Nanofiber Formation and Polymerization of Bolalipids with

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Nanofiber Formation and Polymerization of Bolalipids with Diacetylene-Modified Single Alkyl Chains Gesche Graf, Simon Drescher, Annette Meister, Vasil M Garamus, and Alfred Blume J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b11945 • Publication Date (Web): 24 Jan 2019 Downloaded from http://pubs.acs.org on January 29, 2019

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The Journal of Physical Chemistry

1

Nanofiber Formation and Polymerization of Bolalipids with Diacetylene-Modified Single Alkyl Chains

Gesche Graf,a, Simon Drescher,b# Annette Meister,a,c Vasil M. Garamus,d Alfred Blumea* a

Institute of Chemistry, Physical Chemistry, MLU Halle-Wittenberg, Von-Danckelmann-Platz 4, 06120 Halle, Germany b

Institute of Pharmacy, Biophysical Pharmacy, MLU Halle-Wittenberg, WolfgangLangenbeck-Str. 4, 06120 Halle, Germany

c

HALOmem and Institute of Biochemistry and Biotechnology, MLU Halle-Wittenberg, KurtMothes-Str. 3a, 06120 Halle, Germany

d

Helmholtz Zentrum Geesthacht (HZG): Zentrum für Material- und Küstenforschung GmbH, Max-Planck-Str. 1, 21502 Geesthacht, Germany

#present

address: Institute of Pharmacy, University of Greifswald, Friedrich-Ludwig-JahnStr. 17, 17489 Greifswald, Germany RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required according to the journal that you are submitting your paper to)

CORRESPONDING AUTHOR FOOTNOTE *Alfred Blume, Martin Luther University Halle-Wittenberg, Institute of Chemistry, von-Danckelmann-Platz 4, 06120 Halle/Saale, Germany Tel.: +49-345-5525850; Fax: +49-345-5527157; E-mail: [email protected].

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2 Abstract The nanofiber formation in aqueous suspension of two classes of symmetric single‐chain bolaamphiphiles with different polar headgroups and a diacetylene‐modified alkyl chain with a length of 32, 34, and 36 C-atoms was investigated by differential scanning calorimetry (DSC), transmission electron microscopy (TEM), and small angle neutron scattering (SANS). As observed before for other bolalipids with phosphocholine (PC) and dimethylphosphoethanolamine (Me2PE) headgroups, the molecules form fibers when suspended in water at low temperature but disassemble into micellar-like aggregates upon heating. The introduction of a diacetylene group in the middle of the long chain leads to a perturbation of the chain packing so that this fiber-micelle transition occurs at lower temperature compared to the other bolalipids having unmodified alkyl chains. The aim of our project was the introduction of diacetylene groups into the alkyl chains to be able to polymerize the fibers at low temperature. This should enhance the fiber stability and prevent the disassembly into micellar aggregates at higher temperature. Polymerization of the aggregates containing diacetylene-modified bolaamphiphiles can be easily traced by UV/Vis spectroscopy as colored products are formed. We found that polymerization of bolaamphiphiles with PC headgroups leads to a break-down of most fibers into micelle-like aggregates, only some longer fibers segments are still detectable. In contrast, the use of Me2PE headgroups improves the polymerizability and the length of the polymerized fibers. The compound with 36 C-atoms in the chain could be polymerized at low temperature and the fibers remained stable at least up to a temperature of 60 °C. This shows that the perturbation of the chain packing due to the diacetylene groups in the chains can be overcome by elongation of the chains, so that thermostable fibers with a diameter of the length of the bolalipid molecule can be successfully formed.


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3 1. Introduction Molecules containing diacetylene groups in the alkyl chain can be polymerized via irradiation with UV light leading to polydiacetylenes (PDAs). Self-assembly of such molecules into films or membranes and subsequent 1,4-photopolymerization enable the formation of specific aggregates, in which the single molecules are connected via covalent bonds.1-4 Polymerization leads to the generation of a system of conjugated double and triple bonds as backbone of the PDA polymer. The configuration of the bonds is also referred to as the enyne configuration.1 Diacetylene compounds have successfully been utilized to stabilize membrane structures,5-7 including systems of bolaamphiphiles.8-10 The extended, delocalized π-electron system also enables applications of polydiacetylenes as nanowires.11-14 We have shown before that symmetric single-chain bolaamphiphiles with long alkyl chains self-assemble in water into helical fibers leading by network formation of the nanofibers to a hydrogel.15-21 Upon heating, these fibers disassemble into micellar-like aggregates with a concomitant loss of the gel behavior. If symmetrical single-chain bolaamphiphiles with diacetylene-modified spacer chains would be able to also self-assemble into fibers, polymerization could be a suitable way to create fibers in which the single molecules are linked via covalent bonds instead of just hydrophobic interactions. Such polymerized fibers should be stable against break-up at high temperature, as only the conformation of the side chains attached to the polymerized middle part could change with increasing temperature. The covalent bonds in the PDA backbone would then prevent the collapse of the fiber aggregates.19-20 This approach of strengthening a hydrogel by polymerization of the fiber forming monomers has been shown before to be promising.14 A second interesting feature of diacetylene compounds is their ability to form colored phases upon polymerization, e.g., colored Langmuir-Blodgett films in the case of lipids.2, 22-23 This renders them suitable for applications in sensor devices.4, 24-33 Colored phases arise from the presence of π-electron systems that absorb light with wavelengths in the visible spectrum. An ACS Paragon Plus Environment


4 interpretation of the different observable colors with respect to the structure of the PDAs is difficult. However, the presence of a blue phase, a purple phase, and a red phase have been described.2-3,

32

The blue to red transition is in many cases caused by stimuli such as

increasing temperature, changes in pH, or binding of other molecules with a concomitant change in conformation of the PDA backbone. There are at least two interpretations of this color change. (i) With an increase in disorder, the length of the conjugation of the backbone is decreased leading to a shift of the absorption band to a lower wavelength and a red color of the system. (ii) The red phase does not imply more disorder of the chain but is caused by the introduction of a non-planar geometry of the PDA backbone induced by geometrical constraints of the side chains.22 The purple phase seems to be an intermediate state and not a superposition of red and blue phase.2-3, 22, 32 PDAs dissolved in good solvents tend to exhibit a yellow phase.34-35 Diacetylene modification of symmetrical bolaamphiphiles can be achieved in principle in two different ways. Modification in the headgroup region would add functionality of these polymerizable groups to the bolalipids leaving the alkyl chain region untouched.36 Thereby however, the functionalization of the bolaamphiphile headgroups with further groups, such as sulfur containing moieties or structurally different headgroups, would be inhibited. The diacetylene modification of the hydrophobic chains would be an alternative. However, a perturbation of the packing properties of the bolalipids in the fibers could occur. Despite these possible problems we have chosen the chain modification with diacetylene units as the better alternative.


