J . Phys. Chem. 1986, 90, 6597-6600
-*
0
1
I
I
I
50
100
150
200
0 (deg) Figure 2. Comparison of the potential of mean torque as a function of
for the extended- (solid line) and all-atom (broken line) simulations. Standard error of the mean is less than 0.1 kcal/mol at all points. Points are plotted at intervals of l o . No data smoothing has been done. harmonic analysis of an identical model,I0 indicating the importance of anharmonic effects. As seen in Figure 2, the free energy contours are similar for the two models; differences between the two curves are less than 1 kcal/mol. The free energy difference between the gauche and trans conformers for the extended-atom model and all-atom model are 0.87 and 0.71 kcal/mol, respectively. These values are slightly larger than the value of 0.497 f 0.22 kcal/mol16 derived from electron diffraction. The effect of the entropy on the overall conformational statistics can be illustrated by determining the difference in probability of the trans conformation based on the internal energy and that based on the free energy. For the all-atom model, the fraction of trans conformers would be only 55.6% if there were no entropic contribution (AA = AE = 0.55 kcal/mol). Peterson, M. R.; Csizmadia, I. G. J . Am. Chem. Soc. 1978,100,6911. (16) Bradford, W. F.; Fitzwater, S.; Bartell, L. S. J. Mol. Struct. 1977, 38, 185. (15)
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The corresponding figure for the extended-atom model would be 65.5% ( A A = AE = 0.80 kcal/mol). Although the geometry of the all-atom model has bond angles which are different from those found in microwave ~pectroscopy,~’ this model does incorporate explicit methyl group rotations. Loss of rotational freedom in the eclipsed and cis conformations of the all-atom model probably contributes to most features of the entropy surface. In conclusion, the entropy differences between the extendedatom and all-atom butane models illustrate the influence of the model type on calculated results and, more importantly, interpretation of molecular behavior. Since our results differ from those of Karplus and Kushick using harmonic analysis for the calculation of entropy,I0 anharmonic effects, which are known to be important in macromolecule^,^+'^^'^ are also found to be important even in butane. The difference in barrier heights determined by energy minimization’0 and by molecular dynamics (Table I) suggests that parameters for energy minimization studies may not be suitable for molecular dynamics, where averaging over all thermally accessible states takes place.’* The modified umbrella sampling method presented here may represent an important technique for calculating conformational statistics for the refinement of potential functions in small molecules.
Acknowledgment. We thank Wilfred van Gunsteren for use of GROMOS. This research was supported by grants from N I H (AI-00613 to C.D.B. and GM-34015 to S.C.H.) and the NSF (DMB-84-17001 to S.C.H.). (1 7) Harmony, M.; Laurie, V.; Kuczkowski, R.; Schwendeman, R.; Ramsey, D.; Lovas, F.; Lafferty, W.; Maki, A. J . Phys. Chem. Re$ Data 1979, 8, 619. (18) Mao, B.; Pear, M. R.; McCammon, J. A,; Northrup, S. H. Eiopolymers 1982, 21, 1979. (19) Prahhakaran, M.; Harvey, S. C.; McCammon, J. A. Biopolymers 1985, 24, 1189.
A Time-Resolved C I DEP Study of the Photogenerated Camphorquinone Radical Anion: A Case of Dual Singlet and Triplet Precursors M. C. Depew and J. K. S . Wan* Department of Chemistry, Queen’s University, Kingston, Ontario, Canada K7L 3N6 (Received: September 9, 1986)
Photoreduction of camphorquinone in 2-propanol produced electron spin polarized camphorquinone radical anions. The time-resolved electron spin resonance spectra of the spin-polarized radical anions provided the first evidence of dual singlet and triplet precursors in the CIDEP phenomenon. With the results from fluorescence quenching experiments, the time dependence of the CIDEP spectra can be. accounted for qualitatively by the changes of the relative contributions to the polarization among the singlet pair, F and triplet pairs, and the triplet mechanisms.
