Fluorescence dyes and their use as fluorescence markers

Details Fluorescence dyes and their use as fluorescence markers Fluorescence dyes and their use as fluorescence markers

2000-05-11

2003-04-22

Huang, Evelyn Mei (Department: 1625)

Organic compounds -- part of the class 532-570 series

Organic compounds

Heterocyclic carbon compounds containing a hetero ring...

C544S135000, C546S198000, C546S152000, C548S156000, C548S546000, C548S547000, C549S223000

Reexamination Certificate

active

06552199

ABSTRACT:

The claimed invention concerns the production of new fluorescence dyes with an absorption region above 650 nm, their use as biomolecule markers, and the use of the marker-biomolecule conjugates in diagnostic systems.
To perform immunological assays and in DNA analytics markers or labels allowing the quantification of the analyte after the completion of an analyte-specific reaction are needed.
Recently, fluorescence labels in particular have become generally accepted due to their high sensitivity degree. The labeling of an antibody or an oligonucleotide with fluorescence dyes enables thus a direct quantification.
Widespread fluorescence dyes are for example FITC (fluorescein isothiocyanate), FLUOS (fluorescein N-hydroxysuccinimide ester), resorufin and rhodamine labels which do, however, need a relatively high light source for their excitation, such as an argon laser.
The quick development of inexpensive laser diodes with an emission range from 630-780 nm, and which are very appropriate for miniature system arrangements, makes dyes absorbing within these wave length ranges so desirable.
In EP-A-0 543 333 pentacyclic rhodamine dyes suitable for being used as labels are described. The main absorption range of such dyes is ≦660 nm.
In WO 88/047 77 phthalocyanine dyes are described that have more than one functional group so that in the case of conjugation, e.g. with antibodies, they result in cross-linking and product mixtures requiring a high degree of purification.
In DE 195 21 231 new oxazine dyes with an absorption maximum of up to 700 nm and their use as fluorescence markers are described.
The object of the present invention is to provide dyes suitable for coupling with biological molecules having a high quantum yield, absorbing within a spectral region above 650 nm, and exhibiting an unspecific binding as low as possible to biological compounds or to solid phases.
This object is achieved by the invention as characterized in the claims.
The subject matter of the invention are new xanthene derivatives of the general formula I,
wherein R1,R2, R3, R4, R5 can be—independently of each other (or each)—hydrogen, alkyl, aryl, heteroaryl, alkoxy, cyano, hydroxy, halogen, isocyanate, isothiocyanate or carboxyl, sulfonyl and, respectively derivatives thereof such as alkoxycarbonyl, aryloxycarbonyl, active ester, acid halogenides, mixed anhydrides or unsubstituted or substituted amino residues, especially &ohgr;-amino alcohols, &ohgr;-aminocarboxylic acids or &ohgr;-aminohalogen alkanes.
It has been proven to be advantageous to have at least one residue of the group R1,R2, R3, R4, R5 representing a halogen, in particular fluorine or chlorine and at least one residue of the group R1,R2, R3, R4, R5 representing a functional group, preferably an acidic group, particularly preferably a carboxylic acid or a derivative thereof. The residues R6, R7, R8, R9, R10, R11 are—independently of each other—hydrogen, alkyl-, substituted alkyl residues or a halogenide, particularly preferred are hydrogens or alkyl residues with 1-10 C-atoms, or sulfoalkyl residues, especially sulfomethyl residues or chlorine and fluorine residues.
At least one of the residues X or Y is electrophilic. Preferred electrophilic residues are C-nucleophiles from activated methyl aromatics or heteroaromatics like, e.g. heteroarylidenecyanomethine of the type 2-benzthiazolylidenecyanomethine or 2- or 4-quinolinylidenecyanomethine or malonic acid derivates, such as 2-propyldinitril residues or cyanoacetic acid ester.
At least X or Y is such a residue, or X and Y are such residues. These residues X and Y can be identical or different.
Moreover, X can also be amine- or a substituted amine residues, particularly preferred are alkyl-substituted or cyclic amines where Y is selected from the series of the electrophilic residues (in particular heteroarylidenecyanomethine residues or malonic acid derivatives), as they are described above.
