METHODS AND COMPOSITIONS FOR DETECTING OR MEASURING CASPASES OR APOPTOSIS

• US 20190309341A1
• Filed: 06/21/2017
• Published: 10/10/2019
• Est. Priority Date: 06/23/2016
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: GEE, Kyle, ZHOU, Wenjun, HUANG, Shih-Jung, TOMASZEWSKI, Monica
• Granted Patent: US 10,941,435 B2
• Time in Patent Office: Days
• CPC Class Codes: C07K 5/101 the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
• CPC Class Codes: C07K 5/1021 with the first amino acid being acidic
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: G01N 2333/96466 Cysteine endopeptidases (3.4.22)
• CPC Class Codes: G01N 2510/00 Detection of programmed cell death, i.e. apoptosis
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

Provided herein are compounds, enzyme substrates, compositions, kits, uses, and methods for detecting the presence or absence of a caspase enzyme, measuring the activity of a caspase enzyme, or detecting the presence or absence of apoptosis. The detection or measurement can occur through intracellular cleavage of a compound or enzyme substrate, which can lead to an increase in fluorescence, e.g., in the violet or red channel, through liberation of a nucleic acid binding dye from a peptide, such as liberation of a DNA-binding dye from a negatively charged peptide comprising a sequence recognized and cleaved by a caspase

Claims

1. A method for detecting the presence or absence of a caspase enzyme in a cell or mixture of cells, measuring the activity of a caspase enzyme in a cell or mixture of cells, or detecting the presence or absence of apoptosis in a cell or mixture of cells, the method comprising the steps of:

a) incubating the cell or mixture of cells with a compound of structural formula (I);

2. The method according to claim 1, wherein:

a) each R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂ and R₁₃ is independently H, halogen, or alkyl;

b) each R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂ and R₁₃ is independently H, alkyl, or substituted alkyl;

c) each R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂ and R₁₃ is independently H or alkyl;

d) at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂ and R₁₃ are H;

or

e) each R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂ and R₁₃ is H.

3. The method according to any one of claims 1-2, wherein:

a) X is —

N(R)—

wherein R is H, halogen, alkyl, or substituted alkyl;

b) X is —

N(R)—

wherein R is alkyl or substituted alkyl;

c) X is —

N(R)—

wherein R is alkyl;

d) X is —

N(R)—

wherein R is methyl, ethyl, propyl, or isopropyl;

e) X is —

N(R)—

wherein R is methyl or ethyl;

or

f) X is —

N(CH₃)—

.

4. The method according to any one of claims 1-3, wherein:

a) Y is alkyl;

b) Y is methyl, ethyl, propyl, or isopropyl;

c) Y is methyl or ethyl;

or

d) Y is methyl.

5. The method according to claim 1, wherein the compound is selected from the group consisting of:

13. The method according to any one of claims 1-12, wherein the activity of the caspase enzyme is measured.

14. The method according to any one of claims 1-13, wherein the presence or absence of apoptosis is detected.

15. The method according to any one of claims 1-14, wherein the presence or absence of the caspase enzyme is detected.

16. The method according to any one of claims 1-15, further comprising measuring a fluorescent signal before delivering the compound to the cell or mixture of cells.

17. The method according to any one of claims 1-16, wherein the compound is cleaved by a caspase enzyme.

18. The method according to claim 17, wherein the caspase enzyme is caspase-1, caspase-3, caspase-6, caspase-7, caspase-8 or caspase-9.

19. The method according to any one of claims 1-18, further comprising the step of incubating the cell or mixture of cells with an apoptosis inducer.

20. The method according to any one of claims 1-19, further comprising the step of incubating the cell or mixture of cells with a caspase inhibitor.

21. The method according to any one of claims 1-20, wherein the fluorescent signal is detected by flow cytometry.

22. The method according to any one of claims 1-21, wherein the fluorescent signal is detected by fluorescence microscopy.

23. The method according to any one of claims 1-4, 5-8, or 12-22, wherein the peptide comprises an amino acid sequence of:

a) X₂EX₁D (SEQ ID NO.

     5), wherein X₂ is W, Y, I, L, D, or V, and X₁ is any amino acid;

b) X₃DEX₁D (SEQ ID NO.

     6), wherein X₃ is V or L and X₁ is any amino acid;

c) DX₁VD (SEQ ID NO.

