Fluorescent phospholipase A 2 indicators

• US 8,513,451 B2
• Filed: 05/30/2008
• Issued: 08/20/2013
• Est. Priority Date: 05/30/2007
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Kang, Hee Chol
• Primary Examiner(s): Puttlitz, Karl J
• Application Number:
  US12/602,388
• Publication Number:
  US 20100203564A1
• Time in Patent Office: 1,908 Days
• Field of Search: None
• US Class Current: 558/72
• CPC Class Codes: C09B 23/04 one >CH- group, e.g. cyanines, isocyanines, pseudocyanines
• CPC Class Codes: C09B 57/10 Metal complexes of organic compounds not being dyes in uncomplexed form
• CPC Class Codes: C09B 69/001 Dyes containing an onium group attached to the dye skeleton via a bridge
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: G01N 33/531 Production of immunochemical test materials
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes

Abstract

Compositions, methods of synthesis and applications of phospholipase A₂ (PLA₂) specific enzyme substrates which exhibit fluorescence resonance energy transfer (FRET) are described. The compounds generally have the structure: (I) wherein, the variables are described throughout the application. These novel compounds provide a sensitive method to monitor real time PLA₂ specific enzyme activities in various cells, tissues and small organisms with fluorescence-ratiometric analysis.

Claims

1. A compound of the formula:

2. The compound of claim 1, wherein D¹ is a donor and D² is an acceptor.

3. The compound of claim 1, wherein D² is substituted with an aryl or heteroaryl group.

4. The compound of claim 1, wherein D¹ is:

5. The compound of claim 3, wherein D² is:

6. The compound of claim 1, wherein X is —

O—

.

7. The compound of claim 1, wherein L¹ is a single covalent bond, or a covalent linkage that is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1-30 nonhydrogen atoms selected from the group consisting of C, N, P, O and S;

and are composed of any combination of ether, thioether, amine, ester, carboxamide, sulfonamide, hydrazide bonds and aromatic or heteroaromatic bonds.

8. The compound of claim 1, wherein L¹ is -alkyl- or -substituted alkyl-.

9. The compound of claim 1, wherein L² is a single covalent bond, or a covalent linkage that is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1-30 nonhydrogen atoms selected from the group consisting of C, N, P, O and S;

and are composed of any combination of ether, thioether, amine, ester, carboxamide, sulfonamide, hydrazide bonds and aromatic or heteroaromatic bonds.

10. The compound of claim 1, wherein L² is -alkyl- or -substituted alkyl-.

11. The compound of claim 1, wherein L¹ and L² are independently —

C_3-8 alkyl-.

32. The compound of claim 1, wherein the compound has the formula:

12. A compound of formula:

13. The compound of claim 12, wherein R¹, R², R³, R⁴, R⁵ and R⁶ are each independently selected from the group consisting of H, halo, alkyl or substituted alkyl.

14. The compound of claim 12, wherein R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each independently selected from the group consisting of H, aryl, substituted aryl, heteroaryl, substituted heteroaryl.

15. The compound of claim 14, wherein at least one of R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² is aryl, substituted aryl, heteroaryl, substituted heteroaryl.

16. The method of claim 12, wherein R⁶ is heteroaryl.

17. The method of claim 12, wherein R⁶ is thiophenyl.

18. The method of claim 12, wherein R¹, R², R³, R⁴ and R⁵ are H.

19. The method of claim 12, wherein R¹⁰ and R¹² are methyl.

20. The method of claim 12, wherein R⁷, R⁸, R⁹, and R¹¹ are H.

21. The compound of claim 12, wherein L¹ and L² are each independently a single covalent bond, or a covalent linkage that is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1-30 nonhydrogen atoms selected from the group consisting of C, N, P, O and S;

and are composed of any combination of ether, thioether, amine, ester, carboxamide, sulfonamide, hydrazide bonds and aromatic or heteroaromatic bonds.

22. The compound of claim 12, wherein L¹ and L² are each independently -alkyl- or -substituted alkyl-.

23. The compound of claim 12, wherein L¹ and L² are independently —

C_3-8 alkyl-.

24. A compound of formula:

25. The compound of claim 24, wherein L¹ and L² are each independently a single covalent bond, or a covalent linkage that is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1-30 nonhydrogen atoms selected from the group consisting of C, N, P, O and S;

and are composed of any combination of ether, thioether, amine, ester, carboxamide, sulfonamide, hydrazide bonds and aromatic or heteroaromatic bonds.

26. The compound of claim 24, wherein L¹ and L² are each independently -alkyl- or -substituted alkyl-.

27. The compound of claim 24, wherein L¹ and L² are independently —

C_3-8 alkyl-.

28. A method for detecting phospholipase A₂ (PLA₂) activity in a sample, comprising:

contacting the sample with a compound of formula;

29. The method of claim 28, wherein the sample comprises cells.

30. The method of claim 28, wherein the incubating step comprises incubating the sample and compound for sufficient time to allow the compound to enter a cell.

31. A kit for detecting phospholipase A₂ (PLA₂) activity, comprising:

a compound of formula;