Temperature-Dependent In-Plane Structure Formation of anX‑Shaped Bolapolyphile within Lipid BilayersBob-Dan Lechner,† Helgard Ebert,‡ Marko Prehm,†,‡ Stefan Werner,# Annette Meister,§ Gerd Hause,∥

Andre Beerlink,∇ Kay Saalwachter,⊥ Kirsten Bacia,# Carsten Tschierske,‡ and Alfred Blume*,†

†Institut fur Chemie - Physikalische Chemie and ‡Institut fur Chemie - Organische Chemie, Martin-Luther-UniversitatHalle-Wittenberg, D-06120 Halle (Saale), Germany§Zentrum fur Struktur und Dynamik der Proteine (MZP) and ∥Abteilung Elektronenmikroskopie, BiozentrumMartin-Luther-Universitat Halle-Wittenberg, D-06120 Halle (Saale), Germany⊥Institut fur Physik - NMR and #ZIK HALOmem, Martin-Luther-Universitat Halle-Wittenberg, D-06120 Halle (Saale), Germany∇Hamburger Synchrotronstrahlungslabor (HASYLAB), Deutsches Elektronen−Synchrotron DESY, D-22607 Hamburg, Germany

*S Supporting Information

ABSTRACT: Polyphilic compound B12 is an X-shapedmolecule with a stiff aromatic core, flexible aliphatic sidechains, and hydrophilic end groups. Forming a thermotropictriangular honeycomb phase in the bulk between 177 and 182°C but no lyotropic phases, it is designed to fit into DPPC orDMPC lipid bilayers, in which it phase separates at roomtemperature, as observed in giant unilamellar vesicles (GUVs)by fluorescence microscopy. TEM investigations of bilayeraggregates support the incorporation of B12 into intactmembranes. The temperature-dependent behavior of themixed samples was followed by differential scanningcalorimetry (DSC), FT-IR spectroscopy, fluorescence spec-troscopy, and X-ray scattering. DSC results support in-membrane phase separation, where a reduced main transition and newB12-related transitions indicate the incorporation of lipids into the B12-rich phase. The phase separation was confirmed by X-rayscattering, where two different lamellar repeat distances are visible over a wide temperature range. Polarized ATR-FTIR andfluorescence anisotropy experiments support the transmembrane orientation of B12, and FT-IR spectra further prove a stepwise“melting” of the lipid chains. The data suggest that in the B12-rich domains the DPPC chains are still rigid and the B12 moleculesinteract with each other via π−π interactions. All results obtained at temperatures above 75 °C confirm the formation of a single,homogeneously mixed phase with freely mobile B12 molecules.

1. INTRODUCTION

Cell membranes are a complex mixture of lipids and proteins,with the lipids building up the permeability barrier and theproteins providing the controlled functions. Because of thearchitectural complexity of biological membranes, modelsystems are usually used to study their properties and structure.Phospholipids such as phosphatidylcholines are often used asmodel membranes in the form of vesicular systems or orientedsolid supported bilayers. Interactions of biomolecules such asproteins or peptides with model membranes have widely beeninvestigated during the past few decades. The underlyingprinciples of structure formation and phase change uponincorporation or adsorption of dopant molecules are still amatter of intensive research. Besides naturally occurringproteins and peptides, the interaction of purely syntheticmolecules with model membranes has also been studied. Thesesynthetic molecules are often optimized for interactions with orintercalation into membranes by having an amphiphilic naturewith lipophilic and hydrophilic moieties.

One class of amphiphilic synthetic molecules are blockcopolymers. The well-studied class of pluronics, i.e., PEO-PPO-PEO (poly(ethylene oxide)-poly(propylene oxide)-poly-(ethylene oxide)) triblock copolymers, interacts with lipidmembranes via polar interactions in the headgroup region butcan also insert into lipid bilayers provided they are in the liquid-crystalline state.1 Other amphiphilic block copolymers with ahydrophobic block such as poly(isobutylene)-b-poly(ethyleneoxide) PIB-b-PEO have been shown to be incorporated mainlyinto the hydrophobic inner region of the membranes,2

stabilizing the membrane gel phase.3,4 Also, smaller moleculessuch as elongated oligospiroketals carrying a hydrophobicbackbone and several terminal groups, which have the shape ofmolecular rods, can be incorporated into model as well asbiological membranes.5 In the membrane, these rods adopt a

Received: December 17, 2014Revised: February 18, 2015Published: February 19, 2015

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trans-membrane orientation.6 Other molecular rods composedof molecules with an octaphenylene backbone carrying variousadditional moieties connected to the aromatic rings and twosmall polar groups at the end were also synthesized. Thesemolecules could be incorporated into membranes formingchannels.7,8

We recently reported on the aggregation of a T-shapedamphiphile (A6/6) composed of a hydrophobic rigid terphenylbackbone and a hydrophilic lateral chain and its interactionwith lipid membranes.9 In lipid mixtures, this T-shapedamphiphile disrupts the membrane and leads to the formationof planar bilayer patches of hexagonal symmetry (bicelles) withthe amphiphile molecules bordering the membrane edges, thusreducing the unfavorable interaction of the hydrophobic lipidalkyl chains with water.In this article, we present detailed results on the unique

interaction of a new type of X-shaped polyphilic molecule,B12,10 with lipid membranes. It is built of a rigid π-conjugatedoligo(phenylene ethynylene) backbone with two laterallyattached flexible and lipophilic alkyl chains (n-OC12H25) atopposite sides of the central benzene ring of the rodlike coreand terminated by hydrophilic glycerol groups at both ends,giving the molecule an X-shaped, bolaamphiphile-like structure(Figure 1a).11 As shown below, during self-assembly in

phospholipid membranes all three distinct units segregateinto different domains, and the rodlike cores especially tend toorganize into densely packed π-stacked nanodomains within thelipid matrix; therefore, we consider compound B12 to be apolyphilic (more specifically, triphilic) molecule.12 The proper-ties of other oligo(phenylene ethynylenes) with at least twohydrophilic groups have been reported by various groups,however mostly with lateral hydrophilic groups and withoutaddressing their properties in lipid bilayer membranes.13−17

The aromatic backbone length of the newly designedmolecules is 3.2 nm and is thus in the range of the typicalthickness of the lipophilic part of a phospholipid bilayer.18,19

The total length between the ends of the glycerol units is,depending on the assumed conformation, in the range of 4.0 to4.4 nm (Figure 1). This molecule combines the concepts ofrigidity and flexibility and moieties with different philicities, i.e.,the core being lipophilic, rigid, and amenable to π−π stackinginteractions, the side chains being lipophilic but flexible, and theheadgroups being hydrophilic and capable of hydrogenbonding. The length of the lateral alkyl chains was chosen insuch a way that in an arrangement parallel to one-half of the

aromatic core these chains completely cover this core segmentbut do not reach/disturb the hydrophilic glycerol groups(Figure S1).Bolapolyphile B12 as a bulk substance forms a small range of

an enantiotropic columnar liquid-crystalline phase withtriangular honeycomb structure between 177 and 182 °C.Although B12 shows no solubility in water and also does notform lyotropic mesophases with water due to its smallhydrophilic headgroups, we could observe a remarkable affinityand miscibility with phospholipid model-membrane systems.As shown in our first short report on this novel substance,10

B12 spontaneously self-organizes in lipid membranes made of1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), formingunique snowflakelike structures with 6-fold symmetry in giantunilamellar vesicles made by electroformation. It was shownthat these structures consist of a mixed phase of three to fiveDPPC molecules per B12 molecule, in which the DPPCmolecules are stabilized in an ordered state even above themain lipid phase transition at around 42 °C while the B12molecules are organized in a rigid π−π stacked arrangement.Here, we now present an in-depth investigation of thethermotropic phase behavior of B12 in DPPC and theassociated structural features. In addition to B12/DPPCsystems, we also studied the interaction of B12 with theshorter-chain myristoyl analogue DMPC and with POPC,which has one unsaturated oleoyl chain. These experimentswere performed to test whether changes in bilayer thickness orchanges in fluidity caused by an unsaturated chain have aneffect on the interactions and the resulting thermotropicbehavior. The structural features were studied by means oftransmission electron microscopy and X-ray scattering experi-ments, while the phase behavior, molecular conformations, andorientations were investigated by differential scanning calorim-etry and IR and fluorescence spectroscopy techniques,respectively.

2. EXPERIMENTAL SECTION2.1. Phospholipids and B12. The synthesis, purification, and

analytical data of B12 have been reported in ref 10. Lipids DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), and POPC (1-palmitoyl-2-oleyl-sn-glyc-ero-3-phosphocholine) were purchased from Genzyme Pharmaceut-icals (Liestal, Switzerland), purity >99%, and used without furtherpurification.

2.2. Preparation of Phospholipid/Polyphile Mixtures. Phos-pholipids and B12 were premixed in chloroform prior to suspension inwater. Stock solutions of DPPC, DMPC, POPC (c = 10 mM), andB12 (c = 2 mM) in chloroform (HPLC grade, Carl Roth GmbH,Karlsruhe, Germany) were not stored for longer than 4 weeks.Aliquots of the stock solutions of the lipid and B12 were then mixed toreach the desired molar ratio. All experiments were carried out usingultrapure water (H2O, Millipore, with a conductivity of <0.055 μS/cmand a total organic carbon content (TOC) of <5 ppm) or D2O (CarlRoth GmbH, Karlsruhe, Germany) where a pD = 6.77 value wasadjusted using small amounts of DCl and NaOD (Carl Roth GmbH,Karlsruhe, Germany).

2.3. Methods. 2.3.1. Polarizing Microscopy. The transitiontemperatures of pure B12 were confirmed by polarizing microscopywith an Optiphot 2 polarizing microscope (Nikon). Temperature wascontrolled with a heating stage (FP82HT, Mettler-Toledo) and acontrol unit (FP90, Mettler-Toledo).

