Dioxetane chemiluminescence

The recent studies of 1,2-dioxetane chemiluminescence, ECL, and CIEEL have brought significant advances to the field of chemiluminescence. The relatively simple nature of these processes has allowed attention to be focused on the nature of chemiexcitation and much has been learned. In addition, these relatively simple systems subsequently have been proposed as key intermediates, key steps or key sequences in many more complicated chemi- and bioluminescent systems. 

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. 

Despite the clear implication of the involvement of intramolecular electron transfer in the chemiluminescence of certain dioxetanes, there have been no clear examples of intermolecular electron exchange luminescence processes with dioxetanes. In a recent note, however, Wilson (1979) reports the observation of catalysis of the chemiluminescence of tetramethoxy-1,2-dioxetane by rubrene and, most surprisingly, by 9,10-dicyanoanthracene. While catalysis by the added fluorescers was not kinetically discernible, a lowering of the activation energy for chemiluminescence was observed. These results were interpreted not in terms of an actual electron transfer with the formation of radical ions, but rather in terms of charge transfer interactions between fluorescer and dioxetane in the collision complex. In any event, these results certainly emphasize the need for caution in considering the fluorescer as a passive energy acceptor in dioxetane chemiluminescence. 

While the unimolecular chemiluminescence of dioxetanones appears to fall easily within the framework of conventional dioxetane chemiluminescence, the chemiluminescence of dioxetanones in the presence of certain fluorescers falls resoundingly outside that framework. Adam et al. (1974) noted that the addition of rubrene to solutions of dimethyldioxetanone gave a yield of light twenty times that obtained when an equivalent concentration of 9,10-diphenylanthracene was added. Importantly, the apparent dissimilarity between rubrene and diphenylanthracene is inexplicable by any conventional mechanism of dioxetane decomposition. Also, significantly, Adam et al. (1974) observed an increase in the first-order decay constant of the dioxetanone with the addition of rubrene, an observation for which they did not offer an explanation. Sawaki and Ogata (1977) also observed an unusual dependence of the chemiluminescence yield on the identity of added fluorescer in the base-catalyzed decomposition of or-hydroperoxyesters, for which a dioxetanone intermediate was proposed (25). 

Beck S, Koster H. Applications of dioxetane chemiluminescent probes to molecular biology. Anal Chem 2000 45 2258-70. 

FIGURE 5.2 Dioxetane chemiluminescence reaction pathway. A, AMPPD adamantyl 1,2-dioxetane phosphate) when X = H or CSPD when X = Cl B, dioxetane anion C, adamantanone D, methyl-oxybenzoate anion and E, light emission at 477 nm. 

The activation energy in this case varied from 87 to 93 kJ/mol in different solvents. From the temperature dependence, several competitive reaction paths for this dimethyl-dioxetanone decomposition were deduced, all having a biradical as first intermediate. Heavy-atom effects often play a role in dioxetan chemiluminescence. If DBA is used as fluorescer, the quantum yield is markedly greater than that observed when DPA is used - although the latter has a fluorescence efficiency of 0.89, compared with 0.1 for DBA. In both cases triplet-singlet energy transfer is the origin of the chemiluminescence. 

The chemiluminescence of luminol and the cyclic hydrazides of aromatic and heterocyclic acids is one of the classical and perhaps still most studied of chemiluminescence reactions [1-6]. The mechanism is much more complicated than that of the more recently discovered dioxetans (Chap. V). It is therefore perhaps not surprising that the latter have attracted a far greater amount of research effort in recent times since the excitation step in dioxetan chemiluminescence offers a more acceptable interpretation of experimental results. 

Chemical off—on switching of the chemiluminescence of a 1,2-dioxetane (9-benzyhdene-10-methylacridan-l,2-dioxetane [66762-83-2] (9)) was first described in 1980 (33). No chemiluminescence was observed when excess acetic acid was added to (9) but chemiluminescence was recovered when triethylamine was added. The off—on switching was attributed to reversible protonation of the nitrogen lone pair and modulation of chemically induced electron-exchange luminescence (CIEEL). Base-induced decomposition of a 1,2-dioxetane of 2-phen5l-3-(4 -hydroxyphenyl)-l,4-dioxetane (10) by deprotonation of the phenoHc hydroxy group has also been described (34). 

In addition to ready thermal decomposition, 1,2-dioxetanes are also rapidly decomposed by transition metals (39), amines, and electron-donor olefins (10). However, these catalytic reactions are not chemiluminescent as determined by the temperature drop kinetic method. 

A number of chemiluminescent reactions may proceed through unstable dioxetane intermediates (12,43). For example, the classical chemiluminescent reactions of lophine [484-47-9] (18), lucigenin [2315-97-7] (20), and transannular peroxide decomposition. Classical chemiluminescence from lophine (18), where R = CgH, is derived from its reaction with oxygen in aqueous alkaline dimethyl sulfoxide or by reaction with hydrogen peroxide and a cooxidant such as sodium hypochlorite or potassium ferricyanide (44). The hydroperoxide (19) has been isolated and independentiy emits light in basic ethanol (45). 

Classical chemiluminescence from lucigenin (20) is obtained from its reaction with hydrogen peroxide in water at a pH of about 10 Qc is reported to be about 0.5% based on lucigenin, but 1.6% based on the product A/-methylacridone which is formed in low yield (46). Lucigenin dioxetane (17) has been prepared by singlet oxygen addition to an electron-rich olefin (16) at low temperature (47). Thermal decomposition of (17) gives of 1.6% (47). 

Dioxetane decomposition has also been proposed to account for chemiluminescence from other reactions (43), including gas-phase reactions of singlet oxygen with ethylene and vinyl ethers (53). 

Subsequent studies (63,64) suggested that the nature of the chemical activation process was a one-electron oxidation of the fluorescer by (27) followed by decomposition of the dioxetanedione radical anion to a carbon dioxide radical anion. Back electron transfer to the radical cation of the fluorescer produced the excited state which emitted the luminescence characteristic of the fluorescent state of the emitter. The chemical activation mechanism was patterned after the CIEEL mechanism proposed for dioxetanones and dioxetanes discussed earher (65). Additional support for the CIEEL mechanism, was furnished by demonstration (66) that a linear correlation existed between the singlet excitation energy of the fluorescer and the chemiluminescence intensity which had been shown earher with dimethyl dioxetanone (67). 

The first detailed investigation of the reaction kinetics was reported in 1984 (68). The reaction of bis(pentachlorophenyl) oxalate [1173-75-7] (PCPO) and hydrogen peroxide cataly2ed by sodium saUcylate in chlorobenzene produced chemiluminescence from diphenylamine (DPA) as a simple time—intensity profile from which a chemiluminescence decay rate constant could be determined. These studies demonstrated a first-order dependence for both PCPO and hydrogen peroxide and a zero-order dependence on the fluorescer in accord with an earher study (9). Furthermore, the chemiluminescence quantum efficiencies Qc) are dependent on the ease of oxidation of the fluorescer, an unstable, short-hved intermediate (r = 0.5 /is) serves as the chemical activator, and such a short-hved species "is not consistent with attempts to identify a relatively stable dioxetane as the intermediate" (68). 

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