J . Phys. Chem. 1990, 94, 4439-4446 ,so,
Y
X
CHa
Figure 9. Possible orientation of rings in the electrostatic pair of [ FeTMPyP(DMSO)lSt and [ZnTSPcIC corresponding to calculated positions of iron as shown in Figure 8. An assumed interplanar separation of 4 A in an approximately parallel plane orientation gives the iron position (4.5,2.0,6.1).47 The rotational configuration is suggested by pyrrole ring overlap.
A on these curves. An iron-zinc distance of about 7.6 A results for these orientations. A schematic of the cofacially oriented molecules with the iron atom positioned by the estimated coordinates given here is shown in Figure 9. This orientation shows
4439
the possible K system overlap in the pyrrole groups of each ring. The extent of x overlap between the two ring systems (as measured by the lateral shift and area overlaps) is comparable to the weakly interacting group W defined by Scheidt and Lee." Thus, we believe that the interaction pictured in Figure 9 represents a significant X-T contribution to the electrostatic pair. The effects of the bulky peripheral groups may also influence the overall structure of the electrostatic pair as, for example, in the ruffled K cation radical dimer derivatives of ZnTPPC104.48,49 Conclusions. The electrostatic pairing interactions of [ F ~ T M P Y P ( D M S O ) ~and ] ~ +[ZnTSPc]" in DMSO solution are consistent with reported results for similar systems in their 1:1 combination stoichiometry, their large extent of association, and their electronic spectral changes. The association dynamics are consistent with the loss of a solvent ligand by [FeTMPyP(DMS0)2]S+in the formation of the pair and give an exchange lifetime that is quite long when compared to simple ion-pairing interactions or to coordination of ligands at a labile site. The structural assessments allow approximate placement of the iron atom and consequently the [FeTMPyP(DMSO)IS+plane in the electrostatic pair. The resulting proposed structure is consistent with a cofacially oriented dimeric system displaying modest X-A interactions. Acknowledgment. We thank Professor W. L. Reynolds for the gift of Na4[H2TSPc]and Professors A. G. Lappin, D. M. Stanbury, and F. A. Walker for helpful discussions. We thank Dr. Habib Nasri for assistance with the high ionic strength homoaggregation experiments. We gratefully acknowledge the National Institutes of Health for funding through Grant GM3840 1 - 16. (48) Song, H.; Rath, N. P.; Reed, C. A,; Scheidt, W. R. Inorg. Chem. 1989, 28, 1839-1847. (49) Spaulding, L. D.; Eller, P. G.;Bertrand, J. A,; Felton, R. H. J . Am. Chem. SOC.1974, 96, 982-987.
Ground- and Excited-State Conformational Heterogeneity of the 2'-Naphthylbutadiene Chromophore of a Fluorescent Cholesterol Analogue Probet Jacinta Drew,f*.l Francesco Zerbetto,ll,# Arthur G.Szabo,*'*and Peter Morands Division of Biological Sciences and Division of Chemistry, National Research Council of Canada, Ottawa, Ontario, Canada, K I A OR6, and Ottawa-Carleton Chemistry Institute, University of Ottawa, Ottawa, Ontario, Canada, K I N 9B4 (Received: August 28, 1989; I n Final Form: November 27, 1989)
Steady-state, time-resolved, and temperature-dependent fluorescence studies, together with molecular orbital calculations, have been used to investigate the photophysical behavior of the 2'-naphthyldiene side chain of a novel fluorescent cholesterol analogue with the aim of establishing the utility of this molecule as a cell membrane probe. The photophysics is largely governed by the presence of two ground-state conformers, Le., the s-cis and s-trans rotamers about the single bond adjacent to the naphthyl group. The presence of this ground-state conformational equilibrium could prove useful in monitoring changes in membrane order. An excited-state viscosity-dependent process of the s-cis conformer also shows promise to be exploited to study membrane dynamics.
Introduction Polyenes are the subject of very active research due to their involvement as chromophores in biological systems' and their industrial applications as conductors,2 and also because the un-
'Issued as NRCC No. 31240. t Division
of Biological Sciences, NRCC.
(University of Ottawa.
Present address: Russell-Grimwade School of Biochemistry, University of Melbourne, Parkville, Victoria 3052, Australia. 11 Division of Chemistry, NRCC. # Research Associate.
0022-3654/90/2094-4439$02.50/0
derstanding of their properties and dynamics is a critical benchmark for chemical t h e ~ r y . a,w-Diphenylpolyenes ~ exhibit most of the properties of the parent linear compounds, except that they are generally more suitable for UV spectroscopic studies because of their higher quantum yields of fluorescence. This has resulted (1) Callender, R. In Biological Events Probed by Ultrafast Spectroscopy; Alfano, R. R., Ed.; Academic: New York, 1982; p 239. (2) Stotheim, T. J., Ed. Handbook of Conducting Polymers; Marcel Dekker: New York, 1986. ( 3 ) Hudson, B. S . ; Kohler, B. E.; Schulten, K. In Excited States; Lim, E. C., Ed.; Academic: New York, 1982; Vol. 6 , p I .
