584
Langmuir 1991, 7, 584-589
Cast Polydiacetylene Films from Diacetylenic Glutamate Lipids Thauming Kuo and David F. O'Brien* C.S. Marvel Laboratories, Department
of Chemistry, The University of Arizona, Tucson, Arizona 85721
Received March 13, 1990. I n Final Form: September 17, 1990 The efficiency of retention of the molecular order present in bilayer membranes upon the casting of multilayer films was evaluated with diacetylenic lipids. Comparison of the properties of both bilayers and cast films shows a close correspondence of the photoreactivity of the diacetylenes, as well as the absorption and thermochromic properties of the polydiacetylenes. These results demonstrate that sufficient molecular order is retained in the cast films to permit the efficient topochemical photopolymerization of diacetylenes. The casting technique was most successful at retaining molecular order, if the experiment is performed below the lipid phase transition temperature of the hydrated lipid bilayers. Introduction Well-ordered multilayer films of amphiphiles are frequently prepared by Langmuir-Blodgett techniques. Kunitake and co-workers have recently described a simpler alternative approach for the formation of multilayer films of identical Aqueous dispersions of bilayer vesicles prepared from the amphiphile were cast onto the solid substrate of choice, where they were allowed to dry slowly. The dry films could be removed from the substrate for characterization and use. Examination of the films by both X-ray scattering (edge-on) and differential scanning calorimetry (DSC) indicate that they are composed of ordered multilayers.1-3 This method is somewhat similar to ultracentrifugation techniques for the stacking of multibilayers for X-ray studies of lipid bilayers.6 However, the Kunitake approach converts bilayers into multilayer films by allowing the lipids to slowly concentrate as the water evaporates. The attractiveness of cast films as an alternative to some LB films depends on the retention of order in the lipid assembly during the casting procedure. In order to test the extent of molecular order in cast multilayer films, we designed and synthesized a diacetylenic lipid based on a glutamate backbone and then prepared cast films from aqueous dispersions of this new lipid.' The topotactic photopolymerization of diacetylenes is acutely sensitive to the molecular order of crystals8 or supramolecular assemblies of monomeric diacetylenes; e.g. bilayer membranes of lipid diacetylenes are polymerizable only below the lipid phase transition temperatures of the membrane.g Furthermore, the disordering of lipid chain packing, which occurs on sonication to form small unilamellar vesicles, results in an inhibition of polydiacetylene (PDA) forma(1) Nakashima, N.; Ando, R.; Kunitake, T. Chem. Lett. 1983, 1577. (2) Shimomura, M.; Ando, R.; Kunitake, T. Ber. Bunsen-Ges. Phys. Chem. 1983,87, 1134. (3) Kunitake, T.; Shimomura, M.; Kajiyama, T.; Harada, A.; Okuyama, K.; Takayanagi, M. Thin Solid Films 1984, 121, L89. (4) Nakashima, N.; Kunitake, M.; Kunitake, T.; Tone, S.; Kajiyama, T. Macromolecules 1985, 18, 1515. (5) Higashi, N.; Kajiyama, T.; Kunitake, T.; Prass, W.; Ringsdorf, H.; Takahara, A. Macromolecules 1987, 20, 29. (6) Levine, Y. K.; Wilkins, M. H. F. Nature (London),New Biol. 1971, 230 69. Clark, N. A,; Rothschild, K. J. Methods Enzymol. 1982,88,326. (7) Kuo, T.; O'Brien, D. F. J . Am. Chem. SOC.1988, 110, 7571. Kuo, T.; O'Brien, D. F. Macromolecules 1990, 23, 3225. (8) Wegner, G. Makromol. Chem. 1972, 154, 35. (9) O'Brien, D. F.; Whitesides, T. H.; Klingbiel, R. T. J . Polym. Sci., Polym. Lett. Ed. 1981, 19, 95.
