pubs.acs.org/Langmuir © 2009 American Chemical Society
Effect of the Structure of Bile Salt Aggregates on the Binding of Aromatic Guests and the Accessibility of Anions† Rui Li, Eric Carpentier, Edward D. Newell, Lana M. Olague, Eve Heafey, Chang Yihwa, and Cornelia Bohne* Department of Chemistry, University of Victoria, P.O. Box 3065, Victoria, British Columbia V8W 3 V6, Canada Received May 21, 2009. Revised Manuscript Received July 2, 2009 The binding of naphthalene (Np), 1-ethylnaphthalene (EtNp), acenaphthene (AcN), and 1-naphthyl-1-ethanol (NpOH) as guests to the aggregates of sodium cholate (NaCh), taurocholate (NaTC), deoxycholate (NaDC), and deoxytaurocholate (NaTDC) was studied with the objective of determining how the structure of the bile salts affects the binding dynamics of guests and quenchers with the bile salt aggregates. Time-resolved and steady-state fluorescence experiments were used to determine the binding efficiency of the guests with the aggregates and were also employed to investigate the quenching of the singlet excited state of the guests by iodide anions. Quenching studies of the triplet excited states using laser flash photolysis were employed to determine the accessibility to the aggregate of nitrite anions, used as quenchers, and the dissociation rate constants of the guests from the bile salt aggregates. The binding efficiency of the guests to NaDC and NaTDC is higher than for NaCh and NaTC, and the protection efficiency is also higher for NaDC and NaTDC, in line with the larger aggregates formed for the latter bile salts. The formation of aggregates is in part driven by the structure of the guest, where an increased protection efficiency and residence time can be achieved by the introduction of short alkyl substituents (AcN or EtNp vs Np). NpOH was shown to be located in a very different environment in all four bile salts when compared to AcN, EtNp, and Np, suggesting that hydrogen bonding plays an important role in the formation of the aggregate around NpOH.
Introduction Bile salts (Chart 1) are amphiphilic molecules that aggregate in solution.1-9 The convex face of the bile salt monomer is hydrophobic, and the hydroxyl groups located on the concave face render this face more hydrophilic. Bile salts continuously aggregate as the monomer concentration is raised,5,8-14 and depending on the experimental conditions, a high polydispersity was observed for the size distribution of the aggregates.5,11,15-17 Several models were proposed for the aggregation of bile salts. † Part of the “Langmuir 25th Year: Molecular and macromolecular selfassemblies” special issue. *Corresponding author. Phone: 1-250-721-7151. Fax: 1-250-721-7147. E-mail:
[email protected]. Web:http://www.foto.chem.uvic.ca/.
(1) Hinze, W. L.; Hu, W.; Quina, F. H.; Mohammadzai, I. U. In Organized Assemblies in Chemical Analysis; Hinze, W. L., Ed.; JAI Press Inc.: Stamford, CT, 2000; Vol. 2, pp 1-70. (2) Hofmann, A. F.; Mysels, K. J. Colloids Surf. 1988, 30, 145–173. (3) Kawamura, H.; Murata, Y.; Yamaguchi, T.; Igimi, H.; Tanaka, M.; Sugihara, G.; Kratohvil, J. P. J. Phys. Chem. 1989, 93, 3321–3326. (4) Kratohvil, J. P. Adv. Colloid Interface Sci. 1986, 26, 131–154. (5) Mazer, N. A.; Carey, M. C.; Kwasnick, R. F.; Benedek, G. B. Biochemistry 1979, 18, 3064–3075. (6) O’Connor, C. J.; Wallace, R. G. Adv. Colloid Interface Sci. 1985, 22, 1–111. (7) Roda, A.; Hofmann, A. F.; Mysels, K. J. J. Biol. Chem. 1983, 258, 6362– 6370. (8) Small, D. M. In The Bile Salts; Nair, P. P., Kritchevsky, D., Eds.; Plenum Press: New York, 1971; Vol. 1, pp 249-256. (9) Small, D. M.; Penkett, S. A.; Chapman, D. Biochim. Biophys. Acta 1969, 176, 178–189. (10) D’Archivio, A. A.; Galantini, L.; Tettamanti, E. J. Phys. Chem. B 2000, 104, 9255–9259. (11) Djavanbakht, A.; Kale, K. M.; Zana, R. J. Colloid Interface. Sci. 1977, 59, 139–148. (12) Lindman, B.; Kamenka, N.; Fabre, H.; Ulmius, J.; Wieloch, T. J. Colloid Interface Sci. 1980, 73, 556–565. (13) Matsuoka, K.; Moroi, Y. Biochim. Biophys. Acta 2002, 1580, 189–199. (14) Sugioka, H.; Moroi, Y. Biochim. Biophys. Acta 1998, 1394, 99–110. (15) Mukerjee, P.; Cardinal, J. R. J. Pharm. Sci. 1976, 65, 882–886. (16) Li, G.; McGown, L. B. J. Phys. Chem. 1993, 97, 6745–6752. (17) Li, G.; McGown, L. B. J. Phys. Chem. 1994, 98, 13711–13719.
