Heteroaryl substituted benzothiazole dioxetanes

Details Heteroaryl substituted benzothiazole dioxetanes Heteroaryl substituted benzothiazole dioxetanes

2001-09-05

2003-12-09

Ceperley, Mary E. (Department: 1641)

Chemistry: analytical and immunological testing

Optical result

With fluorescence or luminescence

C435S007400, C435S007900, C436S544000, C544S333000, C546S167000, C546S270100, C548S152000, C548S156000, C548S159000

Reexamination Certificate

active

06660529

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to improved chemiluminescent 1,2-dioxetane compounds. More particularly, this invention relates to improved enzymatically cleavable chemiluminescent 1,2-dioxetane compounds that contain enzymatically removable labile groups. Such labile groups prevent the molecule from decomposing to produce light, i.e., visible light or light detectable by appropriate instrumentation, until an appropriate enzyme is added to remove the labile group.
One enzyme molecule can affect the removal, through a catalytic cycle, of its complimentary labile group from thousands of enzymatically cleavable chemiluminescent 1,2-dioxetane molecules. This is a marked contrast to the situation with chemically cleavable chemiluminescent 1,2-dioxetanes, where one molecule of a chemical cleaving agent is needed to remove the complimentary labile group from each dioxetane molecule.
Enzymatically cleavable light-producing, 1,2-dioxetane compounds will usually also contain stabilizing groups, such as an adamantylidene group spiro bonded to the dioxetane ring's 3-carbon atom, that will aid in preventing the dioxetane compound from undergoing spontaneous decomposition at room temperature (about 25° C.) before the bond by which the enzymatically cleavable labile group is attached to the remainder of the molecule is intentionally cleaved. Wieringa. et al.,
Tetahedron Letters,
169 (1972), and McCapra, et al.,
J. Chem. Soc., Chem
. Comm., 944 (1977). These stabilizing groups thus permit such dioxetanes to be stored for exceptionally long periods of time before use, e.g., for from about 12 months to as much as about 12 years at temperatures ranging from about 4° C. to about as much as 30 C., without undergoing substantial decomposition.
This invention further relates to the incorporation of its dioxetane molecules in art-recognized immunoassays, chemical assays and nucleic acid probe assays, and to their use as direct chemical/physical probes for studying the molecular structure or micro structures of various micromolecules, synthetic polymers, proteins, nucleic acids, catalytic antibodies, and the like, to permit an analyte (e.g., a chemical or biological substance whose presence, amount or structure is being determined) to be identified or quantified.
2. Background of the Invention
The use of 1,2-dioxetanes as chemiluminescent compounds is well established. These compounds, for example, have been used as reporters and labels in ultra sensitive assays for the detection of a variety of biological materials. By using 1,2-dioxetanes, these assays can be conducted quickly and without resort to exotic conditions or elaborate apparatus. See, for example, U.S. Pat. Nos. 4,931,223; 4,931,569; 4,952,707; 4,956,477; 4,978,614; 5,032,381; 5,145,772; 5,220,005; 5,225,584; 5,326,882; 5,330,900; 5,336,596; and 5,871,938. All of the foregoing are incorporated herein by reference. Other patents commonly assigned with this application have issued, and other applications are pending. Together, this wealth of patent literature addresses 1,2-dioxetanes stabilized by a typically polycyclic group, such as an adamantyl group spiro-bonded to one of the carbons of the dioxetane ring and another moiety (e.g., an aryl group) bonded to the remainder carbon of the dioxetane ring. This moiety is typically electron sensitive. Deprotection of the electron sensitive moiety results in the formation of an anion, generally an oxyanion, which is unstable and decomposes. Through decomposition, the O—O bond in the dioxetane is broken and a photon is generated. The same carbon atom to which this electron sensitive moiety is bonded may bear an alkoxy or other electron-active group.
The first of the dioxetanes of this class to be commercialized was 3-(4-methoxy-spiro(1,2-dioxetane-3,2′-tricyclo(3.3.1.1
