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Nanostructured Nonionic Thymidine Nucleolipid Self-Assembly Materials Xavier Mulet,†,‡ Thomas Kaasgaard,†, Charlotte E. Conn,† Lynne J. Waddington,§ Danielle F. Kennedy,† Asoka Weerawardena,† and Calum J. Drummond*,† †
CSIRO Materials Science and Engineering, Bag 10, Clayton South MDC, VIC 3169, Australia, Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 381 Royal Parade, Parkville, VIC 3052, Australia, and §CSIRO Materials Science and Engineering, 343 Royal Parade, Parkville, VIC 3052, Australia. Current address: Danish Technological Institute, Holbergsvej 10, DK 6000 Kolding, Denmark. )
‡
Received August 24, 2010. Revised Manuscript Received October 12, 2010 Three nucleoside lipids have been synthesized: 30 -oleoylthymidine, 30 ,50 -dioleoylthymidine, and 30 -phytanoylthymidine. Differential scanning calorimetry and X-ray diffraction have been employed to characterize the physical properties of these neat lipids. Polarizing optical microscopy, small-angle X-ray scattering, and cryo-transmission electron microscopy techniques have been used to investigate the phase behavior in aqueous systems. Both oleoyl-based nucleoside lipids adopted a lamellar crystalline phase in the neat form at room temperature, and the phytanoyl derivative exhibited a fluid isotropic phase. Under excess water conditions, the presence of one branched (phytanoyl) or one unsaturated (oleoyl) chain promoted the formation of a liquid-crystalline lamellar phase at physiological temperatures. In contrast, the 30 ,50 -dioleoylthymidine derivative is nonswelling and does not exhibit lyotropic liquid-crystalline phase behavior. The nucleolipids’ propensity for DNA-type binding and recognition has been evaluated by using a monolayer system to measure surface pressure-area isotherms in a Langmuir trough and indicates that the nucleoside base is available for nonspecific hydrogen bonding in the monolayer liquid expanded state for the single-chain nucleolipids but not for the dual-chain amphiphile.
Introduction In a hydrated environment, amphiphiles/lipids may form structured self-assembly materials with a range of potentially useful properties. These properties have led them to be considered as materials with diverse applications ranging from templating and membrane protein crystallization to drug delivery.1-4 In one approach, the self-assembly behavior of amphiphiles may be tuned by varying the hydrophobic chain. Variations in the chain unsaturation, length, and branching may modify the self-assembly phase formed by altering the preferential curvature of the selfassembly system.5 Both the phase behavior and kinetics of phase transformations of synthetic and naturally occurring amphiphiles have been widely studied.6-9 Through manipulating the phase behavior, it is possible to control the nanostructure of amphiphile assemblies in a bottom-up approach. Lyotropic liquid crystals that exhibit the fluid lamellar phase allow the formation of liposomal systems that are already being extensively explored as delivery vehicles for a range of drugs *Corresponding author. E-mail:
[email protected]. (1) Mulet, X.; Kennedy, D. F.; Conn, C. E.; Hawley, A.; Drummond, C. J. Int. J. Pharm. 2010, 395, 290–297. (2) Braun, P. V.; Stupp, S. I. Mater. Res. Bull. 1999, 34, 463–469. (3) Conn, C. E.; Darmanin, C.; Sagnella, S. M.; Mulet, X.; Greaves, T. L.; Varghese, J. N.; Drummond, C. J. Soft Matter 2010, 6, 4828–4837. (4) Conn, C. E.; Darmanin, C.; Sagnella, S. M.; Mulet, X.; Greaves, T. L.; Varghese, J. N.; Drummond, C. J. Soft Matter 2010, 6, 4838–4846. (5) Kaasgaard, T.; Drummond, C. J. Phys. Chem. Chem. Phys. 2006, 8, 4957– 4975. (6) Shearman, G. C.; Ces, O.; Templer, R. H.; Seddon, J. M. J. Phys.: Condens. Matter 2006, 18, S1105–S1124. (7) Seddon, J. M.; Squires, A. M.; Conn, C. E.; Ces, O.; Heron, A. J.; Mulet, X.; Shearman, G. C.; Templer, R. H. Philos. Trans. R. Soc., A 2006, 364, 2635–2655. (8) Mulet, X.; Gong, X.; Waddington, L. J.; Drummond, C. J. ACS Nano 2009, 3, 2789–2797. (9) Mulet, X.; Templer, R. H.; Woscholski, R.; Ces, O. Langmuir 2008, 24, 8443–8447. (10) Safinya, C. R.; Ewert, K.; Ahmad, A.; Evans, H. M.; Raviv, U.; Needleman, D. J.; Lin, A. J.; Slack, N. L.; George, C.; Samuel, C. E. Philos. Trans. R. Soc., A 2006, 364, 2573–2596.
