Amino Acid Terminated Polydiacetylene Lipid Microstructures

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Amino Acid Terminated Polydiacetylene Lipid Microstructures: Morphology and Chromatic Transition Quan Cheng,*,† Maki Yamamoto, and Raymond C. Stevens*,‡ Center for Advanced Materials, Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 Received December 9, 1999. In Final Form: March 17, 2000 Novel lipid microstructures with various morphologies have been synthesized using a series of amino acid terminated diacetylenic lipids, and the chromatic transitions of the polymerized microstructures have been characterized by UV-vis and FTIR spectroscopies. The observed morphologies for the microstructures include tubule, helix, ribbon, sheet, braided fiber, and planar platelet, formation of which has shown strong dependency on the chemical structure of the headgroup. All amino acid lipids studied in this work form microstructures that allow polymerization by UV irradiation to yield a blue appearance. Molecular chirality, electrostatic interactions, and hydrogen-bonding interactions in the headgroup region determine the formation of microstructures with twisted features, while nonchiral molecules do not form curved structures. The polymerized lipid microstructures exhibit similar colorimetric properties as observed for related bilayer vesicles, undergoing a blue-to-red color transition in response to thermal and pH changes. Microstructures with hydrophobic headgroup lipids are more sensitive to pH change than those with hydrophilic headgroups. For hydrophilic headgroup lipids, microstructures are more pH-resistant relative to their vesicle counterparts. FTIR studies suggest that thermal and pH-induced chromism of PDA microstructures proceed by different pathways. A mechanism is proposed that links function and change of hydrogen-bonding interactions to the observed chromatic behaviors of the PDA microstructures. In thermochromism, hydrogen-bonding interactions lock in the lipid headgroups so that the temperature-induced gauche-trans conformational transition of the side chains imposes strain on the assembly. In pH-induced chromism, surface ionization and breakdown of hydrogen-bonding interactions lead to reorganization of the headgroups that affects the electron delocalization along the conjugated backbone.

Introduction Supramolecular polydiacetylene (PDA) assemblies (Langmuir-Blodgett (LB) films and bilayer vesicles) have been an attractive platform for developing colorimetric biosensors for pathogenic agents.1-5 The characteristic blue-to-red chromatic transition of PDA assemblies allows for direct detection of biological targets in a “litmus test” fashion. The color change observed in these sensors is different from chromatic transitions caused by mechanical stress,6,7 organic solvent treatment,8 or excess UV irradiation and relies explicitly on biological interactions intentionally defined at the sensing interface. The biointeraction-based chromatic transition, or “biochromism”, requires incorporation of molecular recognition units into the PDA assemblies, either in a covalent1,2 or noncovalent3,4 manner. Target binding to the surface-bound receptor causes changes in electron delocalization along the conjugated polymer backbone, which lead to the color change. Colorimetric sensors using PDA assemblies for cholera toxin,3,4 Escherichia coli enterotoxin,3 influenza virus,1,2 and E. coli bacteria9 have been reported. * To whom correspondence should be addressed. † Phone: (510) 486-4125. Fax: (510) 486-4995. Email: Quan•[email protected]. ‡ Email: [email protected]. Phone: (858) 784-9416. (1) Charych, D. H.; Nagy, J. O.; Spevak, W.; Bednarski, M. D. Science 1993, 261, 585. (2) Reichert, A.; Nagy, J. O.; Spevak, W.; Charych, D. H. J. Am. Chem. Soc. 1995, 117, 829. (3) Charych, D. H.; Cheng, Q.; Reichert, A.; Kuziemko, G.; Stroh, M.; Nagy, J. O.; Spevak, W.; Stevens, R. C. Chem. Biol. 1996, 3, 113. (4) Pan, J. J.; Charych, D. Langmuir 1997, 13, 1365. (5) Cheng, Q.; Stevens, R. C. Adv. Mater. 1997, 9, 481. (6) Tomioka, Y.; Tanaka, N.; Imazeki, S. J. Chem. Phys. 1989, 91, 5694. (7) Nallicheri, R. A.; Rubner, M. F. Macromolecules 1991, 24, 517. (8) Chance, R. R. Macromolecules 1980, 13, 396.

The colorimetric PDA sensors were constructed by selfassembly of diacetylene lipids to form bilayer vesicles or LB films. However, it has been shown that certain diacetylene lipids also form nonspheroidal, nonlamellar assemblies upon hydration.10-20 The most studied lipid is double-chained glycerol-based phosphatidylcholine (DC8,9PC), which forms hollow tubules with a cylindrical morphology.10,11,15,17-20 Microstructure formation typically involves two methods: (1) cooling the multilamellar vesicles (MLVs) through the LR-Lc transition point, which results in mesoscopic structural changes from fluid-layer spherical MLVs in the high-temperature phase to crystalline-layer microstructures in the low-temperature phase, or (2) adding water to a methanol solution of the lipid to force precipitation of the supramolecular assemblies. The latter method has proven to be a convenient way of making long, stable tubules with typical diameters in the sub(9) Ma, Z.; Li, J.; Liu, M.; Cao, J.; Zou, Z.; Tu, J.; Jiang, L. J. Am. Chem. Soc. 1998, 120, 12678. (10) Yager, P.; Schoen, P. E. Mol. Cryst. Liq. Cryst. 1984, 106, 371. (11) Georger, J. H.; Singh, A.; Price, R. R.; Schnur, J. M.; Yager, P.; Schoen, P. E. J. Am. Chem. Soc. 1987, 109, 6169. (12) Frankel, D. A.; O’Brien, D. F. J. Am. Chem. Soc. 1991, 113, 7436. (13) Fuhrhop, J.-H.; Blumtritt, P.; Lehmann, C.; Luger, P. J. Am. Chem. Soc. 1991, 113, 7437. (14) Ihara, H.; Takafuji, M.; Hirayama, C.; O’Brien, D. F. Langmuir 1992, 8, 1548. (15) Schnur, J. M. Science 1993, 262, 1669. (16) Frankel, D. A.; O’Brien, D. F. J. Am. Chem. Soc. 1994, 116, 10057. (17) Schnur, J. M.; Ratna, B. R.; Selinger, J. V.; Singh, A.; Jyothi, G.; Easwaran, K. R. K. Science 1994, 264, 945. (18) Thomas, B. N.; Safinya, C. R.; Plano, R. J.; Clark, N. A. Science 1995, 267, 1635. (19) Thomas, B. N.; Corcoran, R. C.; Cotant, C. L.; Lindemann, C. M.; Kirsch, J. E.; Persichini, P. J. J. Am. Chem. Soc. 1888, 10, 12178. (20) Spector, M. S.; Sellinger, J. V.; Singh, A.; Rodriguez, J. M.; Price, R. R.; Schnur, J. M. Langmuir 1998, 14, 3493.

