US4956477A — Synthesis of 1,2-dioxetanes

Claim 1

Independent Claim 317 words

A process for producing a 1,2-dioxetane compound having the formula: in which T is a spiro-bonded stabilizing group that prevents the dioxetane compound from decomposing before the bond between Y and Z is intentionally cleaved, Y is an aromatic, light-emitting fluorophore-forming fluorescent chromophore group capable of absorbing energy to form an excited energy state from which it emits optically detectable energy to return to its original energy state, Z is hydrogen, a chemically cleavable group or an enzyme-cleavable group, and R is a C 1 -C 20 unbranched or branched, substituted or unsubstituted, saturated or unsaturated alkyl group; a heteroalkyl group or aralkyl group; a substituted or unsubstituted polynuclear or heteropolynuclear aralkyl group; a saturated or unsaturated cycloalkyl group; an N, O or S heteroatom-containing group; or an enzyme-cleavable group; with one or more of T, Y, Z or R being substituted with a group that enhances the water solubility of the 1,2-dioxetane or unsubstituted with such a group, which comprises: reacting a nitrile having the formula: T--C.tbd.N wherein T is the spiro-bonded stabilizing group, with an aromatic Grignard reagent having the formula: XMgYOR', wherein X is a halogen and R' is a substituent as defined for R hereinabove, and acidifying the thus-obtained reaction product with an acid HW, wherein W is the acid's anion, to form the corresponding ketimine salt having the formula: ##STR47## reacting the ketimine salt with an aqueous alcoholic acid to form the corresponding ketone having the formula: ##STR48## reacting the ketone with base plus an R-ylating agent to form the corresponding enol ether having the formula: ##STR49## selectively deetherifying the enol ether to form the corresponding monohydroxy enol ether having the formula: ##STR50## converting the hydroxy group in the monohydroxy enol ether to a Z group to form the corresponding enol ether having the formula: ##STR51## and oxygenating the T=C double bond in this enol ether to give the corresponding 1,2-dioxetane compound.

Claim 2

Dependent Claim 317 words

A process for producing a 1,2-dioxetane compound having the formula: in which T is a spiro-bonded stabilizing group that prevents the dioxetane compound from decomposing before the bond between Y and Z is intentionally cleaved, Y is an aromatic, light-emitting fluorophore-forming fluorescent chromophore group capable of absorbing energy to form an excited energy state from which it emits optically detectable energy to return to its original energy state, Z is hydrogen, a chemically cleavable group or an enzyme-cleavable group, and R is a C 1 -C 20 unbranched or branched, substituted or unsubstituted, saturated or unsaturated alkyl group; a heteroalkyl group or aralkyl group; a substituted or unsubstituted polynuclear or heteropolynuclear aralkyl group; a saturated or unsaturated cycloalkyl group; an N, O or S heteroatom-containing group; or an enzyme-cleavable group; with one or more of T, Y, Z or R being substituted with a group that enhances the water solubility of the 1,2-dioxetane or unsubstituted with such a group, which comprises: reacting a nitrile having the formula: T--C.tbd.N wherein T is the spiro-bonded stabilizing group, with an aromatic Grignard reagent having the formula: XMgYOR', wherein X is a halogen and R' is a substituent as defined for R hereinabove, and acidifying the thus-obtained reaction product with an acid HW, wherein W is the acid's anion, to form the corresponding ketimine salt having the formula: ##STR47## reacting the ketimine salt with an aqueous alcoholic acid to form the corresponding ketone having the formula: ##STR48## reacting the ketone with base plus an R-ylating agent to form the corresponding enol ether having the formula: ##STR49## selectively deetherifying the enol ether to form the corresponding monohydroxy enol ether having the formula: ##STR50## converting the hydroxy group in the monohydroxy enol ether to a Z group to form the corresponding enol ether having the formula: ##STR51## and oxygenating the T=C double bond in this enol ether to give the corresponding 1,2-dioxetane compound.

