Acridan esters oxidation

Although substituted phenols (e.g., para-iodophenol, para-phenylphenol, firefly luciferin, coumaric acid) are popular enhancers, in both luminol and acridan ester oxidation, enhancers with other functional groups [24], e.g., phe-nylboronic acids [25-28], phenothiazines [29], are also useful. As an example the structure of the phenothiazine enhancer used in the Supersignal substrate family is shown in Figure 6. 

In the firefly, the carboxyl group of luciferin is activated. Key features of both compounds include an easily autoxidizable CH group, an activated carbonyl group in juxtaposition, and an oxidation product that is very fluorescent. Studies of the chemistry of the model acridan ester have led to a better understanding of firefly bioluminescence including correct prediction of the structure of the oxidized luciferin product. 

Recently, it was found that certain acridan esters could be oxidized to acridinium esters in a reaction catalyzed by HRP [13, 14], Like the luminol case it was found that the light yield of the reaction could be increased when certain additives (enhancers) were added. 

As in the luminol case, the main role of the enhancer (EnH) seems to be related to turnover of the enzyme, generating enhancer radicals (En rad) in the process that are capable of oxidizing the acridan ester (AcH). The structure of the enhancer obviously is very important. To accelerate HRP turnover, the enhancer must on the one hand be able to rapidly react with the reactive HRP intermediates Cl and especially CII (k2 and k3 large). On the other hand, the oxidized enhancer intermediate (radical or radical cation) must be able to oxidize the acridan ester (light-generating step). This last reaction also depends on the structure of the acridan ester in a very unfavorable case, adding an enhancer for enzyme turnover could actually diminish the light production if k 4 > fct (Fig. 5), i.e., if the enhancer radical would not be able to oxidize the acridan ester. 

Further complicating factors in the choice of an enhancer include degradation of HRP by enhancer radicals [23], pH effects [24] on reduction and oxidation potentials for enhancer and acridan ester, inactivation of enhancer radicals because of dimerization or other reactions, etc. All these, and other, effects of the structures (and because of the kinetics also the concentrations) of enhancer and acridan ester may cause erratic results when optimization studies are conducted. When... 

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. 

The autoxidation of acridan esters has been of great value in the elucidation of the mechanism of luciferin oxidation, and is discussed as a model system for bioluminescence (Chap. (XII)). The following series of compounds which react in a related way summarises the main features which lead to moderately high light emission from the autoxidation of carbonyl compounds. The alkoxycarbamoyl furanones [37], whose chemiluminescent reactions are depicted in the scheme below, show the following useful features. 

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. 

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