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micrometer range.21 Although the mechanism of microstructure formation remains to be fully elucidated, many observations suggest that headgroup conformation (chirality) and hydrogen-bonding interactions play important roles.15,16,22,23 Lipid microstructures exhibit high mechanical stability and additionally have both unique morphology and chemical properties. Applications in filtration, purification, and medical encapsulation have been suggested.15 Some microstructures have demonstrated remarkable enhancement of optical activity and unique dye-binding behavior.24 Unfortunately, little attention has been given to the use of these assemblies as a direct sensing matrix to exploit the blue-to-red colorimetric property of the polydiacetylenes. However, before the chromism of PDA microstructures can be used for sensing purposes, one has to optimize and improve the color development of these microstructures, as they appear to have different properties relative to their bilayer counterparts. Optimization of headgroup functionalization is necessary, as the headgroup has been found to have a strong impact on photopolymerization. For instance, galactonamide- and mannonamide-terminated diacetylenes rapidly produced blue PDAs at high conversion.16 In contrast, polymerization of phosphatidylcholine-terminated DC8,9PC gives red PDAs in relatively low yield. It is worth noting that the galactonamide and mannonamide lipids contain only one acyl chain, while DC8,9PC is a diacyl lipid. Position of the diacetylene group in the lipids is important as well. For D-gluconamides, only lipids with one methylene group between the diacetylene and the amide group polymerize readily.13 Diacetylenic microstructures are advantageous as sensing materials since the topochemical polymerization reaction allows for examination of molecular ordering in the assemblies. In addition, polymerization enhances the stability of these materials, broadening their utility under demanding conditions. In this paper, we report the synthesis of a series of novel PDA microstructures and the characterization of their morphology and chromatic transition using TEM and UVvis and FTIR spectroscopies. The microstructures are formed by single-chained 10,12-pentacosadiynoic acid lipid derivatized with different amino acids (Figure 1). Amino acids were chosen as headgroups to create a compatible surface on the microstructures for protein-related applications. In addition, amino acids are chiral and possess various charges, allowing surface charge and hydrophilicity to be manipulated in a controllable manner. This work is the continuation of a previous study on PDA chromism where the focus was on optical properties of bilayer vesicles formed with these amino acid terminated PDA lipids.25 FTIR is employed to provide molecular information on the chemical interactions occurring on the PDA surface during the chromatic transitions. Comparison between the thermochromic and pH-induced (i.e., surface charge) transitions for both the microstructures and corresponding bilayer vesicles is made, in an attempt to elucidate the mechanism of pH-induced chromism in the microstructures. Additionally, the effects of headgroup structure and hydrophilicity on the morphology and chromatic transitions of the PDA microstructures are discussed. (21) Ratna, B. R.; Baral-Tosh, S.; Kahn, B.; Schnur, J. M.; Rudolph, A. S. Chem. Phys. Lipids 1992, 63, 47. (22) Fuhrhop, J.-H.; Schnieder, P.; Rosenberg, J.; Boekema, E. J. Am. Chem. Soc. 1987, 109, 3387. (23) Fuhrhop, J.-H.; Boettcher, C. J. Am. Chem. Soc. 1990, 112, 1768. (24) Ihara, H.; Hachisako, H.; Hirayama, C.; Yamada, K. Liq. Cryst. 1987, 2, 215. (25) Cheng, Q.; Stevens, R. C. Langmuir 1998, 14, 1974.

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Figure 1. Molecular structure of the amino acid terminated lipids from 10,12-pentacosadiynoic acid (PDA). The amineterminated DMAP lipid was synthesized for comparison experiments.

Experimental Section Lipid Synthesis. Synthesis of the amino acid derivatized diacetylene lipids is described elsewhere.25,26 All the amino acids used in this study are L-configuration. In short, 10,12-pentacosadiynoic acid (PDA) (Farchan Lab, Gainesville, FL) was converted to a succinimidyl ester in the presence of N-hydroxysuccinimide (NHS) and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (DEC) (Aldrich). The ester was reacted with the selected amino acid in a slightly basic THF/water solution for 2 h. After acidification with 1 M HCl, the mixture was extracted with chloroform. The organic phase was dried with MgSO4 and rotoevaporated to yield a white powder. The product was purified on a silica gel column and characterized with NMR spectroscopy; specific products and their characterization follow. L-N-(10,12-Pentacosadiynoyl)glutamic acid (Glu-PDA). 1H NMR (500 MHz, CDCl3 with trace CD3OD): δ 6.61 (1H, d, J ) 7.5 Hz), 4.55 (1H, dd, J ) 8, 5.5 Hz), 2.43 (3H, m), 2.20 (6H, t, J ) 7.5 Hz), 1.99 (1H, m), 1.58 (2H, m), 1.47 (4H, m), 1.33 (m), 1.22 (m), 0.84 (3H, t, J ) 7 Hz). L-N-(10,12-Pentacosadiynoyl)glutamine (Gln-PDA). 1H NMR (500 MHz, CDCl3 with trace CD3OD): δ 4.47 (1H, dd, J ) 8.5, 5.5 Hz), 2.23 (2H, m), 2.18 (6H, m), 2.10 (1H, m), 1.92 (1H, m), 1.56 (2H, m), 1.43 (4H, m), 1.31 (m), 1.19 (m), 0.82 (3H, t, J ) 7 Hz). L-N-(10,12-Pentacosadiynoyl)isoleucine (Ile-PDA). 1H NMR (500 MHz, CDCl3): δ 6.05 (1H, d, J ) 8.5 Hz), 4.63 (1H, dd, J ) 8.5, 5 Hz), 2.25 (8H, m), 1.97 (1H, m), 1.65 (2H, m), 1.52 (4H, m), 1.38 (m), 1.27 (m), 0.97 (3H, d, J ) 2.5 Hz), 0.96 (3H, d, J ) 2.5 Hz), 0.89 (3H, t, J ) 7 Hz). L-N-(10,12-Pentacosadiynoyl)phenylalanine (Phe-PDA). 1H NMR (500 MHz, CDCl3): δ 7.15 (5H, m), 4.74 (1H, m), 3.13 (1H, dd, J ) 15, 5 Hz), 3.00 (1H, m), 2.15 (4H, t, J ) 7.5 Hz), 2.07 (2H, t, J ) 7.5 Hz), 1.43 (6H, m), 1.29 (m), 1.18 (m), 0.80 (3H, t, J ) 5 Hz). L-N-(10,12-Pentacosadiynoyl)methionine (Met-PDA). 1H NMR (500 MHz, CDCl3): δ 4.62 (1H, m), 2.54 (3H, m), 2.22 (6H, t, J ) 7 Hz), 2.08 (3H, s), 2.01 (1H, m), 1.60 (2H, m), 1.49 (4H, m), 1.35 (m), 1.24 (m), 0.86 (3H, t, J ) 7 Hz). (26) Spevak, W. Ph.D. Thesis, University of California at Berkeley, 1993.

