Novel ether substituted fluorescein polyamino acid compounds as fluorescers and quenchers

• US 4,318,846 A
• Filed: 09/07/1979
• Issued: 03/09/1982
• Est. Priority Date: 09/07/1979
• Status: Expired due to Term

• Current Assignee: DADE Behring Beteiligungs GmbH (Siemens AG)
• Original Assignee: Syva Co.
• Inventors: Ullman, Edwin F., Khanna, Pyare
• Primary Examiner(s): Schain, Howard E.
• Application Number:
  US06/073,163
• Time in Patent Office: 914 Days
• Field of Search: 260/112 R, 260/112 B, 260/121, 424/85, 424/88, 525/420, 435/188
• US Class Current: 530/391.5
• CPC Class Codes: C07C 41/26 by introduction of hydroxy or O-metal groups
• CPC Class Codes: C07C 43/23 containing hydroxy or O-metal groups
• CPC Class Codes: C07D 311/82 Xanthenes
• CPC Class Codes: C07D 493/10 Spiro-condensed systems
• CPC Class Codes: C09B 11/08 Phthaleins; Phenolphthaleins; Fluorescein
• CPC Class Codes: C09B 11/26 Triarylmethane dyes in which at least one of the aromatic nuclei is heterocyclic phthaleins C09B11/24
• CPC Class Codes: G01N 33/533 with fluorescent label
• 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
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 530/806 Antigenic peptides or proteins

Abstract

Diether symmetrically substituted fluoresceins are provided having at least one anionic group and a linking functionality. Depending upon the site of substitution, the compounds can be used as fluorescers absorbing at wavelengths in excess of 500 nm or as quenchers, absorbing at wavelengths in excess of 500 nm and exhibiting substantially no fluorescence. The compounds find wide application, particularly as labels in fluorescent immunoassays.

Claims

1. A chromogenic compound capable of donating or accepting electronic energy, which is a 2,7-di(aliphatic chalcogen ether substituted) or 4,5-di(aliphatic chalcogen ether substituted)-9-substituted-6-hydroxy-3H-xanthen-3-one, wherein chalcogen is of atomic number 8 to 16, conjugated to a poly(amino acid).

2. A chromogenic compound according to claim 1, which is a 2,7-di(alkoxy substituted)-9-phenyl-6-hydroxy-3H-xanthen-3-one, conjugated to a poly(amino acid).

3. A chromogenic compound according to claim 1, which is a 4,5-di(alkoxy substituted-9-phenyl-6-hydroxy-3H-xanthen-3-one, conjugated to a poly(amino acid).

4. A compound of the formula:

##STR19## wherein;

the As are the same or different;

the Ds are the same or different;

either the As or the Ds are ethers of the formula --JMX, wherein J is chalcogen of atomic number 8 to 16;

when other than JMX they are hydrogen, or halogen of atomic number 9 to 53;

M is a divalent hydrocarbon group of from 1 to 8 carbon atoms;

W is a poly(amino acid);

one of the Xs is a linking functionality to W;

the other X being hydrogen or non-oxo-carbonyl with the proviso that when X is bonded to an annular carbon atom, X can be halo;

Q is a bond or spacer arm;

Y is halogen of atomic number 9 to 53;

Z is an acidic anionic group;

m is an integer of from 0 to 3;

n is an integer of from 0 to 4, wherein m plus n is not greater than 4;

p is 1 to the molecular weight of W divided by 500.

5. A compound according to claim 4, wherein --JMX is alkoxy of from 1 to 3 carbon atoms.

6. A compound according to claim 5, wherein the As are alkoxy.

7. A compound according to claim 5, wherein the Ds are alkoxy.

8. A compound of the formula:

##STR20## wherein;

the A¹ s are the same and the D¹ s are the same; and

either the A¹ s or the D¹ s are oxyethers of the formula --OR^b X^b ;

when other than --PR^b X^b, they are hydrogen or halogen of atomic number 9 to 53;

wherein R^b is saturated aliphatic hydrocarbon of from 1 to 3 carbon atoms;

one of X^a and X^b is a linking functionality to W¹, wherein said linking functionality including a non-oxo-carbonyl or the sulfur analog thereof;

when other than a linking functionality, X^b is hydrogen or carboxyl and X^a is hydrogen, carboxyl or halo of atomic number 9 to 53;

Q¹ is a bond or a spacer arm, being a bond when X^a is other than a linking functionality;

when Q¹ is a spacer arm, it has a chain of from 1 to 16 atoms which are carbon, oxygen and nitrogen, wherein heteroatoms are spaced apart by at least two carbon atoms when bonded to saturated carbon atoms;

Z¹ is a carboxylic acid or sulfonic acid group;

Y¹ is halogen of atomic number 9 to 53;

m¹ is an integer from 1 to 2;

n¹ is an integer from 0 to 4, wherein m¹ plus n¹ is not greater than 4;

W¹ is a poly (amino acid).

9. A compound according to claim 8, wherein the A¹ s are alkoxy of from 1 to 3 carbon atoms.

10. A compound according to claim 8, wherein the D¹ s are alkoxy of from 1 to 3 carbon atoms.

11. A compound according to any of claims 9 or 10, wherein X^a is a carbonyl.

12. A compound according to the formula:

##STR21## wherein;

either the A² s or the D² s are alkoxy of from 1 to 3 carbon atoms;

when other than alkoxy, they are hydrogen or halogen of atomic number 9 to 53;

Y² is halogen of atomic number 9 to 53;

Z² is carboxyl;

m² is an integer of from 1 to 2;

n² is an integer of from 0 to 4, wherein m² plus n² is not greater than 4;

Q² is a bond or spacer arm of from 1 to 12 atoms in the chain, which are carbon, nitrogen and oxygen;

X^c is a non-oxo-carbonyl containing linking group including the sulfur analog thereof;

W² is a poly(amino acid);

p² is at least 1 and up to the molecular weight of W² divided by 500.

13. A compound according to claim 12, wherein said poly(amino acid) is a hapten of from about 125 to 1000 molecular weight.

14. A compound according to claim 12, wherein said poly(amino acid) is an antigen of at least about 5000 molecular weight.

15. A compound according to any of claims 13 and 14, wherein A² is alkoxy of from 1 to 3 carbon atoms, D² is hydrogen or halogen of atomic 9 to 53, Q² is a bond or spacer arm of from 1 to 12 atoms in the chain, X^c is carbonyl bonded to an amino group of W² to form an amide;

and p² is about 1 to 100.

16. A compound of the formula:

##STR22## wherein W is a poly(amino acid), and n is one to the molecular weight of W divided by 500.

21. A compound according to any of claims 16, 17, 18, 19, or 20, wherein said poly(amino acid) is an antigen.

22. A compound according to any of claims 16, 17, 18, 19, or 20, wherein said poly(amino acid) is an antibody.

17. A compound of the formula:

##STR23## wherein W is a poly(amino acid) and n is 1 to the molecular weight of W divided by 500.

18. A compound of the formula:

##STR24## wherein W is a poly(amino acid) and n is 1 to the molecular weight of W divided by 500.

19. A compound of the formula:

##STR25## wherein W is a poly(amino acid) and n is 1 to the molecular weight of W divided by 500.

20. A compound of the formula:

##STR26## wherein W is a poly(amino acid) and n is 1 to the molecular weight of W divided by 500.