Proc. Natl. Acad. Sci. USA Vol. 75, No. 1, pp. 30-33, January 1978 Chemistry of the : Key steps in the formation of the electronically excited state for model systems (chemiluminescence/electron transfer/dioxetane/fluorescence) JA-YOUNG Koo, STEVEN P. SCHMIDT, AND GARY B. SCHUSTER* Department of Chemistry, Roger Adams Laboratory, University of Illinois, Urbana, Illinois 61801 Communicated by David Y. Curtin, November 10, 1977

ABSTRACT The chemical mechanism for formation of elements of and forms an electronically excited electronically excited-state molecules from the thermal reaction singlet state of the observed emitting amide 3, with very high of dimethyldioxetanone was studied. Light production in the presence of certain easily oxidized aromatic hydrocarbons was efficiency. It is the mechanism of this last step, the chemiexci- found not to conform to the classical mechanistic schemes for tation step, that has not been previously understood and to chemiexcitation. Detailed investigation of the dioxetanone which we will direct our attention. s stem revealed light formation by the recently discovered, (See structure cuts 1, 2, and 3 at top offollowing page.) chemically initiated electron-exchange process. This result is extrapolated to bioluminescent systems. In particular, the key The cyclic peroxidic molecules dioxetane (4) and dioxetanone high-energy molecule involved in firefly luminescence, which (5) has been investigated in a number of elegant synthetic and has been identified as a dioxetanone, is postulated to form ex- mechanistic studies (6). The observation that the thermal re- cited states as a result of intramolecular electron transfer from actions of these high-energy compounds lead to the formation the phenoxythiazole moiety to the dioxetanone. Subsequent of electronically excited carbonyl compounds is taken as strong rapid decarboxylation results in direct formation of an excited circumstantial evidence for the involvement of a similar in- singlet state of the emitting amide. termediate in the bioluminescence of the firefly. The major Bioluminescent organisms are widely distributed throughout difficulty with this interpretation, however, has been the fact terrestrial and aquatic environments. Although the biological that simply substituted dioxetanes and dioxetanones give rise purpose of luminescence varies from species to species, the predominantly to the essentially nonluminescent triplet excited chemical mechanism for generation of the electronically excited state of the carbonyl compound. On the other hand, the natural state, which subsequently emits light, appears to be general in biolumescent systems form the emitting excited singlet state a wide variety of organisms. In nearly all of the bioluminescent with high efficiency, approximating 100%. Application of the processes that have been investigated, high-energy cyclic concepts of chemically initiated electron exchange lumines- peroxide molecules are implicated as providing the energy cence resolves this apparent incongruity. necessary for excited state generation (1). In the study of bioluminescent mechanisms the central concerns have been: EXPERIMENTAL (i) identification of the molecule capable of undergoing a re- General. Chemiluminescence was detected by the photon- action with a free energy change sufficient to permit excited counting technique using an EMI 9813 photomultiplier tube. state generation, (ii) characterization of the emitting species, Spectral resolution was achieved with a Jarrel-Ash/Ebert and (iii) identification of the molecular process that converts 0.25-m grating monochromator. Emission intensities were the high-energy reactant to an electronically excited product corrected for photocathode response and monochromator ef- molecule. ficiency by using the manufacturer's spectral sensitivity data Our recent investigations (2) of chemiluminescence have led and the centers of gravity for the emission spectra. Temperature to the discovery of a general mechanism of excited state for- control was maintained to within ±0.050 by means of an ex- mation identified as chemically initiated electron exchange ternal temperature bath. All solvents were purified by passing luminescence. Studies of exergonic chemical reactions that them through a column of activated alumina followed by dis- model bioluminescent systems now permit us to suggest that tillation. Rubrene, naphthacene, perylene, and 9,10-diphen- this mechanism is operative in the formation of electronically ylanthracene (DPA) was purchased from Aldrich Chemical Co. excited states in living organisms. The light-forming reaction and purified by chromatography on alumina and recrystalli- of the North American firefly (Photinus pyralis) will serve as zation before use. the prototypical case. The conclusions reached from this system Dimethyldioxetanone was prepared according to the pro- are readily extended to other bioluminescent reactions. cedure of Adam and Liu (7). This compound was found to be Many excellent studies of bioluminescence from the firefly remarkably sensitive to minute quantities of impurities. Ex- have led to the characterization of the enzyme/substrate system treme care must be used in purification of solvents and additives involved in the light generation step (3). In summary, the sub- to supress the impurity catalyzed decomposition pathways. strate has been identified as 1 and independently Chemiluminescence of Dimethyldioxetanone in the synthesized. Reaction of 1 with oxygen in the presence of the Presence of Aromatic Hydrocarbons. Solutions of dioxetanone enzyme generates a high-energy content molecule in CH2Cl2 (typical concentration, 1-10 1AM) and the appropriate that has been identified as the dioxetanone (2) by 180 labeling aromatic hydrocarbon (typical concentration, 10-100,uM) were studies (4, 5). In order to produce bioluminescence, 2 loses the prepared. The solutions were deoxygenated by purging with purified, filtered argon for 3 min at 00. The chemiluminescent The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Abbreviations: DPA, 9,10-diphenylanthacene; CIEEL, chemically "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate initiated electron exchange luminescence. this fact. * To whom reprint requests should be addressed. 30 Downloaded by guest on September 27, 2021 Chemistry: Koo et al. Proc. Natl. Acad. Sci. USA 75 (1978) 31 H 0.-0-0 )c3C_