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The most widely adopted model is the primary/secondary aggregation model first proposed by Small and co-workers,8,9 where the bile salt monomers form small primary aggregates containing 2 to 10 monomers and the primary aggregates further interact forming larger secondary aggregates as the concentration of bile salt monomer is raised. An alternate model suggests that aggregation is driven by the formation of hydrogen bonds between the bile salt monomers, leaving the hydrophobic faces exposed to the aqueous phase.18-21 A third approach is to model aggregation as a stepwise process where the types of interactions are not specifically defined.13,14 NMR22,23 and theoretical studies24,25 suggested that the dichotomy between aggregation being driven by hydrophobic interactions or hydrogen bonding is not clear-cut and both types of interactions can play a role when first forming primary aggregates and then leading to the formation of the larger structures. The bile salt concentration for which the onset of aggregation is observed depends on the structure of the bile salt and on the experimental conditions used, such as ionic strength and temperature. The decrease in the number of hydroxyl groups renders the molecules of NaDC and NaTDC more hydrophobic compared to (18) Bonincontro, A.; Briganti, G.; D’Archivio, A. A.; Galantini, L.; Giglio, E. J. Phys. Chem. B 1997, 101, 10303–10309. (19) Bottari, E.; D’Archivio, A. A.; Festa, M. R.; Galantini, L.; Giglio, E. Langmuir 1999, 15, 2996–2998. (20) Campanelli, A. R.; De Sanctis, S. C.; Giglio, E.; Pavel, N. V.; Quagliata, C. J. Inclusion Phenom. Mol. Recognit. Chem. 1989, 7, 391–400. (21) Esposito, G.; Giglio, E.; Pavel, N. V.; Zanobi, A. J. Phys. Chem. 1987, 91, 356–362. (22) Funasaki, N.; Fukuba, M.; Hattori, T.; Ishikawa, S.; Okuno, T.; Hirota, S. Chem. Phys. Lipids 2006, 142, 43–57. (23) Funasaki, N.; Fukuba, M.; Kitagawa, T.; Nomura, M.; Ishikawa, S.; Hirota, S.; Neya, S. J. Phys. Chem. B 2004, 108, 438–443. (24) Partay, L. B.; Sega, M.; Jedlovszky, P. J. Phys. Chem. B 2007, 111, 9886– 9896. (25) Partay, L. B.; Sega, M.; Jedlovszky, P. Langmuir 2007, 23, 12322–12328.
Published on Web 07/16/2009
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Li et al. Chart 1. Structures of Sodium Cholate (NaCh), Sodium Taurocholate (NaTC), Sodium Deoxytaurocholate (NaTDC), and Sodium Deoxycholate (NaDC)
NaCh and NaTC, leading to the formation of aggregates at lower monomer concentrations for NaDC followed in order by NaTDC, NaCh, and NaTC.7,26-29 The aggregates of NaDC and NaTDC were shown to be larger than the aggregates of NaCh and NaTC.5,16,17,26,29 In addition, studies on the mobility of EPR-active probes showed that the structures of the aggregates for NaDC and NaTDC are different from the structures of the aggregates for NaCh and NaTC.3 Theoretical studies suggested that these differences are a reflection of the distribution of hydroxyl groups on the concave surface of the bile salt monomer.24,25 The hydroxyl groups in the case of NaCh define a plane on the concave face of the bile salt framework, whereas for NaDC the two hydroxyl groups and the headgroup form a hydrophilic edge on the molecular framework. As a consequence, NaDC was calculated to form spherical primary aggregates, and NaCh forms disklike or ellipsoid-shaped aggregates. The concentration dependence on the aggregate size was also observed in these simulations, where secondary aggregates with irregular shapes were calculated to form at higher concentrations of bile salts. Bile salt aggregates are interesting host systems in which guests can be included in different environments within the aggregate.30-33 The incorporation of guests into bile salt aggregates alters the selectivity and kinetics for the photoreactivity of the bound guests.34,35 Studies on the binding dynamics of guests with bile salt aggregates and on the accessibility of quenchers to the excited state of guests bound to the aggregates led to a proposal for the presence of two different binding sites.36-39 The size and shape of the guests incorporated into the primary (26) Carey, M. C.; Small, D. M. J. Colloid Interface Sci. 1969, 31, 382–396. (27) Kratohvil, J. P.; Hsu, W. P.; Jacobs, M. A.; Aminabhavi, T. M.; Mukunoki, Y. Colloid Polym. Sci. 1983, 261, 781–785. (28) Meyerhoffer, S. M.; McGown, L. B. Langmuir 1990, 6, 187–191. (29) O’Connor, C. J.; Ch’ng, B. T.; Wallace, R. G. J. Colloid Interface Sci. 1983, 95, 410–419. (30) Bohne, C. In Organized Assemblies in Chemical Analysis; Hinze, W. L., Ed.; JAI Press Inc.: Stamford, CT, 2000; Vol. 2, pp 147-166. (31) Bohne, C. Langmuir 2006, 22, 9100–9111. (32) Kolehmainen, E. J. Colloid Interface Sci. 1985, 105, 273–277. (33) Kolehmainen, E. J. Colloid Interface Sci. 1989, 127, 301–309. (34) Pattabiraman, M.; Kaanumalle, L. S.; Ramamurthy, V. Langmuir 2006, 22, 2185–2192. (35) Rinco, O.; Kleinman, M. H.; Bohne, C. Langmuir 2001, 17, 5781–5790. (36) Amundson, L. L.; Li, R.; Bohne, C. Langmuir 2008, 24, 8491–8500. (37) Ju, C.; Bohne, C. J. Phys. Chem. 1996, 100, 3847–3854. (38) Rinco, O.; Nolet, M.-C.; Ovans, R.; Bohne, C. Photochem. Photobiol. Sci. 2003, 2, 1140–1151. (39) Yihwa, C.; Bohne, C. Photochem. Photobiol. 2007, 83, 494–502.