2.3.2. Transmission Electron Microscopy (TEM). Vitrified speci-mens for cryo-TEM were prepared by a blotting procedure, performedin a chamber with controlled temperature and humidity using a LEICAgrid plunger. A drop of the sample solution (1 mg mL−1) was placedonto an EM grid coated with a holey carbon film (C-flat, Protochips

Figure 1. (A) Chemical structure of X-shaped polyphile B12. (B)Space-filling model of B12.

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Inc., Raleigh, NC). Excess solution was then removed with filter paper,leaving a thin film of the solution spanning the holes of the carbon filmon the EM grid. Vitrification of the thin film was achieved by rapidplunging of the grid into liquid ethane held just above its freezingpoint. The vitrified specimen was kept below 108 K during storage,transfer to the microscope, and investigation. Specimens wereexamined with a Libra 120 Plus instrument (Carl Zeiss MicroscopyGmbH, Oberkochen, Germany), operating at 120 kV. The microscopeis equipped with a Gatan 626 cryotransfer system. Images were takenwith a BM-2k-120 dual-speed on-axis SSCCD camera (TRS,Moorenweis, Germany).Negatively stained samples were prepared by spreading 5 μL of the

dispersion prepared in a similar way as those for DSC measurements(c = 0.1 mM) onto a Cu grid coated with a Formvar film (Plano,Wetzlar, Germany). After 1 min, excess liquid was blotted off withfilter paper, and 5 μL of a 1% aqueous uranyl acetate solution wasplaced on the grid and drained off after 1 min. The dried specimenswere examined with a Zeiss EM 900 transmission electron microscope.This preparation procedure was performed at 20 and 50 °C usingsolutions held and dried at the respective temperature.2.3.3. Differential Scanning Calorimetry (DSC). DSC of pure B12

was performed on a DSC7 (PerkinElmer) with a sample encapsulatedin Al pans (30 μL) using a heating and cooling rate of 10 K/min. Forcalorimetric investigation of the mixed lyotropic systems, a VP-DSC(MicroCal/GE Inc. Northhampton, USA) DSC instrument was usedwith a scan rate of 60 K/h over a temperature range of 2−95 °C.Several scans were made to check for reproducibility of the curves. Fordata processing, the MicroCal Origin software was used aftersubtracting the water/water baseline from the sample thermograms.The aqueous samples were prepared in the following way. Afterpremixing stock solutions of B12 and DPPC to give the desired molarratio, the major part of the solvent was evaporated using a N2 stream.Any residual remaining solvent was then removed in a vacuum dryingoven at 70 °C for at least 3 h. The dry films were then suspended inultrapure water to give a concentration of 2.0−2.5 mM. Thesuspensions were vortex mixed and treated in a bath sonicator at55−65 °C for 30 min. Pure degassed water was used in the referencecell.2.3.4. Attenuated Total Reflection Fourier Transform Infrared

Spectroscopy (ATR-FTIR). Two different techniques for film formationon the internal reflection element (IRE) were used. Vesicle spreadingby fusion of mixed vesicles in an aqueous medium resulted in thinfilms on the IRE. Samples prepared according to this method wereanalyzed using a Bruker BioATR2 unit with a Bruker Tensor27 FT-IRspectrometer. IR spectra were recorded at different temperaturesbetween 10 and 76 °C in steps of 2 K with unpolarized IR light. Thetemperature was adjusted with a Haake Phoenix II thermostat (C25P,Thermo Electron Corporation, Karlsruhe, Germany) and controlledwith Delphi-based home-written software.For the preparation of oriented films, the premixed chloroform

solution of B12 and DPPC (1 mL, 4.4 mM) was applied dropwise tothe hot (60−70 °C) Ge-ATR crystal (sample area of 5 cm × 1 cm, 5internal reflections, nGe = n1 = 4, angle of reflection γ = 45°) to speedup evaporation of the solvent. After drying of the film, several drops ofH2O or D2O were applied to the holder next to the crystal, and theATR cell was tightly closed. The dry film was then hydrated via the gasphase (water vapor) at 60 °C for about 1 to 2 h. Spectra were recordedcontinuously during this time to follow the hydration process. Thehomemade crystal holder for the trapezoidal Ge-crystal reflectionelement was placed into a Bruker ATR1 unit. Single-channel spectra(128 scans) with linearly polarized light with 4 cm−1 resolution wererecorded between 900 and 4000 cm−1 with an aperture of 2.5 mmusing a Bruker IFS66 FT-IR spectrometer (Bruker Optics GmbH,Ettlingen, Germany) equipped with an MCT detector. As reference,single-channel spectra taken from the pure crystal at the respectivetemperatures were used. The absorbance spectra were then calculatedusing the Bruker OPUS 6.5 software. Sample and reference spectrawere measured using perpendicular (s) and parallel (p) polarized IRlight in the range of 10−70 °C with an interval of 2 K. Thetemperature was controlled using the procedure described above.

2.3.5. Adsorption Spectroscopy, Fluorescence Spectroscopy, andConfocal Laser Scanning Fluorescence Microscopy (CFM). Absorp-tion spectra were recorded using an HP 8453 UV−vis diode arrayspectrophotometer (Hewlett-Packard GmbH, Waldbronn, Germany)with a cell holder heated and cooled by Peltier elements. Fluorescencespectra were taken using a Jobin-Yvon Fluoromax II (Horiba JobinYvon Inc., Grasbrunn, Germany) fluorescence spectrometer with a cellholder connected to a NESlab RTE 740 thermostat (ThermoScientific Inc., Schwerte, Germany). For the absorption orfluorescence experiments, quartz cuvettes (Hellma Analytics GbmH,Mullheim, Germany) with a 10 mm path length were used. CFM wascarried out on an LSM 710 setup (Carl Zeiss Microimaging, Jena,Germany) using a C-Apochromat 40×/1.2 N.A. water-immersionobjective. The bolapolyphiles were excited with a diode laser at 405nm; fluorescence emission was collected from 412 to 500 nm. A 561nm, the DPSS laser was used to excite the Rh-PE lipid marker, and thefluorescence emission of the marker was collected from 566 to 681nm.

2.3.6. X-ray Diffraction. X-ray diffraction (XRD) of the bulk samplewas performed on a temperature-controlled heating stage. Ni-filteredand pinhole-collimated Cu Kα radiation was used; the exposure timewas 30 min, and the diffraction patterns were recorded with a 2Ddetector (Vantec-500, Bruker, Germany). We tried to achievealignment via slow cooling (rate 1 K min−1 to 0.1 K min−1) of asmall droplet of the sample on a glass plate.

XRD of the lipid mixtures was performed using three differentsetups. In-house powder diffraction experiments were carried out usingmonochromic Cu Kα1 radiation (λ = 0.154051 nm from a Ge(111)monochromator (Seifert X-ray/GE Inc., Freiberg, Germany) and acurved linear position-sensitive detector (range: 2θ = 0−40°). Sampleswere prepared according to the standard procedure followed bylyophilization and rehydration to give a content of 50 wt % H2O for agood signal-to-noise ratio of the scattering patterns. The pasty sampleswere transferred to glass capillaries which were then flame-sealed.Temperature was controlled by a high-temperature sample holder(STOE & CIE GmbH, Darmstadt, Germany). SAXS and WAXS (s =1−4.7 nm−1) data were collected in the temperature range of −35 and80 °C. The temperature was varied stepwise (heating rate of 1 K/min), and the sample was equilibrated for 5 min at each temperaturebefore data acquisition with 10 min of exposition time perdiffractogram. Each scattering pattern was corrected by the scatteringof an empty capillary. All diffraction patterns of one temperature serieswere then converted to a contour diagram (grayscale intensities).

Experiments using synchrotron radiation (λ = 0.49509 Å) wereperformed at DESY (HASYLAB, Hamburg). Oriented multibilayerstacks were deposited from CHCl3/TFE (1:1) solution on a Si solidsupport. The samples were rehydrated via the gas phase at about 60 °C(relative humidity in the sample chamber 99%). Reflectivity measure-ments were made under specular conditions with a six-circlediffractometer and an angle of incidence αi = 0.2°. Data werecollected using a linear detector (Mythen 1k, DECTRIS Inc., Baden,Switzerland). For grazing incidence X-ray diffraction (GIXD) of thesame samples, a setup with an image plate detector (PerkinElmer flatpanel, 2048 pixels × 2048 pixels,2 PerkinElmer Inc., Rodgau,Germany) was used. The detector-to-sample distance was 1.2 m.

3. RESULTS AND DISCUSSION

3.1. Thermotropic Phase Behavior of Bulk B12. As abulk substance, B12 has a melting point at T = 177 °C (ΔH =62.4 kJ/mol). Above this temperature, a liquid-crystalline (LC)phase is formed which is stable up to T = 182 °C (ΔH = 4.0 kJ/mol), when the transition to the isotropic liquid phase (Iso)occurs. On cooling, the Iso−LC transition takes place at T =180 °C, and at 169 °C, the sample rapidly crystallizes. (ForDSC, see Figure S2.) The optical texture observed betweencrossed polarizers is composed of spherulitic domains andoptically isotropic homeotropic aligned areas and thus indicatesan optically uniaxial columnar LC phase (Figure 2A).

