In situ monitoring of molecular aggregation using circular dichroism.

The aggregation of molecules plays an important role in determining their function. Electron microscopy and other methods can only characterize the variation of microstructure, but are not capable of monitoring conformational changes. These techniques are also complicated, expensive and time-consuming. Here, we demonstrate a simple method to monitor in-situ and in real-time the conformational change of (R)-1,1'-binaphthyl-based polymers during the aggregation process using circular dichroism. Based on results from molecular dynamics simulations and experimental circular dichroism measurements, polymers with "open" binaphthyl rings are found to show stronger aggregation-annihilated circular dichroism effects, with more negative torsion angles between the two naphthalene rings. In contrast, the polymers with "locked" rings show a more restrained aggregation-annihilated circular dichroism effect, with only a slight change of torsion angle. This work provides an approach to monitor molecular aggregation in a simple, accurate, and efficient way.

Introduction

The term “chiral” is used in chemistry to describe molecules which are non-superimposable on their mirror image[1-4]. Chiral molecules are abundant in nature, with many natural chemicals such as amino acids and deoxyribonucleic acid (DNA) exhibiting chirality. There are various ways to characterize chirality, and amongst them circular dichroism (CD) is the most commonly used tool to determine the absolute configuration, both in solution and the solid state, because of its simple operation, high accuracy and efficiency[5-8]. Particularly, CD has been widely used to analyze the secondary structure of optically-active biomacromolecules such as proteins and DNA, which is sensitive to environmental variation. The dynamic processes of these chiral molecules are important to their biological activity. For example, the aggregation of proteins is associated with many pathological conditions[9]. How to monitor the conformational changes during protein aggregation is significant to clarify the working mechanism of biological processes. Traditionally, these processes have been studied by techniques such as electron microscopy and NMR spectroscopy. In these methods, pretreatment of samples is required and the analyses are carried out under harsh conditions[10-17]. Thanks to its capability for in-situ and real-time detection, CD spectroscopy may be a good choice to investigate the relationship between molecular aggregation and conformational change. In previous work, the effect of aggregation-induced circular dichroism has been observed in some self-assembly systems[18]. The electron microscopic results suggested that CD-inactive molecules were induced to generate strong CD signals upon helix formation in the aggregate state. However, the detailed conformational changes of a single molecule during the self-assembly process is difficult to determine using CD spectroscopy since the detected CD signal results from the whole assembly structure.

Recently, an opposite effect called aggregation-annihilated circular dichroism (AACD) was observed in 1,1′-binaphthyl derivatives. Further research revealed that the annihilation might be caused by the conformational change of 1,1′-binaphthyl (BN)[19]. BN is a well-studied chiral molecule[20,21]. Mason et al. have set up a classical model to calculate the dependence of the CD couplet intensity (Δεmax) and Davydov splitting (Δλmax) on the torsion angle (θ) in 1,1′-binaphthyl[6,22,23]. Figure 1a shows the electronic transitions and coupling between the 1Bb and 1Bb′ transitions in 1,1′-binaphthyl, while Fig. 1b demonstrates the simulated CD spectra of (R)-1,1′-binaphthol with θ varying from −45 to −110°. The plots of Δεmax/Δεmax-0 and (Δλmax − Δλmax-0) versus the θ value are shown in Fig. 1c,d, respectively, where Δεmax-0 and Δλmax-0 are the values at θ = −45°. To investigate the rate, the first-order derivatives of d|Δεmax|/d|θ| and d|Δλmax|/d|θ| are also plotted in Fig. 1c,d with blue scatter lines. As suggested by Fig. 1, the maximum |Δεmax| is located at around −65° and the Δλmax gradually decreases when θ evolves from −45° to −110°. The established relationship is expected to determine the absolute configuration of chiral materials using the CD measurement.

