J. Am. Chem. SOC.1989, 111, 2409-2417
2409
as the A-factor remains constant. Structures for the various Mn(CO),+ ions were assumed as noted. M ~ I ( C O ) ~was + assumed to be an octahedral complex with Mn-C bond lengths of 1.87 A and C - 0 bond lengths of 1. I 4 A. Two possibilities are considered for M ~ I ( C O ) ~ +a :C, structure resulting from removal of one C O ligand from octahedral Mn(CO),+ and a rearranged trigonal-bipyramidal structure. The C4, geometry has been predicted by Burdett for a singlet d6 metal p e n t a ~ a r b o n y l .Further ~~ support for the C, geometry comes from X-ray crystallographic dataSoand gas-phase electron diffraction datas1 for H M ~ I ( C O )as ~ , well as modified extended Huckel c a l c u l a t i ~ n son ~ ~Mn(CO)S+. Bond lengths were taken from theory.45 The isoelectronic Cr(CO)S+has also been determined to have C4, geometry on the basis of its infrared spectrum.52 Fe(CO)S+,on the other hand, is believed to be trigonal bipyramidal.I4 For MII(CO)~+,C2,, D4h, and Td geometries were considered. It has been predicted that singlet d6 tetracarbonyls should have the C2, geometry.I4 Infrared spectras3 and C O addition rates4s5 for the isoelectronic Cr(CO), indicated that this molecule has a C2, ground state, while modified extended Huckel calculation^^^ on neutral Mn(CO), also predict this structure. Calculated bond lengths were used.4s Burdett argues, on the basis of theory, that quintet d6 metal tetracarbonyls should have the Td structure.I4 Three geometric possibilities, C,,, D3*, and C, are considered for Mn(CO),+. Burdett has predicted that the C3, d6 metil tricarbonyl has a singlet ground state, while the C2" geometry results in a triplet and the D3h geometry is a quintet. Infrared
spectral data5, for the isoelectronic neutrals Mo(CO), and Cr(CO), are consistent with C3, geometry for both species. Bond lengths were assumed: Mn-C, 1.84 A; C-0, 1.15 A.
(50) La Placa, S. J.; Hamilton, W. C.; Ibers, J. A. Inorg. Chem. 1964, 3, 1491-1 495. (51) Robiette, A. G.; Sheldrick, G. M.; Simpson, R. N. F. J . Mol. Srrucr. 1969, 4, 221-231. (52) Huber, H.; Kundig, E. P.; Ozin, G. A,; Poe, A. J. J . Am. Chem. SOC. 1975, 97, 308-314. (53) Perutz, R. N.; Turner, J. J. J . A m . Chem. Sor. 1975, 97, 480G4804. (54) Elian, M.; Hoffmann, R. Inorg. Chem. 1975, 14, 1058-1076. Also see ref 45.
(55) RRKM calculations were performed using: Hase, W. L.; Bunker, D. L. A General RRKM Program. Grouped harmonic frequency direct counting was used to determine sums and densities of states in the activated complexes, while the Whitten-Rabinovitch a p p r o x i m a t i ~ nwas ~ ~ used for energized molecules. (56) A more recent version of the phase space programs used has been submitted to QCPE: Chesnavich, W. J.; Bass, L.; Grice, M. E.; Song, K.; Webb, D. A. TSTPST: Statistical Theory Package for RRKM/QET/ TST/PST Calculations QCPE, submitted for publication.
Appendix B: Method of Calculation
Fits of theoretical kinetic energy release distributions to the measured experimental distributions were obtained as follows. Initially, a guess was made for the bond energy in question, and this value was used to estimate metastable internal energies assuming the low extreme for log A as discussed above. Decomposition rate constants were calculated using R R K M theory,5s with the assumed bond energy and log A , for a range of ion internal energies. The internal energies were then assigned weights by using the calculated rate constants in eq 6, and phase space calculationss6 were performed using the weighted internal energies. The same bond energy was used in the phase space calculation as was assumed for the R R K M calculation. On the basis of the comparison of the calculated kinetic energy distribution to the experimental distribution, the bond energy was revised and the process was repeated with a new R R K M calculation. This iteration was continued until the bond energy was bracketed for the low log A value, generally to f0.5 kcal/mol. A t that point the high value for log A was assumed, a new guess was made for the bond energy, and iterations were performed until the fit converged on a bond energy a t the high log A value. It is important to note that R R K M theory was used only to determine the metastable internal energies; otherwise, the phase space calculations and fitting procedures did not depend on the R R K M calculations.
