Multiple Stimuli-Responsive Conformational Exchanges of Biphen[3]arene Macrocycle

Conformational exchanges of synthetic macrocyclic acceptors are rather fast, which is rarely studied in the absence of guests. Here, we report multiple stimuli-responsive conformational exchanges between two preexisting conformations of 2,2′,4,4′-tetramethoxyl biphen[3]arene (MeBP3) macrocycle. Structures of these two conformations are both observed in solid state, and characterized by 1H NMR, 13C NMR and 2D NMR in solution. In particular, conformational exchanges can respond to solvents, temperatures, guest binding and acid/base addition. The current system may have a role to play in the construction of molecular switches and other stimuli-responsive systems.

Synthetic macrocycles often have simple structures and fast conformational exchange kinetics [28], which are hard to detect by common kinetic techniques. Although conformational interconversions of macrocyclic acceptors can be observed in molecular recognition processes [29][30][31][32][33][34][35][36], slow exchanges of coexisting conformations of macrocycles have been rarely reported [37]. Developing slow conformational exchange systems of macrocycles can not only apply in the design and synthesis of molecular machines and devices [2,[7][8][9][10] but also function as an important tool for host-guest binding mechanism analysis [37]. The assignment of the binding mechanism is often inconclusive and synthesis of molecular machines and devices [2,[7][8][9][10] but also function as an important tool for host-guest binding mechanism analysis [37]. The assignment of the binding mechanism is often inconclusive in synthetic supramolecular systems because of the fast exchange kinetics [38,39]. Very recently, Jiang et al. demonstrated the strict analysis of the binding mechanism based on a macrocycle with slow conformational exchange [37].

Conformations Analysis
MeBP3 contains three 2,2′,4,4′-tetramethoxyl biphenyl sidewalls and three methylene linkers, which is structurally similar to those synthetic macrocyclic arenes like calix[n]arenes [28,30,[47][48][49], resorc [4]arene [50], cyclotribenzylene [51], pillar[n]arenes [52][53][54] and other biphen[n]arenes [40][41][42][43][44][45][46]. Theoretically, there are two representative conformations for MeBP3, due to flipping of the biphenyl sidewalls ( Figure 1a, MeBP3-I and MeBP3-II). For MeBP3-I, three biphenyl units are arranged in the same orientation, and flipping of one biphenyl panel of MeBP3-I can produce MeBP3-II [55]. Conformer MeBP3-I has D3 symmetry and thus should give rise to two singlets in 1 H NMR for their aromatic signals. Conformer MeBP3-II has C2 symmetry and six singlets in the aromatic regions. If the exchanges between two conformers are slow on the NMR timescale, we can differentiate them in the NMR spectra of MeBP3.  In the 1 H NMR spectrum of MeBP3 (2.0 mM), one set of two broad singlets and the other set of six broad singlets in the aromatic regions were observed (Figure 2a), indicating that MeBP3-I and MeBP3-II are coexisting in CDCl 3 . Two sets of broad singlets indicate that single bonds in biphenyl units rotate fast, but the overall exchanges between MeBP3-I and MeBP3-II were slow on the NMR timescale at room temperature. The 13 C NMR spectrum of MeBP3 in CDCl 3 was tested; six carbon signals for MeBP3-I and 18 carbon signals for MeBP3-II in the aromatic regions were observed (Figure 2b). 1 H-13 C HMQC and HMBC were employed to assign the 1D NMR spectra (Figures S6-S9). The minor species was assigned to MeBP3-II, while the major species was assigned to MeBP3-I, and their percentages were calculated according to the integrals in 1 H NMR spectrum, which are 73% for MeBP3-I and 27% for MeBP3-II at 25 • C in CDCl 3 . We examined the 1 H NMR spectra of MeBP3 in CDCl 3 at a high concentration of 100 mM (Figures S1 and S2). No obvious concentration effect was observed on the distribution of conformers.
Molecules 2020, 25, x FOR PEER REVIEW 3 of 10 In the 1 H NMR spectrum of MeBP3 (2.0 mM), one set of two broad singlets and the other set of six broad singlets in the aromatic regions were observed (Figure 2a), indicating that MeBP3-I and MeBP3-II are coexisting in CDCl3. Two sets of broad singlets indicate that single bonds in biphenyl units rotate fast, but the overall exchanges between MeBP3-I and MeBP3-II were slow on the NMR timescale at room temperature. The 13 C NMR spectrum of MeBP3 in CDCl3 was tested; six carbon signals for MeBP3-I and 18 carbon signals for MeBP3-II in the aromatic regions were observed ( Figure  2b). 1 H-13 C HMQC and HMBC were employed to assign the 1D NMR spectra ( Figure S6 to Figure  S9). The minor species was assigned to MeBP3-II, while the major species was assigned to MeBP3-I, and their percentages were calculated according to the integrals in 1 H NMR spectrum, which are 73% for MeBP3-I and 27% for MeBP3-II at 25 °C in CDCl3. We examined the 1 H NMR spectra of MeBP3 in CDCl3 at a high concentration of 100 mM (Figures S1 and S2). No obvious concentration effect was observed on the distribution of conformers. We obtained two single-crystals of MeBP3 containing CH3CN molecules or cis-1,2dichloroethene (cis-DCE) molecules in unit cells [39], namely, "CH3CN@MeBP3" and "cis-DCE@MeBP3" [46], respectively. The single-crystal X-ray diffraction data suggested that the structure of MeBP3 in CH3CN@MeBP3 could be assigned to MeBP3-I, while the structure of MeBP3 in cis-DCE@MeBP3 was MeBP3-II ( Figure 1b). In the solid state, MeBP3-I exhibits a triangular topology, and three biphenyl sidewalls define a cavity suitable for the linear guests with 5.43 Å diameters [39]. Flipping one biphenyl sidewall in MeBP3-I to the other side can produce MeBP3-II, which was observed in cis-DCE@MeBP3.

