Metal complexes with a charged linker

• US 5,958,783 A
• Filed: 06/20/1996
• Issued: 09/28/1999
• Est. Priority Date: 07/25/1994
• Status: Expired due to Term

• Current Assignee: Roche Diagnostics GmbH (Roche Holding AG)
• Original Assignee: Roche Diagnostics GmbH (Roche Holding AG)
• Inventors: Seidel, Christoph, Josel, Hans-Peter, Upmeier, Barbara, Wienhues, Ursula-Henrike, Hoss, Eva, Ofenloch-Hahnle, Beatus
• Primary Examiner(s): Achutamurthy, Ponnathapura
• Assistant Examiner(s): PONNALURI, PADMASHRI
• Application Number:
  US08/615,278
• Time in Patent Office: 1,195 Days
• Field of Search: 436/172, 436/518, 436/800, 436/84, 436/92, 436/546, 436/536, 436/537, 556/136, 252/301.16, 252/301.33, 252/301.4 R, 546/10
• US Class Current: 436/84
• CPC Class Codes: C07F 15/0053 without a metal-carbon linkage
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C12N 2740/16122 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2770/24222 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/6878 in eptitope analysis
• CPC Class Codes: G01N 33/92 involving lipids, e.g. cholesterol , lipoproteins, or their receptors steroid hormones G01N33/743

Abstract

The present invention concerns new metal complexes with a charged linker and their use as luminescent marker groups in an immunoassay.

Claims

1. A metal complex having the formula:

space="preserve" listing-type="equation"> M(L.sub.1 L.sub.2 L.sub.3)!.sub.n - Y-X!.sub.m

wherein

M is a divalent or trivalent metal cation selected from the group consisting of a rare earth metal cation and a transition metal cation;

L₁, L₂ and L₃ are the same or different, and each is a ligand having at least two nitrogen-containing heterocyclic rings, wherein L₁, L₂ and L₃ are bound to the metal cation via nitrogen atoms;

Y is a linker, connected to at least one of L₁, L₂ and L₃, the linker containing at least one charge carrier independently selected from the group consisting of a positive charge carrier and a negative charge carrier;

X is a reactive functional group;

n is 1 to 10; and

m is 1 to 6.

2. The metal complex as claimed in claim 1, wherein M is selected from the group consisting of ruthenium ion, rhenium ion, osmium ion, chromium ion and iridium ion.

3. The metal complex as claimed in claim 1, wherein M is a ruthenium ion.

4. The metal complex as claimed in claim 1, wherein each of L₁, L₂ and L₃ independently contains a ring system selected from the group consisting of bipyridine and phenanthroline.

5. The metal complex as claimed in claim 1, wherein X is selected from the group consisting of carboxylic acid halogenide, carboxylic acid anhydride, active ester, maleimide and a group which is capable of being photo-activated.

6. The metal complex as claimed in claim 1, wherein the metal complex further comprises at least one counterion A which balances the charge carried by the at least one charge carrier.

7. The metal complex as claimed in claim 6, wherein A is at least one group selected from the group consisting of hexafluorophosphate, trifluoroacetate and tetrafluoroborate.

8. The metal complex as claimed in claim 1, wherein Y is a linker containing at least one negative charge carrier selected from the group consisting of phosphate, phosphonate, sulfonate and carboxylate.

9. The metal complex as claimed in claim 1, wherein Y is a linker containing at least one carboxylate group.

10. The metal complex as claimed in claim 1, wherein Y is a linker containing at least one positive charge carrier selected from the group consisting of amino and substituted amino group.

11. The metal complex claimed in claim 10, wherein the at least one positive charge carrier serves as an electron donor for the metal complex.

12. The metal complex as claimed in claim 1, wherein the linker contains 1 to 10 charge carriers.

13. The metal complex as claimed in claim 1, wherein the linker contains 2 to 8 charge carriers.

14. The metal complex as claimed in claim 1, wherein the linker comprises a plurality of aminocarboxylic acid units which are linked together via peptide bonds.

15. The metal complex as claimed in claim 14, wherein the plurality of aminocarboxylic acid units are derived from polyfunctional aminocarboxylic acids which contain at least one free charge carrier after incorporation into the linker.