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Article Chart 2. Structures of Naphthalene (Np), 1-Ethylnaphthalene (EtNp), Acenaphthene (AcN), and 1-Naphthyl-1-ethanol (NpOH)
aggregates affect both the accessibility of quenchers to the bound guest and the residence time of the guest inside the aggregate.36,40,41 The trends for the residence time and the protection efficiencies from quenching with changes in the structure of the guests are not the same, suggesting that the mechanisms for the entry of anions into the aggregate and the exit of the guest are different. Finally, there is no correlation between the residence time of the guests and their hydrophobicity, as is observed for conventional micelles such as those formed with sodium dodecyl sulfate. This lack of correlation indicates that specific interactions, probably related to the packing of the bile salt monomer around guests, determine the lifetime of the hostguest complex. The presence of more than one type of binding site and the specificity of binding to the primary aggregate make bile salt aggregates potential supramolecular systems where the binding of different molecules to different binding sites could be explored to alter bimolecular reactivity. In this respect, the addition of acetonitrile and ethylene glycol as cosolvents was shown to alter the residence time of guests in the primary aggregates.39,42 A different way of altering the binding dynamics of the guest is by changing the structure of the bile salt monomer. Preliminary studies for the binding dynamics of Np and xanthone showed that the dynamics were fastest with aggregates of NaTC followed by aggregates of NaCh and NaDC.37 A similar trend was observed for EtNp and NpOH in the presence of 40 mM NaCh, NaTDC, and NaDC.42 No systematic studies were performed to understand the effect of bile salt structure on the guest binding dynamics and protection. For this study, two dihydroxyl bile salts (NaDC and NaTDC) and two trihydroxyl bile salts (NaCh and NaTC) were chosen to determine the effect of different numbers of hydroxyl groups and different headgroups on the binding behavior of guests. AcN and EtNp (Chart 2) were chosen as the best-protected guests for which the slowest dissociation from the primary aggregates of NaCh had been determined. Np was chosen because its dynamics are faster than those for AcN and EtNp.36-38 NpOH was chosen as a guest representative of the binding to the secondary aggregates.38
Experimental Section Materials. Sodium chloride (analytical reagent, BDH), sodium cholate (NaCh; 98%, Aldrich; 99+%, Sigma), sodium deoxycholate (NaDC; 98+%, Fluka; g98%, Aldrich), sodium taurocholate (NaTC; 97%, Aldrich), sodium deoxytaurocholate (NaTDC; 95%, Aldrich; g94%, Fluka; g95%, Calbiochem), naphthalene (Np; 99+%, Aldrich), 1-ethylnaphthalene (EtNp; (40) Waissbluth, O. L.; Morales, M. C.; Bohne, C. Photochem. Photobiol. 2006, 82, 1030–1038. (41) Ju, C.; Bohne, C. Photochem. Photobiol. 1996, 63, 60–67. (42) Yihwa, C.; Quina, F. H.; Bohne, C. Langmuir 2004, 20, 9983–9991.
