Chemistry: analytical and immunological testing – Involving producing or treating antigen or hapten – Producing labeled antigens
Reexamination Certificate
2000-08-21
2002-10-08
Ceperley, Mary E. (Department: 1641)
Chemistry: analytical and immunological testing
Involving producing or treating antigen or hapten
Producing labeled antigens
C530S391300, C530S402000, C530S406000, C549S221000, C556S446000, C568S660000
Reexamination Certificate
active
06461876
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to chemiluminescent compounds. More particularly, the present invention concerns stable, triggerable chemiluminescent 1,2-dioxetanes. Even more particularly, the present invention concerns new chemiluminescent 1,2-dioxetane compounds derived from the oxidation of novel alkenes prepared by the coupling of substituted aromatic esters or ketones and spiro-fused ketones with or without a T-electron system or a carbon-carbon double bond in the ring.
2. Prior Art
Chemiluminescent compounds, their preparation and their uses have been long documented in the prior art. These “high energy” molecules store sufficient energy to generate, or fragmentation, electronically excited carbonyl products which are responsible for the observed chemiluminescence. Dioxetanes and especially, 1,2-dioxitanes and eminently useful to detect the presence, as well as the absence, of certain enzymes in fluids such as blood and the like because of their chemiluminescence. Thus, 1,2 dioxetanes are eminently useful in doing medical assays.
Generally, 1,2-dioxetanes are thermally labile substances having a wide range of stability which decompose on heating and emit light, and correspond to the following formula (1):
Where each R corresponds to any one of a multitude of organic moieties widely reported in the prior art, as detailed herebelow. As noted these 1,2-dioxetanes have a wide range of stability. For example, the prior art, as found in: (a) K. W. Lee, L. A. Singer and K. D. Legg, J.Org.Chem., 41, 2685(1976); (b) F. McCapra, I. Beheshti, A. Burford, R. A. Hanu and K. A. Zaklika, J.Chem.Soc.,Chem.Commun., 944(1977); and (c) J. H. Wieringa, J. Strating, H. Wynberg and W. Adam, Tet. Lett., 169 (1972); respectively, disclose the following 1,2-dioxetanes of different stability:
Although these high energy compounds are all spiro-substituted 1,2-dioxetanes, spiroadamantane substitution exerts a tremendous stabilizing effect on these four-membered ring peroxides. The lower activation energy (EA) of the dioxetanes of formulae (3) and (4) above is explained by the donation of charge from nitrogen to the dioxetane ring. The dioxetane of formula (5) above decomposes at 150° C. and has a half life at 25° C. more than 20 years.
A 1,2-dioxetane (6) below, dispiro[adamantane2,3′-[1,2-]dioxetane-4′,9′-fluorene] was isolated as crystals and described by W. Adam and L. A. A. Encarnacion, Chem. Ber., 115, 2592 (1982).
The stability of 1,2-dioxetanes (5) and (6) was described on the basis of bulky and rigid spiro nature of the adamantane group.
The first stable and enzymatic triggerable 1,2-dioxetane was synthesised by the oxidation of (6-acetoxy-2-naphthyl) methoxy methyleneadamantane as reported by A. P. Schaap, R. S. Handley and B. P. Giri, Tet. lett., 935 (1987). This 1,2-dioxetane utlizes aryl esterase emzyme to catalyze the cleavage of the acetate group of a naphthylacetate-substituted-1,2-dioxetane and produce chemiluminescence in aqueous buffers at ambient temperature by the following sequence:
Several other stabilized 1,2-dioxetanes and their use as enzyme substrates have been disclosed in the literature. See, inter alia, A. P. Schaap, T. S. Chen, R. S. Handley, R. DeSilva and B. P. Giri, Tet. Lett., 1155(1987); A. P. Schaap, M. D. Sandison and R. S. Handley, Tet. Lett., 1159 (1987); U.S. Pat. Nos. 4,962,192; 4,978,614; 5,386,017; 5,721,370, the disclosures of which are hereby incorporated by reference.