16. The metal complex as claimed in claim 14, wherein the plurality of aminocarboxylic acid units are derived from trifunctional aminocarboxylic acids which contain

(a) one amino group and two carboxylate groups, or

(b) two amino groups and one carboxylate group.

17. The metal complex as claimed in claim 16, wherein the trifunctional aminocarboxylic acids are selected from the group consisting of lysine, ornithine, hydroxylysine, aspartic acid and glutamic acid.

18. The metal complex as claimed in claim 1, wherein the metal complex has the formula:

##STR5## wherein M, Y and X are as stated above; and

R₁ through R₆, insofar as they are not bound to --Y--X, are each independently selected from the group consisting of hydrogen, C₁ -C₅ alkyl and at least one hydrophilic substituent which is selected from the group consisting of C₂ -C₃ alkyleneoxy, C₂ -C₃ alkylenethio, C₂ -C₃ alkyleneamino, a group of formula --NR--W and a group of formula --O--W, wherein W is an organic residue containing 2 to 10 carbon atoms and 2 to 6 hydroxy groups, and R is hydrogen or C₁ -C₅ alkyl,

wherein at least one of R₁ through R₆ is bound to --Y--X, and is represented by the formula --(CH₂)_s --C(═

O)--NH--(CH₂)₂ --Y--X, wherein s is 0 to 6.

19. The metal complex as claimed in claim 1, wherein the metal complex has the formula:

##STR6## wherein M, Y, X and n are as defined above; and

R₁ through R₃ are each independently selected from the group consisting of hydrogen, C₁ -C6 alkyl and at least one hydrophilic group which is selected from the group consisting of C₂ -C₃ alkyleneoxy, C2-C₃ alkylenethio, C₂ -C₃ alkyleneamino, a group of formula --NR--W and a group of formula --O--W, wherein W is an organic residue containing 2 to 10 carbon atoms and 2 to 6 hydroxy groups, and R is hydrogen or C₁ -C₅ alkyl.

20. A conjugate, comprising the metal complex as claimed in claim 1 coupled with a biological substance.

21. The conjugate as claimed in claim 20, wherein the biological substance is selected from the group consisting of biotin, an antibody, an antibody fragment, a nucleic acid, a polypeptide antigen, an immunological reactive peptide and a hapten.

22. In an immunological detection method wherein a metal complex is bound to an immunological binding partner and used as a marker group to quantitatively or qualitatively determine an analyte in a sample solution, the improvement comprising using the marker group of claim 1 as the marker group.

23. The method of claim 22, wherein the marker group is determined by luminescence.

24. The method of claim 22, wherein the marker group is determined by electrochemiluminescence.

25. In an immunological detection method wherein a metal complex is bound to an immunological binding partner and used as a marker group to quantitatively or qualitatively determine an analyte in a sample solution, the improvement comprising using the conjugate of claim 20 as the marker group.

26. The method of claim 25, wherein the marker group is determined by luminescence.

27. The method of claim 25, wherein the marker group is determined by electrochemiluminescence.

28. In a nucleic acid hybridization detection method wherein a metal complex is bound to a probe and used as a marker group to quantitatively or qualitatively determine a nucleic acid in a sample solution, the improvement comprising using the marker group of claim 1 as the marker group.

29. The method of claim 28, wherein the marker group is determined by luminescence.

30. The method of claim 28, wherein the marker group is determined by electrochemiluminescence.

31. In a nucleic acid hybridization detection method wherein a metal complex is bound to a probe and used as a marker group to quantitatively or qualitatively determine a nucleic acid in a sample solution, the improvement comprising using the conjugate of claim 20 as the marker group.

32. The method of claim 31, wherein the marker group is determined by luminescence.

33. The method of claim 31, wherein the marker group is determined by electrochemiluminescence.

34. The metal complex as claimed in claim 18, wherein the metal complex further comprises at least one counterion A which balances the charge carried by the at least one charge carrier.

35. The metal complex as claimed in claim 19, wherein the metal complex further comprises at least one counterion A which balances the charge carried by the at least one charge carrier.