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98+%, Aldrich), acenaphthene (AcN; 99%, Aldrich), 1naphthyl-1-ethanol (NpOH; 99+%, Fluka), methanol (spectrograde, ACP), ethanol (95%, Commercial Alcohols), and nitrous oxide (USP, Praxair) were used as received. The purity of the aromatic compounds was checked by GC and was greater than 99%. Sodium nitrite (97%, Aldrich) was recrystallized once from deionized water. Initial steady-state quenching experiments were performed with sodium iodide (reagent, ACP) that was shown to contain a minor light-absorbing impurity leading to higher Stern-Volmer constants. All time-resolved fluorescence quenching studies were performed with sodium iodide (99.5%, Aldrich), which was used as received. Deionized water (Sybron Barnstead system) was used for sample preparation. Equipment. Absorption spectra were recorded with a Cary 1 or Cary 5 spectrophotometer at room temperature. Fluorescence spectra were collected with a PTI QM-2 spectrometer, and fluorescence decays were measured with a Edinburgh OB920 single-photon counter (Supporting Information). The excitation wavelength was 290 nm for all guests except for AcN, which was excited either at 280 or 290 nm. All fluorescence experiments were performed at 20.0 ( 0.5 °C. The decays for the time-resolved fluorescence measurements were fit to an exponential function (i = 1) or to a sum of exponential functions according to eq 1, where Ai is the preexponential factor for each emissive species with a different lifetime. The sum of the Ai values is by definition unity. The parameter ki is the decay rate constant of each emissive species, which is equal to the inverse of the singlet excited state lifetime. Fits of experimental data to eq 1 were considered to be acceptable when the χ2 values were between 0.9 and 1.2. In addition, a visual inspection of the randomness of the residuals and the autocorrelation was used to judge the goodness of the fits.43 In some cases, the experimental decays were fit with the FAST software from Edinburgh, which provides the ability to use constant values for the pre-exponential factors when fitting a decay. i X IðtÞ ¼ Io Ai e -ki t ð1Þ 1
room temperature. NaDC is the bile salt most prone to gelate. Previous experiments for NaDC concentrations of 40 mM in the presence of 0.2 M NaCl showed that after the temperatureannealing procedure there was no change within 24 h for the results of the studies for the guest binding dynamics with NaDC aggregates, suggesting that no significant gelation occurred.39 The samples for the laser flash photolysis experiments were deoxygenated by bubbling with N2O for 20 min; N2O captures any solvated electrons formed in a photoionization process during the laser excitation.47
Results The use of photophysical techniques to study the dynamics of host-guest complexes relies on the principle that the molecule formed upon excitation (i.e. the excited state) has a finite lifetime.31,48 The lifetimes of singlet excited states of organic molecules are usually short; therefore, singlet excited states have a low probability of moving from inside the host system to the aqueous phase. The fluorescence characteristics of the guests (i.e., emission spectra and lifetimes) are reporters of the environment surrounding the fluorophore. The singlet excited state lifetimes for the guests used in this work are too short (e100 ns) for relocation of the excited state to occur between the bile salt aggregate and the aqueous phase,36,38,41 and the singlet excited states of the guests are located either in the bile salt aggregate or in the aqueous phase with no guest exchange occurring between these two environments. The fluorescence decays for the guests in water followed a monoexponential function as expected for a fluorophore in a homogeneous medium, with lifetimes for the excited guests in water of 35.4 ( 0.1 ns for Np, 29.4 ( 0.1 ns for EtNp (Figure 1), 24.5 ( 0.1 ns for AcN, and 24.4 ( 0.1 ns for NpOH. The singlet excited guests bound to the bile salt aggregate have longer lifetimes than in the aqueous phase. The fluorescence decays did not follow a monoexponential function when a significant amount of guest was located both in the aqueous phase and in the bile salt aggregate, and in this case, the fluorescence decays were fit to the sum of two exponentials (eq 1, i=2). A monoexponential decay in the presence of bile salt indicated that most guest molecules were included in the aggregate. The comparison of the quenching of the guest’s fluorescence in the aqueous phase and when located inside the bile salt aggregate provides information on how much protection the bile salt aggregate provides for the bound guest. The rate constants (kq) for the quenching of the singlet excited states of the guests by iodide anions were determined from the dependence of the ratio of lifetimes in the absence of quencher (τ0) and presence of quencher (τ) with the quencher concentration [Q], where the Stern-Volmer constant (Ksv) is equal to the product of kq and τ0 (eq 2). τ0 ð2Þ ¼ 1 þ Ksv ½Q ¼ 1 þ kq τ0 ½Q τ
The laser flash photolysis system employed was previously described.44,45 The excitation source was a Spectra Physics GCR12 Nd:YAG laser at 266 nm (e20 mJ/pulse). A Xe arc lamp was used as a monitoring beam to measure the kinetics of transients. A minimum of five decay traces were averaged for each measurement. The monitoring wavelengths for the triplet excited states of the guests were 420 nm for Np, EtNp, and NpOH and 425 nm for AcN. Sample Preparation. Bile salts were dissolved in aqueous 0.2 M NaCl solutions. Guest molecule stock solutions were prepared by dissolving the compounds in methanol (∼0.02 M). The appropriate amount of the guest stock solution was injected into aqueous solutions using a gastight glass syringe. The concentrations of guests for fluorescence experiments were between 10 and 40 μM, giving an absorbance of less than 0.1 at the excitation wavelength. The concentration of guests for laser flash photolysis experiments was higher (50-250 μM), giving an absorbance value between 0.2 and 0.5 at 266 nm. Fresh quencher stock solutions (NaI and NaNO2) were prepared daily by dissolving the salts in water (0.01-5 M). Iodide anions were used as quenchers for the singlet excited states of the guests whereas the nitrite anions were employed as quenchers for the triplet excited states. All solutions containing bile salts were heated (g50 °C) for 30 min to disrupt any gel formation46 and were then cooled to
The iodide anion was used as the quencher because it is expected to interact more weakly with the negatively charged bile salt aggregates than are neutral or positively charged quenchers. Iodide anions were previously shown to quench the singlet excited states of aromatic molecules.41,49,50 Quenching of the guests in water led to the shortening of the singlet excited state lifetimes
(43) Bohne, C.; Redmond, R. W.; Scaiano, J. C. In Photochemistry in Organized and Constrained Media; Ramamurthy, V., Ed.; VCH Publishers: New York, 1991, pp 79-132. (44) Liao, Y.; Bohne, C. J. Phys. Chem. 1996, 100, 734–743. (45) Okano, L. T.; Barros, T. C.; Chou, D. T. H.; Bennet, A. J.; Bohne, C. J. Phys. Chem. B 2001, 105, 2122–2128. (46) Li, Y.; Holzwarth, J. F.; Bohne, C. Langmuir 2000, 16, 2038–2041.
