Langmuir 1990,6, 1408-1416
1408
Dynamic and Static Fluorescence from O-(1-Pyrenyl)alkanoic Acids and “Nonanchored”Pyrenyl Molecules as Probes of Local Environments and Phase Changes in the Gel and Middle Phases of Aqueous Potassium Stearate, Rubidium Stearate, and Potassium Stearate/l-Octadecanoll Roseann M. Jenkins and Richard G . Weiss* Department of Chemistry, Georgetown University, Washington, DC 20057 Received October 10, 1989. In Final Form: March 14, 1990 Static and dynamic fluorescence from three w-( 1-pyreny1)alkanoicacids (( 1-pyreny1)aceticacid, 4 4 1pyreny1)butanoic acid, and 12-(l-pyrenyl)dodecanoicacid), 1-dodecylpyrene,and pyrene, present in very low concentrations within nitrogen-saturated model bilayers, have been used to determine the local environment experienced by pyrenyl groups embedded at various positions in organized layers. The surfactant systems are the gel and middle phases of 50% by weight aqueous potassium stearate, rubidium stearate, and 1/1potassium stearate/l-octadecanol. The results indicate that not all the “anchored”probes adopt extended conformations in the well-ordered gel phases and that chain melting attending the gelmiddle phase transitions can be followed in detail from variations in probe fluorescence lifetimes. Surprisingly, structural changes that occur during the gel-middle phase transition can lead to very large increases in probe lifetimes even though one might anticipate a decrease in the total system order. An explanation of this and other unexpected results (based upon changes in the concentration of aqueous quenchers accessible to pyrenyl singlets) is advanced.
Introduction Understanding the mechanism of molecular transport mediated by aqueous carriers in biological bilayers is complicated by differences among components in a membrane, varying interactions of the components with the lipophilic dopants, and irregular depth profiles for water within the bilayers.2 Model bilayers can be much less complex and, therefore, capable of yielding more easily interpreted data on dopant transport and permeability. We have investigated the depth profiles of water in the nitrogen-saturated gel a n d middle phases (and t h e transition region between them) of model bilayers comprised of potassium stearate (KS), rubidium stearate (RS), and 1/1 (mol/mol) potassium stearate/loctadecanol (KSO) by monitoring the fluorescencelifetimes (7f)and 11/13vibronic ratios (in emission) of three ~ ( 1 pyreny1)alkanoic acids whose lengths place the pyrenyl groups near a layer interface ((1-pyreny1)aceticacid, PAA), somewhat removed from it (4-(l-pyrenyl)butanoicacid, PBA), and near the ends of vicinal stearate anions (12(1-pyreny1)dodecanoic acid, PDA) when the polymethylene chains are fully extended (Figure 1). These results are compared with those obtained in isotropic media (water and tridecane) and by using pyrenyl probes that lack a polar group capable of anchoring a t a water-lipophile interface (pyrene (PY) and 1-dodecylpyrene (DP)). Pyrene and several of its derivatives have been exploited previously as fluorescence probes to study the microenvironments of other organized media, such as micelle~,S-~ (1) Part 40 in our series Liquid-Crystalline Solvents as Mechanistic Probes. For Part 39, see: Nunez, A.; Weiss, R. G. Boletin de la Sociedad Chilena de Quimica 1990, 36.3. (2) Jain, M. K. The Biomolecular Lipid Membrane; Van Nostrand Reinhold: New York, 1972.
K
+
D
)
“O)
0
j
wKS PDA PBA
PA A Figure 1. Length comparisons of extended KS, PDA, PBA, and PAA. microemulsions,6 monolayer^,^^^ and tubule^.^ A useful fluorescence probe should reside in a definable site, and its emissive properties must reflect the nature of that site. The local environment of a pyrenyl group may be probed by monitoring the ffuorescence lifetime which shortens with increasing polaritylOJ1 and 11/13,the ratio of the intensities of the first and third vibronic bands of monomeric emission, ( 3 )Jay, J.; Johnston, L. J.; Scaiano, J. C. Chem. Phys. Lett. 1988,148, 517. (4)Roelants, E.; De Schryver, F. C. Langmuir 1987,3,209. ( 5 ) Waka, Y.; Hamamoto, K.; Mataga, N. Photochem. Photobiol.1980, 32, 21. (6) Atik, S. S.; Thomas, J. K. J. Am. Chem. SOC.1982, 104, 5868. (7) Subramanian, R.; Patterson, L. K. J . Phys. Chem. 1985,89,1202. (8)Fujihira, M.; Nishiyama, K.; Hamaguchi, Y.; Tatsu, Y. Chem. Lett. 1987, 253. (9)Sonnenschein, M. F.; Weiss, R. G. Photochen. Photobiol., in press. (10) Birks, J. B.;Lumb, M. D.; Munro, I. H. Proc. R. SOC.London A 1963, 275, 575. (11)Birks, J. B.;Lumb, M. D.; Munro, I. H. Proc. R. SOC.London A 1964,280, 289.
0 1990 American Chemical Society
w-(1-Pyreny1)alkanoicAcids as Probes
Figure 2. Sideview of amphiphile ordering in a layer of KS, RS, and KSO gel phases ( 0 ,carboxylate; -, lipophilic chain; A, hydroxyl). which correlates with solvent polarity.12J3 Attachment of a fatty acid side chain to the 1-position of pyrene permits the highly polar carboxylate group to act as an anchor at a water interface, enabling the probe to lie a t various points within the lipophilic portion of the gel and middle phases of KS, RS, and KSO. The distance of the pyrenyl group from a bilayer-water interface can then be inferred if the polymethylene chain of the acid is fully extended. Previous studies have demonstrated that flexible rod-like dopants usually adopt preferentially extended conformations within bilayers.14-16 The lower temperature noncrystalline phases of KS, RS, and KSO are gel-like and form even in high concentrations of water. KS and RS gels have been studied extensively by using X-ray diffractionl7J8 and deuterium nuclear magnetic resonance (2H NMR).’9s20 Both methods charcterize the gels as continuously swelled, interdigitated bilayers whose polar head groups are hexagonally packed a t the aqueous interface and whose lipophilic chains are essentially all-trans. In essence, the layers are smectic B-like. The length of a bilayer in RS and KS gels is slightly longer than an extended stearic acid molecule. Although an RS bilayer is about 1A longer than a KS bilayer (Figure 2), the density of chains appears similiar. The polymethylene chains rotate about their long axes and are deformed by collective bending and torsional modes which occur with the greatest probability near the least ordered methyl groups. The middle methylene segments display the greatest order followed by those near the polar head groups. Chain bending is reported to be independent of water content in the gel phase but is slightly dependent on temperature. With increasing temperature, these gels are transformed to a lyotropic nematic liquid-crystalline (12) Kalyanaeundaram, K.; Thomas, J. K. J. Am. Chem. SOC.1977,99, 2039. (13) Dong, D. C.; Winnik, M. A. Phot0che.m. Photobiol. 1982,35,17. (14) Shobha,J.; Srmivas, V.; Balasubramanian,D. J.Phys. Chem. 1989, 93, 17. (15) Lala, A. K.; Dixit, R. R.; Koppaka, V.; Patel, S. Biochemistry 1988, 27, 8981. (16) Treanor, R. L.; Weies, R. G. J . Am. Chem. SOC.1986,108, 3137. (17) (a) Vincent, J. M.; Skoulios, A. Acta Crystallogr. 1966,20, 432. (b) Vincent, J. M.; Skoulios, A. Acta Crystallogr. 1966,20, 441. (18) Skoulios, A.; Guillon, D. Mol. Cryst. Liq. Cryst. 1988, 165, 317. (19) Mely, B.; Charvolin, J. Chem. Phys. Lipids 1977,19,43. (20) (a) Jeffrey, K. R.; Wong,’T. C.; Tulloch, A. P. Mol. Phys. 1984, 52,289. (B)Jeffery, K. R.; Wong, T. C.; Tulloch, A. P. Mol. Phys. 1984, 52, 307.
