Controlling Helical Asymmetry in Supramolecular Copolymers by In Situ Chemical Modification

Amplification of asymmetry in complex molecular systems results from a delicate interplay of chiral supramolecular structures and their chemical reactivity. In this work, we show how the helicity of supramolecular assemblies can be controlled by performing a non-stereoselective methylation reaction on comonomers. By methylating chiral glutamic acid side chains in benzene-1,3,5-tricarboxamide (BTA) derivatives to form methyl esters, the assembly properties are modulated. As reacted comonomers, the methyl ester-BTAs induce a stronger bias in the screw-sense of helical fibers predominantly composed of stacked achiral alkyl-BTA monomers. Hence, applying the in situ methylation in a system with the glutamic acid-BTA comonomer induces asymmetry amplification. Moreover, mixing small quantities of enantiomers of glutamic acid-BTA and glutamate methyl ester-BTA in the presence of the achiral alkyl-BTAs leads to deracemization and inversion of the helical structures in solution via the in situ reaction toward a thermodynamic equilibrium. Theoretical modeling suggests that the observed effects are caused by enhanced comonomer interactions after the chemical modification. Our presented methodology enables on-demand control over asymmetry in ordered functional supramolecular materials.


■ INTRODUCTION
The origin of homochirality in nature is a remarkable and active research topic. Mechanisms that could cause symmetry breaking and asymmetry amplification are widely discussed, yet experimental substantiation is lacking. 1−5 An exception is the Soai reaction, which shows unprecedented forms of amplification of molecular asymmetry in a chemical system. 6−8 Asymmetry is widespread in nature via the enantioselective use of L-amino acids and D-(deoxy)ribose to produce homochiral superstructures like α-helices and deoxyribonucleic acid (DNA) double helices through supramolecular interactions. 9,10 The out-of-equilibrium structures derived from these complex systems are maintained by dissipative processes (i.e., energyconsuming covalent reactions) that affect enantioselective noncovalent interactions between macromolecules. 11,12 Perfect examples are the formation and disintegration of microtubuli during cell proliferation and the recruitment of molecular chaperones to prevent the formation of the thermodynamically favored plaques by amyloid-β precursor proteins. 13,14 In synthetic chemistry too, numerous reports have applied in situ covalent reactions to alter supramolecular assembly properties of monomers, resulting in switchable, adaptive, and even oscillating molecular systems. 12,15−21 Likewise, in situ covalent reactions on the constituents of noncovalent structures can be effective in studying the amplification of asymmetry. 19,22,23 However, such studies are scarce in literature while these contribute to elucidate the mechanisms leading to homochirality and provide new methodologies to incorporate supramolecular asymmetry in purely synthetic systems.
Supramolecular polymers are of interest in amplification of asymmetry studies due to the ability to form dynamic chiral M or P helices that change handedness in response to external factors. 24−32 One supramolecular polymer platform consists of benzene-1,3,5-tricarboxamide (BTA), which is widely studied due to its modularity. 33−35 These discotic molecules form helical supramolecular polymers in apolar solvents through π−π stacking and intermolecular triple hydrogen bonding. A helical bias, i.e., the preference to adopt P or M helicity, is often introduced via the intrinsic molecular chirality of the monomers. Thus, supramolecular polymer systems demonstrate how molecular chirality is translated into macroscopic asymmetry. Consequently, the inherent asymmetry of supramolecular polymer systems can be transferred into reaction products via asymmetric metal and organocatalysis. 36−44 Methods to study the amplification of asymmetry in macromolecules were originally developed by Green and coworkers and were later translated to supramolecular polymer systems. 45−51 The first method is the Sergeant-and-Soldiers experiment, in which the majority of the system consists of achiral monomers that can adopt a preferred M or P helicity by doping the system with chiral monomers. The second method is the Majority-Rules experiment, in which the net helicity is governed by mixtures of the two enantiomers. The principle relies on the mismatch penalty, an intrinsic value that describes the penalty for a monomer to enter a stack of its unfavored helicity. The combination of the two methods is a Diluted Majority-Rules experiment, in which an enantiomeric excess (ee) is introduced in the sergeant fraction of a Sergeant-and-Soldiers experiment. 52−54 The abovementioned experiments result in nonlinear translation of molecular chirality into macroscopic asymmetry and fully rely on thermodynamic equilibria that result from mixing of (a)chiral comonomers. So far, two reports have demonstrated the combination of supramolecular polymers with covalent reactions to amplify the supramolecular asymmetry. 22,23 Herein, in situ reactivity is combined with the Sergeant-and-Soldiers principle to trap the supramolecular chirality into molecular chirality, enhancing the ee of supramolecular monomers. As a consequence, the asymmetry of the aggregated species is amplified. We propose that performing the reaction directly on the sergeant instead of the soldier would lead to a more effective and robust amplification of asymmetry on the supramolecular structures in thermodynamic equilibrium.
