passive and active role of fluorescers in the chemiluminescence of tetramethoxydioxetane

Phnmwhmisrry und Phorohioloyy. Vol. 30. pp. I77 10 183 8 Pergamon Press Lld 1979. Printed in Great Britain

W.1 1-X655;79/0701-0177102.~XJ’O

RESEARCH NOTE PASSIVE A N D ACTIVE ROLE OF FLUORESCERS I N THE

CHEMILUMINESCENCE OF TETRAMETHOXYDIOXETANE

TH~RESE WILSON The Biological Laboratories, Harvard University, Cambridge, MA 02138, U.S.A.

(Received 5 February 1919; uccepted 17 February 1979)

Abstract-Tests for the possible catalytic role of fluorescers in the chemiluminescent decomposition of dioxetanes are discussed and applied to the case of tetramethoxydioxetane. This dioxetane, which can undergo direct thermal decomposition yielding excited methyl carbonate with subsequent excitation of fluorescers (for example 9,lOdibromoanthracene. DBA), is shown to be sensitive also to catalysis by fluorescers such as rubrene. 9,IO-diphenyl-anthracene (DPA) and 9, IOdicyanoanthracene (DCNA). The possibility of a charge-transfer mechanism of chemiluminescent catalysis is suggested.

INTRODUCTION

The thermal decomposition of dioxetanes could once be wishfully regarded as an ideally simple unimolecular reaction leading to excited carbonyl products. The emission from these carbonyls could be observed directly, or the intensity and color of the emission could be altered by adding suitable fluorescers behaving as ‘passive’ acceptors of the excitation energy via energy transfer. Some of these fluorescers displayed useful spin selectivity. Thus, 9,lMiphenylanthracene (DPA) and perylene, for example, were branded as amplifiers of singlet energy only; whereas others, like 9,IO-dibro- moanthracene (DBA), could in addition receive triplet energy and convert it to singlet energy and fluorescence (references in Wilson, 1976; Turro et a/., 1974). This ideally simple picture was soon marred, however, by the finding that several catalytic pro- cesses could destroy the dioxetanes without formation of excited states: among these catalysts are traces of transition metals (Wilson, 1973; Bartlett et a/., 1973), amines and other good electron donors (Lee and Wil- son, 1973), as well as the excited ketones or fluorescers themselves, in a process which regenerates the excitation (Wilson and Schaap, 1971; Lechtken et a/., 1973). In all cases, the role of the catalyst is to open a parallel pathway of decomposition having an apparent low activation energy.

It is clear now, thanks mainly to the work of Koo and Schuster (1977, 1978), McCapra et nl. (1978), and Zaklika et a/ . (1978), that more important types of catalyses can also take place which are not dark pro- cesses but, to the contrary, result in an increase of the quantum yield of singlet products. This is of particular interest in view of the long-standing problem of reconciling the results on the small and stable dioxetanes, which tend to favor triplet products, with the situation which must prevail in the large and very unstable dioxetanones; these compounds are the likely energy-rich intermediates in some biolumines- cences, and they must, therefore, generate singlet

products in high yields (Koo et a/., 1978; see also Baumstark et a/., 1976; Hastings and Wilson, 1976). Koo and Schuster’s (1978) work on diphenoyl peroxide, for example, shows how aromatic hydrocarbons of low oxidation potential, such as rubrene, can act as powerful catalysts of the decomposition of this peroxide and be raised to their excited singlet state in the catalytic process.

Of special interest are systems where the two mechanisms of fluorescers’ excitation, passive or active, can occur in competition. One such system is dimethyl- dioxetanone. In 1974, Adam et a/. reported on the tremendous chemiluminescence enhancement observed with rubrene, an effect which is now inter- preted as the result of a catalytic step (Schmidt and Schuster, 1978; Adam et al., 1978):

(with F being rubrene, perylene, DPA or other easily oxidizable hydrocarbons). Yet in 1974 also, Turro et al. showed that the thermolysis of this dioxetanone generates a good yield of triplet acetone, which can be intercepted by DBA:

Thus, in this system two competitive pathways of excited states generation can occur in parallel: a unimolecular (2) and a bimolecular reaction (1). This situation is reflected in the activation energies. While the non-catalyzed decomposition has an activation energy E, of - 100 kJ/mol ( - 24 kcal), the catalytic process with perylene as ‘activator’ has an E , of -67 kJ only (Schmidt and Schuster, 1978).