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5

Figure 1: (A) Chemical structure of PC-C32diAc-PC (R = CH3, x= 14), PC-C34diAc-PC (R = CH3, x = 15), PC-C36diAc-PC (R = CH3, x = 16), Me2PE-C32diAc-Me2PE (R = H, x = 14), Me2PE-C34diAc-Me2PE (R = H, x = 15), and Me2PE-C36diAc-Me2PE (R = H, x = 16). (B) CPK models of PC-C34-PC (top) and PC-C34diAc-PC (bottom). The chemical structure of the diacetylene-modified bolaamphiphiles used in this work is shown in Figure 1A. The insertion of two neighboring triple bonds into the center of the spacer/alkyl chain leads to a perturbation of the otherwise regular C-C-C bond angle of 113.3°. The bond angle of 180° for triple bonds results in a linear segment in the middle of the spacer chain. Corey-Pauling-Koltun (CPK) models for comparison of the chain structure of the bolaamphiphiles PC-C34-PC and PC-C34diAc-PC are shown in Figure 1B. The length of the alkyl chain is hardly affected by changes of the bond structure. However, the two triple bonds increase the rigidity and lead to a slight kink, i.e. a lateral displacement of chain segments. This kink may result in problems in self-assembly, as the packing is more restrained than in the case of pure all-trans alkyl chains without triple bonds. In addition, the polarity in the middle part of the chain increases slightly due to the high electron density of the triple bonds. We have shown before that modifications in the middle part of the alkyl chains can lead to significant changes in aggregation behavior, as fiber ACS Paragon Plus Environment


6 formation is very sensitive to perturbations of the optimal packing of the alkyl chains.16, 18, 3740

We therefore expected a priori changes in aggregation behavior due to the perturbation of

the chain packing in the fibers. To overcome this perturbation we have successfully used in the past longer alkyl chains and/or changed the bolalipid headgroup.16, 18, 37, 41 In this study we used the same strategy. Despite the structural changes, i.e. the slight destabilization of the fiber structure caused by the diacetylene groups, the modified bolaamphiphiles open new possibilities for stabilization of the aggregates by polymerization, i.e. formation of covalent bonds between the single molecules. 2. Materials and Methods 2.1. Materials. Symmetrical, diacetylene-modified bolaamphiphiles with phosphocholine (PC) or dimethylphosphoethanolamine (Me2PE) headgroups and spacer chain lengths from 30 to 36 carbon atoms were synthesized according to the procedure described recently.42 Solvents and buffer salts were purchased from Carl Roth GmbH + Co. KG (Karlsruhe, Germany) and used as received. Ultra-pure water was used from a Millipore Milli-Q A10 system (Millipore GmbH, Schwalbach, Germany). 2.2. Sample preparation. The appropriate amount of bolalipid was suspended in water (PC headgroup) or acetate buffer at pH 5 (Me2PE headgroup). To achieve a homogeneous suspension the samples were heated above 70 °C three times and vortexed. 2.3. Differential Scanning Calorimetry (DSC) measurements were carried out with a MicroCal VP-DSC (MicroCal Inc., Northampton, USA). The bolaamphiphiles were suspended in water or acetate buffer (pH 5) using the concentration c = 1 mg ml-1. Water or buffer was used as reference. Measurements were carried out using heating rates of 20 °C min-1 in the temperature interval from 2 to 95 °C. To check the reproducibility at least three consecutive heating and cooling scans were recorded of each sample. The water-water


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7 or buffer-buffer baseline was subtracted from the thermograms of the samples, and the DSC scans were evaluated using Origin 8.0 software. 2.4. Transmission Electron Microscopy (TEM) images were recorded with a Zeiss EM 900 transmission electron microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany). 5 µl of the sample solution (c = 0.1 - 0.03 mg ml-1) were spread on a copper grid coated with Formvar film. After 1 minute the excess solution was blotted off with filter paper. The samples were stained with 1% uranyl acetate (5 µl) solution, which was drained off after 1 minute. The samples were dried over night at 30 °C. For the samples prepared below ambient temperature the components were stored (24 h) and prepared in a cold room (5 °C). They were dried for 2 days at 5 °C and were kept in a desiccator at ambient temperature until the images were recorded. 2.5. Small Angle Neutron Scattering (SANS). Experiments were carried out using the SANS-1 instrument at the FRG 1 research reactor, Helmholtz Zentrum Geesthacht, Germany. The bolaamphiphiles were suspended in deuterated water or in deuterated acetate puffer at pH 5 (10 mM) at a concentration of 1 mg ml-1 and the suspensions were filtered through a membrane filter of 0.45 m pore size at 80 °C. The samples were placed in a thermostated sample holder for isothermal conditions (ΔT = ± 0.5 K) in quartz cuvettes with a path length of 5 mm. Four sample-to-detector distances were employed to cover the range of scattering vectors q from 0.05 to 2.5 nm-1. Transmission of the samples was approx. 70 % and the contribution of multiple scattering could be considered negligible. The raw scattering data were corrected for the background from the solvent, sample cell, and other sources using conventional procedures. Subsequently, the scattering data were analyzed by model independent approach using the Indirect Fourier Transform Method43 and by fitting of spherical, ellipsoidal and cylindrical models.44 A detailed description of this process was given before.45-46



