# US 7,745,645 B2 · Sulfonamide derivatives of xanthene compounds

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- Number: US 7,745,645 B2
- Document ID: US7745645B2
- Status: Granted
- Kind: B2
- Application number: 11/625,379
- Filed: 2007-01-22
- Published: 2008-07-24
- Granted: 2010-06-29
- Earliest claimed: 2007-01-22
- Assignees: Pierce Biotechnology, Inc.
- Inventors: Wilhelm G. Frank; Matthias S. Wenzel; Peter T. Czerney; Surbhi Desai; Greg Hermanson
- Web page: https://patentgrep.com/patent/US7745645B2

## CPC

- C07D 311/82: Xanthenes
- G01N 33/582: {with fluorescent label}
- Y10S 436/80: Fluorescent dyes, e.g. rhodamine
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- 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)
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

Compounds having the general of formula I and/or formula II wherein R1 and R2 are the same or different and are independently selected from the group consisting of —H, —C1-C18-alkyl or -ω-sulfoalkyl; X and Y are the same or different and are independently selected from the group consisting of —O−, —OH, —SH, —NH—NH2, —F, —Cl, —Br, —I, —O-Su (succinimidyl/sulfosuccinimidyl), —O-STP (4-sulfo-2,3,5,6-tetrafluorophenyl), —O-TFP (2,3,5,6-tetrafluorophenyl), —O-benzotriazole, -benzotriazole, —NR—CO—CH2—I, —NR2, —NR-biomolecule, —NR-L-COO−, —NR-L-COOH, —NR-L-COO-Su, —NR-L-COO-STP, —NR-L-COO-TFP, —NR-L-CONR2, —NR-L-CO-biomolecule, —NR-L-CO—NH—NH2, —NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-CHO, —NR-L-maleimid, or —NR-L-NH—CO—CH2—I; where R is equal to R1 and R2 and L is selected from the group consisting of a divalent linear (—(CH2)o—, o=1 to 15), crossed, or cyclic alkane group that can be substituted by at least one atom selected from the group consisting of oxygen, substituted nitrogen, and/or sulfur, and the biomolecule is a protein, antibody, nucleotide, oligonucleotide, biotin, or hapten; Z is —O− or OH; U is —O−, —OH, or NH-L-SO2Z; Kat is Li, Na, K, ammonium (mono-, di- or trialkyl) or another cation; An is F, Cl, Br, I, BF4, ClO4, CH3CO2, CF3CO2 or another anion; m is an integer from 1-6 necessary to compensate the negative or positive charge from the dye moiety in formula I or formula II; and n is an integer from 0-12; compositions containing these compounds, and methods using these compounds, are disclosed.

## Claims

### Claim 1

At least one compound of formula I or formula II
wherein
R1 and R2 are the same or different and are independently selected from the group consisting of —H, —C1-C18-alkyl or -ω-sulfoalkyl;
X and Y are the same or different and are independently selected from the group consisting of —O−, —OH, —SH, —NH—NH2, —F, —Cl, —Br, —I, —O-Su (succinimidyl/sulfosuccinimidyl), —O-STP (4-sulfo-2,3,5,6-tetrafluorophenyl), —O-TFP (2,3,5,6-tetrafluorophenyl), —O-benzotriazole, -benzotriazole, —NR—CO—CH2—I, —NR2, —NR-biomolecule, —NR-L-COO−, —NR-L-COOH, —NR-L-COO-Su, —NR-L-COO-STP, —NR-L-COO-TFP, —NR-L-CONR2, —NR-L-CO-biomolecule, —NR-L-CO—NH—NH2, —NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-CHO, —NR-L-maleimid, or —NR-L-NH—CO—CH2—I; where R is equal to R1 and R2 and L is selected from the group consisting of a divalent linear (—(CH2)o—, o=1 to 15), crossed, or cyclic alkane group that can be substituted by at least one atom selected from the group consisting of oxygen, substituted nitrogen, and/or sulfur, and the biomolecule is a protein, antibody, nucleotide, oligonucleotide, biotin, or hapten;
Z is —O− or OH;
U is —O−, —OH or NH-L-SO2Z;
Kat is Li, Na, K, ammonium (mono-, di- or trialkyl) or another cation;
An is F, Cl, Br, I, BF4, ClO4, CH3CO2, CF3CO2 or another anion;
m is an integer from 1-6 necessary to compensate the negative or positive charge from the dye moiety in formula I or formula II; and
n is an integer from 0-12.

### Claim 2

A compound selected from at least one of formula III, formula IV, formula V, formula VI, formula VII, formula VIII, formula IX, formula X, formula XI, formula XII, formula XIII, formula XIV, formula XV, or formula XVI