0 1990 American Chemical Society
4440
The Journal of Physical Chemistry, Vol. 94, No. 11, 1990
Drew et al.
added an equimolar amount of n-butyllithium in hexane. The reaction mixture was allowed to reach ambient temperature and an equimolar amount of 2-naphthaldehyde dissolved in tetrahydrofuran was added followed by the addition of an equimolar amount of potassium n-butoxide in tetrahydrofuran. After stirring at room temperature for 3 h, the reaction mixture was worked up in the usual way6q7and a 71% yield of DN was obtained by chromatography over silica gel with 9: 1 hexane:ethyl acetate as eluant [mp 106 OC; m / z = 208 (M+), 193 (M+-CH3);6 1.89 (s, HO 3 H, C=CCH,), 1.92 (s, 3 H, C=CCH3), 6.08 (d of d, 1 H, J = 11.0, 2.0 Hz, H-3), 6.60 (d, 1 H, J = 15.4 Hz, H-I), 7.14 (d DNC of d, 1 H, J = 15.5, 10.9 Hz, H-2), 7.41 (d of d of d, 1 H, J = 7.4, 7.4, 1.5 Hz, H-7’), 7.46 (d of d of d, 1 H, J = 7.4, 7.4, 1.6 in the widesmead use of 1.6-di~henvl-1,3.5-hexatriene (DPH) as . * Hz, H-89, 7.65 (d of d, 1 H, J = 8.72, 1.7 Hz, H-3’), 7.73 (s, a fluoresceit membrane probe. It can be argued, however, that 1 H, H-l’), 7.76-7.80 (m, 3 h, H4’, H-6’, H-9’). The E-geometry DPH is net the best possible membrane probe since it bears no about the C-1 ,C-2 double bond was confirmed by the observed resemblance to any natural membrane component, and its precise coupling constant of 15.4 Hz for the H-1 proton. A small amount location within the membrane is unknown. Indeed, in model of the Z-isomer, 4-methyl-I -(2’-naphthyl)-( IZ)-3-pentadiene, was membranes, it has been suggested that DPH may adopt orientations both parallel and perpendicular to the bilayer n ~ r m a l . ~ ~ also ~ obtained and the observed coupling constant of 9.0 Hz for H-1 is consistent with what has been reported for Z - ~ l e f i n s . ~ In this and in a forthcoming paper, the potential of sterols with 2. Absorption and Fluorescence Measurements. Absorption aryl polyene side chains, as membrane probes, is investigated. and fluorescence excitation and emission spectra were measured These compounds were chosen because they resemble cholesterol on a Varian DMS-200 UV/visible spectrophotometer and an SLM both in their geometry, as judged by comparing space-filling 8000C spectrofluorimeter (equipped with a Neslab RTE- 5DD models, and their amphipathic nature. Since a suitable membrane temperature controller). All fluorescence spectra were collected probe is one which is able to report on differences of polarity with no polarizer in the excitation beam and with an emission and/or fluidity (Le., order and dynamics) within a membrane, polarizer oriented at 35.3’ to the vertical in order to eliminate the spectroscopic and photophysical behavior of a 2’-naphthyldistortions due to Brownian motion.I0 Besides correcting all butadiene sterol has been investigated by experimental and thefluorescence spectra by the signal from appropriate blanks, exoretical techniques. citation spectra were divided by the excitation spectrum obtained Experimental Section from an optically dense solution of rhodamine B in ethylene glycol (Ae,, 640 nm).” No additional correction of emission spectra 1. Materials. The solvents used in the absorption and was made since emission spectral correction factors were found fluorescence studies were 2,2,4-trimethylpentane (TMP, Caledon), to be constant in the region 350-450 nm. methylcyclohexane (MCH, Caledon), 1-butanol (1 -BuOH, Fluorescence quantum yields were measured on solutions of Fisher), and methanol (MeOH, Caledon) and were of the highest low absorbance (50.1), and are relative to a value of 0.32 for purity grade available. Heavy (H) and light (L) mineral oils anthracene in ethanol (Aex 340 nm).I2 Low-temperature (Drug Trading Co.) were purified by silica gel chromatography steady-state and time-resolved fluorescence measurements were (two passes) using silica gel 60 (70-230 mesh, Merck). Kinematic made on solutions in quartz tubes which were placed in a homeviscosities and densities of the two oils, and mixtures thereof (H/L built cryostat. 50/50 v/v, designated as H I L I ; H / L 75/25 v/v, designated as 3. Time-Resolved Fluorescence Measurements. Time-resolved H3LI), were measured by technical staff in the Fuel and Lufluorescence measurements were made by the method of timebricants Laboratory, Division of Mechanical Engineering, National correlated single photon ~ o u n t i n g ,employing ’~ instrumentation Research Council of Canada. All solvents and oils were checked that has been described previ0us1y.l~ However, due to the diffor fluorescent impurities before use. If necessary, in order to ferent excitation wavelengths used in this work, some of the details eliminate oxygen quenching of fluorescence, solutions of DNC are presented here. The excitation source was a dye laser (Spectra and DN were degassed either by several freeze-pumpthaw cycles, Physics 375) synchronously pumped by a model-locked argon ion bubbling with high-purity nitrogen, or by pumping under vacuum laser (Spectra Physics 171-07, beryllium oxide bore, or Spectra (1 mm) in a desiccator for 1 h (oil solutions only). Physics 2030, ceramic bore). Experiments at excitation waveThe synthesis, purification, and high-resolution IH and I3C N MR characterization of [20(22)E,23E)-24-(2’-naphthyl)-cho- lengths of 320 nm were performed by frequency doubling of the 640 nm output rhodamine 6G in ethylene glycol (2 mM), while la-5,20(22),23-trien-3&ol (DNC, see Scheme I) has been reported the 680- and 700-nm outputs of DCM in benzyl alcohol/ethylene p r e v i o ~ s l y . ~4-Methyl*~ 1-(2’-naphthyl)- 1,3-pentadiene (DN), glycol (40:60, 1.5 mM) were frequency doubled to excite samples the dimethylaryldiene analogue of the DNC side chain, was at 340 or 350 nm.15 Typical average power of the 20-ps fwhm synthesized from 2-naphthaldehyde (Aldrich) by a phosphonate dye laser output pulses was 40 mW at 640 nm and 30 mW at 700 Wittig reaction employing the carbanion generated from dinm. Second harmonic generation was achieved by using two ethyl-3-methyl-2-butenyl phosphonate. This latter compound was different angle-tuned crystals (R6G laser lines, Cleveland Crystals prepared by refluxing equimolar amounts of 1-bromo-3KDPI; DCM laser lines, Inrad KDP ‘C’ model 542-120). methyl-2-butene (Aldrich) and triethyl phosphite in the dark and Fluorescence detection was by means of a proximity-type miunder a nitrogen atmosphere for 4 h. The best yield of DN was crochannel plate detector (Hamamatsu RI 564U-0 l ) , after colobtained by using the modified conditions described by Schlosser lection of the fluorescence through a polarizer oriented at 54.7’ and Christmann.s To a cooled solution (-78 “C) of diethyl-3to the verticallo and a Jobin Yvon H-10 monochromator with methyl-2-butenyl phosphonate in anhydrous tetrahydrofuran was either 4- or 2-nm resolution. The data was recorded in either 1024 SCHEME I
(4) van Ginkel, G.; Korstanje, L. J.; van Langen, H.; Levine, Y. K. Faraday Discuss. Chem. SOC.1986,81, 49. (5) Deinum. G.; van Langen, H.; van Ginkel, G.;Levine, Y. K. Biochemistry 1988,27, 852. ( 6 ) Drew, J.; Letellier, M.; Morand, P.; Szabo, A. G.J. Org. Chem. 1987,
52, 4047.
