Biphenyl-Induced Superhelix in l -Phenylalanine-Based Supramolecular Self-Assembly with Dynamic Morphology Transitions

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE5 Aug 2022Biphenyl-Induced Superhelix in l-Phenylalanine-Based Supramolecular Self-Assembly with Dynamic Morphology Transitions Laiben Gao, Yueyue Feng, Chao Xing, Yu Zhao, Meng Sun, Yunqing Zou, Changli Zhao, Xiaoqiu Dou and Chuanliang Feng Laiben Gao State Key Lab of Metal Matrix Composites, School of Materials Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240 Google Scholar More articles by this author , Yueyue Feng College of Chemistry and Materials Science, Shanghai Normal University, Shanghai 200234 Google Scholar More articles by this author , Chao Xing State Key Lab of Metal Matrix Composites, School of Materials Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240 Google Scholar More articles by this author , Yu Zhao School of Materials Science and Engineering, Zhengzhou University, Zhengzhou 450001 Google Scholar More articles by this author , Meng Sun State Key Lab of Metal Matrix Composites, School of Materials Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240 Google Scholar More articles by this author , Yunqing Zou State Key Lab of Metal Matrix Composites, School of Materials Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240 Google Scholar More articles by this author , Changli Zhao State Key Lab of Metal Matrix Composites, School of Materials Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240 Google Scholar More articles by this author , Xiaoqiu Dou *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] State Key Lab of Metal Matrix Composites, School of Materials Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240 Google Scholar More articles by this author and Chuanliang Feng *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] State Key Lab of Metal Matrix Composites, School of Materials Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.021.202101284 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Dynamic transitions of supramolecular assemblies between lower-order structures and higher-order superhelical structures (e.g., double-helical DNA, helical biopolymers) are of vital importance in many physiological processes, but still remain a great challenge to be realized in artificially assembled systems. Herein, a novel biphenyl central core symmetrically coupled with phenylalanine groups drives the construction of the dynamic superhelix. The rotary packing of biphenyl central units allows π–π stacking under the molecular aggregation state, which combines with hydrogen bonding between phenylalanine moieties to contribute to the formation of the superhelix. Notably, the coordination between carboxyl moieties and metal ions enables the in situ morphological transition between the superhelix and nanospheres, which is regulated by the redox reaction. The superhelical fibers mimicking the extracellular matrix exhibit stronger stereospecific interactions with proteins than primary fibers, facilitating cell adhesion and proliferation. Moreover, the dynamic superhelical fibers as cell culture scaffolds can induce cell release via change of morphology from superhelix to nanospheres. This study provides an innovative approach to explore the supramolecular assembly-related biological processes by the dynamic variation of the superstructured helix in artificial systems. Download figure Download PowerPoint Introduction Chiral architectures (e.g., double-helical DNA, triple-helical collagen, α-helix and β-sheet in proteins) are fundamental units in living systems,1–3 typically because dynamic transitions between lower-order structures and higher-order superhelical structures realize many important biological functions.4,5 For instance, decoiling and reforming of DNA superhelixes are the prerequisites for replication and transcription of genomes.6,7 The conformational misfolding or disorders in coiled-coil superhelical structures of proteins may disrupt normal cellular metabolism and increase