Claim 3

Dependent Claim 24 words

A process as recited in claim 1 wherein the nitrile is reacted with an aromatic Grignard reagent at reflux temperature in an ether solvent.

Claim 4

Dependent Claim 282 words

A process for producing a 1,2-dioxetane compound having the formula: ##STR52## in which T is a spiro-bonded stabilizing group that prevents the dioxetane compound from decomposing before the bond between Y and Z is intentionally cleaved, Y is an aromatic, light-emitting fluorophore-forming fluorescent chromophore group capable of absorbing energy to form an excited energy state from which it emits optically detectable energy to return to its original energy state, Z is hydrogen, a chemically cleavable group or an enzyme-cleavable group, and R is a C 1 -C 20 unbranched or branched, substituted or unsubstituted, saturated or unsaturated alkyl group; a heteroalkyl group or aralkyl group; a substituted or unsubstituted polynuclear or heteropolynuclear aralkyl group; a saturated or unsaturated cycloalkyl group; an N, O or S heteroatom-containing group; or an enzyme-cleavable group, with one or more of T, Y, Z or R being substituted with a group that enhances the water solubility of the 1,2-dioxetane or unsubstituted with such a group, which comprises: reacting an aldehyde having the formula: T--CHO with an aromatic organolithium compound having the formulae: R' O Y Li or XMgYOR' and X is halogen wherein R' is a substituent as defined for R hereinabove, and oxidizing the thus-obtained reaction to form the corresponding ketone having the formula: ##STR53## reacting the ketone with base plus an R-ylating agent to form the corresponding enol ether having the formula: ##STR54## selectively deetherifying the enol ether to form the corresponding monohydroxy enol ether having the formula: ##STR55## converting the hydroxy group in the monohydroxy enol ether to a Z group to form the corresponding enol ether having the formula: ##STR56## and oxygenating the T═C double bond ether to give the corresponding 1,2-dioxetane compound.

Claim 5

Dependent Claim 282 words

A process for producing a 1,2-dioxetane compound having the formula: ##STR52## in which T is a spiro-bonded stabilizing group that prevents the dioxetane compound from decomposing before the bond between Y and Z is intentionally cleaved, Y is an aromatic, light-emitting fluorophore-forming fluorescent chromophore group capable of absorbing energy to form an excited energy state from which it emits optically detectable energy to return to its original energy state, Z is hydrogen, a chemically cleavable group or an enzyme-cleavable group, and R is a C 1 -C 20 unbranched or branched, substituted or unsubstituted, saturated or unsaturated alkyl group; a heteroalkyl group or aralkyl group; a substituted or unsubstituted polynuclear or heteropolynuclear aralkyl group; a saturated or unsaturated cycloalkyl group; an N, O or S heteroatom-containing group; or an enzyme-cleavable group, with one or more of T, Y, Z or R being substituted with a group that enhances the water solubility of the 1,2-dioxetane or unsubstituted with such a group, which comprises: reacting an aldehyde having the formula: T--CHO with an aromatic organolithium compound having the formulae: R' O Y Li or XMgYOR' and X is halogen wherein R' is a substituent as defined for R hereinabove, and oxidizing the thus-obtained reaction to form the corresponding ketone having the formula: ##STR53## reacting the ketone with base plus an R-ylating agent to form the corresponding enol ether having the formula: ##STR54## selectively deetherifying the enol ether to form the corresponding monohydroxy enol ether having the formula: ##STR55## converting the hydroxy group in the monohydroxy enol ether to a Z group to form the corresponding enol ether having the formula: ##STR56## and oxygenating the T═C double bond ether to give the corresponding 1,2-dioxetane compound.

Claim 6

Dependent Claim 33 words

A process as recited in claim 3 wherein the aldehyde is reacted with an aromatic organolithium compound at a temperature of from about -60° C. to about 0° C. in an ether solvent.