Amino Acid Terminated PDA Lipid Microstructures L-N-(10,12-Pentacosadiynoyl)serine (Ser-PDA). 1H NMR (500 MHz, CDCl3 with trace CD3OD): δ 4.42 (1H, t, J ) 3.5 Hz), 3.86 (1H, b), 3.84 (1H, dd, J ) 11.5, 3.5 Hz), 3.70 (1H, dd, J ) 11.5, 3.5 Hz), 2.13 (2H, t, J ) 7 Hz), 2.11 (4H, t, J ) 7 Hz), 1.51 (2H, m), 1.38 (4H, m), 1.24 (m), 1.13 (m), 0.75 (3H, t, J ) 7 Hz). L-N-(10,12-Pentacosadiynoyl)histidine (His-PDA). 1H NMR (500 MHz, CDCl3): δ 8.51 (1H, s), 7.10 (1H, s), 4.62 (1H, m), 3.18 (1H, m), 3.13 (1H, m), 2.19 (2H, t, J ) 7.5 Hz), 2.14 (4H, t, J ) 7 Hz), 1.50 (2H, m), 1.41 (4H, m), 1.29 (m), 1.16 (m), 0.78 (3H, t, J ) 7 Hz). L-N-(10,12-Pentacosadiynoyl)alanine (Ala-PDA). 1H NMR (500 MHz, CDCl3): δ 7.15 (5H, m), 4.74 (1H, m), 3.13 (1H,dd, J ) 15, 5 Hz), 3.00 (1H, m), 2.15 (4H, t, J ) 7.5 Hz), 2.07 (2H, t, J ) 7.5 Hz), 1.43 (6H, m), 1.29 (m), 1.18 (m), 0.80 (3H, t, J ) 5 Hz). N-(10,12-Pentacosadiynoyl)glycine (Gly-PDA). 1H NMR (500 MHz, CDCl3): δ 3.96 (2H, s), 2.19 (6H, t, J ) 7.5 Hz), 1.59 (2H, m), 1.47 (4H, m), 1.32 (m), 1.21 (m), 0.84 (3H, t, J ) 7 Hz). The amine-terminated lipid for the comparison experiment was synthesized by a slightly different method. The ester (NHSPDA) was redissolved in chloroform and added dropwise to a chloroform solution containing 5-fold excess 3-(dimethylamino)propylamine (DMAP). The resulting solution was allowed to react for 2 h and then rotoevaporated and purified on a silica gel column eluting with 4:1 CHCl3:MeOH. 1H NMR (500 MHz, CDCl3): δ 3.98 (2H, m), 3.10 (2H, t, J ) 6.5 Hz), 2.89 (2H, t, J ) 7 Hz), 2.66 (6H, s), 1.79 (2H, m), 1.69 (4H, m), 1.42 (2H, m), 1.32 (4H, m), 1.84 (m), 1.07 (m), 0.69 (3H, t, J ) 7 Hz). Mass spectrum (EI+) 458.7 (M+). Lipid Microstructures and Vesicles. A chloroform solution containing 5 mg of the appropriate lipid was rotoevaporated to dryness in a 20 mL vial. The lipid was then dissolved in 5 mL of methanol. The methanol solution was then warmed in a 60 °C water bath. With vigorous stirring, 10 mL of deionized water prewarmed to 60 °C was added dropwise to the methanol solution. The solution was allowed to cool to room temperature and subsequently stored at 4 °C overnight. Prior to colorimetric characterization, the sample was dialyzed against water using Spectra/Por (Spectrum Lab, Laguna Hills, CA) membrane tubing (12k m.w. cutoff) to remove residual methanol. An alternate method, which is widely used in preparation of diacetylenic aldonamide microstructures,16 is to heat the lipid in water to the boiling point and then allow the solution to cool to room temperature. We have compared the efficiency of these two methods for forming microstructures. The boiling water method yielded similar results for lipids with hydrophilic amino acid headgroups. However, it failed to generate microstructures using lipids with hydrophobic headgroups (i.e., Phe-, Ile-, and Trpterminated diacetylenic lipids). Therefore, all the microstructures reported here were obtained using the methanol/water method. Lipid bilayer vesicles were obtained by hydration of the diacetylenic lipids using probe sonication. Suspensions containing the appropriate lipid and deionized water (typically 0.6 mg lipid/ mL) were sonicated for 30 min with a 40 W probe sonicator. The resulting clear solution was immediately filtered through a syringe filter (0.8 µm), allowed to cool to room temperature, and stored at 4 °C. Rapid cooling of the hot solution generally resulted in formation of large vesicles that converted to microstructures (precipitation). Photopolymerization of diacetylene microstructures and bilayer vesicles was achieved using a UV cross-linker (254 nm). Samples were pipetted into a 96-well microplate (200 µL each well) and irradiated at 0.3 J/cm2 for 1 min to yield a blue color. Transmission Electron Microscopy. TEM images of the microstructures and bilayer vesicles were obtained using a Zeiss electron microscope operating at 80 kV. Owing to the high electron density of PDAs, the microstructures can be directly viewed on the Cu grid without the need for staining. For bilayer vesicles, uranyl acetate was used in sample staining to provide better imaging quality. UV-Vis and FTIR Spectroscopies. Quantification of the colorimetric transition for both microstructures and bilayer vesicles was obtained using a Shimadzu UV-1601 spectrometer. FTIR spectra were obtained using a Perkin-Elmer System 2000 FTIR spectrometer. Samples were prepared by depositing a thin film of the microstructures onto a 0.27 mm thick silicon wafer.