(47) Janata, E.; Schuler, R. H. J. Phys. Chem. 1982, 86, 2078–2084. (48) Kleinman, M. H.; Bohne, C. In Molecular and Supramolecular Photochemistry; Ramamurthy, V., Schanze, K. S., Eds.; Marcel Dekker: New York, 1997; Vol. 1, pp 391-466. (49) DeToma, R. P.; Cowan, D. O. J. Am. Chem. Soc. 1975, 97, 3283–3299. (50) Chen, M.; Gr€atzel, M.; Thomas, J. K. J. Am. Chem. Soc. 1975, 97, 2052– 2057.
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Figure 1. Decay of the fluorescence of EtNp (λex = 290 nm) in water (black, λem=333 nm) and in the presence of 10 mM NaTC (blue, λem=340 nm). The red lines correspond to the fit of the data to a monoexponential function (water) and to the sum of two exponentials (NaTC). The instrument response function is shown in green. The residuals between the experimental data and the fits are shown in the middle panel for the decay in water and in the lower panel for the decay in the presence of NaTC.
(Figure S1 in Supporting Information) and to linear quenching plots (eq 2). The quenching rate constants were determined to be (5.40 ( 0.09) 109 M-1 s-1 for Np, (4.73 ( 0.07) 109 M-1 s-1 for EtNp, (3.30 ( 0.04) 109 M-1 s-1 for AcN, and (5.3 ( 0.2) 109 M-1 s-1 for NpOH. These values are close to the rate constant for a diffusion-controlled process (6.5 109 M-1 s-1) in aqueous solution at 20 °C.51 These high quenching rate constants indicate that once the encounter complex is formed the intrinsic rate constant within the encounter complex leading to quenching is high. Under this condition, the quenching rate constants can be equated to the association rate constant of the quencher with the aggregate where the singlet excited state is located. In the presence of bile salt, the kq values were determined from the fit of the data to eq 2 for the lifetime values assigned to the excited guest bound to the bile salt aggregate. For the fits of the emission decays to the sum of two exponentials, one lifetime corresponded to the emission of the guest located in the aqueous phase whereas the second longer lifetime corresponded to the emission from the guest bound to the aggregate. With the addition of iodide anions, the guest in water is quenched more efficiently than the guest in the aggregate, increasing the differentiation between the lifetimes of the guest in water and in the aggregate (Figure S2 in Supporting Information). The lifetimes for the singlet excited states of the guest in water were fixed to the values measured in water and calculated from eq 2 when iodide anions were present. This procedure led to a higher precision for the determined lifetimes and for the pre-exponential factors associated with each lifetime (eq 1). The quenching plots for the fluorescence of the guests bound to the bile salt aggregates were linear (Figure S3 in Supporting Information), but their slopes were much smaller than for the quenching of the guests’ fluorescence in water. This result indicates that the quenching efficiency decreased when the guests were incorporated into the bile salt aggregates because the access of the iodide anions to the interior of the aggregate is restricted. The values for the quenching rate constants were measured in the presence of 10 and 40 mM bile salt (Table 1). This choice of (51) Montalti, M.; Credi, A.; Prodi, L.; Gandolfi, M. T. Photochemistry, 3rd ed.; CRC Press: Boca Raton, FL, 2006.
