Claims
- 1. A compound of formula I:
- 2. The compound of claim 1 wherein R6 contains from 1 to 50 atoms selected from the group consisting of C, N, O, S, P and halogen atoms.
- 3. The compound of claim 2 wherein R6 is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl and aralkyl groups.
- 4. The compound of claim 1 which is selected from the group consisting of compounds of formula II and compounds of formula III:
- 5. The compound of claim 4 wherein R1 is selected from the group consisting of C1 to C4 alkyl groups and a benzyl group.
- 6. The compound of claim 4 having the formula:
- 7. The compound of claim 6 wherein at least one of R7 to R14 is an alkoxy group and the remaining of R7 to R14 are hydrogen.
- 8. The compound of claim 6 having the formula:
- 9. The compound of claim 8 wherein M is Na and n is 2.
- 10. The compound of claim 6 having the formula:
- 11. The compound of claim 10 wherein M is Na and n is 2.
- 12. A compound having the formula:
- 13. A compound having the formula:
- 14. A compound having the formula:
- 15. A compound having the formula:
- 16. A compound having the formula:
- 17. A compound having the formula:
- 18. A compound having the formula:
- 19. A reagent composition which produces chemiluminescence in the presence of a phosphatase enzyme which comprises in an aqueous solution:
a) a compound of formula I: 50 which reacts with the phosphatase enzyme wherein Het is a heterocyclic ring system comprising at least one five or six-membered ring which comprises at least one heteroatom selected from the group consisting of N, O and S atoms, wherein Z is selected from the group consisting of O and S atoms, wherein R6 is an organic group which allows chemiluminescence to be produced, wherein each M is independently selected from H and a cationic center and wherein n is a number which satisfies electroneutrality; and b) at least one surfactant enhancer in an amount effective to enhance the chemiluminescence.
- 20. The composition of claim 19 in which the compound of formula I is selected from the group consisting of compounds of formula II and compounds of formula III:
- 21. The composition of claim 20 in which the compound of formula I has the formula:
- 22. The composition of claim 21 wherein at least one of R7 to R14 is an alkoxy group and the remaining of R7 to R14 are hydrogen.
- 23. The composition of claim 21 wherein the compound of formula I has the formula:
- 24. The composition of claim 21 wherein the compound has the formula:
- 25. The composition of claim 21 wherein the compound has the formula:
- 26. The composition of claim 19 wherein the surfactant enhancer is selected from polymeric quaternary ammonium and phosphonium salts, monomeric quaternary ammonium and phosphonium salts and dicationic quaternary ammonium and phosphonium salts.
- 27. The composition of claim 19 wherein the surfactant enhancer is a copolymer of a vinylbenzyltributylphosphonium salt and a vinylbenzyltrioctylphosphonium salt.
- 28. The composition of claim 19 wherein copolymer comprises vinylbenzyltributylphosphonium salt and vinylbenzyltrioctylphosphonium salt groups in a ratio of about 3 to 1.
- 29. The composition of claim 19 further comprising a magnesium salt in an amount effective to promote the reaction.
- 30. A method for producing chemiluminescence which comprises reacting a phosphatase enzyme with at least one compound of formula I:
- 31. The method of claim 30 wherein the compound of formula I is selected from the group consisting of compounds of formula II and compounds of formula III:
- 32. The method of claim 30 wherein the compound of formula I has the formula:
- 33. The method of claim 32 wherein at least one of R7 to R14 is an alkoxy group and the remaining of R7 to R14 are hydrogen.
- 34. The method of claim 32 wherein the compound of formula I has the formula:
- 35. The method of claim 32 wherein the compound of formula I has the formula:
- 36. The method of claim 32 wherein the compound of formula I has the formula:
- 37. The method of claim 30 wherein the phosphatase enzyme is selected from the group consisting of bacterial alkaline phosphatase, mammalian alkaline phosphatase, plant acid phosphatase, mammalian acid phosphatase and alkaline phosphatase conjugates.
- 38. The method of claim 37 wherein the alkaline phosphatase conjugate comprises alkaline phosphatase linked to a biological molecule selected from the group consisting of haptens, antibodies, proteins, nucleic acids and oligonucleotides.
- 39. The method of claim 32 wherein the phosphatase enzyme is selected from the group consisting of bacterial alkaline phosphatase, mammalian alkaline phosphatase, plant acid phosphatase, mammalian acid phosphatase and alkaline phosphatase conjugates.