( 7 ) Drew, J.; Brisson, J. R.; Morand, P.; Szabo, A. G. Can. J . Chem. 1987, 65, 1784. (8) Schlosser, M.; Christmann, K. F. Angew. Chem., Inf. Ed. Engl. 1964, I . 112.
(9) Wernly, J.; Lauterwein, J. J . Magn. Reson. 1986,66, 355. (IO) Badea, M. G.;Brand, L. Methods Enzymol. 1979,61, 378. ( I I ) Ghiggino, K. P.; Skilton, P. F.; Thistlethwaite, P. J. J . Photochem. 1985,31, I 1%. (12) Himel, C. M.; Mayer, R. T. Anal. Chem. 1970,42, 130. (13) O’Connor, D. V.;Phillips, D. Time-Correlated Single Photon Counting, Academic: London, 1984. (14) Zuker, M.; Szabo, A. G.; Bramall, L.; Krajcarski, D. T.; Selinger,
Rec. Scr. Instrum. 1985,56, 14. ( 1 5 ) Marason, E. G Opt. Commun. 1981,37, 56.
B.
Conformation of 2'-Naphthylbutadiene Chromophore
The Journal of Physical Chemistry, Vol. 94, No. 11. I990 4441
or 2048 channels of a multichannel analyzer (MCA, Tennecomp) at a resolution of 42.4 ps/channel or 84.8 ps/channel. Usually at least 16000 counts were collected in the maximum channel of the sample decay curve. A blank was measured for each sample (except in the low-temperature experiments) for the same accumulation time, as controlled by a real time clock in the MCA. The instrument response profile (fwhm 80 ps) was determined by measuring the scattered light from a suspension of rabbit liver glycogen (Sigma) in cacodylate buffer (pH 7.0),having an optical density of 0.1 at the excitation wavelength. All fluorescence intensity/time profiles were acquired sequentally and transferred to an IBM 3090/200 VF computer for data analysis. 4 . Time-Resolved Data Analysis. Sample fluorescence decay curves were fitted by iterative convolution (Marquadt algorithm16) of the instrument response profile with trial functions, F,(t), in the form of sums of n exponential terms i- I
r
I
250
n
F,(t) =
1.00
aie-'/rl
(1)
where ai is the preexponential factor associated with fluorescence decay time T ~ .The quality of fitting was judged by the values of the statistical parameters x2, the reduced x2, and SVR (the serial variance ratio)," as well as by inspection of the randomness of the plot of weighted residuals. Once it was determined that T was independent of emission wavelength, curves obtained at various emission wavelengths were analyzed simultaneously so as to improve the accuracy of the fluorescence decay parameters through the principle of overdetermination.I8 Fluorescence decay associated emission spectra (DAS), Di(X,), were computed from the fluorescence decay parametrs at all the emission wavelengths (every 2-5 nm) for which fluorescence decay curves were measured at [bX] = 320 nm, using eq 219 where F[he,](he,) is the intensity
at bm of the steady-state fluorescence spectrum obtained at A[ ], = 320 nm, and [ ] denotes a fixed parameter while ( ) denotes a variable parameter. These DAS represent the emission spectra of different populations of a fluorophore when the various decay times with which they are associated are simply a result of ground-state heterogeneity. In the case where the multiexponential decay arises from an excited-state reaction, the fluorescence decay curves need to be analyzed directly for the various rate constants and "species associated emission spectra" (SAS) computed.20 However, SAS can be extracted from DAS in certain cases, for example in the case of an irreversible excited-state reaction between two species A* and B*.20 Excitation spectra were also resolved into component spectra, Ei(Aex),on the basis of their association with different fluorescence decay times. Specifically, k steady-state excitation spectra, where k 1 n, and fluorescence decay data obtained at these same k emission wavelengths and at a given b, of 320 nm, were used to arrive at indirect excitation decay associated spectra (IEDAS)21through
(16) Marquadt, D. W. J . SOC.Ind. Appl. Moth. 1963, I ! , 431. (17) Durbin, J.; Watson, G. S. Biometriko 1971, 58, I . (18) Knutson, J. R.; Beechem, J. M.; Brand, L. Chem. Phys. Lett. 1983, 102, 501. (19) Donzel, B.; Gauduchon, P.; Wahl, P. J . Am. Chem. SOC.1974, 96, 801.
(20) Knutson, J. R.; Walbridge, D. G.; Brand, L. Biochemistry 1982, 21, 4671.