Introduction The photochemistry of quinones has been extensively used in model systems during the early development of the photoexcited triplet mechanism in the CIDEP (chemically induced dynamic electron polarization) and CIDNP (chemically induced dynamic nuclear polarization) phenomena.’ As well, time-resolved CIDEP techniques have been successfully employed to provide detailed insights in the primary photophysical and photochemical processes involving quinonesS2 In many, if not all, of the reported CIDEP studies involving photoexcited carbonyl compounds, including all (1) For example, see Wan, J. K. S . Adu. Photochem. 1976, 12, 283. (2) For example, see Depew, M. C.; Wan, J. K. S. Quinones II, Patai, S., Rappoport, Z., Eds.; Wiley: London, 1986.
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quinones, the primary photochemical reactions are derived from the polarized triplet states. Because of the highly selective intersystem crossing (isc) processes in the carbonyl compounds, the very large initial polarization from the triplet mechanism usually dominates in the CIDEP observations. Recently, Hirota and co-workers3 have proven the coexistence of the triplet and radical pair mechanisms in the CIDEP phenomenon of the photochemical reaction of acetone with 2-propanol which has been rather controversial for a number of years. Today many chemical and physical processes associated with the triplet CIDEP have been investigated. These include the examples of anisotropic isc pro(3) Yamauchi, 2367.
s.;Tominaga, K.; Hirota, N. J . Phys. Chem. 1986, 90,
0 1986 American Chemical Society
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The Journal of Physical Chemistry, Vol. 90, No. 25, 1986
c e ~ s e s , ~the 3 ~ energy-transfer processes?’ and the photosensitization processes.’ On the other hand, studies of CIDEP from excited singlet photochemical reactions have been few. Only two recent investigations concerning the photodecompositions of the pyridinyl radical dimers’s9 and of tetraphenylhydrazine’O have confirmed that the CIDEP of the primary radicals resulted from their excited singlet-state reactions. Neither of these involves carbonyl compounds. We report here the first case of a carbonyl photo-CIDEP system involving the carbonyl excited singlet state. This is the photohepreduction of camphorquinone (1,7,7-trimethylbicyclo[2.2.1] tane-2,3-dione) in 2-propanol. At short delay times after the laser flash the photogenerated camphorquinone radical anions exhibited strong A/E polarization, superimposed on a total emission component; the latter grew in its relative contribution with time. From the circular dichroism study,I’ the dione chromophore in camphorquinone is expected to be nearly planar-a configuration similar to some of the 1,2-quinones whose polarized radical anions have been shown to derive from triplet reactions.2 Unlike the quinones and other a-diketones, camphorquinone photochemistry appears to be much less understood. One photochemical investigationI2 showed that camphorquinone is extremely photochemically inert in the absence of oxygen and in a large variety of solvents, including alcohols, CC14, and benzene. In the presence of oxygen, camphorquinone appears to undergo both oxidation to the camphoric anhydride and reduction to mixtures of acy1oins.l2 However, details of the excited state involved have not been delineated. The present CIDEP results, coupled with the fluorescence quenching experiments, will provide some insights to these questions.