     7), wherein X₁ is any amino acid;

d) DX₂EX₁D (SEQ ID NO.

     8), wherein X₂ is W, Y, I, L, or V, and X₁ is any amino acid;

or

e) DX₂VX₁D (SEQ ID NO.

     20), wherein X₂ is W, Y, I, L, or V, and X₁ is any amino acid;

optionally wherein the amino acid sequence of a), b), c), d), or e) is the entire amino acid sequence of the peptide or the C-terminal amino acid sequence of the peptide.

24. The method according to any one of claims 1-4, 5-8, or 12-23, wherein the peptide comprises an amino acid sequence of WEHD (SEQ ID NO. 9), YVAD (SEQ ID NO. 10), YVHD (SEQ ID NO. 11), VDVAD (SEQ ID NO. 12), LEHD (SEQ ID NO. 2), IETD (SEQ ID NO. 13), VEID (SEQ ID NO. 14), DEVD (SEQ ID NO. 1), LEVD (SEQ ID NO. 15), or AEVD (SEQ ID NO. 16), optionally wherein the amino acid sequence is the entire amino acid sequence of the peptide or the C-terminal amino acid sequence of the peptide.

25. The method according to any one of claims 1-4, 5-8, or 12-24, wherein the peptide is a substrate for cleavage by at least one caspase, has a length of 5 or more amino acids, and comprises an aspartic acid in the P₅ position.

26. The method according to any one of claims 1-4, 5-8, or 10-25, wherein the peptide comprises an amino acid sequence of DIETD (SEQ ID NO. 17), DLEHD (SEQ ID NO. 18), or DYVAD (SEQ ID NO. 19), optionally wherein the amino acid sequence is the entire amino acid sequence of the peptide or the C-terminal amino acid sequence of the peptide.

27. The method according to any one of claims 1-4, 5-8, or 10-26, wherein the peptide:

a) further comprises one or more additional amino acid residues C-terminally;

b) further comprises one or more additional amino acid residues N-terminally;

c) further comprises one or more additional amino acid residues both C-terminally and N-terminally;

d) further comprises 1 to 100, 1 to 50, 1 to 20, 1 to 10, 1 to 5, 5, 4, 3, 2, or 1 additional amino acid residues C-terminally;

e) further comprises 1 to 100, 1 to 50, 1 to 20, 1 to 10, 1 to 5, 5, 4, 3, 2, or 1 additional amino acid residues N-terminally;

or

f) further comprises 1 to 100, 1 to 50, 1 to 20, 1 to 10, 1 to 5, 5, 4, 3, 2, or 1 additional amino acid residues both C-terminally and N-terminally.

28. The method according to any one of claims 1-4, 5-8, or 10-27, wherein the peptide has a sequence with a total length of 4 to 200 amino acid residues, 4 to 100 amino acid residues, 4 to 50 amino acid residues, 5 to 200 amino acid residues, 5 to 100 amino acid residues, 5 to 50 amino acid residues, 20 to 50 amino acid residues, or 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues.

29. The method according to any one of claims 1-4, 5-8, or 10-28, wherein the peptide comprises an N-terminal acyl.

30. The method according to claim 29, wherein the N-terminal acyl is an acetyl or carboxybenzyl.

31. The method according to any one of claims 1-4, 5-8, or 10-30, wherein L is:

a) —

N(R_N1)—

[CH₂]_n—

, wherein n ranges from 1 to 10 and R_N1 is H or optionally substituted C_1-6 alkyl;

b) —

N(R_N1)-L_a-, wherein L_a is a C_2-10 alkylene optionally interrupted by one or both of —

N(R_N1)—

C(O)—

or —

N⁺(R_N2)₂—

, wherein each R_N1 is independently H or optionally substituted C_1-6 alkyl and each R_N2 is independently an optionally substituted C_1-6 alkyl;

c) —

C(O)—

[CH₂]_n—

, wherein n ranges from 1 to 10;

d) —

N(R_N1)—

[CH₂]_n—

C(O)—

, wherein n ranges from 1 to 10 and R_N1 is H or optionally substituted C_1-6 alkyl;

e) —

C(O)—

[CH₂]_n—

C(O)—

, wherein n ranges from 1 to 10;

f) —

[CH₂]_n—

N(R_N2)₂—

[CH₂]_n—

, wherein each R_N2 is independently an optionally substituted C_1-6 alkyl;

g) —

NH—

[CH₂]₂—

;

h) —

NH—

[CH₂]₄—

;

i) —

NH—

[CH₂]₅—

;

or

j) —

NH—

[CH₂]₂—

NH—

C(O)—

CH₂—

N(CH₃)₂—

[CH₂]₂—

.

