Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2000-07-27
2004-08-31
Gitomer, Ralph (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C546S102000
Reexamination Certificate
active
06783948
ABSTRACT:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
N/A
FIELD OF THE INVENTION
This invention relates to novel chemiluminescent compounds that are substrates of hydrolytic enzymes, the chemiluminescent products of which have distinctly different light emission characteristics (i.e. emission wavelength, kinetics, or quantum yield). This invention also relates to a light-releasing reagent composition that reacts preferentially with the chemiluminescent substrate or with the chemiluminescent product in the mixture of the two, to generate a discernible signal that can be quantified. This invention further relates to detection methods comprising a novel acridinium-based chemiluminescent substrate, a hydrolytic enzyme and a signal-releasing reagent. This invention furthermore relates to detection devices in conjunction with the use of novel chemiluminescent substrates and hydrolytic enzymes, which include a red-sensitive photomultiplier tube or a charge-coupled device, and a light-filtering device to maximize the detection of chemiluminescent product signal. Further, this invention relates to the use of these novel chemiluminescent substrates in assays to detect, quantitatively or qualitatively, a hydrolytic enzyme of interest that is present either as a label or as a marker of a biological sample. Finally, this invention relates to the process and intermediates for the preparation of these novel chemiluminescent substrates.
BACKGROUND OF THE INVENTION
The detection of hydrolytic enzymes has been extensively used in diagnostic assays ranging from immunoassays, nucleic acid assays, receptor assays, and other assays, primarily due to their high sensitivity and non-radioactivity. The hydrolytic enzymes include phosphatases, glycosidases, peptidases, proteases, and esterases. By far, most commonly used are phosphatases and glycosidases. For instance, alkaline phosphatase has been extensively used as a label in various enzyme-linked immunosorbent assays (ELISAs) due to its high turn-over rate, excellent thermal stability and ease of use. Many glycosidases, such as &bgr;-galactosidase and &bgr;-glucuronidase, have also been used in ELISA due to their very high selectivity for the hydrolysis of their preferred substrates. On the other hand, some hydrolytic enzymes play important functions by themselves in biological processes of the human body and microorganisms. Therefore, direct detection of these markers is another important aspect of diagnostics.
In connection with the detection of hydrolytic enzymes, there are three types of substrates: chromogenic, fluorogenic and chemiluminescent substrates. Among them, chemilumi-nescent substrates offer the best enzyme detection sensitivity due to the intrinsic advantages of higher detectability of chemiluminescent product, or lower substrate and instrumental backgrounds, and less interference from biological samples. Therefore, there has been a steady trend towards developing chemiluminescent substrates and applying them in a variety of diagnostics.
Stable Dioxetanes
One class of widely used chemiluminescent substrates for hydrolytic enzymes are stable dioxetanes (Bronstein et al, U.S. Pat. Nos. 4,931,223, 5,112,960, 5,145,772, 5,326,882; Schaap et al, U.S. Pat. Nos. 5,892,064, 4,959,182, 5,004,565; Akhavan-Tafti et al, U.S. Pat. No. 5,721,370). Here, the thermally stable protective group on the phenolic moiety of the dioxetane substrates is cleaved by a hydrolytic enzyme of interest, such as alkaline phosphatase (AP) or &bgr;-galactosidase, depending on whether the protective group is a phosphoryl or &bgr;-D-galactopyranosidyl group. The newly generated dioxetane phenoxide anion undergoes auto-decomposition to a methoxycarbonylphenoxide in an electronically excited state. The latter then emits light at &lgr;max ~470 nm.
In an aqueous environment where virtually all biological assays are performed, the decomposition of the dioxetanes produces chemiluminescence in a very low quantum yield, typically about 0.01%, and a slow kinetics with t
1/2
1~10 minutes. This is quite different from the decomposition of the dioxetanes in an organic environment. For instance, the dioxetane having the phenol moiety protected by an acetyl group or a silyl group, upon treatment with a base or fluoride, exhibits quantum yield up to 25% in DMSO and 9.4% in acetonitrile, respectively, and t
1/2
is about 5 sec at 25° C. (Schaap, et al: Tetrahedron Letters, 28 (11), 1155, 1987, and WO 90/07511 A).