- 40. The method of claim 39 wherein the alkaline phosphatase conjugate comprises alkaline phosphatase linked to a biological molecule selected from the group consisting of haptens, antibodies, proteins, nucleic acids and oligonucleotides.
- 41. The method of any of claims 34, 35 or 36 wherein the phosphatase enzyme is selected from the group consisting of bacterial alkaline phosphatase, mammalian alkaline phosphatase, plant acid phosphatase, mammalian acid phosphatase and alkaline phosphatase conjugates.
- 42. The method of claim 41 wherein the alkaline phosphatase conjugate comprises alkaline phosphatase linked to a biological molecule selected from the group consisting of haptens, antibodies, proteins, nucleic acids and oligonucleotides.
- 43. A method for detecting an analyte in a sample by a chemiluminescent assay procedure which comprises:
(a) reacting a phosphatase enzyme with at least one compound of formula I to produce chemiluminescence for detecting the analyte, 62 wherein in the compound of formula I, Het is a heterocyclic ring system comprising at least one five or six-membered ring which comprises at least one heteroatom selected from the group consisting of N, O and S atoms, wherein Z is selected from the group consisting of O and S atoms, wherein R6 is an organic group which allows chemiluminescence to be produced, wherein each M is independently selected from H and a cationic center and wherein n is a number which satisfies electroneutrality; (b) detecting the chemiluminescence; and (c) relating the amount of the chemiluminescence to the amount of the analyte.
- 44. The method of claim 43 wherein the compound of formula I is selected from the group consisting of compounds of formula II and compounds of formula III
- 45. The method of claim 43 wherein the compound of formula I has the formula:
- 46. The method of claim 45 wherein at least one of R7 to R14 is an alkoxy group and the remaining of R7 to R14 are hydrogen.
- 47. The method of claim 43 wherein the compound of formula I has the formula:
- 48. The method of claim 43 wherein the compound of formula I has the formula:
- 49. The method of claim 43 wherein the compound of formula I has the formula:
- 50. The method of claim 43 wherein the analyte to be detected is the phosphatase enzyme.
- 51. The method of claim 43 wherein the analyte to be detected is an inhibitor of the phosphatase enzyme.
- 52. The method of claim 43 further comprising reacting the analyte in the sample with an analyte binding compound which specifically binds with the analyte wherein the analyte-binding compound is labeled with alkaline phosphatase.
- 53. The method of claim 52 wherein the analyte-binding compound is selected from the group consisting of antibodies, antigens, haptens and nucleic acids.
- 54. The method of claim 45 further comprising reacting the analyte in the sample with an analyte binding compound which specifically binds with the analyte and at least one phosphatase-labeled specific binding substance for the analyte binding compound.
- 55. The method of any of claims 47, 48 or 49 further comprising reacting the analyte in the sample with an analyte binding compound which specifically binds with the analyte and at least one phosphatase-labeled specific binding substance for the analyte binding compound.
- 56. The method of claim 43 further comprising reacting the analyte in the sample with:
(a) a labeled analyte binding compound comprising an analyte binding compound which specifically binds with the analyte and at least one second specific binding substance; and (b) a phosphatase-labeled binding partner for the second specific binding substance.
- 57. The method of claim 43 wherein the detection is performed on a membrane.
- 58. The method of claim 57 wherein the membrane is selected from the group consisting of a nitrocellulose membrane, a polyvinylidene difluoride membrane and a nylon membrane.
- 59. The method of claim 43 further comprising providing the compound of formula I in a reagent composition comprising, in an aqueous solution, the compound of formula I and a surfactant enhancer in an amount effective to enhance the chemiluminescence.
- 60. The method of claim 45 further comprising providing the compound of formula I in a reagent composition comprising, in an aqueous solution, the compound of formula I and a surfactant enhancer in an amount effective to enhance the chemiluminescence.
- 61. The method of any of claim 47, 48 or 49 further comprising providing the compound of formula I in a reagent composition comprising, in an aqueous solution, the compound of formula I and a surfactant enhancer in an amount effective to enhance the chemiluminescence.
- 62. The method of claim 59 wherein the surfactant enhancer is a copolymer of a vinylbenzyltributylphosphonium salt and a vinylbenzyltrioctylphosphonium salt.
- 63. The method of claim 43 further comprising:
(a) reacting the compound of formula I with the phosphatase enzyme in a buffer at a first pH for a first period of time; (b) adding a strongly basic trigger solution to the buffer solution to raise the pH of the buffer to a second pH for inducing the chemiluminescence; and (c) measuring the chemiluminescence.