Fig. 1

Simulated chiroptical properties of binaphthyl. a Schematic of polarization directions of the main electronic transition moments and the torsion angle θ of (R)−1,1′-binaphthyl derivatives. b Simulated circular dichroism spectra of (R)-1,1′-binaphthol with θ varying from −45 to −110° in 1° intervals. c Plot of Δεmax/Δεmax-0 versus θ (red line) and the first-order derivative of d|Δεmax|/d|θ| (blue line), Δεmax = negative circular dichroism couplet intensity at ~220 nm and Δεmax-0 = Δεmax at θ = −45°. d Plots of Δλmax − Δλmax-0 versus θ (red line) and the first-order derivative of d|Δλmax|/d|θ| (blue line). The red dots in Fig. 1c,d were smoothed using the Savitzky–Golay method. Δλmax = Davydov splitting width shown in Fig. 1b according to the Mason model[23], Δλmax-0 = Δλmax at θ = −45°. Calculated with CAM-B3LYP/6-31 G*, Nstates = 30, Gaussian 09 Program

In this work, four BN-based chiral polymers are synthesized through simple Suzuki coupling reactions. For the polymers with “open” BN units, the AACD effect is clearly observed. However, the annihilation is restrained once the BN units are “locked”. Combining the experimental CD data with the simulation results shown in Fig. 1, the process of molecular aggregation can be monitored simply and accurately. Meanwhile, the relevant molecular dynamics simulation proves that this method is reliable.

Results

Synthesis and characterization

The phenomenon of aggregation-induced emission (AIE) was coined by Tang et al. in 2001. AIE luminogens (AIEgens) such as tetraphenylethylene (TPE) usually possess propeller-shaped structures and show no or weak photoluminescence (PL) when molecularly dissolved in their “good” solvent. However, their emission can be dramatically enhanced once aggregates are formed by adding ‘poor’ solvent. Thus, AIE exhibits an exact opposite photophysical phenomenon to the traditional aggregation-caused quenching effect discovered by Förster in 1954. Further study has revealed that the restriction of intramolecular motion (RIM) is the cause of the AIE effect. Since TPE possesses attractive PL properties in the aggregate state, it is considered to be a promising candidate to construct chiral materials with unique chiroptical properties[24-26]. The CD signal of binaphthyl and PL signal of TPE can be used as a dual signal to monitor molecular aggregation. In this work, four (R)-1,1′-binaphthyl and TPE-based polymers (P-1 to P-4) were synthesized through simple Suzuki coupling reactions (Fig. 2a). The detailed synthetic procedures are given in Supplementary Note 1. In P-1 and P-2, the TPE unit is linked to an “open” binaphthyl ring at the 6- and 3-position, respectively. P-3 and P-4 are structurally similar to P-1 and P-2, respectively but the binaphthyl ring is locked. The structures of all polymers were characterized by 1H and 13C NMR spectroscopy with satisfactory results (Supplementary Fig. 1–8). Gel permeation chromatography revealed that their weight-average molecular weight is 11900, 9200, 14000, 4800 and polydispersity is 2.53, 1.87, 2.17, 2.00, respectively (Supplementary Table 1). Thermogravimetric analysis (TGA) revealed that all polymers show 5% weight loss at high temperatures of up to 420 °C (Fig. 2b), suggesting that they are thermally stable.

Fig. 2

Structural and thermodynamic information. a Chemical structures of polymers P-1, P-2, P-3, and P-4, and their b thermogravimetric analysis (TGA) thermograms recorded under N2 at a heating rate of 10 °C/min

Circular dichroism

According to previous studies, it is anticipated that P-1 and P-2 show aggregation-annihilated circular dichroism (AACD) effects, while this effect should be restrained in P-3 and P-4 as the 1,1′-binaphthyl moiety is locked by the methylene group. The CD spectra of these polymers in solution and aggregate state were first investigated. All the polymers were molecularly dissolved in tetrahydrofuran (THF). When a poor solvent for the polymer was gradually added in their THF solutions, they started to form aggregates. Then, CD spectra of different polymers at different water fraction (fw) were measured. All the polymers show a strong CD signal in pure THF solution. However, the CD signals of P-1 and P-2 at fw = 90% are much weaker than those of pure THF solution (Fig. 3a, b). Under the same conditions, the CD signals of P-3 and P-4 also become lower at high fw but the extent is much weaker than in P-1 and P-2 (Fig. 3c, d). As expected, both P-1 and P-2 show an AACD effect, which suggests that the torsion angle θ changes during the aggregation process. Once the binaphthyl ring is locked by the methylene group, the rigid structures of P-3 and P-4 preserve the CD couplet intensity even in the aggregate state.

