, MeCN/water+0.05 M TEAB, 10 mL/min, 5-30% MeCN over 20 min) to afford 2 mg of dye 52 as red solid (88% yield of pentakis triethylammonium salt, according to .sup.1H-NMR).

    [0323] HR-MS: C.sub.25H.sub.29N.sub.4O.sub.18P.sub.3S.sub.3, found 860.9760 [M−H].sup.−, calculated 860.9779.

    [0324] .sup.1H NMR (400 MHz, D.sub.2O) δ 9.05 (s, 1H), 8.97 (d, J=9.8 Hz, 1H), 8.72 (d, 1H), 8.63 (d, J=7.0 Hz, 1H), 8.44 (d, J=16.6 Hz, 1H), 8.14 (s, 1H), 4.23-4.13 (m, 6H), 4.04-3.89 (m, 6H), 3.17 (q, 30H), 1.26 (t, J=7.3, 0.6 Hz, 45H).

    [0325] λ.sub.max (absorption)=476 nm (H.sub.2O), λ.sub.max (emission)=543 nm (H.sub.2O; excitation at 450 nm); Stocks shift 67 nm, fluorescence lifetime 5.9 ns (H.sub.2O), fluorescence quantum yield: 0.8 (absolute value in H.sub.2O). See FIG. 26.

    ##STR00068##

    [0326] A 1.5 mL Eppendorf vial was charged with dye 52 (2 mg, 1.5 μmol), glucose (5 equiv., 7.5 μmol, 1.5 mg) and malonic acid (10 equiv., 15 μL of 1 M solution in DMSO). The sample was stirred at 40° C. for 1 h (Eppendorf ThermoMixer®), and then water and DMSO were removed under reduced pressure (p<0.2 mbar) in lyophilizer. A solution of 2-picoline-borane complex (10 equiv., 15 μL of 1 M solution in DMSO) was added to the residue, and the samples were shaken at 40° C. for 16 h (Eppendorf ThermoMixer®). The product was isolated by preparative HPLC with UV-VIS detection (MeCN/TEAB 0.05 M in water, 5:95.fwdarw.30:70 in 20 min, detected at 500 nm).

    [0327] HR-MS: C.sub.31H.sub.41N.sub.4O.sub.23P.sub.3S.sub.3, found 1025.3522 [M−H].sup.−, calculated 1025.3529.

    [0328] λ.sub.max (absorption)=505 nm (H.sub.2O), λ.sub.max (emission)=565 nm (H.sub.2O; excitation at 485 nm); Stocks shift 60 nm, fluorescence lifetime 5.8 ns (H.sub.2O), fluorescence quantum yield: 0.85 (absolute value in H.sub.2O). See FIG. 27.

    Example 6

    Conjugation of Aminopyrene Dyes with Sugars (“Sugar Ladders”)

    [0329] General Procedure:

    [0330] 1.5 mL Eppendorf vial was charged with a dye (compound 40 or APTS, 10 μL of 0.1 M solution in water), a dextran ladder (1.0 mg, maltodextrin oligosaccharides—DP2 to DP15, Carbosynth), and malonic acid (10 equiv., 10 μL of 1 M solution in DMSO). The samples were shaken at 40° C. for 1 h (Eppendorf ThermoMixer®), and then solvents were removed under reduced pressure (p<0.2 mbar) in lyophilizer. A solution of 2-picoline-borane complex (10 equiv., 10 μL of 1 M solution in DMSO) was added, and the samples were shaken at 40° C. for 16 h (Eppendorf ThermoMixer®). The products were isolated by preparative HPLC with UV-VIS detection (MeCN/aq. TEAB 5:95.fwdarw.30:70 in 20 min detected at 500 nm) or FC (C18, 15C18AQ-F0025 multi-use cartridge, MeCN/aq. TEAB 5:95.fwdarw.30:70).

    [0331] Analytical HPLC: Kinetex, 5 μm C18 100, 25 cm×4.6 mm, MeCN/aq. TEAB 5:95.fwdarw.30:70 in 20 min detected at 500 nm, 1.2 mL/min;

    [0332] Preparative HPLC: Kinetex, 5 μm C18 100, 25 cm×10 mm, MeCN/aq. TEAB 5:95.fwdarw.30:70 in 20 min detected at 500 nm, 4 mL/min;

    [0333] Flash chromatography: RP (C18) cartridge, 15C18AQ-F0025 (Interchim), MeCN/aq. TEAB 5:95.fwdarw.30:70