US 12,174,197 B2
Sulfonated 2(7)-aminoacridone and 1-aminopyrene dyes and their use as fluorescent tags, in particular for carbohydrate analysis
Stefan W. Hell, Goettingen (DE); Vladimir N. Belov, Goettingen (DE); Matthias Bischoff, Dortmund (DE); Dirk Meineke, Cologne (DE); Laura Thomas, Wustrow (DE); Gyuzel Mitronova, Goettingen (DE); Elizaveta Savicheva, Goettingen (DE); Kirill Kolmakov, Magdeburg (DE); Marvin J. Boehm, Uslar (DE); Erdmann Rapp, Magdeburg (DE); and René Hennig, Magdeburg (DE)
Assigned to Max-Planck-Gesellschaft zur Foerderung der Wissenschaften e. V., Munich (DE)
Appl. No. 17/424,415
Filed by Max-Planck-Gesellschaft zur Foerderung der Wissenschaften e. V., Munich (DE)
PCT Filed Jan. 21, 2019, PCT No. PCT/EP2019/051396
§ 371(c)(1), (2) Date Jul. 20, 2021,
PCT Pub. No. WO2020/151804, PCT Pub. Date Jul. 30, 2020.
Prior Publication US 2022/0107325 A1, Apr. 7, 2022
Int. Cl. C09B 15/00 (2006.01); C09B 57/00 (2006.01); G01N 33/58 (2006.01)
CPC G01N 33/582 (2013.01) [C09B 15/00 (2013.01); C09B 57/001 (2013.01); G01N 2400/10 (2013.01)] 20 Claims
OG exemplary drawing
 
1. A fluorescent dye having multiple ionizable and/or negatively charged groups which is selected from the group consisting of compounds of the following general Formula B or of salts thereof:

OG Complex Work Unit Chemistry
wherein R1 and/or R2 are independent from each other and may represent:
H, deuterium (D), alkyl or deutero-substituted alkyl;
R1-R2 may form a four-, five-, six-, or seven-membered non-aromatic carbocycle with an additional primary amino group NH2, secondary amino group NHRa, where Ra═C1-C6 alkyl, or hydroxyl group OH attached to one carbon atom of this carbocycle; or
R1-R2 may form a four-, five-, six-, or seven-membered non-aromatic heterocycle with an additional heteroatom included in the heterocycle;
a hydroxyalkyl group (CH2)mOH, where m=1-12, with a straight or branched alkyl chain;
one of R1 or R2 groups may be a carbonate or carbamate derivative (CH2)mOCOOR4 or (CH2)mNHCOOR4, where m=1-12 and R4=methyl, ethyl, 2-chloroethyl, N-succinimidyl, sulfo-N-succinimidyl, 1-oxybenzotriazolyl, a phenyl group or a substituted phenyl group;
(CH2)mNRaRb, where m=1-12, with a straight or branched alkyl chain; Ra, Rb are independent from each other and may be H, or optionally substituted C1-C4 alkyl group(s);
one of R1 or R2 groups may be a primary amino group to form aryl hydrazines Ar-NR6NH2 where Ar is the entire pyrene residue in Formula B and R6═H or alkyl;
one of R1 or R2 groups may be a hydroxy group to form aryl hydroxylamines Ar-NR7OH where Ar is an entire pyrene residue in Formula B and R7═H or alkyl;
one of R1 or R2 groups may be an alkyloxyamino group (CH2)nONH2 with n=1-12;
one of R1 or R2 groups may be CO(CH2)nCOOR, with n=1-5 and a straight or branched alkyl chain (CH2)n and with R8 selected from H, straight or branched C1-C6 alkyl, CH2CN, 2- and 4-nitrophenyl, 2,3,5,6-tetrafluorophenyl, pentachlorophenyl, pentafluoro-phenyl, N-succinimidyl, sulfo-N-succinimidyl, 1-oxybenzo-triazolyl;
further, one of R1 or R2 may be (CH2)nCONHR9, with n=1-5 and R9═H, C1-C6 alkyl, (CH2)mN3, (CH2)m—N-maleimido, (CH2)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;
wherein linker L may be divalent or polyvalent and comprises at least one carbon atom and may represent alkyl, heteroalkyl, perfluoroalkyl, alkyloxy, single or multiple difluoromethyl (CF2), and alkene or alkine moieties in any combinations, at any occurrence, linear or branched, with a length ranging from C1 to C12;
the linker L may also include a carbonyl (CH2CO, CF2CO) moiety, also as a part of an amide group; or
the linker L may also comprise or contain a residue of 1,3,5-triazine, thus providing two attachment points for group X;
X denotes a solubilizing and/or ionizable anion-providing moiety,
further, the anion-providing moiety is optionally linked through non-aromatic O, N and S-containing heterocycles, or, alternatively, one of the groups X optionally bears any of the moieties listed above for groups R1 and R2, also with any type of linkage listed for group L, and independently from other substituents;
with the proviso that in all compounds represented by Formula B three or six negatively charged groups are present in the residues X of Formula B under basic conditions, and the negatively charged groups represent at least partially deprotonated residues of ionizable groups selected from the following: OH, SH, COOH, SO3H, OSO3H, OP(O)(OH)2, OP(O)(OH)Ra, where Ra═C1-C4 alkyl or substituted C1-C4 alkyl, P(O)(OH)2, and P(O)(OH)Ra, where Ra═C1-C4 alkyl or substituted C1-C4 alkyl.