Tests to determine whether a fluorescer is playing an active or a passive role will be outlined here and

177

178 THERESE WILSON

applied to preliminary results obtained with tetramethoxydioxetane. This will serve as background for a brief discussion of possible catalytic mechanisms.

THE SIMPLIFIED KINETICS

Consider that dioxetane D decomposes according to Scheme I ; the multiplicity of the excited carbonyl A* is left unspecified and the fluorescence yield of F is unity.

(3 )

(4)

A*-A ( 5 )

(6)

( 7 )

21 I D-A* + A

L T A* + F- F* + A

A

Pk., D + F-F* + 2A

hr F* - F + hv.

SCHEME 1

a and fl are the efficiencies of production of A* in the unimolecular reaction (3) and of F* in the catalytic process (6), respectively. The intensity of chemiluminescence is given by

and the overall rate of decomposition of D is

k,,, = CDI [k , + k,,,[Fl 1 . (9)

In the absence of catalysis (k,,, = 0), the intensity will first increase with the concentration of fluorescer, then level off at saturation. How soon this saturation will appear depends on the relative magnitude of k, and k,,[F]. If a fluorescer acts as a catalyst and’if

then the intensity I will vary linearly with [F]. These are two limiting cases, which are also reflected in the overall reaction rates. In the absence of catalysis, the rates are independent of the concentration of fluorescer, whereas if the catalysis dominates, the rates will be proportional to the fluorescer concentration, as shown by Koo and Schuster (1977, 1978). Thus, the dependence of the intensities and rates on the concentration of fluorescer provides us with two criteria by which t o determine whether a given fluorescer is playing a passive or an active role. The most sensitive and incisive test, however, is the effect of temperature, especially the comparison of the activation energies of the overall reaction based on the rates of chemiluminescence decay with or without fluorescer (Ed,F or E,), with the activation energy based on chemiluminescence intensities,

(see Materials and Methods). Three possibilities (listed in Table 1) can be distinguished, according to the passive or active role played by the fluorescer in a given solvent. The third case where E, > E,,, is illustrated in Fig. 1 for a hypothetical dioxetane; lines a and b have slopes equal to E , and t o E,,, . In principle, at high enough temperature (for example between 1/T= 1 and 1/T= 2 in Fig. l), the non-catalyzed process will be so dominant that the measured E,,, will be found equal to E,. At very low temperatures, E,,, will be constant and lower than E,. At intermediate temperatures, the Arrhenius plot will show curvature and the measured E,,, will depend on the temperature, tending to E, at high temperatures and to E,,, at low temperatures. If k, + k,,,[F], then the activation energy based on the rates in the presence of fluorescer, Eil,F, will be equal to E,,, [as observed by Koo and Schuster (1978) for diphenoyl peroxide in CH,C12 with different fluorescers: Ea,F = E,,, < E,]. If a fluorescer is only a poor catalyst of rates and of chemiluminescence (low /?kJ, then the most sensitive test for catalytic effec! is the measurement of Echl at low temperatures, where the uncatalyzed path is minimized. This test is specially useful with the very stable dioxetanes, which decom-

Table 1. Passive or active role of fluorescer

Test Conclusion

Rates: k,,,, is independent of [F] k,,, is proportional to [F]

Intensities : linear plot 1/1 vs 1/[F] linear plot I vs [F]

Activation energies*: E, = E d , ~ = E c h i

Ea.F < Ea = Echl

= Ech! < E d

No catalysis (energy transfer) Dark or chemiluminescent catalysis

No catalysis Chemiluminescent catalysis

No catalysis Dark catalysis Chemiluminescent catalysis

*Ed is the activation energy based on reaction rates without fluorescer, Ed.F on rates with fluorescer, Ec,, on chemiluminescence intensities with fluorescer.

Research Note I79

Ea a t [F] : 0

10

'. '.

l I # . * . a I I I I . . . .

