This invention is in the general field of assay detection systems, such as chemiluminescent detection systems for binding assays. Various reagents and formats are used to detect an event that indicates the presence or amount of an analyte in a mixture. For example, various assays rely on the use of specific analyte binding partners to react with (bind to) the analyte. The binding event is detected in a variety of ways, often by a binding reagent that is labeled in some way with a detectable label. Immunoassays and hybridization assays are just two of the formats that use such labels. These assays can include such diverse labels as radioisotopes, colloidal gold, and enzymatic generation of a light absorbing or emitting compound. One advantage of enzymatic labels is their catalytic function—a single enzyme molecule catalytically generates many molecules of the enzyme product to be detected.
Two classes of activated acridinecarboxylic acid have been previously described: acridinecarbonyl halides and carboxylic acid anhydrides of acridinecarboxylic acid. 9-Acridine percarboxylic acid was first described in 1965 by Rauhut, Sheehan, et al at American Cyanamid (Rauhut, M. M.; Sheehan, D., Clarke, R. A.; Roberts, B. G.; and Semsel, A. M.; Journal of Organic Chemistry, 30, pp. 3587-3592 (1965)) as a product of the reaction between peroxide, pyridine, and 9-acridinecarbonyl chloride. Most of this paper focuses on the chemiluminescence of 9-chlorocarbonyl-10-methylacridinium chloride, but does mention that the above reaction produces chemiluminescence, that acridone was a product of the reaction, and that the chemiluminescent spectrum was consistent with acridone being the emitter. U.S. Pat. Nos. 3,352,791 and 3,539,574 issued based on this research. In U.S. Pat. No. 3,352,791 it is mentioned that 9-acridinecarbonyl chloride was first isolated by Samdahl and Weider (Samdahl and Weider, Bull. Soc. Chim. [5], 2, 2008 (1935)), but was not identified in that work as being chemiluminescent. 9-Acridinecarbonyl chloride was thus the first activated derivative of 9-acridinecarboxylic acid known. However, it is not very stable in water and would not be expected to be a suitable reagent for use in most diagnostic assays. In addition to claiming the acid chloride derivative of 9-acridinecarboxylic acid, the bromide and the fluoride were claimed in these two patents. They also claim carboxylic acid anhydride derivatives of 9-acridinecarboxylic acid. Derivatives of these compounds with substitutions on the acridine rings for all of these compounds are also claimed. Only some of these derivatives (for example, the fluoride and possibly sterically hindered anhydrides) would be expected to be even moderately stable in aqueous media. The main use envisioned for these compounds is as light sources in situations where other light sources might be hazardous. Cyalume light sticks are one product that resulted from this research, but they apparently use derivatives of oxalic acid rather than acridinecarboxylic acid derivatives, presumably because of a higher quantum yield.
In neither of these patents nor the paper covering this research is there any mention of using these compounds as analytical reagents. There is no discussion of whether the chemiluminescent output is proportional to H2O2 concentration. In fact, the peroxide concentrations used to produce the chemiluminescence are generally greater than 50 mM, thus well above the levels required for use in sensitive diagnostic assays. No discussion of their potential use in measuring enzyme activity was included. They do specifically refer to the lack of stability of these derivatives in aqueous media and mention that “if water is added first to the acridine compound, the peroxide should be added reasonably soon thereafter to obtain optimum results.” (Sheehan, D., Clarke, R. A., and Rauhut, M. M., U.S. Pat. No. 3,352,791, column 9, lines 12 to 14). No attention was paid in the bioanalytical community to possible applications of these compounds, perhaps because of anticipated problems with stability in water. Others have examined the chemiluminescence of such compounds (White, Emil H.; Roswell, David F.; Dupont, Andrea C.; Wilson, Alan A., Journal of the American Chemical Society, 109 pp. 5189-5196 (1987)); of the phenyl ester of 9-acridinecarboxylic acid and of various hydrazides of 9-acridinecarboxylic acid (Rapaport, Eliezer; Cass, Malcolm W.; and White, Emil H. Journal of the American Chemical Society, 94 pp. 3153-3159 (1972)).
It is likely that the conditions required for initiating chemiluminescence may have discouraged further examination of these compounds as potential bioanalytical labels. Moreover, the reported quantum yield for many of these derivatives is low. Also, the success of the N-alkylacridinium esters as labels may have overemphasized the importance of substitution on the heterocyclic nitrogen of the acridine ring as a source of chemiluminescent potency. In any case no one has described the use of any of these compounds as reagents for analysis of samples containing peroxide compounds or in enzyme immunoassays or in assays using oligonucleotide probes.