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and genes.10 Structures with 2D nanostructure such as the inverse hexagonal phase11 and 3D networks such as the inverse bicontinuous cubic phase have additional advantages of high loading capacity and controlled release, particularly in the form of particulate hexosome and cubosome dispersions.8,12 In the case of nucleolipids, a further dimension of self-assembly potentially arises from the possible contribution to interfacial behavior induced by specific recognition properties between complementary bases. In contrast to classical amphiphiles, nucleolipids can possess a highly informative polar headgroup (adenosine, thymidine, cytidine, guanosine, uracil, or analogs) with additional H-bonding and π-stacking capacity that may specifically interact with other nucleobases. Nucleolipids have begun to generate interest as novel materials, with a concise and comprehensive review of recent advances in the field recently being published by Gissot et al.13 Nucleolipids can be used to transfect genes to cells to correct a defective gene, to introduce new genes, or to knock down a gene.13 This allows the investigation of biological processes, the manufacture of proteins, and potentially the treatment of many diseases. Several factors affect the ability of nucleolipid systems to be used in gene therapy applications: the cellular uptake of particles, how the orientation and availability of the headgroup affect the binding of the complementary base pair, the release profile, and therapeutic advantages or disadvantages. Nontoxic transfection reagents derived from nucleolipids with high transfection efficiencies have been developed.14,15 (11) Seddon, J. M. Biochim. Biophys. Acta 1990, 1031, 1–69. (12) Drummond, C. J.; Fong, C. Curr. Opin. Colloid Interface Sci. 1999, 4, 449– 456. (13) Gissot, A.; Camplo, M.; Grinstaff, M. W.; Barthelemy, P. Org. Biomol. Chem. 2008, 6, 1324–1333. (14) Moreau, L.; Barthelemy, P.; Li, Y. G.; Luo, D.; Prata, C. A. H.; Grinstaff, M. W. Mol. Biosyst. 2005, 1, 260–264. (15) Chabaud, P.; Camplo, M.; Payet, D.; Serin, G.; Moreau, L.; Barthelemy, P.; Grinstaff, M. W. Bioconjugate Chem. 2006, 17, 466–472.
Published on Web 11/08/2010
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Table 1. Summary of Nucleolipids Synthesized by Other Researchers with Details of the Structure, Self Assembly Properties, and DNA Strand Interaction with the Nucleolipid System
A variety of techniques have been used to synthesize artificial nucleolipids as highlighted by Rosemeyer.16 To readily allow direct comparison with the nucleolipids presented in the current work, the structures and aggregate types of systems of particular relevance to the study have been summarized in Table 1. (16) Rosemeyer, H. Chem. Biodiversity 2005, 2, 977–1062.
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Banchelli et al. recently found that 1,2-dioctanoyl-phosphatidyladenosine micelles complex with ss-polyuridylic acid (polyU) to form hexagonal mesophases in which the biopolymer is confined between cylinders.17 This is a nonintuitive complexation (17) Banchelli, M.; Berti, D.; Baglioni, P. Angew. Chem., Int. Ed. 2007, 46, 3070– 3073.
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Figure 1. Nucleolipids investigated are shown on the left. Right-hand-side displays minimized configurations of 30 -oleoylthymidine, 30 -phytanoylthymidine, and 30 ,50 -dioleoylthymidine using an MM2 force field with Chem3D Pro. It shows that the modulation of the headgroup orientation can lead to significant changes in the lattice parameter of any phase observed and will influence the ability to bind complementary base pairs present in solution.
