Chemiluminescence activators

Finally, in activated chemiluminescence, an added compound also leads to an enhancement of the emission intensity however, in contrast with the indirect CL, this compound, now called activator (ACT), is directly involved in the excitation process and not just excited by an energy transfer process from a formerly generated excited product (Scheme 5). Activated CL should be considered in two distinct cases. In the first case, it involves the reaction of an isolated HEI, such as 1,2-dioxetanone (2), and the occurrence of a direct interaction of the ACT with this peroxide can be deduced from the kinetics of the transformation. The observed rate constant (kobs) in peroxide decomposition is expected to increase in the presence of the ACT and a hnear dependence of kobs on the ACT concentration is observed experimentally. The rate constant for the interaction of ACT with peroxide ( 2) is obtained from the inclination of the linear correlation between obs and the ACT concentration and the intercept gives the rate constant for the unimolec-ular decomposition ( 1) of this peroxide (Scheme 5). The emission observed in every case is the fluorescence of the singlet excited ACT" ° . ... 

The magnitude of km, the experimentally determined bimolecular rate constant for chemiluminescence, is related to several of the rate constant specified in Fig. 8. The data on the hydrocarbon- or amine-activated chemiluminescence indicated that kJ0 > k ACT. Thus simple analysis of the kinetics yields (33), where Kn is the equilibrium constant for complex... 

The involvement of the CIEEL process in the thermolysis of [21] immediately offers new insight into many previously perplexing proposals of dioxetane or dioxetanone intermediacy in various chemi- and bio-luminescent reactions. For example, the discovery of activated chemiluminescence for [21], and the finding that intramolecular electron transfer can generate a very high yield of electronically excited singlet (Horn et al., 1978-79), prompts speculation that an intramolecular version (34) of the CIEEL mechanism is... 

The rates of thermolysis (37) of the peroxyesters in argon-purged benzene can be followed conveniently by the direct, indirect, or activated chemiluminescence. In all of the cases reported peroxyesters in benzene solution show clean first-order reaction for low initial peroxide concentrations (10-5-10-3 M). The activation parameters for the peroxyester thermolyses reveal some important details of the reaction mechanism. The activation enthalpy obtained for peroxyester [28] is quite similar to that reported by Hiatt... 

Activated chemiluminescence is observed from these secondary peroxy-esters as well. When the thermolysis of peroxyacetate [281 in benzene solution is carried out in the presence of a small amount of an easily oxidized substance the course of the reaction is changed. For example, addition of N,N-dimethyldihydrodibenzol[ac]phenazine (DMAC) to peroxyester [28] in benzene accelerates the rate of reaction and causes the generation of a modest yield of singlet excited DMAC. This is evidenced by the chemiluminescence emission spectrum which is identical to the fluorescence spectrum of DMAC obtained under similar conditions. Spectroscopic measurements indicate that the DMAC is not consumed in its reaction with peroxyester 28 even when the peroxyester is present in thirty-fold excess. The products of the reaction in the presence of DMAC remain acetophenone and acetic acid. These observations indicate that DMAC is a true catalyst for the reaction of peroxyacetate 28. The results of these experiments with DMAC, plotted according to (27) give k2 = 9.73 x 10-2 M-1 s-1. 

The thermal decompositions are first order and usually unimolecular. A variety of experimental methods can. be used to follow the rates, which include direct chemiluminescence of the excited carbonyl product (A),14 50,93,96 activated chemiluminescence by energy transfer of the excited carbonyl to an efficient fluorescer (B),14c,l2a,94 9< dioxetane consumption or carbonyl product formation by NMR spectroscopy (C),l2M4b c iodometric titration of the cyclic peroxide (DVJ, Ub,c and infrared spectroscopy of a-peroxylactone consumption or carbonyl product formation (E).2,22,38 The method of choice depends on the particular system, but usually several techniques can be employed. 

Ekins RP, Chu F, Micallef J (1989) High specific activity chemiluminescent and fluorescent markers their potential application to high sensitivity and multi-analyte immunoassays. J Biolumin Chemilumin 4 59-78... 