- 64. The method of claim 63 wherein the first pH is in the range 5.0-9.51 wherein the pH of the trigger solution is greater than about 11 and the first period of time is from about 1 seconds to about 10 minutes.
- 65. The method of claim 63 wherein the basic trigger solution contains a surfactant enhancer.
- 66. The method of claim 65 wherein the enhancer is a copolymer of a vinylbenzyltributylphosphonium salt and a vinylbenzyltrioctylphosphonium salt.
- 67. A reagent composition which produces chemiluminescence in the presence of a phosphatase enzyme which comprises in an aqueous solution:
a) a compound of formula I: 68 which reacts with the phosphatase enzyme wherein Het is a heterocyclic ring system comprising at least one five or six-membered ring which comprises at least one heteroatom selected from the group consisting of N, O and S atoms, wherein Z is selected from the group consisting of O and S atoms, wherein R6 is an organic group which allows chemiluminescence to be produced, wherein each M is independently selected from H and a cationic center and wherein n is a number which satisfies electroneutrality; and b) a cationic aromatic compound in an amount effective to increase the chemiluminescence compared to that generated in the absence of the cationic aromatic compound.
- 68. The composition of claim 67 wherein R6 contains from 1 to 50 atoms selected from the group consisting of C, N, O, S, P and halogen atoms.
- 69. The composition of claim 68 wherein R6 is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl and aralkyl groups.
- 70. The composition of claim 67 in which the compound of formula I is selected from the group consisting of compounds of formulas:
- 71. The composition of claim 67 in which the compound of formula I has the formula:
- 72. The composition of claim 71 wherein the compound of formula I has the formula:
- 73. The composition of claim 72 wherein the compound of formula I has the formula:
- 74. The composition of any one of claims 67, 68, 69, 70, 71, 72, or 73 wherein the cationic aromatic compound is selected from the group consisting of cyanine dyes, carbocyanine dyes, azo dyes, acridinium derivatives, methylene blue, Nile Blue, IR-1040, lucigenin and paraquat dichloride.
- 75. The composition of claim 67 further comprising an anionic surfactant in an amount effective to increase the speed with which maximum chemiluminescence intensity is reached and a non-ionic surfactant in an amount effective to increase the amount of chemiluminescence.
- 76. The composition of claim 75 wherein the anionic surfactant is selected from alkylsulfates containing at least 10 carbon atoms and alkylsulfonates containing at least 10 carbon atoms.
- 77. The composition of claim 75 wherein the non-ionic surfactant is selected from polyoxyethylenated alkylphenols, polyoxyethylenated alcohols, polyoxyethylenated ethers and polyoxyethylenated sorbitol esters.
- 78. The composition of claim 75 additionally comprising a sulfite salt in an amount effective to reduce chemiluminescence produced by the composition in the absence of a phosphatase enzyme.
- 79. The composition of claim 76 wherein the anionic surfactant is sodium dodecyl sulfate.
- 80. The composition of claim 78 wherein the sulfite salt is sodium sulfite.
- 81. A method for producing chemiluminescence which comprises reacting a phosphatase enzyme with
a) at least one compound of formula I: 73 wherein Het is a heterocyclic ring system comprising at least one five or six-membered ring which comprises at least one heteroatom selected from the group consisting of N, O and S atoms, wherein Z is selected from the group consisting of O and S atoms, wherein R6 is an organic group which allows chemiluminescence to be produced, wherein each M is independently selected from H and a cationic center and wherein n is a number which satisfies electroneutrality; and b) a cationic aromatic compound in an amount effective to increase the chemiluminescence compared to that generated in the absence of the cationic aromatic compound.
- 82. The method of claim 81 wherein R6 contains from 1 to 50 atoms selected from the group consisting of C, N, O, S, P and halogen atoms.
- 83. The method of claim 82 wherein R6 is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl and aralkyl groups.
- 84. The method of claim 81 wherein the compound of formula I is selected from the group consisting of compounds of formulas:
- 85. The method of claim 84 wherein the compound of formula I has the formula:
- 86. The method of claim 85 wherein the compound of formula I has the formula:
- 87. The method of claim 86 wherein the compound of formula I has the formula:
- 88. The method of any of claims 81, 82, 83, 84, 85, 86 or 87 wherein the phosphatase enzyme is selected from the group consisting of bacterial alkaline phosphatase, mammalian alkaline phosphatase, plant acid phosphatase, mammalian acid phosphatase and alkaline phosphatase conjugates.