I
280
I
I
310
I
1
340
370
400
Wavelength (nm) Figure 1. Absorption spectra of DN (-) and DNC (---) in MeOH.
The k excitation spectra make up the I matrix while the entries in the M matrix are the a,[320](A,,)~~products, with the sum of these products at a given bmhaving previously been normalized to the steady-state fluorescence intensity F[320](&,). The desired IEDAS are simply the columns of the E(Aex,n)matrix and were determined by a linear least-squares analysis. Computational Methods In a series of papers,22 it has been shown that the combined use of two semiempirical methods, namely QCFF/PI (quantum consistent force field for 7 electrons23)and CNDO/S (complete neglect of differential overlap/spectroscopic parametri~ation),~~ is very effective to study zero-, first-, and second-order properties of electronic transitions. The applications included polyenes and aromatic compounds and derivatives thereof. The QCFF/PI method is used to optimize the molecular geometries. This method treats ?T electrons in a quantum chemical way through a self-consistentfield (SCF) followed by configuration interaction (CI). The u bonds and nonbonded interactions are accounted for by empirical potential functions. The CNDO/S method is an all-valence electron Hamiltonian parameterized for spectroscopic properties. Here, it was used in two formulations: when only the gross features of the electronic spectrum were being investigated, the two-electron integrals were calculated with the Mataga-Nishimoto equation25and the calculation of the configuration interaction was limited to singly excited configurations. The Pariser-Parr formula26and both singly and doubly excited configurations were used when the location of the low-lying polyenic-like3 doubly excited state was critical. In both cases, all the mr* excitations were explicitly included in the calculations. This is mandatory to obtain the exact position of the doubly excited state. Results and Discussion An assessment of the possibility of exploiting DNC as a membrane probe can only be reached through an understanding (21) Willis, K. J.; Szabo, A. G.; Drew, J.; Zuker, M.; Ridgeway, J. Biophys. J . Submitted for publication. (22) Orlandi, G.;Poggi, G.; Zerbetto, F. Chem. Phys. h r t . 1985, 115, 253. Orlandi, G.; Zerbetto, F. Chem. Phys. Lett. 1985, 120, 140. Orlandi, G.; Zerbetto, F. Chem. Phys. 1986, 108, 187. Orlandi, G.; Zerbetto, F. Chem. Phys. 1986, 108, 197. Zerbetto, F.; Zgierski, M. Z. Chem. Phys. 1986, 110, 421. Orlandi, G.; Zerbetto, F. Chem. Phys. 1987, 113, 167. Zerbetto, F.; Zgierski, M. 2.; Orlandi, G.; Marconi, G . J . Chem. Phys. 1987, 87, 2505. Negri, F.; Orlandi, G.;Zerbetto, F. Chem. Phys. Lett. 1988, 144, 31. Orlandi, G.; Zerbetto, F. Chem. Phys. 1988, 123, 175. Zerbetto, F.; Zgierski, M. Z. Chem. Phys. 1988, 127, 17. Zerbetto, F.; Zgierski, M. Z. Chem. Phys. Lett. 1988, 153, 436. (23) Warshel, A.; Karplus, 'M. J . Am. Chem. SOC. 1972, 94, 5612. Warshel, A. In Modern Theoretical Chemistry; Segal, A,, Ed.; Plenum: New York, 1977; Vol. 7, part A, p 133. (24) Del Bene, J.; Jam, H. H. J . Chem. Phys. 1968, 48, 1807. (25) Nishimoto, K.; Mataga, N . Z . Phys. Chem. (Frankfurt) 1957, 12, 335. (26) Pariser, P. J . Chem. Phys. 1953, 21, 568.
4442
The Journal of Physical Chemistry, Vol. 94, No. 11, 1990
Drew et al.
I w t a t i o n HawlenEtli (mi) I
250
280
I
I
310
1
I
340
1
370
1 400
Wavelength (nm) Figure 2. Absorption spectra of DNC in MeOH (-)
and in n-BuOH
Figure 4. The surface of fluorescence intensity versus excitation and emission wavelengths for DNC in TMP at 20 OC. 1.00,
(---).
W a v e l e n g t h (nm) 260
300
400
500
W a v e l e n g t h (nm)
Figure 3. Fluorescence excitation (Aem 390 nm) and emission (A,, 320 nm) spectra of DNC in TMP.
of its spectroscopic and photophysical properties. As a first step, the absorption spectrum of DNC was measured and compared with that of the parent molecule DN. The spectra in methanol are presented in Figure I . As expected, the chromophore of DNC is the naphthyldiene side chain. The overall effect of the steroid moiety is simply to shift the absorption spectrum to lower energies, as is usually the case when alkyl groups are introduced into conjugated molecules. The spectra show that SI is a weak state which could, in principle, be identified with either a polyenic 2A,--like state or a naphthalenic Lb-like state. Figure 2 shows the absorption spectra of DNC in methanol and 1-butanol. The band position is sensitive to solvent polarizability and shifts to longer wavelengths with the increase of solvent refractive index (methanol, n20D = 1.3288, I-butanol, n20D = 1.3993). Solvent polarity, on the other hand, has little effect as judged by the similarity of the abosrption spectrum of DNC in I-butanol and 2,2,4-trimethylpentane (TMP), two solvents of similar refractive index but different polarities. Fluorescence excitation and emission spectra of DNC in TMP are presented in Figure 3. It is readily seen that the low-intensity state at 368 nm is responsible for the emission. The 3D surface of DNC fluorescence intensity versus excitation and emission wavelengths is shown in Figure 4. Sections of this surface are given in Figure 5 and Figure 6 . It is interesting to note that a variation of &, of only 10 nm (from 360 to 370 nm) has a strong effect on the shape of the fluorescence excitation spectrum (Figure 5). This feature, together with the qualitatively different emission spectra obtained at A,, = 320 and 355 nm, and the analogy with the widely studied 1-phenyl-2-(2’-naphthyl)ethene (PNE)27-36point (27) Scheck, Y. 9.;Kovalenko, N . P.; Alfimov, M. V. J . Lumin. 1977, I S . 157
Figure 5. Emission wavelength dependence of excitation spectrum of DNC in TMP (peak normalized);&, 360 nm (A), A,, 370 nm (B), hm 390 nm (C),A,, 410 nm (D).
