Chemiluminescence peroxide intermediates

As mentioned earlier (see p. 122) the previously postulated dioxetane intermediate in firefly bioluminescence has been challenged as no 180 is in-corporated in the carbon dioxide released during oxidation of firefly luciferin with 18C>2. In view of the crucial significance of the 180. experiments De Luca and Dempsey 202> rigorously examined the reliability of their tracer method. They conclude from their experiments that all available evidence is in favour of a linear, not a cyclic peroxide intermediate — in contrast to Cypridina bioluminescence where at least part of the reaction proceeds via a cyclic peroxide (dioxetane) as concluded from the incorporation of 180 into the carbon dioxide evolved 202,203). However, the dioxetane intermediate is not absolutely excluded as there is the possibility of a non-chemiluminescent hydrolytic cleavage of the four-membered ring 204>. 

Another well-known CL amplifier, which is also frequently used for superoxide detection in biological systems, is luminol (5-amino-2,3-dihydro- 1,4-phthalazinedione). It has been proposed that luminol semiquinone reacts with superoxide to form the peroxide intermediate, whose decomposition is accompanied by chemiluminescence [62]. 

Since its discovery by Chandross and to this day, peroxy-oxalate chemiluminescence has been controversial because of its enormous complexity in view of the many alternative steps involved in this process. The principal mechanistic feature of the peroxy-oxalate chemiluminescence pertains to the base-catalyzed (commonly imidazole) reaction of an activated aryl oxalate with hydrogen peroxide in the presence of a chemiluminescent activator, usually a highly fluorescent aromatic hydrocarbon with a low oxidation potential . A variety of putative high-energy peroxide intermediates have been proposed for the generation of the excited states . In the context of the present chapter, it is of import to mention that recent work provides experimental evidence for the intervention of the 1,2-dioxetanedione 18 (Scheme 11) as the high-energy species responsible for the chemiexcitation. Furthermore, clear-cut experimental data favor the CIEEL mechanism as a rationalization of the peroxy-oxalate chemiluminescence . 

Intramolecular electron transfer initiated peroxide decomposition. . 1236 HIGH-EFFICIENCY ORGANIC CHEMILUMINESCENT REACTIONS INVOLVING PEROXIDE INTERMEDIATES. 1238... 

VI. HIGH-EFFICIENCY ORGANIC CHEMILUMINESCENT REACTIONS INVOLVING PEROXIDE INTERMEDIATES... 

In this part of the chapter we will give a more detailed description of some highly efficient organic chemiluminescence systems, which occur with the involvement of peroxide intermediates. We have chosen to begin the subject with the well-known and widely applied luminol oxidation and will show that, even though this reaction has been exhaustively studied, several critical points in its mechanism remain unclear and are still the subject of... 

In aprotic media, only molecular oxygen and a strong base are needed to produce chemiluminescence from luminol. In such media, an important intermediate in the reaction is the dianion of luminol, which can be oxidized by oxygen, resulting in the formation of the diazaquinone 27 and deprotonated hydrogen peroxide. The subsequent nucleophilic attack by hydrogen peroxide dianion to one of the diazaquinone carbonyls gives rise to the formation of a metastable peroxidic intermediate that, by several steps, results in chemiexcited 3-aminophthalate (Scheme 16)123,174, iso ... 

In the presence of hydrogen peroxide and base, acridinium salts lead to chemiluminescence emission. Acridans, in their reduced forms, are able to react directly with oxygen in aprotic solvents with 4>cl up to 10% . Scheme 31 shows the proposed mechanism for chemiluminescence of 9-cyano-lO-methylacridan and 9-cyano-lO-methylacridinium salt in the presence of oxidant and base, which postulates the cyclic peroxidic intermediate 44. 

Merenyi O Lind J S. Role of a peroxide intermediate in the chemiluminescence of luminol. A mechanistic study. J Am Chem Soc 1980 102 5830-5. 

The mechanism is also consistent with the observation that cyclic diacyl hydrazines are far more chemiluminescent than similar acyclic compounds. The bicyclic peroxide intermediate shown in Scheme 6 may be precluded by rupture of one C-N bond after the addition of the hydrogen peroxide anion, but before the formation of an anti-aromatic endoperoxide that decomposes to form the emitter. Obtaining hard evidence for the existence of these... 

Most chemiluminescent and bioluminescent reactions require oxygen. Likewise, most produce an electronically excited emitting species through the decomposition of a peroxide of one sort or another. In the experiment that follows, a chemiluminescent reaction that involves the decomposition of a peroxide intermediate is described. 

In many chemiluminescent reactions of peroxides, two carbonyl groups are formed simultaneously by decomposition of an intermediate such as compound (1) ... 

