Membrane transportable fluorescent substrates

• US 6,933,126 B2
• Filed: 09/19/2002
• Issued: 08/23/2005
• 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.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Kosson, Rosanne
• Application Number:
  US10/246,678
• Publication Number:
  US 20030103902A1
• Time in Patent Office: 1,069 Days
• Field of Search: 435/14, 435/207, 424/486, 424/78.08
• US Class Current: 435/14
• 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 an ionic complex of an enzyme active fluorescent substrate and a carrier molecule;

wherein the fluorescent substrate comprises one or more anionic charge groups and the carrier molecule comprises one or more cationic charge groups, and wherein the complex is formed by ionic charge interactions between the one or more anionic charge groups of the fluorescent substrate and the one or more cationic charge groups of the 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 one or more anionic charge groups are selected from the group consisting of phosphates, phosphonates, carboxylates, sulfates, sulfonates, phenolates, boronates, and carbonates.

4. The fluorescent substrate delivery system of claim 1, wherein the cationic charge groups comprise protonated polyamino groups.

5. The fluorescent substrate delivery system of claim 1, 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 substituents.

10. The fluorescent substrate delivery system of claim 1, wherein the fluorescent substrate further comprises lipophilic substituents capable of anchoring the substrate to a cell membrane.

11. The fluorescent substrate delivery system of claim 1, wherein the fluorescent substrate further comprises chloromethyl substituents.

12. The fluorescent substrate delivery system of claim 1, wherein the fluorescent substrate has a structure as represented by general formula I or general formula II:

wherein Y represents an anionic or polyanionic group and X represents an enzyme cleavable group.

13. The fluorescent substrate delivery system of claim 12, wherein Y comprises an anionic group selected from the group consisting of COOH, COO⁻

, SO₃⁻

, OSO₃⁻

, PO₃²⁻

, OPO₃²⁻

, CONHOH, CONHO⁻

, and poly(maleic acid).

14. The fluorescent substrate delivery system of claim 12, 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.

15. The fluorescent substrate delivery system of claim 12, wherein each X is independently a phosphate having negative charges or a glucuronide having negative charges.

16. The fluorescent substrate delivery system of claim 1, wherein the fluorescent substrate has a structure as represented by general formula III or the general formula IV:

wherein X represents an enzyme cleavable group.

17. The fluorescent substrate delivery system of claim 16, 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.

18. The fluorescent substrate delivery system of claim 16, wherein each X is independently a phosphate having negative charges or a glucuronide having negative charges.

19. The fluorescent substrate delivery system of claim 1, wherein the fluorescent substrate has a structure as represented by general formula V or the general formula VI:

wherein Y represents an anionic or polyanionic group, X represents an enzyme cleavable group, each Z independently represents Cl, F, H or SO₃⁻

, and n is an integer from 0 to 10.

20. The fluorescent substrate delivery system of claim 19, wherein Y comprises an anionic group selected from the group consisting of COOH, COO⁻

, SO₃⁻

, OSO₃⁻

, PO₃²⁻

, OPO₃²⁻

, CONHOH, CONHO⁻

, and poly(maleic acid).

21. The fluorescent substrate delivery system of claim 19, 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.

22. The fluorescent substrate delivery system of claim 19, wherein each X is independently a phosphate having negative charges or a glucuronide having negative charges.

23. The fluorescent substrate delivery system of claim 1, wherein the fluorescent substrate has a structure as represented by general formula VII:

wherein Y represents an anionic or polyanionic group, X represents an enzyme cleavable group, each Z independently represents Cl, F, H or SO₃⁻

, and n is an integer from 0 to 10.

24. The fluorescent substrate delivery system of claim 23, wherein Y comprises an anionic group selected from the group consisting of COOH, COO⁻

, SO₃⁻

, OSO₃⁻

, PO₃²⁻

, OPO₃²⁻

, CONHOH, CONHO⁻

, and poly(maleic acid).

25. The fluorescent substrate delivery system of claim 23, 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.

26. The fluorescent substrate delivery system of claim 23, wherein each X is independently a phosphate having negative charges or a glucuronide having negative charges.

27. The fluorescent substrate delivery system OF claim 1, wherein the fluorescent substrate has a structure as represented by general formula VIII or general formula IX:

wherein X represents an enzyme cleavable group.

28. The fluorescent substrate delivery system of claim 27, wherein the enzyme cleavable group is a β

-galactoside residue.

29. The fluorescent substrate delivery system of claim 1, wherein the fluorescent substrate has a structure as represented by general formula X or general formula XI:

wherein X represents an enzyme cleavable group and each “

A”

substituent independently represents —

CH₂CO₂H or —

CH(CH₂CO₂H)₂.

30. The fluorescent substrate delivery system of claim 1, wherein the fluorescent substrate has a structure as represented by general formula XII:

wherein X represents an enzyme cleavable group.

31. The fluorescent substrate delivery system of claim 30, wherein the enzyme cleavable group is a β

-galactoside residue.

32. The fluorescent substrate delivery system of claim 1, wherein the fluorescent substrate has a structure as represented by general formula XIII or the general formula XIV:

wherein X represents an enzyme cleavable group.

33. The fluorescent substrate delivery system of claim 32, wherein the enzyme cleavable group is a β

-galactoside residue.

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

a) contacting a sample comprising the cell with a composition comprising an ionic complex of an enzyme active fluorescent substrate and a carrier molecule, wherein the fluorescent substrate comprises one or more anionic charge groups and the carrier molecule comprises one or more cationic charge groups, and wherein the complex is formed by ionic charge interactions between the one or more anionic charge groups of the fluorescent substrate and the one or more cationic charge groups of the carrier molecule; and

b) 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.

7. The method of claim 6, wherein the enzyme cleavable group is cleavable by an enzyme selected from the group consisting of glycosidases, esterases, proteases, oxidases, peptidases and phosphatases.

8. The method of claim 6, wherein the enzyme cleavable group is cleavable by β

-galactosidase.

9. The method of claim 6, 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.