E m i s s i o n W a v e l e n g t h (nm)
Figure 6. Excitation wavelength dependence of emission spectrum of DNC in TMP (peak normalized); A,, 320 nm (A), A,, 340 nm (B), A,, 355 nm ( C ) .
toward the existence of several conformers of DNC in the ground state. (28) Haas, E.; Fischer, G.; Fischer, E. J . f h y s . Chem. 1978, 82, 1638. (29) Birks, J. 9.;Bartocci, G.; Aloisi, G. G.; Dellonte, S.; Barigelletti, F. Chem. f h y s . 1980, 51, 113. (30) Fischer, E. J . Mol. Srrucr. 1982, 84, 219. (31) Mazzucato, U. Pure Appl. Chem. 1982,54, 1705. (32) Matthews, A. C.; Sakurovs, R.; Ghiggino, K.P. J. fhorochem. 1982, 19, 235. (33) Bartocci, G.; Masetti, F.; Mazzucato, U.; Marconi, G. J . Chem. Soc., Faraday Trans. 2 1984,80, 1093. (34) Saltiel, J.; Eaker, D. W.J . Am. Chem. SOC.1984, 106, 7624. (35) Bartocci, G.; Mazzucato, U.; Masetti, F.; Aloisi, G. G. Chem. Phys. 1986, 101, 461.
Conformation of 2'-Naphthylbutadiene Chromophore
The Journal of Physical Chemistry, Vol. 94, No. 11, 1990 4443
-
TABLE I: Fluorescence Quantum Yields, bf, for DNC in Various Solvents at Excitation Wavelength, X,
8000
-
solvent TMP
c1 1
5
n-BuOH
MeOH HlLO
2ooo1
A,,
temp,' OC 20.1 20.1 20.1 20. I 20. I 20.1 20.1 20.1 20.0 0.5 10.0 19.9 29.9
bP
nm 310 3 20 340 355 310 320 340 350 340 340 340 340 340
0.41 0.43 0.45 0.45 0.24 0.26 0.26 0.24 0.12 0.65 0.58 0.51 0.44
0 f 0 . 1 OC. *&5% error. 35
0
1
I
70
105
175
140
time ( ns) \\\,
Figure 7. Fluorescence decay profile of D N C in T M P (& 320 nm, A,,
410 nm, T = 20.5 "C)measured at channel width 84.8 ps/channel. The instrument response function is the width of the line on the rising edge. A
i
3
L 0
0
.B
8000
I
I
I
I
I
400
800
1200
1600
2000
channe!s
3
0
B
20
10
30
40
50
60
70
80
90
time ( ns ) Figure 9. Fluorescence decay profile of D N in M C H (Aex 320 nm, ,A,
390 nm, T = 20.5 " C ) measured at channel width 84.8 ps/channel. A
2
;
0
0
.a
I
I
I
I
1
400
800
1200
1600
2000
channels
3
C
B
W
.-F0
s
I
I
0
460
800
1200
16b0
20'00
cncnne!s
Figure 8. Plot of the weighted residuals/rmt mean square of residuals for the best (A) single-exponential fit ( x 2 = 11.1, SVR = 0.02); (B) double-exponential fit ( x 2 = 1.15, SVR = 1.52); (C) triple-exponential fit ( x 2 = 1.02, SVR = 1.90) for the fluorescence decay profile in Figure 7.
A straightforward proof of ground-state heterogeneity would be a marked variation of fluorescence quantum yield, @f with kx. This, coupled with the emission spectra of Figure 6, would demonstrate that different DNC conformers absorb and emit at different wavelengths. Fluorescence quantum yields of DNC in various solvents at different A,, are shown in Table I. The observed invariance does not preclude the possibility of a ground (36)Muszkat, K.A.; Wismontski-Knittel,T. Chem. Phys. Lett. 1981,83, 87.
I
t
I
I
I
1
0
200
400
600
800
1000
channels Figure 10. Plot of the weighted residuals/root mean square of residuals
for the best (A) single-exponential fit ( x 2 = 8.09, SVR = 0.02); (B) double-exponential fit ( x 2 = 1.04, SVR = 1.78) for the fluorescence decay profile in Figure 9.
state equilibrium as long as the conformers have similar values. The marked differences of $f with solvent polarity suggest that DNC may be useful as a membrane polarity probe. The analysis of time-resolved fluorescence data provides further evidence for ground state conformational heterogeneity. Fluorescence decay profiles of DNC, under a number of different experimental conditions, were successfully fitted only by a triple exponential decay function (see Table I1 and Figures 7 and 8). C#J~
Drew et al.
The Journal of Physical Chemistry, Vol. 94, No. 11, 1990
4444
TABLE 11: Lifetimes, rI,and Normalized Preexponential Factors, q,of DNC in Various Solvents at Excitation and Emission Wavelengths,
kx
and x,
solvent
temp,
O C
TMP
20.5
n-BuOH
19.9 19.9 20.2
MeOH MCH HILO
X ,/,
nm
19.9 19.9 19.9 19.9 20 20 20 20 20
ns
r2, ns
69.5‘ 69.5 69.5 66.1 63.9 27.72 27.72 27.64 27.27 27.27 27.27 14.36 14.42 20.32 20.50 55.50
5.42 5.42 5.42 5.29 5.30 2.54 2.54 2.57 2.56 2.56 2.56 1.71 1.80 3.99 4.1 1 4.68
T,,
320/360 320/370 320/390 3401390 350/390 320/370 320/390 340/390 350/370 350/390 350/410 320/410 320/410 320/390 320/390 320/390
ns
r4,ns
0.34 0.34 0.34 0.20 0.41 0.11 0.1 1 0.1 1 0.082 0.082 0.082
0.02b 0.02 0.02 0.02 0.02
73.