32. The method according to any one of the preceding claims, further comprising incubating the cell or mixture of cells with at least one, two, three, or four additional fluorescent molecules, wherein the at least one, two, three, or four additional fluorescent molecules are spectrally distinguishable from at least one of the compound of structural formula (I) or the compound of structural formula (II), and measuring fluorescent signals from the at least one, two, three, or four additional fluorescent molecules.

33. The method according to claim 32, wherein (i) the cell or mixture of cells is incubated with a compound of structural formula (I) and the at least one, two, three, or four additional fluorescent molecules have near infrared, red, orange, yellow, green, or cyan emission maxima;

or (ii) the cell or mixture of cells is incubated with a compound of structural formula (II) and the at least one, two, three, or four additional fluorescent molecules have near infrared, yellow, green, cyan, blue, or violet emission maxima.

34. The method of any one of the preceding claims, wherein the cell or mixture of cells expresses or comprises a construct encoding at least one, two, three, or four fluorescent proteins, optionally wherein at least one, two, three, or four of the fluorescent proteins are fusion proteins, and the at least one, two, three, or four fluorescent proteins are spectrally distinguishable from at least one of the compound of structural formula (I) or the compound of structural formula (II).

35. The method according to claim 34, wherein (i) the cell or mixture of cells is incubated with a compound of structural formula (I) and the at least one, two, three, or four fluorescent proteins have near infrared, red, orange, yellow, green, or cyan emission maxima;

or (ii) the cell or mixture of cells is incubated with a compound of structural formula (II) and the at least one, two, three, or four additional fluorescent proteins have near infrared, yellow, green, cyan, or blue emission maxima.

58. A kit for detecting the presence or absence of a caspase enzyme in a cell or mixture of cells, measuring activity of a caspase enzyme within a cell or a mixture of cells, or detecting the presence or absence of apoptosis, the kit comprising:

a) the compound or enzyme substrate of any one of claims 36-57;

b) at least one of an organic solvent;

c) a desiccant; and

d) optionally, instructions for use according to the method of any one of claims 1-35.

59. The kit according to claim 58, wherein the compound or enzyme substrate is of structural formula (I):

60. The kit according to claim 58, wherein the compound is selected from the group consisting of:

61. The kit according to claim 58, wherein the compound is a compound of structural formula (II):

62. The kit according to claim 58, wherein the compound is selected from the group consisting of:

63. The kit according to claim 58, wherein the kit comprises an enzyme substrate comprising a peptide, and the peptide comprises a sequence of DX₂EX₁D (SEQ ID NO. 8) or DX₂VX₁D (SEQ ID NO. 20), wherein X₂ is W, Y, I, L, or V, and X₁ is any amino acid.

64. The kit according to any one of claims 58-63, wherein the organic solvent comprises DMSO.

6. A method for detecting the presence or absence of a caspase enzyme in a cell or mixture of cells, measuring the activity of a caspase enzyme in a cell or mixture of cells, or detecting the presence or absence of apoptosis in a cell or mixture of cells, the method comprising the steps of:

a) incubating the cell or mixture of cells with a compound of structural formula (II);

7. The method according to claim 6, wherein:

a) R₁₄ is —

OH;

b) R₁₅ is —

OH;

c) R₁₆ is —

NH₂;

d) R₁₄ is —

OH and R₁₅ is —

OH;

e) R₁₄ is —

OH and R₁₆ is —

NH₂;

f) R₁₅ is —

OH and R₁₆ is —

NH₂;

or

g) R₁₄ is —

OH, R₁₅ is —

OH, and R₁₅ is —

NH₂.

8. The method according to any one of claims 6-7, wherein:

a) R′

is H or alkyl;

b) R′

is H or methyl, ethyl, propyl, or isopropyl;

or

c) R′

is H.