2 4 6 8 10 12

most ly uncat . c reaction - m o s t l y c a t . 1 I T

Figure 1. Hypothetical Arrhenius plot of chemiluminescence intensities in the presence of a fluorescer F acting

as catalyst of the thermolysis of a dioxetane.

pose at such slow rates in normal conditions of solvents and fluorescer concentrations that modest in- creases in rates are not easily detectable. O n the other hand, with very efficient catalysts, E,,, will appear constant throughout the whole range of accessible tern perat ures.

MATERIALS AND METHODS

Tetramethoxydioxetane and tetramethyldioxetane were prepared as reported previously (Wilson et al., 1976). The aromatic hydrocarbons used as fluorescers, purchased from Aldrich, were recrystallized and vacuum sublimed. The solvent was high purity o-xylene 'Distilled in Glass' obtained from Burdick and Jackson, Inc,; the solutions were air- equilibrated. The chemiluminescence measurements were made with the apparatus described previously, except that the very weak luminescences emitted at low temperatures were measured by photon counting (Ortec discriminator and counter). The temperature of the solutions, contained in pyrex reaction tubes (5 mm i.d., 0.6 m/ solution volume), was controlled by a Peltier device. In the measurement of intensities vs fluorescer concentrations, appropriate cut- off filters, selecting the long wavelength tail of the fluorescence, were used to minimize the errors due to reabsorp- tion at high concentrations. The activation energies based on chemiluminescence intensities, E,,,, were obtained from the ratios of intensities emitted by a solution at two temperatures, on the assumption that the concentration of dioxetane (and fluorescer) remains constant; this condition is approximated by working in a range of temperatures such that the dioxetane decomposes only very slowly (Steinmetzer er al., 1974). or by changing the temperature of the solution sufficiently fast that the decomposition of dioxetane is negligible during the temperature change (Wilson and Schaap. 1971).

RESULTS AND DISCUSSION

Tetramethoxydioxetane (TMOD) has been shown

t o be considerably more stable than tetramet hyldioxe- tane (TMD), even though the activation energies of these two dioxetanes are similar (TMOD: E , = 119.7kJ, log A = 12.9; T M D : E,, = 115.5kJ. log A = 14.1; Wilson et a/., 1976).

New results with TMOD, given below, now indi- cate that this symmetric dioxetane can, in fact, decompose according to the type of dual mechanism outlined in Scheme I. There is no question that triplet carbonate, or at least a long-lived excited species able to transfer energy t o DBA, is a product of the thermolysis. The plot of intensity vs DBA concentration shows indeed the saturation expected from reactions 3, 4 and 7, while the plot of 1/1 vs l/[DBA] is linear (Fig. 2). The long-lived donor in the energy transfer step can also be quenched by low concentrations of dienes. But in contrast, when the fluorescer is rubrene, a plot of intensity vs concentration remains linear even at the highest rubrene concentration; at the same time, the rates are markedly accelerated by rubrene (Fig. 3). The third test for the active role of rubrene is positive also: the activation energy of chemiluminescence, Ech,, is only - 89 kJ (average value) throughout the range 2Cb9O"C (Table 2).

With DPA, the temperature dependence of the chemiluminescence intensity is that expected from

2 4 6 8 10 12x10-

D B A conc., M

I I 1 I I 1 I 2 4 6 8 10 12 x 1 0 :

11 D B A c o n c . , M -' Figure 2. Effect of DBA concentration on the initial intensity of chemiluminescence from TMOD (-0.4mM) in o-xylene at 25°C (with Wratten cut-off filter No. 3). In- tensities in arbitrary units. In (b), the ratio Y intercept/

slope is 370 M - ' .

I80 T ~ R E S E WILSON

2 4 6 8 10

Rubrene c o n c . , x M

Figure 3. Effect of rubrene concentration on the chemiluminescence from TMOD in o-xylene. (a) Initial chemiluminescence intensity in arbitrary units vs rubrene concentration, with Wratten cut-off filter

No. 23A. (b) Rate of intensity decay vs rubrene concentration: TMOD conc.: -0.15mM.

two competitive pathways of excitation as represented by the curve plot of Fig. 1. E,,, is appreciably smaller at a low temperature than at a high temperature (Table 2). Thus, rubrene and DPA are undoubtedly not 'passive' fluorescers here. Both are easily oxidizable and rubrene, with the lower oxidation potential of the two, is also the more active catalytically, as in the two systems investigated by Schuster (Koo and Schuster, 1978; Schmidt and Schuster, 1978). One might expect that, here also, the catalytic effect of fluorescers will be related t o their oxidation potential. Results with 1, 3-diphenylisobenzofuran, which has a low oxidation potential, support this view: E,,, is only -92kJ (between 65 and 81°C) and this Buor- escer also strongly catalyzes the rates of decomposition of TMOD. Note that with T M D these fluor-

escers all give the same 'normal' value of E,,, expected from passive fluorescers.