tion.lO Thus, the efficient formation of PDAs may be used as a sensitive chemical test of lipid chain order. Both bilayer vesicles and cast films formed from the diacetylenic glutamate lipid (1) were photosensitive and readily gave highly colored PDAs.7 The efficient formation of PDA in the cast films indicated that the molecular order present in the bilayer vesicles was largely retained in the films. In the case of cast multilayer films, the uniform formation of PDAs across the whole exposed area of the film demonstrates that the order detected previously a t the edge of films by X-ray is not limited to the edge. This suggests that the lipids are arranged in multilayers within the films. However, the absorption maximum of the PDA formed in the cast films of 1 was shifted to shorter wavelength from the value observed for the PDA formed by photopolymerization of vesicles of 1 (640 nm). Since the absorption maxima of PDAs are indicative of the polymer chain length and/or the order of the polymer structure,ll this observed shift indicates the PDA chains are shorter or somewhat more disordered in the cast films in the bilayers of 1. In order to examine whether this difference in PDA structure was an inherent consequence of the casting procedure or more specifically related to lipid 1, we have prepared three additional diacetylenic glutamate lipids (2-4). Each of the new lipids is similar to lipid 1, except for structural modifications of the hydrophilic region designed to raise the lipid phase transition temperature (T,) to above the casting temperature (rt). The T , of hydrated lipids shifts to higher temperatures as the water content is decreased;12therefore the casting of lipids, e.g. 1, whose T , is below rt, will cause the membranes to pass through the lipid crystalline to gel transition. The dehydration induced excursion across the transition may result in some lipid disordering that is retained in the cast film. In this report, the properties of bilayer membranes and cast films of each of these new lipids are compared, both before and after PDA formation. Results Synthesis. The synthesis schemes are shown below. Bis(docosa-10,12-diynyl)L-glutamate (5) was prepared (10) Lopez, E.; O'Brien, D. F.; Whitesides, T. H. J . Am. Chem. SOC. 1982, 104, 305. (11) Tieke,B.;Lieser,G.; Wegner, G.J.Polym.Sci.,Polym. Chem.Ed. 1979 77 _ _ _. _. ,, l G R l
(12) Chapman, D. In Form and Function of Phospholipids, 2nd ed.; Ansell, G. B., Hawthorne,J. N., Dawson, R. M. C., Eds.; Elsevier Scientific Publishing Co.: Amsterdam, 1973; pp 117-142.
0743-7463/91/2407-0584$02.50/0 0 1991 American Chemical Society
Langmuir, Vol. 7, No. 3, 1991 585 Scheme I
1: n=5
n=10
2:
3 7
4
2
according to the procedure reported earlier.7 Bis(docosa10,12-diynyl) N-(11-triethylammonioundecanoy1)-L-glutamate bromide (2) was synthesized first by the reaction of 5 and 11-bromoundecanoyl chloride (6) to give bis(docosa- 10,12-diynyl)N - (11-bromoundecanoy1)-L-glutamate (7) followed by amination of 7 with triethylamine (Scheme I). Bis(docosa-10,12-diyyl)N-(6-trimethylammoniohexanoy1)-L-glutamate bromide (3) was prepared by amination with trimethylamine of bis(docosa-10,12-diyyl)N46bromohexanoy1)-L-glutamate (8) prepared previously' (Scheme 11). In order to synthesize a glutamate lipid (4) with an Scheme I11 Br+ -C Br aromatic substituent in the head group, 444-bromobuCH302CeOH CH302C+O-(-CHp-)rBr toxy)benzoyl chloride (11) was prepared as outlined in KOH, EtOH Scheme I11 and detailed in the Experimental Section. Bis9 (docosa-10,12-diynyl) N - [(4-(4-bromobutoxy)benzoyl)-~glutamate bromide (4) was then synthesized by the reaction of 5 and 11 to give 12, followed by amination with triethylamine. Careful analyses of 3 and 4 show minor amounts of the respective impurities Me3NH+Br- and 10 Et3NH+Br-, which could not be completely removed by column chromatography (Scheme IV). Solid-state Polymerization. Diacetylene lipids, 2,3, and 4, and diacetylene intermediates, 7 and 12, are all photopolymerizable in solid state. These white crystals turned purple or blue after a few seconds of irradiation 11 with a 254-nm low-pressure mercury lamp (Pen Ray) at a distance of 8 cm. The hydrogen-bond-forming amide groups and the polar head groups in these diacetylene state. The temperature ranges for the mesophases are molecules provide strong interaction between the monolisted in Table I. meric diacetylene molecules and appear to favor a crystal lattice arrangement suitable for topochemical polymeriVesicle Preparation and Polymerization. Lipid bization as suggested by Wegner.8 layer vesicles of 1,2, 3, or 4 were prepared by hydration Thermotropic Liquid Crystalline Behavior of the of a thin film of 2 pmol of the respective lipids with 2 mL Lipids. While it was known that aqueous dispersions of of water (Milli-Q). The hydrated bilayers were further lipid bilayer vesicles are lyotropic liquid crystals (IC), thervortexed or briefly sonicated a t 45 "C in a thermostated and ammonium motropic IC properties of pho~pholipidsl~ cup-horn sonicator (Heat System, W-380) to give lipid salts14 have also been reported in the literature. Lipids suspensions. Hydrated lipids frequently form bilayer 1,3, and 4 each showed thermotropic IC behavior in solid assemblies that exhibit both gel and liquid crystalline (13) Demus, D.;Demus, H.; Zaschke, H.FlussigeKristalle in Tabellen; phases. Each of these lipids shows similar behavior. The VEB Deutscher Verlag fur Grundstoffindustrie: Leipzig, 1974, p 322. midpoints of the respective phase transitions (T,)for the (14) Kelker, H.; Hatz, R. Handbook ofLiquid Crystals;VerlagChemie: vortexed lipid bilayers are reported in Table 11. The values Deerfield Beach, FL, 1980; p 60.
