1.2- Dioxetanones mechanism

The carbonyl compound (43) has also been synthesi2ed, and its fluorescence spectmm has been shown to match the bioluminescence spectmm under equivalent conditions (214). The details of the excitation step are unclear and a dioxetanone mechanism (59,215) may apply to the reaction. 

FMNH2 requirement in bacterial luminescence Crystallization of Cypridina luciferin Crystallization of firefly luciferin Cypridina luciferin in fishes the first cross reaction discovered Structure of firefly luciferin Discovery of aequorin and GFP (green fluorescent protein) Structure of Cypridina luciferin Concept of photoprotein Structure of Latia luciferin Dioxetanone mechanism proposed in firefly and Cypridina luminescence... 

The quenching of peroxidized luminol chemiluminescence by reduced pyridine nucleotides has been reported. Neither superoxide nor hydroxyl radical scavengers were found to quench the chemiluminescence of luminol in the presence of horseradish peroxidase and H2O2. Both chemi- and bio-luminescence of firefly luciferin have been investigated and a dioxetanone mechanism proposed for the light-producing pathway. ... 

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). 

Fig. 1.12 Mechanism of the bioluminescence reaction of firefly luciferin catalyzed by firefly luciferase. Luciferin is probably in the dianion form when bound to luciferase. Luciferase-bound luciferin is converted into an adenylate in the presence of ATP and Mg2+, splitting off pyrophosphate (PP). The adenylate is oxygenated in the presence of oxygen (air) forming a peroxide intermediate A, which forms a dioxetanone intermediate B by splitting off AMP. The decomposition of intermediate B produces the excited state of oxyluciferin monoanion (Cl) or dianion (C2). When the energy levels of the excited states fall to the ground states, Cl and C2 emit red light (Amax 615 nm) and yellow-green light (Amax 560 nm), respectively.

Contrary to the dioxetanone pathway, DeLuca and Dempsey (1970) proposed a mechanism of the bioluminescence reaction that involves a multiple linear bond cleavage of luciferin peroxide... 

However, the linear bond cleavage hypothesis of the firefly bioluminescence was made invalid in 1977. It was clearly shown by Shimomura et al. (1977) that one O atom of the CO2 produced is derived from molecular oxygen, not from the solvent water, using the same 180-labeling technique as used by DeLuca and Dempsey. The result was verified by Wannlund et al. (1978). Thus it was confirmed that the firefly bioluminescence reaction involves the dioxetanone pathway. Incidentally, there is currently no known bioluminescence system that involves a splitting of CO2 by the linear bond cleavage mechanism. 

Fig. 3.3.4 Reaction mechanism of the coelenterazine bioluminescence showing two possible routes of peroxide decomposition, the dioxetanone pathway (upper route) and linear decomposition pathway (lower route). The Oplopborus bioluminescence takes place via the dioxetanone pathway. The light emitter is considered to be the amide-anion of coelenteramide (see Section 5.4).

One is the concerted decomposition of a dioxetanone structure that is proposed for the chemiluminescence and bioluminescence of both firefly luciferin (Hopkins et al., 1967 McCapra et al., 1968 Shimomura et al., 1977) and Cypridina luciferin (McCapra and Chang, 1967 Shimomura and Johnson, 1971). The other is the linear decomposition mechanism that has been proposed for the bioluminescence reaction of fireflies by DeLuca and Dempsey (1970), but not substantiated. In the case of the Oplopborus bioluminescence, investigation of the reaction pathway by 180-labeling experiments has shown that one O atom of the product CO2 derives from molecular oxygen, indicating that the dioxetanone pathway takes place in this bioluminescence system as well (Shimomura et al., 1978). It appears that the involvement of a dioxetane intermediate is quite widespread in bioluminescence. 

The decomposition of dioxetanone may involve the chemically initiated electron-exchange luminescence (CIEEL) mechanism (McCapra, 1977 Koo et al., 1978). In the CIEEL mechanism, the singlet excited state amide anion is formed upon charge annihilation of the two radical species that are produced by the decomposition of dioxetanone. According to McCapra (1997), however, the mechanism has various shortfalls if it is applied to bioluminescence reactions. It should also be pointed out that the amide anion of coelenteramide can take various resonance structures involving the N-C-N-C-O linkage, even if it is not specifically mentioned. 

