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Langmuir 1997, 13, 4498-4501
Thermal Modulation of Photoisomerization in Double-Azobenzene-Chain Liposomes Robert A. Moss* and Weiguo Jiang Department of Chemistry, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08903 Received March 20, 1997. In Final Form: June 17, 1997X Cationic double-azobenzene-chain lipid 5 undergoes efficient trans f cis photoisomerization in MeCN solution and in 1:9 coliposomes with bisether lipid 2 at temperatures above 20 °C. However, in 1:1 5/2 coaggregates, or in pure liposomal 5, the extent of photoisomerization is very sensitive to temperature; for example, it is limited to e10% below 40 °C. Dispersed 1:9 coliposomes of 5 and 2 spontaneously segregate into domains rich in the individual lipids. Thermal cis f trans isomerization of 5 in MeCN solution and in 1:9 5/2 coliposomes proceeds readily between 20 and 60 °C with activation energies of 19-20 kcal/mol. The photoisomerization of 5 in pure liposomes or in 1:1 5/2 coliposomes is less efficient than that of its single-azobenzene-chain analogue, 1, under comparable conditions.
The readily induced, reversible trans f cis photoisomerization and thermal reversion of azobenzene (AB) derivatives make them a subject of continuing interest.1 The spatial requirements, absorption spectra, and dipole moments of the trans and cis AB isomers differ sufficiently, so that the pair can function as a two-position ‘molecular switch’. Utilization of this principle is fostered by the assembly of AB-containing lipids into aggregates that function as molecular devices with photoresponsive properties. Kunitake’s group, in particular, has pioneered the correlation between the molecular structure of AB lipids and the morphology and spectroscopic properties of their aggregates, especially bilayer assemblies.1bc,2-5 Recently reported aggregate functions that have been controlled or modified by linkage to trans/cis AB isomerization include permeability,1a,6,7 surface potential,8 fluorescence and absorbance,4 liquid crystal phase modification,9 H-bonding,10 and micellar aggregation.11 Additionally, the AB UV absorption maxima are sensitive to stacking of the AB chromophores, so that one can continuously monitor the relative distribution and orientation of AB monomers within aggregates.1b,c,4 Most previously studied AB lipids are single-chain molecules or, if double-chain, carry only a single AB residue.5,12 This reflects concern that the trans f cis AB photoisomerization would be sterically inhibited if there were insufficient room available for the more spatially demanding cis isomer.1a,4,13,14 In this regard, our continuX
ing interest in the relation of lipid molecular structure to intraliposomal dynamics15 led us to study cationic AB lipid 1, both in holoaggregates and in coliposomes with bisether lipid 2.16
Efficient AB photo isomerizations and thermal AB isomerizations of 1 were observed at ambient temperatures in liposomes of varied composition; evidence of steric inhibition to isomerization was absent, even in gel phase holoaggregates of 1.16 Similarly, Osa reported reversible trans f cis photoisomerization of the neutral, head groupfunctionalized AB lipid 3 in LB films.14 Here, the hydrocarbon chains created sufficient free volume within the membrane to permit the isomerizations, although monoalkyl chain analogues of 3 resisted photoisomerization.8
Abstract published in Advance ACS Abstracts, August 1, 1997.
