Evidence supporting a 1,2-dioxetanone as an intermediate in the benzofuran-2(3H)-one chemiluminescence

Abstract

The mechanism of the chemiluminescent reaction of ethyl (5-fluoro-2-oxo-2,3-dihydrobenzofuran-3-yl) carbamate (a 2-coumaranone derivative) with a base and molecular oxygen was investigated. New evidence from the reaction kinetics and absorption/emission profiles was obtained, supporting the existence of a 1,2-dioxetanone as an intermediate: (i) its characteristic activation parameters (ΔH^≠ = 7.2 ± 0.1 kcal mol⁻¹; ΔS^≠ = −45 ± 5 cal K⁻¹ mol⁻¹) indicating a high degree of thermal instability and (ii) its bimolecular decomposition rate constant for the reaction with perylene. The newly developed methodology has been shown to be suitable for determining the reactivity of such thermally unstable peroxides, which are very difficult to prepare and isolate, using this alternative approach of in situ generation of a 1,2-dioxetanone.

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Introduction

The role of organic peroxides in chemiluminescence is crucial at the level of chemiexcitation, i.e. the formation of species at the excited electronic state by means of a chemical reaction. Within the large pool of chemiluminescent and bioluminescent reactions, one can name the presence of the model peroxides 1,2-dioxetanes (1), 1,2-dioxetanones (2) or 1,2-dioxetanedione (3) as almost obligatory.¹

1,2-Dioxetanones

1,2-Dioxetanones are postulated intermediates in firefly bioluminescence² and in chemiluminescence of acridinium esters,³ among many other chemiluminescent systems. The synthesis and isolation of those compounds are very difficult, due to their reactivity towards nucleophiles and electrophiles, and their thermal instability; it is thus an arduous task to work with such substances and study them from the chemical reactivity point of view.⁴

The CIEEL mechanism

In chemiluminescent systems, the peroxide is normally a high-energy species which is somehow able to transfer its energy to an acceptor; however, research made in this field in the 1970s showed that the acceptor is indeed an activator (ACT), since in many cases it is able to catalyse the decomposition of the peroxide. Schuster and coworkers proposed a theory able to explain this activation of peroxides by ACTs (in this case fluorescent aromatic polycyclic hydrocarbons with low oxidation potentials) in a series of papers where he proposed the occurrence of the intermolecular CIEEL (Chemically Initiated Electron Exchange Luminescence) mechanism.⁵ Many following papers have confirmed this proposition of Schuster's group, widening the scope of this mechanism, including systems where chemiexcitation may occur through a charge-transfer process;⁶ it seems clear by now that the CIEEL theory is well supported by experimental evidence.^7,8

Decomposition of 1,2-dioxetanones

The decomposition of 1,2-dioxetanones, resulting in light emission, may take place via two main pathways (Scheme 1).⁹ The first one is by the thermal cleavage of such unstable compounds, a unimolecular process with a rate constant k_D. The yields of singlet excited state production by this process are usually very low (Φ_S < 0.01%); therefore, it is not a good model for firefly bioluminescence (Photinus pyralis), with a recently redetermined emission quantum yield of Φ_BL = 41%.²

Scheme 1 Possible interactions of a 1,2-dioxetanone producing excited states. The bimolecular decomposition is based on the intermolecular CIEEL mechanism proposition.

The second possible mechanism is by a bimolecular reaction with an ACT, an intermolecular version of the CIEEL mechanism.¹⁰ The yields of excited singlet states in this case are much higher, with Φ_S ∼ 10%. The interaction between a peroxide like 2 and an ACT consists of five elementary steps: (i) interaction of the ACT and the peroxide inside a solvent cage, forming a charge-transfer type encounter complex, a process with an equilibrium constant K_CT; (ii) electron transfer from the ACT to the peroxide, forming a pair of radical ions (k_ET), as the rate-determining step for the reaction; (iii) cleavage of the C–C bond eliminating CO₂ in an irreversible process (k_CV); (iv) electron back-transfer from the radical anion to the radical cation, forming the ACT at the excited singlet state (k_EBT); (v) decay of the excited ACT to the ground state resulting in light emission (k_F).