Fig. 3

Circular dichroism spectra. a P-1, b P-2, c P-3, and d P-4 in THF/water mixtures with different water fractions (fw). Concentration: 10−4 M

The effects of water on the CD couplet molar ellipticity [Θ] and wavelength splitting (Δλmax) have been intensively studied. [Θ] is a parameter to describe the molar ellipticity and is usually obtained from experiments. Δε is the symbol of molar circular dichroism collected from theoretical calculation. The equation of [Θ] ≈ 3300 Δε indicates that [Θ] is proportional to the CD couplet intensity Δε. The plot of [Θ]/[Θ]0 against fw is depicted in Fig. 4a, where [Θ]0 is the molar ellipticity at fw = 0% and the values of CD molar ellipticity were acquired from peaks at 280, 240, 280, and 250 nm for P-1, P-2, P-3 and P-4, respectively. It is clear that the CD signals of P-1 and P-2 are tremendously annihilated. Compared to [Θ]0, only 30% of [Θ] remains at fw = 90%. However, for P-3 and P-4, their [Θ] stays at ~ 80% of the initial value even at fw = 90%. These results are in good agreement with our hypothesis that the locked structures of P-3 and P-4 restrict the rotation of the naphthalene rings to suppress the AACD effect. By careful examination of the CD spectra in Fig. 3, it was found that the wavelength splitting (Δλmax) changes continuously when water is added into the polymer solutions. To make it clear, the plots of Δλs versus fw are presented in Fig. 4b, where Δλs = Δλmax – Δλmax-0 and Δλmax-0 = wavelength splitting at fw = 0%. It was found that the Δλs of P-1 keeps decreasing during the process of aggregation. In contrast, for the “locked” polymers (P-3 and P-4), their Δλs increases slightly with increasing fw. It is worth mentioning that the positive splitting peak of P-2 is located in a fairly short-wavelength region, which is even shorter than the cut-off wavelength of THF. Therefore, the Δλs could not be calculated. As the previous study found that the CD couplet intensity and wavelength splitting showed a direct relationship with the torsion angle θ, careful analysis on all these experimental results could provide valuable insights into the conformational change during the aggregation process.

Fig. 4

Experimental analysis of chiroptical properties within the process of aggregation. a Plots of [Θ]/[Θ]0 versus the composite of P-1 to P-4 in THF/water mixtures at a concentration 10−4 M. [Θ]0 = molar ellipticity at water fraction (fw) = 0%. The values of [Θ] were acquired from the peaks at 280, 240, 280, and 250 nm for P-1, P-2, P-3, and P-4, respectively. b Plots of Δλs versus fw, where Δλs = Δλmax – Δλmax-0 and Δλmax-0 = wavelength splitting at fw = 0%

Discussion

To further study the relationship between CD annihilation and the conformational change during the aggregation process, molecular dynamics (MD) simulation of the polymers was performed in THF and water, respectively. Polymers P-1 and P-3 were selected as representatives for the simulation and the details of the calculation are shown in the Methods section. Figure 5a, e show the single P-1 and P-3 molecules in THF and simulated aggregates with 30 P-1 and P-3 molecules in water are demonstrated in Fig. 5b, f. The MD simulation results indicate that P-1 shows a broad distribution of dihedral angle θ but P-3 exhibits a narrow distribution. For example, the full width at half maximum (FWHM) of P-1 is 35° and 45° in THF and water, respectively. However, the “locked” polymer P-3 only possesses a 10° FWHM in both THF and water, suggestive of its higher rigidity. For P-1, the most probable conformation in THF is at θ = −76°. When aggregates are formed in water, the θ of part of the conformation decreases from −76° to −102° though the most probable conformation is still located at ~75°. Meanwhile, compared with the most probable conformation in THF, only a 2° increase in θ from −52° to −50° is observed in water for P-3. The maintenance of conformation in P-3 could be attributed to the restriction from the “locked” binaphthyl.