(1) Reviews: (a) Anzai, J.-I.; Osa, T. Tetrahedron 1994, 50, 4039. (b) Kunitake, T. Angew. Chem., Int. Ed. Engl. 1992, 31, 709. (c) Shimomura, M.; Ando, R.; Kunitake, T. Ber. Bunsen-ges. Phys. Chem. 1983, 87, 1134. (2) Kunitake, T.; Okahata, Y.; Shimomura, M.; Yasunami, S.-i.; Takarabe, K. J. Am. Chem. Soc. 1981, 103, 5401. (3) Kunitake, T.; Ishikawa, J.; Shimoumura, M.; Okawa, H. J. Am. Chem. Soc. 1986, 108, 327. (4) Shimomura, M.; Kunitake, T. J. Am. Chem. Soc. 1987, 109, 5175. (5) Ishikawa, Y.; Kuwahara, H.; Kunitake, T. J. Am. Chem. Soc. 1994, 116, 5579. (6) Tanaka, M.; Sato, T.; Yonezawa, Y. Langmuir 1995, 11, 2834. (7) Song, X.; Perlstein, J.; Whitten, D. G. J. Am. Chem. Soc. 1995, 117, 7816. (8) Maack, J.; Ahuja, R. C.; Tachibana, H. J. Phys. Chem. 1995, 99, 9210. Ahuja, R. C.; Maack, J.; Tachibana, H. J. Phys. Chem. 1995, 99, 9221. (9) Ikeda, T.; Tsutsumi, O. Science 1995, 268, 1873. Kurihara, S.; Ikeda, T.; Sasaki, T.; Kim, H.-B.; Tazuke, S. Chem. Commun. 1990, 1751. (10) Rosengaus, J.: Willner, I. J. Phys. Org. Chem. 1995, 8, 54. See also: Lahav, M.; Ranjit, K. T.; Katz, E.; Willner, I. Chem. Commun 1997, 259. (11) Higuchi, M.; Minoura, N.; Kinoshita, T. Chem. Lett. 1994, 277. (12) For recent examples, see: Everaars, M. D.; Marcelis, A. T. M.; Sudholter, E. J. R. Liebigs Ann./Recueil 1997, 21.
S0743-7463(97)00300-4 CCC: $14.00
It is of obvious interest to study AB lipids that carry two AB-functionalized chains, which should afford aggregates of even greater spatial sensitivity to AB isomerization. Whitten described several zwitterionic doubleAB-chain phosphatidylcholines (e.g., 4) that do undergo photoisomerizations, either as holoaggregates or as coliposomes with dipalmitoylphosphatidylcholine (DPPC).7 However, major morphological changes, including deaggregation, accompanied the AB photoisomerizations, and the isomerization efficiencies were described as “variable” and “somewhat slower when the azobenzene (was) aggregated in aqueous solution either pure or in mixtures with DPPC.”7 In contrast, decreased photo(13) Kunitake, T.; et al. (Nippon Kagaku Kaishi 1988, 1001) reported that trans f cis AB photoisomerization was inhibited in closely-packed LB membranes; cited in ref 1a, p 4057. (14) Anzai, J.-I.; Sugaya, N.; Osa, T. J. Chem. Soc., Perkin Trans. 2 1994, 1987 and refs 2-4 therein. (15) Moss, R. A. Pure Appl. Chem. 1994, 66, 851. (16) Moss, R. A.; Jiang, W. Langmuir 1995, 11, 4217.
© 1997 American Chemical Society
Letters
isomerization efficiency was not observed at ambient temperatures with single AB-chain aggregates of 1.16
Now we have prepared the cationic double-AB-chain lipid 5. Although related to single-AB-chain lipid 1, the photochemical and aggregation behavior of 5 is distinct and unusual. Here we describe the photochemical and thermal isomerizations, spontaneous intraaggregate domain formation, and temperature dependence of the trans/ cis AB photostationary states of lipid 5. Synthesis. Bisazobenzene lipid 5 was prepared from commercially available 1,2-dihydroxy-3-(dimethylamino)propane (6), and the previously described16 AB derivative 7. Diol 6 was converted to its disodium alkoxide (excess
NaH/THF) and then reacted with an excess of 7 in THF, added by syringe pump (1 h, refluxing THF under N2, then reflux for 3 days). The dimethylamino analogue of 5, thus obtained, was purified by silica gel chromatography, affording 17.5% of analytically pure material. Quaternization (saturated MeBr in acetone, sealed tube, 10 h) then gave 84% of lipid 5, whose structure is supported by an appropriate 1H NMR spectrum17 and a satisfactory elemental analysis (C, H, N, hemihydrate). Lipid 5 was examined in holoaggregates and in coaggregates with cationic bisether lipid 2,16 cationic bisester lipid 818 or 9,19 or zwitterionic DPPC (10).