Langmuir, Vol. 6, No. 8, 1990 1409 phase (the middle phase) that persists to above 100 O C . In it, stearate anions associate into cylindrical micelles of undefined length in which the polymethylene chains fill the interior and the polar head groups are projected a t the water interface.21 As a result, order along a stearate chain decreases from the carboxylate head group to the terminal methyl.20 The cylindrical micelles are parallel on average in hexagonally packed two-dimensional arrays, separated from each other by water. The diameter of each cylinder is somewhat less than twice the length of two conformationally extended stearate molecules, as expected, since packing considerations require some chain bending. The mean interface area per chain increases with increasing temperature, as does water content, as a result of increasing chain disorder caused by twisting about C-C single bonds. The structure of the KSO gel phase has been characterized by X-ray diffraction.22 It consists of noninterdigitated bilayers in which stearate anions and l-octadecanol molecules alternate within a layer. The length of the bilayer is slightly shorter than two stearic acid molecules (Figure 2). The bilayers are separated from each other by water, and within the bilayers the alkyl chains are hexagonally packed (i.e., smectic B-like, as with KS and RS gels). 2H NMR results indicate that the KSO gel has greater disorder than the KS gel in the region of the methyl groups;23 however, KSO gels exhibit fewer, relatively more ordered methylene families along an alkyl chain, indicating that, overall, the KSO gel is more ordered than either the KS or RS gel. Although the higher temperature phase of KSO has not been studied, it appears to be composed of cylindrical micelles, also. The process by which the layered gel phases transform to the cylindrical middle phase has not (to our knowledge) been studied structurally. An attractive hypothesis is that temperature-induced undulation^^^ lead to layer foldover and scission. The bulk transitions occur over a defined, relatively broad temperature range upon heating. Local transitions (i.e., “microscopic”transformations in the vicinity of a probe molecule) may be observed a t somewhat different temperatures2s28 since local order may change either more or less abruptly than bulk and the probe itself must create added disorder in neighboring amphiphiles. This is especially true of a bulky probe like pyrene whose van der Waals width is ca. 2.5 times that of a methylene group. Since the probe concentrations are frequently low M in this work), their influence on bulk measurements of transitions (e.g., differential scanning calorimetry curves) can be negligible even though their local influence may be q u i t e drastic. We will refer t o microscopically determined phase transitions simply as “phase transitions”; normal or bulk transitions will be denoted specifically. The initial and final temperatures of a micrpcopic phase transition are defined operationally. They are the temperatures in a heating cycle that initiate and end excursions in Tf from normal Arrhenius behavior (21) Luzzati, V.; Mustacchi, H.; Skoulios, A,; Husson, F. Acta Crystallogr. 1960,13, 660. (22) Vincent, J. M.; Skoulios, A. Acta Crystallogr. 1966,20,447. (23) Treanor, R. L., Ph.D. Dissertation, Georgetown University, 1987. (24) Janiak, M. J.; Small, D. M.; Shipley, G. G. Biochemistry 1976,15, 4575. (25) Treanor, R. L.; Weiss, R. G. Tetrahedron 1987,48, 1371. (26) Gogoll, A,; Schafer, H. J. Justus Liebigs Ann. Chem. 1987,7,589. (27) Zimmerman, R. G.; Liu, J. H.; Weiss, R. G. J. Am. Chem. SOC. 1986,108,5264. (28) Anderson, V. C.; Weiss, R. G. J. Am. Chem. SOC.1984,106,6628. (29) Marconelli, M.; Strauss, H. L.; Snyder, R. G. J . Phys. Chem. 1986, 89, 5260. (30) (a) Weiss, R. G.; Treanor, R. L.; Nunez, A. Pure Appl. Chem. 1988, 60,999. (b) Nunez, A.; Weiss, R. G., unpublished results.
1410 Langmuir, Vol. 6, No. 8, 1990
(vide infra; such lifetimes are reported in the text with corresponding temperatures). Since its carboxylate behaves as an "anchor" at an aqueous interface, the pyrenyl group of PAA must reside near the layer head groups and, thus, in a region relatively accessible to water: the water-hydrocarbon contact in lessordered micellar solutions that contain no disturbing probes is approximately two fully exposed methylene groups per a m ~ h i p h i l e .The ~ ~ longer linking chain of PBA,when extended, permits pyrenyl groups to be near the middle portion of a lipophilic chain region. In KS and RS gel phases, an extended polymethylene chain of PDA would place its carboxylate and pyrenyl groups near the opposite interfaces of a n interdigitated bilayer. Using similar reasoning, the pyrenyl group of PDA would reside near the bilayer midplane of a KSO gel and near the center of a cylindrical micelle in any of the three middle phases. Since PY and DP possess no "anchors", their location within the phases is subject to greater conjecture. In principle, pyrenyl groups from such molecules may prefer to reside near a layer interface, in the hydrophobic middle of a layer, or (in the case of middle phases and the KSO gel) a t a bilayer midplane. With all probes and within a gel or middle phase, our experiments indicate that t h e permeability of water molecules into t h e lipophilic region increases with increasing temperature and, therefore, with disorder (as in biological bilayers). However, upon heating at or near t h e bulk gel-middle phase transition, most probe fluorescence lifetimes unexpectedly increase, indicating decreased accessibility of water (and water-soluble quenchers) to the lumophores. We attribute this decrease, at least in part, to increased flexibility of the polymethylene chains, allowing them to surround and protect the pyrenyl groups more efficiently. In some solvent systems, important changes in probe location may also accompany the phase transitions and lead to significant changes in 7f. Such d o p a n t - i n d u c e d "microscopic" phase transitions undoubtedly occur in biological membranes and may be important mediating factors in molecular transport across them.
