Chemiluminescence, dioxetanes

In the past few years two rather distinct classes of chemiluminescent dioxetanes have become evident. Alkyl, alkoxy, and simple aryl substituted dioxetanes, which includes the earliest dioxetanes prepared, are characterized... 

Chemical off—on switching of the chemiluminescence of a 1,2-dioxetane (9-benzyhdene-10-methylacridan-l,2-dioxetane [66762-83-2] (9)) was first described in 1980 (33). No chemiluminescence was observed when excess acetic acid was added to (9) but chemiluminescence was recovered when triethylamine was added. The off—on switching was attributed to reversible protonation of the nitrogen lone pair and modulation of chemically induced electron-exchange luminescence (CIEEL). Base-induced decomposition of a 1,2-dioxetane of 2-phen5l-3-(4 -hydroxyphenyl)-l,4-dioxetane (10) by deprotonation of the phenoHc hydroxy group has also been described (34). 

In addition to ready thermal decomposition, 1,2-dioxetanes are also rapidly decomposed by transition metals (39), amines, and electron-donor olefins (10). However, these catalytic reactions are not chemiluminescent as determined by the temperature drop kinetic method. 

A number of chemiluminescent reactions may proceed through unstable dioxetane intermediates (12,43). For example, the classical chemiluminescent reactions of lophine [484-47-9] (18), lucigenin [2315-97-7] (20), and transannular peroxide decomposition. Classical chemiluminescence from lophine (18), where R = CgH, is derived from its reaction with oxygen in aqueous alkaline dimethyl sulfoxide or by reaction with hydrogen peroxide and a cooxidant such as sodium hypochlorite or potassium ferricyanide (44). The hydroperoxide (19) has been isolated and independentiy emits light in basic ethanol (45). 

Classical chemiluminescence from lucigenin (20) is obtained from its reaction with hydrogen peroxide in water at a pH of about 10 Qc is reported to be about 0.5% based on lucigenin, but 1.6% based on the product A/-methylacridone which is formed in low yield (46). Lucigenin dioxetane (17) has been prepared by singlet oxygen addition to an electron-rich olefin (16) at low temperature (47). Thermal decomposition of (17) gives of 1.6% (47). 

Dioxetane decomposition has also been proposed to account for chemiluminescence from other reactions (43), including gas-phase reactions of singlet oxygen with ethylene and vinyl ethers (53). 

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). 

Addition of fluorescent energy acceptors such as 9,10-dibromoanthracene substantially increases chemiluminescence intensity by transferring excitation energy (132,133), as is the case with dioxetanes. 

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

One is the concerted decomposition of a dioxetanone structure that is proposed for the chemiluminescence and bioluminescence of both firefly luciferin (Hopkins et al., 1967 McCapra et al., 1968 Shimomura et al., 1977) and Cypridina luciferin (McCapra and Chang, 1967 Shimomura and Johnson, 1971). The other is the linear decomposition mechanism that has been proposed for the bioluminescence reaction of fireflies by DeLuca and Dempsey (1970), but not substantiated. In the case of the Oplopborus bioluminescence, investigation of the reaction pathway by 180-labeling experiments has shown that one O atom of the product CO2 derives from molecular oxygen, indicating that the dioxetanone pathway takes place in this bioluminescence system as well (Shimomura et al., 1978). It appears that the involvement of a dioxetane intermediate is quite widespread in bioluminescence. 

Alkaline phosphatase-labeled probes are synthesized so that 18 bases are complementary to sequences on the arms of the bDNA. Three hybridization sites are located on each branch for a total binding capacity of 45 labeled probes per bDNA molecule. The alkaline phosphatase catalyzes the dephosphorylation of chemiluminescent substrate, dioxetane (Lumi-Phos Plus, Lumigen, Detroit, MI). The intensity of the light emission is measured with a plate luminometer as relative luminescent units. 

In 1982, the Schaap group demonstrated that chemiluminescence can be induced by the addition of a base to dioxetanes bearing a phenolic substituent [11]. Herein, the same group presents a method utilizing aryl esterase to catalyze the cleavage of a naphthyl acetate-substituted dioxetane in aqueous buffer at ambient... 

