Use of metallo-porphyrin conjugates for the detection of biological substances

• US 6,004,530 A
• Filed: 05/30/1997
• Issued: 12/21/1999
• Est. Priority Date: 06/04/1996
• Status: Expired due to Term

• Current Assignee: Roche Diagnostics GmbH (Roche Holding AG)
• Original Assignee: Roche Diagnostics GmbH (Roche Holding AG)
• Inventors: Tanke, Hans, De Haas, Richard Robert, Van Gijlswijk, Rob, Sagner, Gregor
• Primary Examiner(s): Shah, Mukund J.
• Assistant Examiner(s): TRUONG, TAMTHOM NGO
• Application Number:
  US08/866,043
• Time in Patent Office: 935 Days
• Field of Search: 424/9.61, 424/1.65, 424/1.85, 540/145, 540/121, 540/122
• US Class Current: 424/1.65
• CPC Class Codes: C07D 487/22 in which the condensed system contains four or more hetero rings
• CPC Class Codes: G01N 33/533 with fluorescent label

Abstract

The present invention refers to a process for the preparation of an activated metallo-porphyrin derivative and to novel activated metallo-porphyrin derivatives obtainable by said process. Further, the present invention refers to conjugates of metallo-porphyrins with molecules having at least one primary amino group, especially biomolecules. The conjugates are used in a procedure for the detection of a biological substance, especially in an immunoassay or a nucleic acid hybridization assay.

Claims

1. A process for the preparation of a metallo-porphyrin derivative having the general formula (I):

space="preserve" listing-type="equation">M P--(RCOOX).sub.n (I)

wherein M is palladium or platinum,

P is a porphyrin moiety,

X is a succinimide residue, a thiophenyl residue, an aryl residue or a vinyl residue which is covalently bound to the porphyrin moiety via R, wherein R is independently in each occurrence a chemical bond, or a linker group, and

n is from 1 to 4,

the process comprising reacting a metallo-porphyrin having the general formula (II);

space="preserve" listing-type="equation">M P--(RCOOH).sub.m (II)

wherein M, P and R are defined as above and m is 1-4, with a carbodiimide, 1-hydroxybenzothiazole and an active ester coupling agent which comprises a succinimide residue, a thiophenyl residue, an aryl residue or a vinyl residue, in an organic solvent, to obtain the metallo-porphyrin derivative of formula (I).

2. The process according to claim 1,

wherein R is a chemical bond, a C₁ -C₂₀ alkylene group which may contain heteroatoms, an arylene group, an aralkylene group or an alkarylene group.

3. The process according to claim 1,

wherein n is from 2 to 3.

4. The process according to claim 1,

wherein the carbodiimide is dicyclohexylcarbodiimide.

5. The process according to claim 1,

wherein the active ester coupling agent is N-hydroxysuccinimide.

6. The process according to claim 1,

wherein the molar ratio of carboxylic acid groups to be activated (n) to carbodiimide to 1-hydroxybenzothiazole to active ester coupling agent is about 1:

1;

1;

1.

7. The process according to claim 1,

wherein the organic solvent is dry dimethylformamide.

8. The process according to claim 1,

wherein the metallo-porphyrin (II) is Pd- or Pt-3,8,13,18-tetramethyl-21H,23H-porphine-2,7,12,17-tetrapropionic acid.

9. The process of claim 1,

wherein the metallo-porphyrin (II) is Pd- or Pt-meso-tetra (4-carboxyphenyl) porphine.

10. A metallo-porphyrin derivative having the general formula (I):

space="preserve" listing-type="equation">M P--(RCOOX).sub.n (I)

wherein M is palladium or platinum,

P is a porphyrin moiety,

X is a succinimide residue, a thiophenyl residue, an aryl residue or a vinyl residue which is covalently bound to the porphyrin moiety via R, wherein R is independently in each occurrence a chemical bond or a linker group and

n is from 1 to 4.

11. The activated metallo-porphyrin derivative according to claim 10, wherein n is 2 or 3.

12. A process for the preparation of a metallo-porphyrin conjugate, comprising

coupling an activated metallo-porphyrin derivative to a molecule having at least one primary amino group, wherein the metallo-porphyrin derivative has the general formula (I):

space="preserve" listing-type="equation">M P--(RCOOX).sub.n (I)

wherein M is palladium or platinum, P is a porphyrin moiety, X is a succinimide residue, a thiophenyl residue, an aryl residue or a vinyl residue which is covalently bound to the porphyrin moiety via R, wherein R is independently in each occurrence a chemical bond or a linker group and n is from 1 to 4,

wherein at least one bond is formed between an activated carboxylic acid ester group of the activated metallo-porphyrin derivative and the at least one primary amino group.

13. The process according to claim 12,

wherein the molecule having at least one primary amino group is a polymer.

14. The process according to claim 12,

wherein the molecule having at least one primary amino group is a biomolecule.

15. The process according to claim 14,

wherein said biomolecule is selected from the group comprising biogenic amines, hormones, vitamins, metabolites, nucleosides, nucleotides, oligo-nucleotides, nucleic acids, peptides, polypeptides, glycoproteins, saccharides, lipids, cellular and viral components, cells and viruses.

16. The process according to claim 14,

wherein said biomolecule is selected from the group comprising tyramine, allylamine-modified nucleotides, immunoglobulins, immunoglobulin fragments and avidin.

17. A process comprising bringing a sample into contact with a conjugate obtained by the process of claim 12 and detecting the presence of said conjugate.

18. The process according to claim 17, wherein said procedure comprises a detection of the conjugate by phosphorescence.

19. The process according to claim 17, wherein said detection comprises a time-resolved measurement.

20. The process according to claim 17, wherein said procedure comprises an immunoassay.

21. The process according to claim 17, wherein said procedure comprises a nucleic acid hybridization assay.

22. The process according to claim 17, wherein said procedure comprises an in situ and/or in vitro measurement in an autofluorescing environment.

23. The process according to claim 22, wherein said procedure comprises an in situ immunohistochemical, immunocytochemical or hybridization measurement.

24. The process according to claim 23, wherein the detection is carried out by microscope.

25. The process according to claim 22, wherein the detection is carried out on an autofluorescing membrane.

26. The process according to claim 22, wherein said procedure is a FACS-analysis.

27. The process according to claim 17, wherein said procedure comprises an oxygen measurement.

28. A method as recited in claim 17, wherein said step of detecting the presence of said conjugate comprises detecting an amount of said conjugate and/or detecting localization of said conjugate.

29. A process for the preparation of a metallo-porphyrin conjugate, comprising

coupling an activated metallo-porphyrin derivative to a molecule having at least one primary amino group, wherein the metallo-porphyrin derivative has the general formula (I):

space="preserve" listing-type="equation">M P--(RCOOX).sub.n (I)

wherein M is palladium or platinum, P is a porphyrin moiety, X is a succinimide residue, a thiophenyl residue, an aryl residue or a vinyl residue which is covalently bound to the porphyrin moiety via R, wherein R is independently in each occurrence a chemical bond, or a linker group, and n is from 1 to 4, obtained by reacting a metallo-porphyrin having the general formula (II);

space="preserve" listing-type="equation">M P--(RCOOH).sub.m (II)

wherein M, P and R are defined as above and m is 1-4, with a carbodiimide, 1-hydroxybenzothiazole and an active ester coupling agent which comprises a succinimide residue, a thiophenyl residue, an aryl residue or a vinyl residue, in an organic solvent,

wherein at least one bond is formed between an activated carboxylic acid ester group of the activated metallo-porphyrin derivative and the at least one primary amino group.