Many papers and over 30 patents have previously referred to the chemiluminescence of the acridinium esters and related compounds (e.g. the sulfonamides) and much effort has been expended to design better labels or ones different enough from the original ester to be patentable. The original work on acridinium esters was done by F. McCapra's group (McCapra, Frank; Richardson, D. G.; and Chang, Y. C., Photochemistry and Photobiology, 4, pp 1111-1121 (1965)), and the American Cyanamid group mentioned earlier. The application of acridinium esters to immunoassays began with the publication of the synthesis and use of an acridinium ester containing an N-oxysuccinimide ester group that facilitated attachment of the acridinium ester to proteins and other biomolecules, especially those with alkylamine groups. Weeks, Ian; Beheshti, Iraj; McCapra, Frank; Campbell, Anthony K.; and Woodhead, J. Stuart, Clinical Chemistry, 29, pp 1474-1479 (1983). The group at the Welsh National School of Medicine received a patent covering acridinium esters as labeling reagents. (Campbell, Anthony K.; Woodhead, J. Stuart; and Weeks, Ian, U.K. Patent 2,112,779 B, (1982 Dec. 8); Campbell, Anthony K.; Woodhead, James S.; and Weeks, Ian, U.S. Pat. No. 4,946,958). Other patents which have issued directed to modified derivatives include those issued to Ciba-Corning (U.S. Pat. Nos. 4,745,181; 4,918,192; 4,927,769; 5,093,270; 5,110,932; 5,227,489; 5,241,070; 5,395,752); Abbott Laboratories (U.S. Pat. Nos. 5,468,646; 5,543,524; 5,565,570); Gen-Probe (U.S. Pat. No. 4,950,613); Mochida Pharmaceutical Co. (U.S. Pat. Nos. 5,438,139; 5,521,103; 5,594,112); Amoco (U.S. Pat. No. 5,155,216); London Diagnostics (U.S. Pat. Nos. 5,281,712; 5,283,334; 5,284,951; 5,284,952; 5,290,936; 5,321,136; 5,338,847); and Nichols Institute (U.S. Pat. No. 5,395,938). Modifications have included including groups to hinder sterically approaches to the ester group (U.S. Pat. No. 4,745,181) and thus to increase stability in aqueous media such as on the phenyl ring ortho to the ester (U.S. Pat. Nos. 4,745,181; 5,284,951) or on the acridine ring(s) peri to the ester (U.S. Pat. No. 5,321,136). Substituent groups have also been added to each of the rings to improve solubility in water (U.S. Pat. Nos. 5,227,489; 5,281,712) to make the leaving group a better leaving group or to increase the rate of attack by peroxide on the ester. Substituents have been added to the acridine ring(s) to improve the luminescent properties of the product acridone. Acridinium esters with different oxyaryl leaving groups with or without substituents have been made and so have derivatives with sulfonamide groups (U.S. Pat. No. 5,468,646) replacing the regular phenoxy leaving group. Various coupling groups have been added to the acridine ring(s), to the leaving group (the aryl or alkyl ester group or the sulfonamide group) (U.S. Pat. Nos. 5,241,070; 5,283,334), or to the alkyl or aryl group quaternizing the heterocyclic nitrogen (U.S. Pat. No. 5,438,139). The heterocyclic acridine nitrogen has also been quaternized with O−, or O-alkyl (Septak, M., J. of Biolum. and Chemilum., 4, pp 351-356 (1989)) Other leaving groups include hydroxamates, eneamides, thiolesters, thioesters, and activated exocyclic arylamides. Different heterocycles replacing the acridinium moiety (such as phenanthridinium and quinolinium) have been claimed. Virtually all of these groups have used acridinecarbonyl chloride derivatives in the synthesis of their modified compounds, but none have taught the use of acridinecarbonyl halides or carboxylic acid anhydride derivatives of acridinecarboxylic acid as peroxide detectors.
Several patents disclose acridan derivatives. Some of these acridans are true acridans with a hydrogen at the 9-position which require oxygen rather than peroxide for initiation of the chemiluminescent reaction or oxidation back to the acridinium form, but others are adducts which revert to the acridinium form with slight changes in condition such as addition of acid or dilution.
None of the above patents discuss the use of acridine derivatives. Some do apparently claim acridine derivatives, but in the form of the acridinium salt with the ring nitrogen protonated and carrying a positive charge (see claim 2 in U.S. Pat. No. 5,155,216, for example). Under the low pH conditions required to protonate the acridine ring nitrogen and give the acridinium form, peroxide would be virtually completely protonated and thus not a particularly good nucleophile and with the regular phenyl (or modified phenyl) ester leaving group the acridinium salt would not be expected to be very chemiluminescent. In fact the standard phenyl ester of acridinecarboxylic acid is not very chemiluminescent under most conditions. In no instance is an acridine compound discussed in examples with experimental results given.
Despite the large effort expended in the prior art to improve acridinium esters, none of these groups/companies has realized (discovered) that the activated acridinecarboxylic acid derivatives disclosed herein are useful chemiluminescent reagents.
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