Because the charge-transfer reaction is the slowest, it is the rate-determining step of the whole process. So the observed rate constant (k_obs) is defined by the sum of the rates of the unimolecular decomposition (k_D) and the bimolecular decomposition k_CAT [ACT].

2-Coumaranone chemiluminescence

The chemiluminescence of 2-coumaranone derivatives like 4 was first described by Lofthouse et al. in 1979;¹¹ a simple mixture of the compounds with air-saturated DMF produced a bright emission which lasted for a couple of hours; the same reaction was also observed in other polar solvents, which require the addition of non-nucleophilic bases. However, the mechanism behind this emission has been only tentatively suggested, without being strongly backed by evidence; only recently, in an article by Schramm et al., a systematic approach for the mechanism of the reaction has been addressed.¹²

It is assumed that the chemiluminescent step involves the formation and decomposition of the 1,2-dioxetanone 5 (Scheme 2), formed after an unknown number of steps, probably by a radicalic mechanism. The intermediate 5 has never been isolated or characterized, and therefore its existence is just a suggestion at this point.

Scheme 2 Reaction of the studied 2-coumaranone with a base and oxygen forming the intermediate 1,2-dioxetanone, which decays emitting light.

Experimental section

General materials

The ethyl (5-fluoro-2-oxo-2,3-dihydrobenzofuran-3-yl) carbamate 4 was prepared following the method of Schramm et al.¹² Perylene (PER, Acros, 99+%) was used as received. Acetonitrile (ACN, Aldrich, anhydrous, 99.8%) was stored over molecular sieves (Aldrich, 3 Å, beads 8–12 mesh). 1,8-Diazabicyclo-[5.4.0]undec-7-ene (DBU, Aldrich, 98%) was used as received.

The quartz cuvettes used (Hellma, QS suprasil) had a volume of 3.0 mL and an optical path of 10.00 mm, with two polished sides for absorption assays and four sides for fluorescence assays.

Kinetic assays

All kinetic assays were carried out in fluorescence or absorption quartz cuvettes. The standard assay was done by filling the cuvette with 3.0 mL of ACN or the solution of PER in ACN of the desired concentration. This cuvette was left for 5 minutes in the cell holder thermostatized at 25.0 °C, and 20 μL of a 15 mM solution of 4 in ACN was injected, the resulting concentration in the cuvette being [4] = 0.1 mM. The reaction was started with the injection of 5 μL of DBU, and data acquisition was started after a quick shake made by hand (∼3 s). The kinetics was followed for at least five half-lives, and the obtained k_obs values are the means of at least three experiments.

Results

The solvent chosen to conduct all assays was acetonitrile (ACN); even if it was shown that dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) led to higher emission quantum yields, in ACN the decomposition time-profile of the high-energy intermediate shows a clean first-order decay. The high solubility of oxygen (1.9 mM at 0.213 bar O₂) in ACN¹³ is also another advantage, since using 0.1 mM of 4 we set a pseudo-first order condition throughout the experiments.¹⁴

UV-Vis absorption studies

A solution of 4 in ACN is stable throughout a standard experiment procedure, as the spectra acquired within one hour showed no difference. Compound 4 absorbs in the UV region (λ_max = 280 nm; ε = 2.07 × 10³ M⁻¹ cm⁻¹ in ACN), which are somehow characteristic parameters for this class of substances. However, upon addition of DBU (which absorbs in wavelengths lower than 270 nm), a new transient absorption band is formed with fairly complex spectroscopic features, with λ_max = 290 and 332 nm; the compound responsible for this absorption, hereafter called A, decays forming another compound, B, which this time absorbs at even higher wavelengths (λ_max = 378 nm); this last one is stable throughout the experiment duration. An important point to be noted from the UV spectra is the presence of an isosbestic point at λ = 356 nm; also, as the absorption of those three species is well spaced, they can be followed separately (Fig. 1).

Fig. 1 Absorption spectra of the 2-coumaranone 4, intermediate A and the final product B.