Interaction of Triplet Sensitizers with Chlorophyll: Formation of Singlet Chlorophyll' Cornelia Bohne2 and J. C. Scaiano* Contribution from the Division of Chemistry, National Research Council of Canada, Ottawa, Ontario, Canada K1 A OR6. Received August 9, 1988 Abstract: The interaction of several triplet sensitizers with chlorophyll a (Chla) has been examined using laser techniques. For the carbonyl sensitizers (with triplet energies >53 kcal/mol) it was possible to measure the quenching rate constants; these were systematically 3 1O I o M-I s-'. In the cases of acetone, benzophenone, and p-methoxyacetophenone the quenching process leads to the formation of the fluorescent singlet state of Chla. For benzophenone ( k , = 2.4 X 1O'O M-I SKI)approximately 3% of the quenching events lead to the formation of excited Chla. Several sensitizers (decafluorobenzophenone, benzil, and fluorenone) do not induce Chla fluorescence (or do it very inefficiently) in spite of having triplet energies above the SI level of Chla. In light of our results the most probable mechanism involves energy transfer from the triplet sensitizer to an upper triplet state of Chla (3Chla**) which can undergo reverse intersystem crossing to the singlet manifold of Chla and thus induce fluorescence. The inefficient sensitizers are those where electron transfer between the excited singlet of Chla or )Chla** and ground-state sensitizers is energetically favorable, leading to rapid in-cage quenching of the initially formed excited states of Chla. Formation of radical-ion pair between the triplet sensitizer and Chla followed by the generation of singlet Chla in the recombination of the radical ions could not be completely discarded.
Chlorophyll a (Chla) fluorescence can be induced by enzymatically generated triplet carbonyl compounds. This process has been observed for Chla in various media, such as aqueous or micellar solutions, when part of chloroplasts or thylakoid fractions, and when bound to serum albumin^.^^^ Two possible mechanisms ( I ) Issued as NRCC-29983.
(2) Postdoctoral Fellow from the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNP,), Brazil.
0002-7863/89/ 15 1 1-2409$0l.50/0
were suggested: (i) triplet-triplet energy transfer to an upper triplet of Chla and a subsequent population of its SI state by reverse intersystem crossing (RISC),3 or (ii) radical-ion pair formation between the triplet sensitizer and Chla followed by (3) Bohne, C.; Campa, A,; Cilento, G.; Nassi, L.; Villablanca, M . Anal. Biochem. 1986, 155, 1. (4) Bohne, C.; Faljoni-Alario, A,; Cilento, G. Photochem. Photobiol. 1988, 48. 341.
Published 1989 by the American Chemical Society
2410 J . Am. Chem. SOC..Vol. 111, No. 7, 1989 generation of excited Chla in t h e radical-ion recombination rea~tion.~ Examples of tripletsinglet energy transfer in which an acceptor is excited through a RISC mechanism are well known in the case of 9, I O - d i b r o m o a n t h r a ~ e n e , ~and - ~ ~ it has been proposed for xanthene dyes containing heavy-atom substituents." RISC processes have also been reported in molecules that do not contain heavy atoms; such examples include isoalloxazines'* and a n thracene and its derivatives.13 T h e elucidation of the details of a net triplet-singlet energy transfer mechanism is important, given the spin-forbidden nature of the process. Beside this primary interest, a better understanding of this process could lead to a more accurate definition of the requirements for t h e detection of triplet species in biological systems using Chla luminescence a s a probe. It has been shown that chlorophylls in isolated chloroplasts (from spinach), or when this organelle is part of Euglena gracillis (a protozoa), show induced fluorescence a n d a r e bleached in t h e presence of enzymatically generated triplet specie^.^.^^ In this paper we report the results of a study of the interaction of triplet sensitizers with Chla, which has been shown to induce Chla fluorescence. In the case of the Chlalbenzophenone system we have also been able to estimate t h e efficiency of t h e tripletsinglet energy transfer process.
