Bright fluorochromes based on multimerization of fluorescent dyes

Patent metadata

Field	Value
Publication number	US-2016347907-A1
Application number	US-201514723505-A
Country	US
Kind code	A1
Filing date	May 28, 2015
Priority date	May 28, 2015
Publication date	Dec 1, 2016
Grant date	—

Abstract

The invention is directed to a fluorescent dye according to the general formula I:

with
C is a core moiety comprising 20 to 200 atoms; S same or different ether residues comprising 1 to 10 carbon atoms; n is an integer ranging from 2 to 500; m is an integer ranging from 0 to 500; x is an integer ranging from 2 to 50; y is an integer ranging from 1 to 50; R same or different residue comprising a reactive group capable of forming a covalent bond with a biomolecule; F same or different fluorophores covalently bound to (S) n .

The fluorescent dyes can be conjugated to a biomolecule and used for flow cytometry and/or by fluorescence microscopy.

First claim

What is claimed is:

1 . A fluorescent dye according to the formula I:

wherein
C is a core moiety comprising 20 to 200 atoms;
S same or different ether residues comprising 1 to 10 carbon atoms;
n is an integer ranging from 2 to 500;
m is an integer ranging from 0 to 500;
x is an integer ranging from 2 to 50;
y is an integer ranging from 1 to 50;
R same or different residue comprising a reactive group capable of forming a covalent bond with a biomolecule;
F same or different fluorophores covalently bound to (S) n .

2 . The fluorescent dye according to claim 1 , wherein the fluorophore F is selected from the group consisting of xanthene dyes, rhodamine dyes, coumarine dyes, cyanine dyes, pyrene dyes, oxazine dyes, pyridyl oxazole dyes and pyrromethene dyes.

3 . The fluorescent dye according to claim 1 , wherein the fluorophore F is substituted with one more water solubility imparting substituents selected from the group consisting of sulfonates, phosphonates, phosphates, sulfonamides, polyethers and carbonates.

4 . The fluorescent dye according to claim 1 , wherein the core moiety C is at least one of a polyhydroxy compound, a polyamino compound and a polythio compound.

5 . The fluorescent dye according to claim 1 , wherein the core moiety C is selected from the group consisting of pentaerythritol, dipentaerythritol, tripentaerythritol and hexaglycerol.

6 . The fluorescent dye according to claim 1 , wherein S stands for the residue CH 2 CH 2 O— and n, m are independently integers ranging from 10 to 200.

7 . The fluorescent dye according to claim 1 , wherein R comprises a reactive group selected from the group consisting of N-hydroxysuccinimid ester, tetrafluorophenyl ester, pentafluorophenyl ester, sulfodichlorophenyl ester, imido ester, isothiocyanate, isocyanate, sulfonyl halides, acyl halides, acyl azide, monochlorotriazine, dichlorotriazine, aldehyde, glyoxal, maleimide, iodoacetamide, hydrazine, azidonitrophenyl, phosphoramidite, alkyne, alkyl azide, dienes or allyl groups.

8 . The fluorescent dye according to claim 1 , characterized by the structure of general formula (II)

with Z is at least one of F and R and p is at least one of n and m,
with the provision that general formula (II) comprises at least two F and at least one group R.

9 . The fluorescent dye according to claim 1 , characterized by the structure of general formula (III)

with Z is at least one of F and R, and p is at least one of n and m, with the provision that general formula (II) comprises at least two F and at least one group R.

10 . A fluorescent biomolecule conjugate comprising at least one fluorescent dye according claim 1 , wherein a biomolecule is conjugated via group R to the at least one fluorescent dye, and the biomolecule is selected from group consisting of immunoglobulin, antibody, fragmented antibody, Fab, Fab′, F(ab′) 2 , sdAb, scFv, di-scFv each of naturally or recombinant origin.

11 . A fluorescent biomolecule conjugate comprising at least one fluorescent dye according claim 1 , wherein a biomolecule is conjugated via group R to the at least one fluorescent dye, and the biomolecule is selected from the group consisting of peptide/MHC-complexes, receptors for cell adhesion or costimulatory molecules, receptor ligands, antigens, hapten binders, avidin, streptavidin, neutravidin, aptamers, primers and ligase substrates.

12 . A fluorescent biomolecule conjugate, wherein a biomolecule is conjugated with 2 to 20 fluorescent dyes according to claim 1 , each fluorescent dye comprising between 1 and 10 fluorophores F, inclusive.

13 . A method of analyzing cells or tissue, comprising:
labeling with the fluorescent biomolecule conjugate according to claim 10 ; and performing at least one of flow cytometry and fluorescence microscopy.

Assignees

• Miltenyi Biotec Gmbh

Inventors

• Dose Christian

Classifications

• C08G65/48 Primary

  Polymers modified by chemical after-treatment · CPC title

• C09B69/00

  Dyes not provided for by a single group of this subclass · CPC title

• C08G65/33306

  acyclic · CPC title

• A61K49/0043

  Fluorescein, used in vivo · CPC title

• A61K49/0045

  the fluorescent agent being a peptide or protein used for imaging or diagnosis in vivo · CPC title