The compounds according to the invention provide molecules which are due to their spectral properties (absorption maxima within the region of approx. 650 nm and above as well as emission maxima above 670 nm) very suitable as dyes and particularly as fluorescence dyes. Particularly preferred are absorption maxima above 700 nm. At the same time the spectral properties of the molecules can be changed by the fact that the identity, number and position of the residues with respect to R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 is changed. This way, fluorescence dyes can be produced with almost any absorption and emission maximum above 650 nm. A subject matter of the invention therefore also is the use of the xanthene derivatives according to the invention as a fluorescence dye or as a laser dye.
Among the compounds according to the invention of the general formula I at least one of the residues R1, R2, R3, R4, R5 preferably is present in the form of an activated group suitable for coupling or in the form of a group which can be activated for coupling. Such an activated group is particularly derived from a carboxylic-acid or sulfonic acid group which can be activated, and can for example be an acid ester, an acid anhydride, an acid halogenide, preferably bromide, in particular chloride or an N-hydroxysuccinimide ester or a &ohgr;-alkylhalogenide. Furthermore, such an activated group can for example also be phosphoramidites.
The table below gives some examples of activated groups suitable for coupling. The expert knows more of such groups from the field of conjugate synthesis.
TABLE 1
Activated group
Coupling with
Product
NHS-ester
Amines
Amide
Isothiocyanate
Amines
Thiourea
(mixed) Anhydride
Amines
Amides
Maleinmide
Thiol
Thioether
Thiol
Maleinmide
Thioether
Haloacetyl
Thiol
Thioder
Hydrazines
Aldehyde
Hydrazones
Amines
Aldehyde
Amines (after reduction)
Amines
Reactive carboxylic acid
Amides
Phosphoramidites
Protected nucleotides
Labeled oligonucleotides
For the production of conjugates containing the xanthene derivates according to the invention activated derivatives which are for example suitable for biomolecule labeling or labeling of other analytic reagents can be synthesized. The production of the activated derivatives requires at least one of the groups capable of being activated and located at the residues R1, R2, R3, R4, R5 and, respectively, X or Y, where activation is performed according to standard protocols known to the expert. Generally, activation is carried out with at least one hydroxyl group, amino-, sulfo- or carboxyl group of R1, R2, R3, R4 or R5.
Depending on the subsequent application different reactive groups can be introduced. Phosphoramidites and H-phosphonates can for example be derived from a hydroxyl group. The production of xanthene phosphoramidites and, respectively. H-phosphonates is thus generally performed according to hitherto known protocols (Methods in Mol. Biol. Vol 20. “Protocols for Oligonucleotides and Analogs, Synthesis and Properties”. S. Agrawal Hrsg., Humana Press Totowa, N.J.).
N-hydroxy-succininimide (NHS)-esters are however generally derived from a carboxyl group, maleinimides from an amino group (P. Y. Reddy, Synthesis(1998) 999) or by extension of an activated carboxylic acid with the corresponding &ohgr;-aminoalkylmaleinimide.
The production of NHS-esters is preferably performed according to the method described in EP 0 543 333, where the free carboxylic acid is admixed with NHS in the presence of a condensation reagent such as for example DCC or MEI. The production of xanthene isothiocyanates is preferably carried out by the reaction of amino groups with thiophosgene (Advanced Organic Chemistry, Mc Graw Hill, 2
nd
edition. p. 383, 1997).
The subject matter of the invention are therefore also activated derivatives of the xanthenes according to the invention. The reactive groups of the activated derivatives are preferably phosphoramidite, N-hydroxy-succininimide (NHS)-ester, maleinimide-alkylamide, H-phosphonate or isothiocyanate or &ohgr;-alkylhalogenides.
A further subject matter of the invention are conjugates obtainable by the binding of xanthene compou

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