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Investigation with a λ-retarder plate indicates negativebirefringence, which means that the intramolecular π-conjugation pathway occurs along the aromatic cores of B12and hence these cores are arranged on average perpendicular tothe column long axis (details in Figure S3). XRD investigations(Figure S4 and Table S1) gave only one reflectioncorresponding to d = 3.67 nm, together with the second-order reflex. As no aligned samples could be obtained and alsono hk cross reflections with h and k ≠ 0 could be detected inthe XRD patterns, an unambiguous phase assignment is in thiscase not possible, though a simple lamellar organization with d= 3.67 nm can be excluded on the basis of the optical texture(Figures 2A and S3).Considering the textural features, indicating an optically

uniaxial columnar organization with negative birefringence, andalso considering the LC phase structures of related com-pounds,20,21 it could be assumed that B12 forms an LC phasecomposed of honeycombs formed by the π-conjugated rodlikecores and filled with the lateral alkyl chains. The glycerol groupsform separate polar columns involving the hydrogen-bondingnetworks of the terminal glycerol groups aligned along theedges of the honeycombs, thus stabilizing this structure.22,23

Considering the measured d value, two different honeycombstructures are possible, one with a hexagonal p6mm lattice withahex = 4.24 nm and formed by triangular honeycombs and theother one a simple square lattice (p4mm) with asqu = 3.67 nmcomposed of square honeycombs. In both cases, the side lengthof the honeycombs corresponds to lattice parameters ahex andasqu, respectively. As the side length of the square honeycombwould be smaller than the shortest possible molecular lengthrange Lmin = 4.0 nm (Figure S1), this structure can be excluded.The side length of a triangular honeycomb (4.24 nm) is withinthe limits provided by the molecular length range (4.0−4.4 nm)and corresponds to the structure reported for relatedcompounds with the same core unit but other side chains(model in Figure 2B).20 In this case, the triangular cells have awall thickness approximately equal to the width of a singlearomatic core of B12 (estimations in Table S2), which is also inexcellent agreement with previous observations.24 A hexagonalcolumnar phase (p6mm) with a triangular honeycombstructure, as shown in Figure 2B, is therefore most likely forthis columnar LC phase.3.2. Lyotropic Properties of B12/Lipid Mixtures.

3.2.1. Aggregate Visualization by Transmission ElectronMicroscopy (TEM). While pure compound B12 is not water-soluble and can also not be dispersed in water by ultrasonictreatment, the mixed films of DPPC or DMPC with B12 couldbe readily dispersed by ultrasonication, and almost clearsuspensions were obtained. This observation indicated that

either vesicular structures or other types of small aggregateswere formed after the ultrasonication procedure. Foridentification of the aggregate type, TEM images wererecorded. Two different techniques, TEM with negativelystained dried samples and cryo-TEM with vitrified aqueoussamples, were used. A negatively stained sample of 1:10 mixtureB12/DPPC prepared at room temperature (lipid in the gelphase) shows crinkled vesicles (Figure 3A) resulting from the

drying process during sample preparation. A separation intodomains of different composition, i.e., pure DPPC and a B12-rich domain as observed before by confocal microscopy,10

could not be seen. This is probably due to the fact that thedifference in electron density between the two domains ofdifferent composition is only marginal.The cryo-TEM images of samples quenched from 22 °C

show the same vesicular structures with diameters of between50 and several hundred nanometers as the stained/driedsamples (Figure 3B). Some of the vesicles are faceted asexpected for gel-phase lipids. The more rigid parts may beenriched in B12. Again, no clear indication of domain formationis evident due to the marginal difference in electron densitybetween the domains.The aggregates of the B12/DPPC 1:4 mixture prepared at

room temperature look fundamentally different from those ofthe 1:10 mixture. The images of the negatively stained samplesshow flat sheetlike structures and no vesicles at all (Figure 3C).The sheets are in the form of single or connected rows of discswith diameters of ca. 200 nm. The aggregates are very uniformin electron density and are obviously very rigid as no curvedlayers are seen. The cryo-TEM image of the same 1:4 mixturesshows rather large, flat structures (Figure 3D) of up to severalmicrometers in width and length covering the holes of the film.Also, smaller disklike structures as in the negatively stainedsamples can be observed. The cryo-TEM images indicate thepresence of very thin and flat layers that seem to be quite rigid.

3.2.2. Differential Scanning Calorimetry. The DSCthermograms of different B12/lipid (DPPC or DMPC)mixtures show that for all mixing ratios a thermotropictransition close to the pure lipid’s main transition (Tm(DPPC)= 41 °C, Tm(DMPC) = 24 °C) is present. In addition, several

Figure 2. (A) Texture of the LC phase formed on cooling of B12 at178 °C; completely dark areas represent homeotropically alignedregions (honeycombs perpendicular to the substrate surfaces) andindicate the optical uniaxiality of the columnar phase. (For additionaltextures with the λ plate, see Figure S2.) (B) Model of the organizationof B12 in the LC triangular honeycomb phase.

Figure 3. TEM images of B12/DPPC samples prepared at 22 °C: (A)B12/DPPC 1:10 sample negatively stained with UO2Ac2; (B) cryo-TEM image of the same sample; (C) B12/DPPC 1:4 samplenegatively stained with UO2Ac2; and (D) cryo-TEM image of the samesample.

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transitions at higher temperatures can be seen (Figure 4). Thepeak of the main phase transition of the phospholipidsdecreases with increasing B12 content. This indicates that atlow temperature the system is probably phase-separated withpure phospholipid domains and other domains which are eitherpure B12 domains or are enriched in B12 with residualphospholipid.The area of the peak of the main transition of DPPC

decreases with increasing B12 content, but not in a linearfashion (Figure 4 C,D). For a 1:10 mixture, the value of thetransition enthalpy is reduced to 50%, and for a 1:4 mixture, toca. 20%. For a 1:10 mixture, therefore, 50% of the DPPC islocated in the B12/DPPC domains so that the B12/DPPC ratiois ca. 1:5.5. For a 1:4 mixture, this ratio is only slightly changedto 1:4. For B12/DMPC, the decrease in the transition enthalpywith increasing B12 content is more pronounced but alsononlinear, meaning that the phase-separated domains containmore DMPC. In this case, the peak corresponding to the maintransition of pure DMPC has vanished when the mole fractionof B12 exceeds 0.2.The peaks observed at higher temperature are caused by

thermotropic transitions of B12/PC complexes, i.e., the PCchains in the complexes with B12 become disordered at higher

temperature. The sum over the transition enthalpies of all peaksstays almost constant up to a 1:1 ratio for mixtures with eitherDPPC or DMPC (Figure 4C,D). This indicates that the lipidchains in the B12/PC mixtures reach the same degree ofdisorder at high temperature, only that the melting occurs overa wider temperature range covering the additional transitions athigher temperature. The thermograms are quite reproducible,with repeated temperature cycles (2−95 °C) always resulting inthe same transition temperatures and enthalpies. Metastablestates can therefore be excluded.Usually, three different thermotropic transitions are observed

about 25−30 K higher than the main lipid transition peak atTm. Whereas Tm of the main transition is nearly independent ofcomposition, the pattern of the other peaks changes slightlywith increasing polyphile content, indicating that thecomposition of the phase-separated domains containing B12and PC changes, as concluded from the nonlinear decrease inthe transition enthalpy of the main peak (Figure 4).We also performed DSC experiments with the unsaturated

phospholipid POPC to test whether this “complex formation”of B12 occurs only with PCs with saturated chains. The maintransition temperature for POPC is −2 °C29 and thus notdetectable by our DSC. Also, for this lipid three endothermic

Figure 4. DSC thermograms of different mixtures of B12 with (A) DPPC and (B) DMPC with molar ratios as indicated. Transition enthalpies ofmixtures of B12 with DPPC (C) or DMPC (D). Black: transition enthalpy of the main transition at ca. Tm (41 °C for B12/DPPC or 24 °C for B12/DMPC). Red: Sum of transition enthalpies of the upper three peaks. Blue: total transition enthalpy. All transition enthalpies are based on lipidcontent.

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transitions between 15 and 40 °C were observed, indicatingalso that lipids with unsaturated chains can form “complexes”with B12 (Figure S5).3.2.3. Temperature-Dependent ATR-FTIR Investigations of

B12/PC Mixtures. The DSC experiments presented aboveshowed that several thermotropic transitions are present inmixed samples of DPPC or DMPC with B12. We investigatedthis phenomenon further by infrared spectroscopy. Theseexperiments should give information on the molecular levelabout changes in chain conformation of the lipids andintermolecular interactions with B12.Thin B12/lipid films of different mixing ratios were prepared

by vesicle spreading on a BioATR II Si crystal. To discriminatebetween DPPC and B12 alkyl chains in the IR spectra, DPPC-d62 with perdeuterated alkyl chains was used. Thus, thepositions of the vibrational bands of the B12 CH2 stretchingvibrations υas/s(CH2) and the DPPC-d62 CD stretchingvibrations υas/s(CD2) are well separated in the experimentalIR spectra. The positional changes of these bands and theDPPC-d62 CO carbonyl-stretching vibration were inves-tigated as a function of temperature while continuously heatingor cooling the aqueous sample.Figure 5A shows the CD2 vibrational bands of DPPC-d62 in

the 1:4 B12/DPPC-d62 mixture at different temperatures. Thechanges in band position and width of the symmetric CD2

stretching band due to the melting of the acyl chains of DPPC-d62 are clearly evident. The temperature dependence of the

maximum of the symmetric CD2 vibrational band of DPPC-d62for the two mixtures B12/DPPC-d62 = 1:10 and 1:4 is shown inFigure 5A,B. For the 1:10 mixture, the band position remainsconstant (at 2089 cm−1) for temperatures of between 20 and 39°C. Further heating to 55 °C leads to an increase inwavenumber to 2096 cm−1, indicating the fluidization of thechains due to partial chain melting. This process occurs partiallyat the transition temperature Tm of the pure lipid, but thewavenumber increases further at higher temperature in therange where the first of the additional transitions is seen. Thealmost-linear increase in wavenumber seen above a temperatureof 55 °C is due to the normal wavenumber change observed forfluid chains. For the 1:4 sample, the change of the wavenumberof the maximum of the CD2 stretching band is very similar.Because of the decreased amount of DPPC-d62 in the sample,the data show higher scattering. The changes in wavenumber ofthe CD2 stretching bands are shifted to slightly lowertemperature compared to the DSC peaks. This is due to theperdeuteration of the acyl chains of DPPC which shifts itstransition temperature from the gel to the liquid-crystallinephase from 41.5 to ca. 37 °C.25,26

The band positions of the CH2 stretching bands of the B12lateral alkyl chains (at 20 °C, υs(CH2) = 2852 cm−1 for B12/DPPC-d62 = 1:4 and = 1:10) indicate that the C12 alkyl chainsare already in a fluid state at room temperature, as expected.The band positions increase continuously with temperature(not shown).The lipid CO stretching vibrational band (not shown) is

shifted to slightly lower wavenumber with increasing temper-ature (20 °C, 1741 cm−1 to 70 °C, 1736 cm−1; data notshown), indicating an increase in the degree of hydration of thelipid headgroups at higher temperature. This commonphenomenon is also observed for pure lipid membranes.27,28

Thus, B12 obviously does not interact with the lipid headgroupregion and does not change the hydration state of the lipidheadgroups.

