ENZYME SUBSTRATE COMPRISING A FUNCTIONAL DYE AND ASSOCIATED TECHNOLOGY AND METHODS

• US 20090148386A1
• Filed: 11/21/2006
• Published: 06/11/2009
• Est. Priority Date: 11/30/2005
• Status: Active Grant

• Current Assignee: Biotium, Inc.
• Original Assignee: Biotium, Inc.
• Inventors: Leung, Wai-yee, Mao, Fei, Cen, Hui
• Granted Patent: US 8,092,784 B2
• Time in patent office: Days
• US Class Current: 424/9.600
• CPC Class Codes: C07D 263/60 Naphthoxazoles; Hydrogenated naphthoxazoles
• CPC Class Codes: C07D 417/06 linked by a carbon chain containing only aliphatic carbon atoms
• CPC Class Codes: C07D 501/00 Heterocyclic compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula: , e.g. cephalosporins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

Enzyme substrates and associated technology of the present invention are provided. An enzyme substrate of the invention may comprise a biologically functional fluorescent dye and an enzyme-specific substrate moiety attached in such a way that the functionality of the functional dye is diminished. An enzymatic reaction may cleave at least a portion of the substrate moiety from the enzyme substrate to provide a more functional product dye. This product dye may be nonfluorescent or weakly fluorescent, in general, and relatively fluorescent, in a particular condition, such as when bound to a partner biological molecule or an assembly of partner biological molecules. An enzyme substrate of the present invention may thus be useful in fluorescence detection, and/or in any of a variety of useful applications, such as the detection of enzymatic activity in a cell-free system or in a living cell, the screening of drugs, or the diagnosis of disease.

Claims

1. A substrate selected from a compound having a structural formula, DYE-(B)_m, and a pro-enzyme substrate thereof, wherein DYE, when independent of DYE-(B)_m, comprises a dye selected from a biologically functional fluorescent dye and a fluorogenic dye, the dye being capable of binding to a partner biomolecule, partner biomolecules, or an assembly of partner biomolecules;

wherein m is selected from 1, 2, 3, 4 and 5; and

wherein each of at least one B, independently, comprises an enzyme substrate moiety that is capable of enzymatic transformation comprising cleavage of a bond involving the dye and at least one B, cleavage of a bond within at least one B, or formation of a bond involving at least one B.

2. The substrate of claim 1, wherein the dye comprises a nucleic acid dye, a membrane dye, an organelle dye, or a fluorescent ligand dye.

3. The substrate of claim 1, wherein the dye comprises a fluorogenic dye.

4. The substrate of claim 1, wherein the dye comprises a nucleic acid dye or a membrane dye.

5. The substrate of claim 1, wherein the dye is other than a xanthene dye or a resorufin dye.

6. The substrate of claim 1, wherein m is selected from 1 and 2.

7. The substrate of claim 1, wherein at least one B is selected from a peptide and a glycoside.

8. The substrate of claim 1, comprising at least two B, wherein each of the at least two B is an enzyme substrate moiety for the same enzyme.

9. The substrate of claim 1, wherein the enzymatic transformation comprises phosphorylation catalyzed by a kinase.

10. The substrate of claim 1, wherein the dye is a phenanthrodium dye.

11. The substrate of claim 1, wherein the dye is a phenanthrodium dye having a structural formula,

wherein:

B comprises a peptide or an amino acid;

at least one ligand of ligands R₁ and R₂ comprises B, where the C-terminal carbonyl of B is capable of forming an enzymatically cleavable peptide bond with an amino group of the dye;

if any one ligand of ligands R₁ and R₂ does not comprise B, said any one ligand comprises H;

ligand R₃ comprises a substituted or an unsubstituted C1 to C10 alkyl;

ψ

comprises a cation selected from H+, Na+, K+, NH₄+, N,N,N-triethylammonium, and N,N-diisopropylethylammonium, or an anion selected from a halide, a sulfate, a phosphate, a perchlorate, a hexafluorophosphate, and a tetrafluoroborate; and

d is a number of ψ

sufficient to render overall charge of the substrate neutral.

12. The substrate of claim 11, wherein B comprises an enzyme substrate moiety for a peptidase enzyme.

13. The substrate of claim 12, wherein B comprises an enzyme substrate moiety for an enzyme selected from a caspase, a trypsin, a chymotrypsin, an elastase, a cathepsin B, and a cathepsin L.