Unexpectedly, however, a search for the most inert fluorescer available uncovered a puzzling result. Since nitriles are notoriously resistant to oxidation (see Mann and Barnes, 1970). 9,lO-dicyanoanthracene (DCNA) seemed superbly qualified for passivity. Yet, surprisingly, its activation energy of chemiluminescence turned out to be very temperature depen- dent, more so even than that of DPA (Table 2). At a low temperature, DCNA is actually a better fluorescer with TMOD than DPA, resulting in higher intensities at the same concentration; at a high temperature DCNA and DPA result in the same intensities (Table 3). Note that in contrast with rubrene, no catalytic effect on the reaction rates, as measured

Table 2. Effect of temperature on the chemiluminescence activation energies of TMOD with different fluorescers, in o-xylene

Temp. range* Activation energies,? E,,, (in kJ/mol 4 kJ) ("C) Rubrene DPA DCNA

10-20 103.3 84.9 20-30 83.7 89.1 30-40 81.2 113.0 98.3 40-50 88.7 115.9 108.8 50-60 90.8 120.5 120.1 6CL70 90.8 120.4 120.1 7&80 89.1 121.3 122.2 8&90 91.2 129.7 125.5

*Each measurement involves a temperature change of - 1&20°C, with a mid-tem-

?Each point is the average of several measurements within the same temperature perature within temperature range indicated.

range. Dioxetane concentration ca. 0.2 mM, fluorescer concentration 0.1-1 mM.

Research Note 181

Table 3. Relative chemiluminescence intensities from solutions of TMOD in o-oxylene with different fluorescers

Fluorescer* 86“ 20”

DPA 1 1 DCNA 1.3 3 Rubrenet 5 60

*Concentrations: TMOD: 0.2 mM, fluorescers: 1.1 mM. ?Intensities corrected for the difference in sensitivity of

the photomultiplier tube (EM1 9558B) in the spectral ranges of rubrene and DPA fluorescences ( - 2).

by the decay of the chemiluminescence, can be detected with DCNA, in the range of DCNA concentrations compatible with its solubility in xylene. (However, since at 40°C T M O D has a half-life of 5 3 months, even a 10 fold increase in rate would easily go unnoticed!) Thus, the first test of Table 1 is incon- clusive. The second test (Fig. 4) requires caution too, since an apparent linear plot of I vs [F] could also be obtained in a passive system, if the donor lifetime is short and the highest possible concentration of DCNA is relatively low. It was this failure to ap- preciate the catalytic role of DPA in the chemiluminescence of T M O D which had led to the probably erroneous conclusion that this dioxetane generates a relatively higher yield of singlet products than other stable dioxetanes (Wilson et al., 1976). Singlet excitation yields obtained with DPA ought to be regarded only as upper limits.

Before attempting to interpret the results with TMOD and DCNA, necessary controls were carried out. (a) The fluorescence of DCNA in xylene is independent of temperature, at least between 0 and 85°C. Therefore, the variation of E,,, with temperature is not a trivial consequence of the photophysics of DCNA. (b) Dienes d o not quench the chemiluminescence enhanced b y DCNA; thug DCNA does not

DCNA conc.

Figure 4. Cnect of DCNA concentration on initial intensity of chemiluminescence from TMOD ( -0 .5mM) in o-xylene at 20°C (with Wratten cut-off filter No. 4). In-

tensities in arbitrary units.

acquire its energy from triplet carbonate, as could already be inferred from the linearity of the plots I vs [DCNA]. (c) Between two low temperatures, i.e. 20 and 3WC, the values of EL,, obtained with DCNA are smaller for TMOD than for TMD: and with TMOD, the E,,, is smaller with DCNA than with DPA (Table 2). Clearly, the cause of these differences is not a property of DCNA itself nor of free singlet excited carbonate, but rather of the pair T M O D and DCNA.

