Abstract: Spiroadamantyl dioxetanes bearing an alkoxy substituent, and an aromatic substituent of phenyl or naphthyl on the dioxetane ring can be activated to chemiluminesce if the aromatic substituent bears a moiety designated OX, wherein the X is cleaved by an enzyme with which the dioxetane is permitted to come in contact with. The T.sub. 1/2 kinetics of the chemiluminescent reaction, as well as the signal intensity, or quantum yield of the chemiluminescent reaction, can be altered by selection of an electron-withdrawing or an electron-donating group Z, at positions on the aromatic substituent other than those adjacent the point of attachment to the dioxetane. Signal strength can further be enhanced by recognized chemiluminescent enhancers.

Abstract: Methods are disclosed for purifying chemiluminescent water-soluble 1,2-dioxetane derivatives suitable for use as reporter molecules in a variety of biological analytical systems, including enzyme-linked immunoassays, nucleic acid probe techniques, and structural determinations. The methods are based upon high pressure, medium pressure or low pressure liquid chromatography using as the stationary phase alkaline pH-stable compositions with the chromatographic characteristics of reversed-phase adsorbents, at alkaline pH values, and in the absence of acid-forming compounds or compounds with an unshared pair of electrons. Desalting of substantially pure water soluble 1,2-dioxetane derivatives may be accomplished by the same chromatographic systems or by molecular sieve chromatography systems, but in the absence of salt buffers. Under some circumstances, purification and desalting may be combined in a single chromatographic step.

Abstract: Hydroxyaryl enol ether alkali metal salts having the formula: ##STR1## in which T is a fused, substituted or unsubstituted polycycloalkylidene group, OR.sup.3 is an ether group Y is a light-emitting fluorophore-forming group which will be part of a luminescent substance formed by decomposition of a 1,2-dioxetane subsequently formed from the hydroxyaryl enol ether alkali metal salt, capable of absorbing energy to form an excited state from which it emits optically detectable energy to return to its ground state, and AM.sup.30 is an alkali metal cation, processes for the preparation of these intermediate salts, and their use as starting materials for acylation, phosphorylation and glycosylation reactions to give intermediates reactable to give stable, water-soluble chemiluminescent 1,2-dioketames, particularly ones that are enzymatically cleavable, are disclosed.

Abstract: Methods are disclosed for purifying chemiluminescent water-soluble 1,2-dioxetane derivatives suitable for use as reporter molecules in a variety of biological analytical systems, including enzyme-linked immunoassays, nucleic acid probe techniques, and structural determinations. The methods are based upon high pressure, medium pressure or low pressure liquid chromatography using as the stationary phase alkaline pH-stable compositions with the chromatographic characteristics of reversed-phase adsorbents, at alkaline pH values, and in the absence of acid-forming compounds or compounds with an unshared pair of electrons. Desalting of substantially pure water soluble 1,2-dioxetane derivatives may be accomplished by the same chromatographic systems or by molecular sieve chromatography systems, but in the absence of salt buffers. Under some circumstances, purification and desalting may be combined in a single chromatographic step.

Abstract: A new and improved polymeric membrane for use in biological assays is provided. A blotting assay employing 1,2-dioxetanes as a source of chemiluminescence employs, as an improved membrane, a polymer comprised of at least one monomer of the formula: ##STR1## The membranes reduce background signal, improve sensitivity and reliability.

Abstract: Enzymatically cleavable chemiluminescent 1,2-dioxetane compounds capable of producing light energy when decomposed, substantially stable at room temperature before a bond by which an enzymatically cleavable labile substituent thereof is intentionally cleaved, are disclosed. These compounds can be represented by the formula: ##STR1## wherein: X and X.sup.1 each represent, individually, hydrogen, a hydroxyl group, a halo substituent, an unsubstituted lower alkyl group, a hydroxy (lower) alkyl group, a halo (lower) alkyl group, a phenyl group, a halophenyl group, an alkoxyphenyl group, a hydroxyalkoxy group, a cyano group or an amide group, with at least one of X and X.sup.1 being other than hydrogen; andR.sub.1 and R.sub.2, individually or together, represent an organic substituent that does not interfere with the production of light when the dioxetane compound is enzymatically cleaved and that satisfies the valence of the dioxetane compound's 4-carbon atom, with the provisos that if R.sub.1 and R.sub.

