Diazaquinone Chemiluminescence

Although diazaquinones could be considered in a separate class of chemiluminescent compounds their importance lies in their association with the mechanism of luminol chemiluminescence. It is not yet certain whether they are intermediates under all reaction conditions, but they are clearly implicated. 

In the first publications on luminol chemiluminescence [49] the diazaquinone dehydroluminol (40) was postulated as a reaction intermediate. It was assumed to take two different reaction paths Hydrolysis was thought to give di-imine (41) - a compound unknown at that time - and 3-amino-phthalate. Unreacted diazaquinone was then thought to react with the di-imine formed to give nitrogen and luminol, the latter product formed in the excited state by this redox reaction. 

luminol itself would be the chemiluminescence emitter - an assumption which could not be adequately examined spectroscopically by the primitive apparatus then available. 

In spite of the fact that this mechanism has been proved to be wrong in important details (e.g. the nature of the emitter or the role of di-imine [50]) there is experimental evidence that the diazaquinone as an intermediate may well be involved in the luminol reaction, and in luminol type chemiluminescence in general. 

The kinetic experiments on luminol chemiluminescence in the system water/ alkali/potassium persulfate/hydrogen peroxide [45] were interpreted as showing that a two-electron oxidation product of luminol (i.e. the diazaquinone) was a key intermediate. The kinetics of the chemiluminescent oxidation of 7-dimethyl-amino naphthalene-1,2-dicarboxylic hydrazide led to the same conclusion [51]. 

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The requirement of hydrogen peroxide or oxygen in diazaquinone chemiluminescence appears to be another argument against the hypothesis of Albrecht 128> or of Kautsky and coworkers 132> that the diazaquinone is simply hydrolyzed to yield aminophthalate and diimine which in turn reduces other diazaquinone molecules to luminol in an excited state so that it then emits. 

The question is whether diazaquinones are in fact in the main stream 2> of cyclic diacyl hydrazide chemiluminescence or whether they are only one of the possible intermediates or, finally, whether diazaquinone chemiluminescence is a special type of chemiluminescence reaction although very closely related to the chemiluminescence of the corresponding hydrazides. E. H. White and D. F. Roswell 2> point out that the respective diazaquinone may actually be one of the possible intermediates formed either from the hydrazide dianion (essential in aprotic solvents) or from a hydrazide radical anion (which is the usual species in aqueous media). This is outlined in the following scheme ... 

As early as in his first paper on luminol chemiluminescence H. O. Albrecht 128> postulated that diazaquinone 63 ( dehydroluminol ) is in-... 

The source of chemiluminescence in the oxidation of luminol was explored by Merenyi and co-workers in detail (153). The oxidation of luminol yields aminophthalate as a final product and the reaction proceeds via a series of electron transfer steps. The primary oxidation product is the luminol radical which is transformed into either diazaquinone or the a-hydroxide-hydroperoxide intermediate (a-HHP). The latter oxidation step occurs between the deprotonated form of the luminol radical and O -. The chemiluminescence is due to the decomposition of the mono-anionic form of a-HHP into the final products ... 

An alternative path includes oxidation, in the absence of light, of the diazaquinone with weak chemiluminescence (154). The effect of iron(II) on the luminescent intensity was interpreted by considering that it can efficiently generate the O - radical in a reaction with 02 and, as a consequence, increase the importance of reaction (108) in the overall process (155). 

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 first proposal of a mechanism for chemiluminescent luminol oxidation, Albrecht postulates a bicyclic endoperoxide as the high-energy intermediate. The endoperoxide is presumably formed by nucleophilic attack of hydrogen peroxide monoanion on one of the diazaquinone 27 carbonylic groups to form 28, followed, after deprotonation to 29, by ring closure to 30 (Scheme 21) . 

Steinfatt proposed an alternative mechanism for the formation of excited aminophth-alate, based on the concept of dioxirane-carbene mediated chemiexcitation, which is also attributed to other chemiluminescent systems ° °. After the attack of hydrogen peroxide on the diazaquinone 27 carbonyl carbon, a perhydrolysis step is postulated to result in the intramolecular dioxirane-carbene system (32) in the excited state ° ° . This species presumably rearranges to 3-aminophthalate dianion while still in the singlet-excited state (Scheme 23). Although this is a very interesting mechanistic proposal, it is based on experimental evidence obtained with indirect phthaloyl peroxide chemiluminescence and no further evidence corroborates this proposal. 

Several other chromophores have been used in the development of sensors based upon ECL. For example, the luminol reaction is a conventional chemi-luminence reaction that has been studied in detail and it is believed that the mechanism of the ECL reaction is similar, if not identical, to that of the chemiluminescence. As shown in Fig. 2, the luminol ion undergoes a one-electron oxidation to yield a diazaquinone, which then reacts with peroxide or superoxide ( OOH) to give the excited 3-aminophthalate which has an emission maximum of 425 nm. This reaction is particularly versatile and has been utilized in a variety of ECL assays, many of which have been previously summarized by Knight [1], The luminol ECL reaction can be used for the determination of any species labeled with luminol derivatives, hydrogen peroxide, and other peroxides or enzymatic reactions that produce peroxides. A couple of examples are described later. 

Since the preparation of the first simple diazaquinones [53, 54] (all of them being non-chemiluminescent ) chemiluminescent diazaquinones have been described. It should be pointed out that luminol diazaquinone has not been isolated as yet, nor has it been isolated from luminescent oxidation mixtures, except as the Diels-Alder adduct mentioned above. 

By reacting the sodium salt of luminol with tert-hntyl hypochlorite in dimethyl ether at — 50°C, the very unstable luminol diazaquinone was obtained. It could not be purified to give an analytically pure substance, but it chemiluminesced on treatment with alkaline hydrogen peroxide with the typical blue luminol emission. 

Kinetic experiments, trapping the diazaquinone (44) with cyclopentadiene to give the Diels-Alder-adduct, (45), provided additional experimental proof for the role of diazaquinones in luminol type chemiluminescence. Analogous experimental evidence was provided [59] for he diazaquinone (46). 

It is of course essential to connect the chemistry of the dicizaquinone to hydrazide chemiluminescence if its role as an intermediate is to be confirmed. Cyclopentadiene is an excellent trap for the diazaquinone in this case as evidenced by the large rate constant for the Diels-Alder-reaction (7.7501 M s" at 21 °C). The chemiluminescence of the diazaquinone was therefore compared with that of the hydrazide in dimethylphthalate with anhydrous H2O2 and diethy-lamine as base. Heme was a necessary catalyst for the hydrazide reaction. 

Since there is no effect of H2O2 concentration on the integrated chemiluminescence intensity, the conclusion was drawn that oxygen radical anion activates the luminol radical anions to chemiluminescence, forming the non-radical diazaquinone perhydrate (59) ... 

Under both sets of conditions the diazaquinone is implicated but it is quite possible that it is a by-product, albeit a chemiluminescent one. A reasonable explanation of the need for catalysts in the aqueous system is the greater difficulty in oxidizing the mono-anion (either to the radical or through to the diazaquinone). 

The more easily oxidized dianion, possible only in dipolar aprotic solvents, succumbs to O2 alone. The reaction of the radical (or radical anion) with oxygen may lead to the endoperoxide, and thus join the pathway from the diazaquinone. Other peroxidic products leading to chemiluminescence have not been excluded. 

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