Acridans, chemiluminescence

Figure 15.5 HRP-acridan chemiluminescence assay on both glass and silvered slides. Reproduced from Ana Chem 78 8020-8027, (2006).

Figure 15.15 A) Model BSA-biotin, HRP-streptavidin chemiluminescent assay scheme. B) Acridan chemiluminescence emission as a function of time from HRP modified ass coverslips coated with 1 mM BSA-biotin and I mM HRP-streptavidin and positioned glass substrate geometries with and without 12.3 cm Al triangle 7S nm thick (left). C) Acridan chemiluminescence background emission as a function of time for glass coverslips incubated with 1.5% BSA and 1 mM HRP-streptavidin (control) positioned on glass substrate geometries with and without 12.3 cm Al triangle 75 nm thick shapes (right). All samples were ejqiosed to four 10 second microwave pulses (Mw pulse) at 10% power. Adapted from Anal Chem 79 7042-7052 (2007).

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. 

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... 

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

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. 

Analogously to the firefly luciferin/luciferase system, the general chemiluminescence mechanism postulated for 9-carboxyacridinium derivatives proposes the 1,2-dioxetanone 45 as high-energy intermediate However, this 1,2-dioxetanone is the only intermediate that has not yet been isolated . The cleavage of the peroxidic ring presumably results in the release of CO2 and the formation of an acridan residue in its electronically excited state (Scheme 32). 

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. 

To demonstrate MT-MEC as a useful platform for protein quantification, a simple surface biotin-avidin assay was constructed[15,16]. In the assay, biotinylated-BSA is incubated on both silvered and glass substrates (Figure 15.5). HRP-streptavidin is then added to the surface, locaiizing the enzyme catalyst in close proximity to the silver for MT-MEC. The peroxide and Acridan (iumophore) are then added to initiate the chemiluminescence reaction. While this assay in essence determines BSA concentration, this model assay could indeed be fashioned to both iocaiize and sense other proteins / DNAs of interest. 

Figure 15.7 3D plots of the Acridan assay chemiluminescence emission as a function of time from silvered glass slides (Ag) without (Top) and with low power microwave exposure / pulses (Middle). Bottom - dictographs showing the Acradan emission both before (a) and after a low power microwave pulse (b). Mw -Microwave pulse. The concentration of BSA-Biotin was 1.56 pM. Reproduced from AnalChem 78 8020-8027, (2006). 

Perkizas G, Nikokavouras J. Substituent effect on the chemiluminescence quantum efficiency of some acridan derivatives. Monatsh Chem 1983 114 3-11. 

Fluoride ion causes deproteclion of 36 and the resultant aryloxy anion decomposes with chemiluminescence to diisopropyl ketone and a (3-methoxycarbonyl derivative of the naphthol <97TL411>. Other 1,2-dioxetanes containing an acridane 10-acetate group have been investigated as potential fluorescent probes <97TL841>. 

A second tempting possibility is a mechanism which has been proposed for the chemiluminescent oxidation of certain imidazole , acridane , and indole , derivatives. Its essence is expressed in equation (23), where a 4-ring peroxide collapses, leaving one of the fragments in an electronically excited state. This scheme is attractive... 

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. 

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