pH-Induced Transition Between Single-Chain Macrocyclic Amphiphile and [c2]Daisy Chain-Based Bola-Type Amphiphile and the Related Self-Assembly Behavior in Water

Macrocyclic amphiphiles, a type of amphiphiles synthesized based on macrocyclic compounds, have attracted much attention over the past decades due to their unique superiority in the construction of various functional nanomaterials. The regulation of the state of macrocyclic amphiphiles by introducing stimuli-responsive motif to macrocyclic amphiphiles is an efficient way to extend their applications in diverse fields. Herein, pillararene-based macrocyclic amphiphile H1 was prepared. H1 can act as single-chain amphiphile to self-assemble into micelles in water when the pH was ≥5.0. H1 can be protonated to turn into H2 when pH changed to <5.0. Interestingly, H2 formed [c2]daisy chain-based bola-type supramolecular amphiphile. This bola-type supramolecular amphiphile self-assembled into nanosheets in water. Therefore, pH-induced transition between single-chain macrocyclic amphiphile and bola-type amphiphile and the corresponding self-assembly system based on pillararene in water were constructed.

Pillararenes, the generation of macrocycles next to crown ethers, cyclodextrins, calixarenes, and cucurbiturils, have been widely studied in the past decade (Li et al., 2017;Ping et al., 2017;Sathiyajith et al., 2017;Hua et al., 2018Hua et al., , 2019Chen et al., 2019;Xu et al., 2019). Owing to their facile synthesis, easy functionalization and excellent host-guest recognition property, pillararenes have been widely applied to construct amphiphilic self-assembly systems Xia et al., 2017;Zhang et al., 2018;Xiao et al., 2019). The rigid and symmetric structure SCHEME 1 | Chemical structures of the macrocyclic amphiphiles H1 and H2 and cartoon representation of the [c2]daisy chain-based bola-type supramolecular amphiphile and the pH-responsive self-assembly. of pillararenes make them good candidates for the construction of macrocyclic amphiphiles. Several types of pillararenes-based macrocyclic amphiphiles have been reported up to now: (1) the non-symmetric pillararenes with half hydrophilic groups and half hydrophobic groups from non-symmetric monomers (Yao et al., 2012;Yu et al., 2013); (2) the difunctionalized pillararenebased macrocyclic amphiphiles from copillar[5]arenes (Gao et al., 2013); (3) monofunctionalized pillararene-based macrocyclic amphiphiles by linking hydrophobic tails to symmetric pillararenes (Jie et al., 2014); and (4) the symmetric per-functionalized pillararenes-based amphiphiles (Nierengarten et al., 2013;Chang et al., 2014Chang et al., , 2019aYang et al., 2016;Sun et al., 2018). The obtained macrocyclic amphiphiles from these methods displayed interesting stimuli-responsiveness and applications, indicating the importance of pillararenes-based macrocyclic amphiphiles. Herein, we developed a new efficient way to synthesize pillararenes-based macrocyclic amphiphiles. First, we synthesized a long alkyl-containing copillar[5]arene from previous literature. Then, pH-sensitive morpholine groups were covalently linked to the copillar[5]arene to prepare a single-chain macrocyclic amphiphile H1. Interestingly, H1 transformed into protonated state H2, which formed [c2]daisy chains in water when the pH-value decreased under 5.0. As a result, single-chain amphiphile H1 turned into bola-type supramolecular amphiphile. Moreover, the pH-responsive self-assembly behavior was investigated. Singlechain amphiphile H1 self-assembled into micelles in water. When the value of pH decreased to under 5.0, micelles transformed into nanosheets due to the formation of bola-type supramolecular amphiphiles based on the [c2]daisy chain structure (Scheme 1).

MATERIALS AND METHODS
All reagents were commercially available and used as supplied without further purification. Compounds a were prepared according to published procedures (Shi et al., 2014). NMR spectra were recorded with a Bruker Avance DMX 600 spectrophotometer or a Bruker Avance DMX 400 spectrophotometer. Low-resolution electrospray ionization mass spectra were recorded with a Bruker Esquire 3000 Plus spectrometer. High-resolution mass spectrometry experiments were performed with a Waters UPLC H-Class QDA instrument. The melting points were collected on a SGW X-4 automatic melting point apparatus. The determination of the critical aggregation concentration (CAC) values was carried out on a FE38 instrument. Transmission electron microscopy investigations were carried out on a JEM-1200EX instrument. Atomic force microscopy experiments were performed by a Bruker Multi-Mode 8.0 instrument.

Critical Aggregation Concentration Determination
The CAC determination is based on the dependence of the solution conductivity on the solution concentration. Generally, the slope value of the change in conductivity vs. the concentration above CAC is higher than the slope below the CAC. As a result, the CAC-value is the junction of the conductivityconcentration plot. To measure the CAC of H1 and H2, the conductivities of their solutions at different concentrations (from 0 to 0.16 mM and from 0 to 0.25 mM, respectively) were determined. Therefore, through plotting the changes of the conductivity vs. the concentration, the CAC of H1 and H2 can be obtained.