### Claim 3

A biocompatible dye composition comprising at least one excipient and a compound selected from at least one of formula I or formula II
resulting in a biocompatible dye composition, wherein
R1 and R2 are the same or different and are independently selected from the group consisting of —H, —C1-C18-alkyl or -ω-sulfoalkyl;
X and Y are the same or different and are independently selected from the group consisting of —O−, —OH, —SH, —NH—NH2, —F, —Cl, —Br, —I, —O-Su (succinimidyl/sulfosuccinimidyl), —O-STP (4-sulfo-2,3,5,6-tetrafluorophenyl), —O-TFP (2,3,5,6-tetrafluorophenyl), —O-benzotriazole, -benzotriazole, —NR—CO—CH2—I, —NR2, —NR-biomolecule, —NR-L-COO−, —NR-L-COOH, —NR-L-COO-Su, —NR-L-COO-STP, —NR-L-COO-TFP, —NR-L-CONR2, —NR-L-CO-biomolecule, —NR-L-CO—NH—NH2, —NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-CHO, —NR-L-maleimid, or —NR-L-NH—CO—CH2—I; where R is equal to R1 and R2 and L is selected from the group consisting of a divalent linear (—(CH2)o—, o=1 to 15), crossed, or cyclic alkane group that can be substituted by at least one atom selected from the group consisting of oxygen, substituted nitrogen, and/or sulfur, and the biomolecule is a protein, antibody, nucleotide, oligonucleotide, biotin, or hapten;
Z is —O− or OH;
U is —O−, —OH or NH-L-SO2Z;
Kat is Li, Na, K, ammonium (mono-, di- or trialkyl) or another cation;
An is F, Cl, Br, I, BF4, ClO4, CH3CO2, CF3CO2 or another anion;
m is an integer from 1-6 necessary to compensate the negative or positive charge from the dye moiety in formula I or formula II; and
n is an integer from 0-12.

### Claim 4

A biocompatible dye composition comprising at least one excipient and a compound selected from the group consisting of formula III, formula IV, formula V, formula VI, formula VII, formula VIII, formula IX, formula X, formula XI, formula XII, formula XIII, formula XIV, formula XV, formula XVI, and combinations thereof, resulting in a biocompatible dye composition, wherein

### Claim 5

A method of labelling at least one biomolecule, the method comprising
providing a composition comprising at least one excipient and a compound of at least one of formula I or formula II
in an effective concentration to a biomolecule under conditions sufficient for conjugating the compound to the biomolecule, and
detecting the biomolecule-bound conjugate, wherein
R1 and R2 are the same or different and are independently selected from the group consisting of —H, —C1-C18-alkyl or -ω-sulfoalkyl;
X and Y are the same or different and are independently selected from the group consisting of —O−, —OH, —SH, —NH—NH2, —F, —Cl, —Br, —I, —O-Su (succinimidyl/sulfosuccinimidyl), —O-STP (4-sulfo-2,3,5,6-tetrafluorophenyl), —O-TFP (2,3,5,6-tetrafluorophenyl), —O-benzotriazole, -benzotriazole, —NR—CO—CH2—I, —NR2, —NR-biomolecule, —NR-L-COO−, —NR-L-COOH, —NR-L-COO-Su, —NR-L-COO-STP, —NR-L-COO-TFP, —NR-L-CONR2, —NR-L-CO-biomolecule, —NR-L-CO—NH—NH2, —NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-CHO, —NR-L-maleimid, or —NR-L-NH—CO—CH2—I; where R is equal to R1 and R2 and L is selected from the group consisting of a divalent linear (—(CH2)o—, o=1 to 15), crossed, or cyclic alkane group that can be substituted by at least one atom selected from the group consisting of oxygen, substituted nitrogen, and/or sulfur, and the biomolecule is a protein, antibody, nucleotide, oligonucleotide, biotin, or hapten;
Z is —O− or OH;
U is —O−, —OH or NH-L-SO2Z;
Kat is Li, Na, K, ammonium (mono-, di- or trialkyl) or another cation;
An is F, Cl, Br, I, BF4, ClO4, CH3CO2, CF3CO2 or another anion;
m is an integer from 1-6 necessary to compensate the negative or positive charge from the dye moiety in formula I or formula II; and
n is an integer from 0-12.

### Claim 6

A method of labelling at least one biomolecule, the method comprising
providing a composition comprising at least one excipient and a compound of at least one of formula III, formula IV, formula V, formula VI, formula VII, formula VIII, formula IX, formula X, formula XI, formula XII, formula XIII, formula XIV, formula XV, formula XVI, and combinations thereof, in an effective concentration to a biomolecule under conditions sufficient for conjugating the compound to the biomolecule, and
detecting the biomolecule-bound conjugate, wherein

### Claim 7 (depends on 5, 6)

The method of either claim 5 or claim 6 used in at least one of a protein assay, immunofluorescence assay, singleplex application, or multiplex application.

### Claim 8 (depends on 5, 6)

The method of either claim 5 or claim 6 used in a multiplex application in combination with other fluorescent dyes or conjugates.

### Claim 9 (depends on 5, 6)

The method of either claim 5 or claim 6 wherein the compound is rendered reactive prior to conjugation.

### Claim 10 (depends on 5, 6)

The method of either claim 5 or claim 6 wherein the compound further comprises L-Rt where L is a linker group covalently attached to the compound and Rt is a reactive group that is capable of covalently linking to the biomolecule.

### Claim 11 (depends on 5, 6)

The method of either claim 5 or claim 6 wherein the biomolecule is selected from at least one of a protein, antibody, nucleotide, oligonucleotide, biotin, or hapten.

### Claim 12

The compound of formula V (V03-04115)
in a composition capable of use as a fluorescent dye in a biological assay.

### Claim 13 (depends on 12)

The compound of claim 12 detected as a biomolecule conjugate.

### Claim 14 (depends on 12)

The compound of claim 12 in a kit with instructions for use in a protein assay.