8 2.6. Polymerization experiments 2.6.1. Sample preparation. The bolalipid was suspended in MilliQ water (PC bolalipids) or 10 mM acetate buffer at pH 5 (Me2PE bolalipids) at a concentration of c = 1 or 5 mg ml−1. To achieve a homogenous suspension, the samples were heated and vortexed several times. Afterwards, the suspensions were allowed to equilibrate in the refrigerator at 4 °C for at least seven days to ensure the formation of nanofibers. 2.6.2. Polymerization. The bolalipid suspensions were filled into quartz cuvettes (Quartz SUPRASIL, Hellma Analytics, Müllheim, Germany) with a path length of 10 mm. The suspensions were equilibrated at the appropriate temperature for at least 10 min. The sample suspensions were irradiated with a UV lamp (low-pressure Hg lamp, λ = 254 nm, P = 15 W) at a distance of around 3 cm to achieve polymerization of the diacetylene units. The suspensions were not stirred during the irradiation process but carefully shaken by hand between intervals of irradiation. After predefined time points of irradiation, images were taken from the suspensions and UV/Vis spectra were recorded. For irradiation experiments performed below ambient temperature, an isolated polystyrene box containing an ice/water bath kept at around 0 °C was used for cooling the bolalipid suspension during the irradiation. The isolated box was additionally cooled using dry ice to minimize heat development during UV irradiation. For irradiation experiments performed above ambient temperature, the same isolated polystyrene box was used containing a water bath at the appropriate temperature. Images were taken from the polymerized bolalipid suspensions using a digital camera (Samsung NV24 HD). The images were used without any further image processing. 2.6.3. UV/Vis spectroscopy. UV/Vis absorption spectra of polymerized bolalipid suspensions were recorded with a Cary 4000 spectrophotometer (Agilent Technologies Deutschland GmbH, Böblingen, Germany). The spectrophotometer was equipped with a thermostatting unit for temperature control of the cuvette between 3 and 80 °C (∆T = ± 0.5


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9 K). Pure MilliQ water and acetate buffer, respectively, were used as reference (background) and data were analyzed using OriginPro 8.0. 3. Results and Discussion 3.1. Aggregation Behavior of Diacetylene-Modified Bolaamphiphiles Preliminary studies of the aggregation behavior of PC-C32diAc-PC and Me2PEC32diAc-Me2PE with C32-chains in aqueous suspension showed that aggregates were formed but a detailed analysis was difficult as aggregation occurred only at low temperature close to 0 °C.47-48 We therefore increased the chain length of the bolalipids to enhance the aggregation tendency in water. The aggregation behavior of PC-C34diAc-PC and PC-C36diAc-PC as well as Me2PE-C34diAc-Me2PE and Me2PE-C36diAc-Me2PE was then investigated in aqueous suspension using DSC, SANS, and TEM. Due to limited sample resources, not all measurements could be carried out with all of the diacetylene-modified bolaamphiphiles. Recent results showed that the properties of symmetrical single-chain bolaamphiphiles are very similar for bolaamphiphiles carrying different headgroups or having a modified chain structure. With increasing chain length the stability range of the fiber aggregates is shifted to higher temperature.15, 37, 46 Therefore, the behavior observed for one of the diacetylene containing bolaamphiphiles also enables a prediction of the behavior of the longer or shorter chain analogues. 3.1.1. Transmission electron microscopy (TEM). In the TEM image obtained after negative staining of a PC-C34diAc-PC suspension prepared at a temperature of 5 °C, the presence of some fibrous aggregates with a diameter of 6 nm is evident (white arrows in Figure 2A). However, other aggregate structures are also present. Previous cryo-TEM images of a suspension of PC-C32diAc-PC with the C32-chain revealed only the presence of micellar aggregates at 7 °C.47 The fiber segments are considerably shorter than the fibers formed in PC-C32-PC suspensions.19-20 Apparently, the packing of the alkyl chains in the fibers is perturbed by the diacetylene unit so that other, more unspecific ACS Paragon Plus Environment


10 aggregate structures are more stable. Elongation of the chain stabilizes the fiber aggregates and the fiber-micelle transition is shifted to higher temperature.37 We therefore also imaged the other bolalipids with longer chains and indeed found fiber aggregates not only for bolalipids with PC headgroups but also for those with a Me2PE headgroups, which is also known to lead to a stabilization of the fibers.37

Figure 2: TEM images of suspensions of (A) PC-C34diAc-PC, (B) PC-C36diAc-PC, (C) Me2PE-C34diAc-Me2PE and (D) Me2PE-C36diAc-Me2PE prepared at 5 °C.

TEM images of PC-C36diAc-PC clearly showed the existence of fibers (Figure 2B). Similar fibrous aggregates were also observed in suspensions of Me2PE-C34diAc-Me2PE and Me2PE-C36diAc-Me2PE prepared at 5 °C displayed in Figure 2C and 2D. Whereas for PCC36diAc-PC and Me2PE-C34diAc-Me2PE the fiber structure seemed to be very irregular with a diameter of ca. 8-10 nm, the fibers formed by Me2PE-C36diAc-Me2PE were thinner with ca. 5-6 nm diameter and the fibers were arranged in a parallel fashion over larger distances (Figure 2D). ACS Paragon Plus Environment

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11 3.1.2. Differential scanning calorimetry (DSC). For analogous bolalipids without diacetylene groups, an increase in temperature usually leads to a break-down of the fibers and the formation of micellar-like aggregates which can be easily followed by DSC.15,

20, 37.

The DSC heating scans for three suspensions of PC-

C32diAc-PC,47 PC-C34diAc-PC, and PC-C36diAc-PC are shown in Figure 3A. In each of the DSC curves only one transition is observed at low temperature indicating the transition from fibers to micelles. A

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Figure 3: DSC heating scans of suspensions of (A) PC-C32diAc-PC (black, c = 10 mg ml-1, magnification ×10),47 PC-C34diAc-PC (green, c = 1 mg ml-1, magnification ×5), and PCC36diAc-PC (red, c = 1 mg ml-1) in water. (B) DSC heating scans of suspensions of Me2PEC32diAc-Me2PE (black, c = 5 mg ml-1),47 Me2PE-C34diAc-Me2PE (green, c = 1 mg ml-1), and Me2PE-C36diAc-Me2PE (red, c = 1 mg ml-1) in acetate buffer at pH 5.