Subsequent studies (63,64) suggested that the nature of the chemical activation process was a one-electron oxidation of the fluorescer by (27) followed by decomposition of the dioxetanedione radical anion to a carbon dioxide radical anion. Back electron transfer to the radical cation of the fluorescer produced the excited state which emitted the luminescence characteristic of the fluorescent state of the emitter. The chemical activation mechanism was patterned after the CIEEL mechanism proposed for dioxetanones and dioxetanes discussed earher (65). Additional support for the CIEEL mechanism, was furnished by demonstration (66) that a linear correlation existed between the singlet excitation energy of the fluorescer and the chemiluminescence intensity which had been shown earher with dimethyl dioxetanone (67). 

The first detailed investigation of the reaction kinetics was reported in 1984 (68). The reaction of bis(pentachlorophenyl) oxalate [1173-75-7] (PCPO) and hydrogen peroxide cataly2ed by sodium saUcylate in chlorobenzene produced chemiluminescence from diphenylamine (DPA) as a simple time—intensity profile from which a chemiluminescence decay rate constant could be determined. These studies demonstrated a first-order dependence for both PCPO and hydrogen peroxide and a zero-order dependence on the fluorescer in accord with an earher study (9). Furthermore, the chemiluminescence quantum efficiencies Qc) are dependent on the ease of oxidation of the fluorescer, an unstable, short-hved intermediate (r = 0.5 /is) serves as the chemical activator, and such a short-hved species "is not consistent with attempts to identify a relatively stable dioxetane as the intermediate" (68). 

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. 

Several new oxalates have been developed for use ia analytical appHcations. Bis(2,6-difluorophenyl) oxalate (72) and bis(4-nitro-2-(3,6,9-trioxadecylcarbonyl)phenyl) oxalate (97) have been used ia flow iajection and high performance Hquid chromatography (hplc) as activators for chemiluminescence detectors. These oxalates are generally more stable and show better water solubiUty ia mixed aqueous solvents yet retain the higher efficiencies ( ) of the traditional oxalates employed for chemiluminescence. 

Because the chemiluminescence intensity can be used to monitor the concentration of peroxyl radicals, factors that influence the rate of autooxidation can easily be measured. Included are the rate and activation energy of initiation, rates of chain transfer in cooxidations, the activities of catalysts such as cobalt salts, and the activities of inhibitors (128). 

The use of molecular and atomic beams is especially useful in studying chemiluminescence because the results of single molecular interactions can be observed without the complications that arise from preceding or subsequent energy-transfer coUisions. Such techniques permit determination of active vibrational states in reactants, the population distributions of electronic, vibrational, and rotational excited products, energy thresholds, reaction probabihties, and scattering angles of the products (181). 

The rates and chemiluminescent intensities of atom-transfer reactions are proportional to the concentrations of the reactants, but the intensity is inversely proportional to the concentration of inert gas present. The latter quenches the excited state through coUision with an efficiency dependent on the stmcture of the inert gas. Chemiluminescence Qc increases with temperature, indicating that excitation has a higher activation energy than the ground state... 

Divalent copper, cobalt, nickel, and vanadyl ions promote chemiluminescence from the luminol—hydrogen peroxide reaction, which can be used to determine these metals to concentrations of 1—10 ppb (272,273). The light intensity is generally linear with metal concentration of 10 to 10 M range (272). Manganese(II) can also be determined when an amine is added to increase its reduction potential by stabili2ing Mn (ITT) (272). Since all of these ions are active, ion exchange must be used for deterrnination of a particular metal in mixtures (274). 

Present research is devoted to investigation of application of luminol reactions in heterogeneous systems. Systems of rapid consecutive reactions usable for the determination of biologically active, toxic anions have been studied. Anions were quantitatively converted into chemiluminescing solid or gaseous products detectable on solid / liquid or gas / liquid interface. Methodology developed made it possible to combine concentration of microcomponents with chemiluminescence detection and to achieve high sensitivity of determination. 

A note on the assays of coelenterazine and luciferase activity. The methods for measuring coelenterazine and the corresponding luciferases are given in Appendix C5. Special attention must be paid to the fact that coelenterazine in aqueous buffer solutions spontaneously emits a low level of chemiluminescence in the absence of any luciferase, which is greatly enhanced by the presence of various substances, including egg yolk, BSA and various surfactants (especially, hexadecyltrimethylammonium bromide). Therefore, the utmost care must be taken in the detection and measurement of a low level of... 