586 Langmuir, Vol. 7, No. 3, 1991
Kuo and O'Brien
Scheme IV
1.5 1.4
0 B-r
o-&-NH-cH-coZ-C.C-C-C-
I
CHp-CG
1
0.9
4
:::
CH2
S C H C l J
W
C
I
C
-
C
I
C
1
1
1.3
I
11
-
12
A
I/
I
\
4
Table I. Thermotransition Temperatures of Unhydrated Lipids lipid tm.0 "C ti,b "C 1 35 85 3 42 115 4 41 88
wa.i.npUI
t,, thermotransition temperature from solid to liquid crystal state. * ti, thermotransition temperature from liquid crystal state to isotropic melt.
Table 11. Phase Transition Temperature (T,) of Bilayer Vesicles lipid
T,, "C 18.5 30.5 26 35.5
[nml
Figure 1. Absorption spectra of bilayer vesicles of 2 (0.125 mM) in a 1-cm cuvette at rt. The spectra were recorded after exposure to 254-nm light for the following times: curve 1, 0 s; 2, 15 s; 3, 30 s; 4, 60 s; 5, 180 s; 6, 300 s.
1.2
e
E
4
0.2 0.1
were determined by differential scanning calorimetry (DSC) for each lipid suspension at a heating rate of 20 "C/h. The original lipid structure (1)was modified to increase the T , either by using a longer spacer between the head group and the amide group (2), or by the use of a smaller head group (3), or by the incorporation of an aromatic ring into the spacer link (4). It is well-known that phase transition temperatures are sensitive to lipid chain packing. Thus, the incorporation of unsaturated groups or branched methyl groups into the lipid chains is frequently used to lower the T , to an experimentally convenient temperature (usually below rt). Neither of these approaches was suitable for the diacetylenes; therefore we pursued a strategy of modifying the lipid head group. To our knowledge, this is the first demonstration that the T , may be shifted to lower temperatures by modifying the head group structure from choline (MeSN+) (3) to triethylammonium (Et3N+) (l),while maintaining the same lipid chain structure. This illustrates a convenient new synthetic method to modify lipid packing without changing the hydrophobic chain structure. Bilayer membranes of lipid diacetylenes can only be photopolymerized at temperatures below the lipid phase transition temperat~re.~JO Thus, in contrast to bilayers of 1, lipid bilayer of 2 , 3 , or 4 were photosensitive at rt. The lipid bilayer membranes of 2, 3, and 4 were readily polymerized by irradiation with 254-nm light from a lowpressure mercury lamp. The samples turned purple-blue immediately upon irradiation a t rt. The polymerization process was followed by the UV-visible absorption spectra as illustrated in Figures 1-3. They were recorded by irradiation of aqueous lipid bilayer vesicles of 2 (0.125 mM), 3 (0.25 mM), and 4 (0.125 mM) at various times. Comparsion of the polydiacetylene absorbances recorded after selected irradiation times shows that the order to photoreactivity of lipid bilayers was 2 > 4 > 3 at rt. The
o ! 550
4%
up Wal.nqth
650
750
[nml
Figure 2. Absorption spectra of bilayer vesicles of 3 (0.25 mM) in a 1-cm cuvette at rt. The spectra were recorded after exposure to 254-nm light for the following times: curve 1, 0 s; 2, 30 s; 3, 60 s; 4, 300 s; 5, 600 s; 6, 1200 s.
1.8
1
::: 1
0.2
Figure 3. Absorption spectra of bilayer vesicles of 4 (0.125 mM) in a 1-cm cuvette at rt. The spectra were recorded after exposure to 254-nm light for the following times: curve 1, 0 s; 2, 30 s; 3, 60 s; 4,300 s; 5, 600 s; 6, 1200 s.
bilayers of 2 were about twice as photosensitive as those of 4. A similar small difference was found between 4 and 3. In order to compare the relative photosensitivity of these lipid bilayers with that of 1, they were each cooled to 0 "C and irradiated. The lipid bilayers of 1 were less photosensitive than those of 2 , 3 , and 4 at 0 "C. Since the differences in photoreactivity are relatively small, it is difficult to ascribe the effects to particular structural changes in the molecules. It should be noted that the lipid structure did affect the polydiacetylene absorption maxima (1,630 nm; 2,570,620 nm; 3,610 nm; 4,590 nm).