9. The method according to any one of claims 6-8, wherein the compound is selected from the group consisting of:

10. A method for detecting the presence or absence of a caspase enzyme in a cell or mixture of cells, measuring the activity of a caspase enzyme in a cell or mixture of cells, or detecting the presence or absence of apoptosis in a cell or mixture of cells, the method comprising the steps of:

a) incubating the cell or mixture of cells with a compound comprising a peptide and a fluorogenic dye and having structural formula (III);

peptide-L-dye 

 

(III);

wherein;

the peptide comprises a sequence of DX₂EX₁D (SEQ ID NO.

     8) or DX₂VX₁D (SEQ ID NO.

     20), wherein X₂ is W, Y, I, L, or V, and X₁ is any amino acid;

L is a linker; and

dye is the fluorogenic dye, wherein the fluorogenic dye is able to associate with a nucleic acid when not linked to the peptide, and is able to emit a fluorescent signal upon excitation when associated with a nucleic acid;

b) providing a stimulus to the cell or mixture of cells to elicit a fluorescent signal; and

c) measuring the fluorescent signal, whereby the presence or absence of the caspase enzyme is detected, the activity of the caspase enzyme is measured, or the presence or absence of apoptosis is detected.

11. The method of claim 10, wherein the fluorogenic dye is a rhodamine, dibenzorhodamine, thiazole, thiazole orange, coumarin, or cresyl violet;

optionally wherein the fluorogenic dye is NUCVIEW™

488 or a rhodamine 110 (R110) or rhodamine 600 (R600), the R600 optionally comprising a silicon, germanium, or tin atom at the 10 position of its xanthene ring system;

further optionally wherein the R110 is N-octyloxycarbonyl-R110, N′

-morpholinecarbonyl-R110, or the R600 is SiR600 or 2-Me SiR600.

12. The method of claim 10, wherein fluorogenic dye is a compound having structural formula (IV):

36. A compound selected from the group consisting of:

65. A composition for detecting the presence or absence of a caspase enzyme, the compositions comprsing:

a) one or more of the compounds or enzyme substrates of any of claims 36-57; and

b) a carrier,

wherein the one or more of the compounds or enzyme substrates are present in an amount effective to detect the presence or absence of the caspase enzyme.

66. A composition for detecting the presence or absence of a caspase enzyme, the composition comprising:

(a) one or more of the compounds or enzyme substrates of any of claims 36-57; and

(b) an analyte,

wherein the one or more of the compounds or enzyme substrates are present in an amount effective to detect the presence or absence of the caspase enzyme.

67. A composition for detecting the presence or absence of apoptosis, the compositions comprising:

a) one or more of the compounds or enzyme substrates of any of claims 36-57; and

b) a carrier,

wherein the one or more of the compounds or enzyme substrates are present in an amount effective to detect the presence or absence of apoptosis.

68. A composition for detecting the presence or absence of apoptosis, the composition comprising:

(a) one or more of the compounds or enzyme substrates of any of claims 36-57; and

(b) an analyte,

wherein the one or more of the compounds or enzyme substrates are present in an amount effective to detect the presence or absence of apoptosis.

69. A composition for measuring activity of a caspase enzyme, the compositions comprising:

a) one or more of the compounds or enzyme substrates of any of claims 36-57; and

b) a carrier,

wherein the one or more of the compounds or enzyme substrates are present in an amount effective to measure the activity of the caspase enzyme.

70. A composition for measuring activity of a caspase enzyme, the composition comprising:

(a) one or more of the compounds or enzyme substrates of any of claims 36-57; and

(b) an analyte,

wherein the one or more of the compounds or enzyme substrates are present in an amount effective to measure the activity of the caspase enzyme.

71. The composition according to claim 66, 68, or 70, wherein the analyte is a cell and the compound is located inside the cell.

72. The composition according to claim 65, 67, or 69, wherein the compound is conjugated to a carrier molecule.

73. Use of a compound or enzyme substrate of any of claims 36-57 for detecting the presence or absence of a caspase enzyme in a sample, detecting the presence or absence of apoptosis, or measuring activity of a caspase enzyme.

74. The use of claim 73, wherein the caspase enzyme is an intracellular caspase enzyme, or the presence or absence of apoptosis is detected based on whether or to what extent intracellular cleavage of the compound or enzyme substrate occurs.

