J . Phys. Chem. 1991,95, 2415-2420 concentration after 2 min was calculated to be less than 0.43%. From the slope of the straight line, the k3 value was calculated to be 1.62 X lo2 M-I s-' at pH 9.0 and 25 OC for an oxygen concentration of 0.24 mM, Figure 6 also shows that the value of the ordinate intercept is 0.988, which agrees well with the theoretical value of 1.0 (eq 6). There is disagreement in the literature on the rate constant for the reaction of MBH with 02,16J9 In one study,16 the pseudofirst-order constant was found to be 3.2 X lW3 s-' at pHs 7.6 and 9.0 from which the second-order rate constant can be calculated s-l to be -2 M-' s-l. On the other hand a value of 4.26 X at 25 "C is published,I9which leads to a second-order rate constant of 1.70 X lo2 M-' s-' which is in good agreement with our steady-state value. The rate of reoxidation of MBH could have also been increased in an atmosphere of pure oxygen in our steady-state experiments, but it would have yielded a higher steady-state concentration of MB+, which would have resulted in a lower accuracy of determination of the rate constant of k3 from eq 6. It should be noted that, under our steady-state conditions, from 83 to 91% of the methylene blue is in its oxidized form. The decrease in the concentration of dissolved oxygen was calculated to be less than 4% during 120 s, using k3 < 2 X IO2 M-' s-I, based on the assumption that no oxygen entered the solution by passive diffusion from the gas phase. Under the conditions shown in Figure 3B, where the transport of O2from the gas to liquid phase is very slow, the rate of oxygen consumption becomes greater than the O2 supply and the reaction solution becomes anaerobic. Therefore, the third stage occurs, where the O2concentration drops and some net reduction of MB+ can again be observed. Lazar and Rossm-2ihave recently presented experiments on the highly nonlinear oxidation of NADH catalyzed by HRP in the presence of MB+ under conditions of continuous oxygen supply and a perturbation in the O2concentration. Their results confirm that the mode of supply of reactants to a nonlinear (bio)chemical (19) Engbersen, J. F. J.; Koudijs, A.; van der Plas, H. C. Red. Trau. Chim. Pays-Bas 1986, 105,494. (20) Lazar, G. J.; Ross, J. Science 1990, 247, 189. (21) Lazar, G.J.; Ross, J. J . Chem. Phys. 1990, 92, 3579.
2415
reaction determines or controls concentrations of steady states far from equilibrium, The O2flux from the gas flow into the solution is a critical parameter in these experiments.2a.21 There is a range in the rate of 0 2 supply into the reaction solution which gives oscillations in both the HRP-catalyzed oxidation of NADH' and the MB+-catalyzed oxidation of sulfide.12The oxygen-transfer constant, depending on the reaction volume, the surface area, the rate of stirring, and the flow rate and composition of the reaction mixture of N2and 02,for the peroxidasscatalyzed (in the presence s-' (ref 4), is very close to the of MB+) system, k = 3.5 X reciprocal residence time of k = 3.26 X lo-' s-l in CSTR oscillations of MB+.'"14 Under our experimental conditions, utilized in the aerobic oxidation of NADH by MB' shown in Figure 3, the transport of O2from gas to liquid was too slow (B) or too fast (A) to expect oscillations. Actually, the system can be considered to be closed with respect to the total content of the reactants, but there is no protection for the phase exchange of O2in the cuvette. In an open system containing NADH and MB+ with an appropriate continuous supply of O2or in a CSTR, the conditions for oscillations may be fulfilled. Moreover, if the oxidation of MBH by O2 is indirect and a~tocata1ytic'~J~ the oscillations in an open system where oxygen is entering at a constant rate might be found for the methylene blue catalyzed oxidation of various substrates. One of the most promising could be glucose. It has been demonstratedE that an alkaline solution of glucose and methylene blue upon standing in an open flask turns colorless, indicating that the dye has been reduced by glucose. Shaking the flask will cause the blue color to return as the reduced form of methylene blue is oxidized by atmospheric oxygen. Work on methylene blue catalyzed redox reactions in an open system will be the subject of future research. Acknowledgment. This work was supported by Natural Sciences and Engineering Research Council of Canada operating grant A 1248. Registry No. NADH, 58-68-4;MB,61-73-4;02,7782-44-7. (22) Fenster, A. E.; Harpp, D.N.; Schwarcz, J. A.; Glanville, J. 0. J . Chem. Educ. 1988, 65, 621.