1.4 1.3 1.2
-
1.1 7
1.1 1
0.9
oa 0.7
7
0.6
I
\\
0.3 0.4
0;) 02
0.2
0.1
o !
uo
453
550
,
Langmuir, Vol. 7, No. 3, 1991 587
Films from Diacetylenic Glutamate Lipids
wl
750
*tN.1 Figure 4. Absorption spectra of photopolymerized bilayer vesicles of 3 at r t (11, at 50 "C (2), and after cooling back to rt (3).
The above comparison of photoreactivity was made assuming the optical density a t 254 nm of the vesicles of these diacetylene lipids with similar chemical structures are equivalent a t the same concentration. This appears to be the case for methanol solutions of the lipids, but the absorption of the hydrated lipid vesicles a t 254 nm were difficult to measure due to light scattering caused by the opalescent nature of these aqueous dispersions. Thermochromic Behavior of the Polymerized Vesicles. The polymerized vesicles of 2, 3, and 4 each showed reversible thermochromic behavior. The effect is illustrated in Figure 4 for vesicles of 3. The color was blue a t rt with absorption maximum at 610 nm and turned purple-red as the temperature was increased to 50 "C ,A( at 570 nm). The process was reversible; as the vesicles were cooled down to rt, they returned to the original blue color. The thermochromic phenomenon has been observed in PDA crystals,15J6solutions17J8,multilayers,llJgvesicles,2°p21 and films.16 Chance et a1.18 suggested that temperatureinduced or solvent induced color changes in PDA solutions are indicative of conformational transitions involving ordering of the polymer chains. Conformational transitions disrupt the planarity of the polymer backbone and caused the change in effective conjugation length. Short conjugated PDA molecules show yellow color, while long conjugated molecules are blue. Singh and co-workersZ1reported a reversible thermochromism in photopolymerized phosphatidylcholine vesicles. They suggested that the increased motional freedom of the acyl side chains in PDA bilayers caused by raising the temperature altered the polymer conformation. The thermochromism was reversible because the bilayer morphological changes are thermally reversible. Our observations on vesicles of 2, 3, and 4 are consistent with this explanation. Previously, we reported' that photopolymerized vesicles of 1 showed a two-stage thermochromic phase transition (15) Chance, R.R.;Baughman, R. H.; Muller, H.; Eckhardt, C. J. J . Chem. Phys. 1977, 67, 3616. (16) Chance, R. R.; Patel, G. N.; Witt, J. D. J . Chem. Phys. 1979, 71, 206.
(17) Patel, G.N.;Chance, R. R. J.Polym. Sci., Polym. Lett. Ed. 1978, 16, 607.
(18) Chance, R.R.;Sowa, J. M.;Eckhardt, H.J.Phys. Chem. 1986,90, 3031. (19) Tieke, B.; Enkelmann, V.; Kapp, H.; Lieser, G.; Wegner, G . J. J. Mucromol. Sci., Chem. 1981, A15 ( 5 ) , 1045.
(20) Pons, M.; Johnston, D. S.; Chapman, D. J. Polym. Sci., Polym. Chem. Ed. 1982,20,513. (21) Singh, A.;Thompson, R. B.; Schnur, J. M. J . Am. Chem. SOC. 1986, 208, 2785.
250
uo
4%
6%
WavLnpth [nm]
759
Figure 5. Absorption spectra of a cast multilayer film of 2. The spectra were recorded after exposure to 254-nm light for the following times: curve 1, 0 s; 2, 10 s; 3, 30 s; 4, 60 s; 5, 180 S; 6, 300 s.
