Chiral Display of Pyrenes on a Peptoid Backbone: Conformational Homogeneity of Peptoid Controls Excimer Chirality

Controlling chromophore chirality in three‐dimensional space is crucial for understanding the structure–chiroptical relationship. Such control enables the prediction of ideal materials for use in chiral photonic applications. In this study, optically active multipyrene systems are synthesized on peptoids. Pyrene‐based chiral submonomers, (S)‐ and (R)‐1‐pyrenylethylamine (s1pye and r1pye), are successfully incorporated into peptoids as the respective (S)‐ and (R)‐N‐(1‐pyrenylethyl)glycine (Ns1pye and Nr1pye) units. NMR spectroscopy revealed length‐ and N‐acetylation‐dependent conformational homogeneity enhancement in solution, stabilizing cis‐amides in Ns1pye‐containing peptoids. The X‐ray crystal structure of Ns1pye tetramer displayed a polyproline type‐I (PPI)‐like helix. All of the pyrene‐related absorptions are circular dichroism (CD) active, and the CD signal related to the long‐axis‐polarized transition increased with helical stabilization. Intramolecular excimer generation yielded significant circularly polarized luminescence (CPL) in the excimer emission region. Early‐stage peptoids emitted left‐handed CPL, but upon acquiring a PPI‐like helix character, CPL handedness became inverted. The CPL dissymmetry factor (glum) was comparable or superior (10−2) to that of chiral organic dyes (10−5–10−2). This new class of helical pyrene‐containing peptoids provides a CPL‐active intramolecular excimer, modulating optical activity through peptoid secondary structure homogeneity.


Introduction
Peptoids are a class of non-natural heteropolymers composed of N-substituted glycine backbones. [1]In contrast to peptides, the side chains of peptoids are connected to the amide nitrogen atoms.Therefore, peptoids lack the hydrogen bonds between backbone amides that are responsible for the formation of secondary structures in peptides (Figure 1A).[7][8][9] In particular, a helical conformation can be stabilized by introducing various bulky chiral side chains, including aromatic [8,9] and aliphatic ones. [6,10]Peptoid helices display a propensity to adopt a polyproline type-I (PPI) helix, with a 6 Å pitch distance and three residues per turn. [3]Following the discovery of these helices, peptoids have been shown to mimic diverse natural secondary structures, including loops [11] and turns. [12]The defined structure of peptoids can serve as a basis for their rational design.Studies of peptoid systems bearing membrane-active amphiphiles, [13] metalbinding ligands, [14,15] enantioselective catalysts, [16] energy transfer systems, [17] and electron transfer systems [18] have revealed their structure-driven properties and functions.A broad conformational space has been sampled using various peptoid sequences, mostly for the formation of stable and conformationally homogeneous helices.Initial studies focused on peptoids containing (S)-N-(1-phenylethyl)glycine (Nspe) residues, which provide sufficient bulkiness to induce a cis-amide helix.The Nspe residue has been widely used and has become the gold standard for generating peptoid helices.However, Nspe residues often stabilize both cisand trans-amides when other noncovalent interactions are involved (e.g., to form a threadedloop structure for a nonameric Nspe peptoid). [11]Therefore, peptoid monomers with greater propensity to stabilize the cis-amides are desirable for maintaining homogeneous secondary structures under a variety of conditions.Blackwell and co-workers previously surveyed the sequence-structure relationships using a diverse array of peptoid monomers, which revealed (S)-N-(1-naphthylethyl)glycine (Ns1npe) residues to be particularly effective in promoting formation of the cis-rotamer. [19]he peptoid helices were primarily formed because of the substantial steric hindrance between the bulky naphthalenecontaining side chains, which mediated a significant cis-amide-inducing effect with PPI-like structural features. [9]irst used in peptoids by Blackwell's group, naphthalene has often been exploited to induce helical structures, [20] as a fluorescence emitter, [21] and to generate excimers. [22]However, understanding the photophysical properties of peptoids based on their structure poses a challenge owing to the lack of clear relationships between the secondary structures of peptoids and their spectral characteristics.
Circular dichroism (CD), for example, is a valuable method for determining the secondary structures of peptides, peptoids, and proteins. [2,11]Exciton-coupled signals at approximately 192 nm and 202 nm and a negative transition signal at approximately 220 nm are characteristic signatures of Nspe-based helical peptoids.However, the CD spectra of naphthalene-containing peptoids are difficult to interpret on an individual component basis because the UV absorption of naphthalene overlaps with that of the backbone amide groups.It is envisioned that the replacement of naphthalene with pyrene would enhance the understanding of how the secondary structure affects the CD profile in the presence of the CD-active luminophore.Furthermore, the extended conjugation system allows for longer-wavelength emission, opening the way to the development of peptoids as fluorescence emission controllers.
It is hypothesized that if the chiral environment offered by the helical peptoid could allow chiral absorption by the luminophore, the chiral emission would also be affected by the environment.One important phenomenon for understanding chiral emission properties is the circularly polarized luminescence (CPL), which refers to a difference in the emission intensity between left-and right-handed circularly polarized light.The discovery of efficient CPL emitters has enabled improvements in various technical applications, including biological sensing [23] and imaging, [24] data encryption, [25] and enantioselective synthesis. [26,27]CPL from chiral luminophores provides a facile method for producing circularly polarized light, and various organic materials have recently been reported to display CPL activity after incorporating chirality into luminescent molecules.
[30][31] In an effort to overcome this limitation, co-assembled structures of organic molecules have been investigated, leading to higher dissymmetry factors. [32,33]The resulting exciton coupling can improve the CPL dissymmetry in highly ordered molecularly stacked systems.However, the construction of such systems requires a large number of molecules, and their arrangement must be directed toward the enhancement of dissymmetry, which is difficult to modulate and predict.Therefore, a powerful CPL amplification strategy could be the construction of a CPL emission system based on intramolecular interactions between luminophores that are controllable via the secondary structure of an appropriate scaffold molecule.In this regard, peptoid helices offer the potential to enhance the dissymmetry by tailoring the building blocks to obtain defined secondary structures in which the distance and angle of the chromophores can be controlled.
In this work, pyrene-containing helix-inducing peptoid submonomers were synthesized and the pyrene moieties were helically displayed on peptoid backbones with varying degrees of structural homogeneity.A comprehensive structural study was conducted using NMR spectroscopy and X-ray crystallography.Photophysical characterization was performed by absorption and CD analysis for the ground state and by fluorescence and CPL analysis for the excited state.This work provides the first example of CPL-generating peptoids reported to date, and an unprecedented chiral inversion with increasing structural homogeneity is demonstrated.The results suggest that chromophore-containing peptoids can serve as promising optically active scaffolds for understanding the relationship between structure and optical properties.