CONCLUSIONS

The results obtained with TMOD serve-to empha- size how cautious one need be in considering any fluorescer as a passive acceptor of energy. It is obvious that this dioxetane is very vulnerable to chemiluminescent catalysis. It is clear also that easily oxidizable aromatic hydrocarbons make excellent catalysts. Taking the activation energy of chemiluminescence EL,, as a measure of /3k,,, (Eq. 8; the smaller EL,,, the larger Bk,,,), there is a strong sugges- tion that catalytic and electrondonating abilities are directly related. This, of course, has been the observa- tion, particularly well documented in the case of diphenoyl peroxide, which led Koo and Schuster (1977, 1978) to formulate an elegant mechanism of chemiluminescence described as “chemically initiated electron exchange luminescence”, or CIEEL. Accord- ing to their hypothesis, the rate determining step is the transfer of an electron from the fluorescer to the peroxide, even in as non-polar a solvent as benzene, causing the radical anion of the peroxide to break at the 0-0 bond and eliminate a neutral molecule of CO,. The remaining radical-anion fragment, a stronger reductant than the original peroxide anion, then reacts in the original solvent cage with the fluorescer cation; it is this exothermic ion-annihilation

‘which is regarded as the excitation process. The results presented here d o not rule out a similar

mechanism for the catalysis of the decomposition of T M O D by rubrene, for example. However, the effect of DCNA simply does not fit the pattern, and would then require a separate ad hoc hypothesis. Alterna- tively, one might regard the catalyses by rubrene and by DCNA as two manifestations of a same effect, i.e. the weakening of the 0-0 bond of the dioxetane through charge-transfer interactions with the fluorescer ‘in the collision complex’. Either the electrondonating properties of rubrene or the electron-with- drawing properties of DCNA would come to play; in neither case would an electron be actually trans- ferred with formation of radical-ions, at least not in xylene. The weakening of the 0-0 bond would lead to the cleavage of the dioxetane, with a lower activation energy requirement than in the absence of fluorescer, and to the concomitant excitation of the fluorescer molecule which took part in the charge- transfer interaction. Although there is no established precedent for such process, the energetics of T M O D are compatible with this interpretation.

I82 T H L R ~ E WILSON

The role of charge-transfer interactions had been proposed earlier t o explain the 'dark' catalysis of the thermolysis of cis-diethoxydioxetane by arnines and otefins of low ionization potential (Lee and Wilson, 1973). O n the other hand, the catalytic effect of transi-

facilitate the dioxetane cleavage with simultaneous excitation of the fluorescer needs much further testing. The effects of solvents and fluorescer concentration are being investigated.

" not fluorescent. for his continuing interest and am grateful to Dr. David

The hypothesis that charge-transfer interactions Nicoli for the design and construction of the Peltier device.

REFERENCES

Adam, W., G. A. Simpson and F. Yany Adam, W., 0. Cueto and F. Yany Bartlett, P. D., A. L. Baumstark and M. E. Landis (1974) J . Am. Chem. SOC. 96, 5557-5558. Baumstark, A. L., T. Wilson, M. E. Landis and P. D. Bartlett (1976) Hastings, J. W. and T. Wilson (1976) Koo, J-y. and G. B. Schuster (1977) J. Am. Chem. Sac. 99, 6107-6109. Koo, J-y. and G. B. Schuster (1978) J . Am. Chem. SOC. 100, 449-503. Koo, J-y., S. P. Schmidt and G. B. Schuster (1978) Lechtken, P., A. Yekta and N. J. Turro (1973) Lee, D. C.-S. and T. Wilson (1973) In Chemiluminescence and Bioluminescence (Edited by M. J .

Cormier, D. M. Hercules and J . Lee), pp. 265281. Plenum Press, New York. Mann, C. K. and K. K. Barnes Electrochemical Reartiom in Non-Aqueous Systems, p. 335.

Marcel Dekker, New York. McCapra, F., I. Beheshti, A. Burford, R. A. Hann and K. A. Zaklika (1977) J.C.S. Chem. Commun.