14. The substrate claim 11, wherein when B comprises a charged peptide, ligand R₃ comprises a substituted or an unsubstituted C4 to a C10 alkyl.

15. The substrate of claim 11, wherein when B comprises a charged peptide, ligand R₃ comprises a substituted or an unsubstituted C4 to a C8 alkyl.

16. The substrate of claim 11, wherein when B comprises a neutral peptide, ligand R₃ comprises a substituted or an unsubstituted C1 to a C6 alkyl.

17. The substrate of claim 11, wherein B comprises an enzyme substrate moiety for a caspase-3 enzyme and ligand R₂ comprises H.

18. The substrate of claim 17, wherein B comprises Z-DEVD (SEQ ID NO:

1).

19. The substrate of claim 11, wherein B comprises an enzyme substrate moiety for an elastase enzyme, ligand R₁ comprises B, and ligand R₂ comprises B.

20. The substrate of claim 19, wherein B comprises Z-AA-.

21. The substrate of claim 1, wherein the dye is a phenanthrodium dye having a structural formula,

wherein:

L comprises an aliphatic C2 to about C10 linker, optionally comprising an aryl, and optionally comprising at least one hetero atom of heteroatoms O, S, N, F, and Cl;

W comprises at least one atom selected from atoms O, NH and S;

if B comprises an amino acid or a peptide, B is attached to W via a C-linkage;

ψ

comprises a cation selected from H+, Na+, K+, NH₄+, N,N,N-triethylammonium, and N,N-diisopropylethylammonium, or an anion selected from a halide, a sulfate, a phosphate, a perchlorate, a hexafluorophosphate, and a tetrafluoroborate; and

d is a number of ψ

sufficient to render overall charge of the substrate neutral.

22. The substrate of claim 21, wherein B comprises a peptide that comprises at least two enzymatically removable negative charges.

23. The substrate of claim 22, wherein at least one enzymatically removable negative charge of the at least two enzymatically removable negative charges is from a modifying group that is covalently attached to B.

24. The substrate of claim 21, wherein B comprises a peptide that comprises a fluorescence quencher.

25. The substrate of claim 21, comprising a covalent bond involving B and W, wherein B comprises an enzyme substrate moiety for a β

-lactamase enzyme having a structural formula,

wherein;

A comprises a fluorescence quencher, a substituted C1 to C20 alkyl, or a substituted C1 to C20 aryl, optionally comprising at least one hetero atom, and optionally comprising at least one negatively charged group; and

c is selected from 0, 1 and 2.

26. The substrate of claim 1, wherein the dye comprises an asymmetric cyanine dye.

27. The substrate of claim 1, wherein the dye comprises an asymmetric cyanine dye having a structural formula,

wherein:

the dotted line represents atoms of at least one fused aromatic ring, optionally comprising at least one quaternized or unquaternized nitrogen;

X comprises O or S;

n is selected from 0, 1, 2 and 3;

ψ

comprises a cation selected from H+, Na+, K+, NH₄+, N,N,N-triethylammonium, and N,N-diisopropylethylammonium, or an anion selected from a halide, a sulfate, a phosphate, a perchlorate, a hexafluorophosphate, and a tetrafluoroborate;

d is a number of ψ

sufficient to render overall charge of the substrate neutral;

at least one ligand, but no more than two ligands, of ligands R₄, R₅, R₆ and R₇ has a structural formula, -L-W—

B, wherein;

L comprises an aliphatic C2 to about C10 linker, optionally comprising an aryl, and optionally comprising at least one hetero atom of heteroatoms O, S, N, F, and Cl;

W comprises at least one atom selected from atoms O, NH and S; and

if B comprises an amino acid or a peptide, B is attached to W via a C-linkage;

when ligand R₄ does not comprise -L-W—

B, ligand R₄ comprises H;

a C1 to about C6 alkyl;

a C1 to about C6 alkoxy;

a halogen;

or an aryl meta to X, wherein the aryl optionally comprises at least one hetero atom of hetero atoms N, O and S;

when ligand R₅ does not comprise -L-W—

B, ligand R₅ comprises a C1 to about C6 alkyl;

when ligand R₆ does not comprise -L-W—

B, ligand R₆ comprises H;

a C1 to about C10 alkyl, wherein the alkyl optionally comprises at least one hetero atom of hetero atoms N, O and S;