Radlatlon Chemlstry of Cyanine Dyes: Oxidation and Reduction of Merocyanlne 540 Anthony Harriman,* Center for Fast Kinetics Research, University of Texas at Austin, Austin, Texas 78712
Lian C. T. Shoute, and P. Neta Chemical Kinetics Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899 (Received: August 2, 1990)
Merwyanine 540 (MC) shows promise as a treatment for certain types of leukemia. It is shown that MC readily undergoes one-electron reduction under pulse radiolytic conditions. The r-radical anion, produced by reduction with hydrated electrons and 2-hydroxypropyl radicals, disproportionates rapidly (k = 1.9 X lo9 M-' s-') under anaerobic conditions but reduces O2 to superoxide ions (k = 1.6 X lo9 M-l s-I) in aerated solution. The dye reacts with trichloromethylperoxyl radicals (k = 9 X lo* M-I s-I) to form several products, one of which is believed to be an adduct formed by addition of CC1300' to the bridgehead carbon atom of the benzoxazole subunit. This species decays via first-order kinetics (k = 4.0 X lo3s-I) under pulse radiolytic conditions to form cleavage products. A second primary product is believed to arise from addition of CC1302* to the polymethine chain to form an cy-amino carbon-centered radical capable of reducing O2to superoxide ions. Preliminary studies indicate that the breakdown products are cytotoxic and could be important intermediates for the known antiviral activity of MC.
Cyanine dyes have found prominent use as sensitizers for photographic film,' as laser dyes? as membrane-staining
and, more recently, as reagents in biomedicine! The cyanines possess an intense, relatively narrow absorption band, the position
0022-3654/91/2095-2415$02.50/00 1991 American Chemical Society
2416 The Journal of Physical Chemistry, Vol. 95. No. 6, 1991
of which can be modulated by simple structural modification of the chromophore.' The photophysical properties tend to be dominated by rapid isomerization of the (po1y)methine group upon promotion to the first excited singlet ~ t a t e .This ~ isomerization serves to decrease the fluorescence lifetime, keeping triplet-state formation to a minimum, but, because of the inherently high radiative rate constant, fluorescence yields can be quite high. Despite the immense interest that has been given to the cyanine dyes, little attention has been paid to the redox chemistry of these compounds. Byers et a1.6 first described the photosensitized oxidative breakdown of cyanine dyes. It was found6that cyanines possessing low redox potentials were readily oxidized by singlet molecular oxygen to produce carbonyl fragments consistent with 1,2-addition of O2across one of the bonds in the methine chain. Other workers' found that cyanine dyes were destroyed by hydroperoxyl radicals, thus enabling the compounds to be used as antioxidants for protection of polymers. Doizi and Mialocq* showed that 3,3'diethyloxadicarbocyanine (DODCI, a popular laser dye) was photooxidized when irradiated in the presence of electron acceptors such as benzoquinone or methylviologen. Similar behavior has been observed with merocyanine 5409and with kryptocyanine.I0 Because of their widespread use in photography, it is well-known" that cyanine dyes can inject electrons into the conduction band of appropriate semiconductors upon illumination with visible light. The photoreduction of cyanine dyes using ascorbic acid as electron donor has been studied,12and the photoreduction of merocyanine 540 under sacrificial conditions has been described briefly.I3 fJ