Aggregates. Aggregates and coaggregates of 5 were created by immersion probe sonication (60 W, 5 min, 58 °C) of CHCl3-evaporated lipid or colipid films in 0.01 M pH 8.0 aqueous Tris buffer, 0.01 M in KCl. After sonication the solutions were cooled to 25 °C and filtered through 0.8 µm Millex (Millipore) filters. The hydrodynamic diameters of the aggregates were investigated by dynamic light scattering on 5 × 10-4 M lipid preparations.20 The mean diameters observed were (17) NMR of 5 (200 MHz, δ, CDCl3): 0.94 (t, 6H, 2CH3CH2), 1.44 (m, 8H, 2CH3(CH2)2), 1.81 (m, 12H, OCH2CH2), 3.4-3.8 (s + m, 16H, NMe3 + 3CH2OC + CHO), 4.0-4.2 (m, 10H, CH2N + 4CH2OAr), 6.95, 7.00, 7.83, 7.87, (2 × A2B2, J ∼ 9 Hz, 16H, aromatic). (18) Moss, R. A.; Ganguli, S.; Okumura, Y.; Fujita, T. J. Am. Chem. Soc. 1990, 112, 6391. (19) Moss, R. A.; Bhattacharya, S. J. Phys. Org. Chem. 1992, 5, 467. (20) See: Moss, R. A.; Bhattacharya, S.; Chatterjee, S. J. Am. Chem. Soc. 1989, 111, 3680 for experimental details.
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46, 52, and 35 nm for aggregates of pure 5, 1:1 5/2, and 1:9 5/2, respectively, sizes consistent with unilamellar liposomes. However, the aggregates obtained by sonication of pure 5, nominally 46 nm in diameter, did not readily pass through a 200 nm Millex-PF filter. Similar difficulties were encountered by Whitten with zwitterionic (trans) double-AB-chain PC holoaggregates, one of which was found to consist of extended flat bilayers.7 It is therefore possible that our holoaggregates of 5 are not liposomes, although the coaggregates formed from 5 and 2 are likely to be liposomal.7 Differential scanning calorimetry (DSC) of the AB aggregates employed 5 mM lipid in Tris buffer solution, prepared as above, and scanned from 1 to 99 °C at 0.8 °C/min with a Setaram micro-DSC instrument. Holoaggregates of 5 exhibited a gel to liquid crystal transition (Tc) at 68 °C, considerably higher than that of holo-1, its single-AB-chain analogue (Tc ) 30 °C).16 Coaggregated (1:1) 5/2 exhibits thermal transitions at 55 and 68.5 °C (with a pretransition at 50 °C) indicating segregation into intraaggregate domains of 2 and 5, respectively (hololiposomal 2 has Tc ) 56 °C16). AB domain formation (stacked or clustered AB lipids) is also indicated by UV spectroscopy for the 1:1 5/2 coaggregates (see below). Coliposomal 1:9 5/2 gave only the thermal transitions associated with 2; the 68 °C feature characteristic of 5 was absent, suggesting that this AB lipid was well dispersed in the bilayers of bisether lipid 2. This conclusion is supported by the UV studies below, although timedependent AB segregation does slowly occur in these 1:9 coaggregates. Absorption maxima of freshly prepared holoaggregates of 5, as well as 1:1 coaggregates of 5/2, 5/8, and 5/10, in aqueous Tris buffer at 60 °C, afforded AB absorption maxima at 321-324 nm, revealing the formation of “faceto-face” stacked AB domains of 5.1b,c,4 For holo-5, of course, the blue-shifted AB maximum is expected, but for the 1:1 coaggregates it indicates intraaggregate “sorting” into AB and non-AB lipid domains. AB clustering was also observed in the 1:1 5/2 system by DSC (see above). Related phenomena have been reported by Kunitake1c,4,21 and by this laboratory in our study of lipid 1.16 In contrast, 1:9 coliposomes of 5/2, 5/8, 5/9, and 5/10 initially absorb at λmax 357-360 nm, reflecting the general dispersal of 5 within the bilayers, and in agreement with the DSC results. However, these 1:9 bilayers are metastable: over a period of several hours at 60 °C the 5/2 coliposomes undergo AB clustering, as shown by decreasing 358 nm absorbance concomitant with increasing absorbance at 327 nm. Similar spontaneous segregation is shown by 1:9 coliposomal 5/8 and 5/9, whereas the DPPC coliposome (1:9 5/10) is stable in its AB-dispersed form (360 nm) for at least 12 h. We find it significant that spontaneous sorting of cationic AB lipid 5 occurs in cationic host liposomes of 2, 8, and 9 but not in zwitterionic DPPC host bilayers. Cationcation head-group repulsions facilitate the mobility and ultimate stacking of AB lipid 5 in the former cases, whereas the zwitterionic head groups of DPPC obviate this driving force and, depending on the specific mutual arrangements of the lipids, might even provide stabilizing interactions with the cationic headgroups of 5. A variation on this theme is offered by our observation that a 1:9 mixture of holo-5 and holo-10 aggregates readily fuses at 60 °C (λmax (21) Kunitake, T.; Ihara, H.; Okahata, Y. J. Am. Chem. Soc. 1983, 105, 6070 and references cited therein. A reviewer suggests that the sorting process may involve formation of separate AB liposomes and non-AB liposomes rather than intraliposomal segregation. The latter process, however, is precedented,1c,4,21 whereas the former separation seems less likely on entropic grounds.