A study of the kinetics for the formation of A and B was carried out, and matching rate constants were found for the decomposition of A and the formation of B. Those constants (k_obs = 0.01 s⁻¹ and −0.01 s⁻¹ at 25 °C) were shown to be independent from the base concentration. From the determination of the rate constant for the formation of B in different temperatures, an Eyring plot was obtained which led to the determination of the activation parameters for this process, which are: ΔH^≠ = 7.85 ± 0.05 kcal mol⁻¹; ΔS^≠ = −41 ± 1 cal K⁻¹ mol⁻¹; ΔG^≠ = 20.1 ± 0.3 kcal mol⁻¹ at 25 °C.

Emission studies

The addition of DBU to a solution of 4 in ACN showed a bright blue chemiluminescence emission that could be readily observed even in daylight. The rate constant for this emission has been shown to be independent of the concentration of the base (k_obs ∼ 0.01 s⁻¹ at 25 °C); through an Eyring plot the following activation parameters were obtained: ΔH^≠ = 7.2 ± 0.1 kcal mol⁻¹; ΔS^≠ = −45 ± 5 cal K⁻¹ mol⁻¹; ΔG^≠ = 20 ± 2 kcal mol⁻¹ at 25 °C.

To confirm the proposed mechanism operating at the chemiexcitation process and also to get clues about the structure of the high-energy intermediate, this chemiluminescent reaction of 4 triggered by the addition of DBU was also studied in different concentrations of perylene (PER) as an activator.

First, emission spectra of the chemiluminescence of 4 in the absence and presence of PER were acquired (data not shown). In the absence of PER, the chemiluminescent emission spectrum has a broad-shaped profile, with a maximum centred at ca. 440 nm; this profile of chemiluminescence matches exactly the fluorescence of the spent reaction mixture. With the addition of PER, the chemiluminescent emission spectrum changes radically, turning into a fine structured emission which, this time, matches the fluorescence of PER.

A linear correlation was indeed observed between the light emission rate constant and the concentration of PER, and a bimolecular rate of k_bim = 3.05 ± 0.15 M⁻¹ s⁻¹ (R = 0.996) could be obtained (Table 1), together with the values for the relative quantum yield determined by integration of the chemiluminescent emission (Fig. 2).

Fig. 2 Linear dependence of the observed rate constant (k_obs) with the perylene concentration.

Table 1 Observed rate constants (k_obs) and relative quantum yields (Φ_CL) for the chemiluminescent emission of 4 triggered by DBU in different concentrations of perylene

[PER] (mM)	k obs (s^−1)	Φ CL ^a (%)
Conditions: Acetonitrile, 25 °C, [4] = 0.1 mM, [DBU] = 10 mM.a Standard deviation of all measurements is <2%.
0	0.0108 ± 0.0002	91
0.1	0.0108 ± 0.0001	100
0.2	0.0111 ± 0.0001	99
0.4	0.0117 ± 0.0001	96
0.6	0.0124 ± 0.0001	91
0.8	0.0131 ± 0.0002	86
1.0	0.0135 ± 0.0003	83

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Discussion

It seems to be certain from experiments in the absence of perylene that k_obs for light emission, i.e. the decay rate of the emission, is the rate constant for the decomposition of the intermediate 5, being the rate-determining step.

Since the rate constants for the emission decay and the formation of B or decomposition of A, measured respectively in a fluorescence- and a UV-Vis spectrometer, are essentially the same; we can address A as intermediate 5, while B is the decomposition product.

The activation parameters (Table 2) were obtained by following the kinetics of emission and absorption in different temperatures; matching values for the activation enthalpy and entropy, as well as for the Gibbs free energy, were indeed obtained.

Table 2 Activation parameters for the decomposition of intermediate 5 followed by absorption or light emission^a

	ΔH^≠ (kcal mol^−1)	ΔS^≠ (cal mol^−1 K^−1)	ΔG^≠ at 25 °C (kcal mol^−1)
a Conditions: Acetonitrile, 25 °C, [4] = 0.1 mM, [DBU] = 10 mM.
Absorption	7.85 ± 0.05	−41 ± 1	20.1 ± 0.3
Emission	7.2 ± 0.1	−45 ± 5	20 ± 2

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A relatively low value for the activation enthalpy was observed, which is as expected since the peroxidic intermediate 5, a 1,2-dioxetanone, should be very thermal labile. However, a surprisingly very high value for the activation entropy was obtained, indicating that the mechanism of such decomposition is entropically controlled.