Chemiluminescence is defined as the production of light by chemical reactions. This light is cold , which means that it is not caused by vibrations of atoms and/or molecules involved in the reaction but by direct transformation of chemical into electronic energy. For earlier discussions of this problem, see 7 9h Recent approaches towards a general theory of chemiluminescence are based on the relatively simple electron-transfer reactions occurring in aromatic radical-ion chemiluminescence reactions 10> and on considerations of molecular orbital symmetry as applied to 1.2-dioxetane derivatives, which very probably play a key role in a large number of organic chemiluminescence reactions 11>. 

Me Capra in particular proposed n> that the chemiluminescence reactions of a large number of organic compounds had this concerted dioxetane decomposition step as key reaction in the production of electronically excited products, namely acridinium salts 25,26,27) indolylperoxides 28>, activated oxalic esters 29>, diphenyl carbene 30>, tetrakis-dimethylamino-ethylene 31 32>, lucigenin 33>, and substituted imidazoles 23>. 

The recently discovered preparative methods for the synthesis of 1.2-dioxetane derivatives (see Section V.) have made these compounds and their chemiluminescent decomposition the subject of especially intensive study. 

As mentioned in Section II. C., the concerted bond cleavage of 1.2-dioxetane derivatives has been proposed to be of general importance in respect of the excitation step of a large number of chemiluminescence reactions. The first experimental results concerning simple dioxetanes were obtained by M. M. Rauhut and coworkers in their work on activated oxalic ester chemiluminescence 24>. From experimental data on the reaction of e.g. bis (2.4-dinitrophenyl)oxalate with hydrogen peroxide in the presence of rubrene, they concluded that 1.2-dioxetanedione... 

Alkoxy-dioxetanes undergo chemiluminescent thermolysis in the pres-... 

A peculiar effect was observed in the decomposition of 19 a with anthracene as fluorescer when oxygen was carefully removed from the solutions an increase of the chemiluminescence decay rate and of the dioxetane cleavage resulted. It was suggested that this was due to a catalytic effect of triplet anthracene (formed by energy transfer from triplet formate) on the decomposition of the dioxetane. When oxygen is present, triplet anthracene is quenched. Whether such a catalytic effect of triplet anthracene or similar compounds on dioxetane cleavage actually exists has not yet been fully established positive effects were observed by M. M. Rauhut and coworkers 24> in oxalate chemiluminescence and by S. Mazur and C. S. Foote 80> in the chemiluminescent decomposition of tetramethoxy-dioxetane, where zinc tetraphenylporphy-rin seems to exert a catalytic effect. However, the decomposition of trimethyl dioxetane exhibits no fluorescer catalysis 78h... 

Dioxetan-ones appear to be intermediates in the chemiluminescent reaction of singlet oxygen with ketenes, in the presence of fluorescers 81> ... 

The dioxetane derivative 79 may be formed as intermediate in the brilliant chemiluminescence reaction between 10,10 -dimethyl-9,9 -bi-acridylidene and excited-singlet oxygen 125>. Chemiluminescence also occurs when potassium cyanide is added to lucigenin solutions in the... 

The bioluminescence of the American firefly (Photinus pyralis) is certainly the best-known bioluminescent reaction, thanks to the work of Me Elroy and coworkers and E. H. White and his group (for references see P, p. 138, 6,168,169)) The substrate of this enzyme-catalyzed chemiluminescent oxidation is the benzothiazole derivative 107 (Photinus luciferin) which yields the ketone 109 in a decarboxylation reaction. The concept of a concerted cleavage of a dioxetane derivative has been applied to this reaction 170> (see Section II. C.). Recent experiments with 18C>2 have challenged this concept, as no 180-containing carbon dioxide was detected from the oxidation of 107 171>. 

Certain Schiff bases, i.e. 122, were synthesized as model compounds for Latia luciferin. This compound exhibits strong blue chemiluminescence ( max 385 nm) on oxidation with oxygen in DMSO/potassium t.-butylate, the main products being acetone and 2-formamido pyridine 124. The mechanism suggested by Me Capra and Wrigglesworth includes the concerted bond cleavage of a dioxetane derivative 123. 

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