Here, we introduce a noncovalent BTA-based Sergeant-and-Soldiers system comprising chiral sergeants that are functionalized with a glutamic acid residue as a handlebar for in situ covalent methylation (Scheme 1a). We demonstrate that the methylation immediately results in the amplification of asymmetry on the helical structures in solution (Scheme 1b), which can be monitored by electronic circular dichroism (CD) spectroscopy. By selectively mixing enantiomers of the sergeant before and after methylation, we can control amplification and inversion of asymmetry, as well as deracemization upon performing the in situ reaction.
■ RESULTS AND DISCUSSION Supramolecular Homopolymerization of Glu-BTA and Glu(OMe)-BTA. We started our investigation by characterizing the individual homoaggregates of our target molecules to highlight differences in their relative stability. Glu-BTA was synthesized via amidation of an asymmetric monoacid precursor with a dodecyl-extended glutamic acid (Scheme 1a). The optimization, synthetic procedure, and characterization of this specific molecular design are discussed in the supporting information (Figures S1 and S2). We chose the asymmetric design to promote the formation of elongated one-dimensional (1D) structures and decrease the morphological transition from fibers to dimers, which has been demonstrated for symmetric ester-BTAs. 55 The carboxylic acid moiety in the glutamic acid side chain was expected to interfere with the intermolecular hydrogen bonding and retard supramolecular polymerization, as shown by kinetic evolution experiments ( Figure S3a). 56,57 Moreover, this carboxylic acid was identified as a target for chemical modification, of which the reaction product was expected not to interfere with the intermolecular hydrogen bonding. Thus, we aimed to in situ methylate the carboxylic acid to promote supramolecular polymerization and found that (trimethylsilyl)diazomethane ((TMS)CHN 2 ) was reactive and selective in the desired assembly conditions ( Figure S1). Methylation with (TMS)-CHN 2 yielded the stable product Glu(OMe)-BTA (Scheme 1a), 58 which allowed us to characterize both molecules in their thermodynamically stable state.