Spontaneous light emission from fresh material. For mycelium, the two data shown are scraped mycelium / mycelium on agar before scraping. cChemiluminescence activity of the extract in the presence of Fe + and H2O2. dThe extract was activated with methylamine, and then chemiluminescence activity was measured. A minus sign (—) indicates an unsuccessful quantitation due to various causes. 

Panal is stable in aqueous media (pH 1-5) at room temperature, except for a gradual hydration that can be reversed with an aqueous acid. Panal is activated into chemiluminescent compounds upon treatment with the salts of ammonia or primary amines. 

Fig. 9.4 Time course of the chemiluminescence reaction of (NH SO t -activated panal at pH values 4.5, 5.0, 5.5, and 6.0, in 3 ml of 10 mM acetate buffer in the presence of lOmg of CTAB, 20 pi of 0.1 M FeSC>4, and 20 pi of 10% H2O2 and at pH 8.0, in 3 ml of 50 mM Tris-HCl buffer containing 0.18 mM EDTA, 10 mg of CTAB, lOmg of NaHCC>3, 20pi of 0.1 M FeSC>4, and 20pi of 10% H2O2. All at 25°C. From Shimomura, 1989, with permission from the American Society for Photobiology.

Properties of PS-A and PS-B (Shimomura, 1991b Shimomura et al., 1993b). Both PS-A and PS-B are colorless viscous liquid, and their absorption spectra resemble that of panal (Fig. 9.6). By NMR analysis and mass spectrometry, PS-A and PS-B are found to be 1-O-decanoylpanal and 1-O-dodecanoylpanal, respectively. As a minor component, 1-O-tetradecanoylpanal has also been isolated. PS-A and PS-B gain chemiluminescence activity when treated with the salt of primary amines (see below for the conditions). Taking the activity obtained with methylamine as 100%, the activities obtained with other amines were ethylamine, 38% ethanolamine, 10% propylamine, 20% hexylamine, 3% and decylamine, 1%. 

The activation product of an equal-amount mixture of PS-A and PS-B gave four major chemiluminescent compounds, designated PM-1, PM-2, PM-3 and PM-4 in an approximate ratio of 1 2 2 1. The activation product of PS-A gave only PM-1, and that of PS-B gave only PM-4. 

PMs are orange-colored, with an absorption maximum at 488 nm (Fig. 9.6). The absorption characteristics and chemiluminescence activities of those compounds are shown in Table 9.4. All PMs are brightly fluorescent in yellow in organic solvents and also in aqueous solutions containing a surfactant (emission maxima 520-530 nm). The chemiluminescence spectra of PMs are significantly affected by the... 

Table 9.4 Absorption Characteristics and Chemiluminescence Activities of Various Compounds derived from the Fruiting Bodies of Panellus stipticus (Shimomura, 1991b)...

Compound Absorption Maximum (nm) (in methanol) Chemiluminescence Activity (1012 quanta/mg) after Activation with ... 

In the activation of PS-A and PS-B, treatment with methylamine resulted in a considerably higher chemiluminescence activity than with other amines. In the case of K-1, however, a significantly higher chemiluminescence activity was obtained with (NH SC or with hexy-lamine than with methylamine. In spite of this finding, methylamine was used in the activation of K-1 to prepare a model compound of PM. 

Fig. 9.10 Absorption spectra of K-1 (a model compound) and its chemiluminescent methylamine-activation products KM-1 and KM-2. All in methanol.

Fig. 9.13 Absorption spectrum of one of the luciferin precursors of Mycena cit-ricolor in methanol (dash-dot line, A.max 369 nm). The absorption and fluorescence emission spectra of the decylamine-activation product of the same precursor in neutral aqueous solution (solid lines abs. Amax 372 nm and fl. Xmax 460 nm), and in ethanol (broken lines abs. Amax 375 nm and fl. Amax 522 nm). The chemiluminescence spectrum of the same activation product (dotted line, A.max 580 nm). The dotted line (7max 320 nm) is the absorption spectrum of M. citricolor natural luciferin reported by Kuwabara and Wassink (1966).

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