# US 8,785,637 B2 · Carboxamide-substituted dyes for analytical applications

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- Number: US 8,785,637 B2
- Document ID: US8785637B2
- Status: Granted
- Kind: B2
- Application number: 13/962,736
- Filed: 2013-08-08
- Published: 2013-12-05
- Granted: 2014-07-22
- Earliest claimed: 2002-12-18
- Assignees: ATTO-TEC GmbH
- Applicants: ATTO-TEC GmbH
- Inventors: Jutta Arden-Jacob; Karl-Heinz Drexhage; Monika Hamers-Schneider; Norbert Kemnitzer; Alexander Zilles
- Web page: https://patentgrep.com/patent/US8785637B2

## CPC

- C09B 57/00: Other synthetic dyes of known constitution
- C07H 21/00: Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C09B 11/04: derived from triarylmethanes {, i.e. central C-atom is substituted by amino, cyano, alkyl}
- G01N 21/6486: {Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence)}
- Y10T 436/143333: Saccharide [e.g., DNA, etc.]
- C09B: ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES {, e.g. PIGMENTS}; MORDANTS; LAKES (fermentation or enzyme using processes to synthesise a desired chemical compound C12P)
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

The present invention relates to carboxamide-substituted dyes, the production and use of such dyes as labeling groups in analytics.

## Claims

### Claim 1

A carboxamide-substituted dye of the formula (I)
in which
Y=sulfur, selenium, a direct linkage or is NRc;
R1, R3, R4 are independently hydrogen, halogen, —O⊖, a hydroxyl group, thiol group, amino group, ammonium group, sulfo group, phospho group, nitro group, carbonyl group, carboxyl group, a carboxylic acid group, a nitrile group, isonitrile group, cyanate group, isocyanate group, thiocyanate group, isothiocyanate group or a straight-chain, branched or cyclic saturated or unsaturated hydrocarbon group having up to 40 carbon atoms;
Rc independently is as defined for R1, R3, R4;
in which
R7, R8, R9 independently are hydrogen or a straight-chain, branched or cyclic saturated or unsaturated hydrocarbon group having up to 40 carbon atoms; or
R1 together with R2 is
in which
R10, R11, R13 are as defined for R1, R3, R4;
in which
R16, R17, R18 are as defined for R7, R8, R9;
R5, R6, independently are a straight-chain, branched or cyclic saturated or unsaturated hydrocarbon moiety having up to 40 carbon atoms;
Cyc1 is selected from aromatic, heteroaromatic, quinoidal and cycloaliphatic rings;
Cyc2 is selected from aromatic, heteroaromatic, quinoidal and cycloaliphatic rings;
each of said moieties in the dye of the formula (I) being able to form a ring system with one or more neighboring moieties; and
X being one or more mono- or multivalent anions, when required for balancing the charge.

### Claim 2 (depends on 1)

The carboxamide-substituted dye as claimed in claim 1, in which Cyc2 is a nitrogen-containing heterocycle or a ring system substituted with at least one amino group
or/and
or,
together with
in which
in which R7, R8; R10, R11, R13 and R16, R17 are as defined in claim 1.

### Claim 3 (depends on 1)

The carboxamide-substituted dye as claimed in claim 1, in which Cyc2 in the formula (I) has a structure (A), (B), (C), (D), (E), (F), (G), (H) or (J),
in which R in each case independently is defined as R1, R3, R4 in claim 1; R19, R20 and R22, R23 are independently defined as R7, R8 in claim 1; and R21 is defined as R7 in claim 1 and the dashed lines are optionally double bonds in the presence of which the moieties bound via a dashed line are absent.

### Claim 4 (depends on 1)

The carboxamide-substituted dye as claimed in claim 1, in which Cyc1 is substituted or unsubstituted phenyl, naphthyl, pyridyl or cyclohexyl.

### Claim 5 (depends on 1)

The carboxamide-substituted dye as claimed in claim 1, in which
where R7 and R8 are as defined in claim 1.

### Claim 6 (depends on 5)

The carboxamide-substituted dye as claimed in claim 5, in which R1 is bridged with R8 or R3 is bridged with R7 or R1 is bridged with R8 and R3 is bridged with R7 forming a ring system.