1 2 3

4 5

0 + Co2 0 6 7 intensity was measured by integrating the total light emission Chemically Initiated Electron Exchange Luminescence. at the maximum wavelength for the aromatic hydrocarbon In the course of our studies of the thermal reactions of high- fluorescence. energy content molecules, we established that the chemilu- minescence from diphenoyl peroxide (6) did not conform to RESULTS AND DISCUSSION either of the conventional mechanisms for chemical light for- Conventional Mechanisms for Chemiluminescence. Pre- mation. The thermolytic conversion of peroxide 6 to benzo- vious studies of chemiluminescent phenomena have led to the coumarin (7), in ca 70% yield, occurs with no significant gen- evolution of two broad classes of mechanisms for chemiexci- eration of an electronically excited state of 7. However, it was tation. In the first, thermal rearrangement of a high-energy found that inclusion of any one of several easily oxidized aro- content molecule, reacting directly or through an intermediate, matic hydrocarbons in the reaction solution led to efficient generates an electronically excited state of a product molecule. chemiexcitation of the hydrocarbon and bright light emission. Emission of a photon of light from the excited molecule results Moreover, it was demonstrated that the aromatic hydrocarbon in observable direct chemiluminescence. Alternatively, the exerted a powerful catalytic effect on the decomposition of 6. excited product molecule may enter into a bimolecular energy In addition, it was shown that it is this catalytic path that gives transfer reaction with a suitable energy acceptor. This process rise to the chemiluminescence. Significantly, the rate at which results in formation of the electronically excited acceptor which, the aromatic hydrocarbon catalyzes chemiexcitation, and the in a subsequent step, emits a photon of light, producing indirect actual yield of electronically excited states, correlates with the chemiluminescence. This sequence is shown schematically in one-electron oxidation potential of the aromatic hydrocarbon Fig. 1. (Fig. 3). Thus, we have identified the initiating step of this re- The second general mechanism for chemiexcitation that has action sequence as an activated electron transfer from the ar- been well documented is electrogenerated chemiluminescence omatic hydrocarbon to diphenoyl peroxide. Subsequent rapid (8). In this approach a radical cation (usually generated by decarboxylation and ring closure generate the radical anion of electrochemical oxidation at a suitable anode) and a radical benzocoumarin within the same solvent cage as the radical anion (typically the result of reduction at a cathode) form a cation of the aromatic hydrocarbon. Charge annihilation of diffusive encounter pair. Charge annihilation in the encounter these species then generates the luminescing excited state of the ion pair results in the generation of an electronically excited aromatic hydrocarbon. This reaction sequence is detailed in Fig. state. Subsequent emission of light from this excited state, or 4. Further convincing evidence that this reaction proceeds one derived from it, results in detectable luminescence. This through a charge annihilation involving benzocoumarin radical general is shown in 2. anion is derived from the observation of exciplex emission when pathway schematically Fig. triphenylamine is the electron donor. The chemiluminescent