A value of ca. −40 cal mol⁻¹ K⁻¹ for the activation entropy could indicate a bimolecular process in which there is an encounter of two different species at the transition state; however, it does not seem to be the case, since a unimolecular decomposition mechanism was postulated for the process of light emission from the decomposition of 1,2-dioxetanones.¹⁵ Another possibility is the occurrence of a major structural rearrangement of the compound just before the rate-determining step. This would occur at the molecular geometry level, without prominent cleavage of chemical bonds.

Highly negative activation entropies for the unimolecular decomposition of 1,2-dioxetanones have already been observed, and there is not any accepted explanation for this phenomenon. Baader and de Oliveira¹⁶ proposed the existence of a dark decomposition pathway, which is catalyzed by impurities (mainly metal cations), which may be the reason for those negative values of entropy, since the process would have a multimolecular rate-determining step. No formal proposal of this idea has been published until now, although it is backed by experimental evidence; therefore, it remains just a proposition.

The fact that some peroxides may undergo an intramolecular type CIEEL mechanism, producing light emission in high yields, seems to be not the present case. Noting that 5 is a phenolate, able to transfer its charge to the peroxidic ring triggering a chemiluminescent reaction cascade, the ΔS^≠ value is far too low when compared to similar systems.¹⁷ In addition, when the phenolate is located at the ortho position relative to the peroxide, the quantum yields are then very low,¹⁸ and both the situations seem not to be the case here.

The suggestion of a 1,2-dioxetanone as an intermediate in the chemiluminescence of 2-coumaranones should be seriously considered, since the intermediate does interact with an added activator, as other 1,2-dioxetanones do with reasonable rates. The reactivity of 5 with perylene has the value of the bimolecular rate constant k_CAT = 3.05 ± 0.15 M⁻¹ s⁻¹. This constant has also been determined by other authors for the 1,2-dioxetanones 6 (3,3-dimethyl-1,2-dioxetanone), 7 (spiro-cyclopentyl-1,2-dioxetanone) and 8 (spiro-adamantyl-1,2-dioxetanone) under slightly different conditions (Table 3).

Table 3 First order decomposition rates (k_D) and second-order rates (k_CAT) with perylene for some 1,2-dioxetanones

Entry	Structure	k D (10^−3 s^−1)	k CAT (M^−1 s^−1)
a Determined in this work: ACN, 25 °C. b Ref. 9, determined in CH2Cl2 at 25 °C. c Ref. 19, determined in CH2Cl2 at 27 °C. d Ref. 20, determined in toluene at 25 °C. e Ref. 16, determined in toluene at 50 °C.
5		10.8 ± 0.01^a	3.05 ± 0.15^a
6		2 ± 1^b	0.247 ± 0.030^c
7		1.0 ± 0.1^d	1.26 ± 0.06^d
8		0.25^e	1.81 ± 0.09^e

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The decrease of the emission quantum yields with an increase of PER concentration (Table 1) could be explained by the occurrence of its bimolecular interaction with 5. As the PER concentration increases, the proportion of intermolecular CIEEL increases too; however, this type of interaction has markedly low emission quantum yields,⁹ additionally supporting the proposed general mechanism. A complete determination of the emission (Φ_CL) and singlet quantum yields (Φ_S) would contribute to confirming the suggestions made in this paper.

Conclusions

In this work we were able to obtain kinetic data for the chemiluminescent reaction of ethyl (5-fluoro-2-oxo-2,3-dihydrobenzofuran-3-yl) carbamate with a base and oxygen. They point to the existence of a peroxidic intermediate, which is probably a 1,2-dioxetanone with low thermal stability. The data obtained also confirmed the viability of generating 1,2-dioxetanones and studying their reactivity towards reducing compounds in situ without the need of isolation and purification.

Acknowledgements

The authors thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; LFMLC 2012/02428-5 and FHB 2012/13807-7) for financial support.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Kinetic data in all conditions used. See DOI: 10.1039/c3pp50345c

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