Experimental Section The recent configuration of our CIDEP apparatus was described e1~ewhere.l~In addition to the capability of simultaneous detection of polarized and thermalized radicals in a single scan, we have further modified the instrumentation so that the dc signal was fed simultaneously to both the boxcar integrator (APR Model 162) and a nanosecond transient recorder (Nicolet Model 204) interfaced separately to a Nicolet 1180 computer. This modification allowed the “instant” snapshot recording of the transient decay of selected hyperfine lines at predetermined magnetic fields immediately after the laser flash, while simultaneously the total spectrum of the polarized radicals was recorded by the boxcar integrator which permits variation of both the selected delay times as well as the integration times. In a typical experiment, the integration time was 0.5 ps while the delay times varied between 1.5 and 9 M S . The Lambda Physik EMG 101 MSC XeCl excimer laser (308 nm, 10 Hz) was used as the photochemical source. Camphorquinone in 2-propanol exhibits two visible/UV absorption bands at -468 and -300 nm, respectively. Both bands originate from the coupling and splitting of the two carbonyl groups n,r* transitions.” Thus, the XeCl excimer laser excites the camphorquinone to the second n,a* band, S,. Fluorescence experiments were performed in a Varian SF330 spectrofluorimeter in a double-beam configuration. Camphor(4) Terazima, M.; Yamauchi, S.;Hirota, N. J . Phys. Chem. 1985, 89, 1220. (5) Imamura, T.; Onitsuka, 0.;Murai, H.; Obi, K. J . Phys. Chem. 1985, 89, 4921. ( 6 ) Weir, D.; Wan, J. K. S . J . Am. Chem. SOC.1984, 106, 427. Wan, J. K. S.; Dobkowski, J.; Turro, N. J. Chem. Phys. Lett. 1986, 131, 129. (7) Imamura, T.; Onitsuka, 0.;Murai, H.; Obi, K. J . Phys. Chem. 1985, 88, 4028. (8) Akiyama, K.; Tero-Kubota, S.: Ikegami, Y.; Ikenoue, T. J . Am. Chem. SOC.1984, 106, 8322. (9) Ikegami, Y . Rev. Chem. Intermed. 1986, 7 , 91. (10) Matsuda, K.; Murai, H.; I’Haya, Y. Chem. Phys. Lett. 1986, 125, 577. (11) Lightner, D. A.; Crist, B. V.; Flores, M. J. J . Chem. Soc., Chem. Commun. 1980, 273. (12) Meinwald, J.; Klingele, H. 0. J . Am. Chem. SOC.1966, 88, 2071. (13) Kam, E.; Craw, M. C.: Depew, M. C.; Wan, J. K. S . J . Map.Reson.
1986, 67. 556.
Figure 1. The time-dependent CIDEP spectra of CQ’-produced by laser M solution of CQ in 2-propanol, recorded at flash photolysis of a (a) 2, (b) 5 , and (c) 8 ps after the flash. The inserts in the figure represent the “instant” snapshot of the dc signal transient decay at the selected magnetic field. In the 8-ps delay spectrum (c), the insert showed the absorptive transient decay at the low-field peak while the integrated spectrum was totally emissive.
quinone was purchased from Aldrich Chemical Co. and purified by sublimation before use. All solvents, including the triethylamine were distilled.
Results and Discussion The cw photolysis of a degassed 2-propanol solution containing M camphorquinone (CQ) led to a strong ESR signal consisting of four main multiplets. The hyperfine parameters and assignments of the CQ‘- are indeed identical with those reported by RussellI4 and Kuwata.15 The interesting features of CQ’polarization observed in laser flash-time resolved CIDEP experiments will now be described.
8tn,jo(2 H) = 0.15 G
The Time-Resolved CIDEP Observations in the Photoreduction of CQ in 2-Propanol. A typical CIDEP spectrum of CQ’- obtained at 2 ps after its generation by the laser flash is given in Figure 1A. Additional transient decays of the dc signal immediately after the laser flash at selected hyperfine fields (lines) obtained simultaneously are shown in the inserts. This latter information is valuable as it provided the true “initial” pattern uncomplicated by the effects of altering the sampling delay and integration times. This initial polarization exhibited by the CQ’clearly showed an A/E pattern. For delay times of 2 ps and less, the integrated CQ’-CIDEP spectrum clearly showed an A/E pattern. In the range of 2- to 5-ps delay times, the CQ’- spectra progressively emphasized the total emission component which was (14) Russell, G. A,; Chang, K. Y. J . Am. Chem. SOC.1965, 87, 4381. (15) Tosa, R.; Kotake, Y.; Okazaki, M.; Shikata, H.; Kuwata, K. Bull. Chem. SOC.Jpn. 1980, 53, 1747.