Synthesis of Peptoid Oligomers
Pyrene-containing Nα chiral submonomers ((S)-1-pyrenylethylamine, s1pye; (R)-1-pyrenylethylamine, r1pye) were synthesized by a previously reported method [34] using Ellman's chiral auxiliaries (tert-butanesulfinamides) (Scheme S1, Supporting Information).Peptoids can be obtained via solid-phase [35] or solution-phase [9,10,36] submonomer synthesis procedures.In the case of the (S)-N-(1-pyrenylethyl)glycine (Ns1pye) homooligomers, a conventional solid-phase peptoid synthesis approach, using bromoacetic acid/DIC coupling, did not allow for bromoacetylation following the second residue, necessitating the use of previously reported solution-phase synthetic methods. [9,10]For the C-terminal end group, the tert-butyl ester was selected to avoid intramolecular hydrogen bonding, which is known to have an impact on the peptoid secondary structure. [11]In addition, previous studies have demonstrated that C-terminal tert-butyl esters possess the ability to promote crystallization. [9,10,20]Peptoids were synthesized with and without an N-acetyl group to investigate the influence of N-terminal capping on the conformational homogeneity of the secondary structure (Figure 1B). [37]The synthesis began with a substitution reaction between tert-butyl bromoacetate and s1pye to yield monomer 1.The peptoids were then elongated by repeating a reaction cycle comprising acylation with bromoacetyl bromide followed by displacement with s1pye.After each displacement step, N-terminal acetylation was conducted using acetic anhydride to afford peptoids 1 0 -7 0 (Figure S2, Supporting Information, Table 1).