3.2.4. Polarized ATR-FTIR of Oriented Hydrated MixedFilms of B12/DPPC-d62. The thick films cast from chloroformsolution were hydrated with water vapor via the gas phase toperform ATR experiments. Using polarized light, spectra similarto those obtained by unpolarized light were observed (Figure6). From the CD2 vibrational bands taken with s- and p-polarized light, we could determine a dichroic ratio RATR of 0.94for the symmetric stretching band. This indicates a highorientation of the DPPC molecules with a tilt angle of 17°relative to the membrane normal. This is typical of almost pureDPPC with less than 100% hydration. The two B12 ringvibrations at 1519 and 1609 cm−1 were very pronounced andadequately separated to use them for the determination of theorientation of B12. These ring vibrations were assigned by DFTcalculations on a similar compound, namely, B4 with shorterchains. The results of these calculations and the bandassignments are shown in Figure S6. In the spectra obtainedwith p- and s-polarized light, these bands differ significantly inintensity and integral size. Calculating dichroic ratios RATR

using the integral areas of the bands results in RATR values of 15or even higher for the band at 1519 cm−1. In spectra taken withp-polarized light, the vibrational band at 1609 cm−1 shows onlya very weak intensity. Therefore, it is difficult to calculatereliable integral areas and thus RATR values for this band (Figure6). The RATR for this band is thus even higher than 15. Thisindicates that the B12 molecules are highly oriented in themembrane with a tilt angle of less than 30°. As we know that

Figure 5. ATR-FTIR spectroscopy. (A) CD2 bands of B12/DPPC-d62= 1:4 at different temperatures and (B) plots of the DSC thermogramusing normal DPPC (solid line) and the positions of the symmetricDPPC-d62 CD2 stretching vibration (circles) of a 1:10 B12/DPPC-d62(black line and symbol) and a 1:4 B12/DPPC-d62 mixture (red lineand symbol).

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the B12 molecules are not homogeneously distributed withinthe lipid membrane from the DSC results but form aggregatesor complexes distributed within the membrane, we assume thatthis angle represents mainly the orientation of the B12molecule within the B12-enriched domains.3.2.5. Fluorescence Spectra of B12/DPPC. While the FT-IR

spectra documented the molecular properties of DPPC in themixture with B12 as a function of temperature showing a two-step behavior of changes in conformational properties of thelipid chains, the fluorescence properties of B12 in DPPCmembranes can give information on the aggregation behaviorof B12 in the plane of the membrane. B12 shows bluefluorescence in organic solvents due to the long conjugated πsystem of the backbone of the molecule. The DSC and FT-IRdata presented above lead to the proposition, together with theconfocal laser scanning images of GUVs of B12/DPPCmixtures, that at room temperature a phase separation occursin the plane of the membrane leading to starlike domainsenriched in B12.10 Figure 7A shows a confocal fluorescenceimage (CFM) of a GUV of a B12/DPPC (1:10) mixture.The fluorescence spectrum of B12 in chloroform is

characterized by an emission maximum at 428 nm with ashoulder at 449 nm (Figure S7 and Figure 7B, dashed blackline). When B12 is incorporated into DPPC vesicles, theemission maximum is clearly shifted to a longer wavelengthwith the highest intensity at 466 nm, and shoulders at lowerwavelength (444 nm) and also at longer wavelength (486 nm).This large red shift of the emission is a clear indication of thepresence of π−π interactions between the aromatic rings of B12when incorporated into DPPC bilayers.29−33 When the vesiclesare heated, the fluorescence emission spectrum changes are inthe beginning only marginal, with the shorter-wavelengthshoulder increasing only slightly in intensity. When thetemperature reaches 75 °C, i.e., the system is above alltransitions observed by DSC or FT-IR, the fluorescencespectrum is nearly identical to the spectrum recorded inchloroform solution, indicating that the B12 molecules are nowmolecularly dispersed in the plane of the bilayer, i.e., no phaseseparation of B12 is present any more. This agrees well with theprevious observation that at room temperature the B12

molecules are rigid and well packed, which suggests that B12molecules may form extended filaments with parallel π facesand with the lateral alkyl chains interacting and preferentiallymixing with the alkyl chains of the DPPC molecules.10 High-resolution 13C NMR studies going beyond the 1H NMR

Figure 6. Section of the p- (black line) and s-polarized (red line)ATR-FTIR spectrum of a B12/DPPC-d62 = 1:4 mixture, cast fromchloroform solution and hydrated via water vapor (gas phase). Thegreen frame marks the region magnified in the inset. The highlightedvibrational bands at 1518 and 1609 cm−1 were determined by DFTcomputations to be in-plane ring vibrational bands of the B12backbone (Supporting Information).

Figure 7. (A) Confocal fluorescence micrograph showing thesignature of its self-organization in snowflakelike domains (greenautofluorescence) within giant unilamellar vesicles made of B12/DPPC (1:10). (B) Fluorescence emission spectra of B12 in sonicatedvesicles composed of B12/DPPC (1:10) as a function of temperature.The fluorescence spectrum of B12 in chloroform is shown forcomparison (black dotted line). (C) Fluorescence anisotropy r of B12in DPPC as a function of temperature and DSC thermograms (straightline, c(DPPC) = 2.0 mM) of mixtures B12/DPPC = 1:10 (black) and1:4 (blue) in H2O; the dashed lines were drawn to guide the eye.Excitation wavelength λex = 342 nm and detection wavelength λem =466 nm.

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experiments reported in ref 10 in fact indicate that the motionof the aromatic cores is restricted to π flips, which is a commonmotif in π−π stacked aromatic molecules (Achilles et al.,unpublished results). At high temperature, this two-dimen-sional arrangement breaks down and the B12 molecules aremore or less homogeneously distributed in the bilayer plane.Temperature-dependent fluorescence depolarization meas-

urements give information on the dynamics of reorientation ofB12 in DPPC vesicles. This method, usually using addedfluorescence probes such as diphenylhexatriene, has beenextensively used to study order and dynamics in mem-branes.34−37 In our case, the fluorescence anisotropy of B12itself can be studied without the need for additional probes.The temperature-dependent measurements of B12 in DPPCshow that the fluorescence anisotropy r does not change duringthe phase transition of almost pure DPPC at 41 °C butdecreases only at higher temperature in the temperature rangeof the additional DSC transitions (Figure 7C). Thus,fluorescence anisotropy data support the previous IR andNMR spectroscopic data that only at high temperature do theB12 molecules become mobile due to the breakdown of theaggregates stabilized at room temperature by π−π interactionsbetween the aromatic moieties.10

3.2.6. X-ray Scattering Experiments. The phase behaviorand the demixing phenomena found by DSC, ATR spectros-copy, fluorescence spectroscopy, and also by confocalfluorescence microscopy on GUVs10 were further investigatedusing temperature-dependent X-ray powder diffractometry togain more insight into the structural organization of the B12/DPPC system as a function of temperature. Two B12/DPPCsamples of different compositions (B12:/DPPC = 1:10 and1:4) were prepared within a capillary, and temperature-dependent powder patterns of each sample were recordedand displayed in so-called contour plots (Figure S8). In thesmall-angle X-ray scattering region (SAXS), up to fourreflections with equidistant positions are visible, indicatingthe presence of a lamellar structure. The wide-angle X-rayscattering (WAXS) region shows two strong reflections, the(020) and the (110) reflections from the chain lattice of thelipids. From these reflections, the chain packing mode and tiltangle of the chains can be determined to be similar to those ofpure DPPC.The repeat distance (membrane plus interlamellar water

layer thickness) within the multilayer stacks was determined atdifferent temperatures. At room temperature, the lamellarrepeat distances of the mixtures are slightly larger with d (1:10)= 7.25 nm and d (1:4) = 7.14 nm (Figure 8) compared to thedistance found for pure DPPC bilayers (d = 6.35 nm at 20 °Cin the Lβ′- phase and −6.70 nm at 50 °C in the Lα phase38).This means that interlamellar excess water layers are increasedin thickness and/or the molecular tilt has decreased.In the WAXS region, the reciprocal spacings s of 2.33 nm−1