Fig. 5

The atomistic models of P-1 and P-3 in molecular dynamics simulations. a, e Show single P-1 and P-3 molecules in THF solution. b, f Show 30 P-1 and P-3 molecules aggregated in water solution. For clarity, the solvent molecules for THF and water are not shown here. c, d Show the relative Boltzmann populations of torsion angle θ in various conformers of P-1 in THF and water, respectively. g, h Show the relative Boltzmann populations of θ in various conformers of P-3 in THF and water, respectively. FWHM = full width at half maximum

As suggested by the classical model of BN constructed by Mason (Fig. 1), the Δεmax and Δλmax decrease simultaneously when θ decreases from −76° to −102°. However, when θ is slightly increased from −52° to −50°, the Δεmax decreases but Δλmax becomes higher. Expectedly, the simulated change of Δεmax and Δλmax from THF to water coincides with the experimental plots shown in Fig. 4. The conformational change along with molecular aggregation is schematically summarized in Fig. 6. From solution to aggregate, the θ in “open” polymers (P-1 and P-2) becomes more negative and part of the conformers relax from cisoid to transoid. On the contrary, the θ in “locked” polymers (P-3 and P-4) increases slightly and the cisoid conformation is preserved throughout the whole aggregation process.

Fig. 6

Schematic representation of the conformational change of (R)−1,1′-binaphthyl moieties during the aggregation process

The photophysical properties of the polymers were also studied. The UV spectra of P-1, P-2, P-3, and P-4 in THF exhibit an absorption maximum (λabs) at 347, 333, 337, and 328 nm, respectively. On the other hand, in THF/water mixture (v/v, 7/3), their emission maximum (λem) is 506, 498, 499, and 492 nm, respectively (Fig. 7). The photophysical properties and relevant simulation results are summarized in Table 1. From the λabs and λem values, the order of conjugation is: P-1 > P-3 > P-2 > P-4, which is well matched with the calculated energy gap (3.186, 3.204, 3.312, and 3.321 eV for P-1, P-3, P-2 and P-4, respectively).

Fig. 7

Photophysical properties of these four polymers. a Ultraviolet absorption spectra of P-1, P-2, P-3, and P-4 in THF. b Normalized photoluminescence (PL) spectra of P-1, P-2, P-3, and P-4 in THF/water mixture (v/v, 7:3). Concentration: 10−5 M. λex(nm): 355 (P-1), 345 (P-2), 345 (P-3), and 340 (P-4). λabs = absorption maximum, λem = emission maximum

Table 1

Photophysical properties and simulation results. Calculated by B3LYP/6-31 G(d), Gaussian 09 program

Polymer	λabs (nm)	λem (nm)	Eg (eV)	θ (°)	β (°)
P-1	347	506	3.186	−92.4	36.0
P-2	333	498	3.312	−78.8	44.5
P-3	337	499	3.204	−49.0	36.7
P-4	328	492	3.321	−51.2	49.5

λabs and λem absorption maximum and emission maximum in THF and THF/water mixture (v/v, 7:3), respectively, Eg calculated energy gap, θ calculated torsion angle between two naphthalene rings, β torsion angle between the naphthalene and phenyl ring of TPE moiety. DFT calculations were carried out to optimize the ground-state conformation of these polymers in the gas phase. To simplify the calculation, only two repeating units were chosen to perform the optimization (Supplementary Fig. 9 and Supplementary Table 2–5)

Study of the λabs/λem change could be another tool to monitor molecular aggregation. TPE is a well-studied molecule with aggregation-induced emission characteristics. Previous studies have shown that the λabs/λem of TPE shifts little when its molecules aggregate. So, the relationship between λabs and β/θ was further investigated by theoretical calculation. A semi empirical CIS/ZINDO method was applied with NStates = 10 and all the calculations were performed using the Gaussian 09 program. The simulation results are shown in Fig. 8 and Supplementary Fig. 10-11. In “open” polymers such as P-1, the λabs shows almost no change when θ decreases from −80° to −110°. However, an obvious 5 nm hypochromic shift of λabs is observed when β increases from 26° to 46° (Fig. 8a–c). This indicates that the λabs/λem value could be used to monitor the change of β in P-1. However, in “locked” P-3, both β and θ contribute mainly to λabs. As shown in Fig. 8d–f, decreasing θ from −45° to −55° or increasing β from 32° to 42° results in a 1 nm hypochromic shift of λabs. This suggests that the change of λabs/λem in P-3 should be ascribed to the variation of β and θ. P-2 shows the same effect as P-1, and P-4 and P-3 also exhibit similar behaviors.