Experimental Section Materials. Stearic acid (9996, Aldrich), 1-octadecanol(9996, Aldrich), rubidium hydroxide (9976, anhydrous, Strem Chemicals), potassium hydroxide (87.5% , ACS Certified, Fischer), quinine bisulfate (laboratory grade, Fischer), methylene chloride (HPLC grade), chloroform (ACS grade, 0.75% ethanol as preservative), methanol (absolute, Photorex), hexane (Photorex), n-tridecane (99%, Humphrey Chemical Co.), pentane (spectrophotometric grade, Fischer), and boron trifluoridemethanol complex (50% by weight, Aldrich) were used as received. Pyrene (PY) (Aldrich, 99%)was recrystallized 3 times from 95% ethanol and sublimed: mp 150-151 "C (1% mp 149151 "C32). 12-(l-Pyrenyl)dodecanoic acid (PDA) (mp 105-109 "C) and 1-dodecylpyrene (DP) (mp 70-71 "C; lit. mp 76 0C33) were purchased from Molecular Probes, Inc. and used as received. 4-(1-Pyreny1)butanoic acid (PBA) from Molecular Probes, Inc. was recrystallized twice from toluene and once from acetic acid: mp 189-190 "C (lit. mp 190-190.5 0C34). (1Pyreny1)acetic acid (PAA), mp 222-223 "C (lit. mp 222.5-223.0 "C35), was prepared via the acetylation of pyrene to form (31)Halle, B.; Carlstrom, G. J. Phys. Chem. 1981, 85, 2142. (32) Birks, J. B.; Kazzazz, A. A.; King, T. A. Proc. R. SOC.London A
1966.291.556. (33) Proske, T.; Fischer, C.-H.; Gratzel, M.; Henglein, A. Ber. BunsenGes. Phys. Chem. 1977,81,816. (34) Fieser, L. F.;Fieser, M.; Hershberg, E. B. J . Am. Chem. SOC.1936, 58. 1463.~ . . ~,~ (35) Bachman, W. E.; Carmack, M. J.Am. Chem. SOC.1941,63,2494.
Jenkins and Weiss
l-acetylpyrene,36 followed by the Willgerodt reaction and hydrolysis.35 The purity of the w-(1-pyreny1)alkanoicacids, determined by gas chromatography on the methyl esters to be 98% (PAA), 98% (PBA), and 97% (PDA), was performed with a HewlettPackard 5890 capillary gas chromatograph equipped with a flame ionization detector. Relative peak intensities were measured with a Hewlett Packard 3393A integrator. w-(1-Pyreny1)alkanoic acids were first esterified by using a boron trifluoridemethanol complex,37 dissolved in pentane, and injected onto an Alltech wide-bore RSL-300 capillary column (10 m X 0.53 mm) coated with 1.2-pm poly(phenylmethylsi1oxane) (OV-17, Alltech).38 The purity of DP was determined to be 96% by the same gas chromatography conditions. Probe Solutions. An aliquot (1 mL) of a methylene chloride solution containing 10-4 M PAA and PBA was transferred to a 10-mLvolumetric flask, and the solvent was evaporated under a nitrogen atmosphere. Distilled and deionized water was added to the mark, and the solution was sonicated for 20 min and then deoxygenated by chilling in an ice bath and bubbling with Nz for 5 min. An aliquot (1 mL) of M PBA or PDA in hexane was transferred to a 10-mL volumetric flask that was filled to the mark with hexane (for gel preparations) or evaporated to dryness, filled to the mark with tridecane, and bubbled with Nz for 5 min. These deoxygenating procedures led to reproducible fluorescencedecays that did not change when the NZbubbling period was extended. For use in gel studies, 10-5 M PY or DP solutions in hexane were prepared as above. Potassium stearate (KS) and rubidium stearate (RS) were each prepared by the slow addition of 1 equiv of stearic acid (dissolved in methanol) to an equivalent amount of alkali metal hydroxide dissolved in methanol.20 The crystals were obtained by evaporating the solvent, filtering, washing with anhydrous ether, drying, and recrystallization from ethanol. The gel samples were prepared by weighing a portion of the finely ground stearate or 50/50 (mol/mol) potassium stearate/ 1octadecanol and adding an equal amount (w/w) of 10-5 M PAA or PBA in water. For gels containing PDA, PY, or DP, an aliquot of the appropriate hexane solution was evaporated to dryness with a stream of nitrogen, and equal amounts of finely ground stearate or 50/50 (mol/mol) potassium stearate/loctadecanol and water were added. All samples were N2 saturated by bubbling for 5 min, flame-sealed in 2-mm (i.d.) rectangular Pyrex tubes, and suspended in boiling water for at least 8 h. (As before, longer bubbling times did not affect the photophysical properties of the probes.) Upon cooling, a clear, yet viscous, gel was formed.39 Each new batch of stearate was checked with a Koffler hot-stage microscope for proper gelmiddle (bulk) phase transition temperatures. They were found to be 48-54 "C for KS, 74-77 "C for KSO, and 48-56 "C for RS, within 10.5 "C of the literature value^.^^^^^ Fluorescence lifetimes and Z1/Z3 fluorescenceratios were collected only on heating cycles to avoid hysteresis. Instrumentation. Fluorescence lifetimes were measured with an Ortec single photon counting system described elsewhere.40 The excitation light was filtered with a Corning 737 band-pass filter and focused onto the sample cell sitting at a 45" angle to the light source. Photons emitted from the front face of the sample cell were passed through a wavelength filter and then focused onto a RCA 8850 head-on photomultiplier tube placed 90" to the excitation light. Pyrenyl monomer emission was observed through a Corion 400-nm cutoff filter, and the pyrenyl excimer emission was observed through a Corning OG505 filter (transmission 0.1% at 500 nm, 50% at 520 nm, 99% at 560 nm). Data were analyzed with a weighted, nonlinear least-squares Fortran program written by Dr. Mark (36) Kosak, A. 1.; Hartough, H. D. Org. Synth. 1955, 3, 14. (37) Morrison, W. R.; Smith, L. M. J. Lipid Res. 1964,5,600. (38) Hresko, R. C.; Markello, T. C.; Barrenholz, Y.; Thompson, T. E. Chem. Phys. Lipids 1985,38,263. (39) The actual probe concentrations are 5 X 1o-B M. We assume that virtually all of the probe molecules reside in the lipophilic portions, therefore, the concentrations are consldered, for practical purposes, to be IO+ M. (40) Sonnenschein, M. F.; Weiss, R. G. J.Phys. Chem. 1988,92,6828.