(110) and 2.43 nm−1 (020) for 1:10 mixture and 2.31 nm−1

(110) and 2.47 nm−1 (020) for the 1:4 mixture indicate thepresence of an orthorhombic (symmetry group Pbnm)herringbone lattice39 with tilted chains characteristic of bilayersin an Lβ′ phase at room temperature. On cooling, an increase insplitting of the two WAXS reflections is observed, indicating afurther change in chain packing. When the sample is furthercooled to below 0 °C, the crystallization of excess water leads tothe appearance of sharp ice reflections (Figure S8).40 Thefreezing of interlamellar water leads to reduced membranerepeat distances (d = 6.06 nm for the 1:10 mixture and d = 6.13

nm for the 1:4 mixture). After heating the sample again toroom temperature, the initial repeat distance and thusmembrane structure are restored, indicating a reversible processof excess water freezing (dehydration/hydration).At higher temperatures, the two WAXS reflections change

into a broad scattering peak indicating the transition from thegel- to the fluid-phase membrane. For the 1:10 mixture, thisoccurs at 42 °C, coincident with the main transition at Tmobserved by DSC. For the 1:4 mixture, this occurs at a highertemperature of 50 °C simultaneously with the first high-temperature transition peak in the DSC thermograms. Abovethis transition, still-intense WAXS peaks are visible at slightlydifferent reciprocal spacings. For the 1:10 mixture, other WAXSpeaks appear (sT>42°C (1:10) = 2.25 and 2.39 nm−1). Thesereflections indicate an ordered lipid chain packing but in adifferent geometry compared to that of the normal Lβ′ phasewith tilted chains (Figure 8).For the 1:4 mixture, this transition is not that distinct

because the WAXS reflections are broad and the changes aresmall (sT>50°C (1:4) = 2.25, 2.37, and 2.40 nm−1 for the 1:4mixture). There is even a third WAXS peak present whichmight be due to the alkyl chain packing of a second (lamellar)ordered phase (Lβ, rich in B12, while the lipid-rich phasealready adopts a fluid Lα phase) or an underlying tricliniclattice. Above 68 °C, fluid phases are formed. In the WAXSregion, no sharp reflections but only a halo is visible. The

Figure 8. One-dimensional scattering patterns selected at differenttemperatures from the contour plots shown in Figure S8 for mixturesof (A) B12/DPPC = 1:10 and (B) B12/DPPC = 1:4. Temperaturesare indicated in the graph. Scattering curves were taken as slices fromthe contour plots in Figure S8. Samples were prepared with 50 wt %H2O in glass capillaries.

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membrane thickness is decreased further to d = 6.37 nm (1:10)and 5.57 nm (1:4).To overcome the resolution problems in the SAXS region

with our in-house X-ray setup, high-resolution X-ray diffractionexperiments with ordered multibilayer samples were carried outwith synchrotron radiation at the DESY HASYLAB. B12/DPPC samples with mixing ratios of 1:10 and 1:4 wereinvestigated at different temperatures. Reflectometry measure-ments reveal the presence of two independently scatteringlamellar structures for both mixtures. For the 1:10 mixture,these are observed in the two-phase region between 42 and 67°C and for the 1:4 mixture for all temperatures lower than 72°C (Figure 9). The diffractograms exhibit two independent sets

of equidistant peaks. (For indexing, see blue and red dashes.)This proves the hypothesis that in the two-phase region twoimmiscible lamellar phases of different composition are presentin both samples.The molecular organization of the phases of the 1:10 mixture

was also investigated using grazing incidence X-ray diffrac-tometry (GIXD, Figure 10). The scattering pattern at 25 °Cshows lamellar reflections along the scattering vector qz(parallel to the membrane stack normal) and the reflectionsfrom the chain lattice on the equator. However, intensities offthe equator are also evident in the 2D patterns in the WAXSregion (red arrow), indicating that the structure probablyconsists of a coexistence of domains with tilted chains asalready concluded from the appearance of two wide-anglereflections (110) and (020) and domains with nontilted chainswith respect to the membrane normal giving the reflections onthe equator. Although no demixing in the qz direction by meansof reflectometry can be found, a lateral demixing of the lamellaeoccurs at 25 °C. Two of the WAXS reflections populate well-known positions of the DPPC alkyl chain reflexions at s(020) =2.28 nm−1 and s(110) = 2.38 nm−1 as observed with theunoriented samples. At a higher temperature of 48 °C, beforethe high-temperature peaks, the WAXS reflections out of theequator have turned into a diffuse halo (red arrow) and onlythe sharp reflections on the equator are still present,corresponding to s values of 2.23 and 2.37 nm−1, similar tothe values seen for the unoriented sample (see above andFigure S9). These results prove our assumptions obtained fromnonoriented samples that at this temperature the sampleconsists of partially molten domains probably containing onlyDPPC and domains with a B12/DPPC mixture, where theDPPC chains are ordered but packed in a different latticecompared to the packing in the Lβ′phase.In the high-temperature phase at 75 °C, above all transitions

in the DSC, no sharp WAXS reflections are present, but only abroad halo at sT=75°C = 2.13 nm−1 (red arrow) indicatingunordered lipid alkyl chains. Most likely, at this temperature afluid membrane phase (Lα) is formed, whereas the B12 andDPPC molecules are homogeneously mixed.

Figure 9. X-ray reflectometry curves (SAXS) of aqueous mixtures ofB12/DPPC with ratios of 1:10 (left) and 1:4 (right) at 25 °C (top), 48°C (C, D), 75 °C (E), and 78 °C (F). Two lamellar lattices areindexed for B−D with blue (lattice 1) and red (lattice 2) verticaldashes, and a single lamellar lattice is indexed for A, E, and F with bluevertical dashes.

Figure 10. GIXD patterns of an aqueous mixture of B12/DPPC = 1:10 at 25 °C (A), 48 °C (B), and 75 °C (C). Oriented and hydrated multilayerstacks on a Si solid support at 99% relative humidity. Because of the strong intensity decay with increasing scattering vector (qz, qr), the color codingsfor the SAXS and WAXS regions of each GIXD image have been scaled differently and are therefore separated by a gap.

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4. SUMMARY AND CONCLUSIONS

Polyphilic molecule B12 can be stably incorporated into bilayermembranes composed of DPPC or DMPC at differentconcentrations. When the phospholipids are in the gel phase,a tendency for phase separation into almost pure phospholipiddomains and domains with a certain ratio of B12 to DPPC isobserved when only 10 mol % B12 is present. In samples with ahigher B12 content, the amount of pure DPPC decreased butnot in a linear fashion. Using confocal fluorescence microscopyof mixed GUVs, a large-scale phase separation could bevisualized as described before.10 In electron microscopy imagesof smaller vesicles presented here, this phase separation was notvisible due to a lack of contrast between the different domainsof different composition. In the DSC curves, severalendothermic high-temperature transitions are visible besidesthe phase transition due to the almost pure DPPC domainsindicating that the mobilization of the different components ofthe B12-DPPC “complex” occurs in several steps. This could bequalitatively verified by FT-IR spectroscopic investigationsusing DPPC with perdeuterated chains in the mixture with B12.X-ray investigations of oriented bilayers also showed the

coexistence of these two phases, one almost pure DPPC andthe other one enriched in B12, by the appearance of twolamellar repeat distances over a wide temperature range up to70 °C. In the WAXS region also, the coexistence of diffractionsof two coexisting chain lattices could be seen, the almostunperturbed one of the pure DPPC domains with tiltedmolecules and diffractions from the B12-DPPC “complex” withnontilted DPPC molecules. These reflections disappeared andwere converted into a diffuse halo when the system was heatedinto the high-temperature liquid-crystalline phase. Therefore,above all thermotropic transitions a homogeneous bilayer phaseis formed and no phase separation is observed.In summary, we could show that B12 can be incorporated

into DPPC and DMPC bilayers but phase separates at roomtemperature into domains consisting of almost pure PC anddomains with a PC/B12 ratio of 3−4:1 in the case of DPPC.When B12 is incorporated into DMPC vesicles, a similarbehavior is observed. Because of a better matching of the lengthof B12 and the thickness of the DMPC bilayer, the B12concentration at which the phase transition of pure DMPCvanishes is lower than for mixtures with DPPC; i.e., above amolar fraction of B12 of 0.25 only the high-temperature DSCpeaks remain. The study shows that despite the drasticallydifferent chemical structure of the two components B12 andDPPC, the bilayer phase with phase separation in the plane isremarkably stable over a wide temperature range where thephospholipid component undergoes first a phase transition intoa fluid Lα phase before the mixed domains of B12 and DPPCundergo a stepwise mobilization and finally melt, where an Lα

phase with a homogeneous distribution of the B12 molecules isformed. At all temperatures, the B12 molecules are oriented ina trans-membrane fashion, but their angle of orientation andtheir mobility change with temperature. At low temperature,the complexes of B12 with DPPC or DMPC but also withPOPC are very stable despite the differences in molecularstructure among the three lipid compounds, as the DSC scansare very reproducible and the same thermotropic peaks arealways seen in consecutive heating scans (not shown). As theTEM images show, the lamellar structure is retained and veryextended lamellae with little tendency to bend are formed. Thiscan be attributed to the stiff core of the B12 molecules ordering

the PC molecules in their neighborhood and increasing thebending stiffness due to a rivetlike action holding the twomonolayers together.Overall, the self-assembly of X-shaped bolapolyphile B12 is