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Results and Discussion Lipid microstructures. The amino acid headgroups of the diacetylene lipids studied in this work provide the requisite chirality, hydrophilicity, and hydrogen-bonding interactions for the formation of various microstructures. The linkage of the headgroups to the diacetylenic chains is via an amide bond (Figure 1). Previous research postulated that the type of bond between the headgroup and lipid side chain might determine the type of structure formed. For example, Ihara et al. observed that esterlinked lipids tended to form vesicles, whereas lipids with amide bonds were more likely to form helical aggregates.14 We, however, have shown that both vesicles25 and helical microstructures can be formed by the same amide-linked amino acid diacetylenic lipids. It appears that the chemical structure of the headgroup and the specific preparation protocol used, rather than the type of bond between the lipid side chain and headgroup, play more important roles in determining the morphologies of the microstructures formed. Figure 2 shows the TEM images of microstructures made using Glu-PDA and Gln-PDA lipids. For Glu-PDA, after cooling at 4 °C overnight, the methanol/water solution becomes turbid and gradually aggregates to form a gel. The gel consists of fibers and ribbons, twisted and untwisted, with lengths varying from several to hundreds of micrometers. Branching is common, and occasionally a knot can be found that could be either a starting nucleation site or a twisting joint (Figure 2A). Unlike other microstructures, the cooling rate does not appear to significantly affect the formation of Glu-PDA assemblies. Under UV irradiation, Glu-PDA microstructures readily polymerize to give a dark blue color. Gln-PDA lipid forms similar ribbon-shaped microstructures (Figure 2B). However, the assemblies are more uniform in width and length than observed for Glu-PDA. The typical width of the ribbons is ca. 150 nm, and the thickness was estimated to be around 8 nm, corresponding to roughly four layers of lipid. Some of the ribbons were found to twist to helical structures, similar to those found with Glu-PDA (Figure 2C). Since the Gln-PDA ribbons were more uniform in shape, attempts were made to investigate the possible twisting mechanism that generated these assemblies. Interestingly, the twisting process appears to begin with formation of some knots, as if the ribbons were pinched (Figure 2C). The knots occur periodically, seemingly pointing to an early stage of helical formation. The “matured” helixes found in these TEM images, on the other hand, have much smaller distance between “knots”. The results seem to suggest that helix growth could start with formation of periodical knots, with long separations along the ribbons. The subsequent division of the segment by additional knots stimulates further twisting of the ribbons, resulting in a helical appearance. Figure 3 shows the TEM images for His-, Gly-, Phe-, and DMAP-PDA assemblies. For His-PDA, well-defined helical assemblies were readily obtained (Figure 3A). The average diameter of the helix is 60 nm, while the length can extend to several micrometers. Branching is again quite visible, and the helix has a right-handed twist. In addition to helixes, a large number of planar platelets were present. For Phe-PDA, where the headgroup is hydrophobic and bulky, only planar platelets were obtained, even though the headgroup is chiral (Figure 3B). To further explore the effect of headgroup chirality on the morphology of the microstructures formed, we synthesized structurally similar compounds that did not contain chiral

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Figure 2. TEM images of Glu-PDA and Gln-PDA microstructures. (A) Glu-PDA; (B) and (C) Gln-PDA. The bar is 0.6 µm.

center(s), for a comparison experiment. The amino acid terminated Gly-PDA lipid produces sheets and platelets, with no observed twisting or curvature (Figure 3C). For the amine-terminated, nonchiral lipid DMAP-PDA, once again only sheets and platelets were observed (Figure 3D). Apparently, nonchiral headgroups tend to produce microstructures without twisted features. However, it remains to be elucidated why the chiral Phe-PDA lipids fail to produce microstructures other than sheets and platelets. Figure 4 shows the microstructures of various morphologies made with Ser-, Met-, and Ile-PDA lipids. Similar to the phosphatidylcholine-terminated DC8,9PC, Ser-PDA lipids form open-ended tubular assemblies (Figure 4A). The diameter of tubules is around 0.2 µm. TEM studies indicate that a significant amount of sheets is present in the sample, suggesting that the formation of Ser-PDA tubules is through a rolling-up mechanism.27 This argument is further supported by the observation of the layers at the open end of the tubules wrapped around a central aqueous core. The entrapment of aqueous solution was visible for some of the tubules. By estimation, the tubular wall thickness is about 20 nm, corresponding to roughly 10 layers or 5 bilayers of the lipid. Met-PDA lipids form mostly flat, ribbonlike structures, even though the lipid headgroup is chiral (Figure 4B). However, as compared to Gln-PDA, where ribbons are much more uniformly formed, the Met-PDA ribbons vary significantly in terms of width and length. It is interesting that we were able to observe an unusual twisted structure that connects to normal, flat ribbons. Given its enormous dimension (0.4 (27) Fuhrhop, J.-H.; Schnieder, P.; Boekema, E.; Helfrich, W. J. Am. Chem. Soc. 1988, 110, 2861.