NMR Analysis
Conformational homogeneity of peptoids were evaluated by NMR spectroscopy.K cis/trans is an indicative parameter to quantify the homogeneity of PPI-like peptoid helices, which are composed of backbone cis-amides (Figure 2).Following previously described methods, [11,38] the peaks corresponding to cisand trans-amide rotamers were identified via 2D 1 H-1 H NOESY and 2D 1 H- 13 C HSQC experiments.Then, the integrals of the rotamer-related peaks in the 1D 1 H NMR and 2D 1 H-1 H COSY spectra were used to calculate the K cis/trans values (Table 2).The Ns1pye homooligomer successfully stabilized the cis-rotamer upon chain elongation.The overall backbone amide K cis/trans value of 2 0 in acetonitrile was 11.4, which is the highest value reported for any peptoid dimer to date.Trimeric peptoid 3 0 exhibited a K cis/trans value of greater than 19, which surpasses that previously reported for the naphthylbased peptoid Ac-(Ns1npe) 3 -COOtBu (K cis/trans = 16.0). [9]The bulky pyrene-containing side chain led to a general trend of increasing K cis/trans values with increasing peptoid length.Compared with naphthalene, pyrene possesses a lower LUMO, which should promote the noncovalent interaction between the nonbonding orbital of the backbone carbonyl group and the π* orbital of pyrene (nπ* Ar effect). [7]Compared with 1 0 -5 0 , peptoids 1-5 displayed significantly decreased K cis/trans values.Unlike peptides, the side chains of peptoids are attached to the nitrogen atoms; thus, the first residue from the N-terminus is susceptible to nitrogen inversion, resulting in conformational heterogeneity of the overall structure.The N-terminal acetylation in 1 0 -5 0 clearly suppressed the trans-rotamer population at the N-terminus to afford enhanced conformational homogeneity.The K cis/trans values of peptoids 6, 6 0 , 7, and 7 0 could not be measured on account of their limited solubility in acetonitrile.

X-ray Crystallography
Peptoids 1, 2, and 1 0 -4 0 were crystallized (the conditions are given in Table S3, Supporting Information) and their solid-state structures were determined by single-crystal X-ray crystallography (Figure 3). [39]The development of the peptoid helix was visualized using the crystal structures of peptoids 1 0 -4 0 (Figure 3A).Peptoid 4 0 exhibited a pitch of %6 Å and approximately three residues per turn, in accordance with previous studies. [9,10]he torsional angles of peptoids 1, 2, and 1 0 -4 0 are listed in Determined by integration of 2D 1 H-1 H COSY spectra.b) Determined by integration of 1D 1 H NMR spectra.
Table 3. Deviation of the amide bond planarity was observed, as demonstrated by the ω angles ranging from À15.5°to 1.5°.Nonplanar amide bonds have been reported previously for constrained cyclic peptoids [12] and helical peptoids bearing bulky side chains [9,10] ; the deviation observed for the pyrene-containing side chain s1pye may be attributable to its similarity to the latter case.Notably, the nπ* Ar interaction was not observed in the solid state.Unlike the solution state, a condensed solid-state environment may prioritize the packing of molecules over their intramolecular interactions.For example, intermolecular π-π stacking between the pyrene side chains was observed for peptoids 1 and 4 0 (Figure S3, Supporting Information).Similar structural features were observed for the previously reported naphthylbased peptoids. [9]The structural difference between the solid and solution states was observed for the dimeric peptoids.Specifically, peptoid 2 exhibited a cis-amide structure (Figure 3C), which was not a major conformation in solution (K cis/trans = 0.7 in acetonitrile).Compared with peptoid 2 0 , the structure of peptoid 2 featured a longer distance between the two pyrene moieties, demonstrating the flexibility of the peptoid without N-acetyl capping.