a halogen;

a C1 to C10 alkoxy, wherein the alkoxy optionally comprises at least one hetero atom of hetero atoms N, O, and S;

a C1 to C10 alkylmercapto, wherein the alkylmercapto comprises at least one hetero atom of hetero atoms N, O, and S;

a C2 to about C12 dialkylamino, wherein the dalkylamino optionally comprises at least one hetero atom of hetero atoms N, O, and S;

a substituted or an unsubstituted aryl, wherein the aryl optionally comprises 1 to 3 hetero atom(s) of hetero atoms N, O, S and a halogen;

when ligand R₇ does not comprise -L-W—

B, ligand R₇ comprises H;

a C1 to about C10 alkyl, optionally comprising an aryl, and optionally comprising at least one hetero atom of hetero atoms N, O, and S;

a substituted or an unsubstituted aryl, optionally containing 1 to 3 hetero atom(s) of hetero atoms N, O, S and a halogen; and

independently, each ligand of ligands R₈ and R₉ comprises H;

a C1 to about C10 alkyl, optionally comprising at least one hetero atom of hetero atoms N, O, and S;

a halogen;

a C1 to C10 alkoxy, wherein the alkoxy optionally comprises at least one hetero atom of hetero atoms N, O, and S;

a C1 to C10 alkylmercapto, wherein the alkylmercapto optionally comprises at least one hetero atom of hetero atoms N, O, and S;

a C2 to about C12 dialkylamino, optionally comprising at least one hetero atom of hetero atoms N, O, and S;

or a substituted or an unsubstituted aryl, optionally comprising 1 to 3 hetero atom(s) of hetero atoms halogen, N, O and S;

or ligand R₈ and ligand R₉, in combination, form a fused aromatic ring, wherein the ring is optionally substituted by at least one substituent of a C1-C2 alkyl substituent, a C1-C2 alkoxy substituent, a C1-C2 alkyl-mercapto substituent, and a halogen substituent.

28. The substrate of claim 27, wherein:

when ligand R₄ does not comprise -L-W—

B, ligand R₄ comprises H;

a methoxy meta to X;

or an aryl meta to X, wherein the aryl optionally comprises at least one hetero atom of hetero atoms N, O and S;

when ligand R₅ does not comprise -L-W—

B, ligand R₅ comprises a methyl;

when ligand R₆ does not comprise -L-W—

B, ligand R₆ comprises H;

a C1 to about C6 alkyl;

a C1 to about C6 alkoxy;

a C1 to about C6 alkylmercapto;

a C2 to about C12 dialkylamino, optionally comprising one N;

or a substituted or an unsubstituted aryl, optionally comprising 1 to 3 hetero atom(s) of hetero atoms N, O and S;

when ligand R₇ does not comprise -L-W—

B, ligand R₇ comprises a C1 to about C3 alkyl, optionally comprising an aryl; and

each ligand of ligands R₈ and R₉ comprises H;

or ligand R₈ and ligand R₉, in combination, form a fused 6-membered ring, wherein the ring is optionally substituted by at least one substituent of a methyl substituent, a methoxy substituent, a methylmercapto substituent, and a halogen substituent.

29. The substrate of claim 27, wherein only one ligand of ligands R₅ and R₇ has a structural formula -L-W—

B.

30. The substrate of claim 27, wherein only ligand R₇ has a structural formula -L-W—

B.

31. The substrate of claim 27, comprising an asymmetric cyanine dye having a structural formula,

wherein:

n is selected from 0, 1, 2 and 3;

n′

is selected from 1, 2, 3, 4 and 5;

m′

is selected from 0 and 1;

m″

is 1 when m′

is 0;

m″

is selected from 0, 2, 3 and 4, when m′

is 1;

X comprises O or S;

each ligand of ligands R₈ and R₉ comprises H;

or ligand R₈ and ligand R₉, in combination, form a fused benzene ring.

32. The substrate of claim 31, wherein B comprises a peptide that comprises at least two enzymatically removable negative charges.

33. The substrate of claim 32, wherein at least one enzymatically removable negative charge of the at least two enzymatically removable negative charges is from a modifying group that is covalently attached to B.

34. The substrate of claim 31, wherein B comprises a peptide that comprises a fluorescence quencher, wherein the fluorescence quencher is sufficient for removal from the dye upon enzymatic transformation of the substrate.