µm in diameter), the twisting may involve multiple ribbons interacting in a coherent fashion. Ile-PDA lipids form unique braided fibers that connect to each other in a giant network (Figure 4C,D). The diameter of the individual fiber (helix) is 65 nm, similar to that observed for His-PDA. Networking microstructures were previously observed in some special N-diacetylenic gluconamide lipids13 and doped DC8,9PC.28 It is worth noting that Ile-PDA is the only amino acid terminated lipid studied here that has two chiral centers. From Figure 4, it appears that the conversion to twisted fibers is rather complete in Ile-PDA, as even the smallest pieces of assemblies occur as twisted fragments (Figure 4D). The relationship between lipid molecular structure and the preferential morphology of the resulting supramolecular assemblies is poorly understood. However, extensive experimental studies have indicated that headgroup conformation and hydrogen-bonding interactions are among the most important controlling factors. Hydrogen bonding in aldonamides has been suggested to control the packing of those lipids in extended “bilayer” sheets, which grow to a point where the balance of force (dipolar, chiral bilayer packing, and edge effects) causes the curvature of the assembly to change.16 Spontaneous torsion of the edges during formation of helicity and the twisting or rolling-up mechanism of planar bilayer sheets from diastereomeric and enantiomeric lipids have been mentioned.23,27 Applying these “rules” to the amino acid terminated diacetylene lipids to account for the variety of morphologies observed in the microstructures is difficult. There seems no clear correlation between the tubules of Ser-PDA, the (28) Svenson, S.; Messersmith, P. B. Langmuir 1999, 15, 4464.

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Figure 3. TEM images of (A) His-PDA, (B) Phe-PDA, (C) Gly-PDA, and (D) DMAP-PDA microstructures. The bar is 0.8 µm.

ribbons of Glu-PDA, and the braided fibers of Ile-PDA. In addition, electrostatic interactions should play an important role in the microstructure growth for these charged headgroup lipids. The different degree of hydration of the different amino acids in aqueous phase could be another controlling factor, since large, hydrophilic headgroup size can cause curvature, as observed by Fuhrhop and coworkers.23 For all the microstructures studied here, extensive hydrogen-bonding interactions exist, from a predicted high extent for Glu-PDA and Gln-PDA to a low extent for Phe-PDA, where the bulky aromatic ring may effectively prevent intermolecular hydrogen bonding. Interestingly, Phe-PDA is the only chiral amino acid terminated lipid that exclusively forms platelets. These results suggest that aside from a balance of dipolar forces involved in bilayer curvature,16 a balance of headgroup size, and possibly hydrophilicity, to maintain an appropriate degree of interactions (bonding) is critical to inducing structural curvature. Colorimetric Properties of PDA Microstructures. As discussed earlier, supramolecular assemblies of polydiacetylenes have unique chemical and optical properties. The polymerization, known as a topochemical process, requires optimal packing of the diacetylenic segments to allow propagation of the conjugated backbone across the assembly.29 The polymer absorbs light strongly at around 650 nm, giving the material a blue appearance. Upon perturbations such as heat, organic solvent, and mechanical stress, the backbone is distorted, leading to chromic transition from blue to red color.29 Considerable studies (29) See, for example: Polydiacetylenes. Advances in Polymer Science; Bloor, D., Chance, R. R., Eds.; Martinus Nijhoff, Boston, MA, 1985; Vol. 63.

have been recently reported on chromism of PDA bilayer vesicles and LB thin films.30 Microstructures formed by diacetylene lipids possess intrinsic features as organized assemblies that allow topochemical reactions and polymerization to take place. It is worth noting that all the amino acid terminated diacetylene microstructures studied here are polymerizable to form blue PDAs. Only hydrophilic amino acid terminated lipids can readily form bilayer vesicles and allow polymerization, whereas hydrophobic amino acid terminated lipids do not form vesicles.25 It was observed that the intensity of the initial blue color varies with the specific amino acid headgroup present. Hydrophilic amino acids produce the darkest blue appearance, while hydrophobic amino acids (Ile- and Phe-) have a barely noticeable blue appearance. Assemblies with ribbon morphology show the highest color intensity, followed by (in decreasing order) sheets, helixes, tubules, planar platelets, and braids. The colorimetric properties of PDA microstructures were investigated, with an emphasis on thermochromism and solution pH-induced chromism. Though the latter is more relevant to sensor research, thermochromism, which has received considerable attention in the past, can provide molecular insight into the factors that influence the chromatic transition, and more importantly, can provide a useful comparison to the far less studied pH-induced chromism. Figure 5A shows the UV spectra of polymeric His-PDA microstructures at different pH values. Similar to what was observed for amino acid terminated PDA vesicles,25 a change in solution pH triggers the chromatic (30) Okada, S.; Peng, S.; Spevak, W.; Charych, D. Acc. Chem. Res. 1998, 31, 229 and references therein.

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Figure 4. TEM images of microstructures made from (A) Ser-PDA, (B) Met-PDA, (C) and (D) Ile-PDA. The bar on the left side is the scale for the two images on the left column and is 2 µm. The bar on the right side for the Ile-PDA images is 0.5 µm.

transition in PDA microstructures. For the blue form of the microstructure, the maximum absorbance occurs at 646 nm (excitonic band) with a shoulder peak at 592 nm (vibronic absorption). The maximum absorbance varies slightly ((10 nm) for the amino acid terminated microstructures with different headgroups. When the His-PDA microstructure is treated with 0.1 M NaOH, the maximum absorbance shifts to 542 nm, while the vibronic shoulder shifts to 502 nm. The color change is irreversible, similar to what was observed for His-PDA vesicles.25 It is worth noting that the maximal absorbance peaks for both the blue and red forms of His-PDA are identical for both the microstructure and liposome forms, regardless of the method used to trigger the color change (thermally or by a change in pH). A quantitative analysis of the observed chromatic transitions was conducted by analyzing the colorimetric response (CR) as a function of solution pH.3 The CR is defined as the percent change in the maximum adsorption at 646 nm (blue color) with respect to the total absorption at both 542 nm (red color) and 646 nm. Figure 5B shows the CR vs pH for various amino acid terminated PDA microstructures. Sigmoidal curves were obtained for all the amino acid PDA microstructures studied, indicating a blue to red transition with raised pH. However, the pH required for the transition to occur varies, dependent on the specific amino acid headgroups present in the lipid microstructure. This difference can be analyzed by the CR50 values, which are defined as the pH values required to achieve 50% of the maximal color transition.25 The CR50 values for the hydrophilic amino acids (Glu, Gln and His) all fall between pH 10 and 11. The values for Ala and Phe, on the other hand, are much lower (pH 7.1 and 8.6,