Voyta et al (U.S. Pat. No. 5,145,772) disclosed a method of intermolecular enhancement of quantum yield of the dioxetane products using polymeric ammonium salts, which provide a hydrophobic environment for the phenoxide produced by the enzyme.
Akhavan-Tafti et al reported methods of intermolecular enhancement of quantum yield of the dioxetane products using polymeric phosphonium salts (U.S. Pat. No. 5,393,469) and dicationic surfactants (U.S. Pat. Nos. 5,451,347, 5,484,556).
Schaap et al (U.S. Pat. Nos. 4,959,182, 5,004,565) disclosed another method for increasing quantum yield of the dioxetane products using fluorescent co-surfactants as energy acceptors. The resonance energy embodied in the excited phenoxide produced by the enzyme is effectively transferred to the fluorescent co-surfactants. Instead of emitting light at &lgr;max 470 nm characteristic of the dioxetane, this system emits light at &lgr;max 530 nm as a result of energy transfer to the highly efficient fluorophore, fluorescein.
Another approach for improving quantum yield of the dioxetanes, disclosed by Schaap in U.S. Pat. No. 5,013,827, is to covalently attach a fluorophore having high quantum yield to the light emitting phenoxide moiety. The resonance energy from the excited phenoxide is intramolecularly transferred to the attached fluorophore. The latter in turn emits light at its own wavelength. It is claimed that such dioxetane-fluorophore conjugates exhibit quantum yield as high as 2%.
Wang et al in WO 94/10258 unveiled a class of electron-rich, aryl-substituted dioxetane compounds in which the aryl group is poly-substituted with a suitable electron-donating group so that intense luminescence is observed.
Akhavan-Tafti et al in U.S. Pat. No. 5,721,370 provided a group of stable chemiluminescent dioxetane compounds with improved water solubility and storage stability. The compounds are substituted with two or more hydrophilic groups disposed on the dioxetane structure and an additional fluorine atom or lower alkyl group.
Schaap et al in U.S. Pat. No. 5,892,064 disclosed a class of chemiluminescent dioxetane compounds substituted on the dioxetane ring with two nonspirofused alkyl groups.
Urdea at al, in EP Application 0401001 A2, described another sub-class of dioxetane compounds that can be triggered by sequential treatment with two different activating enzymes to generate light. The system rests on the principle that the dioxetane substrates have two protecting groups that can are removed sequentially by different processes to produce an excited phenoxide, and the removal of the first protecting group is triggered by the enzyme used as a label in the assay.
Luminol Substrates
Sasamoto at al [Chem. Pharm. Bull., 38(5), 1323 (1990) and Chem. Pharm. Bull., 39(2), 411 (1991)] reported that o-aminophthalhydrazide-N-acetyl-&bgr;-D-glucosaminide (Luminol-NAG) and 4′-(6′-diethylaminobenzofuranyl)-phthalhydrazide-N-acetyl-&bgr;-D-glucosaminide, both being the non-luminescent forms of luminol, are substrates of N-acetyl-&bgr;-D-glucosaminidase. Upon the action of the enzyme on these substrates, luminol or luminol derivative is generated, which then can be detected by triggering with 0.1% hydrogen peroxide and a peroxidase (POD) or no Fe(III)-TCPP complex catalyst to release a chemiluminescent signal.
Enzyme-modulated Protected Enhancer and Anti-enhancer
U.S. Pat. No. 5,306,621 to Kricka disclosed that light intensity of certain peroxidase-catalyzed chemiluminescent reactions can be modulated by AP that acts on a pro-enhancer or a pro-anti-enhancer. For example, the intensity of a ch
Jiang Qingping
Law Say-Jong
Natrajan Anand
Sharpe David J.
Wong Wen-Jee
Bayer Corporation
Gitomer Ralph
Weingarten Schurgin, Gagnebin & Lebovici LLP
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