Aromatic Residue Positioning Influences Helical Peptoid Structure in Aqueous Solution

Water-soluble peptidomimetics, including peptoids, are promising functional surrogates for biologically relevant, amphiphilic, helical peptides. Twenty amphiphilic peptoid hexamers with predicted helical structures were designed, prepared, and studied using circular dichroism (CD) spectroscopy. The site-specific contributions of aromatic and charged residues to the helical structure of peptoid hexamers in aqueous solution was evaluated, revealing that aromatic residue positioning most significantly impacts structure.

Peptoids (N-substituted glycine oligomers) have many advantages that enable their widespread study and application as biomimetic scaffolds.The sequence-specific synthesis of monodisperse peptoid analogues bearing peptide-like functional groups is inexpensive and efficient. 1,2Moreover, peptoids can overcome peptides' pharmacokinetic liabilities: peptoids exhibit proteolytic stability, 3 are not immunogenic, 4 and have useful cell permeability properties. 56][17] Of these, a peptoid helix that resembles the polyproline type I (PPI) helix 6 is among the most well-studied structures.Water-soluble PPI helical peptoids have found application as antimicrobials, [18][19][20] for example, and modulating the secondary structure has been shown to impact the selectivity for bacterial cells over eukaryotic cells. 19An understanding of how to design and tune peptoid conformations in water is essential to expand peptoids' functional biomimicry.
Inducing peptoid three-dimensional structure, including PPI helical structure, originates from controlling backbone dihedral angles. 21,22Choice of N-substituent (side chain) influences the peptoid backbone amide bond () conformation (Figure 1), and  in turn can promote structure, including the all-cis-amide PPI helix.Model peptoid monomers bearing the chiral 1-naphthylethyl side chain favor the cis conformation (K cis/trans of 6.27 in CD 3 CN) to minimize steric repulsion between the bulky N-substituent and the adjacent (i -1) backbone methylene. 21Tertiary ammonium (N,N-disubstituted)-2-aminoethyl side chains also favor the cis-amide (K cis/trans as high as 8.1 in D 2 O). 23For these residues, the cis-amide conformation minimizes unfavorable steric interactions and allows intramolecular H-bonds between the side chain and both the i and i -1 carbonyl oxygens. 23Inclusion of strong cis-amide-promoting residues is an effective design strategy for generating PPI helical peptoids. 11In this work, we sought to clarify the position-specific contributions of peptoid residues to amphiphilic PPI helix structure in aqueous solution.High-resolution structural studies of linear peptoids are few; 6,7,11 peptoid secondary structure in solution is commonly inferred from compara-