merocyanine 540
In this paper, we describe the oxidation and reduction of merocyanine 540 under steady-state and pulsed radiolytic conditions in aqueous alcohol solution. This dye is of great topical interest because of its preferential binding to certain leukemia cells;' it is currently undergoing phase 1 clinical trials for treatment of leukemia." The mechanism for its in vivo reactivity remains unknown but may involve electron-transfer reaction^.^-^^ It is shown here that both oxidation and reduction steps occur readily to give unstable intermediate species. The possible role of the redox products in chemotherapeutic treatment of leukemia is considered. (1) Hamer, F. M. The Cyanine Dyes and Related Compounds; WileyInterscience: New York, 1964; Chapter 14. (2) Meyer, M.; Mialocq, J.-C.; Perly, B. J . Phys. Chem. 1990, 94, 98. (3) Dragsten, P. R.; Webb, W. W. Biochemistry 1978, 17, 5228. (4) Sieber, F. Phorochcm. Photobiol. 1987, 46, 1035. (5) Tsukada, M.; Mineo, Y.; Itoh, K. J . Phys. Chem. 1989, 93, 7989. ( 6 ) BYCIS, 0.W.; Gross, S.; Henrichs. P. M. Photochem. Photobiol. 1976, 23, 31. (7) Fukuzumi, K.; Ikeda, N . J . Am. Oil Chem. Soc. 1971, 48, 384. (8) Doizi, D.; Mialocq. J.-C. J . Phys. Chem. 1987, 91, 3524. (9) Davila, J.; Harriman, A.; Gulliya, K. S. Photochem. Phorobiol. 1991, 53, 1. (10) Harriman, A.; Luengo. G.; Gulliya, K. S. Photochem. Phorobiol. 1990,52,135. (1 1) Tani, T.; Suzumoto, T.; Ohzeki, K. J. Phys. Chem. 1990, 94, 1298. (1 2) (a) Krasnovskii, A.: Drozdova, N . Dokl. Phys. Chem. (Engl. Trawl.) 1962, 145, 507. (b) Bourdon, J.; Durante, M. Bull. Chim. Soc. Belg. 1962, 71, 907. (13) Sarna, T.; Pilas, B.; Lambert, C.; Land, E. J.; Truscott, T. G.Photochem. Photobiol. Suppl. 1990, 51, 42s. (14) Sieber, F.;OBrien, J. M.; Krueger, G.J.; Schokr, S.L.; Burns, W. H.; Sharkis, S.S.;Sensenbrenner, L. L. Photochem. Photobiol. 1987, 46.707. (15) Davila, J.; Gulliya. K. S.;Harriman, A. J. Chem. Soc., Chem. Commun. 1989. 121 5.
Harriman et al.
Experimental Section16 Merocyanine 540 (MC) was obtained from Sigma Chemicals and was purified by column chromatography on neutral silica with methanol/hexane mixtures as eluant. The dye appeared pure (single component) on paper electrophoresis and on thin-layer chromatography (neutral silica gel with aqueous butanol as eluant). Samples were further analyzed by elemental composition, IH NMR in CD30D solution, and fast-atom-bombardment mass spectrometry. In dilute methanol solution the absorption maximum is located at 554 nm with a molar extinction coefficient of 120OOO M-' cm-' . Merocyanine 540 is stable on prolonged storage as a solution in 1/1 aqueous 2-propanol provided it is kept in the dark; slow decomposition occurs if illuminated with visible or UV light. All solvents and other reagents were of the highest available commercial purity and were used as received. Pulse radiolysis experiments were made with the NIST Febetron 705 accelerator which