Claim 7

Dependent Claim 52 words

A process as recited in either claim 1 or claim 3 wherein the ketone is reacted with an R-ylating agent at a temperature of 0° to 60° C. in a polar aprotic solvent in the presence of an alkali metal hydride or an alkali metal alkoxide to produce the corresponding enol ether.

Claim 8

Dependent Claim 36 words

A process as recited in either claim 1 or claim 3 wherein the enol ether having the formula: ##STR57## is deetherified using sodium thioethoxide in a polar aprotic solvent to produce the corresponding monohydroxy enol ether.

Claim 9

Dependent Claim 107 words

A process as recited in either claim 1 or claim 3 wherein the monohydroxy enol ether is converted to a corresponding alkali metal salt in a polar aprotic solvent, the alkali metal salt is reacted with a phosphorous compound having the formula: ##STR58## wherein X is an electronegative leaving group, in a polar aprotic solvent, the resulting phosphate triester is deprotected using a cyanide salt at a temperature of from 15° to 60° C. to give the corresponding cyanoethyl phosphate diester monosalt, and the monosalt is subjected to beta-elimination in a base at a temperature of 25° to 100° C. to give the corresponding phosphate ester disalt.

Claim 10

Dependent Claim 12 words

A process as recited in claim 7 wherein X is a halogen.

Claim 11

Dependent Claim 12 words

A process as recited in claim 8 wherein the halogen is chlorine.

Claim 12

Dependent Claim 92 words

A process as recited in either claim 1 or claim 3 wherein the monohydroxy enol ether is reacted in a Lewis base with a compound having the formula: ##STR59## wherein X is an electronegative leaving group, in an aprotic solvent, the resulting phosphate triester is deprotected using a cyanide salt at a temperature of from 15° to 60° C. to give the corresponding cyanoethyl phosphate diester monosalt, and the monosalt is subjected to beta-elimination in a base at a temperature of 25° to 100° C. to give the corresponding phosphate ester disalt.

Claim 13

Dependent Claim 12 words

A process as recited in claim 10 wherein X is a halogen

Claim 14

Dependent Claim 12 words

A process as recited in claim 11 wherein the halogen is chlorine.

Claim 15

Dependent Claim 15 words

A process as recited in claim 10 wherein the Lewis base is a tertiary amine.

Claim 16

Dependent Claim 31 words

A process as recited in either claim 1 or claim 3, wherein the double bond in the enol ether having the formula: ##STR60## is photooxygenated to give the corresponding 1,2-dioxetane derivative.

Claim 17

Dependent Claim 27 words

A process as recited in claim 14 wherein photooxygenation is carried out using molecular oxygen, a photosensitizer and light at a temperature at or below 0° C.

Claim 18

Dependent Claim 112 words

A process as recited in either claim 1 or claim 3 wherein the monohydroxy enol ether is reacted with a compound having the formula: ##STR61## wherein X is an electronegative leaving group and R 2 are R 3 each independently cyano, nitrophenyl, dinitrophenyl, arylsulfonyl or alkylsulfonyl, in the presence of a Lewis base in an aprotic organic solvent, to give the corresponding phosphate triester having the formula: ##STR62## and reacting this phosphate triester with a base M + OH - wherein the cation M + is an alkali metal, ammonium, or C 1 -C 7 alkyl, aralkyl, aryl or heterocyclic cation, to give the corresponding phosphate ester disalt having the formula: ##STR63##

Claim 19

Dependent Claim 66 words

A process as recited in either claim 1 or claim 3 wherein the monohydroxy enol ether is reacted with a compound having the formula: ##STR64## wherein X is an electronegative leaving group and R 4 is independently a substituent as defined for R hereinabove, in the presence of a Lewis base in an aprotic organic solvent, to give the corresponding phosphate diester having the formula: ##STR65##

Claim 20

Dependent Claim 31 words

A process as recited in either claim 1 or 3 wherein T is a polycycloalkylidene group, Y is phenylene or naphthylene and the R-ylating agent is dimethyl sulfate or diethyl sulfate.

Claim 21

Dependent Claim 11 words

A process as recited in claim 18 wherein T is adamant-2-ylidene.