Homopolymerizations of Glu-BTA and Glu(OMe)-BTA in methylcyclohexane (MCH) were characterized at room temperature by Fourier transform infrared (FTIR) and CD spectroscopy. The FTIR spectra show absorption bands at 3236 cm −1 (bonded N−H stretch), 1643 cm −1 (bonded C�O amide I vibrational mode), and 1562 cm −1 (bonded C�O amide II vibrational mode) that are typical for threefold hydrogen bonding between amides of the BTA core in supramolecular polymers (Figure 1a). 59 These absorption bands were not observed for 1 mM samples in acetonitrile ( Figure S3b). Furthermore, the band at 1747 cm −1 can be ascribed to an unbound ester C�O, indicating no formation of dimeric aggregates. 55 The shoulder at 1716 cm −1 in the spectrum of Glu-BTA could be ascribed to bonded carboxylic acid moieties, indicating a bifurcated hydrogen bond of the amide C�O in the aggregated state. 60 The CD spectra of 50 μM samples of both Glu-BTA and Glu(OMe)-BTA exhibit Cotton effects with positive maxima at 222 nm ( Figure 1b). These spectra are indicative of the presence of single helical BTA fibers with one preferred handedness. The calculated dissymmetry factor (g-factor) at room temperature is larger for homopolymers of Glu(OMe)-BTA compared to Glu-BTA ( Figure S5). We observed elongation temperatures (T e ) of 42 and 57°C for Glu-BTA and Glu(OMe)-BTA, respectively, by conducting variabletemperature CD measurements (Figure 1c). These measurements suggest that both BTA polymers form via a nucleation− elongation cooperative mechanism. 33,61,62 Thermodynamic analysis was performed to quantify the stability of both homopolymers. The temperature-dependent data was subjected to Van't Hoff analysis and fitting procedures using thermodynamic mass-balance models (for details, see the Supporting Information). 63 The extracted thermodynamic parameters and the resulting binding constants at 20°C in the elongation phase of homopolymerization (K e ) from these two methods were comparable. The calculated K e values of Glu-BTA and Glu(OMe)-BTA were 2 × 10 5 and 5 × 10 5 , respectively, confirming that polymers of Glu(OMe)-BTA are more stable.
Copolymerization of Chiral and Achiral BTAs. We studied the role of Glu-BTA and Glu(OMe)-BTA in Sergeantand-Soldiers experiments to explore the potential for asymmetry amplification by in situ chemical modification of the sergeant. In separate experiments, the sergeants Glu-BTA and Glu(OMe)-BTA were mixed with a-BTA soldiers in ratios between 1 and 20 mol % (Scheme 1a). The CD profiles of these mixtures show the characteristic shape of Sergeant-and-Soldiers BTA copolymers: a maximum at 217 nm and a shoulder at 245 nm as a result of different packing compared to homopolymers of Glu-BTA and Glu(OMe)-BTA ( Figure  2a). 50 The intercalation of Glu-BTA and Glu(OMe)-BTA into polymers of a-BTA was further corroborated by FTIR spectroscopy, as both copolymer mixtures exhibited identical spectra of polymeric aggregates ( Figure S8a). These spectra did not demonstrate a significant proportion of chain ends, indicating that neither Glu-BTA or Glu(OMe)-BTA is effective as a chain capper. The CD signal at 222 nm is plotted over the sergeant range for both experiments to demonstrate the net helicity of the thermodynamic equilibrium state at each composition (Figure 2b). The graph clarifies that a larger helical bias is introduced in the screw-sense of the copolymers by sergeant Glu(OMe)-BTA compared to Glu-BTA. Temperature-dependent CD and ultraviolet−visible (UV−vis) measurements provide mechanistic details on the intercalation of sergeants into stacks of soldiers. In the presence of 5 mol % Glu(OMe)-BTA, the helical bias is introduced at a temperature of 73°C, corresponding to the T e of a-BTA supramolecular polymerization. Hence, the intercalation of this sergeant occurs simultaneously with supramolecular polymerization of the soldiers (Figure 2c). Intercalation of the sergeant Glu-BTA seems to be lagged by 10°C from the T e of the soldiers' homopolymerization. Apparently, the carboxylic acid moiety of the sergeant Glu-

BTA disfavors intercalation in soldiers' stacks in comparison to
Glu(OMe)-BTA. This was further corroborated by conducting a Sergeant-and-Soldiers experiment at variable temperature with the tert-butyl protected precursor BTA as the sergeant (see the Supporting Information, compound 4). The temperature-dependent data perfectly overlaps with that of Glu-(OMe)-BTA as the sergeant (Figure S8b), despite the sterically demanding tert-butyl moiety. The differences between Glu-BTA and Glu(OMe)-BTA as sergeants prompted us to investigate the effect of sergeant ee on the asymmetry of copolymers. For this purpose, we conducted Diluted Majority-Rules experiments, where we vary the enantiomeric composition of the sergeant while keeping the sergeant fraction constant at 5 mol % (Figure 3a). The CD at 222 nm is plotted over the range of sergeant ee and presented in Figure 3b. For Glu-BTA, the relation between ee and CD follows a linear trend. This is typical for self-sorting of the two sergeant enantiomers into stacks of their favored helicity, as the enantiomers in minority do not adopt the helicity of the enantiomers in majority. 54 In contrast, the nonlinear trend resulting from enantiomeric mixtures of Glu(OMe)-BTA sergeants indicates a Majority-Rules behavior, i.e., the enantiomers in minority can intercalate into stacks of the unfavored helicity.
