Chemiluminescent doubletriggered 1,2-dioxetanes

Claim 2

A stable 1,2-dioxetane of the formula: where R₁, R₂, and R₃ are passive organic groups that allow the dioxetane to decompose and produce light when Z and Y are removed, with the proviso that R₁ and R₂ may be joined to form a cyclic moiety, Z is a first protecting group that can be removed by a first deprotecting process, Y is a second protecting group that can be removed by a second deprotecting process, and X is an organic group which upon removal of Z and Y renders the dioxetane susceptible to said decomposition, with the proviso that X may be joined to R₃ to form a cyclic moiety.

Claim 3

The dioxetane of claim 1 wherein R₁ and R₂ are joined and form a carbo cyclic group of 6 to 30 carbon atoms, R₃ contains 1-18 carbon atoms and is alkyl, alkoxy, monocyclic aryloxy, dialkyl or aryl amino, trialkyl or aryl siloxy, Z is an organic group that is cleavable from the dioxetane by a first enzymatic reaction, Y is a moiety that is cleavable from the dioxetane by a second enzymatic reaction, and X is an aryloxy group.

Claim 4

The dioxetane of claim 1 wherein R₁ and R₂ are joined to form an adamantane ring, R₃ is methoxy, X is phenoxy substituted with 0 to 2 Cl or I groups, Y is phosphate and Z is 2-methyl-4-hydroxy naphthyl.

Claim 5

The dioxetane of claim 1 wherein R₁ and R₂ are joined to form an adamantane ring, R₃ contains 1-18 carbon atoms and is alkyl, alkoxy, monocyclic aryloxy, dialkyl or aryl amino, trialkyl or aryl siloxy, X is and YZ is selected from the group consisting of where R₄ is -(CH₂)₄-NH₂, or R₅ is CH₃-, (CH₃)₂CH- , CH₃CH₂-(CH₃)CHCH₂-, or

Claim 6

A method for generating light which comprises: (a) providing a stable 1,2-dioxetane of claim 1, 2, 3 or 4, (b) removing the first protecting group Z, and (c) removing the second protecting group Y.

Claim 7

The method of claim 5 wherein R₁ and R₂ are joined to form an adamantane ring, R₃ is methoxy, X is phenoxy substituted with 0-2 Cl or I groups, Y is phosphate and Z is 2-methyl-4-hydroxy naphthyl, the first protecting group is removed with horseradish peroxidase, and the second protecting group is removed with alkaline phosphatase.

Claim 8

A chemiluminescent method for detecting a component in a sample suspected of containing the component wherein the material is detected via a label carried by a ligand that complexes or binds to the component comprising (a) binding a labeled ligand to the component, (b) contacting the component-labeled ligand complex with a dioxetane of claim 1, 2, 3 or 4 wherein Z is removed through reaction with the label on the complex, (c) contacting the partly deprotected dioxetane with an activating agent that removes Y to produce an unstable oxide intermediate which decomposes to form light, and (d) detecting said light.

Claim 9

The method of claim 7 wherein the component is a single stranded nucleic acid molecule and the ligand is a nucleic acid molecule that hybridizes to the component.

Claim 10

A chemiluminescent signal generating reagent kit for use in generating a chemiluminescent signal on an assay for a component of a sample wherein the component is detected via an enzymatic label carried by a ligand that specifically complexes or bonds to the component comprising in packaged combination: (a) a first container containing a dioxetane of the formula where R₁, and R₂, and R₃ are passive organic groups that allow the dioxetane to decompose and produce light when Z and Y are removed, with the proviso that R₁ and R₂ may be joined to form a cyclic moiety, Z is a first protecting group that can be removed by reaction with said enzymatic label, Y is a protecting group that can be removed by reaction with a deprotecting agent other than enzymatic label, and X is an organic group which upon removal of Z and Y renders the dioxetane susceptible to said decomposition, with the proviso that X may be joined to R₃ to form a cyclic moiety, whereby Z is removed through reaction with the label on the complex; and (b) a second container containing said deprotecting agent.

Claim 11

A chemiluminescent signal generating reagent kit for use in generating a chemiluminescent signal on an assay for a component of a sample wherein the component is detected via an enzymatic label carried by a ligand that specifically complexes or bonds to the component comprising in packaged combination: (a) a first container containing a dioxetane of the formula where R₁, and R₂, and R₃ are passive organic groups that allow the dioxetane to decompose and produce light when Z and Y are removed, with the proviso that R₁ and R₂ may be joined to form a cyclic moiety, Z is a first protecting group that can be removed by reaction with said enzymatic label, Y is a protecting group that can be removed by reaction with a deprotecting agent other than enzymatic label, and X is an organic group which upon removal of Z and Y renders the dioxetane susceptible to said decomposition, with the proviso that X may be joined to R₃ to form a cyclic moiety, whereby Z is removed through reaction with the label on the complex; and (b) a second container containing said deprotecting agent.