- 89. The method of claim 88 wherein the alkaline phosphatase conjugate comprises alkaline phosphatase linked to a biological molecule selected from the group consisting of haptens, antibodies, proteins, nucleic acids and oligonucleotides.
- 90. The method of any of claims 81, 82, 83, 84, 85, 86 or 87 further comprising providing the compound of formula I and the cationic aromatic compound in a reagent composition which composition further comprises, in an aqueous solution, an anionic surfactant in an amount effective to increase the speed with which maximum chemiluminescence intensity is reached and a non-ionic surfactant in an amount effective to increase the chemiluminescence.
- 91. A method for detecting an analyte in a sample by a chemiluminescent assay procedure which comprises:
(a) reacting a phosphatase enzyme with at least one compound of formula I in the presence of a cationic aromatic compound to produce chemiluminescence for detecting the analyte, 78 wherein in the compound of formula I, Het is a heterocyclic ring system comprising at least one five or six-membered ring which comprises at least one heteroatom selected from the group consisting of N, O and S atoms, wherein Z is selected from the group consisting of O and S atoms, wherein R6 is an organic group which allows chemiluminescence to be produced, wherein each M is independently selected from H and a cationic center and wherein n is a number which satisfies electroneutrality; (b) detecting the chemiluminescence; and (c) relating the amount of the chemiluminescence to the amount of the analyte.
- 92. The method of claim 91 wherein R6 contains from 1 to 50 atoms selected from the group consisting of C, N, O, S, P and halogen atoms.
- 93. The method of claim 92 wherein R6 is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl and aralkyl groups.
- 94. The method of claim 91 wherein the compound of formula I is selected from the group consisting of compounds of formulas:
- 95. The method of claim 91 wherein the compound of formula I has the formula:
- 96. The method of claim 95 wherein the compound of formula I has the formula:
- 97. The method of claim 96 wherein the compound of formula I has the formula:
- 98. The method of any one of claims 91, 92, 93, 94, 95, 96 or 97 wherein the analyte to be detected is the phosphatase enzyme.
- 99. The method of any one of claims 91, 92, 93, 94, 95, 96 or 97 wherein the analyte to be detected is an inhibitor of the phosphatase enzyme.
- 100. The method of any one of claims 91, 92, 93, 94, 95, 96 or 97 further comprising reacting the analyte in the sample with a labeled analyte binding compound which specifically binds with the analyte wherein the analyte-binding compound is labeled with alkaline phosphatase.
- 101. The method of claim 100 wherein the analyte-binding compound is selected from the group consisting of antibodies, antigens, haptens and nucleic acids.
- 102. The method of any one of claims 91, 92, 93, 94, 95, 96 or 97 further comprising reacting the analyte in the sample with an analyte binding compound which specifically binds with the analyte and at least one phosphatase-labeled specific binding substance for the analyte binding compound.
- 103. The method of any one of claims 91, 92, 93, 94, 95, 96 or 97 further comprising reacting the analyte in the sample with:
(a) a labeled analyte binding compound comprising an analyte binding compound which specifically binds with the analyte and at least one second specific binding substance; and (b) a phosphatase-labeled binding partner for the second specific binding substance.
- 104. The method of claim 91 wherein the detection is performed on a membrane.
- 105. The method of claim 104 wherein the membrane is selected from the group consisting of a nitrocellulose membrane, a polyvinylidene difluoride membrane and a nylon membrane.
- 106. The method of any one of claims 91, 92, 93, 94, 95, 96 or 97 further comprising providing the compound of formula I and the cationic aromatic compound in a reagent composition which composition further comprises, in an aqueous solution, an anionic surfactant in an amount effective to increase the speed with which maximum chemiluminescence intensity is reached and a non-ionic surfactant in an amount effective to increase the chemiluminescence.