35. The substrate of claim 31, comprising a covalent bond involving B and W, wherein B comprises an enzyme substrate moiety for a β

-lactamase enzyme having a structural formula,

wherein;

A comprises a fluorescence quencher, a substituted C1 to C20 alkyl, or a substituted C1 to C20 aryl, optionally comprising at least one hetero atom, and optionally comprising at least one negatively charged group; and

c is selected from 0, 1 and 2.

36. The substrate of claim 31, wherein B comprises an enzyme substrate moiety for a caspase enzyme.

37. The substrate of claim 31, wherein n is selected from 0, 1 and 2;

and m″

is 2, when m′

is 1.

38. The substrate of claim 31, wherein B comprises an enzyme substrate moiety for a caspase enzyme selected from Ac-DEVD (SEQ ID NO:

1)-;

Ac-LEED (SEQ ID NO;

     3)-;

Ac-LEHD (SEQ ID NO;

     7)-;

Ac—

VDVAD (SEQ ID NO;

     8)-;

Ac—

VEID (SEQ ID NO;

     2)-;

Ac—

IETD (SEQ ID NO;

     9)-; and

Suc-YVAD (SEQ ID NO;

     10)-.

39. The substrate of claim 31, wherein B comprises an enzyme substrate moiety for a membrane-bound enzyme, wherein the enzyme substrate moiety is hydrophobic.

40. The substrate of claim 39, wherein the substrate moiety comprises GGVVIATVK (SEQ ID NO:

11).

41. The substrate of claim 34, wherein the fluorescence quencher is attached to the N-terminal of an enzyme substrate moiety for a caspase enzyme.

42. The substrate of claim 31, wherein B comprises a glycosidyl.

43. The substrate of claim 31, wherein B comprises a β

-D-glucuronidyl or a β

-D-galactopyranosidyl.

44. The substrate of claim 1, wherein the dye comprises an asymmetric cyanine dye having a structural formula,

, wherein:

n is selected from 0, 1, 2 and 3;

n′

is selected from 1, 2, 3, 4 and 5;

m′

is selected from 0 and 1;

m″

is 1, when m′

is 0;

m is selected from 0, 2, 3 and 4, when m is 1;

q is selected from 1, 2, 3, 4 and 5;

q′

is selected from 0 and 1;

q″

is 1, when q′

is 0;

q″

is selected from 0, 2, 3 and 4, when q′

is 1;

X comprises O or S;

each ligand of ligands R₈ and R₉ comprises H;

or ligand R₈ and ligand R₉, in combination, form a fused benzene ring;

W comprises at least one atom selected from atoms O, NH and S;

if B comprises an amino acid or a peptide, B is attached to W via a C-linkage;

ψ

comprises a cation selected from H+, Na+, K+, NH₄+, N,N,N-triethylammonium, and N,N-diisopropylethylammonium, or an anion selected from a halide, a sulfate, a phosphate, a perchlorate, a hexafluorophosphate, and a tetrafluoroborate; and

d is a number of ψ

sufficient to render overall charge of the substrate neutral.

45. The substrate of claim 44, wherein the dye has a structural formula,

46. The substrate of claim 44, wherein W comprises the atom 0 and B comprises a β

-D-galactopyranosidyl, a β

-D-glucuronidyl, or a phosphoryl.

47. The substrate of claim 1, wherein the dye comprises a membrane dye selected from a symmetric cyanine dye, an asymmetric cyanine dye, a merocyanine dye, and a styryl dye.

48. The substrate of claim 1, comprising a cyanine dye having a structural formula,

wherein:

n is selected from 1, 2 and 3;

independently, each ligand of ligands R₁ and R₄ comprises H or Cl;

ligand R₂ comprises a C1 to C12 alkyl;

a C7 to C12 arylalkyl;

or has a structural formula, -L-W—

B;

X comprises (CH₃)₂C, O, S, or NR₃, where ligand R₃ comprises a C1 to C12 alkyl;

a C7 to C12 arylalkyl;

or has a structural formula, -L-W—

B;

Y comprises (CH₃)₂C, O, S, or NR₃, where ligand R₃ comprises a C1 to C12 alkyl;

a C7 to C12 arylalkyl;

or has a structural formula, -L-W—

B;

each X and Y, independently, may be (CH₃)₂C, O, S, or NR₃, where ligand R₃ may be a C1 to C12 alkyl, a C7 to C12 arylalkyl, or a -L-W—