Solvent and Temperature Effects in Conformational Exchanges
Molecular conformations are closely related to solvents and temperatures. We therefore tested the conformational exchanges of MeBP3 in different solvents and temperatures. MeBP3-I and MeBP3-II also coexist in CD2Cl2, acetone-d6, acetonitrile-d3, toluene-d8 and xylene-d10, but their percentages were different (for MeBP3-I is 87%, 88%, 86%, 85% and 80%, respectively) at room temperature ( Figure 3b). However, in high polarity solvent such as DMSO-d6, no signals for MeBP3-II were observed ( Figure S4). We obtained two single-crystals of MeBP3 containing CH 3 CN molecules or cis-1,2-dichloroethene (cis-DCE) molecules in unit cells [39], namely, "CH 3 CN@MeBP3" and "cis-DCE@MeBP3" [46], respectively. The single-crystal X-ray diffraction data suggested that the structure of MeBP3 in CH 3 CN@MeBP3 could be assigned to MeBP3-I, while the structure of MeBP3 in cis-DCE@MeBP3 was MeBP3-II ( Figure 1b). In the solid state, MeBP3-I exhibits a triangular topology, and three biphenyl sidewalls define a cavity suitable for the linear guests with 5.43 Å diameters [39]. Flipping one biphenyl sidewall in MeBP3-I to the other side can produce MeBP3-II, which was observed in cis-DCE@MeBP3.

Solvent and Temperature Effects in Conformational Exchanges
Molecular conformations are closely related to solvents and temperatures. We therefore tested the conformational exchanges of MeBP3 in different solvents and temperatures. MeBP3-I and MeBP3-II also coexist in CD 2 Cl 2 , acetone-d 6 , acetonitrile-d 3 , toluene-d 8 and xylene-d 10 , but their percentages were different (for MeBP3-I is 87%, 88%, 86%, 85% and 80%, respectively) at room temperature ( Figure 3b). However, in high polarity solvent such as DMSO-d 6 , no signals for MeBP3-II were observed ( Figure S4). Variable-temperature 1 H NMR spectra were also examined in different solvents. At relatively high temperatures (≥50 °C), only signals for MeBP3-I were observed in all selected solvents ( Figure  3c, Figures S11-S16 and Table S1), suggesting that MeBP3-I is more thermodynamically stable than MeBP3-II. Decreasing the temperatures of MeBP3's toluene-d8 solution from 100 °C to −60 °C led to gradual transformations of the chemical equilibrium ( Figure 3c). For example, at 25 °C, the percentage of MeBP3-I was 85%, while the percentage of MeBP3-II was 61% at −60 °C. That is, in low polarity solvent of toluene-d8, the chemical equilibrium leaned toward the MeBP3-II at low temperatures. However, at high temperatures, MeBP3-I was the dominant species (Table S1). In contrast, in midpolarity solvents such as CDCl3, CD2Cl2, acetone-d6 and acetonitrile-d3, MeBP3-I was always the main conformer (Table S1). One possible reason is that the temperature-triggered conformational exchange is a solvation-driven process, CDCl3, CD2Cl2, acetone-d6 and acetonitrile-d3 can solvate MeBP3, thereby freezing the changes of chemical equilibrium [21].