because both systems have a negative charge.18 Additionally, nucleolipid-nucleolipid interactions have been studied in different types of supramolecular systems including micelles,19 vesicles,20 and monolayers.21 A transfection agent, based on a cationic nucleoside phosphocholine amphiphile that forms vesicles when complexed with DNA, resulting in a high transfection efficacy, has been described by Moreau et al.14 Barthelemy et al. (18) Campins, N.; Dieudonne, P.; Grinstaff, M. W.; Barthelemy, P. New J. Chem. 2007, 31, 1928–1934. (19) Nowick, J. S.; Cao, T.; Noronha, G. J. Am. Chem. Soc. 1994, 116, 3285– 3289. (20) Onda, M.; Yoshihara, K.; Koyano, H.; Ariga, K.; Kunitake, T. J. Am. Chem. Soc. 1996, 118, 8524–8530. (21) Li, C.; Huang, J. G.; Liang, Y. Q. Langmuir 2000, 16, 7701–7707. (22) Arigon, J.; Prata, C. A. H.; Grinstaff, M. W.; Barthelemy, P. Bioconjugate Chem. 2005, 16, 864–872. (23) Barthelemy, P.; Prata, C. A. H.; Filocamo, S. F.; Immoos, C. E.; Maynor, B. W.; Hashmi, S. A. N.; Lee, S. J.; Grinstaff, M. W. Chem. Commun. 2005, 10, 1261–1263.
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have synthesized nonionic,22,23 zwitterionic,24,25 anionic,18 and cationic26 nucleolipids. In this article, we report a study of three nonionic nucleoside lipids: 30 -mono- and 30 ,50 -dioleoylthymidine and 30 -phytanoylthymidine. The structures of the nucleolipids are shown in Figure 1. Our reported synthesis route (provided in the Supporting Information) illustrates a facile pathway to a wide diversity of nucleolipids. Differential scanning calorimetry (DSC), X-ray diffraction (XRD), small-angle X-ray scattering (SAXS), cross-polarized optical microscopy (X-POM), cryo-transmission electron microscopy (cryo-TEM), and surface pressure-area isotherm results demonstrate that both the thymidine headgroup and the amphiphile (24) Moreau, L.; Barthelemy, P.; El Maataoui, M.; Grinstaff, M. W. J. Am. Chem. Soc. 2004, 126, 7533–7539. (25) Moreau, L.; Grinstaff, M. W.; Barthelemy, P. Tetrahedron Lett. 2005, 46, 1593–1596. (26) Barthelemy, P.; Camplo, M. MRS Bull. 2005, 30, 647–653.
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chains influence the properties of the self-assembly materials formed by the nucleolipids leading to zero-curvature lamellar self-assembled aggregates. In previous work, oleoyl and phytanyl nonionic amphiphiles have frequently led to self-assembled aggregates with zero or inverse curvature.27-30
Materials and Methods Synthesis of Thymidine Nucleolipids. A detailed description of the synthesis procedures for the three nucleolipids is given in the Supporting Information. Materials. Anhydrous pyridine (99.8%), anhydrous toluene (99.8%), cyclohexane (99%), thymidine (99%), oleic acid (99%), and Na2SO4 were purchased from Sigma-Aldrich (Castle Hill, New South Wales, Australia). Methanol and dichloromethane were purchased from Merck (Darmstadt, Germany). 4,40 -Dimethoxytrityl chloride (98%) and dicyclohexylcarbodiimide were purchased from Alfa Aesar (Ward Hill, MA). Dimethylamino4-pyridine was purchased from Novabiochem Merck (Darmstadt, Germany). All solvents and reagents were used without further purification. All water used in the systems was Milli-Q water (18.2 MΩ/cm-1). Cross-Polarized Optical Microscopy (X-POM). X-POM observations of samples were performed on an Olympus BX51 polarized light microscope equipped with a Mettler heating stage (FP90 and FP82HT). Heating scans were performed at 5 °C/min unless otherwise identified. Samples were prepared by melting powders where necessary to achieve a clean interface to allow water penetration scans to be performed with 100 magnification for all images. The samples (approximately 5 mg) were mounted between a glass microscope slide and a coverslip and were flooded through the addition of one or two drops of water. Penetration of the water at the interface allowed the observation of changing phases at different water concentrations and at different temperatures. Differential Scanning Calorimetry (DSC). DSC scans were carried out with a Mettler-Toledo DSC calorimeter (DSC-30). An appropriate amount of material was added to the bottom of each pan (3.00-5.00 mg). The pans were then sealed, and their lids were pierced. An identical