the risk of some diseases including Alzheimer’s and Parkinson’s.8,9 Despite the fact that the transformation of chiral structures has been modulated in some supramolecular assembled systems via external stimuli such as light,10,11 pH,12,13 ions,14,15 temperature,16,17 and solvent,18,19 the helical structures involved are only simple helixes due to the rigorous molecular packing mode required for transition from one-dimensional (1D) helical assemblies to higher-order superhelical structures.20 Therefore, it is a great challenge to fabricate higher-level superhelical assemblies and accomplish reversible transformation between initial assemblies and superhelical structures, which is the key to accurately mimic complex chiral structures related to diverse biological functions. The stimuli-responsive supramolecular assembly has attracted considerable attention to the construction and regulation of hierarchical micro-/nanostructures due to the flexibility and versatility of reversible noncovalent interactions between building blocks.21–24 For example, Liu et al.25 reported the morphological switching of self-assembled pyrene-conjugated histidine between nanofibers with P-chirality and nanospheres with M-chirality by adding and removing Zn2+. Zhang and co-workers26 reported that polymerization-induced chiral self-assembly of azobenzene-containing block copolymers could drive a reversible chiral-archiral switching through alternating light irradiation and heating–cooling treatment. Lee et al.27 reported the supramolecular nanotubes that underwent a reversible contraction-expansion motion accompanied by an inversion of helical chirality upon temperature change. However, among the reported examples, morphological transition and chirality switching mainly focus on the primary assemblies, and dynamic regulation of superhelical chiral structures with higher-order assemblies remains challenging so far. Therefore, it is highly desirable to break through the barrier of the molecular packing mode between low-order assembled structures and higher-order superhelical structures, which is necessary and important for unveiling the fundamental role that chiral structural transformation plays in physiological activities. Inspired by l-amino acids as the basic unit constituting proteins with chiral conformations in life,28–30 we have successfully utilized C2-symmetric 1,4-benzenedicarboxamide l-phenylalanine derivatives to construct helical nanofibers with primary chiral structures through directional hydrogen-bonding interaction between l-phenylalanine moieties (see our previous work referenced here).31–35 Herein, a novel biphenyl central core (representative molecule: FLPPFL) replacing individual phenyl (representative molecule: FLPFL) drives the rotary packing of central moieties in the molecular aggregation state (Figures 1a and 1c). The rotary packing of biphenyl allows π–π stacking between central phenyl rings, which cooperates with hydrogen bonding between phenylalanine moieties to realize the superhelical nanofibers. Notably, the morphological interconversion between the superhelical structures and nanospheres can be achieved by redox reaction (Figure 1b), and the reversible morphological transition can be repeated for several cycles. The superhelical fibers mimicking extracellular matrix (ECM) display stronger protein adsorption than primary fibers, leading to promoted cell adhesion and proliferation. Moreover, the dynamic superhelical fibers can serve as cell culture scaffolds to induce cell detachment without proteolytic enzyme treatment. This study not only provides an innovative approach to fabricate biomimetic superhelical structures via rotary arrangement of building blocks, but also presents an important step forward toward the mimicking of dynamic superstructures in artificial supramolecular assemblies. Figure 1 | (a) Schematic illustration of self-assembly route of FLPPFL. (b) The controllable and reversible morphological transition of FLPPFL-Fe2+and FLPPFL-Fe3+ can be in situ achieved by redox reaction. (c) Chemical