B;

L comprises a C2 to C12 aliphatic linker, optionally comprising an aryl, and optionally comprising at least one hetero atom of hetero atoms O and N;

W comprises at least one atom selected from atoms O, C═

O, NH and S;

B comprises a charged amino acid that is attached to the dye via a C-linkage or an N-linkage;

a charged peptide that is attached to the dye via a C-linkage or an N-linkage, the peptide optionally comprising a fluorescence quencher;

an enzyme substrate moiety for a β

-lactamase enzyme;

a phosphoryl;

a sulfuryl;

a β

-D-glucuronidyl;

a β

-D-galactopyranosidyl;

an α

-D-galactopyranosidyl;

an α

-D-mannopyranosidyl;

an α

-D-glucopyranosidyl;

a β

-D-glucopyranosidyl;

a X-β

-D-cellobiosidyl;

a N-acetyl-β

-D-galactosaminidyl;

a N-acetyl-β

-D-glucosaminidyl;

a phosphorylated or an unphosphorylated phosphatidylinositol;

a glycosylated phosphatidylinositol;

an adenosine-5′

-phosphate;

or a nucleoside-3′

-phosphate;

a number of -L-W—

B is selected from 1 and 2;

ψ

comprises a cation selected from H+, Na+, K+, NH₄+, N,N,N-triethylammonium, and N,N-diisopropylethylammonium, or an anion selected from a halide, a sulfate, a phosphate, a perchlorate, a hexafluorophosphate, and a tetrafluoroborate; and

d is a number of ψ

sufficient to render overall charge of the substrate neutral.

49. The substrate of claim 48, wherein ligand R₁ comprises Cl;

ligand R₂ comprises methyl;

X comprises NR₃, where ligand R₃ is a benzyl;

Y comprises (CH₃)₂C, O or S;

ligand R₄ comprises H; and

-L-W—

B—

has a structural formula,

wherein;

when n′

is 0, n″

is selected from 1 and 2; and

when n′

is 1, n″

is selected from 0, 1 and 2.

50. The substrate of claim 48, wherein n′

is 1;

n″

is 0;

W comprises NH;

and B comprises a charged peptide that is attached to the dye via a C-linkage.

51. The substrate of claim 48, wherein B comprises a negatively charged peptide.

52. The substrate of claim 48, wherein B comprises an enzyme substrate moiety for a caspase enzyme.

53. The substrate of claim 48, wherein B comprises an enzyme substrate moiety for a caspase enzyme selected from Ac-DEVD (SEQ ID NO:

1)-;

Ac-LEED (SEQ ID NO;

     3)-;

Ac-LEHD (SEQ ID NO;

     7)-;

Ac—

VDVAD (SEQ ID NO;

     8)-;

Ac—

VEID (SEQ ID NO;

     2)-;

Ac—

IETD (SEQ ID NO;

     9)-;

or Suc-YVAD (SEQ ID NO;

     10)-.

54. The substrate of claim 48, wherein B comprises a peptide that comprises a fluorescence quencher, wherein the fluorescence quencher is attached to the N-terminal of an enzyme substrate moiety for a caspase enzyme.

55. The substrate of claim 1, comprising a styryl dye having a structural formula,

f is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

n is selected from 1, 2 and 3;

n′

is selected from 1, 2, 3, 4 and 5;

m′

is selected from 0 and 1;

m″

is selected from 1, 2, 3 and 4, when m′

is 0;

m″

is selected from 0, 2, 3 and 4, when m′

is 1;

W comprises at least one atom selected from atoms O, C═

O, NH and S;

B comprises a charged amino acid that is attached to the dye via a C-linkage or an N-linkage;

a charged peptide that is attached to the dye via a C-linkage or an N-linkage, the peptide optionally comprising a fluorescence quencher;

an enzyme substrate moiety for a β

-lactamase enzyme;

a phosphoryl;

a sulfuryl;

a β

-D-glucuronidyl;

a β

-D-galactopyranosidyl;

an α

-D-galactopyranosidyl;

an α

-D-mannopyranosidyl;

an α

-D-glucopyranosidyl;

a β

-D-glucopyranosidyl;

a Xβ

-D-cellobiosidyl;

a N-acetyl-β

-D-galactosaminidyl;

a N-acetyl-β

-D-glucosaminidyl;

a phosphorylated or an unphosphorylated phosphatidylinositol;

a glycosylated phosphatidylinositol;

an adenosine-5′

-phosphate;

or a nucleoside-3′

-phosphate;

ψ

comprises a cation selected from H+, Na+, K+, NH₄+, N,N,N-triethylammonium, and N,N-diisopropylethylammonium, or an anion selected from a halide, a sulfate, a phosphate, a perchlorate, a hexafluorophosphate, and a tetrafluoroborate; and

d is a number of ψ

sufficient to render overall charge of the substrate neutral.