Transmission Electron Microscopy Experiments
The self-assembled structures of H1 and H2 were investigated by TEM. A solution of 1.00 × 10 −4 M H1 was made first in water. The samples of H1 were prepared by drop coating this solution onto a carbon-coated copper grid. The solution of H2 were obtained by adding hydrochloride acid to the solution of H1. Then, the TEM samples of H2 was prepared by drop coating the solution on a carbon-coated copper grid. TEM experiments were performed on a JEM-1200EX instrument.

Atomic Force Microscopy Experiments
The self-assembled structure H2 was investigated by atomic force microscopy (AFM). A solution of 1.00 × 10 −4 M H1 was prepared in water. The solution of H2 were obtained by adding hydrochloride acid to the solution of H1. Then, the TEM samples of H2 was prepared by drop coating the solution on a Si substrate. AFM experiments were carried out on a Bruker Multi-Mode 8.0 instrument.

RESULTS AND DISCUSSIONS 1 H NMR Spectroscopy Experiments
First, 1 H NMR spectroscopy experiments were performed to study the pH-induced transition between H1 and H2. Owing to the poor solubility of H1 in water, the 1 H NMR experiments of H1 was done in the mixture of D 2 O and CD 3 CN (Figures 1A-C). With the decrease in the pH-value of the aqueous solution of H1, the morpholine groups on H1 were protonated, and H1 changed into H2. Therefore, the peaks corresponding to the protons on H2 were quite different from that of H1. Figures 1D-G, the signals for protons H 2g and H 2h appeared in upfield comparing to the protons H 1g and H 1h on H1. In addition, the signals for the protons H 2i -H 2k on the alkyl chain of H2 appeared in upfield and splitted compared to the related protons on H1. This phenomenon was because the alkyl chain threaded into the cavity of H2, forming cyclic oligomers. To investigate whether the specific structures of H2 occur only at low concentrations, concentrationdependent 1 H NMR experiments were carried out. As shown in Figure 2, with the increase in the concentration from 0.500 to 5.00 mM, the peaks related to H2 did not show changes. In addition, at first, we assumed that H2 can also act as monomers for supramolecular polymers like other systems (Shi et al., 2014). However, H2 showed poorer solubility in water than other monomers. That is why the concentration-dependent 1 H NMR experiments were only done in the range of 0.500-5.00 mM. Therefore, the conclusion can be drawn that H2 formed [2]daisy chains in water, which did not changed with its concentration .

2D Nuclear Overhauser Effect Spectroscopy Study
2D nuclear overhauser effect spectroscopy (NOESY) was performed to monitor the formation of the [c2]daisy chain based on H2. As shown in Figure 3, NOE correlation signals were observed between the protons H 2a on the phenyl rings and H 2k on the alkyl chain (A), between H 2b on the methylene bridge and H 2k -H 2j on the alkyl chain (B), and between protons H 2g , H 2h on the morpholine groups and H 2i -H 2k on the alkyl chain (C), suggesting that the alkyl chain thread into the cavity of H2. In addition, NOE correlation signals were also observed between alkyl chains, including the signals between protons H 2j3 and H 2k (D) and between protons H 2j4 and H 2k (E), confirming the formation of the [c2]daisy chain based on H2.

Critical Aggregation Concentration Determinations
The CACs of H1 and H2 were measured. As shown in Figure 4, the CACs of H1 and H2 were measured to be 3.69 × 10 −6 M and 2.67 × 10 −5 M, respectively, using the concentration-dependent conductivity measurements.  Frontiers in Chemistry | www.frontiersin.org

Transmission Electron Microscopy and Atomic Force Microscopy Investigations
The self-assembly morphologies in water were investigated by TEM and AFM. As shown in Figure 5a, H1 formed micelles with an average diameter of ∼6 nm, which was near to the length of two H1 molecules. After adding hydrochloride acid to the solution of H1 to adjust the pH-value to 4.0, H1 turned into H2, the micelles changed into nanosheets (Figure 5b). After further addition of sodium hydroxide to the solution of H2, the nanosheets turned back to micelles (Figure 5c). AFM experiments were also carried out to investigate the selfassembled morphology by H2. As shown in Figures 5d,e, the nanosheet morphology was confirmed and the wall thickness was ∼3.425 nm from AFM results, which was about the extended length of the [c2]daisy chain, suggesting that the nanosheets had a bilayer wall.

CONCLUSION
In summary, a pillararene-based macrocyclic amphiphile H1 was prepared. H1 can act as a single-chain amphiphile and self-assembled into micelles in water. After changing the pH of the solution of H1 to below 5.0, the single-chain amphiphiles turned into [c2]daisy chain-based bola-type supramolecular amphiphiles. As a result, the micelles turned into nanosheets when self-assembling in water. This pH-induced transition between macrocyclic singlechain amphiphile and [c2]daisy chain-based bola-type supramolecular amphiphiles based on pillararenes was first reported, providing a new strategy to tailor the structure and self-assembly property of macrocyclic amphiphiles. The corresponding pH-responsive self-assembly system provides a promising candidate for advanced material such as controlled release, drug delivery systems, and surface modification.

DATA AVAILABILITY STATEMENT
Compound characterization, full synthetic details for this study are included in the article/Supplementary Material.

AUTHOR CONTRIBUTIONS
PW, RW, and DX made contributions to the experiments.