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The Journal of Physical Chemistry, Vol. 90, No. 25, 1986 6599
superimposed on the initial A / E pattern (Figure 1B). At a long delay time of 8 f l , the spectrum was now “almost” in total emission (Figure 1C). The initial A / E pattern can be explained by the excited singlet reaction followed by singlet radical pair S-To mechanism: CQ + 308 nm CQ(S2) (1)
A
1
-
The involvement of CQ(S,) singlet state in the primary processes will be confirmed in a subsequent fluorescence study. Its participation in the generation of CQ’- radical anions is strongly evidenced by the CIDEP results. The reaction rate of CQ(Sz) to yield CQ’- is expected to be fast, since it must compete with the fluorescence process which in solution usually is in the s range. We regret that currently we cannot measure directly the fluorescence lifetimes of C Q in solution. At short delay times and for the transient decays immediately after the laser flash, the A/E pattern cannot be accounted for by any triplet mechanism, nor by triplet geminate pair or F-pair S-To radical pair mechanism.16 However, a careful examination of the relative intensities of the hyperfine components of the polarized CQ’-, particularly in the 2-5-ps delay times, revealed a second polarization component superimposed on the initial A / E pattern. This second component is a total emission pattern. Under the experimental conditions and the small hyperfine coupling constants of CQ’-, it is extremely unlikely that the S-T, mixing in the radical pair mechanism could be operative to yield an all emissive spectrum.l’ In the absence of a third theory, we are thus left only with the triplet mechanism to account for the all emissive polarization. The immediate implication is that the CQ‘ is not exclusively generated by the excited singlet reaction and selective intersystem crossing from either Sz or SI to the triplet manifold may also have occurred to make possible a triplet precursor for the CQ’- formation: CQ(S2) w
IC
* CQ(S,)
It is interesting to note that the nature of the C Q triplet polarization is all emissive in the present experiment and to compare this result with all other quinones reported in literature (emissive) and some aliphatic ketones which are mainly all absorptive in the triplet p o l a r i ~ a t i o n . ~ , ~Camphorquinone, ~*’~ which has a rigid structure and co-planar dione chromophores, thus appears to behave more like a 1,2-quinone than an a-diketone in this respect. In order to shed some light on the nature of the triplet polarization in CQ’-, three separate series of experiments were performed. The first one involved a photosensitization (energy transfer) experiment using benzophenone as the sensitizer:
+ 308 nm -kPh2CO(Tl)* Ph$O(T,)* + C Q CQ(T1)* + PhzCO Ph2C0
CQ(T1)*
2-propanol
(4)
I
E*+€
Since the all emissive triplet character of benzophenone is well established, the photosensitization would yield also an all emissive CQ’ radical anion. The photosensitized CIDEP spectrum of CQ‘is shown in Figure 2A. Here, at a 2-ps delay time, the spectrum consists of an all emissive component superimposed on an E / A pattern which is associated with the triplet pair S-To radical pair mechanism. This experiment clearly demonstrated that the triplet is the precursor for the CQ’- polarization. (16) For example, see Monchick, L.; Adrian, F. J. J . Chem. Phys. 1978,
68, 4316. (17) Adrian, F. J.; Monchick, L. J . Chem. Phys. 1979, 71, 2600. 1980,
Figure 2. The CIDEP spectra of CQ’- recorded at
2-ps delay after its production by (a) benzophenone photosensitization and (b) photolysis in the presence of Et,N. Both spectra can be analyzed by the superposition of an all emissive triplet polarization (E*) and a triplet-pair polarization (E/A). The inset confirmed the initial triplet mechanism dominated immediately after the flash.