Mechanistic Insights through Simulations of Supramolecular Copolymers. We used mathematical massbalance models of two-and three-component copolymerization to simulate the Sergeant-and-Soldiers and Diluted Majority-Rules experiments. 63 These simulations allowed us to examine the effect of the binding constant (K e ) and mismatch penalty (MMP) of both chiral sergeants Glu-BTA and Glu(OMe)-BTA on the asymmetry of copolymers. The simulated Sergeant-and-Soldiers ( Figure S10) and Diluted Majority-Rules experiments (Figure 4a,b) show that variations in the K e and MMP affect the helical bias in a similar manner. For both simulated experiments, an increase in K e or MMP  gives a stronger asymmetry amplification as recognized by the increased nonlinear response of CD with respect to the sergeant ee. Previous reports have shown that BTA-based Diluted Majority-Rules experiments follow Sergeant-and-Soldiers thermodynamic behavior, similar to that indicated by our simulations. 51 However, these simulations do not provide insights into the distinct contribution of either the sergeant's K e or MMP in asymmetry amplification. In contrast, simulations of regular Majority-Rules experiments, i.e., the copolymerization of two enantiomers, did show an opposing effect on the asymmetry of copolymers between variations in K e or MMP (Figure 4c,d). Now, an increase in K e also increases the nonlinearity of the asymmetry with respect to ee, whereas an increase in MMP reduces this nonlinearity. Thus, stronger intermolecular interactions enhance the amplification of asymmetry while diminished mixing of enantiomers decreases copolymer asymmetry. The simulations suggest that we can decouple the individual contributions of K e and  MMP by conducting Majority-Rules experiments with the enantiomers of Glu-BTA and Glu(OMe)-BTA ( Figure S11). These experimental results showed a stronger asymmetry amplification for Glu(OMe)-BTA compared to Glu-BTA, indicating either a higher K e or a lower MMP for homopolymerization of Glu(OMe)-BTA with respect to Glu-BTA. Comparing these findings with the Diluted Majority-Rules experiments revealed that the experimental results are in line with simulations, in which we vary K e , but in contradiction with simulations, in which we vary MMP. Therefore, the combination of experiments and simulations elucidates that methylation of Glu-BTA into Glu(OMe)-BTA results in an increase in K e , i.e., stronger binding between sergeant and soldiers. This stronger interaction leads to an enhanced translation of molecular chirality into supramolecular asymmetry in thermodynamically stable copolymers.
Controlling Asymmetry via In Situ Methylation. The difference in K e for Glu-BTA and Glu(OMe)-BTA to bind to soldier stacks was exploited for the purpose of on-demand amplification of asymmetry. The in situ reaction procedure was optimized as described in the SI (Figures S12−S14). The expected amplification can be expressed as the ΔCD, which is calculated as the difference in CD value at 222 nm between the copolymer systems with Glu-BTA versus Glu(OMe)-BTA as the sergeant ( Figure S9). First, we optimized the in situ reaction conditions to be compatible with the conditions for supramolecular polymerization as described in the Supporting Information. Next, applying our optimized reaction procedure to Sergeant-and-Soldiers mixtures of Glu-BTA and a-BTA copolymers resulted in amplification of asymmetry (black and orange arrows in Figure 5a). The kinetic traces show a strong increase in CD signal within the first hours of the reaction, which gradually slows down over the following reaction time course (Figure 5b). After 16 h, the in situ reaction sample starting at 2 mol % Glu-BTA reached 96% of the calculated ΔCD. The in situ reaction starting with 1 mol % Glu-BTA proceeded slower, but the CD measurement after 4 days revealed that 85% of the calculated ΔCD was reached ( Figure  5c). Therefore, both in situ experiments demonstrate good conversion of Glu-BTA into Glu(OMe)-BTA via their distinct effect on the asymmetry of primarily soldier-containing copolymers.