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concentration was based on previous work,37,38 where for NaCh at 10 mM mostly primary aggregates were formed whereas at 40 mM primary and secondary aggregates were present in solution. The quenching rate constants were much lower for Np, AcN, and EtNp than the rate constant observed for NpOH. The pre-exponential factors associated with each exponential term in the fluorescence decays (Ai) are related to the concentration of each species with a different lifetime. Changes in the Ai values indicate that the relative concentration of a particular species either increases or decreases. The pre-exponential factors for the species assigned to the guest in water or bound to the aggregates did not change as the concentration of iodide anions was increased. This result is consistent with the assumption made above that the singlet excited states of the guests are too shortlived for relocation of the excited state to occur between the aggregate and the aqueous phase. The values of A2, which correspond to the fraction of guest in the bile salt aggregate, increased as the concentration of each bile salt was raised from 10 to 40 mM because the concentration of binding sites in the aggregates was higher as the bile salt monomer concentration was raised (Table 2). For 10 mM bile salt, the fraction of each guest included in the aggregates is higher for the dihydroxyl bile salts (NaTDC and NaDC) than observed for the trihydroxyl bile salts (NaTC and NaCh) (Table 2). For each bile salt, the highest degree of incorporation was observed for EtNp and AcN followed by that for Np, whereas for NpOH much lower incorporation was observed. At a concentration of 40 mM bile salt, Np, EtNp, and AcN are mostly incorporated into the aggregates, whereas for NpOH an appreciable amount of the guest is still located in the aqueous phase. Steady-state fluorescence quenching experiments were performed to establish if static quenching occurred and to determine the concentration threshold for which guests are protected by the bile salt aggregates. The singlet excited states of bile salt-bound guests can, in principle, be quenched by two different mechanisms (i.e., dynamic or static quenching). Dynamic quenching is the mechanism where the quencher has to diffuse to the excited state before deactivation of the excited state occurs. Static quenching in supramolecular systems occurs when the excited guest is formed in an environment that also contains the quencher (e.g., the same binding site in the aggregate), leading to immediate deactivation of the excited state. An analysis of the intensity and lifetime ratios in the absence and presence of quencher showed that only dynamic quenching occurred in the systems studied. (See Supporting Information for details.) This result eliminates the possibility of the incorporation of iodide anions with the guest in the same binding site and ensures that in the time-resolved studies all guest species in the aggregate are included in the kinetics analysis. If static quenching had occurred, then it would not have been apparent in the time-resolved studies and a guest population bound to the bile salt aggregates would not have been sampled. Steady-state quenching experiments were employed to determine the bile salt concentration for which an onset in the protection of the guest was observed, indicating the formation of aggregates. Such an onset is indicated by either a sharp decrease in the Ksv values as the bile salt concentration is raised or the observation of curved quenching plots (see below). Linear quenching plots were observed for the quenching of the steadystate fluorescence when most of the guest molecules were either located in the aqueous phase or bound to the aggregates. When most of the guests were located in the aqueous phase, the Ksv values were similar to those observed in water, whereas when most of the guests were located in the bile salt aggregates the Ksv values were much lower and did not show any further decrease DOI: 10.1021/la901826y
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Table 1. Rate Constants for the Quenching of the Singlet Excited States (kq) of the Guests in the Presence of 10 and 40 mM Bile Salta kq/109 M-1 s-1 guest ([bile salt]/mM)
NaTC
NaCh
NaTDC
NaDC
Np (10) 0.43 ( 0.03 0.22 ( 0.02 0.044 ( 0.001 0.034 ( 0.006 EtNp (10) 0.242 ( 0.009 0.11 ( 0.01 0.023 ( 0.001 0.012 ( 0.001 AcN(10) 0.13 ( 0.01 0.053 ( 0.005 0.019 ( 0.002 0.007 ( 0.001 NpOH (10) 1.0 ( 0.2 0.9 ( 0.2 0.35 ( 0.03 0.4 ( 0.1 Np (40) 0.37 ( 0.01 0.177 ( 0.007 0.054 ( 0.008 0.034 ( 0.005 EtNp (40) 0.22 ( 0.01 0.106 ( 0.008 0.025 ( 0.003 0.014 ( 0.001 AcN(40) 0.108 ( 0.002 0.039 ( 0.002 0.0122 ( 0.009 0.0043 ( 0.0009 NpOH (40) 0.80 ( 0.07 0.7 ( 0.1 0.25 ( 0.03 0.34 ( 0.09 a Errors correspond to the error propagation from the Ksv values and lifetime values in the absence of quencher (Tables S1-S4 in the Supporting Information).
Table 2. Pre-exponential Factors (A2) for the Fluorescence from Guests Located in the Bile Salt Aggregatesa A2 guest ([bile salt]/mM)
NaTC
NaCh
NaTDC
NaDC
Np (10) 0.24 ( 0.04 0.17 ( 0.04 0.6 ( 0.1 0.73 ( 0.05 EtNp (10) 0.64 ( 0.08 0.4 ( 0.1 0.89 ( 0.04 0.96 ( 0.02 AcN(10) 0.53 ( 0.04 0.35 ( 0.04 0.91 ( 0.04 0.95 ( 0.01 NpOH (10) 0.09 ( 0.03 0.06 ( 0.02 0.27 ( 0.07 0.27 ( 0.03 Np (40) 0.83 ( 0.04 0.86 ( 0.01 0.82 ( 0.05 0.92 ( 0.02 EtNp (40) 0.95 ( 0.01 0.94 ( 0.01 1 1 AcN(40) 0.94 ( 0.02 1 1 1 NpOH (40) 0.58 ( 0.05 0.60 ( 0.09 0.69 ( 0.03 0.6 ( 0.1 a A2 =1 indicates that only one emissive species was present. Errors correspond to standard deviations from the average value determined at all iodide anion concentrations.