delivers single 50-ns pulses of 2-MeV electrons. The course of reaction was followed by optical spectroscopy, as described previously." Signals were detected with an RCA 4840 photomultiplier tube, digitized with a Tektronix 7612 recorder, and transferred to a microcomputer for subsequent analysis. Transient differential absorption spectra were recorded point by point, using appropriate glass narrow-band-pass filters to avoid undue photolysis of MC. For reduction studies, MC (9 pM)was dissolved in 1/ 1 aqueous 2-propanol and the solutions were purged thoroughly with Ar. Under such conditions, the major reducing species are hydrated electrons (ew-) and 2-hydroxypropyl radicals ((CH3),COH) as produced in the primary radiation processes.18 For the oxidation studies, MC (9 pM) was dissolved in aerated 1/1 aqueous 2-propanol containing 0.05 M CC14. Under such conditions, the primary radiolytic species are converted into the CC130< peroxyl radi~a1s.I~This radical is known to be a strong one-electron oxidant (Eo >1 V vs NHE)20 and to oxidize many organic substrates at a high rate.2' Steady-state radiolyses were carried out in a Gammacell 220 6oco source with a dose rate of 135 Gy/min using solutions similar to those used for pulse radiolysis studies. The course of reaction was followed by absorption spectroscopy using a Cary 2 19 spectrophotometer. Material for chemical analysis was produced by radiolysis of MC (1 mg/mL) in Ar-saturated 9/1 aqueous 2-propanol (for reductions) or aerated 1/ 1 aqueous 2-propanol containing 0.01 M CC14 (for oxidations). After radiolysis, the solvent was removed under vacuum and the residue was subject to reversed-phase HPLC and TLC analyses using authentic materials for calibration purposes. IH NMR studies were made in CD30D solution with TMS as internal standard using a 360-MHz instrument. Infrared spectra were recorded as KBr pellets, and resonance Raman measurements were made in C H 3 0 H using an argon ion laser excitation source. Cytotoxicity studies were made with acute promyelocytic leukemia cell line HL-60 cells maintained in RPMI-1640 supplemented with 10% fetal bovine serum, 10 pg/mL gentamicin, and 0.25 mM L-glutamine at 37 OC in a humidified atmosphere of 5% C02 in air. Cells were maintained in log phase with >95% viability by using the trypan blue dye exclusion method.22
Results and Discussion General Properties of MC. In 111 aqueous 2-propanol the absorption maximum of MC is located at 556 nm, and Beer's law is followed over a wide concentration range (0-8 X lo4 M). (16) The mention of commercial equipment or material does not imply recognition or endorsement by the National Institute of Standards and Technology nor does it imply that the material or equipment identified are necessarily the best available for the purpose. (17) Neta, P.; Huie, R. E. J. Phys. Chem. 1985, 89, 1783. (18) (a) Neta, P. Adu. Phys. Org. Chem. 1976, 12, 223. (b) Swallow, A. J. Prog. React. Kiner. 1978, 9, 195. (19) Brault, D.; Neta, P. J . Phys. Chem. 1984,88, 2857. (20) Neta, P.; Harriman, A. J. Chem. Soc., Faraday Trans. 2 1985,81, 123. (21) Neta, P.; Huie, R. E.; Ross,A. B. J . Phys. Chem. Re/. Dara 1990, 19, 413. (22) Gulliya, K. S.; Pervaiz, S. Blood 1989, 73, 1059.