Of the many types of bioluminescence in nature, that of the firefly represents the most thoroughly studied and best understood biological luminescent process. The molecular mechanism of light emission by the firefly was elucidated in the 1960s in which a dioxetanone (a-peroxy lactone) was proposed as an intermediate, formed by the luciferase-catalyzed enzymatic oxidation of the firefly luciferin with molecular oxygen (Scheme 15). This biological reaction constitutes one of the most efficient luminescent processes known to date . Hence, it is not surprising that the luciferin-luciferase system finds wide use... 

The CIEEL mechanism has been utilized to explain the catalyzed decomposition of several cyclic and linear peroxides, including diphenoyl peroxide (4), peroxyesters and 1,2-dioxetanones. Special interest has focused on this mechanism when it was utilized to explain the efficient excited state formation in the chemiexcitation step of the firefly s luciferin/luciferase bio luminescence. However, doubts have been voiced more recently about the validity of this mechanistic scheme, due to divergences about the... 

The unimolecular decomposition of 1,2-dioxetanes and 1,2-dioxetanones (a-peroxylac-tones) is the simplest and most exhaustively studied example of a thermal reaction that leads to the formation, in this case in a single elementary step, of the electronically excited state of one of the product molecules. The mechanism of this transformation was studied intensively in the 1970s and early 1980s and several hundreds of 1,2-dioxetane derivatives and some 1,2-dioxetanones were synthesized and their activation parameters and CL quantum yields determined. Thermal decomposition of these cyclic peroxides leads mainly to the formation of triplet-excited carbonyl products in up to 30% yields. However, formation of singlet excited products occurs in significantly lower yields (below... 

Intensive studies in the field of mechanistic CL by several research groups have resulted in the description of a large variety of peroxides which, in the presence of appropriate activators, show decomposition in an activated CL process and might involve the CIEEL mechanism . Even before the formulation of the CIEEL mechanism, Rauhut s research group obtained evidence of the involvement of electron-transfer processes in the excitation step of the peroxyoxalate CL. Results obtained in the activated CL of diphe-noyl peroxide (4) led to the formulation of this chemiexcitation mechanism , and several 1,2-dioxetanones (a-peroxylactones), such as 3,3-dimethyl-l,2-dioxetanone (9) and the first a-peroxylactone synthesized, 3-ierr-butyl-l,2-dioxetanone (14), have been shown to possess similar CL properties, compatible with the CIEEL mechanism Furthermore, the CL properties of secondary peroxyesters, such as 1-phenethylperoxy acetate (15) , peroxylates (16) , o-xylylene peroxide (17) , malonyl peroxides... 

Analogously to the firefly luciferin/luciferase system, the general chemiluminescence mechanism postulated for 9-carboxyacridinium derivatives proposes the 1,2-dioxetanone 45 as high-energy intermediate However, this 1,2-dioxetanone is the only intermediate that has not yet been isolated . The cleavage of the peroxidic ring presumably results in the release of CO2 and the formation of an acridan residue in its electronically excited state (Scheme 32). 

The chemiluminescence of dioxetanones is of particular interest due to their postulated intermediacy in several bioluminescence reactions, including that of the firefly, the sea pansy Renilla, and the ostracod crustacean Cypridina (Fig. 4). The generalized mechanism for these bioluminescence reactions (22),... 

In addition to their implication as reactive intermediates in bioluminescence, dioxetanones have been proposed as key intermediates in several chemiluminescent systems. Most notable are the chemiluminescent oxidation reaction of acridan esters [19] and the chemiluminescent reaction of the related acridinium salts [20] (Rauhut et al., 1965a McCapra et al., 1977). Both reactions are quite efficient at generating singlet excited states (pCE = 10% and 2% respectively) and, owing to the elegant work of McCapra and others, are among the best understood complex chemiluminescent reaction mechanisms. 

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). 

The proposed mechanism for the activator-catalyzed chemiluminescence of dimethyldioxetanone is the general mechanism identified as chemically initiated electron-exchange luminescence (Schmidt and Schuster, 1978a Adam et al., 1978). The CIEEL sequence as applied to dimethyldioxetanone is shown in Fig. 8. In short, the light-generating sequence is initiated by electron transfer from the activator (act) to the dioxetanone. Subsequent decarboxylation gives acetone radical anion. Annihilation of acetone radical anion and activator radical cation generates the excited state of the activator. 

The involvement of the CIEEL process in the thermolysis of [21] immediately offers new insight into many previously perplexing proposals of dioxetane or dioxetanone intermediacy in various chemi- and bio-luminescent reactions. For example, the discovery of activated chemiluminescence for [21], and the finding that intramolecular electron transfer can generate a very high yield of electronically excited singlet (Horn et al., 1978-79), prompts speculation that an intramolecular version (34) of the CIEEL mechanism is... 

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