dominated by the core−core interactions which strongly favor adense packing and parallel alignment of the long oligo-(phenylene ethynylene) cores. Not only the Meier−Saupe-type interactions (reduction of excluded volume) and attractiveπ−π and C−H−π interactions between the rodlike units32,41−43but also the strong segregation of these rigid units from theflexible alkyl chains (rigid−flexible incompatibility)22,23 shouldcontribute to this effect. Simultaneously, as two sides of themolecule are shielded by the lateral alkyl chains, the possiblemodes of packing are restricted, mainly leaving the option of alinear side-by-side stacking in ribbons (strings). In thethermotropic LC phase of the bulk material, this unavoidablyleads to a triangular honeycomb structure with the long lateralalkyl chains filling the space inside the resulting triangularhoneycomb cells (Figure 2). Though a swelling of the alkylchain domains by simple n-alkanes (C32H66) appears not to bepossible (no uptake of the hydrocarbon was observed and thephase transitions were not influenced in the contact region),these bolaamphiphilic molecules are capable of interacting withlipids, probably as in this case the interaction can take placesimultaneously with the polar groups as well as with thelipophilic segments. The much increased contribution of thepolar ends and alkyl chains in these mixtures modifies theirpreferred mode of self-assembly. The honeycombs break up,and a mixed organization of lipid and B12 in a commonstructure with a trans-membrane orientation of the aromaticcores becomes dominating. Even in the presence of the lipids,the core−core interactions seem to be retained and B12-enriched domains segregate from almost pure DPPC or DMPCdomains at low B12 content of the bilayers. It is reasonable toassume that the aromatic cores are organized in strings tomaximize the core−core interactions. The lateral chains,together with the alkyl chains of the lipids, isolate these strings.This separation of B12-enriched and almost pure PC regionswas also observed in GUVs, which is in this case indicated bythe formation of star-shaped B12-rich domains with hexagonalsymmetry.10 We proposed in this study that the hexagonalsymmetry results from a hexagonal honeycomb structure. Incontrast to the triangular honeycombs, where the orientation ofthe molecules is parallel to the 2D lattice, in the hexagonalhoneycomb it is perpendicular to the hexagonal lattice (trans-membrane). These honeycombs could be considered to resultfrom a fusion of individual strings. At higher temperature in theLα phase, the increased mobility prevents the formation of aregular array, leading to irregularly distributed strings (orclusters) or even single B12 molecules as a pronounced shift inthe fluorescence emission to shorter wavelength is observed,indicating vanishing π−π interactions.These investigations show that a stable incorporation of long

and stiff π-conjugated systems, i.e., charge-carrier conductingrods, into lipid membranes is possible due to the fact that π−πinteractions can stabilize the trans-membrane orientation of thebolapolyphiles in the bilayer. This is of wider interest formolecular electronics in well-organized hybrid structures andsuperstructures,43−47 especially for the addressing of cells byexternal electrical signals as required in device-coupling sensors,stimulators, or chips with living cells or protocells.48,49

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■ ASSOCIATED CONTENT*S Supporting InformationSupplemental methods, details of the thermotropic behavior ofB12, DSC curves of POPC/B12 lipid dispersions, FT-IRspectra and band assignments of B12, fluorescence spectra ofB12, and additional X-ray data on B12/DPPC mixtures. Thismaterial is available free of charge via Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONNotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe thank the Deutsche Forschungsgemeinschaft (DFG) forfinancial support in the framework of the Forschergruppe 1145.

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Fleischer, B., Eds.; Methods in Enzymology; Academic Press: SanDiego, CA, 1989; Vol. 172, pp 462−471.(35) Jahnig, F. Structural order of lipids and proteins in membranes:Evaluation of fluorescence anisotropy data. Proc. Natl. Acad. Sci. U.S.A.1979, 76, 6361−6365.(36) Van Blitterswijk, W. J.; Van Hoeven, R. P.; Van Der Meer, B. W.Lipid structural order parameters (reciprocal of fluidity) inbiomembranes derived from steady-state fluorescence polarizationmeasurements. Biochim. Biophys. Acta 1981, 644, 323−332.(37) Lentz, B. R.; Moore, B. M.; Barrow, D. A. Light-scatteringeffects in the measurement of membrane microviscosity withdiphenylhexatriene. Biophys. J. 1979, 25, 489−494.(38) Nagle, J. F.; Tristram-Nagle, S. Structure of lipid bilayers.Biochim. Biophys. Acta 2000, 1469, 159−195.(39) Forster, G.; Meister, A.; Blume, A. Chain packing modes incrystalline surfactant and lipid bilayers. Curr. Opin. Colloid Interface Sci.2001, 6, 294−302.(40) Brill, R.; Tippe, A. Gitterparameter von Eis I bei tiefenTemperaturen. Acta Crystallogr. 1967, 23, 343−345.(41) Handbook of Liquid Crystals, 2nd ed.; VCH-Wiley: Weinheim,2014.(42) Steed, J. W.; Atwood, J. L. Supramolecular Chemistry, 2nd ed.;Wiley: Chichester, 2009.(43) Klosterman, J. K.; Yamauchi, Y.; Fujita, M. Engineering discretestacks of aromatic molecules. Chem. Soc. Rev. 2009, 38, 1714−1725.(44) Petty, M. C. Molecular Electronics: From Principles to Practice;Wiley: Chichester, 2008.(45) Arrhenius, T. S.; Blanchard-Desce, M.; Dvolaitzky, M.; Lehn, J.-M.; Malthete, J. Molecular devices: Caroviologens as an approach tomolecular wiressynthesis and incorporation into vesicle membranes.Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 5355−5359.(46) Fuhrhop, J. H.; Krull, M.; Schulz, A.; Moebius, D. Bolaformamphiphiles with a rigid hydrophobic bixin core in surface monolayersand lipid membranes. Langmuir 1990, 6, 497−505.(47) Visoly-Fisher, I.; Daie, K.; Terazono, Y.; Herrero, C.; Fungo, F.;Otero, L.; Durantini, E.; Silber, J. J.; Sereno, L.; Gust, D.; Moore, T. A.;Moore, A. L.; Lindsay, S. M. Conductance of a biomolecular wire. Proc.Natl. Acad. Sci. U.S.A. 2006, 103, 8686−8690.(48) Duan, X.; Fu, T. M.; Liu, J.; Lieber, C. M. Nanoelectronics-biology frontier: From nanoscopic probes for action potentialrecording in live cells to three-dimensional cyborg tissues. NanoToday 2013, 8, 351−373.(49) Voelker, M.; Fromherz, P. Signal transmission from individualmammalian nerve cell to field-effect transistor. Small 2005, 1, 206−210.

Langmuir Article

DOI: 10.1021/la504903dLangmuir 2015, 31, 2839−2850

2850

S1

Su

ppor

ting

Info

rmat

ion

Tem

pera

ture

dep

ende

nt in

-pla

ne st

ruct

ure

form

atio

n of

an

X-

shap

ed b

olap

olyp

hile

with

in li

pid

bila

yers

B

ob-D

an L

echn

er† , H

elga

rd E

bert§ , M

arko

Pre

hm†,

§ , Stef

an W

erne

r�, A

nnet

te M

eist

er�

, Ger

d

Hau

se‡ , A

ndré

Bee

rlink

&, K

ay S

aalw

ächt

er# , K

irste

n B

acia

�, C

arst

en T

schi

ersk

e§ , Alfr

ed

Blu

me† *

S2

Bul

k pr

oper

ties o

f B12

Fi

gure

S1.

CPK

mod

els

of B

12 s

how

ing

the

mol

ecul

ar c

onfo

rmat

ions

with

A)

the

shor

test

an

d B

) the

long

est p

ossi

ble

dist

ance

bet

wee

n th

e te

rmin

i of t

he g

lyce

rol g

roup

s of B

12.

Figu

re S

2. D

SC h

eatin

g an

d co

olin

g tra

ces

of b

ulk

B12

(10

K m

in-1

) with

pea

k te

mpe

ratu

res

and

enth

alpy

val

ues (

in p

aren

thes

es, �

H/k

J mol

-1) o

f the

pha

se tr

ansi

tions

.

A

B

L min =

4.0

nm

L max

= 4

.4 n

m

S3

Figu

re S

3. T

extu

res

of t

he L

C p

hase

of

bulk

B12

bet

wee

n cr

osse

d po

lariz

ers:

a)

at T

=

178

°C, b

) sam

e te

xtur

e w

ith �

-ret

arde

r pla

te; c

) tex

ture

as

obse

rved

at T

= 1

81 °

C d

urin

g th

e Is

o-LC

tra

nsiti

on w

ith �

-ret

arde

r pl

ate,

red

are

as a

re r

esid

ues

of t

he i

sotro

pic

liqui

d; t

he

orie

ntat

ions

of t

he h

igh

and

low

inde

x ax

es o

f the

indi

catri

x ar

e sh

own

in th

e in

sets

; d) s

how

s th

e or

gani

zatio

n of

the

mol

ecul

es i

n th

e sp

heru

litic

dom

ains

; th

e ci

rcle

s in

dica

te t

he

orie

ntat

ion

of t

he h

oney

com

b cy

linde

rs, t

he b

old

blac

k lin

es i

ndic

ate

the

orie

ntat

ion

of t

he

arom

atic

cor

es i

n th

e cy

linde

rs.

In t

he s

pher

uliti

c do

mai

ns t

he c

ylin

ders

are

org

aniz

ed i

n ci

rcle

s aro

und

the

cent

re; m

olec

ules

with

the �-

conj

ugat

ion

path

way

alo

ng th

e hi

gh in

dex

axis

le

ad to

a b

lue

shift

, tho

se w

ith th

e �-

conj

ugat

ion

path

way

per

pend

icul

ar to

the

high

inde

x ax

is

give

a y

ello

w s

hift.

Bec

ause

blu

e/gr

een

colo

r is

in th

e no

rthea

st a

nd s

outh

wes

t qua

dran

ts th

e al

ignm

ent o

f th

e �-

conj

ugat

ion

path

way

, i.e

. the

orie

ntat

ion

of th

e lo

ng a

xes

of th

e ar

omat

ic

core

s, is

per

pend

icul

ar to

the

cylin

der l

ong

axis

(neg

ativ

e bi

refr

inge

nce)

.

S4

Figu

re S

4. X

RD

pat

tern

s of

the

Col

hex

phas

e of

bul

k B

12 a

t T

= 17

9 °C

(m

easu

red

upon

co

olin

g): a

, b) o

rigin

al d

iffra

ctio

n pa

ttern

s (a

ttem

pted

alig

nmen

t) an

d c,

d) c

orre

spon

ding

2�-

scan

; a, c

) wid

e an

gle

and

b, d

) sm

all a

ngle

.