0.02
0.48 0.20 0.67
0.05
“1
0.258 0.100 0.170 0.032 0.005 0.280 0.264 0.107 0.007 0.011 0.01 1 0.320 0.206 0.19 0.158 0.154
In this and in the following tables the error in the lifetimes is in the last decimal place shown. reported for completeness since it arises from an instrumental artifact
TABLE 111: Decay Times, T,, and Normalized Preexpooential Factors, aB of DN, in Various Solvents at Room Temperature solvent MCH MCH MeOH MeOH
_____
X,,/~,m.
nm 320/390 320/390 320/390 320/390
TI?
ns 31.8 32.1 28.6 28.7
72,
ns
739
ns 6.97 7.04 0.10 3.75 3.78 0.022
a,
0.150 0.127 0.282 0.222
CY,
as
x2
0.850 1.04 0.725 0.148 1.00 0.718 0.99 0.565 0.213 0.98
SVR 1.78 1.94 1.87 1.89
TABLE IV: Fluorescence Decay Parameters“ of DNC in n-BuOH at L o w Temoeratures (X, 320 nm. h 370 nm)
ff2
ff3
0.409 0.789 0.704 0.914 0.942 0.406 0.501 0.820 0.436 0.614 0.649 0.680 0.406 0.81 0.670 0.585
0.333 0.111 0.127 0.054 0.053 0.314 0.235 0.073 -0.556 -0.375 -0.340
-50 -81
-105
0.282 (0.836) 43.78 4.08 0.74 0.02 0.346 (0.877) 50.04 4.46 0.90 0.02 0.398 (0.91 1) 52.67 4.59 1.20 0.02 0.417 (0.924)
0.572 (0.159) 0.474 (0.112) 0.380 (0.078) 0.315 (0.061)
0.146 (0.006) 0.180 (0.008) 0.222 (0.009) 0.268 (0.013)
0.172 0.260
SVR 1.99 1.99 1.99 2.00 1.84 1.97 1.97 1.94 1.81 1.81 1.81 1.42 1.96 1.38 1.82 1.92
this and in the following tables r4 is only
TABLE V Fluorescence Decay Parameters of DNC in Mineral Oils as a Function of Viscositv (h.350 nm. h 370 nm) temp, 7, rI, 72, 7 3 , oil OC CP ns ns ns al a2 aj x2 SVR HILO” 30.3 100 43.4 4.47 0.42 0.011 0.890 0.100 1.04 1.86 59 42.1 4.42 1.35 0.011 0.945 0.044 1.03 1.90 H3Ll 30.7 HILI 30.7 38 42.3 4.40 0.83 0.011 0.980 0.009 1.12 1.75 OThere are large errors associated with the data in the solvent at this temperature, since only -650 channels at 84.8 ps/channel were used.
SCHEME I1
A 19.9 27.70 2.55 0.23 0.03
0.388
x2 1.03 1.03 1.03 1.01 1.03 1.03 1.03 1.02 1.25 1.25 1.25 1.26 1.02 1.15 1.00 1.01
B
k%,
C
*
1.03 1.97 kA
*
1.08 2.03
A ‘ +
1.57 1.74
O 3 are the fractional fluorescence values of each ith component, where 3,
= ffrT,/Xal7I.
(In some cases a fourth component improved the statistics of fitting, but the origin of this short-lived component probably lies in an instrumental artifact since it is of the order of one channel width.) In order to gain some understanding of the origin of the three fluorescence decay components, fluorescence decay profiles of DN were also measured (see Figures 9 and 10, and Table 111). In this latter case, satisfactory fits were obtained with doubleexponential functions. It can be concluded that the first and the second decay processes are intrinsic to the naphthyldiene fluorophore while the third is induced by the introduction of the sterol group. The simplest way to account for the multiexponential decay is to associate a different species with each exponential term: three species (viz., A, B, C) for DNC and two for DN. The species could be either different excited states of the same molecule or different conformers. On the basis of the steady-state results presented earlier, the latter hypothesis is preferred. It was with this “a different conformer for each different exponential” model, together with the further simplifying assumption that the conformers do not undergo equilibration in the excited state (NEER ~rinciple),~’ that the kinetics of the aforementioned PNE was successfully analyzed. Attractive as it is, the NEER principle may not be the best explanation of the photophysics of DNC because of the different number of decay components of DNC and DN;38*39 thus, the (37) Jacobs. H. J. C.: Havinga, E. Adu. fhorochem. 1979, 11. 305
4
A
1.43 1.91
k,
*
kC’
+
B
temperature dependence of the fluorescence decay parameters of DNC was investigated (Table IV). All three decay times increased with decreasing temperature because of the obvious reduced efficiency of nonradiative processes at lower temperature. More important, however, is the fact that the preexponential factors and a, increased while a2decreased as the temperature was lowered. A study of the viscosity effect at constant temperature (see Table V) showed that, of the decay times, only T~ is affected by solvent viscosity. The preexponential factor a I is viscosity-invariant while a2decreases and a, increases with increasing viscosity. Comparison of Tables 11, 111, and V shows the following: (1) the species associated with T , is the most stable, and its photophysics is viscosity independent; (2) the negative preexponential factor a3,obtained at A,, = 350 nm and various A,, (see Table 11), indicates that the species associated with the short T~ is involved in an excited-state process; (3) the variation of T~ with viscosity demonstrates that this process is viscosity-dependent; (4) the opposite variations of a2 and a3 with viscosity show that the excited-state process involves an interconversion of the species associated with T* and T 3 ; ( 5 ) and finally the insensitivity of f 2 to viscosity shows that the reaction is irreversible and that the NEER principle does indeed hold. (38) Mazurenko, Y. T.; Udaltsov, V. S.; Cherkasov, A. S. Opt. Spectrosc.