Claim 22

Dependent Claim 79 words

A process as recited in either claim 1 or claim 3 wherein the monohydroxy enol ether is converted to a corresponding alkali metal salt in a polar aprotic solvent, the alkali metal salt is reacted with a phosphite compound having the formula: ##STR66## wherein X is an electronegative leaving group, in a polar aprotic solvent, and the resulting phosphite enol ether is oxygenated by molecular oxygen in the presence of light to a 1,2-dioxetane derivative of the formula: ##STR67##

Claim 23

Dependent Claim 12 words

A process as recited in claim 20 wherein X is a halogen.

Claim 24

Dependent Claim 12 words

A process as recited in claim 21 wherein the halogen is chloride.

Claim 25

Dependent Claim 158 words

A process for producing a 1,2-dioxetane compound having the formula: ##STR68## wherein T is a polycycloalkylidene group, R is a C 1 -C 20 unbranched or branched, substituted or unsubstituted, saturated or unsaturated alkyl group; a heteroalkyl group or aralkyl group; a substituted or unsubstituted polynuclear or heteropolynuclear aralkyl group; a saturated or unsaturated cycloalkyl group; an N, O or S heteroatom-containing group; or an enzyme-cleavable group; Y is an aromatic, light-emitting fluorophore-forming fluorescent chromophore group capable of absorbing energy to form an excited energy state from which it emits optically detectable energy to return to its original energy state, and Z is an O-glycosidically linked sugar molecule, which comprises reacting a monohydroxy enol ether having the formula: ##STR69## with a tetra-O-acyl-D-hexopyranosyl halide to give a compound having the formula: ##STR70## hydrolyzing the O-tetraacetate groups in the D-hexopyranoside tetraacetate to give a compound having the formula: ##STR71## and oxygenating the double bond to give the corresponding 1,2-dioxetane compound.

Claim 26

Dependent Claim 158 words

A process for producing a 1,2-dioxetane compound having the formula: ##STR68## wherein T is a polycycloalkylidene group, R is a C 1 -C 20 unbranched or branched, substituted or unsubstituted, saturated or unsaturated alkyl group; a heteroalkyl group or aralkyl group; a substituted or unsubstituted polynuclear or heteropolynuclear aralkyl group; a saturated or unsaturated cycloalkyl group; an N, O or S heteroatom-containing group; or an enzyme-cleavable group; Y is an aromatic, light-emitting fluorophore-forming fluorescent chromophore group capable of absorbing energy to form an excited energy state from which it emits optically detectable energy to return to its original energy state, and Z is an O-glycosidically linked sugar molecule, which comprises reacting a monohydroxy enol ether having the formula: ##STR69## with a tetra-O-acyl-D-hexopyranosyl halide to give a compound having the formula: ##STR70## hydrolyzing the O-tetraacetate groups in the D-hexopyranoside tetraacetate to give a compound having the formula: ##STR71## and oxygenating the double bond to give the corresponding 1,2-dioxetane compound.

Claim 27

Dependent Claim 37 words

A process as recited in claim 23 wherein oxygenation of the double bond is carried out by photooxygenation with molecular oxygen, light and a photosensitizer at a temperature at or below 0° C. in a halogenated solvent.

Claim 28

Dependent Claim 34 words

A process as recited in claim 23 wherein T is adamant-2-ylidene, R is methyl or ethyl, Y is phenylene or naphthylene, and the D-hexopyranoside is α-D-glucopyranoside, β-D-glucopyranoside, α-D-galactopyranoside, β-D-galactopyranoside, β-D-fucopyranoside, β-D-mannopyranoside, α-D-mannopyranoside or β-D-glucopyranosiduronate.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	1993-09-10	1994-03-12	1994-09-11	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	1997-09-10	1998-03-12	1998-09-11	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2001-09-10	2002-03-12	2002-09-11	$7700.00	Window Closed / Lapsed

Synthesis of 1,2-dioxetanes

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