1.2 1.1 1
e
c
i
0.9 0.1 0.7 0.6 0.5 0.4 0.3 0.2 0.1
uo
I
4x
550
Ww-
wl
7%
bml
Figure 6. Absorption spectra of a cast multilayer film of 3. The spectra were recorded after exposure to 254-nm light for the following times: curve 1,0 s; 2, 10 s; 3,30 s; 4,60 s; 5, 180 s; 6, 300 s. when the temperature was elevated. In the first stage, the vesicles turned from blue (0 " C ) to orange-red (22 " C ) and the change was irreversible; in the second stage, they turned from orange-red (22 " C ) to yellow-orange (50 "C) and the change was reversible. The reason for the different thermochromic behavior of polymerized vesicles of 1compared to photopolymerized vesicles of 2,3,and 4 is uncertain at this time. Multilayer Film Casting and Polymerization. A few drops of the bilayer vesicles were spread on a clean glass slide and allowed to dry slowly over two days to give thin transparent films, which were then vacuum dried. When the cast diacetylene films were irradiated at rt by UV light, the films immediately became blue and the color intensified with continuous irradiation indicating the formation of polydiacetylene. Figures 5-7 show the absorption spectra obtained by irradiation of cast multilayer films for various times. The absorption maxima of photopolymerized multilayer films are 570 and 620 nm (2), 570 and 620 nm (3), and 590 nm (4), which were very similar to the absorption maxima found for the polymerized vesicles in Figures 1-3, respectively. When a higher concentration of lipid bilayer vesicles ( 5 mM) was used for casting, the resulting PDA films were thick enough to be peeled off the glass after irradiation to give deep blue, flexible, free standing films (thickness = 10 pm). The films are not soluble in common organic solvents, such as chloroform, THF, DMF, and DMSO. ThermochromicBehavior of Cast Multilayer Films. Similar to the polymerized vesicles, the cast PDA films
Kuo and O'Brien
588 Langmuir, Vol. 7, No. 3, 1991
were cooled in air to rt and irradiated with 254-nm light; no color change or partial color change was observed. It appeared that the order of lipid bilayers was disrupted at higher temperatures and the resulting cast films did not have sufficient order to undergo topochemical polymerization.
2w
sw
ua -w
i
h l
Figure 7. Absorption spectra of a cast multilayer film of 4. The spectra were recorded after exposure to 254-nm light for the following times: curve 1, 0 s; 2, 10 s; 3, 30 s; 4, 60 s; 5 , 120 s; 6, 180 s. Table 111. Absorption Maxima (nm) of Polydiacetylenes in Lipid Bilayer Vesicles and Cast Multilayer Films lipid bilayer vesicles cast film Temperature 22 "C 2 570,620 570,620 3 570,610 570,620 4 590 590 Temperature 50 "C 2 545 540 3 545 540 4 540 540
also showed reversible thermochromic behavior. The films were blue at rt and turned purple-red when heated to 50 OC.
Thermochromic behavior of solution-cast films of urethane-substituted PDAs (poly-3BCMU and poly-4BCMU) had been observed by Chance et a1.16 They suggested that color changes were caused by the disruption of intramolecular hydrogen bonding and the destabilization of the planar polymer conformation. We have also demonstrated the reversible thermochromism of cast multilayer films of 1.7
The relaxation process of this reversible thermochromism of cast films appeared to be very fast. For example, when a free standing film of 1, 2, 3, or 4 was rapidly heated in air with a heat gun, the color changed from purple-blue to orange-red but immediately returned to the original color when the heating source was removed. Correlation of the Absorption Spectra of Vesicles and Cast Films. The absorption maxima in the UVvisible spectra of polydiacetylenes in lipid bilayer vesicles and cast multilayer films are summarized in Table 111. The bilayer vesicles and cast multilayer films of each lipid show similar absorption maxima a t rt, as well as after heating to 50 OC. These data indicate that the molecular order of lipid bilayers was retained during the formation of cast multilayer films. Consequently, the absorption maxima were only affected by the chemical structures of the lipid bilayers but not affected by their degree of hydration. Effect of Temperatures on Film Casting. The PDA films were typically cast by drying the lipid bilayer vesicles in air at rt. Attempts made to remove water at higher temperatures did not yield light-sensitive cast films. Thus, lipid bilayer vesicles of 3 (2.5 mM) and 4 (1 mM) were spread on glass slides which were placed on a hot bench (Kofler Heizbank) at 50, 60, 80, 100, and 130 f 3 "C, respectively. After drying, the resulting diacetylene films
