Enzymes dioxetane chemiluminescence

The bioluminescence of the American firefly (Photinus pyralis) is certainly the best-known bioluminescent reaction, thanks to the work of Me Elroy and coworkers and E. H. White and his group (for references see P, p. 138, 6,168,169)) The substrate of this enzyme-catalyzed chemiluminescent oxidation is the benzothiazole derivative 107 (Photinus luciferin) which yields the ketone 109 in a decarboxylation reaction. The concept of a concerted cleavage of a dioxetane derivative has been applied to this reaction 170> (see Section II. C.). Recent experiments with 18C>2 have challenged this concept, as no 180-containing carbon dioxide was detected from the oxidation of 107 171>. 

Recently, two major enzyme-catalyzed chemiluminescent reactions have become popular. These use either luminol as a substrate of peroxidase or 3-(2 -spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD) as a substrate of alkaline phosphatase (ALP). 

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

A CL ISH assay for the detection of human papillomavirus (HPV) DNA was developed, in which the hybridization reaction was performed using either digoxigenin-, biotin-, or fluorescein-labeled probes [64], The hybrids were visualized using AP as the enzyme label and a highly sensitive 1,2-dioxetane phosphate as chemiluminescent substrate. This assay was applied to biopsy specimens from different pathologies associated with HPV, which had previously proved positive for HPV DNA by polymerase chain reaction (PCR). The analytical sensitivity was assessed using samples of HeLa and CaSki cell lines, whose content in HPV DNA is known (10-50 copies of HPV 18 DNA in HeLa cells and 400-600 copies... 

Lipid hydroperoxides are also generated in singlet molecular oxygen mediated oxidations and by the action of enzymes such as lipoxygenases and cyclooxygenases. Chemiluminescence (CL) arising from lipid peroxidation has been used as a sensitive detector of oxidative stress both in vitro and in vivo . Several authors have attributed ultra-weak CL associated with lipid peroxidation to the radiative deactivation of O2 and to triplet-excited carbonyls (63, 72) (equations 35 and 36) " . It has been proposed that the latter emitters arise from the thermolysis of dioxetane intermediates (61, 62) (equation 35), endoperoxide (73) (equation 37) and annihilation of aUtoxyl, as well as peroxyl radicals ... 

Dioxetanes, labeled with triggers sensitive to the alkaline-phosphatase enzyme, serve as highly sensitive chemiluminescent probes in numerous bioassays. Current applications include immunoassays, membrane-based detection of proteins and nucleic acids, and microplate-based and array-based nucleic-acid detection. ... 

There is a high interest to know the structural features which endow the thermal stability of dioxetanes since this property makes the dioxetane easy to handle and, if accompanied with high chemiluminescence, usable as enzyme label in bioanalytical, clinical application [111]. In this field,... 

Another chemiluminescent enzyme system is based on the use of stabilized dioxetane substrates. Dio-xetanes are intermediates in many chemiluminescent reactions. It s possible to synthesize stabilized dio-xetanes (phosphatase and p-galactose moieties) that do not spontaneously react. When exposed to the right enzyme (alkaline phosphatase and p-galactosidase, respectively) the dioxetane will be destabilized and spontaneously undergo a chemiluminescence reaction. " ... 

Signal antibodies have most often been radiolabeled (for immunoradiometric assays [IRMA]) with ( 25j) 7,62,i%,344,4i4 labeled with a chemiluminescent (for immunochemilummo-metric assays [ICMA]) compound, such as acridinium ester, or an enzyme (enzyme-linked immunosorbent assay [ELISA] or enzyme immunoassay [EIA]), such as ALP, converting a substrate (1,2-dioxetane phosphate) to a chemiluminescent product. 

Various 1,2-dioxetane derivatives have been synthesized and reported. Among them, AMPPD (Adamantyl Methoxy Phenyl Phosphoryl Dioxetane) is a well known dioxetane bearing a spiroadamantyl group at the 3-position and a phenol phosphate at the 4-position, which is now used for chemiluminescence enzyme immunoassay (CLEIA) using alkaline phosphatase with highly sensitive detection. 