structures of FLPPFL and FLPFL. Download figure Download PowerPoint Experimental Methods Assemblies of F LPPFL and FLPFL For the assemblies of FLPPFL and FLPFL in deionized water, the suspensions of FLPPFL (0.25 mg/mL) and FLPFL(2 mg/mL) were heated to 90–100 °C, respectively. Then the clear solutions were formed. The assemblies of FLPPFL and FLPFL were obtained when the solutions were cooled down to room temperature. For the assemblies of FLPPFL and FLPFL in a mixture of H2O and ethanol (EtOH), deionized water was added to an EtOH solution of FLPPFL and FLPFL (both final concentrations of FLPPFL and FLPFL were 2.0 mg/mL). For the assemblies of FLPPFL-Fe3+/Fe2+ and FLPFL-Fe3+/Fe2+ in a mixture of H2O and EtOH, deionized water containing Fe3+ or Fe2+ ions was added to an EtOH solution of FLPPFL and FLPFL (both final concentrations of FLPPFL and FLPFL were 2.0 mg/mL). Materials characterization 1H NMR spectroscopy was performed on a Bruker AVANCE III 400 instrument (Bruker Corp., Karlsruhe, Baden-Württemberg, Germany) operating at 400 MHz. Dimethyl sulfoxide (DMSO)-d6 was used as the solvent for 1H NMR measurements. Two-dimensional (2D) NMR spectra were recorded on Bruker AVANCE III 600 instrument (Bruker Corp.) operating at 600 MHz. The mixture of EtOH-d6 and D2O was used as the solvent for 2D NMR measurements. Electrospray ionization mass spectrometry (ESI-MS) was recorded on a Bruker impact II instrument (Bruker Corp.). Methanol was used as the solvent for ESI-MS measurements. Circular dichroism (CD)/linear dichroism (LD) spectra were recorded on a JASCO J-1500 CD spectrometer (Japan Spectroscopic Co., Tokyo, Japan). All concentrations of samples for CD/LD tests were 2.0 mg/mL. All scans were performed at a scan speed of 500 nm/min with a data pitch of 0.5 nm at room temperature. Temperature-dependent CD spectra was performed at 1 K/min from 25 to 90 °C. Scanning electron microscopy (SEM) images were obtained using a FEI Quanta 250 microscope (FEI Company, Hillsboro, OR). The samples were prepared by depositing solutions of assemblies on silicon wafers. After drying under vacuum, the samples were coated with a thin layer of Au. Fourier transform infrared (FT-IR) spectra were taken using with a ThermoFisher Scientific Nicolet iS50 instrument (Thermo Fisher Scientific Inc., Waltham, MA). The X-ray diffraction (XRD) patterns of assemblies were recorded on a Bruker D8 Advance instrument (Bruker Corp.). Protein adsorption FLPPFL, FLPPFL-Fe3+, FLPPFL-Fe2+, FLPFL, FLPFL-Fe3+, and FLPFL-Fe2+ assemblies (concentration: 2 mg/mL, volume: 1 mL, solvent: 10% EtOH, Fe3+ and Fe2+: 1.0 equiv) were prepared in 15 mL centrifuge tubes. The solvent and redundant Fe3+ and Fe2+ were removed by centrifuging. A solution (1 mL) of fluorescein isothiocyanate-bovine serum albumin (FITC-BSA) conjugate (0.512 mg/mL) in phosphate-buffered saline (PBS) (pH 7.4) was added in samples, and then the mixture was incubated at room temperature for 15 min. The fluorescence of the upper solution was measured with a Tecan Infinite M200 PRO spectrometer (Tecan Group Ltd., Männedorf, Kanton Zürich, Switzerland) using λex = 493 nm and λem = 550 nm and by correlating these intensity values to calibration. Cell experiments The 96-well plate coated with these structures was prepared by adding 100 μL 1 mg/mL FLPPFL, FLPPFL-Fe2+, FLPPFL-Fe3+ or FLPFL, respectively, in corresponding wells and drying them in the oven. In addition, glutathione (GSH) was mixed with the FLPPFL-Fe2+ assemblies to inhibit the oxidation of Fe2+. 