# US 7,745,640 B2 · Hydrophilic labels for biomolecules

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 7,745,640 B2
- Document ID: US7745640B2
- Status: Granted
- Kind: B2
- Application number: 11/566,699
- Filed: 2006-12-05
- Published: 2007-06-07
- Granted: 2010-06-29
- Earliest claimed: 2005-12-05
- Assignees: Dyomics GmbH
- Inventors: Peter T. Czerney; Wilhelm G. Frank; Frank G. Lehmann; Bernd G. Schweder; Matthias S. Wenzel
- Web page: https://patentgrep.com/patent/US7745640B2

## CPC

- G01N 33/582: {with fluorescent label}
- G01N 33/533: with fluorescent label
- 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)

## Abstract

Compounds, compositions, and methods for optical, including fluorescence optical, determinations useful in labelling biomolecules such as protein and deoxyribonucleic acid for their detection and quantitation. The compounds are diasteromeric cyanines with high hydrophilicity and other desirable properties.

## Claims

### Claim 1

An isolated enantiomeric mixture selected from
diastereomer Ia,
diastereomer Ib,
diastereomer IIa,
or, diastereomer IIb,
whereby diastereomers Ia and Ib are derived from general formula I
and diastereomers IIa and IIb are derived from general formula II,
and whereby each of R1 and R2 is the same or different and is independently selected from the group consisting of an aliphatic and heteroaliphatic group;
each of L1 to L4 is the same or different and is independently selected from the group consisting of a divalent linear (—(—CH2)o—, o=1 to 15), crossed, or cyclic alkylene group which can be substituted by at least one atom selected from the group consisting of oxygen, substituted nitrogen, and sulfur;
X1 is selected from the group consisting of —OH, —SH, —NH2, —NH—NH2, —F, —Cl, —Br, —I, —NHS (hydroxysuccinimidyl/sulfosuccinimidyl), —O-STP (4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-TFP (2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole, —NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH2, —NR-L-NH—NH2, —NR-L-CO2H, —NR-L-CO2—NHS, —NR-L-CO2-STP, —NR-L-CO2-TFP, —NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimid, and —NR-L-NH—CO—CH2—I, R is —H or is equal to R1 or R2, and L is equal to L1 to L4;
each of X2 and X3 is the same or different and is independently selected from the group consisting of hydrogen, alkyl-, tert-alkyl-, aryl-, carboxyaryl-, dicarboxyaryl-, heteroaryl-, cycloalkyl-, heterocycloalkyl-, alkyloxy-, alkylmercapto-, aryloxy, arylmercapto, hydroxy-, amino-, nitro-, and cyano-residues, or is a solubilizing or ionizable substituent selected from the group consisting of —SO3−, —PO32−, —CO2−, tert-ammonium, cyclodextrine, sugar, and combinations thereof;
Kat is a number of Na+, K+, Ca2+, ammonia, or other cation(s) needed to compensate the negative charge brought by the cyanine;
n in formula I is a integer from 0 to 3;
Y in formula II is selected from the group consisting of fluorine, chlorine, bromine, a substituted phenoxy-, and a substituted arylmercapto-(phenyl sulfanyl-) function; and
each of R3 and R4 in formula II is the same or different and is independently an aliphatic or heteroaliphatic group respectively, or forms together the divalent structural element selected from the group consisting of —(CH2)m—, —(CH2)mO(CH2)m′—, —(CH2)mS(CH2)m′—, —(CH2)mCH═CH—, and —OCH═CH— where each of m and m′ is the same or different and is a integer from 2 to 6.

### Claim 2 (depends on 1)

The diastereomer of claim 1 conjugated to a biomolecule selected from at least one of a protein, antibody, enzyme, nucleoside triphosphate (NTP), oligonucleotide, biotin, hapten, cofactor, lectin, antibody binding protein, carotenoid, hormone, neurotransmitter, growth factors, toxin, biological cell, lipid, receptor binding drug, organic polymer carrier material, or inorganic polymeric carrier material.

### Claim 3

A biocompatible dye composition comprising at least one excipient and an isolated enantiomeric mixture selected from
diastereomer Ia,
diastereomer Ib,
diastereomer IIa,
or, diastereomer IIb,
whereby diastereomers Ia and Ib are derived from general formula I
and diastereomers IIa and IIb are derived from general formula II,
and whereby each of R1 and R2 is the same or different and is independently selected from the group consisting of an aliphatic and heteroaliphatic group;
each of L1 to L4 is the same or different and is independently selected from the group consisting of a divalent linear (—(CH2)o—, o=1 to 15), crossed, or cyclic alkylene group which can be substituted by at least one atom selected from the group consisting of oxygen, substituted nitrogen, and sulfur;
X1 is selected from the group consisting of —OH, —SH, —NH2, —NH—NH2, —F, —Cl, —Br, —I, —NHS (hydroxysuccinimidyl/sulfosuccinimidyl), —O-STP (4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-TFP (2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole, —NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH2, —NR-L-NH—NH2, —NR-L-CO2H, —NR-L-CO2—NHS, —NR-L-CO2—STP, —NR-L-CO2-TFP, —NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimid, and —NR-L-NH—CO—CH2—I, R is —H or is equal to R1 or R2′ and L is equal to L1 to L4;
each of X2 and X3 is the same or different and is independently selected from the group consisting of hydrogen, alkyl-, tert-alkyl-, aryl-, carboxyaryl-, dicarboxyaryl-, heteroaryl-, cycloalkyl-, heterocycloalkyl-, alkyloxy-, alkylmercapto-, aryloxy, arylmercapto, hydroxy-, amino-, nitro-, and cyano-residues, or is a solubilizing or ionizable substituent selected from the group consisting of —SO3−, —PO32−, —CO2−, tert-ammonium, cyclodextrine, sugar, and combinations thereof;
Kat is a number of Na+, K+, Ca2+, ammonia, or other cation(s) needed to compensate the negative charge brought by the cyanine;
n in formula I is a integer from 0 to 3;
Y in formula II is selected from the group consisting of fluorine, chlorine, bromine, a substituted phenoxy-, and a substituted arylmercapto-(phenyl sulfanyl-) function; and
each of R3 and R4 in formula II is the same or different and is independently an aliphatic or heteroaliphatic group respectively, or forms together the divalent structural element selected from the group consisting of —(CH2)m—, —(CH2)mO(CH2)m′—, —(CH2)mS(CH2)m′—, —(CH2)mCH═CH—, and —OCH═CH— where each of m and m′ is the same or different and is a integer from 2 to 6.