Whereas for PC-C36diAc-PC a large DSC peak is seen at a temperature of ca. 18 °C, endothermic transitions are barely visible for the two shorter compounds PC-C32diAc-PC,47 and PC-C34diAc-PC. The temperature of the transition peak increases from 6 to 11.8 to 18.5 °C with increasing chain length. The enthalpy of the transition almost vanishes for the two shorter chain analogues. Upon cooling, the transitions of the suspensions of PC-C34diAcPC and PC-C36diAc-PC show a hysteresis to lower temperature (not shown). DSC heating scans of suspensions of Me2PE-C32diAc-Me2PE,47 Me2PE-C34diAcMe2PE, and Me2PE-C36diAc-Me2PE in acetate buffer at pH 5 are shown in Figure 3B. As for ACS Paragon Plus Environment


12 analogous bolaamphiphiles with PC headgroups, an increase of the transition temperature with increasing chain length from 14.3 to 19.7 to 29.5 °C is observed. The higher transition temperatures of the analogues with the Me2PE headgroups compared to bolalipids with PC headgroups have been consistently observed for all bolalipids and can be explained by the additional stabilization of the fiber structure via hydrogen bonds between the Me2PE headgroups at the fiber surface.49 The DSC curve for Me2PE-C36diAc-Me2PE is somewhat different, as in addition to the transition peak at 29.5 °C a broad underlying peak between 15 and 40 °C is found (Figure 3B, red curve). The occurrence of different aggregate types, such as bundles of fibers and single fiber strands having different transition temperatures, are possibly the reason for this behavior. 3.1.3. Small angle neutron scattering (SANS). SANS measurements give further information on the structure of aggregates in aqueous suspensions. The results of neutron scattering experiments at 5 and 30 °C with a suspension of PC-C36diAc-PC in heavy water are shown in Figure 4A. The TEM images in Figure 2B clearly showed fiber structures at low temperature. In agreements with this finding, the scattering data for the sample at 5 °C are typical for fibers found also for other bolalipids.45-46 Aggregates of different shape can be distinguished using SANS data via slope analysis, i.e., the approximation of scattering intensities as (d(q)/dΩ)c−1 ~ q−α. In general, a value α of 1 points to elongated aggregates (one dimension is much larger than two others, a value of 2 stands for disc-like structures (two dimensions are much larger than the third one), a value of almost zero points to small spherical aggregates.44 In the present case, the values of α at low and intermediate q interval decrease from near 1 to near zero at higher temperature indicating the transformation from fibers into smaller aggregates. The data obtained for the samples at 5 and 30 °C were fitted with the IFT method and the model of cylinders and spheres, respectively (Table 1). The fits are in good agreement with the experimental data and lead to the conclusion that the PC-C36diAc-PC molecules indeed ACS Paragon Plus Environment

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13 form fibers below the DSC transition peak as seen in the TEM images. The transition at 18.5 °C is connected with the break-up of these fibers into spherical micelles. Compared to the fibers of PC-C36-PC without diacetylene groups, the fibers formed by the analogue PCC36diAc-PC are very similar. However, the thermal stability of these fibers is significantly decreased. The bolaamphiphiles PC-C34-PC and PC-C36-PC show the presence of an additional fiber region (fibers II) as was also found for Me2PE-C32-Me2PE and its analogues with longer chains.49 This transition is not present in suspensions of PC-C34diAc-PC and PCC36diAc-PC. Furthermore, no micelle-micelle transition as for PC-C32-PC and PC-C36-PC was observed.19-20, 49 This shows, that the modification of the bolalipid alkyl chain leads to changes in aggregation behavior due to perturbations in chain packing. Similar effects were observed before for bolalipids with alkyl chains modified by the introduction of sulfur or oxygen atoms.16, 18

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Figure 4: (A) SANS data (squares) and IFT fits (solid lines) of the SANS data of a PCC36diAc-PC suspension in D2O with a concentration c = 1 mg ml-1 at 5 °C (black) and 30 °C (red). (B) Me2PE-C36diAc-Me2PE (c = 1 mg ml-1) in acetate buffer at 5 (black) and 55 °C (red).



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14 Table 1: Results of IFT fits of the SANS data of PC-C36diAc-PC suspended in D2O (c = 1 mg ml-1) at 5 and 30 °C and of Me2PE-C36diAc-Me2PE suspended in acetate buffer (c = 1 mg ml-1) at 5 and 55 °C.a

bolalipid

T/ °C

aggregate shape

Dmax / nm

Nagg / nm-1 Nagg per micelle 9 25

ML / g cm-1 M/g

Rg or RCS,g / nmb

R or RCS / nmb

PC-C36 5 fibers 5 1.30∙10-13 1.7 ± 0.2 2.5 ± 0.3 -20 30 spheres 6.5 3.56∙10 2.3 ± 0.2 3.0 ± 0.3 diAc-PC PC-C3625 fibers 5.5 10 1.46∙10-13 1.9 ± 0.2 2.6 ± 0.3 PC49 bundles Me2PE5 15 12 1.70∙10-13 4.0± 0.3 5.6 ± 0.4 of fibers C36diAc55 fibers 4.5 7 9.46∙10-14 1.5 ± 0.2 2.3 ± 0.3 Me2PE Me2PE-C3670 fibers 5.5 11 1.51∙10-13 1.7 ± 0.2 2.3 ± 0.3 Me2PE24 aD max: maximal size or cross-section of aggregate, M: mass, ML: mass per unit length, Nagg: aggregation number, Rg: and R radius of gyration and radius of spheres, RSC,g and RSC radius of gyration of cross-section and radius of cross section for fibers and bundles of fibers. bDeviation values contain statistical and systematic error contributions. Systematic error contributions are calculated according to Feigin and Svergun50 and are approximately equal to (µ/2.7)4(qRg)4, where µ corresponds to the shape factor (ranging from 2.4, spheres, to 4, rods) and q is the minimal value of scattering vector used in the analysis.

SANS experiments were also carried out with a suspension of Me2PE-C36diAc-Me2PE in deuterated acetate buffer at 5 and 55 °C. The SANS data and IFT fits are presented in Figure 4B. The IFT fits for data at 5 and 55 °C were done using the model of infinitely long cylinders and are in good agreement with the experimental data (Table 1). The pair distance distribution function of cylindrical cross section obtained from the analysis of the scattering curves taken at 5 °C is asymmetric and has three maxima around 2, 8, and 14 nm (not shown). This indicates that the aggregates in the suspension have different radii which can be explained by the formation of bundles of fiber strands. Fibers with larger radii of cross section are also obtained by a fit of the scattering data at 5 °C with the model of long cylinders. The radius is approximately twice as high as determined for other bolaamphiphile systems (Table 2)45-46 and is an average over all aggregates in the suspension. The TEM images shown in Figure 2D support these results as they show fibers with a range ACS Paragon Plus Environment

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15 of radii for the Me2PE-C36diAc-Me2PE suspension at 5 °C, supporting the interpretation of the DSC results, i.e. the observation of sharper overlapping transition peaks with a broad underlying peak. 3.2. Polymerization of Diacetylene-Modified Bolaamphiphiles Diacetylene containing bolaamphiphiles self-assemble into fibers below the temperature of the DSC transition peak where they dissociate into micellar aggregates. Polymerization using UV irradiation is a way to examine the possibility for developing thermostable fibers via the formation of covalent bonds between the single molecules that form the fibers. It is well known that the polymerization process is very sensitive to the distance and relative orientation of the diacetylene units. Both parameters were shown to determine the reactivity of the diacetylene monomers.22, 51 A distance between 0.47 and 0.52 nm and an inclination angle of 45° between the diacetylene axes are necessary for an efficient polymerization reaction.52 This reaction leads to an alternating double-triple bond (enyne) structure with changed orientation of the side chains. Figure 5 shows a scheme for the polymerization of diacetylene-modified bolaamphiphile chains and CPK models for PC-C34diAc-PC molecules.