37. An enzyme substrate comprising:

a) a fluorogenic dye moiety of structural formula (I);

38. The enzyme substrate according to claim 37, wherein:

a) each R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂ and R₁₃ is independently H, halogen, or alkyl;

b) each R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂ and R₁₃ is independently H, alkyl, or substituted alkyl;

c) each R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂ and R₁₃ is independently H or alkyl;

d) at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂ and R₁₃ are H;

or

e) each R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂ and R₁₃ is H.

39. The enzyme substrate according to any one of claims 37-38, wherein:

a) X is —

N(R)—

wherein R is H, halogen, alkyl, or substituted alkyl;

b) X is —

N(R)—

wherein R is alkyl or substituted alkyl;

c) X is —

N(R)—

wherein R is alkyl;

d) X is —

N(R)—

wherein R is methyl, ethyl, propyl, or isopropyl;

e) X is —

N(R)—

wherein R is methyl or ethyl;

or

f) X is —

N(CH₃)—

.

40. The enzyme substrate according to any one of claims 37-39, wherein:

a) Y is alkyl;

b) Y is methyl, ethyl, propyl, or isopropyl;

c) Y is methyl or ethyl;

or

d) Y is methyl.

44. The enzyme substrate according to any one of claims 37-43, wherein the peptide comprises an amino acid sequence of:

a) X₂EX₁D (SEQ ID NO.

     5), wherein X₂ is W, Y, I, L, D, or V, and X₁ is any amino acid;

b) X₃DEX₁D (SEQ ID NO.

     6), wherein X₃ is V or L and X₁ is any amino acid;

c) DX₁VD (SEQ ID NO.

     7), wherein X₁ is any amino acid;

d) DX₂EX₁D (SEQ ID NO.

     8), wherein X₂ is W, Y, I, L, or V, and X₁ is any amino acid, or

e) DX₂VX₁D (SEQ ID NO.

     20), wherein X₂ is W, Y, I, L, or V, and X₁ is any amino acid;

optionally wherein the amino acid sequence of a), b), c), d), or e) is the entire amino acid sequence of the peptide or the C-terminal amino acid sequence of the peptide.

45. The enzyme substrate according to any one of claims 37-44, wherein the peptide comprises or is attached to a caspase cleavage site.

46. The enzyme substrate according to any one of claims 37-45, wherein the peptide comprises the amino acid sequence WEHD (SEQ ID NO. 9), YVAD (SEQ ID NO. 10), YVHD (SEQ ID NO. 11), VDVAD (SEQ ID NO. 12), LEHD (SEQ ID NO. 2), IETD (SEQ ID NO. 13), VEID (SEQ ID NO. 14), DEVD (SEQ ID NO. 1), LEVD (SEQ ID NO. 15), or AEVD (SEQ ID NO. 16), optionally wherein the amino acid sequence is the entire amino acid sequence of the peptide or the C-terminal amino acid sequence of the peptide.

47. The enzyme substrate according to any one of claims 37-46, wherein the peptide is a substrate for cleavage by at least one caspase, has a length of 5 or more amino acids, and comprises an aspartic acid in the P₅ position.

49. The enzyme substrate of claim 47, wherein the fluorogenic dye is a rhodamine, dibenzorhodamine, thiazole, thiazole orange, coumarin, or cresyl violet;

optionally wherein the fluorogenic dye is NUCVIEW™

488 or a rhodamine 110 (R110) or rhodamine 600 (R600), the R600 optionally comprising a silicon, germanium, or tin atom at the 10 position of its xanthene ring system;

further optionally wherein the R110 is N-octyloxycarbonyl-R110, N′

-morpholinecarbonyl-R110, or the R600 is SiR600 or 2-Me SiR600.

51. The enzyme substrate according to any one of claims 37-50, wherein the peptide comprises the amino acid sequence of DIETD (SEQ ID NO. 17), DLEHD (SEQ ID NO. 18), or DYVAD (SEQ ID NO. 19), optionally wherein the amino acid sequence is the entire amino acid sequence of the peptide or the C-terminal amino acid sequence of the peptide.