reference DSC pan was run simultaneously. Sealed aluminum pans were used with heating rates set to 2.5 °C/ min from -150 to 300 °C. The reported results refer to heating scans. Enthalpies were obtained by integration of the transition peaks. Small-Angle X-ray Scattering (SAXS). SAXS measurements were carried out using two specialized in-house, custombuilt SAXS beamlines. The first is coupled to a copper target Bede Microsource (Durham, U.K.) X-ray generator with integrated glass polycapillary X-ray focusing optics. The Ni-filtered Cu KR radiation (λ = 1.54 A˚) was cut down with 300 μm pinholes. X-ray diffraction images were acquired on an X-ray-intensified chargecoupled device Gemstar detector (Photonic Science, East Sussex, U.K.). Samples were prepared in flame-sealed glass capillaries with a 1.5 mm diameter. The sample holder has computer-controlled Peltier-based temperature control over a range of 10-120 °C with a precision of (0.05 °C. The second is an Osmic SAXS camera based on a MicroSource microfocusing copper X-ray source coupled with Confocal Max-Flux (CMF) optics. The X-ray source must be operated at 45 kV and 0.66 mA to ensure that the source is coupled correctly to the optics providing radiation at λ = 1.54 A˚. A custom-made “wet cell” composed of a Teflon or steel washer with mica windows on either side was used. The X-ray path length (27) Sagnella, S. M.; Conn, C. E.; Krodkiewska, I.; Drummond, C. J. Soft Matter 2009, 5, 4823–4834. (28) Sagnella, S. M.; Conn, C. E.; Moghaddam, M.; Seddon, J. M.; Drummond, C. J. Langmuir 2010, 26, 3084–3094. (29) Liu, G. Z.; Conn, C. E.; Drummond, C. J. J. Phys. Chem. B 2009, 113, 15949–15959. (30) Conn, C. E.; Panchagnula, V.; Weerawardena, A.; Waddington, L. J.; Kennedy, D. F.; Drummond, C. J. Langmuir 2010, 26, 6240–6249.
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Mulet et al. through the cell is approximately 1 mm. Temperature control was achieved using a recirculating water bath. Fuji BAS image plates (BAS-MS2025) are used with a Fuji BAS-5000 image plate reader as the main detection system. Lattice parameters are calibrated using silver behenate with a repeat lamellar spacing of 58.38 A˚. Samples were prepared by the addition of water to the desired level to a known amount of lipid (typically 10.0 mg). This sample was stirred and allowed to equilibrate for several (at least 4) hours to ensure sample homogenization. These samples were then loaded into the wet-cell holder described above. For all samples, the equilibration times were 20 min at each temperature. SAXS Analysis. Diffraction patterns were analyzed with the AXcess software package developed by Dr. Andrew Heron at Imperial College London.7 The estimated accuracy of the layer spacing is (0.15 A˚. X-ray Diffraction (XRD). XRD patterns were obtained using the powder diffraction beamline at the Australian Synchrotron.35 Each sample (5-10 mg) was mounted between 0.15 mm Mylar film windows (Sietronics Pty Ltd., ACT) and were contained within a custom-designed sample stage, mounted on the ω axis of the diffractometer, and were measured in transmission mode. A 1 mm 0.8 mm beam was aligned to a reference point on the stage containing an R-alumina standard. The beam energy was set at 12.39847 keV with a slit size of 2 mm 0.8 mm at 29 °C; the sample was rocked between -0.5 and þ0.5 ω with 3 cycles per acquisition. X-ray diffraction data from 2 to 82° at ambient temperature were collected on a Mythen II microstrip detector. Analyses were performed on the collected XRD data for each sample using Bruker XRD search match program EVA. Langmuir Trough. Langmuir trough experiments were run on a laboratory-built instrument. Polyadenosine and polythymidine were obtained from Geneworks (SA, Australia) at PCR quality and used without further purification. Doubly distilled water with a resistance of 18.2 mΩ was used for all subphase preparations. A solution of each nucleolipid (20 μM) was prepared in HPLC-grade chloroform (Merck). To deposit a monolayer, 8 μL of solution was placed on the surface of the Langmuir trough aqueous phase. Monolayers were prepared on an in-house-built Langmuir trough with a mica Wilhelmy plate as a surface-pressure sensor. One barrier compressed the monolayer