Fluorescent cyanine labels containing a sulfamido linker arm

Details Fluorescent cyanine labels containing a sulfamido linker arm Fluorescent cyanine labels containing a sulfamido linker arm

2000-06-30

2002-09-10

Jones, W. Gary (Department: 1634)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C536S024200, C536S023100, C536S025320, C514S112000, C514S222200, C514S224200

Reexamination Certificate

active

06448008

ABSTRACT:

CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims priority to European Patent Application No. 99112696.2 filed Jul. 2,1999.
FIELD OF THE INVENTION
The present invention relates to a new class of fluorescent dyes belonging to the cyanine family. The new fluorescent dyes can be excited using powerful yet inexpensive light emitting diodes and diode lasers; they exhibit good water solubility and can be attached or conjugated to a wide variety of molecules or surfaces for labelling purposes.
BACKGROUND OF THE INVENTION
There has been, in recent years, an upsurge in research concerning the fluorescent labelling of biological compounds for the purpose of developing simpler, more sensitive assay methods. In particular, a class of fluorescent dyes has attracted much attention, namely the class of cyanine dyes. While in the past much work on these compounds was focused on obtaining lipophylic materials to be used in photographic processes, the current field of application in the biological sciences requires instead hydrophylic species. Moreover, a new requirement emerged, in the necessity of providing reactive spacer links to be used for the binding of the dyes to biomolecules.
With reference to the basic structure of the compounds such as shown in
FIG. 1
, it can be seen that such linker arms can be generally attached to either the aromatic portion of the molecule, or to the indolenine nitrogens, or to the polymethine bridge forming the Q moiety.
For example, Waggoner at al. described in Cytometry 10, 3-10 (1989) iodoacetamido groups bound to the aromatic frame, for the purpose of labelling thiol containing molecules, and isothiocyanide groups also bound to aromatic frame for labeling amine containing compounds (in Cytometry 10, 11-19 (1989).
Mank and coworkers in Anal. Chem. 67, 1742-1748 (1995) made N-hydroxysuccinimide (NHS) ester cyanine dyes derivatives from carboxyl groups or carboxymethyl groups attached to the aromatic frame, for precolumn derivatisation of amines in liquid chromatography. The labelled amines could then be detected with ultra high sensitivity by visible diode laser-induced fluorescence.
Waggoner and co-workers developed cyanine labels in which the reactive NHS function is connected to the indolenine nitrogen by an alkyl chain (Mujumdar et al., Bioconjugate Chem. 4, 105-111, 1993). This approach was also followed by Brush and Erie (U.S. Pat. No. 5,808,044) who disclosed a method for making cyanine phosphoramidites useful in nucleotide labelling.
Patonay and co-workers described a tricarbocyanine class of cyanine dye containing isothiocyanide groups attached to the polymethine bridge via a thiophenol linker for the binding of molecules with the amino functionality in J. Org. Chem, 57, 4578-4580 (1992).
While the previous approaches achieved various degrees of success, in many cases they introduced unwanted side effects in the dyes. For example, when the reactive group is directly attached to aromatic frame, the fluorescence efficiency of the dye is negatively affected as shown in Anal. Chem. 67, 1742-1748 (1995). In other cases, the existence of a flexible side chain directly attached to the indolenine N allows the labeled molecule to come into close contact with the chromophoric polymethine chain: this can also perturb or negatively affect the fluorescence of the dye by intermolecular quenching. Finally, some approaches (e.g. Organic Chem, 57, 4578-4580 (1992) can be used only for particular dyes.


REFERENCES:
patent: 5714386 (1998-02-01), Roederer
patent: 5808044 (1998-09-01), Brush et al.
Mujumdar et al., Bioconjugate Chemistry, vol. 4, No. 2, pp. 105-111, 1993.*
Ernst et al.; “Cyanine Dye Labeling Reagents for Sulfhydryl Groups,”Cytometry10:3-10 (1989).
Mank et al.; “Visible Diode Laser-Induced Fluorescence Detection In Liquid Chromatography After Precolumn Derivatization Of Amines,”Analytical. Chemistry67:10 1742-1748 (May 15, 1995).
Mujumdar et al.; “Cyanine Dye Labeling Reagents Containing Isothiocyanate Groups,”Cytometry10:11-19 (1989).
Mujumdar et al.; “Cyanine Dye Labeling Reagents: Sulfoindocyanine Succinimidyl Esters,”Bioconjugate Chemistry4:2 105-111 (Mar./Apr. 1993).
Strekowski et al.; “Substitution Reactions Of A Nucleofugal Group In Heptamethine Cyanine Dyes. Synthesis Of An Isothiocyanato Derivative For Labeling Of Proteins With A Near-Infrared Chromophore,”J. Org. Chem.57:17 4578-4580 (1992).

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