Guest-Binding and Acid/Base Addition in Conformational Exchanges
Suitable guests may efficiently switch the conformations of acceptors [29][30][31][32][33][34][35][36]. According to MeBP3's cavity size (5.43 Å) [40] and π-electron rich characteristics [40][41][42][43][44][45][46], a linear secondary ammonium guest 1 + was chosen to study the binding properties of the macrocycle. As shown in Figure 4, in the 1 H NMR spectrum of host-guest 1:1 mixtures, no signals for MeBP3-II were found, showing that the conformational exchanges were fixed in the presence of 1 + . Meanwhile, the proton signals of 1 + shifted upfield dramatically (for example, ∆σ = −0.40 ppm for the protons at 2.94 ppm), suggesting that an interpenetrated complex was formed. In other words, MeBP3-I can bind 1 + efficiently, and the host-guest association can fix the conformational exchanges. Furthermore, no signals for free guest and free MeBP3 were detected, suggesting that the guest exchange kinetics of complex 1 + ⸦ MeBP3 was fast on the NMR timescale. This is in contrast to the slow interconversion kinetics between two conformers. Variable-temperature 1 H NMR spectra were also examined in different solvents. At relatively high temperatures (≥50 • C), only signals for MeBP3-I were observed in all selected solvents (Figure 3c, Figures S11-S16 and Table S1), suggesting that MeBP3-I is more thermodynamically stable than MeBP3-II. Decreasing the temperatures of MeBP3's toluene-d 8 solution from 100 • C to −60 • C led to gradual transformations of the chemical equilibrium (Figure 3c). For example, at 25 • C, the percentage of MeBP3-I was 85%, while the percentage of MeBP3-II was 61% at −60 • C. That is, in low polarity solvent of toluene-d 8 , the chemical equilibrium leaned toward the MeBP3-II at low temperatures. However, at high temperatures, MeBP3-I was the dominant species (Table S1). In contrast, in mid-polarity solvents such as CDCl 3 , CD 2 Cl 2 , acetone-d 6 and acetonitrile-d 3 , MeBP3-I was always the main conformer (Table S1). One possible reason is that the temperature-triggered conformational exchange is a solvation-driven process, CDCl 3 , CD 2 Cl 2 , acetone-d 6 and acetonitrile-d 3 can solvate MeBP3, thereby freezing the changes of chemical equilibrium [21].
As mentioned above, the host-guest association can fix the conformational exchanges at room temperature, and the macrocycle favored the conformer MeBP3-II at low temperature intoluene-d8. Next, we investigated the influence of guest-binding toward conformational exchanges in the toluene-d8 solution of MeBP3 at −60 °C. Upon addition of 1.2 equivalents (eq.) of 1 + , the 1 H NMR (Figure 5b) spectra of MeBP3 suggested that the conformational exchanges were nearly fixed. In both instances the absence of resonances in the aromatic regions of 1 H NMR signals suggested that the MeBP3-II nearly disappeared. Then, we expected that the 1 + can be deprotonated with the assistance of organic base n-Pr3N, which would dissociate the host-guest complex and resume the conformational exchanges. Upon further addition of 1.5 eq. n-Pr3N, the NMR spectrum almost recovered the resonances for a mixture of MeBP3-I and MeBP3-II (Figure 5b). After that, with the addition of 1.8 eq. CF3COOH, no 1 H NMR signals for MeBP3-II were found in the mixture solution (Figure 5b). The re-protonation of dibutylamine re-associated the host-guest complex and fixed the conformational exchanges. That is, the conformational exchanges between MeBP3-I and MeBP3-II can be responsive to the guest binding and acid/base addition. This result also suggests that the exchange kinetics of host-guest complex is faster than the conformational exchange. NMR titration experiments were employed to measure the association constants (K 1 , K 2 ) between two conformers of MeBP3 and 1 + [37]. When we gradually titrated 1 + into a CDCl 3 solution of MeBP3 (1.0 mM, Figures S17 and S18), the obvious shifts were observed in both two conformers' 1 H NMR signals, showing that 1 + can be recognized by both MeBP3-I and MeBP3-II. Nonlinear curve-fitting ( Figure S19) gave the association constants of K 1 = (3.50 ± 0.54) × 10 3 M −1 (1 + ⊂ MeBP3-I) and K 2 = (2.27 ± 0.18) × 10 3 M −1 (1 + ⊂ MeBP3-II), respectively, which further indicated that MeBP3-I can bind 1 + more efficiently.
As mentioned above, the host-guest association can fix the conformational exchanges at room temperature, and the macrocycle favored the conformer MeBP3-II at low temperature intoluene-d 8 . Next, we investigated the influence of guest-binding toward conformational exchanges in the toluene-d 8 solution of MeBP3 at −60 • C. Upon addition of 1.2 equivalents (eq.) of 1 + , the 1 H NMR (Figure 5b) spectra of MeBP3 suggested that the conformational exchanges were nearly fixed. In both instances the absence of resonances in the aromatic regions of 1 H NMR signals suggested that the MeBP3-II nearly disappeared. Then, we expected that the 1 + can be deprotonated with the assistance of organic base n-Pr 3 N, which would dissociate the host-guest complex and resume the conformational exchanges. Upon further addition of 1.5 eq. n-Pr 3 N, the NMR spectrum almost recovered the resonances for a mixture of MeBP3-I and MeBP3-II (Figure 5b). After that, with the addition of 1.8 eq. CF 3 COOH, no 1 H NMR signals for MeBP3-II were found in the mixture solution (Figure 5b). The re-protonation of dibutylamine re-associated the host-guest complex and fixed the conformational exchanges. That is, the conformational exchanges between MeBP3-I and MeBP3-II can be responsive to the guest binding and acid/base addition. This result also suggests that the exchange kinetics of host-guest complex is faster than the conformational exchange.