0.01 mg GSH was added in each well. Each group had at least six duplicated wells. The cell culture medium for human umbilical vein endothelial cells (HUVECs) cells was Dulbecco’s modified Eagle medium (DMEM) containing fetal bovine serum (FBS, 10%), penicillin (100 U mL−1), streptomycin (100 μg mL−1), and l-glutamine (2 × 10−3 mL−1). Cell proliferation was quantitatively analyzed using the cell counting kit-8 (CCK-8) assay. The cell incubation time after the addition of CCK-8 solution was 4 h. The absorbance at 450 nm was measured using a microplate reader. The cells were treated using 4% paraformaldehyde, Triton X-100, and 2% BSA in PBS solution. Actin cytoskeleton and cell nuclei were stained with phalloidin-fluorescein and Hoechst 33238, respectively. For live and dead staining, the cultured cells were washed by PBS. Then 200 μL working solution containing 2 × 10−6 M calcein AM and 8 × 10−6 M propidium iodide (PI) in PBS were added into each well, and the cells were incubated at 37 °C for 30 min. Density functional theory calculations All calculations were performed by the Gaussian 09 package. The ground-state geometry of these dimers was optimized using density functional theory (DFT) with the B3LYP functional, Grimme’s dispersion (B3LYP-D3), and the 6-31G (d, p) basis set.36 Then, frequency calculations at the same level of theory were carried out to identify all the stationary points as minima (zero imaginary frequency). Finally, the electronic CD spectrum was also calculated at the same level. The CD spectra were generated by Gaussian broadening of the stick spectra with a full width at half-maximum of 0.5 eV. All the calculations were analyzed by Multiwfn program package.37 Results and Discussion FLPPFL and FLPFL molecules were designed and synthesized based on l-phenylalanine coupled with biphenyl-4,4′-dicarboxylic acid and terephthaloyl chloride, respectively ( Supporting Information Figures S1–S6). The self-assembly behaviors of FLPPFL and FLPFL were investigated in H2O, EtOH, and various H2O/EtOH mixtures (the volume ratios of EtOH were 5, 10, 30, 50, and 70%(v/v), which were labeled as 5, 10, 30, 50, and 70% EtOH, respectively). Heating–cooling and solvent-induced assembly methods were respectively applied in single-solvent (pure H2O or EtOH) and mixed-solvent (H2O/EtOH mixture) systems. The morphology of FLPPFL and FLPFL self-assemblies obtained from various solvents was observed under SEM. FLPPFL molecules aggregated into right-handed helical hollow tubes with about 800 nm in width and about 2 μm in helical pitch, and then these tubes further entangled and formed higher-order helical structures in pure H2O ( Supporting Information Figure S7). Upon adding a small amount of EtOH in H2O (5% and 10% EtOH), FLPPFL initially assembled into right-handed nanofibers instead of hollow tubes, with the diameter of fibers ranging from 800 to 1000 nm and a helical pitch of around 800–2000 nm (Figure 2a and Supporting Information Figures S8a and S8b). The atomic force microscopy (AFM) image also confirmed this helical structure (Figure 2b). The coiling of helical fibers resulted in superhelical bundles with right-handedness. Interestingly, the coiling degree was solvent-dependent, and the decoiling of the superhelical structure was triggered in 30% EtOH ( Supporting Information Figure S8c). When the fraction of EtOH increased to 50%, the helical nanofibers disappeared, and nonhelical nanobelts formed ( Supporting Information Figure S8d). A further increase of EtOH led to the coexistence of nanobelts and nanospheres, and nanobelts totally transformed into nanospheres upon the use of pure EtOH as solvent ( Supporting Information Figures S8e and 8f). For FLPFLmolecules, they generated nanofibers with diameters of about 100–800 nm in pure H2O and H2O/EtOH mixtures. Like FLPPFL assemblies, FLPFL nanofibers had right-handed helical structures in pure H2O and 5%, 10%, and 30% EtOH (Figure 2c and Supporting Information Figures S9 and S10a–S10c), whereas these helixes disappeared in 50% and 70% EtOH ( Supporting Information Figures S10d–S10e). The sea urchin-like clusters with nonhelical short nanofibers were finally formed from pure EtOH ( Supporting Information Figure S10f). Compared with FLPPFL, FLPFL with a flat rigid core of phenyl moiety lacked conformational flexibility, resulting in difficulty in formation of complex superhelix. These results suggested that the biphenyl central unit played a key role in the formation of the superhelix. Figure 2 | (a) SEM and (b) AFM images of FLPPFL in 10% EtOH. (c) SEM image of FLPFL in 