4500 Langmuir, Vol. 13, No. 17, 1997
Figure 1. UV spectra accompanying irradiation of 1:9 coliposomal 5/2 at 350 nm, 60 °C: (1) Initial absorbance of dispersed trans-5 at 358 nm; (2) after 2 min of irradiation; maxima of cis-5 are observed at ∼325 and ∼450 nm; (3) recovery of (dispersed) trans-5 after 45 min at 60 °C; (4) conversion to stacked trans-5, λmax ∼325 nm, completed after ∼10 h at 60 °C. See text for discussion.
of 5 changes from 320 to 356 nm over 7 h) whereas an analogous experiment with 5 and 2 holoaggregates reveals minimal fusion after 10 h at 60 °C. Finally, we note that the spontaneous sorting observed for the 1:9 5/2 coliposomes was not observed with 1:9 coliposomes of 1 and 2.16 Apparently the driving force for clustering is greater with the double-AB-chain lipid 5 than with its single-AB-chain analogue 1. Photoisomerization. At 60 °C, above the Tc of 2, and even at ambient temperature, photoisomerization (Rayonet reactor, 16 8 W, 350 nm lamps, 2 min) of 1:9 coliposomal 5/2 occurs readily, affording a photostationary state (PSS) with ∼55% conversion to cis-5 (∼325 and ∼450 nm).22 Thermal reisomerization to (dispersed) trans-5 occurs over ∼45 min and is followed by the slow (up to 10 h) spontaneous sorting and AB stacking of 5 described above. These changes are well illustrated in Figure 1, where three “states” of 5 are detectable: dispersed trans-5, cis-5, and stacked trans-5.23 The facile photoisomerization of 5 is analogous to that observed for single-AB-chain lipid 1 in coliposomal 216 and reveals no inhibitory effect of the second AB-chain in dispersed bilayers of 5 at 25 °C. Behavior parallel to that displayed in Figure 1 was also seen with 1:9 coliposomes of 5 in ester lipid hosts 8 and 9, as well as in DPPC, where the PSS was ∼90% cis-5 and spontaneous sorting did not occur after cis f trans reisomerization (see above). Coaggregates that were richer in 5 (1:1 5/2 and 5/8, as well as holo-5) also supported smooth trans f cis photoisomerization at 60 °C, resulting in PSS containing ∼50% cis-5 within 2 min.24 Thermal reisomerization readily followed within 30-55 min at 60 °C, except for the 1:1 5/10 coaggregate, where 215 min was required. Again, the zwitterionic lipid 10 appears to stabilize the AB coliposome, even when the AB chains are in their cis configuration. However, at temperatures below 60 °C, photoisomerizations of (e.g.) 1:1 5/2 and holo-5 are significantly (22) The % cis was estimated from 100(A0 - At)/A0, where A0 is the initial absorbance of trans-5 at λmax and At is the corresponding absorbance at time t, or at the PSS. (23) Note that the absorbance of “monomeric” 5 at 358 nm is stronger after photoisomerization to cis-5 and thermal reisomerization to trans-5 than it is in the initial 5/2 coliposome. This suggests the initial presence of some AB clusters of 5 (λmax 321-324 nm) which are dispersed during photoisomerization. (24) The disappearance of trans-5 was followed at the blue-shifted UV maxima of these AB-stacked aggregates. Simultaneous formation of cis-5 could be observed at 450 nm.