Figure 5: Scheme and CPK models of the polymerization reaction of three PC-C34diAc-PC molecules to polymerized pPC-C34diAc-PC. The PDA chain at the bottom right has been terminated by two CH2-groups.



16 The first polymerization experiments were carried out by M. Bastrop with PC-C32diAcPC and Me2PE-C32diAc-Me2PE suspensions and were described in his PhD thesis.47 PCC32diAc-PC suspensions (c = 5 and 10 mg ml-1) turned blue upon UV irradiation at 0 °C or at room temperature, respectively, indicating the formation of the blue PDA “phase”. When the polymerized sample was heated, the color of the suspension changed with temperature and turned from blue to purple to red upon heating from 10 to 30 to 40 °C. This thermochromism was to some extent reversible when the suspension was heated to only 40 °C and subsequently cooled again to 10 °C. No reversibility was observed if the sample was heated to temperatures above 50 °C. In contrast, Me2PE-C32diAc-Me2PE suspensions in acetate buffer at pH 5 (c = 5 mg ml-1) only turned yellow upon UV irradiation at 5 °C and at room temperature.47 This behavior can be explained by the formation of an extended π-conjugated polymer backbone with alternating double and triple bonds (see Figure 5). A planar conformation of the polymer backbone and ordered side chains usually lead to the occurrence of a blue phase.22, 47 The side groups of the diacetylene containing molecules were proven to have a strong impact on the properties and the color of the PDAs. The side chain conformation can lead to a shift of the observed absorption maximum. With increasing temperature, higher flexibility of the side groups and induced rotation of the PDA backbone cause the formation of a red phase. This process also reduces the conjugation length of the polymer.3 The purple phase was described as a transition state in the conversion of the blue into the red phase.53 The formation of yellow phases was observed for PDAs in good solvents and may be ascribed to the presence of partially dissolved polymerized diacetylene bolaamphiphile fibers with a nonplanar PDA backbone.22, 34-35 M. Bastrop explained the absence of blue or red “phases” for polymerized suspensions of Me2PE-C32diAc-Me2PE by the different structure of the aggregates due to the smaller size of the Me2PE headgroup compared to the PC headgroup.47 This results in a changed distance of ACS Paragon Plus Environment

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17 the diacetylene groups and prevents the formation of extended polymerized groups, as this process is very sensitive to structural modifications like decreased distance between the diacetylene groups.22 The polymerization experiments described above were carried out in an isolated box, in which the bolaamphiphile suspension was thermostatted inside an ice/water bath and irradiated with UV light. The samples were taken out of the ice water bath and placed into the UV/Vis spectrometer to record the spectra. We now performed new polymerization experiments with the diacetylene-modified bolaamphiphiles in situ using a different spectrophotometer with a thermostatted cuvette holder, in which the samples could be irradiated directly with the UV lamp prior to the measurement of the absorption spectra. This improved the temperature stability during irradiation and measurement. Suspensions of PC-C32diAc-PC, PC-C34diAc-PC, and PC-C36diAc-PC were irradiated with UV light to achieve polymerization and to investigate the formation of colored “phases”. Me2PE-C34diAc-Me2PE and Me2PE-C36diAc-Me2PE suspensions in acetate buffer at pH 5 were also irradiated with UV light to check polymerizability of the fibers and coloring of the suspensions. Table 2 shows a summary of the experiments together with a qualitative description of the results.



18 Table 2: Results of polymerization experiments with bolalipids with PC or Me2PE headgroups and with diacetylene groups in the alkyl chains of different length C32, C34, and C36.a PC headgroup

Me2PE headgroup

C36

c = 5 mg/mL T < Tm: salmon T↑: yellow (irreversible) T > Tm: yellow c = 1 and 5 mg/mL T < Tm: intense red T↑: yellow-orange (irreversible) T > Tm: faint yellow

Tm = 14.7 °C

T > Tm blue T↑: purple > red > orange > yellow (partially reversible) or slight yellow

Tm = 19.7 °C

T < Tm: blue T↑: purple > red > orange > yellow (partially reversible)

c = 5 mg/mL

Tm = 30.5 °C

C34

Tm = 11.7 °C

C32

Tm ≈ 6 °C

c = 5 and 10 mg/mL

Tm = 18.5 °C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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T < Tm: yellow-orange

T > Tm: slight yellow

c = 5 mg/mL T < Tm: red T↑: yellow (irreversible) T > Tm faint purple c = 1 and 5 mg/mL T < Tm purple T↑: yellow-orange (irreversible) T > Tm: faint purple

aPolymerization

experiments were carried out at temperatures below the transition temperature Tm of the appropriate bolaamphiphile (T < Tm) or above (T > Tm). After UV irradiation, the sample suspension was heated (T↑). A more detailed description of the polymerization of the two bolalipids with the longest alkyl chain but different headgroups is described below. 3.2.1. PC-C36diAc-PC. A PC-C36diAc-PC suspension (c = 1 mg ml-1) thermostatted at 5 °C was irradiated for 30 minutes resulting in a red color of the suspension. The UV/Vis spectrum shows a peak around 500 nm and a shoulder at 550 nm after 2 minutes of irradiation (Figure 6). This indicates a successful polymerization of PC-C36diAc-PC to pPC-C36diAc-PC, the polymeric compound. The peak at higher wavelength decreases with continuing irradiation until only the peak around 500 nm is visible after 30 minutes of irradiation. Continued irradiation leads to a shift ACS Paragon Plus Environment

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19 of the absorption maximum to 430 nm indicated by the orange to yellow color of the suspension.

Figure 6: UV/Vis spectra of a PC-C36diAc-PC suspension (c = 1 mg ml-1) after different irradiation intervals at 5 °C and images of the suspensions after 0, 2, 30, and 70 minutes of irradiation.