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tive studies of peptoids' circular dichroism (CD) spectra.In organic solution, the site-specific effects of helix-promoting aliphatic and aromatic residues on the solution structures of peptoid PPI helical hexamers 24 and heptamers, 25 respectively, have been explored.In both of these studies, the Cterminal residue strongly influenced the peptoid PPI helix structure.However, analogous studies to understand position-specific sequence-structure relationships in watersoluble peptoids are underdeveloped -the helical modulation of 12-residue antimicrobial peptoids reported by the Seo laboratory is the sole example we found. 19Here we report systematic studies of twenty peptoid hexamers designed to adopt amphiphilic helix structures in aqueous solution.The site-specific contributions of varied residues to the putative peptoid structure were explored.Our results reveal that the hydrophobic, aromatic residue identity and position in the sequence have the most significant effect on peptoid structure.
Twelve isomeric peptoid hexamers were initially designed and synthesized to explore the influence of residue positioning on peptoid secondary structure (1-12, Table 1).The peptoids prepared were composed of three residues: the aromatic, hydrophobic residue (S)-N-1-(naphthylethyl)glycine (Ns1npe), N-[(N,N-diisopropyl)2-aminoethyl]glycine (N i Pr 2 ae), a polar residue expected to have a positive charge at neutral pH in aqueous solutions, and (S)-N-(1-carboxyethyl)glycine (Nsce), expected to have a negative charge at neutral pH.Both Ns1npe and N i Pr 2 ae residues have been shown to strongly favor the cis-amide bond conformation; 21,23 as such, we reasoned that they would be strongly PPI-helix-promoting, and the chiral Ns1npe and Nsce residues would enable CD spectroscopic comparisons to other peptoids.Following design principles outlined in the literature, 8,26 the putative helix was expected to display the aromatic, hydrophobic side chains on one face and the polar groups on the other two faces (see PPI helix wheel for 1 in Table 1, for other peptoids in the Supporting Information).For structural comparison, our laboratory has previously reported similar amphiphilic helical structures from 6 to 15 residues in length, including a close analogue of 5. 27 We have observed peptoid self-association of longer, 15residue peptoid amphiphilic PPI helices in water; [27][28][29][30] our work here focused on shorter, nonaggregating sequences.
Isomeric peptoids with predicted amphiphilic PPI helix structure were prepared that varied the specific ordering of residues.Peptoids 1-4 all included Ns1npe aromatic residues at positions 1 (N-terminus) and 4 in the linear sequence, and they vary from one another in the ordering of the polar N i Pr 2 ae and Nsce residues on the other two helix faces.Peptoids 5-8 all included aromatic Ns1npe residues at positions 2 and 5 while varying the placement of the polar residues, and peptoids 9-12 included aromatic Ns1npe residues at positions 3 and 6 (C-terminus).Solid-phase peptoid synthesis and purification were carried out following reported methods, 1,31 and peptoids' identities were con-firmed by mass spectrometry (see the Supporting Information).We did not observe evidence of peptoid aggregation in aqueous solution, e.g., concentration-dependent CD or fluorescence spectral features (see the Supporting Information).
To evaluate structural differences between the peptoids and to compare them with peptoids previously synthesized by our laboratory 27,28 and others, 11,24,25 we acquired their CD spectra in a range of conditions (Figure 2). 32Peptoid PPI helices that comprise different residues have different CD spectra, and side-chain contributions to the CD spectra often obscure the canonical peptide PPI helix CD signature. 6- 12The CD spectra of 1-12 are dominated by features attributed to side-chain contributions of the Ns1npe residues.
Most notably, spectra of peptoids 1-4 in neutral aqueous buffer exhibited uniform features that were not observed in the spectra of 5-12.CD spectra of 1-4 all included split maxima/minima around the naphthalene 1 B b transition: a maximum around 220 nm and a minimum at 230 nm.These spectral features were characteristic of excitonic coupling of the two naphthalene chromophores; in excitonically coupled systems, the amplitude of the peaks and the separation of the bisignate peaks along the wavelength axis (Δ max ) are correlated to the specific orientation of the coupled naphthalene dipoles. 33,34The overlapping features in

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this region suggested that 1-4 all place the naphthalene side chains in a similar arrangement.Other water-soluble helical peptoids and -peptoids bearing analogous naphthalene side chains also exhibited excitonic coupled CD features. 27,28,35At wavelengths below 210 nm, there were no distinct spectral features, and we hypothesized that spectral differences in this region were the result of other sidechain interactions and/or backbone heterogeneity.
Most of the CD spectra of peptoid isomers without an N-terminal Ns1npe residue did not show strong excitoncoupled peaks, suggesting that the internal and C-terminal naphthalenes have less flexibility to interact with the other aromatic group in the sequence.CD spectra of peptoids 6-8, 11, and 12 exhibited broad maxima at 207 nm and sharp minima at 227 nm (7, 8, and 11) or 229 nm (6 and 12).Their similar spectra suggested that dipole-dipole interactions of the naphthalene side chains in peptoids 6-8, 11, and 12 were minimized.We speculated that the broad maximum at 207 nm was analogous to the helical spectral signature of similar magnitude observed for aliphatic peptoid hexamer helices in organic solution. 24The spectrum of 10 was unique from the others in that it exhibited a broad minimum at 227 nm and a broad maximum at 202 nm.Lastly, spectra of peptoids 5 and 9 had red-shifted minima at 233 and 231 nm, respectively, and both had broad maxima around 217 nm.Peptoids 5 and 9 share the four-residue sequence Ns1npe-N i Pr 2 ae-N i Pr 2 ae-Ns1npe, and we posited that this subsequence positioned the naphthalene side chains in similar conformations.None of the peptoids exhibited appreciable CD signals in the near UV wavelengths (see the Supporting Information).Additionally, we observed that CD spectra were not changed appreciably by solution pH changes, despite that the N i Pr 2 ae modestly favors the trans-amide conformation at high pH 23 (see the Supporting Information).We suggest that the Ns1npe residues' conformations most strongly direct the peptoid PPI helix structure in aqueous solution.
The CD spectra of peptoids 1-12 in organic solvents, methanol, and acetonitrile, were compared.Because K cis/trans values are higher in organic solvents than in water, 21,23 spectral features that correlate with more helical structure were expected to be more intense in organic solvents.Correspondingly, spectral features were more intense in organic solvents for most peptoids (see the Supporting Information).The persistence of the excitonic coupling features observed in the spectra of 1-4 in organic solvents is unique from previous observations in longer peptoids. 27,28We posit that the peptoid backbone conformation directed the naphthalene side-chain dipole-dipole interactions, rather than a hydrophobic effect that positioned the naphthalenes in a specific orientation.
The effects of temperature on the aqueous CD spectral features of representative peptoids were also evaluated as shown for 2 in Figure 3 (others in the Supporting Information).For all of the selected peptoids studied, CD signal intensity decreased by roughly half upon heating from 2 °C to 90 °C.This intensity change was consistent with each of these peptoids exhibiting more cis-trans-amide heteroge-