Discussion
In conclusion, we investigated the multiple stimuli-responsive conformational exchanges of 2,2′,4,4′-tetramethoxyl biphen [3]arene. Two preexisting conformers of MeBP3 were found in solutions with slow conformational interconversions. Structures of these two conformers were fully characterized by 1 H-13 C HMQC and HMBC NMR spectra. Besides, solvents, temperatures, guest binding and acid/base addition can dramatically influence the conformational exchanges. In relatively high temperatures, conformer MeBP3-I is the dominant species in all solutions, for example, percentage of MeBP3-I is >95% in CDCl3, acetone-d6, toluene-d8 and xylene-d10. In contrast, MeBP3-II is the main conformation in MeBP3's toluene-d8 solution at −60 °C (61%). Secondary ammonium guest 1 + can enter the cavity of MeBP3 and fix the conformational exchanges, even at −60 °C in toluene-d8, whereas only MeBP3-I was observed in the host-guest mixture's solution. The addition of acid/base can resume/re-fix the conformational interconversions of MeBP3 in the hostguest mixture's solution. This slow conformation-exchange system may have a role to play in the

Discussion
In conclusion, we investigated the multiple stimuli-responsive conformational exchanges of 2,2 ,4,4 -tetramethoxyl biphen [3]arene. Two preexisting conformers of MeBP3 were found in solutions with slow conformational interconversions. Structures of these two conformers were fully characterized by 1 H-13 C HMQC and HMBC NMR spectra. Besides, solvents, temperatures, guest binding and acid/base addition can dramatically influence the conformational exchanges. In relatively high temperatures, conformer MeBP3-I is the dominant species in all solutions, for example, percentage of MeBP3-I is >95% in CDCl 3 , acetone-d 6 , toluene-d 8 and xylene-d 10 . In contrast, MeBP3-II is the main conformation in MeBP3's toluene-d 8 solution at −60 • C (61%). Secondary ammonium guest 1 + can enter the cavity of MeBP3 and fix the conformational exchanges, even at −60 • C in toluene-d 8 , whereas only MeBP3-I was observed in the host-guest mixture's solution. The addition of acid/base can resume/re-fix the conformational interconversions of MeBP3 in the host-guest mixture's solution. This slow conformation-exchange system may have a role to play in the binding mechanism analysis, molecular switches and stimuli-responsive materials. Studies directed to exploring these possibilities are underway.
Author Contributions: C.L. conceived the project. C.L., Y.W. and L.-P.Y. designed the experiments. Y.W. and X.Z. synthesized the MeBP3. Y.W. and L.-P.Y. performed NMR titration experiment. Y.W., C.L., L.C., J.L., X.J. and J.F. analyzed the data. C.L., Y.W. and L.-P.Y. co-wrote the manuscript. All authors discussed and commented on the paper. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the National Natural Science Foundation of China (21971192, 21801125 and 21772118), and funds provided by Tianjin Normal University.

Conflicts of Interest:
The authors declare no conflict of interest.