Letters
Figure 2. Percent cis-5 in photostationary states of holo-5 (0), 1:1 5/2 (b), and 1:9 5/2 (9) as a function of temperature. The PSS is described as 100(A0 - At)/A0,22 which is taken as % cis-5.
impacted. Figure 2 charts the disappearance of trans-5 in these cases, as well as in 1:9 coliposomes of 5/2, as a function of photoisomerization temperature. Although the 1:9 coliposomes afford 50-55% photoisomerization within 2 min at 20-25 °C, the photoisomerizations of 1:1 5/2 and holo-5 are limited to e10% below 40 °C.25 Inefficiency of AB photoisomerization in aggregates has been noted before,4,7 but the sigmoidal dependence of the PSS on temperature demonstrated in Figure 2 is noteworthy. The 1:1 5/2 coaggregates are mainly organized into separate domains of 5 and 2 (see above), so that this coaggregate (and holo-5) features gel phase bilayers of 5 (Tc ) 68 °C) over the temperature range covered in Figure 2. Under these conditions, the PSS of irradiated 5 seems to sensitively reflect the “space” available within the bilayers of 5 for isomerization to the sterically more demanding cis-AB chains. Accordingly, the cis/trans partition of the photoexcited AB units26 increases smoothly with temperature, presumably reporting enhanced mobility within the gel phase bilayers. The PSS reaches a maximum of cis-5 at 60-65 °C, near the Tc of holo-5, but the PSS of 1:1 5/2 is somewhat richer in cis content than the PSS of holo-5 at temperatures between 30 and 55 °C (Figure 2). Presumably there is some dispersal of 5 into the less restricting domains of 2 within the 1:1 coaggregates, where 5 can more readily photoisomerize. At 60 °C, above the Tc of 2 and near the Tc of 5, the PSS of holo-5 becomes identical with that of 1:1 5/2. When 5 is dispersed (1:9) in gel phase bilayers of 2, the sigmoidal PSS/temperature dependence persists (Figure 2), but it is shifted to 0-20 °C and does not intrude at or above ambient temperature. In the absence of bilayers (MeCN solution), we find that both 5 and 1 achieve a ∼90% cis PSS within ∼20 s, even at -20 °C. Single-AB-chain lipid 1, in holoaggregates or 1:1 or 1:9 coaggregates with 2, readily yields a PSS with 45-60% cis-1 at 20 °C, but below 10 °C, the photoisomerization of holo-1 becomes very inefficient; cf., Figure 3. At 20 °C, (25) Control experiments at 35 or 55 °C show that the PSS is reached within 2 min and that the cis-5/trans-5 distributions do not vary over an additional 18 min of irradiation. (26) Isomerization of the excited AB by the spatially concise “azo inversion” mechanism would be appropriate within the restricted confines of the bilayer: Ikeda, T.; Tsutsumi, O. Science 1995, 268, 1873. Rau, H.; Ludecke, E. J. Am. Chem. Soc. 1982, 104, 1616 and references therein.
Letters
Langmuir, Vol. 13, No. 17, 1997 4501 Table 1. Thermal Cis f Trans Isomerization of AB Lipid 5a 105kisom (s-1) temp (°C)
CH3CN solution
1:9 coliposome with 2
21 30 40 50 60
1.60 3.64 8.77 27.1 71.5
3.95 9.62 27.9 79.4 215.2
Ea (kcal/mol) log A (s-1) ∆Sq (eu)b b
Figure 3. Percent cis-1 in photostationary states of holo-1 (0), 1:1 1/2 (4), and 1:9 1/2 (O) as a function of temperature. The % cis-1 is described analogously to % cis-5; see the caption to Figure 2 and ref 22. The correlation for holo-1 is drawn with a sigmoidal curve in analogy to the behavior of holo-5, see Figure 2.