The Journal of Physical Chemistry, Vol. 95, No. 6,1991 2417
Radiation Chemistry of Cyanine Dyes Increasing the amount of water (to a mole fraction greater than 0.9) or moving to less polar solvents causes aggregation or precipitation of the dye as evidenced by extensive broadening of the lowest energy absorption bands and scattering of incident laser light. Absorption specrtra recorded for the lowest energy transition in 1/ 1 aqueous 2-propanol solution cannot be resolved into a simple series of Gaussian profiles. Instead, satisfactory analysis demands the presence of a minimum of two overlapping series of Gaussian peaks. This feature is taken to indicate3 that MC exists in zwitterionic forms. The dipole moment along the transition axis for zwitterionic MC in methanol is calculated23to be 62 D. 'H N M R studies in D 2 0 indicate that alignment of the zwitterionic dipoles causes head-to-tail dimerization whereas in nonpolar solution, such as hexane/methanol mixtures, extensive aggregation occurs. In CD30D, IH N M R studies indicate that MC exists as a monomer, at least below 1V3M, and there is no indication of the presence of nonequilibrating conformers even a t -80 OC. Time-resolved fluorescence studies9 are also consistent with ground-state MC existing either as a single conformer or as a rapidly equilibrating mixture. Resonance Raman studies made in C H 3 0 H show an intense band a t 1627 cm-I, which is taken to indicate24 an all-trans alignment of the polymethine chain. From extrapolation of earlier r e s u l t ~ , 9 .we ~ ~estimate *~~ that the activation barrier for isomerization of the ground state is 185 kJ mol-'. Using this value, with a frequency factor of 10" PI, we expect the Boltzmann concentration of the unstable (presumably cis) isomer to be insignificant in our experiments. Reduction of MC. Under pulse radiolytic conditions in Arsaturated aqueous 2-propanol, MC was reduced by both hydrated electrons and 2-hydroxypropyl radicals. The rate constants for these reactions are expected to be =lolo M-' s-l for reduction by e r i and =1O9 M-l S-I for reduction by (CH3)2COHradicals by comparison with reactions of related compounds.26 Both reductants are one-electron reducing agents, and reaction was carried out with excess MC such that the reduction product can be identified as the s-radical anion of MC (or a protonated version). MC eaq- MC'(1)
+
MC
+ (CH3)2COH
-+
-
MC'-
+ (CH3)2CO + H+
(2)
H+ s MCH' (3) MC'Differential absorption spectral changes monitored by pulse radiolysis (Figure la) indicate bleaching of the MC absorption band centered at 556 nm and appearance of a broad, featureless absorption band centered around 400 nm with a weaker band at 290 nm. Sarna et have also found that the r-radical anion, as formed by photoreduction, absorbs at 400 nm. The s-radical anion decays via second-order kinetics whereby there was a 50% recovery of the MC absorbance a t 556 nm (Figure la). This process is attributed to disproportionation of the s-radical anion, the bimolecular rate constant for which was calculated to be (1.9 f 0.3) x 109 M-1 s-1.
-
+ MC2MC + MCH2
(4)
MC2- + 2H+ s MCH2
(6)
2MC'2MCH'
+
MC
(5)
(23) Kushner, L. M.; Smyth, C. P. J . Am. Chem. Soc. 1949, 71, 1401. (24) (a) Bennett, J. A.; Birge, R. R. J . Chem. Phys. 1980, 73,4234. (b) Wilbrandt, R.; Grossman, W. E. I.; Killough, P. M.; Bennett, J. E.; Hester, R. E. J . Phys. Chem. 1984,88,5964. Problems from fluorescenceemission and the possibility of photoindud isomerization of MC during laser (514.5 nm) excitation limit the amount of reliable information attainable from these studies. (25) (a) Hoebeke, M.; Seret, A.; Piette, J.; van der Vorst, A. J. Photochem. Photobiol., B 1988, 1,437. (b) Hoebeke, M.; Piette, J.; van der Vorst, A. J . Photochem. Photobiol., B 1990. 4, 273. (c) Aramendia, P. F.; Krieg, M.; Nitsch, C.; Bittersmann, E.; Braslavsky, S.E. Photochem. Photobiol. 1988, 48, 187. (d) Aramendia, P. F.; Duchowicz, R.;Scaffardi, L.; Tocho, J. 0. J . Phys. Chem. 1990, 94, 1389. (26) (a) Buxton, G. V.; Greenstock, C. L.; Helman, W. P.; Ross,A. B. J . Phys. Chem. Ref Datu 1988,17,513. (b) Ross,A. B.; Neta, P. Natl. S t u d . ReJ Dot4 Ser. (LIS.,Nut/. Bur. S t u d . ) 1982, 20, 70.
-1504 200
500
400
500
800
c
700
WAVELENGTH/m 2.0,
, .
.
,
.
3
.
. ,.