(USSR)1979, 46, 389.
(39) Flom, S. R.; Nagarajan, V.; Barbara, P. F. J . Phys. Chem. 1986, 90,
2085.
(40) Beechem, J. M.; Ameloot, M.; Brand, L. Anal. Instrum. 1985, 14, 379. (41) Beechem, J. M.; Ameloot, M.; Brand, L. Chem. f h p . Lett. 1985,120, 466.
The Journal of Physical Chemistry, Vol. 94, No. I I , 1990 4445
Conformation of 2'-Naphthylbutadiene Chromophore
E m i s s i o n W a v e l e n g t h (nm) 0
Figure 11. Normalized decay associated emission spectra (DAS) for DNC in TMP at A,, 320 nm, 7 = 69.5 ns (A), 7 = 5.42 ns (C). TABLE VI: Derived Photophysical Parameters of DNC in TMP at 20.5 OC component /1(& rI, 7fr i 340 nm) ns ns #h kf,,s-' k,,,, s-I cp 1 0.718 69.5 164 0.42 6.09 X IO6 8.30 X IO6 0.69 2 0.281 5.42 11.7 0.46 8.55 X IO' 9.90 X IO' 0.31 3 0.002 0.34
0
260
270
280
sbo
290
3io
ko
3jO
A
&Q
360
370
EXCVATION WAVELENGTH (nm)
Figure 12. Unnormalized indirect excitation decay associated spectra (IEDAS) for DNC in TMP at ;L, 390 nm, 7 = 69.5 ns (-); T = 5.42 ns (---). SCHEME 111
Calculated neglecting component 3.
On the basis of these conclusions, the three species A, B, and C are proposed to be interrelated as shown in Scheme 11. A and B are different ground-state conformers of DNC, while C is formed by an irreversible excited-state reaction from B. The kinetic parameters which are associated with this scheme are derived by means of eqs 4-9.
u -NBD
t~ -NBD
S; (Lb) Si (B,)
s3 s: 1
1
k~ + ~
(Y3
C B
kf, + knr,
tc-NBD
(6)
(Yi
=
a2
= (Yc
(8)
= ( Y B - (YC
(9)
(YA
(7)
Conformer A is the more stable form of DNC in the ground state. The nature of the excited-state process between B and C is proposed to be a conformational change from a nonplanar B conformer to a planar C conformer, consistent with the tendency for planarity in excited states of conjugated molecules through increased delocalization. Out-of-plane displacements are known to promote radiationless transitions and this, coupled with the irreversible excited-state reaction, can qualitatively account for the order of magnitude shorter decay time f 3 ( = T ~ )compared with ' T (vC). ~ The nonplanarity of B appears to be a consequence of the presence of the bulky steroid group, since the fluorescence decay of DN is due to only two species, with decay times similar to those of 7, (=s,) and ' T ~( = T ~ )of DNC. With this kinetic scheme, and the associated equations relating the observed preexponential factors aI.aZ,and cy3 to "species-associated" preexponential factors aA,ag,and aC,it is possible to deconvolve the emission spectrum of DNC to obtain species-associated emission spectra (SAS). As a first approximation, the third component (Le., that associated with 7J can be neglected since it accounts for less than 2% of the fluorescence intensity. The SAS for the two species A and C are identical in shape with the DAS associated with T , and 72, respectively, and are shown in Figure 1 I . Indirect excitation decay associated spectra (IEDAS, see Experimental Section) were also deconvoluted from the excitation spectrum of DNC and are shown for 71and 7*in Figure
a -NBD
TABLE VII: Excitation Energies and Oscillator Strengtbs, f, of Low-Lvine Excited Electronic States of the NBD Rotamers rotamer' state E,6 nm E: nm f fd tt-NBD
73=-=-
a-NBD
S; (Lb) S i (8,)
s3 s: ct-NBD
S; (Lb) S i (B,)
s3 s; cc-NBD
S; (Lb) s p " ) s4
329 303 266 238 328 310 264 239 329 306 269 239 328 313 261 240
363 332 281 363 345 280
0.0113 1.1236 0.9870 0.0 163 0.0022 0.7750 1.2183 0.0225 0.0131 0.9906 0.8476 0.0222 0.0038 0.8788 0.9182 0.0438
0.02 1.00 0.63 0.08 0.36 0.50
-
'See Scheme 111 for a diagram of the various rotamers of NBD. *Calculated. CPositionsof the 0-0 bands of the So S,* transitions of tt-DNC and tc-DNC from their respective IEDAS based on the conformational assignment discussed in the text. dRelative c, in arbitrary units, at the 0-0 band of the So S,' transitions of tt-DNC and tcDNC from their IEDAS (Aex 390 nm), arbitrarily assigning f(So-S;, 0-0) for tt-DNC at 1.00.
-
12. An analysis, presented in the Appendix, allowed calculation of photophysical parameters of A and C (see Table VI) from which it is possible to gain insight into the identities of these two species. Molecular orbital calculations were of considerable help in this respect. The geometry optimization of the ground state of the four conformers of the model molecule 1-(2'-naphthyl)-l,3-b~tadiene (NBD, see Scheme 111) leads to planar conformations. This is in keeping with the planarity of s-~is-butadiene.~~ It is expected, however, that alkyl substitution will affect the planarity. (42) Squillacote, M. E.; Sheridan, R. S.; Chapman, 0. L.; Anet, F. A. L. J . Am. Chem. SOC.1979, 101, 3657. Fisher, J. J.; Michl, J. J . Am. Chem. Soe. 1987, 109, 1056.