Langmuir, Vol. 6, No. 8, 1990 1411
w-(1-Pyreny1)alkanoic Acids as Probes Sonnenschein.41 The instrument was calibrated by comparison with the fluorescence lifetime of 10-5 M quinine bisulfate in 1.0 N sulfuric acid. The value obtained was 19.4 ns (lit. 19.2 ns42). Decay curves were reproducible to within *l%on different days with the same samples. Duplicate samples of PBA and PDA in KS were prepared several weeks apart, and their rf values were found to agree quantitatively (A1%). A large scattering component for the gel phase required subtraction of signals from a blank consisting of pyrenyl-free gel phases.40 Peak channel intensities between 3000 and 10000 counts were collected for each temperature, and 235 out of 256 channels were analyzed. Data fits were deemed acceptable when x 2 (1.3 Ix 2 2 OB), the Durbin-Watson parameter (I 1.70) and skew fit (I 0.16) were within the limits shown in parentheses. In all cases, background-correctedfluorescence decays were best fit to a single-exponentialfunction. Fluorescence spectra were recorded on a Spex Fluorolog spectrofluorimeter with a 15O-W/1XBO high-pressure xenon lamp and a Datamate computer. All spectra were corrected for wavelength-dependent variations in lamp intensity by using rhodamine B as a standard. The spectra were recorded in the front-face mode with 0.5-mm monochromator slits. The 11/13 ratios were determined by subtracting the background from the 11 (374.0 f 2.0 nm) and the 13 (385.0 2.0 nm) emission bands and then ratioing the corrected intensities. Samples in both the single-photon counter and the fluorimeter were thermostated (h0.5 "C) by circulating water through a cell block holding the sample. Temperature was monitored with a calibrated thermistor that was in contact with the sample vessels.
Table I. I , / & Fluorescence Ratios for M w-(1-Pyreny1)alkanoic Acids in Nz-Saturated Isotropic Solvents at 25 "C. 11113
solvent
PAA
PBA
PDA
tridecane methylene chloride chloroform
0.90
1.90
2.70
1.40
methanol water
2.33 2.20
2.32 2.91 3.12 2.99
3.52b
Standard deviation is h0.04. Obtained by extrapolation from higher temperature data. 2.40 I
*
Results In isotropic solvents, irradiation of M pyrene does not result in detectable excimer emission.43 Although ground-state aggregation in the anisotropic layered phases could result in excimer formation, no evidence for such a process could be found. Steady-state fluorescence spectra from all of our pyrenyl probes a t M concentrations in isotropic, gel, and middle phases lacked the broad, structureless, long-wavelength emission characteristic of pyrenyl excimers. Fluorescence decays obtained with a Corning OG-505 emission filter (to exclude monomer pyrenyl fluorescence) gave only background signals. Also, data from single-photon counting experiments fit singleexponential decay curves. At higher pyrenyl concentrations (15 X M), excimer was clearly present, as discerned from the static emission spectra. Steady-StateFluorescence Spectroscopy. Our pyrenylalkanoic acids and DP have very similar fluorescence excitation and emission spectra. Their emission spectra in isotropic solutions tend to shift bathochromically as solvent polarity is decreased. Hence, the position of the first vibronic emission band (Il)may be used as a crude indicator of the average of all the local environments of the pyrenyl groups in one anisotropic medium. By this criterion, pyrenyl groups of the three amphiphilic probes prefer relatively nonpolar environments when dissolved in each of the gel and middle phases, since the position of I1 for each probe is nearly identical with that in tridecane: I1 is located at 375.0 (tridecane) and 375.3 nm (gel and middle phases) for PAA, at 375.5 (tridecane) and 375.8 nm (gel and middle phases) for PBA, and at 374.5 (tridecane) and 374.8 nm (gel and middle phases) for PDA. In water, PAA and PBA exhibit I1 a t 374.0 and 373.5 nm, respectively. More detailed insights into variations in pyrenyl environments may be derived from scrutiny of 11/13 vi(41) Sonnenschein, M. F. Ph.D. Dissertation, Georgetown University
1987.
(42) OConnor, D. V.; Meech, S. D.; Phillips, D. Chem. Phys. Lett. 1982, 88.. 22. (43) Birks, J. B.; Dyson, D. J.; Munro, I. H. h o c . R. SOC.London A 1963, 275, 575.
10
30
50
70
'
90
T l'C1
Figure 3. Effect of temperature on the 11/13 fluorescence ratios water; of 10-5 M PAA in isotropic, gel, and middle phases (0, 0 , tridecane; 0 , KS; A, RS;X KSO). bronic emission ratios. However, like the position of II, such ratios report an ensemble average (weighted by mole fraction and emission probability for each environment). As such, they must be interpreted with caution, especially in cases where more than one environment is likely to be o ~ c u p i e d .T~h e fluorescence decays, being singleexponential, are consistent with one type of dopant site for each probe in each of our phases but do not require that a pyrenyl singlet experience an environment of fixed polarity during ita lifetime. The 11/13 ratios for PAA, PBA, PDA, DP, and PY in isotropic solvents of differing polarity are listed in Table I. The ratio for PDA in methanol a t 25 "C was extrapolated from higher temperatures due to solubility problems near room temperature. Each acid behaves like pyrene:12J3 the 11/13 ratios increase with increasing solvent polarity. Additionally, the ratios in a given solvent decrease with decreasing temperature (Figures 3-6).
Fluorescence Lifetimes of Pyrenyl Probes in Isotropic, Gel, and Middle Phases. Isotropic Phases. The effect of temperature on the fluorescence lifetimes of PAA, PBA, and PDA, measured by single-exponential decay kinetics, in isotropic solutions are shown in Figure 7. In all cases, an Arrhenius treatment of Tfis linear (Table 11). The lifetimes a t room temperature are in good agreement with previous work from our and other laboratorie~:~~44~fi generally, fluorescence lifetimes of pyrene and pyrenyl groups are greatly reduced by the introduction of water. A clear manifestation of t h i s effect is demonstrated by Tf of PBA in water and tridecane (Table 11). Another potential quencher are the potassium or rubidium counterions of stearate in the ordered phases. The concentration of counterions in our gel and middle phases
~~
(44) Vaughan, W. M.; Weber, G. Biochemistry 1970,10,464. (45) Geiger, M. W.; Turro, N. J. Photochem. Photobiol. 1977,26,221.
,
Jenkins and Weiss
1412 Langmuir, Vol. 6, No. 8, 1990
2.801
11'13
1
1 4 '
2 001
i *,j
I 60
*
. 50
30
C 8 2
I
3 u
II
~
10
II 0
YO
-
C 6
, Jr
20
. .
