Claims
- 1. A luminescent assay method to detect one or more caspases, comprising:
a) contacting a sample suspected of having one or more caspases with a mixture comprising luciferase and an amino-modified aminoluciferin or a carboxy-terminal protected derivative thereof, wherein the modification is the covalent linkage of a substrate for the caspase to the amino group of aminoluciferin or the derivative thereof via a peptide bond, and wherein the caspase cleaves the substrate at the peptide bond; and b) detecting luminescence in the sample, wherein the luminescent assay is more sensitive than a corresponding assay with a conjugate comprising a fluorophore covalently linked to the substrate or a functional equivalent thereof.
- 2. A luminescent assay method to detect a protease that specifically cleaves a substrate comprising aspartate, comprising:
a) contacting a sample suspected of having one or more aspartate-specific proteases with a mixture comprising luciferase and an amino-modified aminoluciferin or a carboxy-terminal protected derivative thereof, wherein the modification is the covalent linkage of a substrate comprising aspartate to the amino group of aminoluciferin or the derivative thereof via a peptide bond, and wherein the protease cleaves the substrate at the peptide bond; and b) detecting luminescence in the sample, wherein the luminescent assay is more sensitive than a corresponding assay with a conjugate comprising a fluorophore covalently linked to the substrate or a functional equivalent thereof.
- 3. The method of claim 1 or 2 further comprising correlating luminescence with protease concentration or activity.
- 4. The method of claim 1 or 2 which detects a caspase other than caspase 3 or caspase 7.
- 5. The method of claim 1 or 2 which detects caspase 3 or caspase 7.
- 6. The method of claim 1 or 2 wherein the substrate comprises X1-X2-X3-D, wherein X1 is Y, D, L, V, I, A, W, or P; X2 is V or E; and X3 is any amino acid.
- 7. The method of claim 6 wherein X3 is A, V, H, I, or T.
- 8. The method of claim 1 or 2 wherein the substrate comprises DEVD (SEQ ID NO:1).
- 9. The method of claim 1 or 2 wherein the substrate comprises YVAD (SEQ ID NO:2).
- 10. The method of claim 1 or 2 wherein the substrate comprises LEHD (SEQ ID NO:3).
- 11. The method of claim 5 which detects at least 0.2 microunits of caspase.
- 12. The method of claim 1 or 2 which is at least 2 times more sensitive than a corresponding assay with a conjugate comprising rhodamine-110 covalently linked to the substrate.
- 13. The method of claim 1 or 2 wherein the sample is a cell lysate.
- 14. The method of claim 13 wherein the cells are treated with an apoptosis inducing agent prior to lysis.
- 15. The method of claim 1 or 2 wherein the sample comprises intact cells.
- 16. The method of claim 15 wherein the cells are treated with an apoptosis inducing agent.
- 17. A compound comprising aminoluciferin or a carboxy-terminal protected derivative thereof covalently linked via a peptide bond to a substrate for a caspase.
- 18. A compound comprising aminoluciferin or a carboxy-terminal protected derivative thereof covalently linked via a peptide bond to a substrate that specifically cleaves a substrate comprising aspartate.
- 19. The compound of claim 17 or 18 wherein the substrate comprises DEVD (SEQ ID NO:1)
- 20. The compound of claim 17 or 18 wherein the substrate comprises YVAD (SEQ ID NO:2).
- 21. The compound of claim 17 or 18 wherein the substrate comprises LEHD (SEQ ID NO:3).
- 22. The compound of claim 17 or 18 wherein the substrate comprises X1-X2-X3-D, wherein X1 is Y, D, L, V, I, A, W, or P; X2 is V or E; and X3 is any amino acid.
- 23. The compound of claim 22 wherein X3 is A, V, H, I, or T.
- 24. The compound of claim 17 or 18 which is a carboxy-terminal protected derivative of aminoluciferin.
- 25. The compound of claim 24 wherein the carboxy-terminus is protected as a (C1-C6)alkyl ester.
- 26. The compound of claim 24 wherein the carboxy-terminus is protected as a methyl, ethyl, propyl, or tert-butyl ester.
- 27. The compound of claim 24 wherein the N-terminus of the substrate is modified to prevent degradation by aminopeptidases.
- 28. The compound of claim 27 wherein the N-terminus of the substrate is modified with an amino protecting group.
- 29. The compound of claim 24 wherein the carboxy-terminus is protected by coupling to a solid support.
- 30. A method to prepare a carboxy-terminal protected derivative of aminoluciferin, comprising protecting the corresponding acid with a suitable carboxy-protecting group.
- 31. A method to prepare a carboxy-terminal protected derivative of aminoluciferin of claim 17 or 18, comprising protecting the corresponding acid with a suitable carboxy-protecting group.
- 32. The method of claim 30 or 31 wherein the acid is protected as an ester.
- 33. The method of claim 32 wherein the acid is protected by a tert-butyl ester.