Hhv.kngth
I
-
Figure 1. (a) Transient differential absorption spectrum observed (0) 0.6 ms and (v)6 ms after pulse radiolysis of MC in Ar-saturated aqueous 2-propanol. (b) Absorption spectral profile following steadystate radiolysis of MC in Ar-saturated aqueous 2-propanol showing the gradual disappearance of dye. The insert shows the effect of accumulated radiation dose (eV/NA) on the dye concentration that leads to the derived radiation yield of 0.29 pmol J-'.
This behavior is in accord with steady-state radiolyses in which the radiation yield for disappearance of MC was found to be 0.29 pmol J-' (Figure lb). This latter value corresponds to approximately half the total yield of primary reducing radicals in this system.18 The resultant s-dianion may undergo partial or complete protonation with protons formed during the radiation chemistry. The final reduction product, which absorbs weakly a t 290 and 490 nm (Figure lb), is stable over many hours standing under aerobic conditions. Bearing in mind that the benzoxazole and polymethine subunits should be resistant toward reduction, it seems most likely that the first reduction equivalent is localized on the thiobarbituric acid subunit. As such, the electron could be localized at one of the carbonyl functions, forming a semiquinone species which on protonation provides three possible a-amino carbon-centered radicals. Alternately, the electron could be localized on the thio group where protonation will also generate a carbon-centered radical having reducing properties. Huckel molecular orbital calculation^^^ suggest that the most preferred radical center is that having the unpaired electron sited a t the a-hydroxy carbon atom (structure A in Figure 2). This radical should have reducing properties (see later), and by comparison to ketyl and semiquinone radicals,2* it is expected to be susceptible to disproportionation. Addition of the second electron, followed by protonation, is expected to give the alcohol (structure B in Figure 2). Chemical analysis of the irradiation product indicated that the IR peak associated with the carbonyl functions on the thiobarbituric acid ring had decreased in intensity. There was a corresponding appearance of a broad band centered around 3500 cm-' attributable to an 0-H stretching vibration. Further reduction can occur in which the second carbonyl function on the thiobarbituric acid subunit is converted to the corresponding alcohol (structure C). Subsequent reduction can take place a t (27) HMO calculationswere based on an energy minimization procedure assuming an all-trans alignment of the polymethine bridge: Pople, J. A,; Beveridge, D. L. Approximate Molecular Orbital Theory; McGraw-Hill: New York, 1970. (28) Neta, P.; Harriman, A. Photoinduced Electron TruwJec Fox, M . A., Chanon, M., Eds.; Elsevier: Amsterdam, 1988; Part B, Chapter 2.3. p 110.
2418 The Journal of Physical Chemistry, Vol. 95, No. 6, 1991
Harriman et al. 0 0
rl
\
I
W
o
-so
0 -100
200
400
300
500
600
700
WAVELENGTH/m
B
-.6t,
'
" '
300
'
'
"
400
' . '
"
500
'
'
"
"
600
'
''
700
WAVELENGTH/nm Figure 3. (a) Transient differential absorption spectrum observed ( 0 ) 120 gs and (v)6 ms after pulse radiolysis of M C in aerated aqueous 2-propanol containing CClb (b) Corresponding differential absorption spectrum recorded 0.1 s after the pulse.
Figure 2. Reaction scheme showing the stepwise reduction of MC. Note: the side chains have been abbreviated for clarity of presentation.