when the bile salt concentration was raised. When a significant fraction of the guest was located in both environments, the quenching plot showed downward curvature (Figure S5 in the Supporting Information) because at low quencher concentrations the more accessible guest in water was quenched whereas at high concentrations the remaining emitters were the excited guests in the aggregate for which quenching was less efficient. The curved quenching plots were fit to an equation that takes into account two different populations of emitting species leading to the Ksv2 values for the guest in the bile salt and a fraction of the emission intensity (F2) that corresponds to the emission intensity of the guest in the aggregate (Supporting Information, Tables S5-S8). It is important to note that the values for A2 obtained from the time-resolved studies and F2 obtained from the steady-state studies are related, but the latter also includes changes in the emission quantum yields for the bound and free fluorophores. Therefore, only trends for these two parameters are comparable. The dependence of the Stern-Volmer constants on the bile salt concentration is qualitatively the same for Np, AcN, and EtNp, whereas the decrease for the Ksv values in the case of NpOH occurs at much higher bile salt concentrations (Figure 2 and Tables S5-S8 in the Supporting Information). For NaDC and NaTDC, the formation of aggregates and incorporation of the guest were observed at the lowest concentrations studied (3 mM). At this low concentration, a fraction of Np, AcN, and EtNp was incorporated into aggregates that provided similar protection to that observed at the higher bile salt concentrations. In the case of NpOH, a decrease in the Stern-Volmer constant was observed at 5 mM NaDC or NaTDC, suggesting that some interaction occurred between NpOH and these bile salts. In the presence of NaCh or NaTC, the threshold for the observation of aggregates that protect the guests well was 10 mM for Np, AcN, and EtNp, with the exception of Np and NaTC where the threshold was observed to be between 10 and 20 mM NaTC. For NpOH, a decrease in the Ksv2 values was observed, which became prominent at 20 mM bile salt (Figure 2). 13804 DOI: 10.1021/la901826y
Figure 2. Dependence on the concentration of bile salt for the ratio of the Stern-Volmer constants in the presence of bile salt (Ksv2) and in water (Ksv1) for the fluorescence quenching of Np (NaCh, green squares; NaDC, black squares) and NpOH (NaCh, blue circles; NaDC, red circles). The lines were included to guide the eye.
The triplet excited states of the guests that are unreactive toward the host or solvent have lifetimes that are much longer than the lifetimes of the singlet excited states of the same guests. Therefore, the triplet excited states can relocate between the aqueous phase and the interior of the bile salt aggregates, providing an opportunity to measure the association and dissociation rate constants of the triplet guests with the host system31,48,52 (i.e., the bile salt aggregates) (Figure 3). A quencher is employed that primarily resides in the aqueous phase. Nitrite anions quench triplet states by energy transfer,53 and this anion was shown previously to quench the triplet states of Np, EtNp, AcN, and NpOH.36-38 The bile salt aggregate provides a barrier for the quenching of the triplet excited states bound to the aggregate, and for this reason, the quenching rate constant in water (kq) is higher than the quenching rate constant for the triplet state incorporated into the aggregate (kBSa q ). The lifetime of the triplet guest in water is very short at high quencher concentrations, and the exit of the excited guest from the bile salt aggregate becomes rate-limiting. As a consequence, the quenching plot (kobs vs [nitrite]) deviates downward from the linearity observed in water (eq 3).31,48,52,54-56 When the concentration of triplet states in the aqueous phase is relatively small, the decay of the triplet states follows a first-order function and the observed rate constant (kobs) can be related to the association (k+) and dissociation (k-) rate constants of the triplet guest with the bile salt aggregate, the quenching rate constants in water (kq) and in the aggregate (kBSa q ), and the triplet lifetimes in water (k0) and in the aggregate (kBSa) (52) Pace, T. C. S.; Bohne, C. Adv. Phys. Org. Chem. 2008, 42, 167–223. (53) Treinin, A.; Hayon, E. J. Am. Chem. Soc. 1976, 98, 3884–3891. (54) Almgren, M.; Grieser, F.; Thomas, J. K. J. Am. Chem. Soc. 1979, 101, 279– 291. (55) Turro, N. J.; Bolt, J. D.; Kuroda, Y.; Tabushi, I. Photochem. Photobiol. 1982, 35, 69–72. (56) Turro, N. J.; Okubo, T.; Chung, C.-J. J. Am. Chem. Soc. 1982, 104, 1789– 1794.
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Figure 3. Kinetics scheme for the dynamics of the triplet excited guest (red circles) with the bile salt aggregate (squares). The guest binding dynamics are defined by the association (k+) and dissociation (k-) rate constants for the triplet state. The decay of the guest in water is defined by the decay rate constant in the absence of quencher (k0) and the quenching rate constant kq, whereas the decay of the guest in the host is defined by rate constants kBSa and kBSa q .
(eq 4). The concentration of host sites is not known because the aggregation numbers (N) for the primary and secondary sites are not known. For this reason, the concentration of binding sites is expressed as [bile salt]/N. The requirements for the derivation of eq 4 are the following: (i) A quencher is used for which the quenching efficiency for the triplet guest in water (kq) is higher than the quenching efficiency for the triplet excited guest in the bile salt aggregate (kBSa q ). (ii) The concentration of host is sufficiently high to ensure that the concentration of guest in water is low so that steady state can be applied to the rate law for the triplet decay, leading to an expression corresponding to an exponential decay with kobs expressed by eq 4.48,54 kobs ¼ k0 þ kq ½nitrite
Figure 4. Kinetics for the triplet excited state of EtNp monitored at 420 nm in the presence of 40 mM NaCh and in the absence (a) or presence (b) of 0.06 mM nitrite anion. The traces were fit (red lines) to monoexponential decays. The residuals for the fits are shown in the bottom two panels.