The second experiment used the triplet CQ(T1)*as the sensitizer or precursor for the formation of the vitamin E radical. When a toluene solution containing both vitamin E and C Q was flash photolyzed, only the vitamin E radical was observed which exhibited a strong total emission pattern, characteristic of a triplet reaction. The third series of experiments involved the quenching of the CQ(Sz) excited singlet by the addition of amine. Both CIDEP and fluorescence quenching will be discussed in the next section. The Quenching of CQ(S,) by Amines. Although the quenching of camphor fluorescence by amines has been mentioned in literature,20we were not able to find any such reports with camphorquinone. We have therefore carried out a series of fluorescence quenching experiments of camphorquinone in benzene solutions using triethylamine as the quencher. Photoexcitation of camphorquinone in a benzene solution ( M) led to a fluorescence with the maximum emission at 505 nm and the maximum excitation wavelength at 290 nm. Upon addition of various concentrations of triethylamine, the fluorescence intensity was greatly reduced. The results can be fitted to the Stern-Volmer equation
IFo/zF= 1 + rkQ[Q] where IFois the fluorescence intensity in the absence of the quencher, 7 is the lifetime of the excited singlet state, and kQ is the rate constant of the “quenching” reaction (7): CQ(S2) CQ(S,)
L+ CQ(Si)
+ Et,N -k. CQ(So) + Et,N
(7)
At room temperature and in benzene solution, the vlaue of 7kQ is equal to 21.5 f 0.2. The quenching was not carried out in 2-propanol solution, since it is obvious that 2-propanol itself will react (chemical quenching) with the excited singlet C Q to yield CQ’-, as observed in the CIDEP experiments. However, the ability of amine to quench the CQ(S,) fluorescence is clearly demonstrated, although the detailed mechanism involved in the quenching process cannot be determined in the present experiments. Nevertheless, it is reasonable to expect that the addition of amine to a 2-propanol solution of C Q should enhance the C Q triplet reaction by reducing (quenching) the
72, 5186. (18) Grant, A. I.; McLauchlan, K.A. Chem. Phys. Lett. 1983, 101, 120. (19) McLauchlan, K. A,; Ritchie, A. J. D. Mol. Phys. 1985, 56, 141.
E*+A
B
(5)
CQ’+*
~
(20) Rau, H. Chem. Reu. 1983, 83, 535.
J . Phys. Chem. 1986, 90. 6600-6602
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participation of CQ(S2) in the overall photochemical processes. This is indeed consistent with the following CIDEP observation. A typical CIDEP spectrum of CQ’- in 2-propanol with added Et,N and recorded at 2-w~delay time is shown in Figure 2B. The polarization spectrum is characteristic of an E/A pattern (triplet or F-pair) superimposed on a totally emissive component (triplet mechanism). In the absence of amine, the same spectrum (Figure 2) showed a strong A / E pattern (singlet pair) underlined by a weak, total emission component. Combined with the fluorescence quenching information, we have demonstrated the role of amine in reducing the contribution of CQ(S2) reaction and thus confirmed the dual involvements of the CQ(S,) and CQ(T,) in the photoreduction of C Q leading to polarized CQ’-. One single question remains intriguing: In the absence of amine, why does the relative triplet contribution to the CQ’- polarization increase with the delay time when the CQ’- was observed. One may reason qualitatively that the initial contribution of the triplet mechanism and the triplet pair is indeed small; but as time progresses (we are only talking in the time frame of a few microseconds) the “dominant” singlet pairs are rapidly equilibrated to F-pairs. Since the polarization from F-pairs (E/A) is exactly opposite to the singlet pairs (A/E), at a longer delay times, they may cancel each other and the underlining triplet polarization will be relatively enhanced for the observation. Another more likely explanation for this observation results from the likelihood of spin exchange occurring in random radical-radical encounters. If sufficiently rapid relative to spin-lattice relaxation, spin exchange will mediate the hyperfine-state-dependent RPM polar-
ization of the singlet pairs without decreasing the magnitude of the T M polarization, thus accounting for the latter’s apparent enhancement. Unlike the systems of acetone/2-propanol3 and pyruvic/lactic acids,*I the CQ’- spectrum is more complex and not completely resolved in the laser flash photolysis. Furthermore, the relatively poor signal-to-noise ratio in the CIDEP spectra also prevents us from a detailed analysis of the accurate relative contributions from the singlet pair, the triplet pair, and the initial triplet mechanisms. To the best of our knowledge, the photosystem of camphorquinone is a unique case in which both the excited singlet and triplet states participate in the chemical reduction and the resultant radical polarization involved the triplet mechanism as well as both singlet and F-pair radical pair mechanism. As we are further intrigued by the chiralty of camphorquinone, we have produced, by using optically active CQ, and observed optically active CQ’- that showed a Cotton effect in the new band at -310 nm with the same sign and similar magnitude as the K-*, n+ K+*). We parent CQ second electronic band (nhope that in future we will be able to combine the magnetic polarization and the optical polarization to probe the nature of the formation of optically active free radicals.