*
~
ic
,
,
63
0
, 70
.~,
90
80
T 'C
T I'CI
Figure 4. Effect of temperature on the ZI I3 fluorescence ratios of M PBA in isotropic, gel, and mi dle phases (0, water; 0, tridecane; 0, KS; A, RS;X, KSO).
d
Figure 6. Effect of temperature on the 11/13 fluorescence ratios of 10-6 M DP and PY in isotropic, gel, and middle phases (+, DP in tridecane; +, DP in KS; X, DP in KSO; v,PY in KSO).
3 33
m
m
+
i
+
*
*
m
1
x x
2 i
o
IC
30
1 50
4 70
1
90
T i'C)
Figure 5. Effect of temperature on the 11/Z3 fluorescence ratios of 1od M PDA in isotropic, gel, and middle phases (a,methanol; 0, tridecane; 0, KS; A,RS;X, KSO).
is quite high, approximately 3 M in the aqueous parts (assuming regular solutions in the aqueous components) or effectively higher (if the ions are associated with the carboxylate anions). In 0.5 and 3.0 M aqueous potassium hydroxide a t 25 "C,PBA fluorescence lifetimes were 119 and 117 ns, respectively. Since they compare favorably with 7f obtained in water (119 ns), even a very high concentration of potassium ions quenches pyrenyl singlets inefficiently. However, in aqueous 0.5 and 3.0 M rubidium hydroxide a t 25 "C, lifetimes were 98 and 76 ns, respectively, indicating the greater efficiency of rubidium ion as a q ~ e n c h e r . ~ ~ KS Gel and Middle Phases. Fluorescence lifetimes of PAA and PBA in the KS gel are somewhat longer than in water, indicating (similar to the Z1/Z3 ratios) that the pyrenyl singlets experience a somewhat lipophilic environment (Table 11). A typical fit of the fluorescence decay to a single-exponential decay curve is shown in Figure 8. Arrhenius treatments of the lifetime data provide linear fits a t temperatures removed from the phase transition region (Figure 9). At 25 "C, the PDA fluorescence lifetime is slightly longer in the KS gel than in tridecane (Table 11). The corresponding Arrhenius activation energies are near those of PAA and PBA (Figure 9). For all these 4 1 pyreny1)alkanoic acids, E, in the gel phase is lower than in the middle phases. However, only with PDA is there no hint of a lifetime increase upon heating though the (46) The quenching of the singlet excitad state of naphthalene in zeolites by rubidium ions has also been observed: Ramamurthy, V.; Caspar, J. V.; Corbin, D. R.; Eaton, D. F. J. Photochem. Photbiol. A 1989, 50, 157.
m
m
3 13-1
------------I 1
GC28
0 CJ33
1 003'
Ill
i
3334
k
Figure 7. Arrhenius plot of fluorescence lifetimes of nyl probes in isotropic solutions,.( PBA in water; water; A, PDA in tridecane; 0,PBA in tridecane).
M pyre-
+, PAA in
transition region. It is noteworthy that DP, which is about the same length as PDA but lacks a carboxylate anchor, gives analogous results (Figure 9 and Table 111). KSO Gel and Middle Phases. The temperatureinduced variations of PAA and PBA lifetimes in KSO are qualitatively similiar to, but larger in magnitude than, those in KS (Figure 10). Also, a t the lowest temperature examined, the lifetime of PAA or PBA in the KSO gel is actually shorter than in KS or water (Table II), suggesting the presence of a quencher near the pyrenyl groups. Prior to the bulk-determined onset of the middle phase, the lifetimes of PAA and PBA undergo an enormous increase (Figure 10). Similar increases in the 7f of PDA, DP, and PY are found near the phase transition region (Figure 11). A t temperatures far from the bulk gel-middle phase transition, the lifetime data can be fit easily to the Arrhenius equation. The paucity of data points and small temperature range examined mean that KSO middlephase activation energies for all probes a r e only approximate. RS Gel and Middle Phases. PAA lifetime changes in RS phases are qualitatively similar to, but somewhat smaller than, those in KS and KSO (Figure 12). The activation energies, although small, are inverted in magnitude with respect to KS and KSO gel and middle phases (Table 11). This comportment is also followed by PBA and PDA in RS. The 7f values of PBA are much shorter throughout the RS gel phase than in water, but at ca. 25 "C, they are comparable to the values determined in aqueous 3.0 M RbOH. The E , values for PDA in the RS gel (5.5 kcal/mol) and middle (2.5 kcal/mol) phases are much larger than in the
Langmuir, Vol. 6, No. 8,1990 1413
w-(1-Pyreny1)alkanoic Acids as Probes
~
Table 11. Fluorescence Lifetimes and Activation Energies. for Fluorescence Decay of Some w-( 1-Pyreny1)alkanoic Acids in KS, KSO. and RS Ge1.b Middle' Phases, and Isotrodc Phases PAA PBA PDA solvent Tf E. Tf E. Tf E. ~~
~
~~~~
KS
gel middle
142.0 121.8
0.7f 0.1 1.6f 0.1
129.9 129.0
0.6 f 0.1 1.0 f 0.2
199.2 136.9
0.4 f 0.1 1.6f 0.3
110.3 127.2
1.4 f 0.2 1.6 f 0.1
85.2 142.2
0.8 f 0.1 2.5f 0.6
124.0 158.8
0.8 f 0.1 0.9 0.3
79.5 98.3 138.1d 110.7'
1.8f 0.2 1.2 f 0.1 1.1 f 0.1
80.3 100.3 119.2d 103.2, 272.4b 239.W
3.5 f 0.7 2.1 f 0.2 1.1 f 0.1
194.4 178.2
5.5 f 0.3 2.5f 0.1
0.7 f 0.1
192.4b 171.N
0.7f 0.1
KSO
gel middle RS gel middle water tridecane
Lifetimes in ns and activation energies in kcal/mol. 25 OC. At 55 O C for KS, 80 "C for KSO, and 60 Oc for RS. 24 "C. e 64 "C. 160 O C . 16.1
15.3
7
0.0028
0.0034
0,0032
0,0030
ill I K I
0
CHANNEL
255
Figure 8. Dynamic fluorescence decay curve for PBA in KS gel phase at 30 "C. The single-exponential best fit to the decay is represented by the solid line. The reduced residuals are shown at the inset (Tf = 125.0ns, x* = 1.1, DW = 1-90).
corresponding phases of KS. Also, the influences of increasing temperature on the PDA singlet lifetimes in the phase transition regions of RS and KS are very different: the lifetimes in RS increase from 127 ns a t 50 "C to 195 ns at 55 "C,the latter being nearly 60 ns longer than the lifetime measured in KS a t the same temperature. Over the same temperature span, the lifetimes of PDA in KS decrease by nearly 40 ns.