S5

Tab

le S

1. C

ryst

allo

grap

hic

data

of

B12

: 2�

val

ues,

d-sp

acin

gs (

d obs

: ex

perim

enta

l; d c

alc:

calc

ulat

ed)

and

indi

ces

of t

he r

efle

ctio

ns o

f th

e LC

pha

se a

t T

= 17

9 °C

, in

dexe

d w

ith

assu

mpt

ion

of a

hex

agon

al la

ttice

.

2�ob

s/°

d obs

/nm

hk

d c

alc/n

m

d obs

-dca

lc

a hex

/nm

2.41

3.

67

10

3.67

0.

00

4.24

4.

75

1.86

20

1.

84

0.02

19.3

7 0.

46

diff

use

T

able

S2.

Cal

cula

tion

of m

olec

ular

vol

umin

a (V

mol

), vo

lum

ina

of th

e hy

poth

etic

al u

nit c

ell

(Vce

ll), a

vera

ge n

umbe

r of m

olec

ules

in th

is u

nit c

ell (

n cel

l) an

d ca

lcul

ated

ave

rage

num

ber o

f m

olec

ules

in

the

cros

s se

ctio

n of

the

cyl

inde

r w

alls

(n w

all)

for

the

poss

ible

tria

ngul

ar a

nd

squa

re h

oney

com

b ph

ases

of B

12.

Hon

eyco

mb

type

Pla

ne g

roup

a

/nm

V m

ol/n

m3

V cel

l/nm

3 n c

ryst

n liq

u n c

ell

n wal

l

trian

gula

r

p6m

m

4.2

4 1

.474

7.

00

3.56

2.

79

3.17

1.

06

squa

re

p4m

m

3.6

7 1

.474

6.

06

4.11

3.

23

3.67

1.

84

V cel

l = v

olum

e of

the

unit

cell

defin

ed b

y a s

qu2 x

h fo

r the

squa

re p

hase

and

ahe

x2 x si

n(60

°) x

h

for

the

trian

gula

r ho

neyc

omb

phas

e, a

ssum

ing

a he

ight

of

h =

0.45

nm

cor

resp

ondi

ng t

o th

e av

erag

e st

acki

ng d

ista

nce

alon

g th

e co

lum

ns (c

orre

spon

ding

to th

e m

axim

um o

f the

diff

use

XR

D w

ide

angl

e sc

atte

ring)

; V m

ol =

mol

ecul

ar v

olum

e as

cal

cula

ted

usin

g Im

mirz

is c

ryst

al v

olum

e in

crem

ents

[S1

]; n c

ryst =

num

ber

of m

olec

ules

in th

e un

it ce

ll, c

alcu

late

d ac

cord

ing

to n

cell

= V c

ell/V

mol

(ave

rage

pac

king

coe

ffic

ient

in th

e cr

ysta

l is

k =

0.7)

; nliq

u =

num

ber o

f mol

ecul

es in

th

e un

it ce

ll of

an

isot

ropi

c liq

uid

with

an

aver

age

pack

ing

coef

ficie

nt k

= 0

.55,

cal

cula

ted

acco

rdin

g to

nliq

u = 0

.55/

0.7

x n c

ryst[S

2] n

cell

= nu

mbe

r of m

olec

ules

in th

e un

it ce

ll in

the

LC

phas

e es

timat

ed a

s th

e av

erag

e of

that

in th

e n c

ryst a

nd n

liqu;

n wal

l = n

umbe

r of m

olec

ules

in th

e cr

oss s

ectio

n of

the

cylin

der w

alls

as c

alcu

late

d fr

om n

cell.

Bes

ides

the

disc

repa

ncy

betw

een

poss

ible

mol

ecul

ar le

ngth

(see

Fig

. S4)

and

cyl

inde

r siz

e in

th

e sq

uare

latti

ce (3

.67

nm),

also

the

num

ber o

f nw

all =

1.8

4 in

the

squa

re h

oney

com

b ph

ase

is

less

rea

sona

ble

for

X-s

hape

d bo

lapo

lyph

iles

with

alk

yl c

hain

s at

bot

h si

des

of th

e ar

omat

ic

core

. The

se c

hain

s inh

ibit

an e

ffic

ient

bac

k-to

-bac

k pa

ckin

g of

the

core

s, as

wou

ld b

e re

quire

d in

cyl

inde

r wal

ls fo

rmed

by

~2 m

olec

ules

in th

e cr

oss s

ectio

n.

S6

Diff

eren

tial S

cann

ing

calo

rim

etry

Figu

re S

5. D

SC th

erm

ogra

ms

of B

12:P

OPC

= 1

: 4

and

pure

PO

PC in

H2O

; the

lipi

ds m

ain

trans

ition

pea

k is

not

det

ecta

ble

by o

ur se

tup,

but

shou

ld b

e at

Tm

= -2

°C.

18.6 °C

28.9 °C

32.6 °C

1020

3040

50

0,0

0,1

0,2

0,3

0,4

PO

PC

pur

e B

12:P

OPC

= 1

:4

B12:

POPC

= 1

:4 in

H2O

Cp / kcal mol-1 K

-1

Tem

pera

ture

/ o C

S7

Den

sity

func

tiona

l the

ory

calc

ulat

ions

(DFT

).

DFT

cal

cula

tions

wer

e ca

rrie

d ou

t with

the

Gau

ssia

n03

W p

rogr

am p

acka

ge [

S3]

usin

g th

e hy

brid

func

tiona

l B3L

YP.

[S4-

S7] T

he b

asis

set

6-3

11G

(2d,

p) w

as e

mpl

oyed

as

impl

emen

ted

in th

e G

auss

ian

prog

ram

. The

mol

ecul

e ge

omet

ry w

as fu

lly o

ptim

ized

with

out a

ny s

ymm

etry

re

stric

tions

. The

resu

lting

geo

met

ry w

as id

entif

ied

as e

quili

briu

m s

truct

ure

by a

naly

sis

of th

e fo

rce

cons

tant

s of

the

norm

al v

ibra

tiona

l mod

es. T

he p

ositi

ons

of th

e vi

brat

iona

l ban

ds w

ere

obta

ined

by

mul

tiply

ing

the

calc

ulat

ed w

aven

umbe

rs w

ith a

cor

rect

ion

fact

or o

f 0.

96,

as

anha

rmon

ic e

ffec

ts w

ere

not i

nclu

ded

in th

e co

mpu

tatio

n.[S

8-S1

0]

Ass

ignm

ent

of

vibr

atio

nal

band

s of

B

12

usin

g de

nsity

fu

nctio

nal

theo

ry

(DFT

) ca

lcul

atio

ns

To c

orre

ctly

ass

ign

the

band

s of t

he e

xper

imen

tal I

R sp

ectru

m o

f B12

to th

e ac

tual

vib

ratio

nal

mod

es a

nd in

ord

er to

det

erm

ine

the

trans

ition

dip

ole

mom

ents

(TD

M)

of th

ese

vibr

atio

ns,

quan

tum

che

mic

al c

alcu

latio

ns u

sing

DFT

with

hyb

rid f

unct

iona

ls w

ere

carr

ied

out.

To s

ave

calc

ulat

ion

time

the

mol

ecul

e w

as s

impl

ified

by

shor

teni

ng t

he l

ater

al c

hain

s to

(C

H2)

4H

chai

ns (C

4/4)

inst

ead

of th

e (C

H2)

12H

in B

12. T

he re

duct

ion

in le

ngth

of t

he la

tera

l cha

ins w

ill

not

seve

rely

inf

luen

ce t

he c

alcu

late

d vi

brat

iona

l sp

ectru

m,

only

the

alk

yl C

H (

stre

tchi

ng

�as

/s(C

H2)

and

sci

ssor

ing)

vib

ratio

ns w

ill b

e de

crea

sed

in i

nten

sity

. The

com

puta

tion

of t

he

mod

el m

olec

ule

C4/

4 yi

elds

the

posi

tion

and

rela

tive

inte

nsiti

es o

f th

e vi

brat

iona

l ban

ds a

s w

ell a

s the

vec

tor o

f the

TD

M a

nd th

e ac

tual

ly v

ibra

ting

grou

ps.

The

mol

ecul

ar d

irect

or (

MD

) w

as d

efin

ed a

s th

e ve

ctor

alo

ng th

e m

olec

ular

bac

kbon

e, i.

e. a

lin

e co

nnec

ting

the

ethe

r-bo

und

arom

atic

C a

tom

s of

eith

er t

erm

inal

phe

nyl

ring,

A).

The

angl

e �

betw

een

the

vect

or o

f th

e ca

lcul

ated

TD

Ms

and

the

MD

of

the

mol

ecul

e fo

r ea

ch

vibr

atio

nal b

and

coul

d th

en b

e ca

lcul

ated

.

The

band

s at

160

9 cm

-1 (�

= 6

°) a

nd 1

518

cm-1

(� =

13°

) with

the

TDM

nea

rly p

aral

lel t

o th

e M

D c

ould

be

assi

gned

to tw

o in

-pla

ne (a

lmos

t) pa

ralle

l rin

g vi

brat

ions

. All

phen

yl ri

ngs o

f the

m

olec

ular

bac

kbon

e ar

e in

the

sam

e pl

ane

due

to a

cer

tain

deg

ree

of c

onju

gatio

n th

roug

h th

e w

hole

mol

ecul

ar b

ackb

one

(rot

atio

nal b

arrie

rs fo

r the

ring

s are

abo

ut 4

.0 k

J mol

-1).

S8

1600

1500

1400

1300

1200

1100

1000

1607,6

1570,0

1518,9

1488,01467,9

1414,8

1382

1282,7

1246

1215,1

1174,6

1104,2

1041,5

Absorbance / a.u.