### Claim 7 (depends on 6)

The carboxamide-substituted dye as claimed in claim 6, in which the ring system comprises 5- or 6-membered rings.

### Claim 8 (depends on 7)

The carboxamide-substituted dye as claimed in claim 7, in which a ring system of the structure (K), (L), (M), (N) or (O) is formed:
in which R is independently defined as R1, R3, R4 and R7, R8 independently are hydrogen or a straight-chain, branched or cyclic saturated or unsaturated hydrocarbon group having up to 40 carbon atoms,
and the dashed lines are optionally double bonds in the presence of which the moieties bound via a dashed line are absent.

### Claim 9 (depends on 1)

The carboxamide-substituted dye as claimed in claim 1, in which
R2 together with R1 is
where R10-R13 are as defined in claim 1.

### Claim 10 (depends on 9)

The carboxamide-substituted dye as claimed in claim 9, in which
R12=O.

### Claim 11 (depends on 9)

The carboxamide-substituted dye as claimed in claim 9, in which
where R16 and R17 independently are hydrogen or a straight-chain, branched or cyclic saturated or unsaturated hydrocarbon group having up to 40 carbon atoms are independently hydrogen, halogen, O⊖, a hydroxyl group, thiol group, amino group, ammonium group, sulfo group, phospho group, nitro group, carbonyl group, carboxyl group, a carboxylic acid derivative, a nitrile group, isonitrile group, cyanate group, isocyanate group, thiocyanate group, isothiocyanate group or a straight-chain, branched or cyclic saturated or unsaturated hydrocarbon group having up to 40 carbon atoms.

### Claim 12 (depends on 3)

The carboxamide-substituted dye as claimed in claim 3, in which Cyc1 is optionally substituted phenyl, Cyc2 has the structure (E) and Y=S or is NRc and R7 and R3 form a ring system (K), R7 is hydrogen or a straight-chain, branched or cyclic saturated or unsaturated hydrocarbon group having up to 40 carbon atoms, R3 is hydrogen, halogen, —O⊖, a hydroxyl group, thiol group, amino group, ammonium group, sulfa group, phospho group, nitro group, carbonyl group, carboxyl group, a carboxylic acid derivative, a nitrile group, isonitrile group, cyanate group, isocyanate group, thiocyanate group, isothiocyanate group or a straight-chain, branched or cyclic saturated or unsaturated hydrocarbon group having up to 40 carbon atoms.

### Claim 13 (depends on 3)

The carboxamide-substituted dye as claimed in claim 3, in which Cyc1 is optionally substituted phenyl, Cyc2 has the structure (A) and Y=sulfur, selenium, a direct linkage or is NRc, wherein Rc is independently hydrogen, halogen, —O⊖, a hydroxyl group, thiol group, amino group, ammonium group, sulfa group, phospho group, nitro group, carbonyl group, carboxyl group, a carboxylic acid derivative, a nitrile group, isonitrile group, cyanate group, isocyanate group, thiocyanate group, isothiocyanate group or a straight-chain, branched or cyclic saturated or unsaturated hydrocarbon group having up to 40 carbon atoms.

### Claim 14 (depends on 1)

A multichromophore system in which the carboxamide-substituted dye as claimed in claim 1 is coupled via R5 or R6 to one or more further carboxamide-substituted dye molecules as claimed in claim 1, wherein R5 and R6 independently are a straight-chain, branched or cyclic saturated or unsaturated hydrocarbon group having up to 40 carbon atoms.

### Claim 15 (depends on 14)

The multichromophore system as claimed in claim 14, in which coupling takes place on R5 or R6 of the further carboxamide-substituted dyes, R5 and R6 independently are a straight-chain, branched or cyclic saturated or unsaturated hydrocarbon group having up to 40 carbon atoms.

### Claim 16 (depends on 1)

A multichromophore system of the formula (III)
where the moieties are as defined in claim 1, R in each case independently is defined as R1, R3, R4 and R24, R25 and R26, R27 are defined as R7, R8 in claim 1, with n independently being 0, 1, 2 or 3 and m being 0, 1, 2, 3 or 4;
X being one or more mono- or multivalent anions, when required for balancing the charge.

