Membrane transportable fluorescent substrates

• US 20030103902A1
• Filed: 09/19/2002
• Published: 06/05/2003
• Est. Priority Date: 09/19/2001
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Sparks, Alison L.
• Granted Patent: US 6,933,126 B2
• Time in Patent Office: Days
• US Class Current: 424/9.6
• CPC Class Codes: C09B 11/02 derived from diarylmethanes
• CPC Class Codes: C09B 11/08 Phthaleins; Phenolphthaleins; Fluorescein
• CPC Class Codes: C09B 11/10 Amino derivatives of triarylmethanes
• CPC Class Codes: C09B 11/24 Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
• CPC Class Codes: C09B 57/02 Coumarine dyes
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

Intracellular enzyme-activated fluorescent substrates that can be transported into a cell are provided. The membrane transportable fluorescent substrates are complexes (e.g., ionic complexes) formed between an enzyme activated fluorescent substrate and a carrier molecule. The fluorescent substrates can be used in an intracellular assay of enzyme activity and/or expression.

Claims

1. A fluorescent substrate delivery system comprising a complex of an enzyme active fluorescent substrate and a carrier molecule.

2. The fluorescent substrate delivery system of claim 1, wherein the carrier molecule is selected from the group consisting of polylysine, histone, spermidine, polyamidoamine dendrimers, polyethylenimine, polyethylenimine dendrimers, polyvinylpyridinium salts and polyguanidine peptoids.

3. The fluorescent substrate delivery system of claim 1, wherein the fluorescent substrate comprises one or more anionic charge groups and the carrier molecule comprises cationic charge groups, and wherein the complex is formed by ionic charge interactions between the anionic and cationic charge groups.

4. The fluorescent substrate delivery system of claim 3, wherein the one or more anionic charge groups are selected from the group consisting of phosphates, phosphonates, carboxylates, sulfates, sulfonates, phenolates, boronates, and carbonates.

5. The fluorescent substrate delivery system of claim 3, wherein the cationic charge groups comprise protonated polyamino groups.

6. The fluorescent substrate delivery system of claim 1, wherein the fluorescent substrate comprises one or more cationic charge groups and the carrier molecule comprises anionic charge groups, and wherein the complex is formed by ionic charge interactions between the anionic and cationic charge groups.

7. The fluorescent substrate delivery system of claim 1, wherein the carrier molecule comprises a boronic acid group and the fluorescent substrate comprises at least one saccharide group complexed to the boronic acid group.

8. The fluorescent substrate delivery system of claim 7, wherein the saccharide group comprises β

-galactoside, β

-glucuronoside, or β

-mannoside.

9. The fluorescent substrate delivery system of claim 7, wherein the carrier molecule is selected from the group consisting of monomers, polymers and dendrites having boronic acid substituents.

10. The fluorescent substrate delivery system of claim 3, wherein the fluorescent substrate is selected from the group consisting of a fluorescein di-β

-D-galactopyranoside (FDG) having anionic substituents, a 4-methylumbelliferyl β

-D-galactopyranoside (MUG) having anionic substituents, a resorufin β

-D-galactopyranoside having ionic substituents and a 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) β

-D-galactopyranoside (DDAO galactoside) having ionic substituents.

11. The fluorescent substrate delivery system of claim 6, wherein the fluorescent substrate is selected from the group consisting of a fluorescein di-β

-D-galactopyranoside (FDG) having cationic substituents, a 4-methylumbelliferyl β

-D-galactopyranoside (MUG) having cationic substituents, a resorufin β

-D-galactopyranoside having cationic substituents and a 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) β

-D-galactopyranoside (DDAO galactoside) having cationic substituents.

12. A method for detecting the presence and/or measuring the activity of an enzyme in a cell comprising:

contacting a sample comprising the cell with a composition comprising a complex of a carrier molecule and an enzyme active fluorescent substrate comprising a group cleavable by the enzyme; and

detecting fluorescence from the sample;

wherein the presence of fluorescence indicates the presence of the enzyme in the cell and wherein the intensity of fluorescence indicates the activity or expression of the enzyme in the cell.

13. The method of claim 12, wherein the enzyme cleavable group is cleavable by an enzyme selected from the group consisting of glycosidases, esterases, proteases, oxidases, peptidases and phosphatases.

14. The method of claim 12, wherein the enzyme cleavable group is cleavable by β

-galactosidase.