Abstract: Enzymatically cleavable chemiluminescent 1,2-dioxetane compounds capable of producing light energy when decomposed, substantially stable at room temperature before a bond by which an enzymatically cleavable labile substituent thereof is intentionally cleaved, are disclosed. These compounds can be represented by the formula: ##STR1## The corresponding dioxetanes which, instead of being substituted at the 5' or 7', or at the 5' and 7' positions, instead contain a 4' methylene group, are also disclosed, as are intermediates for all these 3-substituted adamant-2'-ylidenedioxetanes, and their use as reporter molecules in assays.

Abstract: Compounds having the formula: ##STR1## wherein T is a polycycloalkylidene group (e.g., adamant-2-ylidene); R is a C.sub.1-20 alkyl, aralkyl or cycloalkyl group; and Y is a fluorescent chromophore (e.g., m-phenylene), produced by reacting a compound having the formula: ##STR2## with an R-ylating agent (e.g., R.sub.2 SO.sub.4) in the presence of an alkali metal alkoxide in a polar aprotic solvent. Also, compounds having the formula: ##STR3## are produced by reacting a compound having the formula: ##STR4## wherein X is an electronegative leaving group (e.g., a halogen anion such as chloride ion) in the presence of a Lewis base (e.g., a trialkyl-amine) dissolved in an aprotic organic solvent (e.g., benzene or toluene). Also, compounds having the formula ##STR5## are produced by reacting a compound of the formula ##STR6## with a tetra-O-acylated-O-hexopyranoside halide, then hydrolyzing off the protective acyl groups.

Abstract: Water soluble naturally-occurring and synthetic enhancer substances, generally macromolecular in nature, for example globular proteins that include hydrophobic regions such as bovine serum albumin, and polymeric quaternary ammonium salts such as poly(vinylbenzyltrimethylammonium chloride), which have the ability to inhibit light-emitting fluorophores resulting from the decomposition of chemiluminescent compounds from releasing energy through non-light emitting pathways, are disclosed as permitting the stabilization, and hence increasing the light intensity, of such light-emitting fluorophores in aqueous media as compared to the intensity of the light emitted by the same quantities of such fluorophores in aqueous media in the absence of such enhancer substances. Any chemiluminescent enzymatically cleavable 1,2-dioxetane, for example 3-(2'-spiroadamantane)-4-methoxy-(3"-phosphoryloxy)phenyl-1,2-dioxetane disodium salt, can be used.

Abstract: Enzymatically cleavable chemiluminescent 1,2-dioxetane compounds capable of producing light energy when decomposed, substantially stable at room temperature before a bond by which an enzymatically cleavable labile substituent thereof is intentionally cleaved, are disclosed. These compounds can be represented by the formula: ##STR1## wherein: X and X.sup.1 each represent, individually, hydrogen, a hydroxyl group, a halo substituent, an unsubstituted lower alkyl group, a hydroxy (lower) alkyl group, a halo (lower) alkyl group, a phenyl group, a halophenyl group, an alkoxyphenyl group, a hydroxyalkoxy group, a cyano group or an amide group, with at least one of X and X.sup.1 being other than hydrogen; andR.sub.1 and R.sub.2, individually or together, represent an organic substituent that does not interfere with the production of light when the dioxetane compound is enzymatically cleaved and that satisfies the valence of the dioxetane compound's 4-carbon atom, with the provisos that if R.sub.1 and R.sub.