### Claim 17 (depends on 1)

A process for preparing the carboxamide-substituted dyes of the formula (I) as claimed in claim 1, comprising the following steps:
(a) converting the carboxyl group of a dye of the formula (II)
in which the moieties are defined as indicated in claim 1, into an activated form;
(b) reacting the activated dye obtained in step (a) with a secondary amine HNR5R6 resulting in the carboxamide-substituted dye of claim 1; and
(c) optionally isolating the carboxamide-substituted dye of the formula (I) obtained in step (b).

### Claim 18 (depends on 17)

The process as claimed in claim 17, in which step (a) is carried out at temperatures of from room temperature to 60° C.

### Claim 19 (depends on 17)

The process as claimed in claim 17, wherein an aprotic solvent is used in the reacting step (b).

### Claim 20 (depends on 17)

The process as claimed in claim 17, in which N-hydroxysuccinimide, N-hydroxyphthalimide, N-hydroxynaphthalimide, O—(N-succinimidyl)-N,N,N′,N′-tetramethyluronim and tetrafluoroborate (TSTU) are used for activation.

### Claim 21 (depends on 1)

A method of using the carboxamide-substituted dye as claimed in claim 1 for qualitative or quantitative determination of an analyte comprising the step of:
coupling the carboxamide-substituted dye of the formula (I) to the analyte to be detected or to a component of a detection reagent or to a support, and
determining the analyte through quantitative or qualitative means.

### Claim 22 (depends on 21)

The method as claimed in claim 21, in which detection comprises at least one of an immunological detection and detection by way of nucleic acid hybridization.

### Claim 23 (depends on 1)

A conjugate of the carboxamide-substituted dye of the formula (I) as claimed in claim 1 and a binding partner.

### Claim 24 (depends on 23)

The conjugate as claimed in claim 23, in which the binding partner is selected from among peptides, polypeptides, nucleic acids, nucleosides, nucleotides, nucleic acid analogs and haptens.

### Claim 25 (depends on 23)

A method of using the conjugate as claimed in claim 23, wherein coupling of the carboxamide-substituted dye takes place to the respective binding partner, and carrying out detection by nucleic acid hybridization processes and immunochemical processes.

### Claim 26 (depends on 21)

The method as claimed in claim 21, in which coupling takes place via the substituents R5 or R6 of the carboxamide-substituted dye of the formula (I), wherein R5, R6, independently are a straight-chain, branched or cyclic saturated or unsaturated hydrocarbon group having up to 40 carbon atoms.

### Claim 27 (depends on 21)

The method as claimed in claim 21, in which coupling is carried out via a covalent bond.

### Claim 28

A method of detecting an analyte using carboxamide-substituted dye comprising the steps of providing one or more compounds of the general formula (I)
wherein
Y=sulfur, selenium, a direct linkage or is NRc;
R1, R3, R4 are independently hydrogen, halogen, —O⊖, a hydroxyl group, thiol group, amino group, ammonium group, sulfa group, phospho group, nitro group, carbonyl group, carboxyl group, a carboxylic acid derivative, a nitrile group, isonitrile group, cyanate group, isocyanate group, thiocyanate group, isothiocyanate group or a straight-chain, branched or cyclic saturated or unsaturated hydrocarbon group having up to 40 carbon atoms;
Rc independently are as defined for R1, R3, R4;
in which
R7, R8, R9 independently are hydrogen or a straight-chain, branched or cyclic saturated or unsaturated hydrocarbon group having up to 40 carbon atoms; or
R1 together with R2 is
in which
R10, R11, R13 are as defined for R1, R3, R4;
in which
R16, R17, R18 are as defined for R7, R8, R9;
R5, R6, independently are a straight-chain, branched or cyclic saturated or unsaturated hydrocarbon moiety having up to 40 carbon atoms;
Cyc1 is an organic moiety which comprises a ring system selected from aromatic, heteroaromatic, quinoidal and cycloaliphatic rings;
Cyc2 is an organic moiety which comprises a ring system selected from aromatic, heteroaromatic, quinoidal and cycloaliphatic rings;
each of said moieties in the dye of the formula (I) forming a ring system with one or more neighboring moieties;
and X being one or more mono- or multivalent anions, when required for balancing the charge;
coupling the carboxamide-substituted dye to the analyte to be detected; and
then determining at least one of the qualitative and quantitative presence of the analyte.