Summary The new diacetylenic glutamate lipids reported here each form photosensitive hydrated bilayer membranes, which have a T , > rt. Multilayer PDA films were obtained by the casting of unpolymerized lipid bilayer vesicles on glass substrates at rt, followed by photopolymerization. Both the polymerized bilayer vesicles and cast PDA films of each lipid showed reversible thermochromic behavior. The efficiency of the polymerization reaction and absorption characteristics of the PDAs in both the bilayers and the films indicate that the molecular order of lipid bilayers was retained in cast multilayer films. These initial chemical tests will need to be supplemented by physical methods. A preliminary X-ray diffraction study on lipid 1 was described by Rhodes et a1.22 Langmuir-Blodgett films have recently attracted considerable attention due to their potential applications in high-tech electronic and photonic materials.23 The cast film technique of Kunitake et al.1-5 appears to be a convenient alternative procedure for the preparation of homogeneous multilayers on solid supports. The authors showed that bilayer characteristics were retained in these cast multilayer films. These results and our prior studies7 demonstrate that sufficient molecular order is retained in the cast films to permit the efficient topochemical photopolymerization of diacetylenes. There is especially close similarity of the PDA characteristics for the PDAs formed in bilayers and cast multilayer films if the hydrated lipid phase transition temperature is greater than the casting temperature. The casting technique appears to be most successful a t retaining molecular order of the original bilayer, if the experiment is always below the T m of the hydrated lipids. Under these circumstances, little disordering occurs during the drying process, because the lipids do not pass through a phase transition during dehydration. Experimental Section Methods. All the experiments were carried out under yellow light. Melting points were taken on a Mel-Temp apparatus unless otherwise indicated. Infrared spectra were recorded on a PerkinElmer 983 spectrometer. NMR spectra were taken on a 250MHz Bruker WM250 spectrometer. Visible absorption spectra were recorded on a Hewlett-Packard diode array 8452A spectrophotometer. Elemental Analyses were performed by Desert Analysis, Tucson, AZ. Chloroform and acetonitrile were dried over PzOs. Distilled water was further purified by a Milli-Q water system, Millipore. Synthesis. Bis(docosa-10,12-diynyl)N-( 11-Bromoundecanoy1)-L-glutamate (7). The 11-bromoundecanoyl chloride 6 (prepared by treatment of 11-bromoundecaroic acid with thionyl chloride) was added to a solution of 0.10 g (0.13 mmol) of 5 in 20 mL of dried chloroform and 0.016 g (0.13 mmol) of N-(dimethy1amino)pyridine and then stirred at r t for 30 min. After the reaction, the mixture was extracted with dilute HCl and 10% sodium bicarbonate. The organic layer was separated and dried, and the solvent was removed to give colorless oil which was crystallized from methanol to give 0.11 g (85%) of 7: mp 36-38 "C; IR (KBr)3350,2919,2847,1726,1640,1536,1462,1246,1180, (22) Rhodes, D. G.; Kuo, T.; O'Brien, D. F. Biophys. J. l990,57,263a. (23) Electronic and Photonic Applications of Polymers; Advances in Chemistry Series 218; Bowden, M. J., Turner, S. R., Eds.; American Chemical Society: Washington, DC, 1988; p 225.
Films from Diacetylenic Glutamate Lipids
Langmuir, Vol. 7, No. 3, 1991 589
