Advanced methods for automated high-performance identification of carbohydrates and carbohydrate mixture composition patterns and systems therefore as well as methods for calibration of multi wavelength fluorescence detection systems therefore, based on new fluorescent dyes

A method for an automated determination and/or identification of carbohydrates and/or carbohydrate mixture composition pattern profiling comprising the steps of:

• a) obtaining a sample containing at least one carbohydrate;
• b) labelling said carbohydrate(s) with a first fluorescent label;
• c) providing a standard of known composition labelled with a second fluorescent label;
• d) determining the migration/retention time(s) of said carbohydrate(s) and the standard of known composition using electrokinetic/chromatographic separation techniques combined with fluorescence or laser induced fluorescence detection;
• e) aligning the migration/retention time(s) to migration/retention time indice(s) based on given standard migration/retention time indice(s) of the standard;
• f) comparing these migration/retention time indice(s) of the carbohydrate(s) with standard migration/retention time indice(s) from a database;
• g) identifying or determining the carbohydrate(s) and/or the carbohydrate mixture composition pattern,
• wherein the standard composition is added to the sample containing the unknown carbohydrate and/or carbohydrate mixture composition, the first fluorescent label and the second fluorescent label are different and wherein the first fluorescent label or the second fluorescent label is a fluorescent dye, preferably having multiple ionizable and/or negatively charged groups
• which is selected from the group consisting of compounds of the following general Formula A and B:
• wherein
• R1, R2, R3, R4, R5 are independent from each other and may represent:
• H, CH3, C2H5, a straight or branched C3-C12, preferably C3-C6, alkyl or perfluoroalkyl group, a phosphonylated alkyl group (CH2)mP(O)(OH)2, where m=1-12, preferably 2-6, with a straight or branched alkyl chain, (CH2)nCOOH, where n=1-12, preferably 1-5, or (CH2)nCOOR6, where n=1-12, preferably 1-5, and
• R6 may be alkyl, in particular C1-C6, CH2CN, benzyl, fluorene-9-yl, polyhalogenoalkyl, polyhalogenophenyl, e.g. tetra- or pentafluorophenyl, pentachlorophenyl, 2- and 4-nitrophenyl, N-succinimidyl, sulfo-N-succinimidyl, 1-oxybenzotriazolyl, or other potentially nucleophile-reactive leaving groups, alkyl sulfonate ((CH2)nSO3H) or alkyl sulfate ((CH2)nOSO3H) where n=1-12, preferably 1-5, and the alkyl chain in any (CH2)n may be straight or branched;
• a hydroxyalkyl group (CH2)mOH or thioalkyl group (CH2)mSH, where m=1-12, preferably 2-6, with a straight or branched alkyl chain, a phosphorylated hydroxyalkyl group (CH2)mOP(O)(OH)2, where m=1-12, preferably 2-6, with a straight or branched alkyl chain;
• one of R1 or R2 groups may be a carbonate or carbamate derivative of (CH2)mOCOOR7 or COOR7, where m=1-12 and R7=methyl, ethyl, tert-butyl, benzyl, fluoren-9-yl, CH2CN, N-succinimidyl, sulfo-N-succinimidyl, 1-oxybenzotriazolyl, phenyl, substituted phenyl group, e.g., 2- or 4-nitrophenyl, pentachlorophenyl, penta-fluorophenyl, 2,3,5,6-tetrafluorophenyl, 2-pyridyl, 4-pyridyl, pyrimid-4-yl;
• (CH2)mNRaRb, where m=1-12, preferably 2-6, with a straight or branched alkyl chain;
• Ra, Rb are independent from each other and represent hydrogen and/or C1-C4 alkyl groups, a hydroxyalkyl group (CH2)mOH, where m=2-6, with a straight or branched alkyl chain, a phosphorylated hydroxyalkyl group
• (CH2)mOP(O)(OH)2, where m=1-12, preferably 2-6, with a straight or branched alkyl chain;
• an alkyl azide (CH2)mN3, where m=m=1-12, preferably 2-6, with a straight or branched alkyl chain;
• R1, R2, R3, R4, R5 may contain a terminal alkyloxyamino group (CH2)mONH2, where m=1-12, preferably 2-6, with a straight or branched alkyl chain, that can include one or multiple alkylamino (CH2)mNH or alkylamido (CH2)mCONH groups in all possible combinations with m=0-12;
• (CH2)nCONHRB, with n=1-12, preferably 1-5;