Fig. 8

Simulated photophysical properties of different conformations. a, b, d, e Simulated absorption spectra of a, b P-1 and d, e P-3 with θ varying from a −80° to −100° and constant β (36°) and d from −45° to −55° and constant β (37°), and β varying from b 26° to 46° and constant θ (−90°) and e from 32° to 42° and constant θ (−50°). c, f Plots of λabs – (λabs)0 versus θ and β. λabs: simulated absorption maximum, (λabs)0: simulated absorption maximum wavelength of P-1 at θ = −90° and β = 36°; and P-3 at θ = −50° and β = 37°. θ = torsion angle between two naphthalene rings, β = torsion angle between the naphthalene and phenyl ring of the TPE moiety. A semi empirical CIS/ZINDO method was applied with NStates = 10 and all calculations were performed using Gaussian 09. For the purposes of a clear demonstration and simplified calculation, the hexyl groups are replaced with methyl groups in P-1

Then, the PL spectra of these polymers were measured in THF/water mixtures with different fw (Fig. 9 & Supplementary Fig. 12-13). All the polymers show no or quite weak emission in pure THF solution. However, their emission is tremendously enhanced upon water addition. For example, the PL intensity (I) of P-1 increases by almost 60 times at fw = 90%, demonstrative of an AIE effect (Fig. 8c)[27-29]. Similarly, the fluorescence quantum yield (ΦF) of the polymers also increases upon aggregate formation and their maximum solid-state ΦF reaches 60% (Supplementary Fig. 14 and Supplementary Table 6). Except for P-2, all the polymers show an insignificant change of λem during aggregation (Fig. 9d). For P-1 and P-2, as the decrease of θ was proved to exhibit negligible impact on λem, so we can conclude that the β value remains almost unchanged from solution to aggregates in P-1. On the other hand, the 14 nm hypsochromic shift of λem in P-2 suggests an increase of β in the aggregate state. In P-3 and P-4, both θ and β play important roles in the λem change. However, the λem stays almost unchanged before and after the aggregation. As suggested by the CD results, only a small increase of θ is observed in P-3 and P-4. We can infer that the β value also suffers small change by aggregation. Among these polymers, it seems that only P-2 shows an obvious increase of β during aggregation presumably due to its crowded structure, and only a slight change of β exists in the other three polymers. In order to verify this conclusion, the distribution of β for P-1 and P-3 obtained from the MD simulation was extracted and the corresponding results are shown in Supplementary Fig. 20-23. Unexpectedly, the dihedral angle β is not fixed both in THF and water. There is an interchange between the conformers with β ≈ 30°, −30°, 150°, and −150°, which indicate the phenyl ring rotation is active in these polymers but with several probable conformers. From the point of view of electronic conjugation, the energy gap almost shows no difference among β = 30°, −30°, 150°, and −150°. Then all the angles of β were transformed and centralized in the range of 0–90°. In this case, the angle with β = 30°, −30°, 150° and −150° could be classified into β = 30°. The processed data are shown in Supplementary Figure 24. From THF to water, the distribution of processed β does not show an obvious change both in P-1 and P-3. Meanwhile, their most probable angle is also similar. For example, P-1 and P-3 shows the highest probability of β at 31° and 32°, respectively, both in THF and water, which is consistent with the PL results. Based on the above analysis, we further conclude that the PL method cannot accurately monitor the conformational change during the process of aggregation, due to the insufficient information provided by the PL spectra. On the contrary, in CD spectra, both molar ellipticity and Davydov splitting could work synergetically to monitor the conformational change, which makes CD spectroscopy a promising tool to monitor molecular aggregation.