at a rate of 36 cm2/min. The chloroform was left to evaporate for at least 10 min prior to obtaining surface pressure-area (π-A) isotherms at room temperature. The polynucleoside was prepared as 0.68 μM stock solutions. Polyadenosine and polythymidine displayed no significant surface activity in the absence of a nucleolipid monolayer. Cryo-Transmission Electron Microscopy. A laboratorybuilt humidity-controlled vitrification system was used to prepare the samples for cryo-TEM. Humidity was kept close to 80% for all experiments. A 4 μL aliquot of the sample was transferred onto a 300-mesh copper grid coated with a lacy Formvar carbon film (ProSciTech, Thuringowa, Queensland, Australia). After 30 s of adsorption time, the grid was blotted manually using Whatman 541 filter paper for 2-10 s. The blotting time was optimized for each sample. The grid was then plunged into liquid ethane cooled by liquid nitrogen. Frozen grids were stored in liquid nitrogen until required. The samples were examined using a Gatan 626 cryoholder (Gatan, Pleasanton, CA) and a Tecnai 12 transmission electron microscope (FEI, Eindhoven, The Netherlands) at an operating voltage of 120 kV. At all times, low-dose procedures were followed using an electron dose of 8-10 electrons/A˚2. Images were recorded using a Megaview III CCD camera and AnalySIS camera control software (Olympus) at magnifications of between 70 000 and 110 000. (31) Berti, D.; Bombelli, F. B.; Fortini, M.; Baglioni, P. J. Phys. Chem. B 2007, 111, 11734–11744. (32) Berti, D.; Luisi, P. L.; Baglioni, P. Colloids Surf., A 2000, 167, 95–103. (33) Khiati, S.; Pierre, N.; Andriamanarivo, S.; Grinstaff, M. W.; Arazam, N.; Nallet, F.; Navailles, L.; Barthelemy, P. Bioconjugate Chem. 2009, 20, 1765–1772. (34) Heiz, C.; Radler, U.; Luisi, P. L. J. Phys. Chem. B 1998, 102, 8686–8691. (35) Wallwork, K. S.; Kennedy, B. J.; Wang, D. AIP Conf. Proc. 2007, 879, 879.
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Table 2. Thermal Transitions Obtained by Differential Scanning Calorimetry for Neat Nucleoplipid Derivatives 30 -Oleoylthymidine, 30 -Phytanoylthymidine, and 30 ,50 -Dioleoylthymidinea
amphiphile
onset transition temp (°C)
transition enthalpy (kJ/mol)
visual melting point (°C)
0
49.1 -0.8 51 3 -oleoylthymidine -8.6 -4.0 (broad peak) -15 30 -phytanoylthymidine 37.4 -43.7 35 30 50 -dioleoylthymidineb a The heating rate was 5 °C/min. Melting points were determined by visual inspection. b The heating rate was 15 °C/min.
The 30 -oleoylthymidine powder was melted onto the grid, and upon cooling to 35 °C, a drop of water was added. The system was then incubated for 2 h to allow the fibrils to develop and then visualized by X-POM.
Results and Discussion The nucleolipids were synthesized as described in the Supporting Information. Their structures are shown in Figure 1. 30 -Oleoylthymidine (1), 30 -phytanoyl-thymidine (2), and 30 ,50 -dioleoyl-thymidine (3) were characterized using DSC, X-POM, SAXS, and XRD. An evaluation of complementary base pair binding was performed using a Langmuir trough to measure the surface pressure versus area (π-A) isotherms for the nucleolipids. Differential Scanning Calorimetry (DSC). The unhydrated 30 -oleoylthymidine (1) sample exhibits an endothermic melting peak at 49.1 °C (Table 2). The melting peak observed for the dioleoyl derivative (3) is lower at 37.4 °C. These peaks can be attributed to a crystal-isotropic transition. Both observed melting points are higher than the melting point for oleic acid (14 °C). The 30 -phytanoylthymidine derivative (2) does not show a sharp crystal-isotropic peak, but rather a broad endothermic peak has been observed with an onset at -8.6 °C . We note that this is lower than the melting point for phytantriol (5-10 °C). X-ray Diffraction (XRD). X-ray diffraction results provided in Figure 2 for the three neat nucleolipids at room temperature show that both neat 30 -oleoylthymidine and 30 ,50 -dioleoylthymidine have a crystalline nature, with a large number of Bragg reflections at higher angles. 