944946. McCapra, F. (1977) J.C.S. Chem. Commun. 946948. Schmidt, S. P. and G. B. Schuster (1978) J. Am. Chem. Sac. 100, 1966-1968. Steinmetzer, H-C., A. Yekta and N. J. Turro (1974) J . Am. Chem. Soc. 96, 282-284. Turro, N. J., P. Lechtken, G. Schuster, J. Orrell, H-C. Steinmetzer and W. Adam

Chem. Sac. 96, 1627-1629. Wilson, T. and A. P. Schaap (1971) J . Am. Chem. SOC. 93, 412W136. Wilson, T., M. E. Landis, A. L. Baumstark and P. D. Bartlett (1973) J . Am. Chem. SOC. 95, 476S4766. Wilson, T., D. E. Golan, M. S. Harris and A. L. Baumstark (1976) J . Am. Chem. SOC. 98, 1086-1091. Wilson, T. (1976) In t . Rev. Sci.: Phys. Chem. Ser. 2 (Edited by D. R. Herschbach), pp. 265322.

Zalika. K. A,, P. A. Burns and A. P. Schaap (1978) J. Am. Chem. SOC. 100, 318-320.

(1974) J . Phys. Chem. 78, 2559-2569. (1978) J . Am. Chem. Sac. 100, 2587-2589.

Tetrahedron Lett. 2397-2400. Photochem. Photobiol. 23, 461-473.

Proc. NatL Acad. Sci. U.S.A. 75, 3633. J . Am. Chem. Soc. 95, 3027-3028.

(1970)

(1974) J. Am.

Butterworth, London.

DISCUSSION

M. KASHA In such energy transfer and fluorescence quenching

studies, when various anthracene derivatives are used such as 9,10dibromo, diphenyl, and dicyano in comparison with unsubstituted anthracene, it would be directly in- formative to have more direct spectroscopic observations available in interpreting quenching constants and activation parameters. It is known that the T2 state of anthracene interchanges in spectral position relative to the S, state merely upon going from the crystalline state into (hy- drocarbon) solution. One can expect equally dramatic interchanges with the kind of substituted derivatives studied, with consequent effects on energy transfer and fluorescence quenching.

T. WILSON I fully agree with Dr. Kasha about the difficulty of inter-

preting data on energy transfer to anthracene derivatives. I beiieve, however, that by taking into account the fluorescence properties of the specific aromatic derivatives in the solvents used (here xyplene) in the temperature range of the dioxetane experiments, and by comparing the chemiluminescence behavior of a particular fluorescer with two dioxetanes (say rubrene with tetramethoxydioxetane vs rubrene with tetramethyldioxetane, in the same conditions

of solvent and temperature), I can be confident that the difference observed in the activation energies of chemiluminescence cannot be attributed to the photophysics of the fluorescer alone, but to the behavior of the particular dioxetane-fluorescer system.

M.A. EL-SAYED Is it possible that at low temperature you have a charge

transfer complex that gives a mechanism that changes with temperature because the nature and equilibrium concentration of the complex changes with temperature?

T. WILSON This is a possibility that I cannot rule out. and should

definitely investigate further. I have looked for and seen no evidence of a charge transfer complex in the UV-visible spectrum, but admittedly this was not a very sensitive test.

F. MCCAPRA M. M. Rauhut in his study of the interaction of peroxy

oxalates with fluorescers has come to a similar conclusion-namely that a charge transfer or exciplex rather than full electron transfer is a likely event in the mechanism. Can you comment on the distinction observed between DBA and dicyanoanthracene?

Research Note 183

T. WILSON

to compare it to DBA since both are electron acceptors rather than donors. rhere is no doubt that the main mode of action of DBA is of triplet energy from methyl carbonate, since: (1) the plots of chemiluminescence inten-

sity versus DBA concentration show saturation [see J. Am. Chem. sot. 9, 1086 (1976)], whereas those with DCNA do not: (2) the chemiluminescence with DBA, in contrast to X N A , can be Wenched by conjugated d i e m : (3) DBA behaves very much like the chelate of europium which we use as trip1et acceptors. '

Actually, One of my re som for trying DCNA

passive and active role of fluorescers in the chemiluminescence of tetramethoxydioxetane

Documents