• R8=H, C1-C6 alkyl, (CH2)mN3, or (CH2)m—N-maleimido, (CH2)m—NH—COCH2X (X=Br or I), with m=1-12, preferably 2-6, and with straight or branched alkyl chains in (CH2)n, (CH2)m and R8;
• a primary amino group, preferably as R1, R2, or R3, which forms aryl hydrazines;
• a hydroxy group, preferably as R2 or R3, which forms aryl hydroxylamines;
• further, one of the residues R1, R2, R3, R4, R5 may represent CH2—C6H4—NH2, COC6H4—NH2, CONHC6H4—NH2 or CSNHC6H4—NH2 with C6H4 being a 1,2-, 1,3- or 1,4-phenylene, COC5H3N—NH2 or CH2—C5H3N—NH2, with C5H3N being pyridin-2,4-diyl, pyridin-2,5-diyl, pyridin-2,6-diyl, or pyridin-3,5-diyl;
• additionally, R2-R3 (R4-R5) may form a four-, five, six-, or seven-membered cycle, or a four-, five, six-, or seven-membered cycle with or without a primary amino group NH2, secondary amino group NHRa, where Ra=C1-C6 alkyl, a hydroxyl group OH, or a phosphorylated hydroxyl group —OP(O)(OH)2 attached to one of the carbon atoms in this cycle;
• optionally R2-R3 (R4-R5) may form a four-, five, six-, or seven-membered heterocycle with an additional 1-3 heteroatoms such as O, N or S included into this heterocycle;
• further, R1 may represent an unsubstituted phenyl group, a phenyl group with one or several electron-donor substituents chosen from the set of OH, SH, NH2, NHRa, NRaRb, RaO, RaS, where Ra and Rb are independent from each other and may be C1-C6 alkyl groups with straight or branched carbon chains, a phenyl group with one or several electron-acceptors chosen from the set of NO2, CN, COH, COOH, CH═CHCN, CH═C(CN)2, SO2Ra, CORa, COORa, CH═CHCORa, CH═CHCOORa, CONHRa, SO2NRaRb, CONRaRb, where Ra and Rb are independent from each other and may be H, or C1-C6 alkyl group(s) with straight or branched carbon chains;
• or R1 may represent a heteroaromatic group;
• with the proviso that in all compounds of Formula A above at least two, preferably at least 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:
• SH, COOH, a sulfonic acid residue SO3H, a primary phosphate group OP(O)(OH)2, a secondary phosphate group OP(O)(OH)Ra, where Ra=C1-C4 alkyl or substituted C1-C4 alkyl, a primary phosphonate group P(O)(OH)2, a secondary phosphonate group P(O)(OH)Ra, where Ra=C1-C4 alkyl or substituted C1-C4 alkyl;
• and compounds of Formula A can exist and can be used as salts, solvates and hydrates, preferably as salts with alkaline metal cations including Na+, Li+, K+ and organic ammonium or organic phosphonium cations;
• wherein R1 and/or R2 are independent from each other and may represent:
• H, CH3, C2H5, a linear or branched C3-C12 alkyl or perfluoroalkyl group, or a substituted C2-C612 alkyl group;
• in particular, (CH2)nCOOR3, where n=1-12, preferably 1-5, R3 may be H, alkyl, in particular C1-C6, CH2CN, benzyl, fluorene-9-yl, polyhalogenoalkyl, polyhalogenophenyl, e.g. tetra- or pentafluorophenyl, pentachlorophenyl, 2- and 4-nitrophenyl, N-succinimidyl, sulfo-N-succinimidyl, 1-oxybenzotriazolyl, or other potentially nucleophile-reactive leaving groups, and the alkyl chain in (CH2)n may be straight or branched;
• and
• R1-R2 may form a four-, five, six-, or seven-membered non-aromatic carbocycle with an additional primary amino group NH2, secondary amino group NHRa, where Ra=C1-C6 alkyl, or hydroxyl group OH attached to one of the carbon atoms in this cycle;
• optionally R1-R2 may form a four-, five, six-, or seven-membered non-aromatic heterocycle with an additional heteroatom such as O, N or S included into this heterocycle;
• a hydroxyalkyl group (CH2)mOH, where m=1-12, preferably 2-6, with a straight or branched alkyl chain;
• one of R1 or R2 groups may be a carbonate or carbamate derivative (CH2)mOCOOR4 or COOR4, where m=1-12 and R4=methyl, ethyl, 2-chloroethyl, N-succinimidyl, sulfo-N-succinimidyl, 1-oxybenzotriazolyl, a phenyl group or substituted phenyl group, e.g., 2- and 4-nitrophenyl, pentachlorophenyl, pentafluorophenyl, 2,3,5,6-tetrafluoro-phenyl, 2-pyridyl, or 4-pyridyl;
• (CH2)mNRaRb, where m=1-12, preferably 2-6, with a straight or branched alkyl chain;
• Ra, Rb are independent from each other and may be H, or optionally substituted C1-C4 alkyl group(s), in particular, one of R1 or R2 groups may be an alkyl azide group (CH2)mN3 with m=2-6 and a straight or branched alkyl chain;