722 cm-I; 'H NMR (CDC13)6 0.88 (t, 6 H, CHsC), 1.26-2.05 (m, sodium bicarbonate. The organic layer was separated and dried, 76 H, CHz), 2.24 (t, 8 H, CHzCr), 2.37 (t, 2 H, CH~COZ), 3.41 and the solvent was removed to give colorless oil which was (t, 2 H, CHzBr), 4. 06 (t, 2 H, CHzO), 4.13 (t, 2 H, CH20),4.62 crystallized from methanol to give 0.19 g (95%) of (12): mp 37(m, 1 H, CH), 6.12 (d, 1H, NH). Anal. Calcd for C,&l~NO5Br: 38 "C; IR (KBr) 3298,2923,2849,1732,1630,1607,1506,1463, C, 72.43; H, 10.06; N, 1.41. Found: C, 72.31; H, 10.41; N, 1.36. 1252, 1208, 1108, 1064, 857, 771, 723 cm-l; lH NMR (CDC13) d Bis (docosa- 10,12-diynyl) N-( 11-triethylammoniounde0.88(t,6H,CH3C),1.26-2.00(m,62H,CH2),2.24(t,8H,CH2C~), canoy1)-L-glutamate Bromide (2). A stirred solution of 0.10 2.45 (t, 2 H, CHzCOz), 3.50 (t, 2 H, CHZBr), 4.03 (t,2 H, CHzO), g (0.10 mmol) of 7 in 20 mL of dry acetonitrile was refluxed for 4.05 (t, 2 H, CHzO), 4.16 (t, 2 H, CHZOPh), 4.80 (m, 1 H, CH), 1 day with an excess of triethylamine. The solvent was then 6.90 (d, 2 H, C&), 7.80 (d, 2 H, C6H4). Anal. Calcd for CGOH92evaporated under reduced pressure, and the resulting oil was NO&: C, 71.86; H, 9.18; N, 1.40. Found: C, 72.00; H, 9.24; N, taken up in a small amount of chloroform for silica gel flash 1.35. chromatography (eluent, chloroform with increasing fraction of methanol, 5 50,10%,20 70): yield 0.07 g (64 70); mp 36-39 "C; IR Bis(docosa- 10,lf-diynyl) N-[(4-( 4-Bromobutoxy)ben(KBr) 3434, 2922, 2850, 1728, 1648, 1536, 1463, 1200, 801, 723 zoyll-L-glutamate Bromide (4). A stirred solution of 0.10 g cm-'; lH NMR (CDC13)6 0.86 (t, 6 H, CHsC), 1.25-2.00 (m, 76 (0.10 mmol) of 12 in 20 mL of dry acetonitrile was refluxed for H, CH2), 1.38 (t, 9 H, CHBC), 2.22 (t, 8 H, CH~CG),2.36 (t, 2 H, a day with an excess of triethylamine. The solvent was then CH&02), 3.46 (4, 8 H, CHZN), 4.03 (t, 2 H, CHzO), 4.10 (t,2 H, evaporated under reduced pressure, and the resulting oil was CH20),4.58 (m, 1 H, CH), 6.38 (d, 1 H, NH). Anal. Calcd for taken up in a small amount of chloroform for silica gel flash C66H115N~05Br:C, 72.33; H, 10.50;N, 2.56. Found: C, 70.26; H, chromatography (eluent, chloroform with increasing amount of 10.36; N, 2.49. methanol, 5%,10%,20%): yield 0.08 g (73%);mp t, 41 "C, ti Bis(docosa-10,12-diynyl)N-(6-Trimethylammoniohex88 "C; IR (KBr) 3442,2918,2848,1726,1630, 1605,1502,1463, anoy1)-L-glutamate Bromide (3). A stirred solution of 0.10 g 1262, 1208,1179,802, 772,723 cm-1; lH NMR (CDC13) 6 0.88 (t, (0.11mmol) of the previously reported7 bis(docosa-10,12-diynyl) 6 H, CHsC), 1.26-2.00 (m, 62 H, CHz), 1.41 (t, 9 H, CHsC), 2.24 N-(6-bromohexanoyl)-~-glutamate (8) in 10 mL of acetonitrile was refluxed for 2 h with an excess of trimethylamine (25% (t,8 H , CH~CE),2.46 (t,2 H, CHzCOz), 3.50 (q,8 H, CHzN), 4.05 aqueous solution). To the mixture was added 30 mL of aceto(t, 2 H, CHZO), 4.14 (t, 2 H, CHZO), 4.16 (t, 2 H, CHZOPh), 4.78 nitrile, and the solvent was evaporated under reduced pressure. (m, 1 H, CH), 6.94 (d, 2 H, C,3H4),7.01 (d, 1 H, NH), 7.81 (d, 2 The resulting oil was taken up in a small amount of chloroform H, C&). Anal. Calcd for C~Hlo7N&Br: C, 71.80; H, 9.70; N, for silica gel flash chromatography (eluent, chloroform with 2.54. Found: C, 67.50; H, 9.94; N, 2.16. increasing fraction of methanol, 570, 1070, 20%): yield 0.08 g Vesicle Preparation. The diacetylenic glutamate lipid (2 (47c, ); mp t , 42 "C, t , 115 "C; IR (KBr) 3442,2922,2847,1725, l642,1536,1463,1261,1207,1096,802,723~m-~;~HNMR(CDCl~) pmol) in chloroform was dried on a rotary evaporator to form a thin film. The film was further dried with a vacuum pump, then 6 0.88 (t, 6 H, CH,C), 1.26-1.66 (m, 64 H, CHz), 2.24 (t, 8 H, hydrated with 2 mL of Milli-Q water, and subsequently soniCH~CE),2.47 (t, 2 H, CHZCOZ), 3.42 (s, 9 H, CHsN), 3.58 (t, 2 cated at 45 "C for 45 s with a cup-horn type sonicator (Heat H, CH2N), 4.05 (t, 2 H, CH20), 4.08 (t, 2 H, CH20), 4.50 (m, 1 H, CH), 7.40 (d, 1 H, NH). Anal. Calcd for C58H~Nz05Br:C, Systems, W-380) to yield a slightly opalescent suspension of 70.80; H, 10.07; N, 2.85. Found: C, 66.52; H, 9.60; N, 2.55. vesicles (1 