ð3Þ
kobs ¼ kBSa þ k þ kBSa q ½nitrite -
k - k þ ½bileNsalt k0 þ kq ½nitrite þ k þ ½bileNsalt
ð4Þ
The quenching rate constants with nitrite anions for the triplet states of Np ((3.6 ( 0.1) 109 M-1 s-1), EtNp ((3.2 ( 0.2) 109 M-1 s-1), and NpOH ((3.0(0.1) 109 M-1 s-1) were determined in water. AcN was not sufficiently soluble in water, and the value for kq in 1:1 ethanol/water was determined to be 9.8 108 M-1 s-1, which is the same value as observed for the quenching of EtNp in 1:1 ethanol/water (9.2 108 M-1 s-1), suggesting that the decrease in the quenching rate constants in the mixed solvent is due to a solvent effect. For this reason, a kq value of 3109 M-1 s-1 was used as the quenching rate constant for triplet AcN by nitrite anions in water. The triplet decays for the guests in all bile salt solutions, with the exception of AcN in the presence of 10 mM NaCh, followed a monoexponential decay at all of the nitrite concentrations investigated (Figure 4). In the case of AcN/NaCh (10 mM), the data could not be fit with eq 4 because the decay for the triplet state did not follow a monoexponential function. Quenching plots were obtained for bile salt concentrations of 10 and 40 mM. Curved quenching plots were observed for Np, AcN, and EtNp, whereas for NpOH curved quenching plots were observed for both concentrations of NaTDC and NaDC but only at 40 mM for NaTC and NaCh. Qualitatively, the farther the curved quenching plot from the linear quenching plot in water (Figure 5), the slower the guest association/dissociation dynamics with the bile salt aggregates. The slowest dynamics were observed for the dihydroxyl bile salts when compared to that of the trihydroxyl bile salts. Langmuir 2009, 25(24), 13800–13808
Figure 5. Quenching plot for triplet Np quenching by nitrite anions in water (black triangles, only points at low quencher concentration are shown for clarity) and in the presence of 10 mM bile salt: NaTC (blue squares), NaCh (black circles), NaTDC (green squares), and NaDC (red circles). The data in water were fit to eq 3, whereas the data in the presence of bile salt were fit to eq 4.
The dependencies of kobs on the quencher concentration in the presence of bile salt were fit to eq 4. The values for k0 and kq were determined independently from studies in water, and kBSa is the triplet lifetime of the guest in the presence of bile salt but in the absence of nitrite anions. These three parameters are fixed in eq 4, and the recovered parameters are k-, kBSa q , and k+/N (Tables S9S12 in the Supporting Information). No discernible trends were observed for k+/N because this parameter has large errors. The were similar for the experiments in the values for k- and kBSa q presence of 10 and 40 mM bile salt considering the experimental errors, and for this reason, they were averaged (Tables 3 and 4). The dissociation rate constants were lowest for EtNp and AcN, followed by Np, and were highest for NpOH. For each guest, the dissociation rate constants were highest for NaTC. In the case of NpOH, it is more difficult to establish a trend because of the larger errors involved. It is important to note that for NpOH no curved quenching plot was observed in the presence of 10 mM NaCh or NaTC, suggesting that at this bile salt concentration NpOH did not interact with the bile salt aggregates. The quenching rate constants for the triplet states of the guests in the bile salt aggregates were higher for the trihydroxyl bile salts than for the dihydroxyl bile salts (Table 4). For experiments where the precision was higher because the quenching plots were better DOI: 10.1021/la901826y
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Table 3. Dissociation Rate Constants for the Triplet Excited States of the Guest from Bile Salt Aggregatesa k-/106 s-1 guest
NaTC
NaCh
NaTDC
NaDC
Np 2.4 ( 0.4 1.4 ( 0.3 0.9 ( 0.2 0.7 ( 0.2 EtNp 0.6 ( 0.4 0.18 ( 0.08 0.3 ( 0.1 0.18 ( 0.08 b,c 0.4 ( 0.2 0.16 ( 0.07 AcN 1.7 ( 0.6 0.4 ( 0.1 5.5 ( 0.5d 1.9 ( 0.8 4(2 NpOH 8 ( 2d a The values correspond to the averages obtained for measurements in the presence of 10 and 40 mM bile salt (Tables S9-S12 in the Supporting Information). b From ref 36. c Average for data collected at 20 and 40 mM NaCh. d Values from experiments with 40 mM bile salt.
Table 4. Quenching Rate Constants by Nitrite Anions for the Triplet Excited States of the Guest Bound to Bile Salt Aggregatesa 7 -1 -1 kBSa s q /10 M
guest
NaTC
NaCh
NaTDC
NaDC
Np 4(2 4(1 1.5 ( 0.2 1.4 ( 0.4 EtNp 5.0 ( 0.8 3.3 ( 0.9 0.9 ( 0.2 0.5 ( 0.1 0.8 ( 0.3 0.7 ( 0.1 AcN 7(3 2.2 ( 0.5b,c ∼2d 6( 2