Fig. 9

Photoluminescence spectra. a P-1 and b P-2 in THF/water mixtures with different water fractions (fw). Concentration: 10−5 M. λex = 355 nm. c Plots of αAIE versus fw, where αAIE = I/I0 and I0 = maximum emission intensity at fw = 0%. Inset: photographs of P-1 in THF/water mixtures with different fw taken under UV illumination. d Plots of λem–(λem)0 versus different fw. λem: emission maximum of each plot, (λem)0: maximum emission wavelength at fw = 0%

In summary, four 1,1′-binaphthyl and TPE-based chiral AIE polymers were designed and synthesized. Polymers P-1 and P-2 with “open” binaphthyl exhibited a typical AACD effect and their CD couplet intensity was largely annihilated in the aggregates. However, the AACD effect was suppressed in P-3 and P-4 as the binaphthyl moieties were locked by methylene. These results indicate that the AACD effect is mainly caused by the twisting of naphthalene rings. The combination of MD simulation and analysis on the change of CD couplet intensity and wavelength splitting during the aggregation process is thus an appealing method for in-situ and real-time monitoring of the conformational change. From the solution to aggregate state, the θ of “open” polymers is largely decreased and part of the conformers relax from cisoid to transoid. On the contrary, the “locked” polymers show a slightly larger θ and their cisoid conformation is preserved during the aggregation process. Thus, CD spectroscopy may be used for real-time and in-situ monitoring of molecular aggregation, especially for biomacromolecular aggregations in physiological conditions.

Methods

Molecular dynamics simulations

Molecular dynamics simulations were performed using the GROMACS software package (version 5.1.5)[30]. The atom types and parameters of P-1 and P-3 were set from the general amber force field[31]. The electrostatic potential of P-1 and P-3 were calculated by Gaussian 09 package[32] and the corresponding partial charges reproducing the electrostatic potential were obtained by the restrained electrostatic potential fit method[33,34]. To mimic the dilute THF solution of P-1 and P-3, the single P-1 and P-3 molecules were placed in a cubic box of pre-equilibrated THF molecules[35-37], respectively. For each system, the box length was 6.5 nm containing one P-1 or P-3 molecule and 2000 THF molecules (see Fig. 5a, e). To simulate the aggregate state of P-1 and P-3, we first placed 30 P-1 or P-3 molecules in a small cubic box with length 4.5 nm. Then we centered the small cubic box in a large cubic box with length 8.0 nm and solvated by the pre-equilibrated TIP3P water molecules (see Fig. 5b, f)[38]. For all the systems, including single molecule THF solution and aggregates of P-1 and P-3, we first carried out energy minimization, followed by 5 ns NPT (T = 300 K and P = 1 bar) simulations to relax the system. After equilibration, we further ran four independent 50 ns production MD simulations for single P-1 and P-3 molecules in THF solution and carried out 6 independent 50 ns production MD simulations for P-1 and P-3 aggregates in water solution, respectively (Supplementary Fig. 15–18). The temperature and pressure were controlled by the velocity rescaling thermostat[39] and Berendsen barostat[40] respectively. The time constants of couplings for both temperature and pressure were 0.1 ps. For the electrostatic interactions, the reciprocal space summation was evaluated by the particle mesh Ewald method[41,42]. The direct space summation was computed at a cutoff distance of 1.2 nm. The cutoff distance of VdW interactions was 1.2 nm. All bond lengths here were constrained via the LINCS algorithm[43]. Periodic boundary conditions were applied in all three directions to minimize the edge effects in a finite system. The time step here was 2 fs. The configurations were stored at a time interval of 40 ps for statistical analysis of dihedral angles and the corresponding free energy calculation. In total, for single molecule THF solution, we collected 25,000 configurations, while for aggregates, we collected 37,500 conformations. The obtained distributions of the dihedral angle and the Gibbs free energy were demonstrated in Fig. 5 and Supplementary Fig. 19, respectively.

Quantum mechanics calculations

All the quantum mechanism calculations here were performed by the Gaussian 09 program. The optimized conformations of these four polymers were calculated in the gas phase by B3LYP/6-31 G(d). To simplify the calculation, only two repeating units were chosen to do the optimization. Calculation on the CD spectra of BINOL was carried out at CAM-B3LYP/6-31 G* level with Nstates = 30. Calculation on the UV spectra of the polymers was performed with semiempirical CIS/ZINDO method with NStates = 10. The CD and UV spectra were processed by Multiwfn 3.6 Program[44].

Peer Review File