56. The substrate of claim 55, wherein f is selected from 1, 2, 3, 4, 5 and 6.

57. The substrate of claim 55, wherein B comprises a charged peptide that is attached to the dye via a C-linkage or an N-linkage.

58. The substrate of claim 55, wherein B comprises an enzyme substrate moiety that comprises a charged peptide that is attached to the dye via a C-linkage.

59. The substrate of claim 55, wherein B comprises an enzyme substrate moiety selected from Ac-DEVD (SEQ ID NO:

1)- and Ac-LEED (SEQ ID NO;

     3)-.

60. The substrate of claim 55, wherein B comprises an enzyme substrate moiety for an enzyme selected from a matrix metalloprotease enzyme, a gelatinase enzyme, and a collagenase enzyme.

61. The substrate of claim 55, wherein B comprises an enzyme substrate moiety that comprises a charged peptide that is attached to the dye via an N-linkage.

62. The substrate of claim 55, wherein B comprises an enzyme substrate moiety, —

PLGLWA-D-R-D-R-D-R—

NH₂.

63. The substrate of claim 55, wherein B comprises an enzyme substrate moiety, —

PQGLEA-D-R-D-R-D-R—

NH₂.

64. The substrate of claim 55, wherein B comprises a peptide that comprises a fluorescence quencher, wherein the fluorescence quencher is sufficient for removal from the dye upon enzymatic transformation of the substrate.

65. The substrate of claim 1, wherein B comprises an enzyme substrate moiety selected from an enzyme substrate moiety for a peptidase enzyme, an enzyme substrate moiety for a histone deacetyltransferase enzyme, an enzyme substrate moiety for phosphatase enzyme, an enzyme substrate moiety for a sulfatase enzyme, an enzyme substrate moiety for an esterase enzyme, an enzyme substrate moiety for a cytochrome P450 enzyme, an enzyme substrate moiety for a glydosidase enzyme, an enzyme substrate moiety for a phospholipase C enzyme, an enzyme substrate moiety for a phosphodiesterase enzyme, an enzyme substrate moiety for a ribonuclease enzyme, and an enzyme substrate moiety for a β

-lactamase enzyme.

66. The substrate of claim 1, wherein B comprises an enzyme substrate moiety selected from an amino acid, a peptide, an α

-amino-protected ε

-N-acetyllysine, a phosphoryl, a sulfuryl, a carbonyl, an alkyl, a β

-D-glucuronidyl, a β

-D-galactopyranosidyl, an α

-D-galacto-pyranosidyl, an α

-D-mannopyranosidyl, an α

-D-glucopyranosidyl, a β

-D-glucopyrano-sidyl, a Xβ

-D-cellobiosidyl, a N-acetyl-β

-D-galactosaminidyl, a N-acetyl-β

-D-glucosaminidyl, a phosphatidylinositol, an adenosine-5′

-phosphate, and a nucleoside-3′

-phosphate.

67. A kit for detecting presence, absence, or activity of an enzyme comprising:

an inhibitor of activity of the enzyme;

optionally, a promoter of activity of the enzyme; and

a substrate of claim 1 for the enzyme.

68. A kit for determining an effect of a substance on an enzyme comprising:

an inhibitor of activity of the enzyme; and

a substrate of claim 1 for the enzyme.

69. A method for detecting presence, absence, or activity of an enzyme in a cell-free system comprising using a substrate of claim 1.

70. A method for detecting presence, absence, or activity of an enzyme in a live cell system comprising using a substrate of claim 1.

71. A method for detecting presence, absence, or activity of an enzyme in a living animal comprising using a substrate of claim 1.

72. A method for determining an effect of a substance on an enzyme comprising using a substrate of claim 1.