respectively). Finally, the amine-terminated DMAP-PDA shows an inverse-sigmoidal response to pH as expected (CR50 ) 4.8). The pH-induced colorimetric transitions for the PDA microstructures show quite different properties compared to the respective bilayer vesicles. For the vesicles, GluPDA was found to be the most base-sensitive; the CR50 for Glu-PDA was observed to be pH 6.3, followed by 8.1 for His-PDA and 9.0 for Gln-PDA.25 The microstructure forms of these lipids, on the other hand, have a close transition range; the CR50 values range from pH 10.4 to 10.7, which is only a variance of 0.3 pH units. The high CR50 values suggest that the microstructures of these lipids are more stable to pH changes relative to their vesicle counterparts. The chromatic transitions do not occur until the materials reach a point where extensive ionization of the microstructure surface significantly alters the lipid packing and triggers the color change. The steep slope observed for these transitions (less than 1.5 pH units change for the Glu-, His-, and Gln-PDA lipids) further supports this argument. In comparison, the colorimetric transition of the Gln-PDA vesicles spans over 6 pH units.25 The colorimetric transition of the amino acid terminated PDA microstructures can also be achieved by thermal treatment (thermochromism). Figure 6A shows the CR of the various lipid microstructures as a function of temperature. Similar sigmoidal curves were obtained for all the lipids, except for Gln-PDA. The temperature required for a 50% CR change (CR50T) varies with the specific headgroup present in the PDA microstructures. The Alaand Phe-terminated lipids are very sensitive to changes in temperature, showing more than 10% CR even at 30 °C. The CR50T values for the hydrophobic Ala- and Phe-

Amino Acid Terminated PDA Lipid Microstructures

Figure 5. Colorimetric response of amino acid terminated polydiacetylene microstructures: (A) UV spectra of His-PDA microstructures in water at 20 °C; (B) colorimetric response (CR) for the polymerized microstructures as a function of solution pH.

lipids are lower (both having values of 45 °C) than the hydrophilic Glu- (49 °C), Gly- (57 °C), and His-PDA lipids (58 °C). Apparently, the Gln-PDA microstructure is the most heat-resistant, requiring the temperature to be as high as 71 °C for the microstructure to convert 50% of its original color. Figure 6B shows the thermochromic CR for the bilayer vesicles containing hydrophilic amino acid terminated lipids. Different from microstructure assemblies, the GlnPDA vesicles now become the most thermally sensitive, while the Glu-PDA vesicles are observed to be the least. The CR50T for Glu-PDA vesicles is 61 °C, much higher than that observed for the corresponding microstructure assembly (49 °C). It is not immediately clear why the observed thermochromism for the vesicles behaves inversely compared to the microstructures of the same lipids. One possible explanation is that the microstructure assemblies and vesicles interact with the aqueous phase in a different fashion, with vesicles having higher membrane fluidity. Glu-PDA vesicles have two COOH groups that can form strongly interacting hydrogen bonds and thus contribute to a higher thermostability while GlnPDA has only one COOH group and one amide group. In the Glu-PDA microstructure, on the other hand, its higher charge density on the more close-packed, rigid, and crystalline-like microstructure surface likely leads to a lower transition temperature.

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Figure 6. Thermochromic behavior of amino acid terminated polydiacetylene assemblies: (A) microstructures; (B) bilayer vesicles.

FTIR Studies on Chromatic Transition of PDA Microstructures. UV-vis spectroscopy and transmission electron microscopy have been used to characterize PDA microstructures, providing information on their morphology and optical properties. However, these methods are confined to revealing the average properties of the materials and are marginally related to submicrometer scale observations. To study the structural changes and headgroup interactions of PDA microstructures during chromatic transitions, we have employed FTIR spectroscopy to investigate the conformational order of the hydrophobic chains, as well as to probe the changes in molecular packing and bonding interactions of the lipid headgroups. Infrared spectroscopy has proven to be an excellent tool for studying thin organic films,31 and various modes of measurement, including reflection-absorption, transmission, and attenuated total reflection have been utilized.32-34 Transmission FTIR is particularly attractive to us since it is suitable for the characterization of (31) Nuzzo, R. G.; Dubois, L. H.; Allara, D. L. J. Am. Chem. Soc. 1990, 112, 558. (32) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559. (33) Ahn, D. J.; Franses, E. I. J. Phys. Chem. 1992, 96, 9952. (34) Smith, E. L.; Porter, M. D. J. Phys. Chem. 1993, 97, 8032.

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Figure 7. FTIR spectra of polymerized microstructures made with Gln-PDA (upper) and Phe-PDA (lower) before and after chromatic transition: (a) original blue form, (b) thermochromic red form, and (c) pH-induced red form.