These several other 1,2-dioxetanes, generally, have the following general structures:
wherein
is a non-active site and which is selected from the group of polycyclic alkyl groups containing 6 to 30 carbon atoms, OX is an oxy group substituted on an aryl ring which forms an unstable oxide intermediate 1,2-dioxetane compound when triggered to remove X by an activating agent selected from the group consisting of an acid, a base, a salt, an enzyme and an inorganic or organic catalyst, and electron donor source, and X is a chemically labile group which is removed by the activating agents to form light and carbonyl containing compounds, R
1
is a lower alkyl containing 1 to 8 carbon atoms, or mixtures thereof, or
where T is a non-active site which is a cycloalkyl or a polycycloalkyl group bonded to the 4-membered ring portion of the dioxetane by a spiro linkage; Y is a fluorescent chromophore; X is a hydrogen, alkyl, aryl arylkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl, or enzyme cleavable group; and Z is hydrogen or an enzyme cleavable group, provided that at least one of X or Z must be an enzyme cleavable group.
The enzyme cleavable 1,2-dioxetanes of formulae (14), (15) and (16) shown below have been commercialized and used in immuno assays, southern blotting, northern blotting, western blotting, plaque/colony lifts and DNA sequencing.
The 1,2-dioxetane of formula (15) with the chloro substitution in the adamantane ring demonstrates better results in DNA sequencing when compared to dioxetane (14). Dioxetane (16) is more soluble in an aqueous system than 1,2-dioxetane (14) and (15) when a CH
3
group is replaced with a CH
2
CH
2
CH
2
COOH.
Other relevant prior art can be found in U.S. Pat. Nos. 5,386,017; 4,962,192; 5,018,827; 5,578,253; 5,004,565; 5,068,339, the disclosures of which are hereby incorporated by reference.
While these prior art compounds provide enzyme cleavable 1,2-dioxetanes, it has been observed that in an aqueous buffer, the luminescence of these molecules is particularity poor, especially when trace amounts of biological materials are sought to be detected. Thus more powerful dioxetanes are needed i.e. dioxetanes having higher levels of chemiluminescence in an aqueous buffer.
SUMMARY OF THE INVENTION
The present invention provides novel 1,2-dioxetanes derived from spiro-fused ketones with or without &pgr;-electrons in the ring or with carbon-carbon double bond(s) in the spiro-fused ring. Additionally, these new dioxetanes have electron donating or withdrawing groups at the four-membered peroxide ring to render these dioxetanes active at all sites.
The 1,2-dioxetanes hereof generally correspond to the formula:
wherein
(1) when Ar—O—Y and OR join together to give an aryl group substituted with an X-oxy group to form a stable 1,2-dioxetane intermediate which is triggerable to form an unstable intermediate oxide, R
2
and R
3
either form
which is either a cyclic, polycyclic or spiro-fused ring containing at least one carbon-carbon double bond or carbon-carbon triple bond in the ring or side chain with or without heteroatoms or
which is a cyclic, polycyclic or spiro-fused ring containing substituted or unsubstituted fused aromatic ring or substituted or unsubstituted aromatic ring attached by linker arms; or
(2) when Ar—O—Y and OR, do not join together
(a) Ar is aryl and may be phenyl, substituted phenyl, naphthyl, substituted naphthyl, anthryl, substituted anthryl or other aromatic or nonaromatic fluorescent or nonfluorescent group; Y is a hydrogen, alkyl, acetate, t-butyldimethylsilyl or an enzyme cleaveable group, an antibody cleaveable group; R
1
is selected from the group consisting of alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl, alkyletheralkyl, alkyletheraryl, alkyl(etheralkyl)
2
, alkyl(etheralkyl)
3
, alkyletherhaloalkyl, alkyl(etherhaloalkyl)
2
, alkylalkene, alkylalkyne, arylalkene, arylalkyne, halogenated alkyl(mono, di, tri or any position in normal or branched or cyclic chain), alkylalcohol, alkylnitrile, alkylamine, alkylacid (mono or dibasic) or the inorganic salts thereof, haloalkylalcohol, haloalkylnitrile, haloalkylamine, haloalkylacid (mono or dibasic) or inorganic salts, linker-flourescent molecule, linker-antibodies, linker-antigen, linker-biotin, linker-avidin, linker-protein or linker-carbohydrates or linker-lipids; when R
2
and R
3
forms either
which is a cyclic, polycyclic or spiro-fused ring containing at least one carbon-carbon double bond or cabon-carbon triple bo
Ceperley Mary E.
Plunkett & Cooney PC
Weintraub Arnold S.
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