Using the substrate of 1,2-dioxetane derivative in combination with an enhancer, we applied a chemiluminescence enzyme immunoassay (CLEIA) for TSH (Thyroid Stimulating Hormone). 

We used an anti-TSH monoclonal antibody bound magnetizable microparticles as solid phase and alkaline phosphatase-labelled anti-TSH monoclonal antibody as a tracer. After 6 min incubation of solid phase, a tracer and sample or calibrator (30 fiL), microparticles were washed to remove unbound materials and were then incubated for 5 min with 50 /iL of chemiluminescence substrate, new 1,2-dioxetane derivative. The amount of enzyme-labeled monoclonal antibody that binds to the microparticles is directly proportional to the TSH concentration in the test sample. 

Bronstein I, Enhanced chemiluminescence form 1,2-dioxetane enzyme substrates membrane effects, macromolecular enhancement, and structure modification. The Spectrum 7,1994 2 10-5. 

Upon an extensive study, we found a new chemiluminescence substrate for the enzyme iimnunoassay using a new 1,2-dioxetane derivative, 5-t-butyl-4,4-dimethyl-l-(3 -phosphoryloxy)phenyl-2,6,7-trioxabicyclo[3.2.0]heptane disodium salt as shown in Scheme l. °... 

Dioxetanes are beginning to have a tremendous impact on chemiluminescent methodologies for clinically relevant analytes. This impact is due to the development of dioxetanes as enzyme substrates in enzyme-amplified assays similar to the type described in Section 1.3.2. The evolution of the current generation of highly efficient substrates merits some comment. Schaap and co-workers (S8) showed that the naphthalene silyl ether shown in Fig. 40a could be triggered by fluoride ions to give chemiluminescence with a quantum yield of 0.01. On the other hand, the /nefa-disubstituted phenyl derivative in Fig. 40b is extremely efficient in a CIEEL chemiluminescence process (for reasons outlined in the previous section) with a quantum efficiency of 0.45. Subsequent studies showed that the naphthalene derivative in Fig. 40a with an 0-acetyl group instead of the silyl ether, could... 

One major advantage of the glow-type emission obtained from dioxetane enzyme substrates is that one can easily record the emission on photographic film (in much the same way that emission is recorded) to obtain qualitative or semi-quantitative data. This technique has been described for a dry-format membrane-based assay for human luteinizing hormone (hLH), in which Polaroid 612 film (ASA 20,000) was used to record the intensity from unknown samples and from a set of standards (B18). A dioxetane-based ELISA method for human growth hormone (somatotropin) has been recently described. The chemiluminescent assay was reported to be superior to a similar fluorescence assay, and the detection limit for hGH was found to be 5 pM (A9). 

Albrecht, S., Ehle, H., Schollberg, K., Bublitz, R., and Horn, A., Chemiluminescent enzyme immunoassay of human growth hormone based on adamantyl dioxetane phenyl phosphate substrate. In Bioluminescence and Chemiluminescence Current Status (P. E. Stanley and L. J. Kricka, eds.), pp. 115-118. Wiley, Chichester, 1991. 

Brestel, E. P., Co-oxidation of luminol by hypochlorite and hydrogen peroxide. Implications for neutrophil chemiluminescence. Biochem. Biophys. Res. Commun. 126, 482-488 (1985). Brolin, S., and Wetteimark, G., Bioluminescence Analysis. VCH Verlag, Weinheim, 1992. Bronstein, L, Chemiluminescent 1,2-dioxetane-based enzyme substrates and their applications. In Luminescence Immunoassay and Molecular Applications (K. Van Dyke and R. Van Dyke, eds.), pp. 255-274. CRC Press, Boca Raton, FL, 1990. 

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