52. The enzyme substrate according to any one of claims 37-51, wherein the peptide:

a) further comprises one or more additional amino acid residues C-terminally;

b) further comprises one or more additional amino acid residues N-terminally;

c) further comprises one or more additional amino acid residues both C-terminally and N-terminally;

d) further comprises 1 to 100, 1 to 50, 1 to 20, 1 to 10, 1 to 5, 5, 4, 3, 2, or 1 additional amino acid residues C-terminally;

e) further comprises 1 to 100, 1 to 50, 1 to 20, 1 to 10, 1 to 5, 5, 4, 3, 2, or 1 additional amino acid residues N-terminally;

or

f) further comprises 1 to 100, 1 to 50, 1 to 20, 1 to 10, 1 to 5, 5, 4, 3, 2, or 1 additional amino acid residues both C-terminally and N-terminally.

53. The enzyme substrate according to any one of claims 37-52, wherein the peptide has a sequence with a total length of 4 to 200 amino acid residues, 4 to 100 amino acid residues, 4 to 50 amino acid residues, 5 to 200 amino acid residues, 5 to 100 amino acid residues, 5 to 50 amino acid residues, 20 to 50 amino acid residues, or 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues.

54. The enzyme substrate according to any one of claims 37-53, wherein the peptide comprises a protected amino acid side-chain.

55. The enzyme substrate according to any one of claims 37-54, wherein the linker is attached to the peptide at a side-chain of an amino acid, an amino terminus of an amino acid or a carboxy terminus of an amino acid.

56. The enzyme substrate according to any one of claims 37-55, wherein L is:

a) —

N(R_N1)—

[CH₂]_n—

, wherein n ranges from 1 to 10 and R_N1 is H or optionally substituted C_1-6 alkyl;

b) —

N(R_N1)-L_a-, wherein La is a C_2-10 alkylene optionally interrupted by one or both of —

N(R_N1)—

C(O)—

or —

N(R_N2)₂—

, wherein each R_N1 is independently H or optionally substituted C_1-6 alkyl and each R_N2 is independently an optionally substituted C_1-6 alkyl;

c) —

C(O)—

[CH₂]_n—

, wherein n ranges from 1 to 10;

d) —

N(R_N1)—

[CH₂]—

C(O)—

, wherein n ranges from 1 to 10 and R_N1 is H or optionally substituted C_1-6 alkyl;

e) —

C(O)—

[CH₂]_n—

C(O)—

, wherein n ranges from 1 to 10;

f) —

[CH₂]—

N(R_N2)₂—

[CH₂]—

, wherein each R_N2 is independently an optionally substituted C_1-6 alkyl;

g) —

NH—

[CH₂]₂—

;

h) —

NH—

[CH₂]₄—

;

i) —

NH—

[CH₂]₅—

;

or

j) —

NH—

[CH₂]₂—

NH—

C(O)—

CH₂—

N(CH₃)₂—

[CH₂]₂—

.

57. The enzyme substrate according to any one of claims 37-56, wherein the enzyme substrate is covalently attached to a solid support.

41. An enzyme substrate comprising:

a) a fluorogenic dye moiety of structural formula (II);

42. The enzyme substrate according to claim 41, wherein:

a) R₁₄ is —

OH;

b) R₁₅ is —

OH;

c) R₁₆ is —

NH₂;

d) R₁₄ is —

OH and R₁₅ is —

OH;

e) R₁₄ is —

OH and R₁₆ is —

NH₂;

f) R₁₅ is —

OH and R₁₆ is —

NH₂;

or

g) R₁₄ is —

OH, R₁₅ is —

OH, and R₁₆ is —

NH₂.

43. The enzyme substrate according to any one of claims 41-42, wherein:

a) R′

is H or alkyl;

b) R′

is H or methyl, ethyl, propyl, or isopropyl;

or

c) R′

is H.

48. An enzyme substrate comprising a peptide and a fluorogenic dye and having structural formula (III):

peptide-L-dye 

 

(III);

wherein;

the peptide comprises a sequence of DX₂EX₁D (SEQ ID NO.

     8) or DX₂VX₁D (SEQ ID NO.

     20), wherein X₂ is W, Y, I, L, or V, and X₁ is any amino acid;

L is a linker; and

dye is the fluorogenic dye, wherein the fluorogenic dye is able to emit a fluorescent signal upon excitation when free from the peptide.

50. The enzyme substrate of claim 48, wherein the fluorogenic dye is a compound having structural formula (IV):