Abstract: A dioxetane that includes a fluorescent chromophore spiro-bound at the 4-carbon of the dioxetane. The dioxetane has the formula ##STR1## where X is CR.sub.7 R.sub.8, O, S, or N-R (where each R.sub.7, R.sub.8, and R, independently, is H, alkyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, cycloheteroalkyl, aralkyl, alkaryl, or an enzyme cleavable group), and each R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, andR.sub.6, independently, is H, an electron-withdrawing group, an electron-donating group, heteroaryl, or an enzyme cleavable group, or groups R.sub.1 -R.sub.6 together form a ring, and T is a substituted or unsubstituted aryl, polyaryl, cycloalkylidene or polycycloalkylidene group spiro-bound at the 3-carbon of the dioxetane.The dioxetane can be decomposed by direct cleavage of the dioxetane 0--0 bond or by cleavage of an enzyme cleavable group bonded to the dioxetane to form a luminescent substance that includes the coumarin portion of the dioxetane.

Abstract: Compounds having the formula: ##STR1## wherein T is a polycycloalkylidene group (e.g., adamant-2-ylidene); R is a C.sub.1-20 alkyl, aralkyl or cycloalkyl group; and Y is a fluorescent chromophore (e.g., m-phenylene), produced by reacting a compound having the formula: ##STR2## with an R-ylating agent (e.g., R.sub.2 SO.sub.4) in the presence of an alkali metal alkoxide in a polar aprotic solvent. Also, compounds having the formula: ##STR3## are produced by reacting a compound having the formula: ##STR4## wherein X is an electronegative leaving group (e.g., a halogen anion such as chloride ion) in the presence of a Lewis base (e.g., a trialkyl-amine) dissolved in an aprotic organic solvent (e.g., benzene or toluene). Also, compounds having the formula ##STR5## are produced by reacting a compound of the formula ##STR6## with a tetra-O-acylated-O-hexopyranoside halide, then hydrolyzing off the protective acyl groups.

Abstract: Chemiluminescent 1,2-dioxetane compounds are disclosed in which the molecule is stabilized at the 3-position on the dioxetane ring against decomposition prior to the molecule's coming in contact with a labile group-removing substance (e.g., an enzyme that will cleave the labile group to cause the molecule to decompose to form at least one light-emitting fluorophore) and substituted at the 4-position on the dioxetane ring with a fused polycyclic ring-containing fluorophore moiety bearing a labile ring substituent whose point of attachment to the fused polycyclic ring, in relation to this ring's point(s) of attachment to the dioxetane ring, is such that the total number of ring atoms separating these points of attachment, including the ring atoms at the points of attachment, is an odd whole number. These odd pattern substituted compounds decompose to emit light of greater intensity and of a different wavelength than that emitted by the corresponding even pattern substituted isomers.

Abstract: Methods are disclosed for purifying chemiluminescent water-soluble 1,2-dioxetane derivatives suitable for use as reporter molecules in a variety of biological analytical systems, including enzyme-linked immunoassays, nucleic acid probe techniques, and structural determinations. The methods are based upon high pressure, medium pressure or low pressure liquid chromatography using as the stationary phase alkaline pH-stable compositions with the chromatographic characteristics of reversed-phase adsorbents, at alkaline pH values, and in the absence of acid-forming compounds or compounds with an unshared pair of electrons. Desalting of substantially pure water soluble 1,2-dioxetane derivatives may be accomplished by the same chromatographic systems or by molecular sieve chromatography systems, but in the absence of salt buffers. Under some circumstances, purification and desalting may be combined in a single chromatographic step.

Abstract: A method for acylating aminoanthraquinone dyes which comprises reducing the dye to the leuco form, reacting the leuco dye with an acylating agent and oxidizing the acylated leuco compound to form the desired acylated dyes. In a preferred embodiment bis 1,4-aminoanthraquinone dyes and bis 1,5,aminoanthraquinones are formylated.