Chemiluminescence peroxyoxalates

The mechanism of chemiluminescence is still being studied and most mechanistic interpretations should be regarded as tentative. Nevertheless, most chemiluminescent reactions can be classified into (/) peroxide decomposition, including biolurninescence and peroxyoxalate chemiluminescence (2) singlet oxygen chemiluminescence and (J) ion radical or electron-transfer chemiluminescence, which includes electrochemiluminescence. 

Peroxyoxalate chemiluminescence is the most efficient nonenzymatic chemiluminescent reaction known. Quantum efficiencies as high as 22—27% have been reported for oxalate esters prepared from 2,4,6-trichlorophenol, 2,4-dinitrophenol, and 3-trif1uoromethy1-4-nitropheno1 (6,76,77) with the duorescers mbrene [517-51-1] (78,79) or 5,12-bis(phenylethynyl)naphthacene [18826-29-4] (79). For most reactions, however, a quantum efficiency of 4% or less is more common with many in the range of lO " to 10 ein/mol (80). The inefficiency in the chemiexcitation process undoubtedly arises from the transfer of energy of the activated peroxyoxalate to the duorescer. The inefficiency in the CIEEL sequence derives from multiple side reactions available to the reactive intermediates in competition with the excited state producing back-electron transfer process. 

Most peroxyoxalate chemiluminescent reactions are catalyzed by bases and the reaction rate, chemiluminescent intensity, and chemiluminescent lifetime can be varied by selection of the base and its concentration. Weak bases such as sodium saUcylate or imidazole are generally preferred (94). 

A number of other chemiluminescent reactions appear to be related to peroxyoxalate chemiluminescence although thek mechanistic details may vary. For example, various chlotinated esters and ethers react with and a fluorescer to emit light (98—101). Other examples have been given... 

In order to optimize the chemiluminescence response, we have investigated the mechanism of the complex reactions leading to chemical generation of chemiluminescence. A new peroxyoxalate-hydrogen peroxide reaction mechanism has emerged from our preliminary studies on the five contributing factors listed above. Two kinetic models are discussed, one for the... 

Applications of the oxalate-hydrogen peroxide chemiluminescence-based and fluorescence-based assays with NDA/CN derivatives to the analysis of amino acids and peptides are included. The sensitivity of the chemiluminescence and fluorescence methods is compared for several analytes. In general, peroxyoxalate chemiluminescence-based methods are 10 to 100 times more sensitive than their fluorescence-based counterparts. The chief limitation of chemiluminescence is that chemical excitation of the fluorophore apparently depends on its structure and oxidation potential. 

These results encouraged further efforts in the development of the peroxyoxalate chemiluminescent method for HPLC-detectors. 

Though we and others (27-29) have demonstrated the utility and the improved sensitivity of the peroxyoxalate chemiluminescence method for analyte detection in RP-HPLC separations for appropriate substrates, a substantial area for Improvement and refinement of the technique remains. We have shown that the reactions of hydrogen peroxide and oxalate esters yield a very complex array of reactive intermediates, some of which activate the fluorophor to its fluorescent state. The mechanism for the ester reaction as well as the process for conversion of the chemical potential energy into electronic (excited state) energy remain to be detailed. Finally, the refinement of the technique for routine application of this sensitive method, including the optimization of the effi-ciencies for each of the contributing factors, is currently a major effort in the Center for Bioanalytical Research. 

DCIA has been used to label numerous proteins and other biomolecules, including phospholipids (Silvius et al., 1987), to study the interaction of mRNA with the 30S ribosomal subunit (Czworkowski et al., 1991), in the investigation of cellular thiol components by flow cytometry (Durand and Olive, 1983), in the detection of carboxylate compounds using peroxyoxalate chemiluminescence (Grayeski and DeVasto, 1987), and for general sulfhydryl labeling (Sippel, 1981). 

Grayeski, M.L., and DeVasto, J.K. (1987) Coumarin derivatizing agents for carboxylic acid detection using peroxyoxalate chemiluminescence with liquid chromatography. Anal. Chem. 59, 1203. 

Mansouri A, Makris DP and Kefalas P. 2005. Determination of hydrogen peroxide scavenging activity of cinnamic and benzoic acids employing a highly sensitive peroxyoxalate chemiluminescence-based assay structure-activity relationships. J Pharm Biomed Anal 39(l-2) 22-26. 

The brilliant emissions resulting from the oxidation of certain oxalic acid derivatives, especially in the presence of a variety of fluorophores, are the bases of the most active area of current interest in CL. This group of chemiluminescent reactions has been classified as peroxyoxalate chemistry because it derives from the excited states formed by the decomposition of cyclic peroxides of oxalic acid derivatives called dioxetanes, dioxetanones, and dioxetanediones. 

The leaving group of the oxalic ester has a strong effect on the efficiency of the peroxyoxalate chemiluminescent system. The electron-attracting power of the substituents on the phenyl rings of the substituted diphenyl oxalates is important to the overall efficiency of the chemiluminescent reactions. Steric effects... 

A wide variety of different classes of fluorescent molecules has been investigated in the peroxyoxalate chemiluminescent systems. Among those screened were fluorescent dyes such as rhodamines and fluoresceins, heterocyclic compounds such as benzoxazoles and benzothiazoles, and a number of polycyclic aromatic hydrocarbons such as anthracenes, tetracenes, and perylenes. The polycyclic aromatic hydrocarbons and some of their amino derivatives appear to be the best acceptors as they combine high fluorescence efficiency with high excitation efficiency in the chemiluminescent reaction [28],... 

Peroxyoxalate chemiluminescence (POCL) was first reported in 1963 by Chan-dross [1], who observed emission of light from a mixture consisting of oxalyl... 

Figure 6 Proposed formation of an alternative C204 structure in the excitation mechanism for peroxyoxalate chemiluminescence.

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