Experimental Section Materials. Chlorophyll a (Chla) from Sigma, acetone (ACS grade from Fisher), acetonitrile (spectroscopic grade from BDH), anthracene from Eastman Kodak, and absolute ethanol (Consolidated Alcohols Ltd.) were used as received. Benzophenone (BP), p-methoxyacetophenone (PMAP), o-hydroxybenzophenone (OHBP), benzil, fluorenone, and 1,2-benzanthracene from Aldrich were recrystallized from ethanol/water. Decafluorobenzophenone (DFBP) from Aldrich was purified by sublimation. Stock solutions of all sensitizers were prepared in acetonitrile, and for Chla the stock solutions were in ethanol or acetonitrile-ethanol (8:l). The concentrations of Chla in acetonitrile were determined spectrophotometrically based on extinction coefficients of 6.9 X lo4 and 8.5 X lo4 M-' cm-' at 662 and 432 nm, respe~tive1y.l~ All experiments were carried out in samples deaerated by nitrogen bubbling for approximately 15 min. General Techniques. UV-visible absorption spectra were obtained on a HP-8451A diode array spectrometer. Some redox properties were determined by Dr. Fred Hartstock (NRC) using cyclic voltammetry. Steady-state (prompt) fluorescence spectra were recorded on a Perkin-Elmer LS-5 spectrofluorimeter controlled by a PE-3600 data station. Fluorescence lifetimes were measured on a PRA single photon counting instrument employing a hydrogen lamp for excitation. Laser Photolysis Experiments. The samples for these experiments were contained in quartz cells constructed of 7 X 7 mm2 Suprasil tubing. Static cells typically contained 2.5 mL of solution. For the flow experiments the irradiation cell was built of the same tubing as above and was connected with Teflon tubing to a reservoir where the solutions were deaerated. Flow rates were high enough to ensure reproducible results for Chla emission intensities. The laser flash photolysis system at NRC and modifications for twolaser experiments have been described earlier.'6~'7 A Lumonics TE-860-2
(5) Kobayashi, S.;Kikuchi, K.; Kokubun, H. Chem. Phys. Lett. 1976,62, 494. (6) Kobayashi, S.; Kikuchi, K.; Kokubun, H. Chem. Phys. 1978,27,399. (7) Kikuchi, K.; Fukumura, H.; Kokubun, H. Chem. Phys. Lett. 1986, Z23, 226.
(8) Wilson, T.; Halpern, A. M. J . A m . Chem. Soc. 1980, 102, 7272. (9) Catalani, L. H.; Wilson, T. J . A m . Chem. Soc. 1987, 109, 7458. (10) McGimpsey, W. G.; Scaiano, J. C. J . Am. Chem. Soc. 1988, 110, 2299. ( 1 I ) Duran, N.; Cilento, G. Photochem. Photobid. 1980, 32, 11 3. (12) Richter, C.; Hub, W.; Traber, R.; Schneider, S. Photochem. Phorob i d . 1987, 45, 671. ( 1 3 ) Fukumura, H.; Kikuchi, K.; Koike, K.; Hiroshi, K. J . Photochem. Photobid. A 1988, 42, 283. (14) De Mello, M. P.; Nascimento, A.L.T.O.; Bohne, C.; Cilento, G. Photochern. Photobiol. 1988, 47, 457. (15) Seely, G. R.; Jensen, R. G. Spectrochim. Acta 1965, 21, 1835. (16) Scaiano, J. C. J . Am. Chem. Soc. 1980, 102, 7747. (17) Scaiano, J. C.; Tanner, M.; Weir, D. J . A m . Chem. SOC.1985, 107, 4396.