4446
The Journal of Physical Chemistry, Vol. 94, No. I I , I990
The excitation energies and oscillator strengths predicted for transitions from the ground state are reported in Table VII. In all the conformers SI is a naphthalenic Lb-like state, while the polyenic 2A --like state is calculated to be 140 nm higher in energy than S, and will not play a role in the spectroscopic properties of these molecules. S2is predicted to be a polyenic B,-like state for all conformers. Comparison of the IEDAS spectra of DNC and the calculated oscillator strengths of NBD (in particular the relative intensities of the So-S2and So-& transitions for a given conformer) support the notion, also dictated by chemical intuition, that the more stable A species is the tt conformer and that C is a planar tc conformer. The one apparent inconsistency between the experimental and the calculated spectra is the intensity of the So-Sl transition. The calculation predicts that the transition is stronger for the tt species. However, the energy gap between SI and S2 is much smaller for the tc as opposed to the tt rotamer (see Figure 12 and Table VII), a feature nicely reproduced by the calculations. As a consequence, intensity borrowing mechanisms will be much more effective for the tc conformer. Since the So-S2transition is very strong, this will result in a stronger So-S, transition for the tc conformer and in a radiative lifetime closer to that of an intense state (Le., shorter). This is indeed the case, since the radiative lifetime of the C species is 1 1.7 ns compared with 164 ns for species A (see Table VI). When an excited-state reaction occurs between two fluorescent species such as is proposed to occur between B and C in Scheme 11, then the kinetic expressions for the fluorescence of B* and C* may be described by eqs IO and 1 1 where y I ,y2, cyc, and as are F B ( t ) = ace-‘/T1
+ aBe-‘/72
Fc(t) = cyc(e-‘/71- e-‘/%)
(43) Birks. J .
function of the inverse of the sixth power of the distance, the observation of an enhancement of the resonance of both protons can only be explained by the coexistence of the tt and tc conformers of DNC. Conclusion
Scheme 11 shows the kinetic scheme that emerges from the analysis of the UV spectroscopy and photophysics of DNC. We believe that this system shows excellent promise for future use as a cell membrane probe. In particular, the viscosity-dependent excited-state process should be able to report on the dynamical freedom of membranes and the presence of a ground-state conformational equilibrium could allow studies of changes of membrane order at a given temperature. Acknowledgment. We thank Marie Letellier for the synthesis of DN. We are also grateful to Willem Siebrand for many interesting discussions. Financial support from a Commonwealth Scholarship (J.D.) and from the Natural Sciences and Engineering Research Council (NSERC) is gratefully acknowledged. Appendix Calculation of the Photophysical Parameters of DNC in TMP. The expression relating the total excitation spectral intensity at a particular he, and fixed A,, E(A,,)[A’,,], to the excited-state fractions f; reads i= I
(10) ( 1 1)
terms made up of the rate constants associated with species B* and C*43 If the process of isomerization of the nonplanar species B* to the planar species C* is irreversible, as we propose, then the decay terms y, and y2 become the actual singlet lifetimes (72 and T ~ of ) C* and B*. Since the fluorescence spectra of B* and C* have a high degree of overlap, the fluorescence decay is a composite of eqs IO and 1 1. The observed preexponentials a2and cy3 are then ac and as - ac, respectively (eqs 8 and 9). These preexponentials are not only related to rate constants associated with each species but also to their spectral intensities. Hence in some cases when there is an interconversion between two species in the excited state, a negative preexponential will not be observed. Such is the case of DNC in TMP. Similar considerations explain why the positive preexponential terms do no equal the negative preexponential in the case of DNC in butanol. The interconversion between species B* and C*, as proposed herein, is strongly suggested both by the results in butanol and by the correlation between the values of a2and a3at different viscosities and temperatures. Other explanations including the proposition that there are three ground-state conformers are not consistent with the observed data and the effects of solvents. Independent corroborative evidence as to the existence of tt and tc conformers of DNC in solution, in the ground state, was obtained from a nuclear Overhauser effect (NOE) experiment. Upon saturation of the IH NMR resonance at 6 7.21 ppm due to 23-H (see Scheme I for numbering), an enhancement was seen of the two resonances at 6 7.71 ppm and 6 7.64 ppm.’ These resonances are due to H-I’ and H-3’, respectively, on the naphthalene ring. Since the NOE occurs by a dipole-dipole interaction which is a 1970.
Drew et al.
B.Photophysics of Aromatic Molecules; Wiley: London,
(12)
where e is the molar absorptivity coefficient of the DNC solution, S the total concentration of the DNC solution, Fi the fluorescence intensity of species associated with q,and n the number of lifetime components to the fluorescence decay. The corresponding equation for a particular IEDAS, Ei(Aex)[A’eml, is Ej(Xex)
[A’,,]
= z( Aex)zf;XAex)Fi[ A’eml
(13)
For a given lifetime associated species, the ratio of IEDAS intensities at two different A,, namely At, and A,! measured at AfeX is
where A is the total absorbance of the DNC solution. The sum of allf;, at a particular A,, must, by definition, be equal to one. If IEDAS and absorption intensities at n different &, are known, these relations allow f;(A’ex) to be calculated. The fluorescence quantum yield for the two major components of the fluorescence decay (neglecting the small contribution of the third component), @h,is given by
Standard photophysical relationships allow determination of the radiative rate, k,-, and the nonradiative rate, knr,. The above kinetic analysis is only strictly valid when there is no excited-state process occurring, in other words, when the multiexponential decay is solely a result of ground-state heterogeneity. This analysis together with the molecular orbital calculations proved useful to obtain an understanding of the disparity in the values of T , and 72.