Discussion Gel Phases. As mentioned previously, 11/13 ratios from pyrenyl groups in anisotropic media may not provide a clear indication of local polarity. However, since the ratios obtained from the gel phases differ for the various probes, we conclude that their pyrenyl groups reside in different average environments. A less certain conclusion concerning any one probe, based upon the similarity of its 11/13ratios, is that the pyrenyl group experiences a rather common polar environment, regardless of the gel constituents. By comparison with isotropic phase derived ratios, pyrenyl of PAA seems to reside in a very polar (water-like) gel environment, pyrenyl of PBA is exposed to fewer polar neighbors, and the pyrenyl groups of PDA, DP, and P Y provide ratios indicative of a nonpolar, hydrocarbon location. Simple models that extend the polymethylene chains of a pyrenylalkanoic acid and leave the carboxylate a t a layer interface are compatible with the 11/13ratios obtained from PAA and PBA and with PDA in the KSO gel: as the
Figure 9. Arrhenius plot of fluorescence lifetimes of lV M pyrenyl probes in KS gel and middle phases (+, PAA; 0,PBA; (0, PDA; A, DP). Arrows indicate the bulk phase transition
temperatures.
Table 111. Fluorescence Lifetimes and Activation Energies. for Fluorescence Decay of DP and PY in Various Phases
DP solvent nonaneb liquid paraffinb cyclohexane
Tf
PY
E.
Tf
E. 1.2 3.0
192.W 132.0d
KS
gel middle KSO
184.5O 133.U
0.8 f 0.1 1.3 f 0.1
*
163.8' 2.2 0.1 169.4O 1.6 f 0.1 gel middle 185.48 1.5 f 0.1 193.78 2.3 f 0.1 Lifetimes in ns and activation energies in kcal/mol. bFoerster, T.; Seidel, H. P. 2. Phys. Chem. 1965, 45, 58. Stevens, B.; Thomaz, M. F.; Jones, J. J. Chem. Phys. 1967, 46, 405. 23 "C,from Anderson, V. C.; Weiss, R. G. J. A m . Chem. SOC.1984,106,6628. 70 "C.e 25 "C.f 60 O C . 8 78 O C .
chain lengths increase, the distance between a layer interface and the pyrenyl probe increases. Similar arguments, which allow DP and PY to be buried in the lipophilic portion of a bilayer, can explain the 1 1 / 1 3 ratios from these probes, also. However, in the interdigitated layers of KS and RS gels, chain extensions of PDA place its pyrenyl and carboxylate groups near opposite interfaces of the same bilayer and predict much higher (polar) I 1 / I3 ratios than those measured. This and relatively large E, calculated in the RS gel phase indicate that the pyre-
Jenkins and Weiss
1414 Langmuir, Vol. 6,No. 8, 1990
16
AA
i n 1 'Tr I
A
I
A
1
A
A A A
. 0027
c
c
0 0C31 I,' ( K
0025
c
0013
3035
Figure 10. Arrhenius plot of fluorescence lifetimes of 10-5 M w-(1-pyreny1)alkanoicacids in KSO gel and middle phases (+, PAA; 0,PBA; A,PDA). Arrows indicate the bulk phase transition temperatures. 16 1
I
~
i
I
A A
0 0027
0 0031
0 00251
0 0035
0 0033
1IT I K
Figure 11. Arrhenius plot of fluorescence lifetimes of M pyrenyl probes in KSO gel and middle phases (X, PY; 0 , DP). Arrows indicate the bulk phase transition temperatures. 11
16
E
,6 3
,
3
4
5 1
4
1;
+
o +
C
1
1
0 0029
l
I
A
A A
1 ,t
A
u
0 0031
0033
0 C015
111 K r
Figure 12. Arrhenius plot of fluorescence lifetimes of M w-(1-pyreny1)alkanoicacids in RS gel and middle phases (+, PAA; 0,PBA; A PDA). Arrows indicate the bulk phase transition temperatures.
ryl group of PDA is well-protected from the efficient rubidium ion quenchen and located far from a layer interface. Further evidence to support this assertion for PDA in both K S a n d R S gels comes from magnitudes of fluorescence lifetimes measured a t a common temperature (25 "C). The lifetimes of PDA in KS and RS gels are significantly longer than in KSO gel and comparable to those obtained in tridecane. They are also longer than the lifetimes of PAA or PBA in the same gel phases. Since Tf of PBA is 80 ns longer than T f of PDA in tridecane at 25 "C, the increased PDA lifetime in the gels must be due to protection of its pyrenyl group from aqueous quenchers and not to an intrinsic property of the two molecules.
Based upon the magnitude of r f , we conclude that the pyrenyl group of DP is somewhat better protected from quenchers by the KS gel than by the KSO gel. Since the midplane of a KSO bilayer (near where pyrenyl of PDA and DP in rod-like conformations might reside) is somewhat disordered, small aqueous pools (and traces of lipophilic impurities, if present) should reside preferentially there. Thus, the polymethylene chains of PDA and, probably, of DP are not extended in the gel phases of KS and RS, allowing the pyrenyl group to retreat from the vicinity of an interface.*O~~~ Given that the 11/13 ratio of PBA is indicative of a less polar pyrenyl environment than for PAA in the gel phases, it is somewhat surprising that the T f of PAA are longer in both KS and KSO gels and nearly identical for the two probes in the RS gel a t 25 "C. At least in part, these differences may be related to intrinsic probe properties in polar media: in water at 25 "C, q o f PAA is 19 ns longer than Tf of PBA. Regardless, the data discussed above and the very low Ea (Tables I1 and 111) suggest that the pyrenyl groups of PAA and PBA in the gel phases are in contact with aqueous pools or channels that lead from a bilayer interior to the aqueous bulk.4s Even subtle reorientation of terminal methyl groups of stearate in the gel and middle phases of RS requires 2.6 kcal/mol and rotation about a long molecular axis in the RS gel phase exhibits a 12.7 kcal/mol barrier.20b Thus, with the possible exception of PBA and PDA in the RS phases, fluorescence quenching appears to depend primarily upon diffusion of aqueous quenchers to pyrenyl groups rather than relocation of the probes. Middle Phases. As in the gel phases, 11/13 ratios from the pyrenyl group of each pyrenylalkanoic acid are similar in the three middle phases but different for each probe. Assuming that a rough correlation exists between 11/13 ratios in isotropic and anisotropic media, the average environment of pyrenyl groups in the pyrenylalkanoicacids follows the same progression as found in the gel phases: the polarity increases from PAA to PBA to PDA. In fact, the 11/13 ratios of PDA and DP in the KS middle phases are actually lower (implying less polar) than in tridecane. Clearly, this is an unreasonable expectation and emphasizes our contention that 11/13 ratios in anisotropic media may depend upon other factors besides local polarity. In the lower temperature domains of the middle phases (e.g., 55 "C for KS and RS and 80 "C for KSO), the Tf of all the probes (except PAA, PDA, and D P in KS) are somewhat longer than in the higher temperature portion of the corresponding gel phases. This and the higher Ea for PAA and PBA in the KS or KSO middle phase than in the gel phase suggest that pyrenyl groups of the probes experience greater protection from aqueous quenchers in the less ordered cylindrical micelles of the middle phase.49 We conclude that the more flexible (melted) alkyl chains of the middle phases are able to solvate better the bulky pyrenyl groups than can the more rod-like chains of the gel phase: the aqueous pools (or channels) near pyrenyl groups of PAA and PBA in the gel phases can be squeezed from the lipophilic layers as the chains adopt shapes that fill the cylindrical volumes of the micelles. Also, since the (47) Zachariasse,K. A,; Kunhle, W.; Weller, A. Chem. Phys. Lett. 1980, 73, 6.