10% EtOH. Scale bar: 1 μm for figure a, 500 nm for figure b, and 1 μm for figure c. (d) CD and UV spectra of FLPPFL and FLPFL in 10% EtOH. (e) Time-dependent CD spectra of FLPPFL at 278 nm and FLPFL at 260 nm in 10% EtOH. (f) VT-CD spectra of FLPPFL at 278 nm in 10% EtOH. (g) XRD patterns of FLPPFL and FLPFL assemblies. (h) FT-IR spectra of FLPPFL and FLPFL assemblies. (i) 1H NMR of FLPPFL in EtOH-d6 and D2O/EtOH-d6 (9:1, v/v) mixtures, respectively. (j) Expanded 2D nuclear Overhauser enhancement spectroscopy (NOESY) spectrum of FLPPFL in D2O/EtOH-d6 (9/1, v/v) mixtures. (k) Optimized structure of FLPPFL dimer obtained by DFT calculations. Download figure Download PowerPoint The effect of the central core on the chiroptical activities of FLPPFL and FLPFL assemblies was further investigated by CD and LD spectroscopy. FLPPFL assemblies in H2O showed a negative Cotton effect at 266 nm and a significant positive Cotton effect at 310 nm ( Supporting Information Figure S11a), with the addition of 10% EtOH, CD signals of FLPPFL reversed (a positive peak at 278 nm and a negative peak at 323 nm in Figure 2d), which corresponded to the transformation of SEM morphologies from helical hollow tubes to nanofibers accompanied by the optical activity inversion. Meanwhile, FLPFL exhibited a positive Cotton effect at 260 nm and a negative Cotton effect at 286 nm in H2O and 10% EtOH (Figure 2d and Supporting Information Figure S11b), which was consistent with the helical structures observed in SEM images. There were obvious CD peaks at 323 nm for FLPPFL and at 286 nm for FLPFL. However, the UV absorption at 323 nm for FLPPFL and at 286 nm for FLPFL almost kept silent (Figure 2d). Besides, no notable peak was observed in LD spectra, indicating that both the FLPPFL and FLPFL assemblies were not macroscopically aligned ( Supporting Information Figure S12). Therefore, the CD signal of FLPPFL at 323 nm and the CD signal of FLPFL at 286 nm should be ascribed to the helical architectures of FLPPFL and FLPFL, implying chiral transformation from the stereogenic center of the phenylalanine motif to the supramolecular assemblies. Furthermore, the CD signals of the dried films [prepared from H2O/EtOH (9/1, v/v)] were consistent with the assemblies in the H2O/EtOH mixtures ( Supporting Information Figure S13), confirming that evaporation of solvent did not change the chiral confirmation of FLPPFL and FLPFL assemblies. Time-dependent CD (Figure 2e and Supporting Information Figure S14) and SEM ( Supporting Information Figure S15) were employed to study the evolution process of the aggregates formed by FLPPFL and FLPFL, respectively. CD signal changes of FLPPFLin 10% EtOH monitored at 276 nm showed a sigmoidal transformation with a lag phase ((black line in Figure 2e and Supporting Information Figure S14a), indicating the nucleation–elongation growth process of the FLPPFL assembly.38–40 Correspondingly, a topological transformation from sphere to nanofiber was observed by SEM after 1 and 10 min ( Supporting Information Figures S15a and S15b). The maximum positive CD signal was detected after around 14 min, which was the mark of the end of the assembling process. However, it was observed that the primary right-handed nanofibers continuously twisted themselves into higher-order helixes with the same helical direction accompanied by the entanglement of the fine nanofibers, as shown in SEM images at 15, 30, 60, and 120 min ( Supporting Information Figures S15c–S15f). These results revealed that the superhelix of FLPPFL arose from the subsequent entanglement of the primary helix. For FLPFL, it took only a few seconds to achieve dynamic equilibrium of the formed assemblies, and afterwards the CD signal and morphology of the assembly remained unchanged (green line in Figure 2e and Supporting Information Figures S14b and S16). Based on these results, it is obvious that the entanglement of the nanofibers plays an important role in the formation of superhelical structures. However, this process is absent during the self-assembly process of FLPFL. For