CD Spectroscopy
The CD spectra of Nspe-based helical peptoids have been extensively investigated. [5,40]The orbital interactions between the backbone carbonyl and side chain phenyl groups result in π!π* Ar (%192 nm and 202 nm) and n!π* Ar (%220 nm) transitions, providing a CD signature that is similar to that of PPI-type helical peptides. [2,5]Subsequently, helical peptoids containing naphthyl- [9] and alkyl-based side chains [6,10] were characterized by NMR spectroscopy and X-Ray crystallography; however, the unusual CD signature of these folded peptoids has yet to be rationalized. [6,9,10]Herein, an attempt has been made to explain the nature of each peak observed in the CD spectra of the pyrenecontaining peptoids.First, the absorption spectra of the pyrene-containing peptoid series are recorded to investigate the electronic and vibrational modes of the pyrene moieties (Figure S5, Supporting Information).The pyrene absorption spectra displayed the distinctive electronic states of 1 L a (S 2 -S 0 , λ max = 344 nm), 1 B b (S 3 -S 0 , λ max = 277 nm), and 1 B a (S 4 -S 0 , λ max = 242 nm), but not the symmetrically forbidden state 1 L b Figure 3. A) Solid-state structures of peptoids 1 0 -4 0 as determined by X-ray crystallography.B) View parallel to the helical axis from the N-terminus of peptoid 4 0 .C) Overlay of the molecular structures of peptoids 2 (beige) and 2 0 (cyan).Table 3. Torsion angles of peptoids 1, 2, and 1 0 -4 0 as determined by X-ray crystallography.
Residue (S 1 -S 0 ). [41]Because the entire set of vibronic states was maintained in the Ns1pye oligomers, the neighboring 1-ethylamine substituent and amide groups had minimal effect on the vibronics of the pyrene moieties.For the pyrene moieties of the Ns1pye oligomers, every absorption ( 1 L a , 1 B b , and 1 B a ) is CD active, and multiple Cotton effects were observed (Figure 4A-C).The through-space coupling of adjacent chromophores in a chiral environment generates exciton-coupled circular dichroism (ECCD), which can be experimentally identified by a bisignate CD couplet. [42,43]Except for the 1, 1 0 , 2, 2 0 , and 3, the negative Cotton effect for the ECCD signals of the 1 L a and 1 B a states gradually increased for both sets of peptoids in a length-dependent manner (Figure 4B,C).These results indicate that the interaction between the long-axis-polarized transition dipoles ( 1 L a and 1 B a ) became stronger as the peptoid length increased from a trimer to a heptamer.The ECCD intensity is proportional to the cross product of the two transition dipoles (i.e., |μ 1 Â μ 2 |) and inversely proportional to the interchromophore distance. [42,43]Therefore, it is assumed that as the peptoid elongated from a trimer to a heptamer, the transition dipoles ( 1 L a and 1 B a ) became located near each other in space and oriented away from a parallel (or anti-parallel) alignment.In contrast, the ECCD signal of the short-axis-polarized transition dipole 1 B b tended to decrease.This suggests that peptoid elongation caused the 1 B b transition dipoles to align parallel to one another, resulting in a decrease in |μ 1 Â μ 2 |.The negative Cotton effect in ECCD indicates a counterclockwise arrangement (i.e., right-handed helical sense) of the pyrene moieties in the peptoid helix, which is in accordance with the X-ray crystal structures of 3 0 and 4 0 (Figure 3).The overall similarity of the CD signatures between peptoids 3-7 and 3 0 -7 0 provides convincing evidence that the Ns1pye oligomers folded into the right-handed helical structure irrespective of the presence or absence of N-acetylation.Similar to the results obtained by NMR spectroscopy (Table 2), a more homogeneous conformation was demonstrated for N-acetylated peptoids based on the stronger CD intensities observed for peptoids 3 0 -7 0 .The N-terminal acetylation holds promise for diminishing the average intramolecular distance between pyrenes, subsequently resulting in an improvement in ECCD signal.For example, heptameric 7 and tetrameric 4 0 exhibited similar CD intensities (Figure 4D).Notably, peptoid 2 afforded a distinctive CD profile with an inverse signal for the 1 B a state, indicating that the optical environment of the pyrene moieties in peptoid 2 was different from those in the longer peptoids.The enantiomers of 2 and 2 0 (i.e., 2 R and 2 0 R ) were synthesized using the r1pye submonomer and found to display CD spectra that were clear mirror images (Figure 4E)

Fluorescence Measurements and Excimer Characterization
The excimer emission represents the electronic perturbation induced by interactions between luminophores in the excited state.Fluorescence spectra were collected at an excitation wavelength of 355 nm (Figure 5).The excimer emission of peptoids 2-7 was observed at an emission wavelength (λ em ) of 458-466 nm, while the N-acetylated peptoids 5 0 -7 0 exhibited excimer emission at 444 nm.Peptoid 2 displayed a unique behavior with early-stage excimer generation (Figure 5A).In the solution state, the heterogeneity of this peptoid brings the two pyrenes into close proximity, facilitating the generation of the excimer state.As shown by both CD and fluorescence spectra, the pyrene arrangement in peptoid 2 did not correlate with that of the longer peptoids.After N-acetylation, excimer formation was suppressed in peptoids 2 0 -4 0 , which was likely attributable to a reduction in the conformational flexibility necessary to generate the excimer state between the pyrene moieties (Figure 5B).A distinct excimer peak appeared for peptoid 5 0 and longer oligomers.

