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. The chemical activation of a fluorescer by the reactions of hydrogen peroxide, a catalyst, and an oxalate ester has been the object of several mechanism studies. It was first proposed in 1967 that peroxyoxalate (26) was converted to dioxetanedione (27), a highly unstable intermediate which served as the chemical activator of the fluorescer (fir) (6,9). 

The lack of independent evidence for dioxetanedione (27) (69) and later results (66,68) have diminished the likelihood that (27) plays any significant role in the chemical excitation process and attention has been redirected to peroxyoxalate (26) and its isomers. More recent studies suggest that more than one intermediate may be required (70) ie, a pool of intermediates has been suggested. 

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... 

Peroxyoxalate chemistry has been used to carry out photochemical reactions but does not appear to produce triplet excited states (91). 

Multi functional initiators where the radical generating functions are in appropriate proximity may decompose in a concerted manner or in a way such that the intermediate species can neither be observed nor isolated. Examples of such behavior are peroxyoxalate esters (see 3.3.2.3.1) and a-hydroperoxy diazenes (e.g. 31), derived peroxyesters (65)2S3 2S4 and bis- and multi-diazenes such as 66.233,250... 

Rate law and activation energy. The oxidation of nitrosobenzene by terf-butyl hydroperoxide is catalyzed by di-terf-butyl peroxyoxalate (TBO) and by a cobalt(II) chelate complex. 

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. 

Figure 7. Absence of a linear correlation of either oxidation potential or singlet energy to chemiluininescence efficiency for the peroxyoxalate reaction.

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. 

The formation of isobutyric acid in the presence of the additives studied, and the results of additional studies (di-tert.-butyl peroxyoxalate/isobutyroal-dehyde/amine), point to the intermediate formation of acyl peroxy radicals. 

Separate experiments in which tert.-butoxy radicals were produced thermally in benzene from di-tert.-butyl peroxyoxalate failed to reveal any direct reaction of these radicals with amine II. Even at higher temperatures (A/ 150°C, dichlorobenzene, +00+ decomposition), the +0 radicals attacked neither amine II nor nitroxide I. The earlier described experiments of ketone photooxidation showed additionally that amine II displays no specially marked reactivity towards peroxy radicals. 

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. 

In 1965, Rauhut et al. [73] reviewed the oxalyl chloride CL system and showed that oxalyl esters could be used for this system instead of oxalyl chloride. Since then, they synthesized a number of oxalates including oxamides and established a new, potent luminescent system, namely the peroxyoxalate CL (PO CL) system. Much work has been carried out to synthesize suitable oxalic compounds. The first study dealing with different reagents was published in 1967 by Rauhut et al. [98] for the American Cyanamid Company with the purpose of developing... 

In the gas and liquid phases, very well-established CL reactions exist that have been chronologically introduced in Chapter 1, together with their mechanisms they will be treated in different chapters of this book. Particularly, some chapters include descriptions of the CL systems and applications in the liquid phase in organic and inorganic analysis (Chapters 5 and 6, respectively), for BL systems (Chapter 10) applications derived from the use of organized media (Chapter 11) the specific study of the mechanism and applications of a widely applied CL system based on the reaction of peroxyoxalates (Chapter 7) kinetics... 

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],... 

Hydrocortisone Digoxin Theophyllin Solid-state peroxyoxalate CL, high-performance liquid chromatography 2 ng/mL 2 ng/mL 4 ng/mL 74... 

---