microstructure assemblies. The PDA microstructures are formed by self-assembly of multiple lipid bilayers. In comparison to LB thin films studied with FTIR, PDA microstructures can provide higher absorption signals for the target functional groups. The relatively large surface area of the samples also allows probing of the surface interactions and hydrogen bonding in the headgroup region. Figure 7 shows the FTIR spectra of polymeric Gln-PDA and Phe-PDA microstructures in their original blue forms, thermochromic red forms (generated by heating at 110 °C for 20 min), and pH-induced red forms (30 s after treatment with 0.1 M NaOH). The choice of the two lipids was aimed at understanding the role of headgroup hydrophilicity (with Gln-PDA being hydrophilic and Phe-PDA being hydrophobic) in causing the observed chromatic transition. In addition, Gln-PDA forms extensive intermolecular hydrogen-bonded structures that are not possible with Phe-PDA. From Figure 7, Gln-PDA shows strong absorption bands at 2920 and 2850 cm-1, which can be attributed to asymmetric νas(CH2) and symmetric νs(CH2) stretching vibrations, respectively. Both red forms, thermochromic

and pH-induced, show the absorption bands at the same wavenumbers. The intensities are reduced relative to the blue form, with the signal for the thermochromic red form decreased by roughly 30% and a smaller decrease observed for the pH-induced red form (19%). The νas(CH2) vibration is known to be conformationsensitive. Changes in frequency and intensity can be used to characterize film ordering and preferential molecular packing.35 Shifts to lower frequencies for the νas(CH2) vibration are indicative of highly ordered conformations, with preferential all-trans configurations.35 Similar conclusions have been drawn for Langmuir monolayers that were studied at different surface pressures using IRRAS.36-38 As for band intensity, Charych and co-workers have previously reported that the intensity of the asym(35) Mendelsohn, R.; Brauner, J. W.; Gericke, A. Annu. Rev. Phys. Chem. 1995, 46, 305. (36) Neumann, V.; Gericke, A.; Huhnerfuss, H. Langmuir 1995, 11, 2206. (37) Huhnerfuss, H.; Neumann, V.; Stine, K. J. Langmuir 1996, 12, 2561. (38) Hoffmann, F.; Huhnerfuss, H.; Stine, K. J. Langmuir 1998, 14, 4525.

Amino Acid Terminated PDA Lipid Microstructures

metric νas(CH2) stretching vibration increases by 14% when PDA monolayers were heated to 70 °C. The authors ascribed this observation to the rotation of a methylene C-C bond pendant to the ene-yne backbone, rendering movement of the C-C-C plane for the pendant alkyl side chain toward the surface normal.39 Apparently, the FTIR results for the PDA microstructures reported here are at variance with these previous observations for the PDA monolayers. We attribute this difference to the random surface orientation of the microstructure assemblies. In microstructures, the impact of such a methylene C-C bond rotation, if any, appears to be trivial. The observed shoulder peak at 2949 cm-1, which disappears in monolayer thermochromic transitions,40 is clearly visible for all three microstructure forms. TEM studies indicate that the integrity of the microstructures remains unchanged in thermochromic or pH-induced transitions, suggesting that the observed microstructure chromism has originated from conformational changes rather than disruption of the assemblies. The weak, broad band observed at 3206 cm-1 can be assigned to the N-H stretching vibration of the amide where the amide proton is hydrogen-bonded to neighboring carbonyl oxygen atoms.41,42 This amide N-H vibration was found to be unaffected by thermal treatment, but to become significantly broadened and shifted toward higher frequency when the pH was raised. More dramatic changes were observed in the region between 1900 and 1300 cm-1. Two strong bands found at 1694 and 1675 cm-1 are assigned to amide carbonyl vibrations (νs(CdO)). The high wavenumber values for these vibrations indicate strong headgroup hydrogen bonding between three carbonyl groups and four different hydrogen atoms.43 The two bands decrease in intensity during thermochromic transitions, but the frequencies remain unchanged. In contrast, during the pH-induced chromatic transitions, these carbonyl vibrational bands shrink significantly in intensity, forming a broad unresolved multibanding pattern. Additionally, a new strong absorption band occurs at 1558 cm-1. A small band at 1796 cm-1 is also affected; this band completely disappears in pH-induced chromatic transitions. The scissoring band of the methylene group at 1466 cm-1 was not affected during any of the chromatic transition events. The dramatic change in the IR spectra for Gln-PDA between 1694 and 1558 cm-1 cannot be explained solely on the basis of a difference in lipid conformations. One must also consider the specific hydrogen bonding interactions in the headgroup region and take into account changes in association or disassociation of hydrogenbonding interactions during pH-induced transitions. As previously mentioned, three carbonyl groups and four hydrogen atoms are available for forming hydrogen bonding, generating over a dozen possibilities for intramolecular interactions. In addition, extensive intermolecular hydrogen-bonding interactions are expected to be present, leading to hydrogen-bonding networks that resembles ringlike structures. Solution pH increases result in the ionization of COOH groups. This change in electrostatics may have two important consequences: leading to breakdown of the hydrogen-bonding network as a result of losing participating hydrogen atoms, and additionally increasing intermolecular distances, owing (39) Lio, A.; Reichert, A.; Ahn, D. J.; Nagy, J. O.; Salmeron, M.; Charych, D. H. Langmuir 1987, 13, 6524. (40) Wenzel, M.; Atkinson, G. H. J. Am. Chem. Soc. 1989, 111, 6123. (41) Jonas, U.; Shah, K.; Norvez, S.; Charych, D. H. J. Am. Chem. Soc. 1999, 121, 4580. (42) Rubner, M. F.; Sandman, D. J.; Velazquez, C. Macromolecules 1987, 20, 1296. (43) Kimura, F.; Umemura, J.; Takenaka, T. Langmuir 1986, 2, 96.