FLPPFLin 10% EtOH, the variable-temperature CD (VT-CD) spectra were further employed to understand the supramolecular assembly mechanism (Figure 2f and Supporting Information Figure S17). Similar to time-dependent CD, VT-CD signal changes of FLPPFL monitored at 278 nm also showed a sigmoidal transformation with a lag phase. Furthermore, a thermal hysteresis was observed by comparing the heating and cooling process at a rate of 5 K/min ( Supporting Information Figure S17). The value of the elongation temperature (Te) in the cooling process was shifted to a higher temperature when the cooling rate was decreased from 8 to 2 K/min. However, no notable effect of heating rate on the Te was observed in the heating process. These results indicated that the self-assembly process was under kinetic control, and the disassembly process occurred under thermodynamic control.41 The curve of aggregation (αAgg) in the heating process fit well with the cooperative model proposed by Meijer, Schenning, and coworkers.42,43 The Te and the elongation enthalpy were calculated to be 349 K and −102 kJ mol−1, respectively, at the given concentration of 3.7 mM ( Supporting Information Figure S18). XRD measurements were employed to reveal the molecular order arrangement of the FLPPFL and FLPFL assemblies. The XRD patterns of FLPPFL prepared from 10% EtOH displayed d-spacings of 1.73, 1.23, 1.00, and 0.86 nm, which corresponded to the ratios of 1, 1/ 2 , 1/ 3 , and 1/2, indicating a body-centered cubic (BCC) packing structure with a d-spacing of 1.73 nm (Figure 2g and Supporting Information Figure S19).44 At the same time, two characteristic XRD peaks at 1.33 and 0.65 nm were obtained for the FLPFL assemblies prepared from 10% EtOH, which corresponded to lamellar aggregates (Figure 2g and Supporting Information Figure S19).45 Given the different molecular arrangements of FLPPFLand FLPFL, it was apparent that the biphenyl central core significantly regulated the molecular packing model. According to the single-crystal structure of FLPFL in the 2004 study by Hemley and Castelletto,44 the individual phenyl core in FLPFL facilitates the formation of extended intermolecular hydrogen-bonds between carboxyl groups in the side chains, leading to lamellar aggregates. BCC packing in FLPPFLassemblies should be ascribed to biphenyl as molecular center. Owing to the flexible nature of biphenyl in FLPPFL, no crystal suitable for X-ray crystallography could be obtained. FT-IR, 1D, and 2D 1H NMR were applied to elucidate the role of biphenyl center in the superhelix. In FT-IR spectra, amide vibrational bands were observed at 1631 cm−1 (amide I) and 1527 cm−1 (amide II), indicating the association of the amide groups induced by hydrogen bonds (Figure 2h). For FLPFL, amide I and amide II were located at 1639 and 1538 cm−1, respectively, suggesting that the hydrogen bonds in FLPFL were weaker than in FLPPFL. In the 1H NMR spectrum, the obvious shift of the Hc proton in FLPPFL (at 4.90 ppm in EtOH-d6 and 4.65 ppm in D2O/EtOH-d6 (9:1, v/v)) further confirmed that the amide group participated in the formation of hydrogen bonds (Figure 2i). Besides, the 1H NMR spectrum in EtOH-d6 showed two signals the biphenyl center (Hb: around 7.66 ppm, Ha: around 7.85 ppm) (Figure 2i). In D2O/EtOH-d6 (9:1, v/v), the Ha proton in the biphenyl center shifted to a higher field, implying that biphenyl centers contributed to π–π stacking (Figure 2i). For FLPFL, the hydrogen bonds formed by an amide group and the π–π stacking in phenyl cores were confirmed by the shift of Hb, and Ha, protons, respectively ( Supporting Information Figure S20). 