Unsymmetrical fluorescein derivatives

• US 4,439,356 A
• Filed: 03/03/1981
• Issued: 03/27/1984
• Est. Priority Date: 03/03/1981
• Status: Expired due to Term

• Current Assignee: DADE Behring Beteiligungs GmbH (Siemens AG)
• Original Assignee: Syva Co.
• Inventors: Khanna, Pyare, Colvin, Warren
• Primary Examiner(s): Kight, III, John
• Assistant Examiner(s): Nutter, Nathan M.
• Application Number:
  US06/240,031
• Time in Patent Office: 1,120 Days
• Field of Search: 260/112 R, 260/112 B, 260/112.5 R, 260/112.7, 260/121, 260/335, 424/8, 424/12, 424/85, 424/88, 435/7, 435/188, 23/230 B, 525/420, 549/388
• US Class Current: 530/350
• CPC Class Codes: C07D 311/82 Xanthenes
• CPC Class Codes: Y10S 435/961 including a step of forming, releasing, or exposing the antigen or forming the hapten-immunogenic carrier complex or the antigen per se

Abstract

Unsymmetrically substituted 6-hydroxy-3H-xanthen-3-ones for use as fluorescers or quenchers, particularly conjugated to provide reagents in competitive protein binding assays. Substituents are aliphatic groups bonded to an annular carbon atom through carbon or oxygen.

Claims

1. A compound of the formula ##STR6## wherein:

one of the γ

s will be of the formula --(O).sub.ε

ρ and

the other is δ

;

wherein ε

is 0 or 1 and ρ

is an aliphatic group of from about 1 to 12 carbon atoms and 0-1 carboxyl groups;

the δ

'"'"'s are the same or different and are hydrogen or halogen of atomic number 17 to 80;

L is a bond or divalent organic radical of up to 20 carbon atoms;

α

may be taken together with Ψ

to define an active group capable of forming a stable covalent bond with carbon, nitrogen or oxygen or is an organic compound, and is at other than the ortho position when L is benzene and Ψ and

α

are taken together to define carboxy;

Ψ

, when not taken together with α

is a linking group to α

;

v is 1 when Ψ

is taken together with α and

is otherwise on the average at least 1 and not more than the molecular weight of α

divided by 500.

with the proviso that when ε

is 0, one of the δ

'"'"'s may be an aliphatic group of from about 1 to 12 carbon atoms asymmetrically substituted in relation to ρ

.

2. A compound according to claim 1 conjugated to a support.

3. A compound of the formula ##STR7## wherein:

one of the Gs is of the formula --(O)_e R and is otherwise D, where e is 0 or 1 and R is an aliphatic group of from 1 to 6 carbon atoms having from 0 to 1 carboxy groups;

Z is carboxy or sulphonoxy;

D is hydrogen or halogen of atom number 17 to 80;

W is a bond or divalent organic radical having up to 10 carbon atoms;

Y may be taken together with A to form an active functionality capable of forming a covalent bond with carbon, nitrogen or oxygen when not taken together with A, Y is a linking functionality;

A when not taken together with Y is a ligand or receptor of at least about 125 daltons;

X is halo of atomic number 9 to 80;

m is 1 to 3;

5. A compound according to claim 3, wherein e is 0.

6. A compound according to claim 3, wherein e is 1.

8. A compound according to claim 6, wherein A'"'"' is taken together with Y'"'"'.

9. A compound according to claims 6 or 7, wherein R'"'"' is methyl.

10. A compound according to claims 6 or 7, wherein at least one of the D'"'"'s and X'"'"'s are chloro.

11. A compound according to claim 6, wherein A'"'"' is a ligand.

12. A compound according to claim 6, wherein A'"'"' is a receptor.

14. A compound according to claim 12, wherein e'"'"' is 0.

15. A compound according to claim 12, wherein e'"'"' is 1.

4. p is 0 to 3, with the sum of m and p being not greater than 4;

n will be 1 when Y and A are taken together or otherwise be on the average 1 to the molecular weight of A divided by 500,

with the proviso that when W is a bond and Y and A are taken together to define carboxy, Y is at other than the ortho position.

7. A compound of the formula ##STR8## wherein:

one of G'"'"' or G" is --(O)_e'"'"' R'"'"' and is otherwise D'"'"', wherein e'"'"' is 0 or 1 and R'"'"' is alkyl of from 1 to 3 carbon atoms;

D'"'"' is hydrogen or halogen of atomic number 17 to 80;

Y'"'"' may be taken together with A'"'"' to define a non-oxo-carbonyl group and is otherwise a non-oxo-carbonyl group forming an amide group with an amino group of A'"'"';

A'"'"' when not taken together with Y'"'"', is a ligand or receptor of at least about 125 daltons;

W'"'"' is a bond or aliphatic linking group of from 1 to 12 atoms other than hydrogen, which are carbon, nitrogen, oxygen or sulfur, there being from 0 to 8 carbon atoms and 0 to 8 heteroatoms, wherein nitrogen is present as amino or amido; and

oxygen and sulfur are present as oxy, oxo or the sulfur analogs thereof;

Z'"'"' is carboxy;

X'"'"' is halo of atomic number 9 to 80;

n'"'"' is 1 when Y'"'"' and A'"'"' are taken together and is otherwise on the average at least 1 to the molecular weight of A'"'"' divided by about 1,000;

m'"'"' is 0 to 2;

p'"'"' is 0 to 3, the sum of m'"'"' and p'"'"' being not greater than 4,

with the proviso that, when W'"'"' is a bond and Y'"'"' and A'"'"' are taken together to define carboxy, Y'"'"' is at other than the ortho position.

13. A compound of the formula ##STR9## wherein:

D'"'"' is hydrogen or chloro;

e'"'"' is 0 or 1;

the sum of m'"'"' and p'"'"' is 0 to 4, wherein m'"'"' is 0 to 2;

A'"'"' is hydroxyl or a ligand or receptor of at least about 125 daltons; and

--COA'"'"' is at other than the ortho position.

16. A compound according to claims 13 and 14, wherein A'"'"' is hydroxyl.

17. A compound according to claims 13 and 14, wherein A'"'"' is a ligand.

19. A compound according to claim 17, wherein e'"'"' is 0.

20. A compound according to claim 17, wherein e'"'"' is 1.

21. A compound according to claims 17 and 18, wherein A'"'"' is hydroxyl.

22. A compound according to claims 13 or 17, bound to a support.

18. A compound of the formula ##STR10## wherein:

e'"'"' is 0 or 1;

m'"'"' is 0 to 2;

p'"'"' is 0 to 3, the sum of m'"'"' and p'"'"' being not greater than 4;

D'"'"' is hydrogen or chloro; and

A'"'"' is hydroxyl or a ligand or receptor of at least about 125 daltons;

with the proviso that, when A'"'"' is hydroxyl, --COA'"'"' is at other than the ortho position.

23. A compound according to claims 18 or 19, wherein A'"'"' is a ligand.