mM). Methyl 4 4 4-Bromobutoxy)benzoate (9). Methyl 4-hydroxCalorimetry. Calorimetric data were obtained with a Miybenzoate (3.04 g, 0.02 mol) was added to a solution of 1.12 g crocal, Inc., MC-2, differential scanning microcalorimeter at a (0.02mol) of potassium hydroxide in 100 mL of absolute ethanol. heating rate of 20 "C/h. Suspensions of extended bilayers were The mixture was stirred for 15 min, followed by the addition of prepared by vortexing lipid 2, 3, or 4 (2 pmol) in purified water 17.2 g (0.08 mol) of 1,4-dibromobutane. The reaction mixture (2 mL) at 45 "C and subsequently transferring to the calorimeter was then refluxed for 45 min, and a white precipitate formed. via a calibrated syringe. The mixture was cooled to rt and the precipitate removed by filtration. The solvent was evaporated from the filtrate under Photopolymerization of t h e Vesicles. Bilayer vesicles of reduced pressure. The residue was taken up in chloroform and 2 (0.125 mM), 3 (0.25 mM), or 4 (0.125mM) were introduced into extracted with lop, sodium hydroxide. The organic layer was a 1-cm quartz cell, the water suspension was then purged with separated and the solvent was removed. The resulting oil was argon, and and the cell was capped tightly. The samples were purified by silica gel flash chromatography (eluent, 10% ethyl irradiated with a low-pressure mercury lamp a t a distance of 4 acetate in hexane) to give 3.5 g (61?) of methyl 4-(4-bromocm for selected times. The polymerization process was monitored butoxy)benzoate: mp 37-39 "C; IR (KBr) 2945,1722,1605,1509, spectrophotometrically. 1433,1317, 1281, 1256, 1170,849,767 cm-l; 'H NMR (CDC13) 6 1.97 (m, 2 H, CH2), 2.08 (m, 2 H, CH2), 3.50 (t,2 H, CHZBr), 3.90 Film Casting and Polymerization. A few drops of the un(s, 3 H, CH30), 4.05 (t, 2 H, CH20), 6.90 (t, 2 H, C6H4), 8.00 (d, polymerized vesicles of 2 (0.5 mM), 3 (0.5 mM), or 4 (0.25 mM) 2 H, C&). Anal. Calcd for C12H1503Br:C, 50.17;H, 5.23. Found: were spread on a portion of glass slides (75 X 25 mm). Thin C, 50.19; H, 5.26. transparent films were formed after drying at rt in air for 2 days. 4-(4-Bromobutoxy)benzoicAcid (10). A stirred solution of The films on glass were further dried under vacuum for 2 h and 1.43 g (5 mmol) of methyl 4-(4-bromobutoxy)benzoate (9) in 50 irradiated by the low-pressure mercury lamp at a distance of 8 mL of ethanol (5OoC) was treated with 0.2 g ( 5 mmol) of sodium cm for selected times. The polymerization process was followed hydroxide and refluxed for 1 h. A white precipitate formed as spectrophotometrically. the mixture was acidified with dilute sulfuric acid. The precipitate was isolated and dried to give 0.54 g (40%)of 4-(4-bromobutoxy)benzoic acid: mp 134-137 "C; IR (KBr) 2947, 1678, Acknowledgment. Acknowledgement is made to the 1602, 1512, 1428, 1254, 1172, 1026, 849, 775 cm-l. Anal. Calcd donors of t h e Petroleum Research Fund, administered by for C11HllOsBr: C, 48.35; H, 4.76. Found: C, 48.59; H, 4.77. t h e American Chemical Society, for t h e partial support of 4-(4-Bromobutoxy)benzoylChloride (11). A solution of this research. 0.136 g (0.5 mmol) of 4-(4-bromobutoxy)benzoicacid in an excess of thionyl chloride was stirred a t rt for 30 min. The excess of thionyl chloride was then removed under pressure. The resulting Registry No. 1, 121225-79-4;2, 121225-81-8;2 (homopoly4-(4-bromobutoxy)benzoylchloride was used in the next step. mer), 121225-82-9;3,128653-08-7;3 (homopolymer), 128653-09Bis(docosa-10,12-diynyl) N-[(4-Bromobutoxy)benzoyl]8; 4,121225-83-0;4 (homopolymer), 121225-84-1;5,121239-55-2; L-glutamate (12). To a solution of 0.15 g (0.20 mmole) of 5 in 6,15949-84-5; 7,131759-83-6; 8,127232-94-4; 9,124064-22-8; 10, 20 mL of dry chloroform was added 4-(4-bromobutoxy)benzoyl 88931-96-8;11,88185-43-7;12,131759-84-7;Et3N, 121-44-8;MesN, chloride (11) and 0.0313 g (0.25 mmol) of N-(dimethylamino)75-50-3;methyl 4-hydroxybenzoate, 99-76-3;1,4-dibromobutane, pyridine. The reaction mixture was stirred at rt for 30min. After 110-52-1. the reaction, the mixture was extracted with dilute HCl and 10A