3.7
)decan)-4-yl)phenyl phosphate, particularly the disodium salt, generally known as AMPPD®. This compound has been commercialized by the assignee of this application, Tropix, Inc. of Bedford, Mass., as well as Lumigen, Inc. of Detroit, Mich. Superior performance of the above-described compounds can be obtained by selective substitution on the spiro-bound adamantane ring. For example, substitution at either bridgehead carbon with an electron active species, such as chlorine, has been found to improve reaction speed and signal to noise ratio (S/N). The chlorine substituted counterpart of AMPPD®, available under the trademark CSPD®, has also been widely commercialized by Tropix. “Third-generation” dioxetane compounds of similar structure, wherein the aryl moiety also bears an electron active substituent, such as chlorine, have been found to afford further improvements in performance. The 1,2-dioxetanes having aryl groups bearing phosphate moieties are available under the trademarks CSPD® and CDP-Star®, both of which are registered trademarks of Tropix, Inc.
Various materials have been used to enhance the chemiluminescent emissions of 1,2-dioxetanes. These materials, commonly referred to as chemiluminescent enhancing agents, include polymeric ammonium, phosphonium or sulphonium salts such as poly[vinyl benzyl(benzyldimethyl ammonium chloride)] (“BDMQ”) and other hetero polar polymers.
It has been observed, however, that chemiluminescent dioxetanes such as AMPPD® in aqueous solution and also in the present chemiluminescent enhancers, may exhibit longer than optimum periods of time to reach constant light emission characteristics. The half-life or “t
1/2
” of the active chemiluminescent species is defined as the time necessary to obtain one-half of the maximum chemiluminescence intensity at constant, steady-state light emission levels. This emission half-life can vary as a function of the stability of the dioxetane oxyanion in various environments. For example, the half-life of AMPPD® at concentrations above 2×10
−5
M in an aqueous solution at a pH 9.5 in the presence of BDMQ has been found to be approximately 7.5 minutes. At 4×10
−3
M in the absence of BDMQ, the t
1/2
of AMPPD® has been found to be approximately 30-60 minutes, while at 2×10
−5
M in an aqueous solution, the t
1/2
of AMPPD® has been found to be about 2.5 minutes.
Chemiluminescent intensity is typically measured after achieving steady state light emission kinetics. Statistically, approximately seven t
1/2
periods are required to reach steady-light emission kinetics. While chemiluminescent intensity can be measured before achieving steady state kinetics, sophisticated thermally-controlled luminometry instrumentation must be used if one wishes to acquire precise data prior to achieving steady-state emission kinetics. Therefore, in assays such as bioassays that employ enzymatically cleavable chemiluminescent 1,2-dioxetanes as reporter molecules, it is desirable to reach steady-state light emission kinetics as quickly as possible.
Furthermore, AMPPD®, in an aqueous buffered solution both in the presence and absence of chemiluminescent enhancers such as BDMQ, exhibits higher than desirable thermal and non-enzymatically activated light emission, or “noise.” Such noise can be attributed to emission from the excited state adamantanone and of the methyl m-oxybenzoate anion derived from the aromatic portion of the AMPPD® molecule. The measured noise level of AMPPD® can be as much as two orders of magnitude above the dark current in a standard luminometer. This noise can therefore limit the levels of detection and prevent the realization of ultimate sensitivity in chemiluminescent assays.
Also, various instruments for detecting chemiluminescent emission such as charge coupled device (CCD) cameras have greater detection sensitivities in the green and red wavelengths. AMPPD® and related dioxetanes typically emit in the shorter wavelengths (e.g., blue wavelengths) of the visible spectrum. Heretofore, polymeric enhancers have been used to “shift” the emission wavelength toward the green or red end of the visible spectrum. It would therefore be desirable to obtain dioxetanes wh

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