CPL Measurement and Chirality Inversion
CPL measurements were performed to examine the excimer chirality of the pyrene-containing peptoids.Peptoids 2-7 and 2 0 -7 0 were found to exhibit CPL activity with a high dissymmetry factor (g lum = 10 À2 , Table 4 and Figure 6).The absence of dissymmetry (or CPL activity) for local emission (<400 nm) indicates that the formation of the chiral excimers is crucial for generating CPL emission in the Ns1pye oligomer system.For this reason, monomeric peptoids 1 and 1 0 did not display CPL activity.The generation of LCPL and RCPL was found to be influenced by the structural homogeneity of the ground state.As the peptoid chain became longer and after N-acetylation, the dissymmetry tended to enhance RCPL (Figure 6).The largest positive g lum value was observed for the dimeric peptoid 2 (g lum = 1.75 Â 10 À2 ), while the largest negative g lum values were found for the conformationally homogeneous peptoids 5 0 -7 0 (g lum = À1.3Â 10 À2 ).The opposite sign of g lum for peptoid 2, compared with the longer peptoids, indicates the opposite chirality of the pyrene excimer.
The unique chiroptical properties of peptoid 2 were also observed in the CD spectra (Figure 4A).The strong intensities of the ECCD peaks for peptoid 2, along with its distinct spectral signature, particularly the positive Cotton effect for the long-axis transitions ( 1 L a and 1 B a ), align well with the CPL results.Peptoids 4 and 5 exhibited wavelength-dependent CPL sign inversion.RCPL and LCPL were recorded at shorter and longer wavelengths, respectively.This implies that these peptoids with moderate conformational homogeneity form multiple excimer conformations in the excited state, each with distinct optical activity.Peptoids 2-7 and 2 0 -7 0 exhibited saturation of luminescence (g lum ) at specific lengths (i.e., the hexamer and pentamer, respectively).Besides the dissymmetry factor, a high excimer quantum yield is another prerequisite for an efficient CPL emitter.In the trimeric and longer peptoids (3-7 and 3 0 -7 0 ), a tendency of increasing quantum yield with greater peptoid length was observed (Table S5, Supporting Information).Therefore, 7 0 can be considered an efficient CPL emitter compared with the other pyrene-containing peptoid helices.The intricate structural features of flexible and multichromophore-containing system make it challenging to undertake a detailed structure-based CPL rationallization.However, these results suggest that a chromophore display strategy based on peptoid helices is a versatile method for generating excimer CPL.

Conclusion
A series of optically active peptoids, displaying multiple pyrene moieties, were synthesized.Characterization of the structures and photophysical properties of the prepared 1-pyrenecontaining peptoids (i.e., Ns1pye oligomers) revealed valuable insights into the relationship between conformational homogeneity and optical activity.The helical propensity of the Ns1pye oligomers is examined using NMR spectroscopy and observed stabilization of the cis-amide backbone upon sequence elongation and N-terminal acetylation.Peptoid 3 0 started to exhibit significant cis-amide stabilization (K cis/trans > 19 in acetonitrile), affording one of the most conformationally stable trimeric peptoids reported to date.The X-Ray crystal structure of the N-acetylated tetramer 4 0 demonstrated the progression of a right-handed PPI-like helix, as confirmed by backbone torsion angles that were similar to those of previously reported PPI-like peptoid helices. [9]his well-defined structure served as the foundation for our interpretation of the CD spectra, providing a practical solution for situations where multiple transitions from the incorporated  chromophores are CD active.In the case of pyrene, the perpendicular property between the long-and short-axis-polarized transitions induced the opposite tendency of change in the ECCD intensity in a length-dependent manner.
The incorporation of multiple pyrene units in the peptoids resulted in not only chiral absorption but also the formation of chiral pyrene excimer fluorescence.Notably, the nonacetylated peptoid dimer 2 exhibited an intramolecular excimer generating LCPL with a significant dissymmetry factor (g lum = 1.75 Â 10 À2 ).N-Acetylation and elongation promoted the formation of a more homogeneous secondary structure, resulting in a more negative CPL (RCPL), which became saturated at the pentamer length (5 0 ).Our results with the Ns1pye homooligomers indicated that flexible short peptoids tend to emit LCPL and long peptoids with structural homogeneity tend to emit RCPL.On the basis of the proposed structurephotophysical property relationship, our peptoid series may broaden the understanding of multichromophoric chiral systems and expand the potential applications of biomimetic scaffolds in the fields of imaging and sensing.

Figure 1 .
Figure 1.A) Chemical structures of peptides and peptoids.B) Peptoids discussed in this study.

Table 2 .
Overall K cis/trans values as determined by integration of the 1 H NMR or 1 H-1 H COSY spectra in CD 3 CN.