Wav

enum

ber /

cm

-1

B12

exp

erim

enta

l B4

c

ompu

ted

fwhh

=10

B4

com

pute

d fw

hh=4

� ip/p(r

ing)

� =

12,

7 °

Relative Absorbance

B� ip

/s(r

ing)

� =

6,1

°

Fi

gure

S6.

A) G

eom

etry

opt

imiz

ed m

olec

ular

stru

ctur

e of

the

B4

(mod

el s

ubst

ance

for B

12);

B)

com

paris

on o

f th

e m

easu

red

infr

ared

spe

ctru

m o

f B

12 (

blac

k, A

TR)

and

the

com

pute

d in

frar

ed s

pect

rum

of B

4 (r

ed, D

FT) b

etw

een

900

cm-1

and

1700

cm-1

; com

pute

d sp

ectru

m w

ith

two

diff

eren

t ful

l wid

th h

alf h

eigh

t (fw

hh) v

alue

s. In

ord

er to

com

pare

the

com

pute

d vi

brat

iona

l ban

ds (f

rom

B4)

to th

e ex

perim

enta

l spe

ctru

m,

pure

B12

was

dep

osite

d on

an

ATR

cry

stal

fro

m a

chl

orof

orm

sol

utio

n an

d th

e in

frar

ed

spec

trum

was

rec

orde

d B

). C

ompa

ring

the

com

pute

d w

ith th

e ex

perim

enta

l spe

ctru

m in

the

ring

vibr

atio

n re

gion

, (10

00-1

700

cm-1

) the

ban

d po

sitio

ns a

nd re

lativ

e in

tens

ity p

rofil

es a

re in

go

od a

gree

men

t to

each

oth

er. E

spec

ially

the

desi

red

ring

vibr

atio

nal b

ands

at 1

609c

m-1

and

15

18 c

m-1

are

dis

tinct

ive

in b

oth

spec

tra a

nd c

ould

the

refo

re b

e us

ed f

or d

emin

ing

the

orie

ntat

ion

of B

12 in

mem

bran

es.

S9

Abs

orba

nce

and

fluor

esce

nce

spec

tra

of B

12:

Figu

re S

7. U

V/V

IS-s

pect

rum

(ful

l bla

ck li

ne, c

= 0.

133

mM

) and

fluo

resc

ence

spe

ctru

m (r

ed

dash

ed l

ine,

c =

1.3

3 μM

) of

B12

in C

HC

l 3: t

he e

xciti

tion

wav

e le

ngth

use

d in

the

LSF

M

expe

rimen

ts i

s in

dica

ted

(�ex

=

dash

ed g

reen

lin

e) t

he e

mis

sion

wav

e le

ngth

ran

ge �

em

(hat

ched

gre

en b

ox )

is a

lso

indi

cate

d.

300

400

500

600

700

0,00

0,02

0,04

0,06

0,08

0,10

0,12

386 nm

334 nm

428 nm

Fluo

resc

ence

spe

ctro

scop

y:ab

sorp

tion

scan

�ex 1

���3

33 n

m, �

ex 2���3

78 n

m

emis

sion

sca

n be

i �ex

= 3

33 n

m

�em 1���4

28 n

m, �

em 2���4

49 n

m

UV-VIS absorption / a. u.

Wav

elen

gth ��

/ nm

B12

in C

HC

l 3

Fluo

resc

ence

-LSM

:�ex

= 4

05 n

m�em

= 4

43 n

m -

545

nm

Fluorescence intensity / a.u.

S10

X-r

ay a

naly

sis o

f B12

:DPP

C=1

:X in

gla

ss c

apill

arie

s

Figu

re S

8. X

-ray

con

tour

plo

ts o

f aq

ueou

s su

spen

sion

s of

B12

:DPP

C 1

:10

(top)

and

1:4

(b

otto

m)

with

50

wt.-

% H

2O; i

n th

e up

per

part,

the

scat

terin

g in

tens

ities

are

plo

tted

in g

rey

scal

es (r

ecip

roca

l spa

cing

axi

s at

the

left

side

) and

in th

e lo

wer

par

t the

tem

pera

ture

pro

file

is

plot

ted

(tem

pera

ture

axi

s at

the

low

er r

ight

sid

e);

arro

ws

poin

ting

upw

ards

mar

k ph

ase

trans

ition

s an

d ar

row

s po

intin

g do

wnw

ards

sho

w th

e fr

eezi

ng a

nd m

eltin

g of

exc

ess

wat

er;

scat

terin

g cu

rves

at 0

°C a

re h

ighl

ight

ed w

ith w

hite

ver

tical

line

s; a

t the

tem

pera

ture

s be

twee

n fr

eezi

ng a

nd m

eltin

g of

wat

er, s

harp

ice

refle

ctio

ns a

ppea

r w

hich

are

inde

x w

ith h

oriz

onta

l ar

row

s at

the

right

bor

der;

SAX

S re

flect

ions

at t

he te

mpe

ratu

res

I-IV

are

mar

ked

with

whi

te

horiz

onta

l das

hes

and

resp

ectiv

e re

flect

ions

of

the

WA

XS

regi

on w

ith y

ello

w o

r re

d da

shes

(tw

o ph

ases

); th

e W

AX

S re

flect

ion

inde

xing

at t

he ri

ght b

orde

r (lo

ng h

oriz

onta

l arr

ows)

refe

r to

the

lip

id r

ich

phas

e; t

he i

mpr

inte

d bo

x sh

ows

the

repe

at d

ista

nces

at

the

mar

ked

tem

pera

ture

(I-I

V).

S11

GIX

D o

f B12

:DPP

C=1

:X o

n or

ient

ed so

lid su

ppor

ted

bila

yers

In th

e W

AX

S re

gion

for a

ll te

mpe

ratu

res

exce

pt th

e hi

gh te

mpe

ratu

re p

hase

, tw

o re

flect

ions

ar

e ve

ry p

rono

unce

d th

at i

ndic

ate

the

(110

) an

d (0

20)

refle

ctio

ns o

f th

e D

PPC

alk

yl c

hain

la

ttice

. Th

e ab

solu

te p

ositi

on o

f th

is p

eaks

are

shi

fting

to

smal

ler

valu

es w

ith i

ncre

asin

g te

mpe

ratu

re. B

esid

es th

ese

refle

ctio

ns, t

wo

or m

ore

addi

tiona

l ref

lect

ions

occ

ur in

dica

ting

the

pres

ence

of a

t lea

st o

ne s

econ

d la

ttice

. Onl

y in

the

high

tem

pera

ture

pha

se n

o sh

arp

refle

ctio

n bu

t onl

y a

broa

d ha

lo is

vis

ible

indi

catin

g th

at a

ll ch

ains

are

flui

d

Figu

re S

9. 1

D s

catte

ring

patte

rns

extra

cted

from

2D

GIX

D d

ata

(left)

and

plo

t of t

he W

AX

S re

flex

posi

tions

as

func

tion

of th

e te

mpe

ratu

re (

right

), da

ta ta

ken

from

GX

ID m

easu

rem

ents

of

orie

nted

and

hyd

rate

d m

ultil

ayer

sam

ples

of t

he m

ixtu

re B

12 :

DPP

C =

1:1

0 on

a S

i sol

id

supp

ort.

2030

4050

6070

1,2

1,4

1,6

1,8

(02

0) r

efle

ctio

n (

110)

ref

lect

ion

alk

yl c

hain

hal

o a

dd. r

efle

ctio

ns

Scattering vector qr / Å-1

Tem

pera

ture

/ °C

1,2

1,4

1,6

1,8

75°C

Scattering intensity I / a.u.

Scat

terin

g ve

ctor

qr /

Å-1

71°C

58°C

B12

: D

PPC

= 1

:10

25°C

48°C

S12

Tab

le S

3. C

ompa

rison

of m

embr

ane

thic

knes

ses

obse

rved

from

refle

ctom

etry

mea

sure

men

ts

with

syn

chro

tron

radi

atio

n us

ing

orie

nted

mul

tilay

er s

ampl

es w

ith 9

9% r

el.

hum

idity

(h

ydra

ted

via

gas

phas

e) a

nd in

-hou

se X

-ray

diff

ract

ion

mea

sure

men

ts w

ith c

apill

ary

sam

ples

at

50

wt.-

% H

2O fo

r the

mix

ture

B12

:DPP

C =

1:1

0 an

d 1:

4; fo

r the

mix

ture

B12

:DPP

C=1

:4 a

t 63

°C a

dro

p in

sca

tterin

g in

tens

ity o

ccur

s be

twee

n 0.

12 Å

-1 a

nd 0

.22

Å-1

and

the

scat

terin

g da

ta w

ere

fitte

d ex

clud

ing

the

seco

nd o

rder

diff

ract

ion

peak

(val

ues a

re it

alic

).

DE

SY d

ata,

ref

lect

omet

ry

In-h

ouse

dat

a

latti

ce 1

la

ttice

2

T

/°C

d HH/n

md/

nmd H

H/n

md/

nmT/°C

d/nm

B12

:DPP

C =

1:1

0 25

4.

60

6.45

-

-10

6.06

48

4.

22

5.24

4.

40

5.70

36

7.

25

58

3.98

5.

21

4.38

5.

76

55

6.99

64

4.

22

5.70

4.

30

5.33

78

6.

37

75

3.88

5.

28

-

B12

:DPP

C =

1:4

25

4.

68

6.41

4.

94

6.66

-2

0 6.

13

48

3.70

5.

51

4.56

6.

16

20

7.14

63

4.

30

5.84

5.

16

5.92

60

7.

00

71

3.96

5.

52

5.16

7.

15

77

5.57

78

3.

78

5.39

-

R

efer

ence

s:

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irzi,

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illam

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enga

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akaj

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