- -
Acknowledgment. This research was supported by the Natural Sciences and Engineering Research Council of Canada. We thank Dr. T. Ho for his assistance in the fluorescence experiments. (21) Choo, K. Y . ; Wan, J. K.
S. J . Am. Chem. Soc.
1975, 97, 7127.
Coupled Transport of Sodium Chloride Driven by Diffusion of Bovine Serum Albumin Derek G. Leaist Department of Chemistry, University of Western Ontario, London, Ontario N6A 5B7, Canada (Received: September 18, 1986)
Magnetically stirred porous-diaphragm cells have been used to measure coupled transport of sodium chloride during diffusion of aqueous bovine serum albumin (BSA) at 25 OC. At normal physiological pH (7.2) and ionic strength (0.15 mol L-I) each mole of diffusing BSA cotransports about 175 mol of NaCl. Coupled flow of NaCl vanishes if the solution pH is adjusted to the isoelectric point (pH 5.4). At lower pH values diffusing BSA produces a counterflow of NaCl. The coupled transport of NaCl is driven primarily by the diffusion-induced electric field along BSA concentration gradients. The direction and magnitude of the coupled flow of NaCl can be estimated if the ionic mobilities and protein charge are known.
Introduction Solutions of biological interest in which proteins diffuse invariably contain dissolved salts and other solutes. Although the effects of pH and ionic strength on protein transport have been documented,’-’ multicomponent diffusion in these solutions has not been explored. Because the mobilities of proteins and typical counterions differ by orders of magnitude, significant electric fields* are generated by protein diffusion in order to prevent charge (1) Creeth, J. M. Biochem. J . 1952, 51, 10. (2) Tinoco, Jr., I.; Lyons, P. A. J . Phys. Chem. 1956, 60, 1342. (3) Wagner, M. L.; Scheraga, H. A. J . Phys. Chem. 1956, 60, 1066. (4) Keller, K. H.; Canales, E. R.; Yum, S . I. J . Phys. Chem. 1971, 75, 379. (5) Raj, T.; Flygare, W. H. Biochem. J . 1974, 13, 3336. (6) Phillies, G.D. J.; Benedek, G. B.; Mazer, N . A. J . Chem. Phys. 1976, 65, 1883. (7) Neal, G.D.; Purich, D. Cannell, D. S. J . Chem. Phys. 1984,80,3469. (8) Leaist, D. G.;Lyons, P . A. J . Phys. Chem. 1982, 86, 1542.
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separation. The diffusion-induced electric field might in turn drive significant coupled flows of other ions in the so hi on^.^ The work reported here was undertaken to measure coupled transport of sodium chloride during diffusion of aqueous bovine serum albumin. It is shown that the diffusing protein can produce large coup1ed flows Of and that the direction Of the flow can be reversed by adjusting the solution pH. This behavior can be understood qualitatively by treating protein-salt solutions as simple mixtures of strong electrolytes.
Experimental Section BSA (Sigma Biochemicals No. A-39 12) was purified by dialysis and then ion exchange.2.” Potentiometric titration of the product (9) Leaist, D. G. J . Colloid Interface Sci. 1986, I l l , 240. (IO) Leaist, D. G.; Lyons, P. A. J . Phys. Chem. 1982, 86, 564. (11) Tanford, C.; Swanson, S. A.; Shore, W. S.J . Am. Chem. Soc. 1955, 77, 6414.
0 1986 American Chemical Society