(48) Luisetti, J.;Mohwald, H.; Balla, H. J. Biochim. Biophys. Acta 1979,
552, 519.
(49) Although the gel phase of KSO is well-characterizad,n*s the middle phase is not. However, the qualitative similarity between the Arrhenius curves in Figures 9 (KSO) and 11 (RS)leads us to believe that the higher temperature phase of KSO is comprised of cylindrical micelles, also. Our subsequent discussions will be based upon that assumption.
w-(1-Pyreny1)alkanoic Acids as Probes diameter of a cylindrical micelle is much greater than the length of an extended stearate, even PDA may, in principle, have an extended polymethylene chain in the middle phases and still experience a nonpolar environment about its pyrenyl group. At the same time, the pyrenylalkanoic acids in RS yield E , that do not follow the trends of KS and KSO. Pyrenyl quenching is more facile in the middle phase than in the gel. Also, the general trend for middle-phase lifetimes of one pyrenylalkanoic acid to increase from RS to KS to KSO is followed consistently except by PDA in RS. While several explanations for these results can be advanced, none is very satisfying. For instance, a lower than bulk concentration of rubidium ions may be included within the pools (or channels) that are near the pyrenyl groups of PAA or PBA in the lipophilic layers. Access of rubidium ions t o pyrenyl groups would be affected in two opposing ways by chain-melting that accompanies middlephase formation: first, it should facilitate diffusion leading to a decrease in E,; second, it should decrease the concentration of metal ions about a pyrenyl group, resulting in increased Tf. However, this suggestion explains neither why t h e magnitude of 7 f from PDA in t h e lower temperature portion of the RS middle phase is near the value measured in tridecane nor why the lifetimes of PAA and PBA at the same temperature in the RS middle phase are slightly lower than those obtained in water. Even if the pyrenyl groups of PDA in KS and RS middle phases are buried so deeply in the lipophilic micellar layers that they are well-protected from quenchers, it is difficult to explain why the RS phase, with its better quencher, allows the significantly longer lifetime. Our general observation that the pyrenyl groups are protected better from aqueous quenchers by the less ordered middle phases than by the more ordered gel phases agrees with our observations employing pyrenylalkanoic acids in small unilamellar vesicles and tubule^.^ Subcynski and Hydeso and Geiger and T ~ r r found o ~ ~ that the permeability of lipid bilayers to the nonpolar quencher, oxygen, increases above the melting transition. Menger e t al.51 using 13C NMR and C a ~ a employing l ~ ~ IR spectroscopy on dopant ketones concluded that the water content in lipid bilayers is much lower than in the much more disordered micelles. Also, Skoulios and Guillonl* have suggested, on the basis of packing considerations, that the permeability of "spherical" and cylindrical micelles to water should be much greater than the permeability of gel bilayers. Clearly, our results do not agree with this suggestion, although it is reasonable to assume that soluteinduced disorder (like that caused by our bulky pyrenyl probes) may facilitate permeability locally, leaving the wellordered layer regions that are free of solute also free of water pools or channels. Phase Transition Region. The conversion of the gel layers to middle-phase cylindrical micelles is not a sharp, first-order transition whether measured as a bulk (by X-ray diffraction17922 or polarized microscopy) or a microscopic (from changes in d o p a n t fluorescence properties) phenomenon. Even upon slow heating, the bulk transition temperature ranges were 6 "C (48-54 "C) for KS, 3 "C (7477 "C) for KSO, and 8 "C (48-56 "C) for RS. They were unaffected by the very small concentrations of pyrenyl p r o b e s e m p l o y e d in t h i s s t u d y . However, t h e microscopically detected transition temperatures changed (50) Subczynski, W. K.; Hyde, J. S. Biophys. J. 1983,41, 283. (51) Menger, F. M.; Aikens. P.: Wood. M..Jr. J. Chem. Soc.. Chem. Commun. 1988, 180. (52) (a) Casal, H. L. J.Am. Chem. SOC. 1988,110,5203. (b) Casal, H. L. J. Phys. Chem. 1989, 93, 4328.