the thio group (structure D). This fully reduced product can be generated directly by treating MC with a slight excess of NaBH4 followed by glacial acetic acid. Thus, it appears that reduction involved stepwise addition of hydrogen atoms to the thiobarbituric acid group. In the presence of low concentrations of 02,the a-radical anion decayed via first-order kinetics to regenerate MC as indicated by measurements made at 400 and 556 nm. Reaction is attributed to electron transfer to O2to form superoxide ions which, under the present conditions, are expected to disproportionate and protonate to generate hydrogen peroxide: MC'-
+ 02
202'-
+ 2H"
-
+ 02'0 2 + H202
MC
(7)
(8) The bimolecular rate constant for the reduction of O2(reaction 7) was determined to be (1.6 f 0.5) X lo9 M-' s-l; Sarna et al. have reported a rate constant of ca. 2 X IO9 M-' s-l for this reaction under similar conditions. Hydrogen peroxide does not react with MC but accumulates in the solution, as shown by polarographic measurements. Oxidation of MC. Many cyanine dyes undergo facile oxidation: especially by hydroperoxyl radicals.' However, it was found that M C was not oxidized by hydrogen peroxide, sodium persulfate, silver sulfate,29 or bromine in 1/1 aqueous methanol. Singlet molecular oxygen, on the other hand, rapidly bleaches M C M T h i s latter process is irreversible and results in formation -w
(29) Addition of excess silver sulfate to an aqueous methanol solution of MC caused aggregation of the dye, which was reversed by dilution or addition of surfactant, but there was no indication that oxidation of the dye occurred under such conditions. (30) These experiments were performed in CD30D solution using anthracene as photosensitizer. Thus, 02-saturated solutions containing anthracene (1 X I@ M) and MC (0-1 mM) were irradiated at 365 nm for short times. The course of reaction was followed by monitoring absorption bands due to MC. Similar destruction of MC occurred in the a k n c e of anthracene, but the reaction required at least 30 times longer irradiation times. Singlet molecular oxygen, 02(lA&was observed as a transient intermediate by nanosecond laser flash excitation (355 nm) with time-resolved near-IR luminescence d e t e c t i ~ n . The ~ ~ ' bimolecular ~ rate constant for reaction between 02(lAi) and MC was measured to be (3 k 1) X lo9 M-I s-I under such conditions.
of a variety of products, including hydroperoxides (measured by colorimetric analysis3') and carbonyl compounds (measured by infrared spectro~copy).~~ Rapid bleaching of MC is also observed upon addition of N-bromosuccinimide in aqueous methanol. In this case, a slight excess of N-bromosuccinimide causes complete decoloration of the dye solution, presumably due to bromination of the polymethine chain. Radiolysis of MC was studied in aerated aqueous 2-propanol containing CCI4 where the primary radiolytic species are rapidly and quantitatively converted into CC130; radi~a1s.I~ This peroxyl radical is known to be a strong one-electron oxidizing agent that can react via an inner-sphere mechanism,33but it can also abstract hydrogen atoms from appropriate substrate^'^ and form adducts without subsequent electron transfer.3s It was found that CC130; radicals reacted with MC with a bimolecular rate constant of (9 f 1) X lo8 M-' s-I. The differential absorption spectrum recorded at the end of the oxidation reaction (Figure 3a) is dominated by bleaching of MC, but there is a weak absorption band located around 420 nm that can be assigned to the reaction product. The radiolytic yield of this product, calculated from the bleaching of MC, is only ~ 0 . pmol 1 J-I. This value corresponds to a yield of about 20% of the initial CC130; radicals, the other radicals being consumed in competing bimolecular reactions,36 and is a conse(3 1) The reagent kit for peroxide determination was obtained from Kamiya Biomed. Co. (Thousand Oaks, CA). The presence of peroxides and/or hydroperoxides in the product solution was demonstrated by using a hemoglobin-catalyzed reaction of the peroxide with lO-(N-methylcarbamoy1)-3,7(dimethylamino)- 10H-phenothiazine. Reaction produces a stoichiometric quantity of methylene blue dye which is readily quantified by absorption spectroscopic detection at 675 nm. Blank experiments were made to determine background levels of peroxides in the solutions. and addition of ferrous sulfate was found to destroy any peroxides produced during photolysis. (32) Even for short irradiation periods reaction between O2(IAJ and MC is nonselective, and according to our HPLC analysis, there are at least four major products. One of these is N-(3-sulfonatopropyl)-2-benmxamlinoneas shown by comparison to an authentic sample prepared according to: Hutchins, J. E. C.; Fife, T. H. J. Am. Chem. Soc. 1973, 95,2282. ( 3 3 ) Alfassi, Z. B.; Harriman, A.; Mosseri, S.; Neta, P. Inf. J. Chem. Kinef. 1986, 18, 1315. (34) (a) Mosseri, S.; Alfassi, Z. B.; Neta, P. Inf. J. Chem. Kinef. 1987. 19,309. (b) Brault, D.; Neta, P.; Patterson, L. K. Chem. Eiol. Inreracf. 1985, 54, 289. (35) (a) Packer, J. E.; Mahood, J. S.; Willson. R. L.; Wolfenden, B. S. Inr. J. Radiaf.Eiol. 1981,39, 135. (b) Shen, X.;Lind, J.; Eriksen, T. E.; Merenyi, G. J . Chem. Soc., Perkin Trans. 2 1989, 555.
