Fluorescent Phospholipase A2 Indicators

• US 20100203564A1
• Filed: 05/30/2008
• Published: 08/12/2010
• 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
• Granted Patent: US 8,513,451 B2
• Time in Patent Office: Days
• US Class Current: 435/18
• 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 comprising:

a substrate for phospholipase A₂ (PLA₂) operably connected through a linker to a FRET pair comprising a fluorescent donor molecule and fluorescent acceptor molecule having a first fluorescent signal, wherein one of the donor molecule or acceptor molecule is cleaved by PLA₂ and not phospholipase A₁ (PLA₁), wherein cleavage by PLA₂ results in a second fluorescent signal;

with the proviso that the FRET pair does not comprise NBD or Nile Red.

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

wherein,

D¹ is a borapolyazaindacene fluorophore;

D² is a borapolyazaindacene fluorophore;

L¹ is a linker;

L² is a linker; and

X is —

O—

, —

S—

or —

NH—

;

or a salt, stereoisomer, or tautomer thereof;

wherein D¹ and D² exhibit fluorescence resonance energy transfer (FRET).

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

4. The compound of claim 2, wherein D¹ is a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY).

5. The compound of claim 2, wherein D² is a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY).

6. The compound of claim 2, wherein D² is substituted with an aryl or heteroaryl group.

8. The compound of claim 2, wherein D² is:

9. The compound of claim 2, wherein X is —

O—

.

10. The compound of claim 2, 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.

11. The compound of claim 2, wherein L¹ is -alkyl- or -substituted alkyl-.

12. The compound of claim 2, 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.

13. The compound of claim 2, wherein L² is -alkyl- or -substituted alkyl-.

14. The compound of claim 2, wherein L¹ and L² are independently —

C_3-8 alkyl-.

7. The compound of claim 7, wherein D¹ is:

15. A compound of formula:

wherein,

L¹ is a linker;

L² is a linker;

R¹, R², R³, R⁴, R⁵ and R⁶ are each independently selected from the group consisting of H, alkyl, substituted alkyl, carbonyl, substituted carbonyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo, hydroxy, nitro, SO₃⁻

, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio, substituted alkylthio, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; and

R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each independently selected from the group consisting of H, alkyl, substituted alkyl, carbonyl, substituted carbonyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo, hydroxy, nitro, SO₃⁻

, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio, substituted alkylthio, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

16. The compound of claim 15, wherein R¹, R², R³, R⁴, R⁵ and R⁶ are each independently selected from the group consisting of H, halo, alkyl or substituted alkyl.

17. The compound of claim 15, wherein R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each independently selected from the group consisting of H, aryl, substituted aryl, heteroaryl, substituted heteroaryl.

18. The compound of claim 17, wherein at least one of R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² is aryl, substituted aryl, heteroaryl, substituted heteroaryl.

19. The method of claim 15, wherein R⁶ is heteroaryl.

20. The method of claim 15, wherein R⁶ is thiophenyl.

21. The method of claim 15, wherein R¹, R², R³, R⁴ and R⁵ are H.

22. The method of claim 15, wherein R¹⁰ and R¹² are methyl.

23. The method of claim 15, wherein R⁷, R⁸, R⁹, and R¹¹ are H.

24. The compound of claim 15, 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.

25. The compound of claim 15, wherein L¹ and L² are each independently -alkyl- or -substituted alkyl-.

26. The compound of claim 15, wherein L¹ and L² are independently —

C_3-8 alkyl-.

27. A compound of formula:

wherein,

L¹ is a linker; and

L² is a linker.

28. The compound of claim 27, 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.

29. The compound of claim 27, wherein L¹ and L² are each independently -alkyl- or -substituted alkyl-.

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

C_3-8 alkyl-.

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

contacting the sample with a compound of formula;

wherein,

D¹ is a borapolyazaindacene fluorophore;

D² is a borapolyazaindacene fluorophore;

L¹ is a linker;

L² is a linker; and

X is —

O—

, —

S—

or —

NH—

;

or a salt, stereoisomer, or tautomer thereof;

wherein D¹ and D² exhibit fluorescence resonance energy transfer (FRET) generating a first fluorescent signal;

incubating the sample and compound for sufficient time for -L²-D² to be cleaved if PLA₂ is in the sample; and

illuminating the sample with an appropriate wavelength, wherein PLA₂ activity is detected by a change in fluorescence to a second fluorescent signal.

32. The method of claim 31, wherein the sample comprises cells.

33. The method of claim 31, wherein the incubating step comprises incubating the sample and compound for sufficient time to allow the compound to enter a cell.

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

a compound of formula;

wherein,

D¹ is a polyazaindacene fluorophore;

D² is a polyazaindacene fluorophore;

L¹ is a linker;

L² is a linker; and

X is —

O—

, —

S—

or —

NH—

;

or a salt, stereoisomer, or tautomer thereof;

wherein D¹ and D² exhibit fluorescence resonance energy transfer (FRET); and

one or more components selected from the group consisting of written instructions, a standard, a control, a vial, an aqueous buffer solution and an organic solvent.