### Claim 4 (depends on 3)

The composition of claim 3 wherein the diastereomer is conjugated to a biomolecule selected from at least one of a protein, antibody, enzyme, nucleoside triphosphate (NTP), oligonucleotide, biotin, hapten, cofactor, lectin, antibody binding protein, carotenoid, hormone, neurotransmitter, growth factors, toxin, biological cell, lipid, receptor binding drug, organic polymeric carrier material, or inorganic polymeric carrier material.

### Claim 5

A method of labeling at least one biomolecule, the method comprising
providing a composition comprising at least one excipient and one isolated enantiomeric mixture in an effective concentration to at least one biomolecule under conditions sufficient for binding the enantiomeric mixture to the biomolecule, and detecting the biomolecule-bound enantiomeric mixture,
where the one isolated enantiomeric mixture is
diastereomer Ia
diastereomer Ib
diastereomer IIa,
or, diastereomer IIb,
whereby diastereomers Ia and Ib are derived from general formula I
and diastereomers IIa and IIb are derived from general formula II,
and whereby
each of R1 and R2 is the same or different and is independently selected from the group consisting of an aliphatic and heteroaliphatic group;
each of L1 to L4 is the same or different and is independently selected from the group consisting of a divalent linear (—(CH2)o—, o=1 to 15), crossed, or cyclic alkylene group which can be substituted by at least one atom selected from the group consisting of oxygen, substituted nitrogen, and sulfur;
X1 is selected from the group consisting of —OH, —SH, —NH2, —NH—NH2, —F, —Cl, —Br, —I, —NHS (hydroxysuccinimidyl/sulfosuccinimidyl), —O-STP (4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-TFP (2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole, —NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH2, —NR-L-NH—NH2, —NR-L-CO2H, —NR-L-CO2—NHS, —NR-L-CO2—STP, —NR-L-CO2-TFP, —NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimid, and —NR-L-NH—CO—CH2—I, R is —H or is equal to R1 or R2′ and L is equal to L1 to L4;
each of X2 and X3 is the same or different and is independently selected from the group consisting of hydrogen, alkyl-, tert-alkyl-, aryl-, carboxyaryl-, dicarboxyaryl-, heteroaryl-, cycloalkyl-, heterocycloalkyl-, alkyloxy-, alkylmercapto-, aryloxy, arylmercapto, hydroxy-, amino-, nitro-, and cyano-residues, or is a solubilizing or ionizable substituent selected from the group consisting of —SO3−, —PO32−, —CO2−, tert-ammonium, cyclodextrine, sugar, and combinations thereof;
Kat is a number of Na+, K+, Ca2+, ammonia, or other cation(s) needed to compensate the negative charge brought by the cyanine;
n in formula I is a integer from 0 to 3;
Y in formula II is selected from the group consisting of fluorine, chlorine, bromine, a substituted phenoxy-, and a substituted arylmercapto-(phenyl sulfanyl-) function; and
each of R3 and R4 in formula II is the same or different and is independently an aliphatic or heteroaliphatic group respectively, or forms together the divalent structural element selected from the group consisting of —(CH2)m—, —(CH2)mO(CH2)m′—, —(CH2)mS(CH2)m′—, —(CH2)mCH═CH—, and —OCH═CH— where each of m and m′ is the same or different and is a integer from 2 to 6.

### Claim 6 (depends on 5)

The method of claim 5 wherein the diastereomer is conjugated to a biomolecule selected from at least one of a protein, antibody, enzyme, nucleoside triphosphate (NTP), oligonucleotide, biotin, hapten, cofactor, lectin, antibody binding protein, carotenoid, hormone, neurotransmitter, growth factors, toxin, biological cell, lipid, receptor binding drug, fluorescent proteins, organic polymer carrier material, or inorganic polymeric carrier material.

### Claim 7 (depends on 5)

The method of claim 5 used in at least one of an immunoassay, hybridization, chromatographic assay, electrophoretic assay, microwell plate based assay, fluorescence resonance energy transfer (FRET) system, high throughput screening, or microarray.

### Claim 8 (depends on 5)

The method of claim 5 wherein detection sensitivity is at least 0.1 ng.

### Claim 9 (depends on 5)

The method of claim 5 wherein protein detection sensitivity is at least 0.1 pg protein.