Polymerization experiments at 5 °C with a PC-C36diAc-PC suspension with a higher concentration of c = 5 mg ml-1, essentially showed the same UV/Vis spectra, only with increased intensity of the absorption. In this case, the suspension was still deep red in color after 100 min of irradiation with an absorption maximum at 500 nm. As more PC-C36diAcPC molecules are present in the suspension it can be expected to take longer for all fibers to polymerize. In this case, the irreversible transition to the yellow “phase” was achieved by heating the suspension to 30 °C. Polymerization experiments with a PC-C36diAc-PC suspension (c = 5 mg ml -1) at a temperature of 25 °C yielded only a light yellow suspension after 50 minutes of irradiation. However, when the suspension was cooled to 5 °C and further irradiated, the formation of a ACS Paragon Plus Environment


20 red “phase” was observed. This indicates that at 25 °C, above the fiber-micelle transition, only very few of the PC-C36diAc-PC molecules could be polymerized, even after 50 min of irradiation, i.e. the chains are too disordered in the micellar aggregates to achieve a regular array of diacetylene units with the propensity for polymerization. DSC experiments of the irradiated samples were also performed. Figure 7A shows for PC-C36diAc-PC the DSC curves before and after 30 min of irradiation for the sample with 1 mg ml-1. The DSC experiments clearly show that the usually observed fiber-micelle transition peak is not present any more, indicating that either no fibers are pres ent, or that the fibers are still present at low temperature but cannot disassemble into micelles connected with an increase in disorder in the chains due to the polymerization of the monomers in the fibers. SANS measurements with pPC-C36diAc-PC were carried out to examine the structure of the polymerized aggregates more closely. The scattering data and the IFT fits are shown in Figure 7B. The slope of the scattering data at small and intermediate q values is smaller than expected for fibrous aggregates (~q−1). The approximation of 3d objects (all dimensions of similar values) was used for the IFT fits that are in good agreement with the experimental data. At 5 °C the model of an ellipsoid of revolution with two semiaxes can also describe the scattering data well. The results of the fits are provided in Table 3. Aggregation numbers Nagg cannot be calculated from the fit results as the molecular mass of the polymerized fibers could not be determined.


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21

-1

before irradiation after 30 min irradiation

10 8 6 4

10

4

10

3

10

2

5° C fit ellipsoid fit sphere 30 °C fit sphere

-1

PC-C36diAc-PC, 1 mg/ml, H2O

12

-1

B

14

(d(q) / d) / c / cm² g

A Cp / kJ mol K

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2 0

10

20

30

40

50

60

70

80

90

0.1

q / nm

Temperature / °C

-1

1

Figure 7: (A) DSC curves of samples of PC-C36diAc-PC before and after irradiation (30 min irradiation, c = 1 mg ml-1). (B) SANS scattering data with IFT fits of pPC-C36diAc-PC at 5 °C and 30 °C after polymerization with UV light at 5 °C. (C) TEM image of a red colored polymerized suspension of pPC-C36diAc-PC at 5 °C prior to the DSC measurement and (D) of a yellow colored polymerized suspension of pPC-C36diAc-PC at 25 °C after the DSC measurement. The bars correspond to 100 nm.

The analysis of the SANS measurements shows that the fiber structure is not preserved after polymerization of the diacetylene bolaamphiphile fibers, supporting the interpretation of the TEM images (Figure 7C and 7D). However, the pair distance distribution function p(r) (not shown) at 5 °C shows an asymmetric distribution indicating the additional presence of larger aggregates than at 30 °C as it can also be deduced from the higher scattering intensity at 5 °C. ACS Paragon Plus Environment


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22 This was seen in the TEM images of the pPC-C36diAc-PC suspension at 5 °C (Figure 7C) as well. The radius of the micelles obtained from the IFT fit at 5 °C is larger than the one usually determined for bolaamphiphile micelles.45-46 This indicates that the aggregates are larger after polymerization than regular micelles and that the fit with the model of ellipsoids is more appropriate in this case. Figure 7C and 7D show TEM images with only marginal differences in the structure of the aggregates. Short fiber segments or elongated micelles that have a diameter of approximately 5 nm appear. This illustrates that the fiber structure formed by PC-C36diAcPC at low temperatures cannot be preserved completely upon polymerization of the diacetylene units as already mentioned above. The TEM image of the red suspension prepared at 5 °C displays only a few longer fiber segments.

Table 3: Results of IFT fits of the SANS measurements with pPC-C36diAc-PC suspended in deuterated water at 5 °C and 30 °C and pMe2PE-C36diAc-Me2PE suspended in acetate buffer at 5 °C. and 30 °C. The data at 30 °C refer to the IFT analysis for the q region from 0.2 nm-1 to 3 nm-1.a bolalipid

T / °C

aggregate shape

Dmax / nm

Rg / nmb

R, a, b / nmb

pPC-C36 diAc-PC

5

ellipsoid

15

4.5 ± 0.2

a = 2.5  0.1 b = 7.9 ± 0.2

30

spheres

6.5

2.3 ± 0.2

2.9 ± 0.2

pMe2PE-C36 diAc-Me2PE

5

fibers

15

4.1  0.2

5.8  0.2

30

fibers and spheres

7.5

2.7  0.2

3.4  0.2

aD

max:

maximal size or cross-section of aggregate, Rg: radius of gyration, R: radius of sphere, a and b: parameters of ellipsoid of revolution with semi axis a,b. bDeviation values contain statistical and systematic error contributions. Systematic error contributions are calculated according to Feigin and Svergun.50 3.2.2. Me2PE-C36diAc-Me2PE. The analogue Me2PE-C36diAc-Me2PE suspended in acetate buffer at pH 5 at the concentration c = 1 mg ml-1 develops a wide spectrum of colors upon irradiation at 10 °C, indicating the formation of the polymerized form pMe2PE-C36diAc-Me2PE. The UV/Vis


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23 absorption spectra first show a very broad absorption maximum from 530 to 580 nm, which results in a purple color of the suspension (see Figure 8). With further irradiation of the suspension, the absorption around 530 nm increases, and an additional shoulder around 470 nm appears. After 45 minutes of irradiation, the absorption is highest around 470 nm, but absorption bands centered at 530 and 580 nm are still present, leading to a red color of the suspension. When the suspension is heated first to 20 °C and further to 50 °C, the color changes irreversibly first to orange and then to yellow, respectively. Irradiation of suspensions of Me2PE-C36diAc-Me2PE at a temperature above the DSC transition peak only resulted in the formation of yellow suspensions.

