Acridan esters enhanced

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... 

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. 

Another substrate for HRP is a stable trifluorophenyl substituted acridan known as Lumigen PS-3, which emits photons at 430 nm. The detection of HRP activity by PS-3 is based on a CL process involving the enzymatic generation of an intermediate acridinium ester. In the presence of hydrogen peroxide and p-iodophenol as enhancer, the action of the acridan substrate with the HRP label generates thousands of acridinium ester intermediates ([AE]- -) per minute, which react with hydrogen peroxide, at alkaline pH, to produce... 

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