15. The method of claim 12, wherein the fluorescent substrate comprises anionic or cationic charge groups which form an ionic complex with oppositely charged groups on the carrier molecule.

16. The method of claim 15, wherein the fluorescent substrate is selected from the group consisting of a fluorescein di-β

-D-galactopyranoside (FDG) having ionic substituents, a 4-methylumbelliferyl β

-D-galactopyranoside (MUG) having ionic substituents, a resorufin β

-D-galactopyranoside having ionic substituents and a 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) β

-D-galactopyranoside (DDAO galactoside) having ionic substituents.

17. A fluorescent substrate comprising at least one ionic group available for complexation through ionic charge interactions with oppositely charged ionic groups on a carrier molecule.

18. The fluorescent substrate of claim 17, wherein the fluorescent substrate is selected from the group consisting of a fluorescein di-β

-D-galactopyranoside (FDG) having ionic substituents, a 4-methylumbelliferyl β

-D-galactopyranoside (MUG) having ionic substituents, a resorufin β

-D-galactopyranoside having ionic substituents and a 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) β

-D-galactopyranoside (DDAO galactoside) having ionic substituents.

19. The fluorescent substrate of claim 17, further comprising lipophilic substituents capable of anchoring the substrate to a cell membrane.

20. The fluorescent substrate of claim 17, further comprising chloromethyl substituents.

21. The fluorescent substrate of claim 17, having a structure as represented by general formula I or general formula II:

22. The fluorescent substrate of claim 21, wherein Y comprises an anionic group selected from the group consisting of COOH, COO⁻

, SO₃⁻

, OSO₃⁻

, PO₃²⁻

, OPO₃²⁻

, CONHOH, CONHO⁻

, and poly(maleic acid).

23. The fluorescent substrate of claim 21, wherein each X is independently selected from the group consisting of a β

-galactoside residue, a β

-glucoside residue, an ester and a phosphate group.

24. The fluorescent substrate of claim 21, wherein each X is independently a phosphate having negative charges or a glucuronide having negative charges.

25. The fluorescent substrate of claim 17, having a structure as represented by general formula III or the general formula IV:

26. The fluorescent substrate of claim 25, wherein each X is independently selected from the group consisting of a β

-galactoside residue, a β

-glucoside residue, an ester and a phosphate group.

27. The fluorescent substrate of claim 25, wherein each X is independently a phosphate having negative charges or a glucuronide having negative charges.

28. The fluorescent substrate of claim 17, having a structure as represented by general formula V or the general formula VI:

29. The fluorescent substrate of claim 28, wherein Y comprises an anionic group selected from the group consisting of COOH, COO⁻

, SO₃⁻

, OSO₃⁻

, PO₃²⁻

, OPO₃²⁻

, CONHOH, CONHO⁻

, and poly(maleic acid).

30. The fluorescent substrate of claim 28, wherein each X is independently selected from the group consisting of a β

-galactoside residue, a β

-glucoside residue, an ester and a phosphate group.

31. The fluorescent substrate of claim 28, wherein each X is independently a phosphate having negative charges or a glucuronide having negative charges.

32. The fluorescent substrate of claim 17, having a structure as represented by general formula VII:

33. The fluorescent substrate of claim 32, wherein Y comprises an anionic group selected from the group consisting of COOH, COO⁻

, SO₃⁻

, OSO₃⁻

, PO₃²⁻

, OPO₃²⁻

, CONHOH, CONHO⁻

, and poly(maleic acid).

34. The fluorescent substrate of claim 32, wherein each X is independently selected from the group consisting of a β

-galactoside residue, a β

-glucoside residue, an ester and a phosphate group.

35. The fluorescent substrate of claim 32, wherein each X is independently a phosphate having negative charges or a glucuronide having negative charges.

36. The fluorescent substrate of claim 17, having a structure as represented by general formula VIII or general formula IX:

37. The fluorescent substrate of claim 36, wherein the enzyme cleavable group is a β

-galactoside residue.

38. The fluorescent substrate of claim 17, having a structure as represented by general formula X or general formula XI:

39. The fluorescent substrate of claim 17, having a structure as represented by general formula XII:

40. The fluorescent substrate of claim 39, wherein the enzyme cleavable group is a β

-galactoside residue.

41. The fluorescent substrate of claim 17, having a structure as represented by general formula XIII or the general formula XIV:

42. The fluorescent substrate of claim 41, wherein the enzyme cleavable group is a β

-galactoside residue.