Figure 8: UV/Vis spectra of a Me2PE-C36diAc-Me2PE suspension in buffer at pH 5 (c = 1 mg ml-1) after different irradiation intervals at 10 °C and subsequent heating to 20 and 50 °C and photographs of the suspension after 0, 2, 18, and 45 minutes of irradiation and subsequent heating of the suspension to 20 and 50 °C.



24

B 4.5

Me2PE-C36diAc-Me2PE,

4.0

1 mg/ml, acetate buffer pH 5 before irradiation after 30 min irradiation

-1

Cp / kJ mol K

-1

3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5

0

10

20

30

40

50

60

70

80

90 100

Temperature / °C

C 10 -1

4

(d(q) / d) / c / cm²g

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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3

10

-4

~q

2

10

1

10

0.1

q / nm

-1

1

Figure 9: (A) TEM image of a pMe2PE-C36diAc-Me2PE (c = 1 mg ml-1) at 25 °C after polymerization at 10 °C and heating to 60 °C. The bar corresponds to 100 nm. (B) DSC curves of samples of Me2PE-C36diAc-Me2PE before and after irradiation (30 min irradiation, c = 1 mg ml-1). (C) Scattering data and IFT fits of SANS measurements of a suspension of pMe2PE-C36diAc-Me2PE in acetate buffer at 5 °C (black) and 30 °C (red). Dashed line shows a slope of −4 for comparison with 30 °C data at lowest q range. A TEM image of the suspension of pMe2PE-C36diAc-Me2PE, taken after polymerization at 10 °C for 45 minutes and then heating up to 60 °C, shows fiber segments with a diameter of 3 nm to 4 nm and a length of up to 100 nm (Figure 9A). This shows that after polymerization the fiber structure is retained though the fibers are shorter than in the non-polymerized sample (see Figure 2D).


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25 Figure 9B shows for Me2PE-C36diAc-Me2PE the DSC curves before and after 30 min of irradiation for the sample with 1 mg ml-1. In contrast to the irradiated PC-C36diAc-PC sample (Figure 7A), a broad transition peak between 20 and 30 °C is observed. Since this transition peak is smaller compared to the transition peak of the non-polymerized sample, the presence of some non-polymerized fiber strands in the polymerized sample suspension is conceivable. The SANS scattering data recorded at 5 °C (Figure 9C) can be fitted well with the IFT method and the model of infinitely long cylinders. However, the pair distance distribution function p(r) (not shown) shows an asymmetric profile again indicating a size distribution of the cross section of fibers in suspension. This is the same effect already observed for the suspension of Me2PE-C36diAc-Me2PE prior to polymerization (Figure 4) and can be explained by the formation of bundles of fibers: Scattering data at 30 °C show an intensity curve that suggests the presence of different aggregate structures in the suspension. At the lowest q range, the scattering intensity follows a slope -4. This means that the scattering arises from a surface of large compact aggregates with smooth and sharp interface. At large q values (q = 0.2 - 3 nm-1) the data can be fitted with the model of spherical micelles. The explanation for this finding is that apparently polymerization was not complete, so that after heating from 5 °C, where only fibers are present, to 30 °C, the fibers partially break down into micellar aggregates and a mixture of micelles and fibers is present (see Table 3). This conclusion is supported by the TEM image in Figure 9A, where also a mixture of short and long fiber segments is seen. However, the fibers were shorter than the ones observed for nonpolymerized Me2PE-C36diAc-Me2PE (see Figure 2D). In any case, these results prove that polymerization does not destroy the fiber structure and that the fibers are stable to some extent at elevated temperatures. As described above, the distance and relative orientation of two adjacent diacetylene groups determine the reactivity of the diacetylene monomer.22 The bolaamphiphile molecules inside the fiber structure show apparently not a sufficient long-range order to enable the ACS Paragon Plus Environment


26 formation of blue colored “phases”. However, red “phases” can be readily observed for the bolalipids with longer chains, indicating that the influence of the side chains on the orientation of the polymer backbone is an important effect. Many transitions from the blue to the red phase, described in other systems, e.g., planar orientations of PDAs such as polymerized pentacosa-10,12-diyonic acid, are irreversible.3, 54 Functional moieties incorporated into the diacetylene compounds induced some reversible phase transitions through additional stabilizing groups such as azobenzene units or hydrogenbonding interactions inside the side chains.3, 13, 55-56 Bolaamphiphiles with PC headgroups and shorter chains show not sufficient long-range order of the chains to be successfully polymerized by irradiation. For the longer chain analogues with C36 chains a polymerization was possible but the fibers seemed not to be stable at higher temperature but fell apart into short aggregates (see Figure 7). Comparing the results of the polymerization experiments of PC-C36diAc-PC with those of Me2PE-C36diAc-Me2PE, a tendency for absorption maxima at higher wavelength could be observed for pMe2PE-C36diAc-Me2PE. This is particularly evident in the suspension of pMe2PE-C36diAc-Me2PE that exhibits an absorption maximum around 580 nm, which is characteristic for the presence of purple phases (see Figure 8).22 Non-polymerized Me2PE-C36diAc-Me2PE shows an increased fiber stability with a higher transition temperature for the fiber-micelle transition (see Figure 3). The reason for this higher stability is the fact that the Me2PE headgroups are able to form hydrogen bonds in their zwitterionic form at pH 5 and thus to stabilize the ordered structure of the chains. Apparently, this also leads to a better long-range order of the molecules within the fibers so that the polymerization reaction can proceed more easily. Nevertheless, the color of the suspension is mostly red, showing that the chains flanking the PDA backbone are not perfectly ordered and that the PDA backbone is not planar. The reason for this is that the steric requirements of the


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27 headgroups with the larger cross-sectional area than the chain lead to an induced twist in the PDA backbone. When polymerized samples are heated to high temperature, the color turns to yellow. This red to yellow transition is irreversible as upon cooling the yellow color persists. The high temperature treatment apparently leads to further disordering of the PDA side chains and a further distortion of the PDA chain leading to an apparent shorter conjugation length of the backbone and a yellow color. The side chains are not long enough to overcome the energy barrier for ordering at lower temperature so that a metastable state persists. In other systems, the thermal reversibility of the blue to red transitions and more stable PDAs were designed by incorporating other conjugated moieties into the side chains. This was shown for an azo chromophore-functionalized diacetylene molecule forming micelles56 and for 10,12-pentacosadiynoic acid films by adding new functional groups enhancing the hydrogen-bonding interactions between the headgroups.55 The pH-dependence of the polymerizability and the color of the corresponding “phases” for pMe2PE-C36diAc-Me2PE were not examined so far. However, as the charge of the Me2PE headgroup was proven to influence the aggregation behavior, an influence of the headgroups protonation state on the polymerizability might be observed as well.17,

21

pH-dependent

coloring of PDA phases has also been described for other systems.3, 57A positive effect on the stability and color of PDAs was found when longer alkyl chains were used due to the high order the alkyl chains can induce.58 This indicates that the use of diacetylene bolaamphiphiles with spacer chains longer than 36 carbon atoms might be an option to improve the polymerizability of the aggregates. Results of DSC measurements with bolalipids with increasing alkyl chain length clearly showed the stabilizing effect.46 For diacetylene-modified bolalipids a similar effect is expected on the stability of the fibers and can then lead to an improvement in the length of the fibers after polymerization.