2D NMR experiments were performed to reveal the spatial arrangement of molecules. Nuclear Overhauser effect (NOE) peaks between Ha and Hd were present in D2O/EtOH-d6 (9:1, v/v; zoomed region in Figure 2j and the entire region in Supporting Information Figure S21; the entire region of COSY spectrum in Supporting Information Figure S22), which suggested that the distance between Ha of biphenyl centers and Hd of benzene rings in phenylalanine sides was less than 5 Å. A similar phenomenon (NOE peak between Ha, of phenyl center and Hc, of benzene rings in D2O/EtOH-d6 [9:1, v/v)] was also observed in FLPFL ( Supporting Information Figures S23 and S24). These results indicated that similar intermolecular interactions (hydrogen bonding and π–π stacking) existed in the assemblies of FLPPFL and FLPFL. Based on the above FT-IR and 1D and 2D 1H NMR results, we speculated that there were two possible molecular packing modes were speculated: (a) parallel packing mode in which intermolecular hydrogen bonds formed between CONH and CONH; (b) slipped packing mode in which intermolecular hydrogen bonds formed between CONH and COOH ( Supporting Information Figure S25). These two possible packing modes of FLPPFL were further analyzed via DFT calculations. Compared to the slipped packing dimer of FLPPFL, the parallel packing dimer displayed much lower binding energy (−170.66 kJ/mol for the parallel packing dimer; −105.02 kJ/mol for the slipped packing dimer, Supporting Information Figure S26), suggesting that the parallel packing model was more reasonable. After optimization of the parallel packing dimer of FLPPFL(Figure 2k), we found that the FLPPFL dimer exhibited a quite different packing arrangement compared to the dimer structure of FLPFL (the optimized structure dimer of FLPFL was reported in the literature46). From the top view of the FLPPFL dimer structure, it is clear that two central biphenyl rings have an around 30° rotation to each other, which facilitates the helical stacking of FLPPFL molecules. The distance of central biphenyl rings is about 3.4 Å, which agrees with the π–π stacking distances (∼0.35 nm) observed by XRD experiments (Figure 2g). Intermolecular hydrogen bond interactions are formed between amide bonds, and the distance of hydrogen bonds is about 1.9 Å. In addition, the distance between Ha and Hd is 4.3 Å, which fits with the NOE peaks observed in FLPPFL assemblies. The calculated CD spectrum of FLPPFL dimer was in good agreement with the experimental results ( Supporting Information Figure S27), which showed that the predicted structure of FLPPFL dimer was reasonable and proper. In comparison to FLPPFLdimers, the central phenyl rings of FLPFL molecules were nearly perpendicular to each other, and intermolecular hydrogen bonds were formed between C=O of carboxylic acid and N–H of the amide groups.46 The above results reveal that although the self-assemblies of FLPPFL and FLPFL are all driven by hydrogen bonding and π–π stacking, different central units lead to different molecular arrangements and morphologies. Herein, the detailed formation process of FLPPFL superhelix is illustrated in Figure 1a, in which rotary packing of central biphenyl units plays an important role during FLPPFL assembly. The rotary packing of biphenyl allows π–π stacking under the molecular aggregation state, which combines with hydrogen bonding between amide groups to stabilize the helical aggregates. Subsequently, the helical aggregates bundle into nanofibers and then the nanofibers twist themselves into the higher-order superhelix. The higher-order superhelix was successfully obtained by regulating the number of aromatic molecules in the core of C2-symmetric molecules; however, this superhelix lacked the dynamic transformation property that is an indispensable feature of many superhelical structures in life (e.g., DNA, RNA, collagen, protein). To endow the FLPPFL superhelix with this dynamic feature, the coordination of –COOH with metal ions47 and the reversible conversion between Fe3+ and Fe2+ were applied. In this respect, the coassemblies with iron ions were prepared from 10% EtOH. For FLPPFL, adding a small quantity of Fe3+ (below 0.3 equiv) first triggered the uncoiling of the superhelical nanofibers ( Supporting Information Figures S28a and S28b). By continually increasing the amount of Fe3+ to 0.5 equiv allowed nanofibers to break up into spherical aggregates, and nanofibers were totally converted to nanospheres until Fe3+ reached 1.0 equiv (Fig