High-energy Thermal Electronically Energy transfer Excited molecule reaction excited state acceptor acceptor A Direct Indirect Ad - B+ B | Light Light or or Cathode Anode photochem ical photochem ical reactI reaction Annihilation Examples: (A B) electronicoly excited (A,B,or Exciplex) 0-0 MO - Picture a) 1,2-Dioxetanes 4A- Lumo - b) Luminol -L ight H2 NH2 °- Homo - -t c) Dewar benzene 1iID (A-) (B+) Excited State FIG. 1. Conventional chemiluminescence of organic molecules. FIG. 2. Electrogenerated chemiluminescence. Downloaded by guest on September 27, 2021 32 Chemistry: Koo et al. Proc. Natl. Acad. Sci. USA 75 (1978)

12. 0

10 ArH 2 , ; g: ArH j 8.).0 ,,,,//I 0 a- 61.0

4~.0 [g:ArH4] + C02 - ArH + 0 N0 2.

Il Il Il Il Il l -0.5 0.6A0.7 l- 08 0.9 10 1.1 1.2 1.3 1.4 Oxidation Potential (eV vs. SCE) FIG. 3. Effect of the oxidation potential of the aromatic hydro- Light carbon on the rate of the induced decomposition of diphenoyl per- FIG. 4. Chemically initiated electron exchange mechanism for oxide.aP In order of decreasing oxidation potential the points are: ru- the chemiluminescence of diphenoyl peroxide. brene, naphthacene, perylene, DPA, chrysene, and pyrene. available to the high-energy bioluminescent intermediate, exciplex emission in this case is identical in all respects to the dioxetanone 3, for chemiexcitation it is immediately clear that emission generated by photoexcitation of mixtures of benzo- this molecule is ideally constituted for excited state production coumarin and triphenylamine. We have designated this se- by intramolecular chemically initiated electron exchange. In quence of reactions leading to light emission "chemically ini- particular, electron transfer from the easily oxidized polycyclic tiated electron exchange luminescence (CIEEL)" (2). heterocycle portion of the molecule to the high-energy dioxe- We have demonstrated the generality of chemiexcitation by tanone moiety followed by rapid decarboxylation will generate the CIEEL mechanism in a number of different systems. Of a charge transfer resonance structure of the excited state of the most relevance to the present discussion of bioluminescent observed emitter. If decarboxylation is faster than spin equili- mechanisms is the dioxetanone system. Initial investigation of bration, the intramolecularity of this process prejudices the dioxetanone thermolysis led to the observation that unimolec- reaction toward the formation of excited states of singlet mul- ular decomposition generates triplet excited carbonyl com- tiplicity, as is observed in the living system. This reaction se- pounds in at least 20 times higher yield than the singlet excited quence is:

0 ( H0:XN N ssN /O).NQN 0 Electron donor Excited singlet state carbonyl compound (9). In a later report, Adam et al. (10) noted Consistent with this mechanistic postulate, it has been ob- that the overall yield of light was greater by a factor of 20 when served that methylation of the phenolic oxygen of firefly lu- rubrene was included in the reaction solution than when DPA ciferin makes the system nonbioluminescent although the was the additive. This result was interpreted as being due to a methylated itself fluoresces efficiently (12). This is the triplet-triplet annihilation reaction. However, triplet-triplet expected result if intramolecular CIEEL is responsible for light annihilation as the cause of the increased light yield is in- production. The methylated substrate is anticipated to be much consistent on theoretical grounds and has recently been shown more difficult to oxidize than the phenoxide anion (13). experimentally not to be possible under the chemiluminescent In summary, we have demonstrated that our recently dis- conditions reported (11). covered CIEEL mechanism for chemical light formation op- We have investigated the chemiluminescent reaction of di- erates in the dioxetanone system. The dioxetanone is a reason- methyldioxetanone. Our results show that the rate of reaction able model for firefly bioluminescence. Application of CIEEL of dioxetanone is dependent upon the presence, concentration, to these bioluminescent systems results in an explanation for and nature of easily oxidized aromatic hydrocarbons. Also, most the observed high singlet yield and the effect of methylation. significantly, for the series of aromatic hydrocarbons DPA, Moreover, chemiexcitation by this path is quite efficient bio- perylene, 9,10-diphenylethynylanthracene, and rubrene, the logically. Nature has built both an effective eletron donor and total light yield is correlated with the oxidation potential of the a high-energy electron acceptor, as well as an efficiently fluo- hydrocarbon. For example, at identical hydrocarbon concen- rescing structure, into the same molecule. Certainly this is an trations in CH2Cl2 solution, the light intensity from rubrene- effective way to ensure high-yield light formation. activated solutions is more than 75 times that from DPA-acti- vated solutions. These observations implicate CIEEL as the G.B.S. is a fellow of the Alfred P. Sloan Foundation. This work was major light-producing reaction pathway for dioxetanones. If supported in part by the Office of Naval Research and in part by a simple electronic energy transfer from the initially formed grant from the Research Corporation. singlet excited state of acetone to the aromatic hydrocarbon was responsible for light production, than all of the aromatic hy- 1. McCapra, F. (1976) "Chemical mechanisms in bioluminescence," drocarbons, when corrected for fluorescence efficiency, should Acc. Chem. Res. 9,201-208. have generated the same photon yield. 2. Koo, J.-Y. & Schuster, G. B. (1977) "Chemically initiated electron Bioluminescence by CIEEL. In considering the options exchange luminescence. A new chemiluminescent reaction path Downloaded by guest on September 27, 2021 Chemistry: Koo et al. Proc. Natl. Acad. Sci. USA 75 (1978) 33 for organic peroxides," J. Am. Chem. Soc., 99, 6107-.6109. 213-264. 3. McElroy, W. D. & DeLuca, M., (1973) in Chemiluminescence 9. Turro, N. J., Lechtken, P., Schuster, G., Orell, J., Steinmetzer, and Bioluminescence, Cormier, M. J., Hercules, D. M. & Lee, H.-C. & Adam, W. (1974) "Indirect chemiluminescence by J. eds. (Plenum Press, New York), pp. 285-313. 1,2-dioxetanes," J. Am. Chem. Soc. 96, 1627-1629. 4. White, E. H., Maino, J. D. & Umbreit, M. (1975) "On the 10. Adam, W., Simpson, G. A. & Yany, F. (1974) "Mechanism of mechanism of firefly luciferin luminescence," J. Am. Chem. Soc. direct and rubrene enhanced chemiluminescence during a- 97, 198-200. peroxylactone decarboxylation," J. Phys. Chem. 78, 2559- 5. Shimomura, O., Goto, T. & Johnson, F. H. (1977) "Source of 2569. oxygen in the CO2 produced in the bioluminescent oxidation of 11. Liu, D. K. K. & Faulkner, L. R. (1977) "P-type delayed fluores- firefly luciferin," Proc. Natl. Acad. Sci. USA 74,2799-2802. cence from rubrene," J. Am. Chem. Soc. 99, 4594-4599. 6. Wilson, T. (1976) Int. Rev. Sci.: Phys. Chem. Ser. Two 9,265- 12. McCapra, F. (1970) "Chemiluminescence of organic com- 311. pounds," Pure Appl. Chem. 24, 611-629. 7. Adam, W. & Liu, J.-C., (1972) "An a-peroxy lactone. Synthesis 13. Mann, C. K., & Barnes, K. K. (1970) Electrochemical Reactions and chemiluminescence," J. Am. Chem. Soc. 94, 2894-2895. in Nonaqueous Systems (Marcel Dekker, New York), pp. 8. Faulkner, L. R. (1976) Int. Rev. Sci.: Phys. Chem. Ser. Two 9, 245-259. Downloaded by guest on September 27, 2021