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to electrostatic repulsion between adjacent COO- groups so that formation of intermolecular hydrogen bonds is no longer possible. It is difficult to assign the Gln-PDA bands unambiguously owing to the complex structure of the headgroup region. By comparison to similar structures in the literature, we believe the band at 1558 cm-1 has mixed contributions from amide II vibration and NH2 scissoring mode.44 These bands are known to be sensitive to conformational changes. The amide II vibration, for instance, disappears with formation of a cyclic conformation.45 This may explain why these bands are absent in the spectra of the original blue and thermochromic-generated red films. The band at 1796 cm-1, uncharacteristically high for a carbonyl vibration in an acid, can be attributed to a high degree stress for the CdO group, and also possibly to H-bonding or H-bonding-associated steric hindrance. The 1796 cm-1 band disappears when pH is raised, consistent with removal of hydrogen atoms in COOH groups. As compared to Gln-PDA, the Phe-PDA microstructures show notably different FTIR spectra (Figure 7, bottom spectra). The asymmetric νas(CH2) and symmetric νs(CH2) stretching vibrations remained virtually unchanged (2921 and 2851 cm-1, respectively). The N-H stretching vibration of the secondary amide located at around 3300 cm-1 remains unchanged during the thermochromic transition but was observed to move to higher frequency when pH is raised. For IR spectra in the 1300-1900 cm-1 region, there is little difference between the blue form and the thermochromic red form. However, when the pH is raised, the amide carbonyl band at 1647 cm-1 becomes broadened and splits into three unresolved bands (1639, 1625, and 1604 cm-1), indicating a complex conformation of the headgroup, as observed in transition metal complexes.46 The lower frequency observed for the carbonyl vibration suggests a conversion from the protonated COOH form to the ionic COO- form; this change is supported by a diminution in intensity of the acid vibration band at 1709 cm-1 and appearance of a band at 1408 cm-1. The latter band is characteristic of the COO- group. The strong bands at 1546 and 1538 cm-1 are believed to be due to amide II and aromatic “benzene-like” vibrations, respectively. The weak band at 1498 cm-1 is assigned to a dCH stretching vibration such as found in benzene. The benzene-like bands appear to be unaffected by either thermal or pH changes. The amide II band, on the other hand, increases in intensity slightly when the sample is treated with base. The difference in the FTIR spectra for thermochromism and pH-induced chromism of PDA microstructures clearly reflects the distinctive driving forces involved in their respective chromatic transitions. Previous studies on thin films suggested that PDA thermochromism is associated with a gauche to trans conformational transition of the methylene groups pendant to the polydiacetylene chain that strains the backbone and causes the chromatic transition.42,47,48 Side chain disorder and entanglement has been ruled out as a potential mechanism for the transitions observed by Charych and co-workers using AFM and FTIR. They found that red lipid forms showed more ordered packing than blue lipid forms.39 For micro(44) Dhamelincourt, P.; Ramirez, F. J. Appl. Spectrosc. 1993, 47, 446. (45) Bellary, L. J. The Infrared Spectra of Complex Molecules; Chapman & Hall: London, 1975. (46) Nakamoto, K. Infrared and Raman Spectra of Inorganic and Coordination Compounds; Wiley: New York, 1986. (47) Tanaka, H.; Gomez, M. A.; Tonelli, A. E.; Thakur, Macromolecules 1989, 22, 1208. (48) Beckham, H. W.; Rubner, M. F. Macromolecules 1993, 26, 5192.

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structure assemblies, we have found no noticeable difference between the νas(CH2) and νs(CH2) stretching vibrations that would suggest significant changes occur in side chain packing during the chromic transitions. More importantly, by comparison with pH-induced transition where the chemical interactions are affected by the headgroup, it is clear that hydrogen bonding in thermochromic red forms remains intact. By considering these FTIR results, we propose that a temperature-induced side chain conformational transition, with retention of headgroup hydrogen-bonding interactions, is responsible for the observed thermochromism of the PDA microstructures. Conserved hydrogen-bonding interactions ensure “locking” of headgroup configurations, imposing significant strain on the side chains that undergo a gauche-trans conformational transition as the temperature is raised. For the pH-induced chromatic transition, however, our FTIR studies clearly indicate that the headgroups undergo significant reorganization as a result of ionization and possible loss of hydrogen-bonding interactions with changes in pH. These changes do not cause a gauche-trans transition as observed in thermochromism, but rather a new conformational adjustment (possibly staggered packing) that accommodates the newly acquired charge distribution. These side chain rearrangements impose backbone strain and cause the chromatic transition. The transition is irreversible since the polymerized microstructures lack the freedom of lipid movement that is required for restoration of the previous conformation.49 This is in agreement with what was previously suggested for the pH-induced chromism of PDA bilayer vesicles.25 Conclusions Novel microstructures with various morphologies have been synthesized using amino acid terminated diacetylene lipids. The chemical structure of the amino acid headgroups was found to have a strong impact on the shape and properties of the resulting assemblies. Different from the bilayer vesicles made with hydrophilic amino acid terminated PDA lipids, all of the microstructures, regardless of headgroup hydrophobicity, can polymerize with UV irradiation to generate blue assemblies with varied intensity. Molecular chirality and hydrogen-bonding interactions in the headgroup regions were found to control the growth of microstructures with twisted features. The (49) Mino, N.; Tamura, H.; Ogawa, K. Langmuir 1992, 8, 594.

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presence of chiral headgroups was found to lead to the formation of twisted assemblies, although the presence of such groups did not necessarily result in the formation of these structures. Nonchiral molecules, however, do not form any curved structures. The colorimetric properties of the microstructures, in general, are similar to those observed for the bilayer vesicles. The polymerized microstructures respond to thermal and pH changes and undergo a blue-to-red chromatic transition. Microstructures with hydrophobic headgroups are more sensitive to pH changes, possibly owing to the instability of the assemblies caused by hydrophobic headgroup packing. Microstructures with hydrophilic amino acids are more resistant to pH change than their vesicle counterparts. FTIR studies revealed that thermal and pH-induced chromism of the PDA microstructures may proceed through very different mechanisms. Hydrogen bonding apparently plays an important role in these changes. We propose that in thermochromism, the hydrogen bonding “locks” the lipid headgroups so that the temperatureinduced gauche-trans conformational transition of the side chains imposes strain on the polymer backbone. In pH-induced chromism, however, the hydrogen-bonding interactions are removed as a result of surface ionization, such that headgroup reorganization generates a stress on the conjugated system. It is unclear whether there is a combined effect from hydrogen bonding and electrostatic interactions as well as conformational changes that act coherently to trigger the chromatic transitions. Future work will be focused on molecular level investigations using atomic resolution techniques to reveal changes associated with the chromatic transitions observed for these amino acid terminated lipid assemblies. Acknowledgment. This work is supported by the Director, Office of Nonproliferation and National Security, Office of Research and Development of the U.S. Department of Energy under Contract No. DE-AC03-76SF00098. We thank San Yu and Rick Burkard for their help in TEM experiments, Jie Song and Mark Davey for critical reading of the manuscript, and Dr. Mark Alper, Program Director of the Center for Advanced Materials, Biomolecular Materials Program, for continued encouragement and support of this research program. LA9916169