Hydrophilic metal complexes

• US 5,981,286 A
• Filed: 01/16/1997
• Issued: 11/09/1999
• 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: Issberner, Jorg, Frommberger, Bruno, Herrmann, Rupert, Josel, Hans-Peter, Vogtle, Fritz, Pappert, Gunter
• Primary Examiner(s): Adams, Donald E.
• Assistant Examiner(s): PONNALURI, PADMASHRI
• Application Number:
  US08/765,452
• Time in Patent Office: 1,027 Days
• Field of Search: 436/172, 436/518, 436/800, 436/84, 436/92, 556/136, 252/700, 252/301.16, 252/301.33, 252/301.4 R, 546/10
• US Class Current: 436/84
• 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
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10T 436/141111 Diverse hetero atoms in same or different rings [e.g., alkaloids, opiates, etc.]

Abstract

The present invention concerns new hydrophilic metal complexes and their use as luminescent marker groups in immunoassays.

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 --X.sub.m A

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;

X is a reactive or activatable functional group selected from the group consisting of an activated carboxylic acid group, a maleimide, a carboxylic acid, a thiol, a halide and a photoactivatable group;

n is 1 to 10;

m is 1 to 6; and

A is at least one negatively charged group,

wherein the metal complex contains at least one hydrophilic group selected from the group consisting of a C₂ -C₃ alkyleneoxy group, a C₂ -C₃ alkylenethio group, a C₂ -C₃ alkyleneamino group and a polyhydroxy group, wherein the at least one hydrophilic group is located at at least one of the following locations (a) and (b);

(a) as a substituent or component of a substituent of one of the ligands L₁, L₂ and L₃ ; and

(b) as a linker or component of a linker which links the group X and one of the ligands L₁, L₂ and L₃.

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 A is selected from the group consisting of a hexafluorophosphate group, a trifluoroacetate group, a tetrafluoroborate group and a halide ion.

6. The metal complex as claimed in claim 1, wherein the polyhydroxy group is a group of formula (IIa) or (IIb)

7. The metal complex as claimed in claim 6, wherein the polyhydroxy group has a formula --NR--C(CH₂ OH)₃, wherein R is as defined in claim 6.

8. The metal complex as claimed in claim 1, wherein the C₂ -C₃ alkyleneoxy group is an ethyleneoxy group, the C₂ -C₃ alkylenethio group is an ethylenethio group, and the C₂ -C₃ alkyleneamino group is an ethyleneamino group.

9. The metal complex as claimed in claim 1, wherein the metal complex contains 1-30 hydrophilic groups each independently selected from the group consisting of a C₂ -C₃ alkyleneoxy group, a C₂ -C₃ alkylenethio group and a C₂ -C₃ alkyleneamino group.

10. The metal complex as claimed in claim 1, wherein the metal complex is of formula (III):

##STR8## wherein M, X and A are as defined in claim 1;

one of R₁ through R₆ is bound to --X; and

the remainder of R₁ through R₆ which are not bound to --X are each independently selected from the group consisting of hydrogen, C₁ -C₅ alkyl and a substituent containing at least one hydrophilic group which is selected from the group consisting of a C₂ -C₃ alkyleneoxy group, a C₂ -C₃ alkylenethio group and a C₂ -C₃ alkyleneamino group, wherein at least one of the remainder of R₁ through R₆ is the substituent containing the at least one hydrophilic group.

11. The metal complex as claimed in claim 1, wherein the metal complex is of formula (IIIa):

##STR9## wherein M, X and A are as defined in claim 1;

R₁ through R₅ are each independently selected from the group consisting of hydrogen, C₁ -C₅ alkyl and a substituent containing at least one hydrophilic group which is selected from the group consisting of a C₂ -C₃ alkyleneoxy group, a C₂ -C₃ alkylenethio group and a C₂ -C₃ alkyleneamino group;

s is 0-6; and

Y is a linker having 1-10 hydrophilic groups each independently selected from the group consisting of a C₂ -C₃ alkyleneoxy group, a C₂ -C₃ alkylenethio group and a C₂ -C₃ alkyleneamino group.