Radiation Chemistry of Cyanine Dyes
The Journal of Physical Chemistry, Vol. 95, NO. 6,1991 2419 MC
+ CC1302'
n
Wavelength - nm Figure 4. Absorption spectral profile showing disappearance of MC (66 pM) during steady-state radiolysis in aerated aqueous 2-propanol containing CCI,. The insert shows the effect of accumulated radiation dose (eV/N,,) on the concentration of dye that gives the derived radiation yield of 0.57 pmol J-I.
t
+
GTHER PRODUCTS
I Figure 5. Partial reaction scheme proposed for the breakdown of MC following reaction with CC1,02' radicals in aqueous 2-propanol. The side chains have been abbreviated for clarity of presentation.
quence of the low concentration of MC (9 pM) that must be used to monitor the reaction by optical spectroscopy. Monitoring at 420 nm showed that the product species decayed via first-order kinetics with a rate constant of (4.0 f 0.6)X IO3 s-' that remained independent of the radiation dose. This reaction did not involve re-formation of MC, as evidenced from the lack of recovery of absorbance at 556 nm, but resulted in formation of a product that absorbed weakly in the 300-350-nm region (Figure 3a). Kinetic measurements made at 330 nm showed an absorbance growth occurring with a first-order rate constant of (1.6 f 0.5) X 1 O3 s-l whereas at 420 nm the signal decayed to leave a residual absorbance. The species absorbing around 330 nm was found to react on much longer time scales, under pulse radiolytic conditions, to form a species with pronounced absorption around 280 nm (Figure 3b). During the pulse radiolysis experiments it was noticed that further bleaching of MC occurred on time scales ranging over many tens of milliseconds. The first-order rate constant derived for this latter process increases with increasing radiation dose for a given concentration of MC. Reaction is attributed to oxidation of MC by radiolytic products such as CCI3O2H.To confirm the reaction with hydroperoxide, we irradiated the solvent mixture without M C to generate the hydroperoxide product, which is known to be stable under such condition^,^^ and then added MC. This treatment caused extensive bleaching of the dye. Overall, the radiolytic bleaching of MC corresponds to a total yield of 0.33 pmol J-I, which is half the initial yield of peroxyl radicals. Steady-state radiolysis of MC (9 pM) in the above solvent system resulted in complete bleaching of the 556-nm absorption band. The product showed absorption in the near-UV region together with a broad absorption band centered around 480 nm. This solution was stable over many days storage at 0 O C , but on further radiolysis both the 330- and the 480-nm absorption bands disappeared to leave only species absorbing in the UV region around 280 nm. Analysis of the final solution by HPLC indicated the presence of at least seven compounds not present in the unirradiated solution. Steady-state radiolysis using a much higher concentration of MC (66 pM) resulted in progressive bleaching of the dye absorption band centered at 556 nm (Figure 4). From the gradual decrease in absorbance at 556 nm as a function of irradiation time, we derived a radiation yield of 0.57 @molJ-I. This value approaches the yield of CCI3O2' peroxyl radicals expected in this system (yield 0.62 pmol J-1),36indicating that the low yield observed under pulse radiolytic conditions stems from incomplete scavenging of CC1302' radicals by MC. Radiolysis of MC in aerated aqueous 2-propanol in the absence of CCI4 resulted in very little bleaching of the dye, the radiolysis yield being