30 -Phytanoylthymidine has a broad amorphous peak at a high angle reflecting its noncrystalline nature at 25 °C. At lower angles, the second and third lamellar reflections of a 27 A˚ repeat lattice can be observed for 30 -oleoylthymidine; this is comparable to the crystalline lamellar phase observed by SAXS (Table 3). Two lamellar reflections corresponding to the phase at 26 A˚ for the 30 ,50 -dioleoylthymidine amphiphile are observed. Cross-Polarized Optical Microscopy (X-POM). Water penetration scans permit the observation of the birefringence of anisotropic lyotropic crystal phases via cross-polarized microscopy, which can be used to provide a rapid assessment of the phase behavior of amphiphile systems. When observed under cross-polarizers, isotropic phases such as inverse bicontinuous cubic phases appear as dark bands and anisotropic phases such as lamellar and hexagonal phases are birefringent with well-characterized textures.36 30 -Oleoylthymidine (1). Three distinct lamellar phases can be observed upon hydration and heating to 37 °C. At this temperature, spherulites or long birefringent filaments appear as shown in Figure 3A. These are stable to 65 °C, after which both the bulk material and strands become isotropic. The nature of these spherulites has been observed using cryo-TEM and is discussed more fully in a later section. Upon cooling, three distinct regions of the lamellar phase are again formed at 35 °C. (36) Rosevear, F. B. J. Am. Oil Chem. Soc. 1954, 31, 628–639.
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Figure 2. Powder X-ray diffraction data from (A) 30 -oleoylthymidine, (B) 30 -phytanoylthymidine, and (C) 30 ,50 -dioleoylthymidine at 25 °C. The 2θ range is from 2 to 30°.
30 -Phytanoylthymidine (2). The optical texture at 37 °C (Figure 3B) shows a lamellar phase in the excess water region of the water penetration scan. The less-hydrated, dry material appears to be isotropic. Increasing the temperature to 60 °C has little effect on the optical texture of the hydrated material, indicating a lack of phase change. Above 60 °C, the lamellar phase appears to melt. 30 ,50 -Dioleoylthymidine (3). The optical texture of the polarizing microscopy images (Figure 3C) suggests that the material is DOI: 10.1021/la103370q
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Table 3. Variation of the Lattice Parameter with Temperature for 30 -Oleoylthymidine Samples with Different Levels of Hydrationa wt % H2O temp/°C
20 lamellar
35 lamellar
lamellar
50 lamellar
lamellar
lamellar
25 32.2 32.1 26.3 32.2 35 26.4 32.3 37 32.3 32.4 32.7 45 32.5 39.2 32.4 39.2 55 38.0 38.7 65 37.8 37.9 75 80 37.2 a The lattice parameter is quoted in A˚ngstroms. The diffractions patterns from which the values are extracted can be seen in Figure S6.
Figure 3. Cross polarized optical microscopy water penetration scans. (A) 30 -Oleoylthymidine showing three lamellar regions at 37 °C, including fibrils grown from the bulk phase. (B) 30 -Phytanoylthymidine water penetration scan at 37 °C, with water infiltration starting at the bottom of the image. (C) 30 ,50 -Dioleoylthymidine at 37 °C in the presence of water.
crystalline and totally separated from water. From 35 °C, the material begins to melt and forms an isotropic phase. The lattice parameter of the phase as observed by SAXS is invariant with temperature, consistent with the hypothesis that the phase observed is a crystalline one. SAXS Analysis. For 30 -oleoylthymidine, three samples (20, 35, and 50 wt % water) were run at a series of discrete temperatures between 25 and 80 °C. For 30 -phytanoylthymidine, one 18420 DOI: 10.1021/la103370q
lamellar 40.5 39.4 39.2 38.3 37.8 37.4
sample was run at 50 wt % hydration at 20, 37, and 65 °C. For 30 ,50 -dioleoylthymidine, one sample was run at 50 wt % hydration at 20, 37, and 65 °C. Representative 1D plots of intensity versus s (s is 1/d where d is the d spacing), stacked to show changes in temperature are shown in Figure S6. The data described below is represented as a “stacked” plot in Figure S6, showing the variation in the diffraction pattern observed over all temperatures studied, allowing changes in the position and intensity of the diffraction peaks with temperature to be more easily visualized. Note that although the program “streaks” out the data this was not a temperature scan but corresponds to discrete diffraction images at various temperatures. 30 -Oleoylthymidine (1). At temperatures