Bohne and Scaiano
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Figure 1. Transient absorption spectra 1.0 ws after the laser pulse (20-ns gate) of triplet Chla alone (I) and in the presence of OHBP (11) or acetone (111). [Chla] = I O pM, [OHBP] = 0.5 mM, and [acetone] = 0.27 M. excimer laser operated with Xe/HCl/He mixtures (308 nm, -5 ns, 6 2 0 mJ/pulse), a Molectron UV-24 nitrogen laser (337.1 nm, -8 ns, 6 1 0 mJ/pulse), or a Candela 500M flash-pumped dye laser (Coumarin 480 or Oxazine perchlorate 720 dyes in 50% aqueous methanol, -250 ns, 100-350 mJ/pulse) were employed for excitation. Three types of detection were employed. For time-resolved experiments the signals from an RCA-4840 photomultiplier were captured by either a Tektronix R7912 transient digitizer interfaced to a PDPl1/23+ computer, or a Transiac 2001 digitizer interfaced to a P D P l l / 7 3 computer via a Camac crate. The latter is better suited for slow kinetics and shares the same optical system as the OMA detector described below. Spectrally resolved data (absorption or emission) were recorded with a Series I11 gated intensified optical multichannel analyzer (OMA) from EG&G operated by a P D P l l / 7 3 computer via an EG&G Model 1461 interface. Detection gates were either 20 ns or a continuously adjustable gate for times in excess of 120 ns. This system has remarkable sensitivity for low levels of emission. The delay between laser excitation and the opening of the OMA gate can be controlled in 10-ns steps. Emission intensities measured with the OMA are relative ones and depend on the alignment of the system. Emission intensity values can only be compared for experiments performed on the same day. Magnetic fields were applied by a "home-made" magnet which allows continuous variation of the field strength up to 5.4 kG.
Results Unless otherwise indicated, all the experiments described below were performed a t room temperature under oxygen-free conditions, using acetonitrile as solvent. Transient Absorption Spectra. T h e characteristic triplet-triplet ( T T ) absorption spectrum of C h l a was produced by direct (308 nm) excitation of a 1.1 X M acetonitrile solution of Chla. This spectrum is shown in Figure 1 and agrees well with literature reports.'* Typical triplet half-lives under our deaerating conditions were -20 f i s . C h l a is not transparent a t 308 nm, where our sensitizers a r e usually excited. Naturally t h e light absorbed by a given concentration of C h l a is less in t h e presence than in t h e absence of triplet sensitizers. In order to evaluate quantitatively our d a t a (vide infra), it is necessary to determine the signal intensity which is due to direct excitation of Chla. While a calculated correction based on Beer's law is possible, such a correction does not take into account that the distribution of transients across the reaction cell is a function of the total absorbance a t the laser wavelength. In order to achieve matched conditions with our sensitizers (same (18) Ford, W. E.; Tollin, G. Photochem. Photobiol. 1982, 35, 809.
J . Am. Chem. SOC..Vol. 1 1 1 , No. 7 , 1989 2411
Formation of Singlet Chlorophyll
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Figure 2. Transient absorption spectra of triplet BP (A) and of the BPIChlu system (B) at 0.2 (I), 1.0 (11, A), and 10 ps (111) after the laser pulse (20-11s gate). [Chla] = 11 pM; [BPI = 10 mM.
Figure 3. Transient absorption spectra of triplet DFBP(A) and of the DFBPIChlu system (B) at 0.2 (I), 1.0 (11), and 10 ps (111) after the laser pulse (20-11sgate). [Chla] = 11 pM; [DFBP] = 1.1 mM.
absorbance a t 308 nm), we have employed o-hydroxybenzophenone (OHBP) as a UV screen; the approach is similar to the one employed in singlet oxygen studies reported from this laboratory.19 O H B P is used as a photochemically inert molecule, taking advantage of the rapid deactivation of its excited state by reversible intramolecular hydrogen transfer; a t room temperature O H B P does not yield any detectable signals in the nanosecond time scale, and does not induce any changes in the emission or ground-state absorption spectra of Chla. Figure 1 also shows a Chla TT absorption spectrum obtained in the presence of OHBP. The approach described above allows the evaluation of signals from direct excitation of Chla under conditions that properly reproduce the absorption and spatial distribution obtained in the presence of sensitizers. When acetone is used as a triplet sensitizer (0.8 total absorbance, 0.6 due to acetone), the concentration of Chla triplet formed in the Chla/acetone system is higher than that formed in a matched ChlaIOHBP sample (Figure I ) . The absorbance signals corresponding to triplet acetone (