- 107. A method for detecting acid phosphatase and alkaline phosphatase in a sample suspected of containing both acid phosphatase and alkaline phosphatase by a chemiluminescent assay procedure which comprises:
(a) reacting the sample with a reagent composition comprising at least one compound of formula I, 83 wherein in the compound of formula I, Het is a heterocyclic ring system comprising at least one five or six-membered ring which comprises at least one heteroatom selected from the group consisting of N, O and S atoms, wherein Z is selected from the group consisting of O and S atoms, wherein R6 is an organic group which allows chemiluminescence to be produced, wherein each M is independently selected from H and a cationic center and wherein n is a number which satisfies electroneutrality, a cationic aromatic compound, an anionic surfactant in an amount effective to increase the speed with which maximum chemiluminescence intensity is reached and a non-ionic surfactant in an amount effective to increase the chemiluminescence; (b) detecting the amount or intensity of chemiluminescence during an initial period; (c) waiting a second period of time until the chemiluminescence has achieved a constant level; (d) detecting the amount or intensity of chemiluminescence during a third period; (e) relating the chemiluminescence in the inital time period to the amount of acid phosphatase; and (f) relating the chemiluminescence in the third time period to the amount of alkaline phosphatase.
- 108. The method of claim 107 wherein the compound of formula I is selected from the group consisting of compounds of formulas:
- 109. The method of claim 107 wherein the compound of formula I has the formula:
- 110. The method of claim 109 wherein the compound of formula I has the formula:
- 111. The method of claim 107 wherein the compound of formula I has the formula:
- 112. A process for the preparation of a compound of formula I:
- 113. The process of claim 112 wherein the step of reacting the enolate of compound VIII with a phosphorylating agent to form a protected enol phosphate IX comprises the steps of:
(a) reacting the enolate of compound VIII with a phosphorus oxyhalide compound POW3, where W is a halogen atom selected from F, Cl, Br and I to form an enol dihalophosphate X having the formula 91 wherein Het, Z and R6 are as defined in compound VIII; and (b) reacting compound X with at least two equivalents of a hydroxylic compound Y—OH to form the protected enol phosphate IX.
- 114. The process of claim 112 wherein the step of reacting the enolate of compound VIII with a phosphorylating agent to form a protected enol phosphate IX comprises reacting the enolate of compound VIII with a phosphorylating agent containing the protecting groups Y and having the formula W—PO(OY)2 and wherein W is a halogen atom selected from F, Cl, Br and I.
- 115. The process of claim 112 wherein the groups Y are selected from the group consisting of lower alkyl groups, substituted lower alkyl groups, phenyl, substituted phenyl and benzyl groups.
- 116. The process of claim 112 wherein the groups Y are connected to form the single group —CH2CH2—.
- 117. The process of claim 112 wherein the deprotecting agent is selected from the group consisting of organic and inorganic bases such as sodium hydroxide, potassium hydroxide, potassium carbonate, sodium methoxide, sodium ethoxide, potassium t-butoxide, ammonium hydroxide, nucleophilic agents such as cyanide ion, fluoride ion.
- 118. The process of claim 112 wherein Y is a CH2CH2CN group and the deprotecting agent is selected from sodium hydroxide and sodium carbonate.
- 119. The process of claim 113 wherein Y is a CH2CH2CN group and the deprotecting agent is selected from sodium hydroxide and sodium carbonate.
- 120. The process of claim 112 wherein the compound of formula I is selected from the group consisting of compounds of formula II and compounds of formula III:
- 121. The process of claim 120 wherein the compound of formula I has the formula:
- 122. A compound of formula IX:
- 123. The compound of claim 122 which is selected from the group consisting of compounds of formulas:
- 124. The compound of claim 123 having the formula:
- 125. The compound of any of claims 122, 123 or 124 wherein Y is a CH2CH2CN group.
- 126. A compound of formula X:
- 127. The compound of claim 126 which is selected from the group consisting of compounds of formulas:
- 128. The compound of claim 127 having the formula:
- 129. The compound of any of claims 126, 127 or 128 wherein W is a Cl atom.
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part of applicant's co-pending U.S. applications Ser. Nos. 08/585,090 filed on Jan. 16, 1996 and 08/683,927 filed on Jul. 19, 1996.
Divisions (1)
|
Number |
Date |
Country |
Parent |
08894143 |
Aug 1997 |
US |
Child |
09539816 |
Mar 2000 |
US |
Continuations (1)
|
Number |
Date |
Country |
Parent |
09539816 |
Mar 2000 |
US |
Child |
10054417 |
Jan 2002 |
US |
Continuation in Parts (2)
|
Number |
Date |
Country |
Parent |
08683927 |
Jul 1996 |
US |
Child |
PCT/US97/00015 |
Jan 1997 |
US |
Parent |
08585090 |
Jan 1996 |
US |
Child |
PCT/US97/00015 |
Jan 1997 |
US |