12. The metal complex as claimed in claim 1, wherein the metal complex is of formula (IIIb):

##STR10## wherein M, X and A are as defined in claim 1;

R₁ through R₅ are each independently selected from the group consisting of hydrogen, C₁ -C₅ alkyl and a substituent containing 1-10 hydrophilic groups each of which is selected from the group consisting of a C₂ -C₃ alkyleneoxy group, a C₂ -C₃ alkylenethio group and a C₂ -C₃ alkyleneamino group, wherein at least one of R₁ through R₅ is the substituent containing the 1-10 hydrophilic groups; and

s is 0-6.

13. The metal complex as claimed in claim 1, wherein the metal complex is of formula (IV):

##STR11## wherein M, Y, X and n are as defined in claim 1;

R₁ through R₃ are each independently selected from the group consisting of hydrogen, C₁ -C₅ alkyl and a substituent containing at least one hydrophilic group which is selected from the group consisting of a C₂ -C₃ alkyleneoxy group, a C₂ -C₃ alkylenethio group and a C₂ -C₃ alkyleneamino group; and

Y is a linker having 1-10 hydrophilic groups each independently selected from the group consisting of a C₂ -C₃ alkyleneoxy group, a C₂ -C₃ alkylenethio group and a C₂ -C₃ alkyleneamino group.

14. The metal complex as claimed in claim 1, wherein the metal complex is of formula (V):

##STR12## wherein M, X and A are as defined in claim 1;

Z is a linker;

R₁ '"'"' through R₄ '"'"' are each independently selected from the group consisting of hydrogen, C₁ -C₅ alkyl and a substituent containing at least one polyhydroxy group, wherein at least one of R₁ '"'"' through R₄ '"'"' is the substituent containing the at least one polyhydroxy group; and

R₅ '"'"' is hydrogen or C₁ -C₅ alkyl.

15. The metal complex as claimed in claim 14, wherein the --OH group of the at least one polyhydroxy group is substituted by a hydrophilic group.

16. The metal complex as claimed in claim 15, wherein the hydrophilic group comprises an element of formula (VIa) or (VIb):

space="preserve" listing-type="equation">--A.sub.1 --NR--W.sub.1 (A.sub.2 --NR--W.sub.2).sub.n '"'"' (VIa)

space="preserve" listing-type="equation">--A.sub.1 --O--W.sub.1 (A.sub.2 --O--W.sub.2).sub.n '"'"' (VIb)

wherein

A₁ and A₂ are each a same or different linker;

W₁ and W₂ are each a same or different organic residue having at least two hydroxy groups;

R is hydrogen or C₁ -C₅ alkyl; and

n'"'"' is 0 or the same number as the number of hydroxy groups on W₁.

17. A conjugate, comprising a metal complex coupled with a biological substance, wherein the metal complex has the formula:

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

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;

X is a reactive or activatable functional group;

n is 1 to 10;

m is 1 to 6; and

A is at least one negatively charged group,

wherein the metal complex contains at least one hydrophilic group selected from the group consisting of a C₂ -C₃ alkyleneoxy group, a C₂ -C₃ alkylenethio group, a C₂ -C₃ alkyleneamino group and a polyhydroxy group, wherein the at least one hydrophilic group is located at at least one of the following locations (a) and (b);

(a) as a substituent or component of a substituent of one of the ligands L₁, L₂ and L₃ ; and

(b) as a linker or component of a linker which links the group X and one of the ligands L₁, L₂ and L₃.

18. The conjugate as claimed in claim 17, wherein the biological substance is selected from the group consisting of biotin, an antibody, an antibody fragment, a nucleic acid, a polypeptide antigen, an immunologically reactive peptide and a hapten.