Chemiluminogenic acridinium salts: A comparison study. Detection of intermediate entities appearing upon light generation

The nine derivatives of acridine-9-carboxylic acid (CMADs) capable for chemiluminescence (CL), representing various classes of compounds were isolated in a chemically pure state (assessed by RP-HPLC) and identified using high resolution mass spectrometry (ESI-QTOF) and magnetic resonance (1H NMR) techniques. Among them are aryl acridinium esters, containing certainly selected and located substituents in both aromatic systems, an acridinium perfluorinated aliphatic ester and an acridinium sulfonamide. The main aim of the study was to compare, under the conditions of their optimal performance, the luminogenic properties of the above molecular systems, such as efficiency and kinetics of emission, limits of detection/quantification as well as stability in aqueous solutions. All of the investigated acridinium salts can serve as CL indicators or fragments of CL labels in modern medical diagnostics (e.g. chemiluminescence and electrochemiluminescence immunoassays (CLIA, ECLIA)), hybridization protection assays of nucleic acids (HPA) as well as other ultra-sensitive luminescence tests. Taking advantage of the flow injection mass analysis, the appearance of unstable cationic intermediates produced upon chemiluminogenic oxidation of selected acridinium salts in an alkaline environment was confirmed for the first time. The observed entities include hydroxyl and hydroperoxide adducts (as radical cations) as well as the acridan spirodioxetanone derivative, making a high energetic and direct precursor of the target emitters, an electronically excited 10-methylacridan-9-ones. The results are supported with quantum chemistry calculations at the DFT level of theory. Theoretical studies have helped to determine the unstable intermediates appearing during in CL in ESI-QTOF experiments. In the group of acridinium ester they allowed to observe the positive linear trend among between the kinetics of CL decay and calculated coefficients of pz LUMO orbitals on the carbon atoms being the reaction centers responsible for generation of light.