Acridans

An interesting variation of this ring expansion has been described in which the acridane meth-anesulfonate 42 undergoes a base-induced rearrangement accompanied by loss of methanesul-fonate ion to yield 10-phenyl-5//-dibenz[6,/]azepine (43).165... 

When acridane 1 is oxidized by dibenzoyl peroxide in propanol/ water in acid or neutral medium, there occurs chemiluminescence whose emission spectrum matches the fluorescence spectrum of acridinium cation (protonated acridine) 2. As radical scavengers have no influence... 

Intense and analytically useful direct chemiluminescence (CL) has been observed from a rather limited group of organic compounds. These include diacylhydraz-ides, indoles, acridines and acridans, polydimethylaminoethylenes, anthracenes, and aroyl peroxides. A substantial number of other kinds of compounds, when... 

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. 

Application of Novel Acridan Esters as Chemiluminogenic Signal Reagents in Immunoassay... 

Bioluminescence, the phenomenon of biological light emission, has fascinated mankind for many centuries. Scientists have been intrigued by it, and for many years have tried to answer simple questions like how and why certain animals and bacteria bioluminesce. This research has led to new insights in (molecular) biology and biochemistry. For example, in the 1960s, McCapra studied the chemical mechanisms of bioluminescence and devised a model for firefly luciferin the acridan esters (Fig. 1) [1-5],... 

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. 

Figure 1 Structures of firefly luciferin and acridan ester.

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. 

In 1994 at the 8th International Symposium on Bioluminescence and Chemiluminescence, Schaap and Akhavan-Tafti presented a new technology for the detection of HRP [13, 19, 20], They proposed the use of certain aromatic acridan esters (see Fig. 4 for the structures) as part of a signal reagent for very sensitive detection... 

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. 

Figure 5 Proposed reactions involved in the HRP-catalyzed chemiluminescent peroxidation of acridan esters.

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... 

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. 

The pH optimum of HRP is around pH 5. Therefore, this would be the pH of choice. Unfortunately this is not the optimal pH for the light-generating reaction (the general base-catalyzed reaction of acridinium ester with hydrogen peroxide). An acridan ester like GZ-11 with a leaving group of low pKa (perfluoro-ferf-butanol has a pKa below 6) is clearly advantageous. 

Acridan esters were synthesized according to the general synthetic scheme depicted in Figure 11. 

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