Langmuir, Vol. 6, No. 8, 1990 1415 by amounts that depended upon the probe type. Their analysis is complicated by the possibility that pyrenyl groups of a probe may change their average locations within a layer during a phase transition. In none of the anisotropic surfactant mixtures did the 11/13 ratios from PAA or PBA undergo a clear change associated with the gel-middle transition (Figures 3 and 4). Since pyrenyl groups from both probes reside relatively close to a layer interface in the gel and middle phases, the lack of effect upon 11/13 was not unexpected. As shown in Figure 5 , distinct changes in t h e t e m p e r a t u r e independence of 11/13 from PDA on solvent phase are apparent but difficult to interpret (vide ante). More definitive, transition-dependent changes in Tf were detected for all of our probes. The lifetime excursions detected a t or near the bulk-phase transition temperatures (Figures 8-11) are not found for the same probes in isotropic solvents. Thus, the changes detected in 7f are environmentally induced and indicate that local (i.e., in the vicinity of the pyrenyl groups) changes in phase structure both precede and follow the bulk transitions. In KS, the initial change in Arrhenius slope associated w i t h t h e c o m m e n c e m e n t of t h e g e l - t o - m i d d l e transformation occurs a t essentially the same temperature (2' z 45 "C) for all probes. The final temperature is less clear (varying from ca. 56 to ca. 62 "C) but definitely depends upon the probe. Even more dramatic are the magnitudes of the excursions in 7f that occur in this temperature regions: as expected of pyrenyl groups near a layer-water interface, the changes in 7f of PAA and PBA are small increases (ca. 2% and 6 % , respectively, of the lifetimes measured a t the initial transition temperature). The pyrenyl groups of PDA and DP are buried more deeply in the lipophilic layers and exhibit very large decreases; if initial lifetime increases occur, they are too small to be detected. These results imply that during the phase transformation, pyrenyl probes initially near aqueous quenchers became more protected from water while those that are initially shielded from water became more exposed to it. The transient increases in Tf from PAA and PBA probably arise as neighboring chains begin to melt and undergo conformational changes that squeeze water from the vicinity of the pyrenyl groups or allow the pyrenyl groups to migrate over larger distances within the layers. Somewhat different comportment is observed for the probes in RS (Figure 12). Again, the initial Arrhenius slope change for the three pyrenylalkanoic acids occurs a t ca. 45 "C. The final changes for PAA and PBA occur near 59 "C, while that for PDA is again a t a lower temperature, ca. 55 "C. Thus, the microscopically detected phase transition region is slightly narrower in RS than in KS, even though the bulk-detected RS transition region is 2 "C broader. Additionally, 7f increases significantly for all three pyrenylalkanoic acids in the transition region, indicating that even the pyrenyl group of PDA experiences environmental changes that diminish the concentration of nearby aqueous quenchers: the lifetimes increase 50% (PAA), 64% (PBA), and 54% (PDA) between the initial and final phase transition temperatures. Since X-ray diffraction17shows that chain packing in undoped KS and RS layers is very similar, it is reasonable to assume that the location of pyrenyl groups of one probe will be similar, regardless of the stearate counterion, and that the pyrenyl groups will suffer similar positional changes during the KS and RS phase transitions. If these assumptions are correct, the differences in the magnitude (and sign) of the Tf changes in this region must be due to the influences of the counterions. The attenuated
1416 Langmuir, Vol. 6, No. 8, 1990 i f increases observed from PAA and PBA in KS can be attributed to the weaker quenching ability of potassium. The question of why ifof PDA suffers a large decrease in the phase transition region of KS (7% of its magnitude at the initial transition temperature) and a large increase in the transition region of RS requires refinement of our previous explanations. As indicated by their much lower i fat 45 "C in RS, pyrenyl groups of PDA are not protected completely from rubidium ions. Even in the presence of a larger concentration of potassium ions in KS at 45 "C, the pyrenyl groups of PDA should be quenched to a much lower extent and, as found, exhibit a much longer lifetime. As the polymethylene chains about the pyrenyl groups in KS and RS begin to melt, they will decrease the local concentration of metal ions (leading to an increase in i f ) and allow the pyrenyl groups greater mobility within a layer (leading to a decrease in i f ) .Since it is the sum of these effects that we measure, their relative importance will determine whether a net increase or decrease in i f is observed in the phase transition region. With the more efficient rubidium ion quencher, the decrease in its local concentration must dominate; with the very weak potassium ion quencher, the greater mobility of pyrenyl, leading to greater access to a layer interface, must be the more important factor. The i f values of all the probes suffer large increases (greater t h a n one-third of their initial transition temperature values) as the temperature is increased throughout the phase transition region of KSO. The initial temperature at which the phase transition is evident differs for the probes and occurs several degrees below the bulkdetected transition onset. The pattern of the onset temperature is not obvious: both PBA and PDA sense the inception of the phase transition a t ca. 50 "C (more than 20 "C below the lower temperature of the bulk-detected phase transition region), while rf of PAA, DP, and PY begins to increase near 60 "C. Since the midplane region, where pyrenyl attached to an extended chain of PDA would reside, is the least organized part of a KSO bilayer,53 it is reasonable that this probe should sense phase changes first. Why the onset temperature of PBA is close to that of PDA but those of DP and PY (which, presumably, are far from a layer interface) experience the same onset temperature as PAA is unclear to us at this time. However, the pyrenyl groups clearly sense changes in their environment (which probably include an expulsion of water) a t several more degrees below the bulk phase transition onset in KSO than when they are placed in KS or RS. In another sense, the higher temperature limit of the phase transition region can also be considered as an indicator of the degree to which a pyrenyl probe disturbs its local environment: the disorganization created by a
(53)(a)Davis, J. H. Biochim.Biophys. Acta 1983,737,117. (b)Tremor,
R.L.; Weiss, R. G . J . Am. Chem. SOC.1988, 110, 2170.
Jenkins and Weiss pyrenyl probe accelerates the transformation of a gel to its middle phase. On this basis, PDA adds the smallest incremental disturbance to its local environment since its final transition temperature is within experimental error of that measured for the bulk. PAA, whose pyrenyl is nearest the highly ordered head-groups of a bilayer, exhibits the largest difference between its microscopic final temperature and that of the bulk (ca. 7 "C) while PBA, whose pyrenyl should occupy a position of intermediate order, exhibits an intermediate depression. The ca. 2 and 5 "C depressions exhibited by DP and PY, respectively, indicate that they reside during the phase transition in regions of a bilayer that are between those of PAA and PDA on average.
Conclusions The local environments experienced by pyrenyl groups appended to different mediating substituents in the layered gel and cylindrical micellar phases of model bilayer systems and the changes that occur during their phase transitions have been followed by analyzing the static and dynamic fluorescence properties of the probes. Depending upon the extended length of the probe and the thickness of the bilayer, the pyrenyl groups experience very different average environments. Chain melting by surfactant molecules surrounding a pyrenyl group can be detected differentially and separately from macroscopically detected phase transitions. This reinforces our contention that the probes do not experience a bulk-averaged environment during their excited singlet lifetimes. In the gel phases, some of the pyrenyl probes are less protected from aqueous quenchers than would be expected on the basis of a simple model in which they act as surfactant surrogates. Other pyrenyl groups, especially those of PDA in KS or RS gels, are more protected, indicating that their polymethylene chains are severely bent. Large increases in fluorescence lifetimes of several probes are found upon heating from the gel to middle phases. Thus, a decrease in local solvent order need not lead to greater quenching of a probe. In our experiments, the probable causes of the lifetime increases in the phase transition region are subtle changes in probe positions and local chain melting leading to more intimate solventsolute interactions and decreased accessibility of water near pyrenyl groups.
Acknowledgment. We thank the National Science Foundation (Grant CHE 88-18873) for its financial support of this work. Dr. Mark Sonnenschein provided valuable insights into several experimental problems. Dr. George S. Hammond is thanked for valuable discussions and suggestions. Supplementary Material Available: Tables I-VI11 of temperature-dependent fluorescence lifetimes ( i f of ) pyrenyl probes in isotropic, gel, and middle phases (7 pages). Ordering information is given on any current masthead page.