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). 

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). 

Weak to moderate chemiluminescence has been reported from a large number of other Hquid-phase oxidation reactions (1,128,136). The Hst includes reactions of carbenes with oxygen (137), phenanthrene quinone with oxygen in alkaline ethanol (138), coumarin derivatives with hydrogen peroxide in acetic acid (139), nitriles with alkaline hydrogen peroxide (140), and reactions that produce electron-accepting radicals such as HO in the presence of carbonate ions (141). In the latter, exemplified by the reaction of h on(II) with H2O2 and KHCO, the carbonate radical anion is probably a key intermediate and may account for many observations of weak chemiluminescence in oxidation reactions. 

To understand how these parameters affected the efficiency of the chemiluminescent reaction, we examined the mechanism originally proposed by Rauhut (26). As shown in Scheme 2, hydrogen peroxide reacts with an oxalate ester, such as 2,4,6-trichlorophenyl oxalate (TCPO), in a two-step process to form a reactive intermediate for which Rauhut suggested structure 1, the 1,2-dioxetanedione. The dioxetanedione then interacts with an acceptor (ACC) to produce two molecules of COj and the excited state of the acceptor. The last stage of the sequence is fluorescence emission from the acceptor. 

Though we and others (27-29) have demonstrated the utility and the improved sensitivity of the peroxyoxalate chemiluminescence method for analyte detection in RP-HPLC separations for appropriate substrates, a substantial area for Improvement and refinement of the technique remains. We have shown that the reactions of hydrogen peroxide and oxalate esters yield a very complex array of reactive intermediates, some of which activate the fluorophor to its fluorescent state. The mechanism for the ester reaction as well as the process for conversion of the chemical potential energy into electronic (excited state) energy remain to be detailed. Finally, the refinement of the technique for routine application of this sensitive method, including the optimization of the effi-ciencies for each of the contributing factors, is currently a major effort in the Center for Bioanalytical Research. 

In the preceding paragraph peroxides were described as key intermediates in autoxidation chemiluminescence. In most cases hydroperoxides were involved. The majority are well-defined compounds (e.g. cumene hydroperoxide), but autoxidation reactions are rather complex and peroxides are only one, though very important type of compound involved. 

DPA) in dimethylphthalate at about 70°, yields a relatively strong blue Umax =435 nm) chemiluminescence the quantum yield is about 7% that of luminol 64>. The emission spectrum matches that of DPA fluorescence so that the available excitation energy is more than 70 kcal/mole. Energy transfer was observed on other fluorescers, e.g. rubrene and fluorescein. The mechansim of the phthaloyl peroxide/fluorescer chemiluminescence reaction very probably involves radicals. Luminol also chemiluminesces when heated with phthaloyl peroxide but only in the presence of base, which suggests another mechanism. The products of phthaloyl peroxide thermolysis are carbon dioxide, benzoic acid, phthalic anhydride, o-phenyl benzoic acid and some other compounds 65>66>. It is not yet known which of them is the key intermediate which transfers its excitation energy to the fluorescer. 

In the case of chemiluminescence occurring on treatment of di-benzanthrone with hypochlorite, as mentioned above, an endo peroxide might well be a key intermediate formed from 4 and singlet oxygen. The emitting species, however, is trichloro-dibenzanthrone, not 4 itself 70h... 

Several N-methyl-9-acridinecarboxylic acid derivatives (e.g., 10-methyl-9-acridinecarboxylic chloride and esters derived therefrom [39]) are chemiluminescent in alkaline aqueous solutions (but not in aprotic solvents). The emission is similar to that seen in the CL of lucigenin and the ultimate product of the reaction is N-methylacridone, leading to the conclusion that the lowest excited singlet state of N-methylacridone is the emitting species [40], In the case of the N-methyl-9-acridinecarboxylates the critical intermediate is believed to be either a linear peroxide [41, 42] or a dioxetanone [43, 44], Reduced acridines (acridanes) such as N-methyl-9-bis (alkoxy) methylacridan [45] also emit N-methylacridone-like CL when oxidized in alkaline, aqueous solutions. Presumably an early step in the oxidation process aromatizes the acridan ring. 

In the case of the experiments performed by Hohman and co-workers [149], the fluoride anion would readily displace the silicon-leaving group. The peroxide anion could then further react via an intramolecular nucleophilic attack, resulting in cyclization to form the reactive intermediate responsible for the chemiluminescence that was observed. A recent kinetic study by Stevani and Baader [150] of the reaction of 4-chlorophenyl-O,O-hydrogen monoperoxyoxalate with various oxygen and nitrogen bases suggested that the intermediate formed must be 1,2-dioxetandione. 

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