Porphyrin derivatives as fluorescent markers for immunoassays

• US 4,614,723 A
• Filed: 05/23/1984
• Issued: 09/30/1986
• Est. Priority Date: 06/03/1983
• Status: Expired due to Fees

• Current Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Original Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Inventors: Schmidt, Dieter, Steffen, Hans
• Primary Examiner(s): Warden, Robert J.
• Assistant Examiner(s): NOT, DEFINED
• Application Number:
  US06/613,127
• Time in Patent Office: 860 Days
• Field of Search: 436/536, 436/800, 436/546, 260/245.91, 546/256
• US Class Current: 436/536
• 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
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

Abstract

There are described novel porphyrin derivatives of the general formula ##STR1## wherein either R¹ signifies the group ##STR2## and R² signifies the group ##STR3## or R¹ signifies the group ##STR4## and R² signifies the group ##STR5## whereby X^- signifies a halogen ion, an arylsulphonate ion, an alkylsulphonate ion or an alkyl sulphate ion, A signifies (C_1-8)-alkylene and R³ signifies (C_1-4)-alkyl,

and sulphonic acid salts of compounds of formula I in which R¹ signifies the group ##STR6## as well as a process for their manufacture and intermediates used in this process.

Because of their water-solubility the novel porphyrin derivatives are suitable as label molecules for highly sensitive fluorescence immuno-assays, especially for time-resolving fluorescence immuno-assays. The coupling of the novel porphyrin derivatives to immunologically-active materials is carried out in the customary manner, for example with a water-soluble carbodiimide derivative.

Claims

1. A porphyrin compound of the formula:

##STR22## wherein R¹ is ##STR23## R² is ##STR24## A is (C_1-8)-alkylene, and the salts thereof, wherein said salts are formed from inorganic or organic bases.

2. A compound according to claim 1 which is 4-[10,15,20-tris(4-sulphophenyl)-21H,23H-porphin-5-yl]-benzoic acid.

3. A compound according to claim 1 which is [4-[10,15,20-tris(4-sulphophenyl)-21H-23H-porphin-5-yl]phenoxy]acetic acid.

4. A compound according to claim 1 which is 6-[4-[10,15,20-tris(4-sulphophenyl)-21H,23H-porphin-5-yl]phenyl]hexanecarboxylic acid.

5. A compound according to claim 1 which is 1-(carboxymethyl)-1'"'"',1",1"'"'"'-trimethyl-4,4'"'"',4",4"'"'"'-(21H,23H-porphin-5,10,15,20-tetrayl)tetrakispyridinium tetraiodide.

15. A compound according to claim 1 wherein said salts are alkali metal salts or ammonium salts.

16. A compound according to claim 15 wherein said salt is the sodium salt.

6. Compounds of the formula ##STR25## wherein R⁴ is selected from the group consisting of --COOH, --A--COOH and --O--A--COOH and A is (C_1-8)-alkylene.

7. Compounds of the formula ##STR26## wherein X^- is selected from the group consisting of halogen ions, benzenesulfonate ions, p-toluenesulfonate ions, (C_1-12)-alkylsulfonate ions and (C_1-12)-alkyl sulphate ions and A is (C_1-8)-alkylene.

8. A fluorescent antibody, consisting essentially of an antibody specific to an antigen to be detected, and, conjugated to said antibody, a fluorescent compound of the formula ##STR27## wherein R¹ is ##STR28## and R² is ##STR29## A is (C_1-8)-alkylene and the salts of said fluorescent compound, wherein said salts are formed from inorganic or organic bases.

9. A fluorescent antibody according to claim 8 wherein the antibody is specific to carcinoembryonal antigen.

10. A fluorescent antibody according to claim 9 wherein a water-soluble carbodiimide derivative couples the antibody to said fluorescent compound.

19. A fluorescent antibody according to claim 10 wherein said carbodiimide derivative is 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide methyl-p-toluenesulfonate.

11. In an immunoassay of the time resolving type which utilizes an antibody conjugated to fluorescent label, the improvement which comprises using as said label a compound of the formula ##STR30## wherein R¹ is ##STR31## and R² is ##STR32## A is (C_1-8)-alkylene and the salts thereof wherein said salts are formed from inorganic or organic bases.

12. A porphyrin compound of the formula:

##STR33## wherein R¹ is ##STR34## R² is ##STR35## X^- is selected from the group consisting of halogen ions, benzene sulfonate ions, p-toluenesulfonate ions, (C_1-12)-alkylsulfonate ions and (C_1-12)-alkyl sulphate ions;

A is (C_1-8)-alkylene; and

R³ is (C_1-4)-alkyl.

13. A compound according to claim 12 wherein R¹ is ##STR36## and R² is ##STR37##

14. A compound according to claim 12 wherein R² is ##STR38##

17. A compound according to claim 12 wherein X^- is p-toluenesulfonate ion.

18. A compound according to claim 12 wherein X^- is halogen ion.

20. A fluorescent antibody consisting essentially of, an antibody specific to an antigen to be detected, and, conjugated to said antibody, a fluorescent compound of the formula ##STR39## wherein R¹ is ##STR40## R² is ##STR41## X^- is selected from the group consisting of halogen ions, benzene sulfonate ions, p-toluenesulfonate ions, (C_1-12)-alkylsulfonate ions and (C_1-12)-alkyl sulphate ions;

R³ is (C_1-4)-alkyl.

21. A fluorescent antibody according to claim 20 wherein the antibody is specific to carcinoembryonal antigen.

22. A fluorescent antibody according to claim 21 wherein a water-soluble carbodiimide derivative couples said antibody to said fluorescent compound.

23. A fluorescent antibody according to claim 22 wherein said carbodiimide derivative is 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide methyl-p-toluenesulfonate.

24. In an immunoassay of the time resolving type which utilizes an antibody conjugated to fluorescent label, the improvement which comprises using as said label a compound of the formula:

##STR42## wherein R¹ is ##STR43## R² is ##STR44## X^- is selected from the group consisting of halogen ions, benzene sulfonate ions, p-toluenesulfonate ions, (C_1-12)-alkylsulfonate ions and (C_1-12)-alkyl sulphate ions;

R³ is (C_1-4)-alkyl.