28 4. Conclusions In this work we could show that diacetylene containing bolaamphiphiles can also form similar fibrous aggregates as their analogues containing only unmodified saturated alkyl chains. However, the structural perturbation induced by the different bond angle of the diacetylene unit compared to the all-trans alkyl chain causes a pronounced reduction of the fiber stability. For the compounds with 32 C atoms and PC headgroups the temperature of the fiber-micelle transition is shifted by ca. 40 °C to a temperature of 6 °C and can only partially be observed by DSC. This destabilization effect can be counterbalanced by the use of overall longer alkyl chains or by the use of headgroups that add to the stabilization of the fibers by hydrogen bonds between the headgroups, such as bolaamphiphiles with Me2PE headgroups. Polymerization of the aggregates containing diacetylene-modified bolaamphiphiles is possible and can be traced by monitoring the development of colored products. Upon polymerization of bolaamphiphiles with PC headgroups, most fibers break-down into micellelike aggregates and only small numbers of longer fibers segments are still detectable. The use of Me2PE headgroups improves the polymerizability and the length of the polymerized fibers. TEM images of a pMe2PE-C36diAc-Me2PE suspension display fibers even after the suspension was heated to 60 °C. This shows that polymerizing the self-assembled fiber structure is a promising way to build thermostable fibers. The formation of colored “phases” connected with the polymerization of diacetylenes via UV light is also apparent in systems of diacetylene bolaamphiphiles. The development of yellow and red “phases” is observed for bolaamphiphiles with PC headgroups. Bolaamphiphiles with Me2PE headgroups additionally show the development of a purple “phase” with an absorption maximum shifted to higher wavelength. Extended irradiation or increase of the temperature lead to an irreversible transition from red or purple “phases” to a yellow “phase”. The formation of yellow “phases” is due to a high degree of disorder in the PDA side chains as the bolalipids in the nanofibers are not as ordered as PDAs in planar ACS Paragon Plus Environment

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29 arrangements such as Langmuir-Blodgett films. Further efforts to increase the chain length of the diacetylene containing bolaamphiphiles have to be made to improve the stability of the fibers during and after polymerization. Acknowledgments This work was supported by grants from the Deutsche Forschungsgemeinschaft (S.D., A.M., A.B.; project Bl 182/19-3 and S.D; project Dr 1024/1-1). The support of Gerd Hause (Biocenter, Martin-Luther-University Halle-Wittenberg) for providing access to the electron microscope facility is greatly appreciated. References 1. Jose, D. A.; Stadlbauer, S.; König, B., Polydiacetylene-Based Colorimetric SelfAssembled Vesicular Receptors for Biological Phosphate Ion Recognition. Chem. Eur. J. 2009, 15, 7404-7412. 2. Reppy, M. A.; Pindzola, B. A., Biosensing with polydiacetylene materials: structures, optical properties and applications. Chem. Commun. 2007, 4317-4338. 3. Sun, X. M.; Chen, T.; Huang, S. Q.; Li, L.; Peng, H. S., Chromatic polydiacetylene with novel sensitivity. Chem. Soc. Rev. 2010, 39, 4244-4257. 4. Lee, J.; Kim, H.-J.; Kim, J., Polydiacetylene Liposome Arrays for Selective Potassium Detection. J. Am. Chem. Soc. 2008, 130, 5010-5011. 5. Johnston, D. S.; Sanghera, S.; Manjon-Rubio, A.; Chapman, D., The formation of polymeric model biomembranes from diacetylenic fatty acids and phospholipids. Biochim. Biophys. Acta 1980, 602, 213-216. 6. Leaver, J.; Alonso, A.; Durrani, A. A.; Chapman, D., The physical properties and photopolymerization of diacetylene-containing phospholipid liposomes. Biochim. Biophys. Acta 1983, 732, 210-218. 7. Hillmann, R.; Viefhues, M.; Goett-Zink, L.; Gilzer, D.; Hellweg, T.; Golzhauser, A.; Kottke, T.; Anselmetti, D., Characterization of Robust and Free-Standing 2DNanomembranes of UV-Polymerized Diacetylene Lipids. Langmuir 2018, 34, 3256-3263. 8. Song, J.; Cheng, Q.; Kopta, S.; Stevens, R. C., Modulating Artificial Membrane Morphology: pH-Induced Chromatic Transition and Nanostructural Transformation of a Bolaamphiphilic Conjugated Polymer from Blue Helical Ribbons to Red Nanofibers. J. Am. Chem. Soc. 2001, 123, 3205-3213. 9. Song, J.; Cheng, Q.; Stevens, R. C., Morphological manipulation of bolaamphiphilic polydiacetylene assemblies by controlled lipid doping. Chem. Phys. Lipids 2002, 114, 203214. 10. Song, J.; Cisar, J. S.; Bertozzi, C. R., Functional self-assembling bolaamphiphilic polydiacetylenes as colorimetric sensor scaffolds. J. Am. Chem. Soc. 2004, 126, 8459-8465. 11. Okawa, Y.; Aono, M., Nanoscale control of chain polymerization. Nature 2001, 409, 683-684. 12. Schenning, A. P. H. J.; Meijer, E. W., Supramolecular electronics; nanowires from self-assembled π-conjugated systems. Chem. Commun. 2005, 3245. 13. Zhou, W. D.; Li, Y. L.; Zhu, D. B., Progress in polydiacetylene nanowires by selfassembly and self-polymerization. Chem.-Asian J. 2007, 2, 222-229. ACS Paragon Plus Environment


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Nanofiber Formation and Polymerization of Bolalipids with

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