the figure reveals a minor variation of the extent of cis-1 at the PSS in the expected order: 1:9 1/2 >1:1 1/2 > holo1. Clearly, aggregated double-AB-chain lipid 5 experiences more difficulty in trans f cis photoisomerization than comparably aggregated single-AB-chain lipid 1. Photoisomerization of 5 is particularly disfavored within gel phase holoaggregates, where the cis/trans distribution at the PSS smoothly tracks the temperature, whereas the photoisomerization of holo-1 is much less difficult at ambient temperature, even in the gel phase. Thermal Reisomerization. The cis f trans thermal reversion of double-AB-chain lipid 5 was briefly examined in 1:9 5/2 coliposomes and in MeCN solution at temperatures between 21 and 60 °C. Because of the complexities attending the trans f cis photoisomerization of 1:1 5/2 and holo-5 at lower temperatures, it was not possible to carry out parallel thermal reisomerization studies in these aggregates. Reisomerizations were studied with PSS mixtures of cis- and trans-5, monitoring the restoration of trans-5 by UV spectroscopy at 358 nm, the λmax of trans-5 dispersed in 2. Excellent first-order kinetics (r > 0.998) were obtained over the temperature range 21-60 °C; rate constants and derived activation parameters appear in Table 1. Surprisingly, reisomerization of cis-5 in 1:9 5/2 coliposomes occurs 2-3 times faster than reisomerization in MeCN solution at each of the five temperatures. The activation parameters reveal that the origin of this effect resides in a significantly less unfavorable ∆Sq for the liposomal isomerization. Possibly, the more spatially demanding cis-527 present in the 1:9 5/2 coliposomes causes
19.1 9.36 -17.7
20.1 10.5 -12.4
a Estimated errors are (3% in k, (5% in E , and (15% in ∆Sq. a At 298 K.
a substantial freezing out of normal vibrations and rotations among neighboring host lipids that is relieved upon cis f trans isomerization, somewhat mitigating the unfavorable ∆Sq of the isomerization itself. No such offsetting factor exists for cis-5 in solution, where the full unfavorable ∆Sq of the isomerization is encountered. For comparison, activation parameters for the cis f trans isomerization of single-AB-chain lipid 1 are Ea ) 22.6 kcal/mol and ∆Sq ) -6.25 eu (1:9 1/2 coliposomes) and Ea ) 21.8 kcal/mol and ∆Sq ) -8.52 eu (MeCN solution).16 The activation energies are similar to those of cis-5 f trans-5, but the activation entropies are significantly less unfavorable, as would be anticipated for the sterically less demanding single-AB-chain lipid. There may be a small differential entropy of activation (2.3 eu) favoring isomerization of coliposomal 1, analogous to the effect observed with 5. Conclusions. Double-AB-chain cationic lipids such as 5 undergo trans f cis photoisomerization readily in solution and in 1:9 coliposomes with cationic bisether lipid 2. However, in 1:1 5/2 or holo-5 aggregates, the photoisomerization is less efficient, and the PSS is strongly temperature dependent between 30 and 60 °C. The photoisomerization of 5 is also less efficient under these conditions than that of its single-AB-chain analogue 1. Dispersed 1:9 coliposomes of 5 and 2 spontaneously sort into segregated domains of each lipid. Thermal cis f trans reisomerization of 5 is more facile in 1:9 5/2 coliposomes than in MeCN solution, aided by a less unfavorable entropy of activation. Acknowledgment. We are grateful to the U.S. Army Research office for financial support. We thank Professor K. Breslauer and his associates, Drs. J. Elliott, G. Kaletunc, and J. Volker, for helpful discussions and the use of their microcalorimeter. LA9703003 (27) The PSS does not exceed 50-60% of the cis AB lipid in the coaggregates, so that there must be mixed populations of 5 with 0, 1, or 2 cis-AB chains. We do not know what the activation parameters would be for the isomerization of all-cis-5.