• one of R1 or R2 may be (CH2)nSO2NR5NH2 with n=1-12, while the substituent R5 can be represented by H, alkyl, hydroxyalkyl or perfluoroalkyl groups C1-C12;
• one of R1 or R2 groups may be a primary amino group to form aryl hydrazines Ar—NR6NH2 where Ar is the entire pyrene residue in Formula B and R6=H or alkyl;
• one of R1 or R2 groups may be a hydroxy group to form aryl hydroxylamines Ar—NR7OH where Ar is the entire pyrene residue in Formula B and R7=H or alkyl;
• one of R1 or R2 groups may contain a terminal alkyloxyamino group (CH2)nONH2 with n=1-12, which can be linked via one or multiple alkylamino (CH2)mNH, alkylamido (CH2)mCONH, alkyl ether or ester group(s) in all possible combinations with m=0-12;
• one of R1 or R2 groups may be CO(CH2)nCOORB, with n=1-5 and a straight or branched alkyl chain (CH2)n and with R8 selected from H, straight or branched C1-C6 alkyl, CH2CN, 2- and 4-nitrophenyl, 2,3,5,6-tetrafluorophenyl, pentachlorophenyl, pentafluoro-phenyl, N-succinimidyl, sulfo-N-succinimidyl, 1-oxybenzotriazolyl;
• further, one of R1 or R2 may be (CH2)nCONHR9, with n=1-5 and R9=H, C1-C6 alkyl, (CH2)mN3, (CH2)m—N-maleimido, (CH2)m—NHCOCH2X (X=Br or I), where m=2-6 and with straight or branched alkyl chains in (CH2)n and R9;
• or one of R1 or R2 may represent CH2—C6H4—NH2, COC6H4—NH2, CONHC6H4—NH2 or CSNHC6H4—NH2 with C6H4 being a 1,2-, 1,3- or 1,4-phenylene, COC5H3N—NH2 or CH2—C5H3N—NH2, with C5H3N being pyridin-2,4-diyl, pyridin-2,5-diyl, pyridin-2,6-diyl, or pyridin-3,5-diyl;
• or one of R1 or R2 may be an alkyl azide (CH)N3 or alkine, in particular propargyl;
• the linker L comprises at least one carbon atom and may comprise alkyl, heteroalkyl, in particular alkyloxy such as CH2OCH2, CH2CH2O CH2CH2OCH2, alkylamino or dialkylamino, particularly diethanolamine or N-methyl (alkyl) monoethanolamine moieties such as N(CH3)CH2CH2O— and N(CH2CH2O—)2, perfluoroalkyl, like single or multiple difluoromethyl (CF2), alkene or alkyne moieties in any combinations, at any occurrence, linear or branched, with the length ranging from C1 to C12;
• the linker L may also include a carbonyl (CH2CO, CF2CO) moiety, also as part of an amide group;
• the linker L may also comprise or contain a residue of 1,3,5-triazine, thus providing two attachment points for group X;
• X denotes a solubilizing and/or ionizable anion-providing moiety, in particular consisting of or including a moiety selected from the group comprising hydroxyalkyl (CH2)nOH, thioalkyl ((CH2)nSH), carboxy alkyl ((CH2)nCO2H), alkyl sulfonate ((CH2)nSO3H), alkyl sulfate ((CH2)nOSO3H), alkyl phosphate ((CH2)nOP(O)(OH)2) or phosphonate ((CH2)nP(O)(OH)2), wherein n is an integer ranging from 0 to 12, or an analogon thereof wherein one or more of the CH2 groups are replaced by CF2,
• further, the anion-providing moieties may be linked by means of non-aromatic O, N and S-containing heterocycles, e. g., piperazines, pipecolines, or, alternatively, one of the groups X may bear any of the moieties listed above for groups R1 and R2, also with any type of linkage listed for group L, and independently from other substituents;
• Compounds of Formula B can exist and can be used as salts, solvates and hydrates, preferably as salts with alkaline metal cations including Na+, Li+, K+ and organic ammonium.
• With the proviso that in all compounds represented by Formula B three or six negatively charged groups are present in the residues X of Formula B under basic conditions, i.e. 7<pH<14, and these negatively charged groups represent at least partially deprotonated residues of ionizable groups selected from the following:
• SH, COOH, SO3H, OP(O)(OH)2, OP(O)(OH)Ra, where Ra=C1-C4 alkyl or substituted C1-C4 alkyl, P(O)(OH)2, P(O)(OH)Ra, where Ra=C1-C4 alkyl or substituted C1-C4 alkyl is provided;
• and compounds of Formula B can exist and can be used as salts, solvates and hydrates, preferably as salts with alkaline metal cations including Na+, Li+, K+ and organic ammonium or organic phosphonium cations;