1.2- Dioxetanes alkaline phosphatase

Fig. 42. Modifications to the adamantyl ring of the dioxetane alkaline phosphatase substrate shown in Fig. 41. Such modified compounds are claimed to give lower background signals in clinical assays (B25,B27).

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). 

Alkaline phosphatase-labeled probes are synthesized so that 18 bases are complementary to sequences on the arms of the bDNA. Three hybridization sites are located on each branch for a total binding capacity of 45 labeled probes per bDNA molecule. The alkaline phosphatase catalyzes the dephosphorylation of chemiluminescent substrate, dioxetane (Lumi-Phos Plus, Lumigen, Detroit, MI). The intensity of the light emission is measured with a plate luminometer as relative luminescent units. 

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). 

Figure 2 illustrates the reaction mechanisms of acridinium ester label probes and alkaline phosphatase probes using dioxetane chemiluminescent detection. Table 2 summarizes approaches for labeling DNA. 

The rational design of effective and efficient dioxetane-based bioanalytical probes requires the in-depth mechanistic understanding of the enzymatically triggered chemiluminescence. In this context, alkaline-phosphatase-triggered CIEEL furnishes a textbook example , which will be examined below in detail. 

A noteworthy feature of the CIEEL phenomenon is the fact that the emission spectrum of the alkaline-phosphatase-triggered dioxetane m-15c coincides with that of the NaOH-triggered, hydroxy-substituted dioxetane m-15e in the absence of enzyme (Figure 4) . 

FIGURE 4. Normalized CIEEL spectmm of the alkaline-phosphatase-triggered ([alkaline phosphatase] = 2 x 10 M) dioxetane m-15c (1.7 x 10 M) in 0.05 M carbonate buffer (pH 9.5), and that of the NaOH-triggered, hydroxy-substituted dioxetane m-15c (3.4 x 10 M, pH 12.7), both at 20 °C. Reprinted with permission from Reference 34b. Copyright (2000) American Chemical Society... 

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. ... 

In the application of alkaline-phosphatase-sensitive, triggerable 1,2-dioxetanes, the nucleic-acid hybridization assay is nowadays quite popular . Such techniques include viral load assays for hepatitis B and C and for human immunodeficiency viruses (HBV,... 

Chemically Initiated Electron Exchange Luminescence (CIEEL) acridinium salts, 1256 alkaline phosphatase, 1193-8 1,2-dioxetanes, 1182-200 firefly bioluminescence, 1191-3 intermolecular, 1213-15, 1231-6 intramolecular, 1214-15, 1236-8 luminol, 1247-8... 

HCV and HIV-1). The bDNA assay53 is being much employed for the quantification of messenger RNA. Moreover, for the detection of viral and pathogenic disorders based on alkaline-phosphatase-sensitive dioxetanes, several assay methods are available these include the Polymerase-Chain-Reaction (PCR) amplification, probe ligation, strand-displacement amplification and the ligase chain reaction53. 

Protection of the phenolic moiety as a phosphate provides substrates such as 58 that are specifically recognized and cleaved by alkaline phosphatase to afford 59 (Scheme 13) <1998PCA5406>. This type of dioxetane is behind many of the commercial assays that are discussed further in Section 2.16.10.1. 

CIEEL is of particular interest for the development of modern chemiluminescent bioassays. The most popular clinical bioassays utilize thermally persistent spiro-adamantyl-substituted dioxetanes with a protected phenolate moiety. These designed 1,2-dioxetanes include an energy source, a fluorophore, and a trigger grouping, and are therefore structurally similar to bioluminescent substrates such as firefly luciferin. Three main commercial dioxetanes 75 are available as one-reagent assays for alkaline phosphatase and are sold under the name of AMPPD (R1 = R2 = H), CSPD (R1 = Cl, R2 = H), and CDP-Star (R1 = R2 = Cl) <2006S1781, 2003ANA279>. These substrates are sensitive to 10 21 mol of alkaline phosphatase in solution. 

Chemiluminescence is the production of light from a chemical reaction. The emitted light is detected with either a luminometer or on photographic film. There are several substrates capable of producing these light emissions and luminol (5-amino-2, 3-dihydro-l, 4-phthalazinedione) and adamantyl 1,2-dioxetane aryl phosphate are two of the most popular.114,115 The former is used with horseradish peroxidase (HRP) coupled antibodies and the latter is used with alkaline phosphatase (AP)-coupled antibodies. Using an AP-cou-pled antibody and adamantyl 1, 2-dioxetane aryl phosphate, Bronstein et al.U5 were able to detect as little as 125 pg of protein in a Western blot. 

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. " ... 

Chemiluminescence assays are ultrasensitive (attomole to zeptomole detection limits) and have wide dynamic ranges. They are now widely used in automated immunoassay and DNA probe assay systems, (e.g., acridinium ester and acri-dinium sulfonamide labels and 1,2-dioxetane substrates for alkaline phosphatase labels and the enhanced-luminol reaction for horseradish peroxidase labels [see Chapter 9]). 

---