- 34. A carboxy-terminal protected aminoluciferin prepared by the method of claim 30 or 31.
- 35. The method of claim 30 or 31 wherein the acid is protected by coupling aminoluciferin to a solid support.
- 36. The method of claim 35 wherein the solid support is a resin suitable for peptide synthesis.
- 37. The method of claim 35 wherein the acid is coupled to the support through an ester linkage.
- 38. A method for preparing a compound of claim 17 or 18 comprising forming an amide bond between a first amino acid or peptide and the amino group of a solid support bound aminoluciferin; and optionally attaching one or more additional amino acids or peptides through peptide bonds to provide the compound.
- 39. The method of claim 38 wherein the solid support bound aminoluciferin is prepared by attaching an N-protected aminoluciferin to a solid support through the carboxy group; and deprotecting the aminoluciferin.
- 40. The method of claim 38 or 39 further comprising removing the compound from the solid support to provide a compound having a free carboxylic acid group.
- 41. The method of claim 39 further comprising protecting the carboxylic acid group with a suitable protecting group.
- 42. The method of claim 1 or 2 wherein the luciferase is a thermostable luciferase.
- 43. A compound comprising aminoluciferin coupled via the carboxyl group to a solid support.
- 44. A compound comprising N-protected aminoluciferin coupled via the carboxyl group to a solid support.
- 45. The compound of claim 43 or 44 wherein the solid support is a resin suitable for peptide synthesis.
- 46. The compound of claim 45 wherein the amino group is protected with an Fmoc or a t-Boc group.
- 47. The method of claim 1 wherein the carboxy-terminal protected derivative thereof is a compound of formula (I):
- 48. The method of claim 2 wherein the carboxy-terminal protected derivative thereof is a compound of formula (I):
- 49. The method of claim 47 or 48 wherein R comprises X1-X2-X3-D, wherein X1 is Y, D, L, V, I, A, W, or P; X2 is V or E; and X3 is any amino acid.
- 50. The method of claim 49 wherein X3 is A, V, H, I, or T.
- 51. The method of claim 49 wherein R comprises DEVD (SEQ ID NO:1).
- 52. The method of claim 49 wherein R comprises YVAD (SEQ ID NO:2).
- 53. The method of claim 49 wherein R comprises LEHD (SEQ ID NO:3).
- 54. The method of claim 47 wherein R′ is (C1-C6)alkyl, phenyl or benzyl.
- 55. The method of claim 47 or 48 wherein R′ is (C1-C6)alkyl.
- 56. The method of claim 47 or 48 wherein R′ is methyl, ethyl, propyl, or tert-butyl.
- 57. A compound of formula (I):
- 58. A compound of formula (I):
- 59. The compound of claim 57 or 58 wherein R comprises X1-X2-X3-D, wherein X1 is Y, D, L, V, I, A, W, or P; X2 is V or E; and X3 is any amino acid.
- 60. The compound of claim 59 wherein X3 is A, V, H, I, or T.
- 61. The compound of claim 59 wherein R comprises DEVD (SEQ ID NO:1).
- 62. The compound of claim 59 wherein R comprises YVAD (SEQ ID NO:2).
- 63. The compound of claim 59 wherein R comprises LEHD (SEQ ID NO:3).
- 64. The compound of claim 57 or 58 wherein R′ is (C1-C6)alkyl, phenyl or benzyl.
- 65. The compound of claim 57 or 58 wherein R′ is (C1-C6)alkyl
- 66. The compound of claim 57 or 58 wherein R′ is methyl, ethyl, propyl, or tert-butyl.
- 67. A method to prepare a compound of formula (I):
- 68. A method to prepare a compound of formula (I):
- 69. A compound of formula (I):
- 70. The compound of claim 69 wherein the solid support is a resin suitable for peptide synthesis.
- 71. A compound of formula (I):
- 72. The compound of claim 71 wherein the solid support is a resin suitable for peptide synthesis.
- 73. The compound of claim 69 or 71 wherein R comprises X1-X2-X3-D, wherein X1 is Y, D, L, V, I, A, W, or P; X2 is V or E; and X3 is any amino acid.
- 74. The compound of claim 73 wherein X3 is A, V, H, I, or T.
- 75. The compound of claim 73 wherein R comprises DEVD (SEQ ID NO:1).
- 76. The compound of claim 73 wherein R comprises YVAD (SEQ ID NO:2).
- 77. The compound of claim 73 wherein R comprises LEHD (SEQ ID NO:3).
CROSS-REFERENCE TO RELATED APPLICATONS
[0001] This application claims the benefit of the filing date of U.S. application Ser. No. 60/353,158, filed Feb. 1, 2002, under 35 U.S.C. § 119(e). The disclosure of U.S. application Ser. No. 60/353,158 is incorporated by reference herein.
Provisional Applications (1)
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Number |
Date |
Country |
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60353158 |
Feb 2002 |
US |