Additional intriguing consequences of the in situ reaction on the macroscopic asymmetry were realized by mixing enantiopure (R)-Glu-BTA with (S)-Glu(OMe)-BTA in a Diluted Majority-Rules system with 95 mol % a-BTA ( Figure  6a). We changed the ratio of (R)-Glu-BTA / (S)-Glu(OMe)-BTA from 1/1 to 1/0 and observed drastically shifted CD values compared to the Diluted Majority-Rules experiments that included only enantiomers of either Glu-BTA or Glu(OMe)-BTA (Figure 6b). Now, the asymmetry is strongly directed by the (S)-Glu(OMe)-BTA sergeant even up to 80% excess of (R)-Glu-BTA sergeant, where a racemic state of the helices in solution is obtained. In these mixtures, the addition of the reagents for in situ methylation will selectively convert enantiomers of Glu-BTA into Glu(OMe)-BTA, as illustrated in Figure 6a. Therefore, the expected transition in the asymmetry (ΔCD) will be toward the measured CD for the Diluted Majority-Rules experiment with enantiomers of Glu(OMe)-BTA. Accordingly, the arrows in Figure 6c illustrate two new effects on the asymmetry that could be observed upon in situ chemical modification: inverting the asymmetry of the major screw-sense (orange arrow) and deracemization of helices in solution (black arrow). Addition of the reagents in a mixture containing 70 mol % (R)-Glu-BTA in the sergeant fraction (an excess of 40%) indeed resulted in an inversion of the CD signal. Here, an absolute CD change of 92 mdeg within 8 h was obtained (Figure 6d, orange trace), which is 85% of the calculated ΔCD ( Figure S9b). Similarly, the introduction of the reagents in the CD-silent mixture containing 90 mol % (R)-Glu-BTA in the sergeant fraction (an excess of 80%) resulted in deracemization. Here, an absolute CD change of 62 mdeg was observed within 8 h (Figure 6d, black trace), which is 82% of the calculated ΔCD. Both in situ reaction experiments exhibited the largest effect on the asymmetry within the first hour, proving effective and quick on-demand control over the asymmetry of supramolecular helices in solution.

■ CONCLUSIONS
We have demonstrated that covalent and noncovalent chemistry can be combined to precisely control the asymmetry in supramolecular polymers under thermodynamic equilibrium. When mixed with achiral comonomers, the covalent methylation of the carboxylic acid side chain of chiral monomers enhanced the intermolecular interactions and thus participation in supramolecular polymerization. The observed macroscopic effect is a stronger helical bias upon chemical conversion of the chiral monomers. Variations in the enantiomeric composition of the chiral comonomer fraction enabled controlled effects on the asymmetry, which are initiation (i.e., deracemization), amplification, and inversion of asymmetry by the addition of chemical reagents.
The presented work is an illustration of how a multistep approach of covalent and noncovalent reaction steps in situ can be employed as a mean to control asymmetric features in molecular systems. We propose that such simple systems can greatly contribute to our understanding of mechanisms toward homochirality in complex systems.

* sı Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.3c03411. Synthetic procedures; materials and methods; additional FTIR, UV−vis, CD spectra; details on thermodynamical analysis and simulations; and details on in situ reaction procedures (PDF)