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neity at higher temperatures.The spectra of 2 exhibited isodichroic points at 213 nm and 225 nm over the temperature range.Because the maxima/minima wavelengths did not change, we speculated that the structural heterogeneity introduced upon heating was not accompanied by a change in electronic interactions of the Ns1npe side chains.No appreciable hysteresis was observed in the CD spectra when the samples were cooled to 2 °C after heating (see the Supporting Information).
Eight peptoids predicted to be less helical than 1-4 were subsequently designed and prepared (Table 2).These included N-(2-aminoethyl)glycine (Nae) residues (13-16) or (S)-N-2-(naphthylethyl)glycine (Ns2npe) residues (17-20)  in lieu of N i Pr 2 ae or Ns1npe, respectively.Both Nae and Ns2npe have reported K cis/trans values lower than those reported for N i Pr 2 ae or Ns1npe. 21,23We anticipated that these substitutions would introduce backbone structural heterogeneity, allowing us to interrogate residue-specific contributions to the structure.

Table 2 Additional Peptoids Prepared
CD spectra of peptoids 13-20 were acquired (Figure 4).In aqueous buffer at neutral pH, CD spectral features of 13-16 were very similar to 1-4, suggesting that this residue substitution has little impact on peptoid helix structure (see the Supporting Information).In contrast, the spectral features of 17-20 were quite different from those of 1-4 (Figure 4), consistent with the unique absorbance and excitonic coupling of the 2-substituted naphthalene in the Ns2npe side chain relative to the Ns1npe naphthalene. 36Additionally, there were many differences in peak intensities and peak wavelengths between 17-20.The spectra of 17 and 20 have minima that are more than twice as intense as the minima for 18 and 19 (and 1-4).Spectra of 17, 19, and 20 all included a minimum at 223 nm, but the minimum in 18 was at 230 nm.The spectra of 19 and 20 also included a modest shoulder minimum at 230 nm.
We interpreted the varied spectral features for 17-20 to be consistent with backbone conformational heterogeneity among these peptoids.Because of their different backbone conformational ensembles, peptoids 17-20 accessed a range of excitonic structures; the changes to minimum peak wavelengths and the presence of shoulders were evidence for these excitonic structures, rather than the bisignate spectral features observed for 1-4.
Our findings here confirm that placement and identity of naphthalene-bearing residues within a peptoid hexamer influence side-chain interactions accessible within the peptoid helical structure.Specifically, we hypothesize that when the Ns1npe or Ns2npe aromatic residue is in the Nterminal position, the side chain has flexibility that allows it to interact with another aromatic side chain.The ordering of charged residues has a more minimal effect on the peptoid structure.Ongoing studies will examine if these observations are unique to peptoids comprising naphthalene-bearing residues.Further, we intend to use other structural studies (e.g., NMR spectroscopy) to detail the structures of these hexamers and of longer and more diversely functionalized peptoids.These results provide insights to guide the future design of short, structured, watersoluble peptoids, and will accelerate peptoids' use in a variety of biomimetic applications.

Figure 2
Figure2CD spectra of peptoids 1-12 exhibit spectral features that are dominated by side-chain interactions and that correlate with aromatic residue placement in the sequence.All peptoids were 100 M in 5 mM citrate-phosphate buffer, pH 7.

Figure 3 D
Figure 3 CD spectra of peptoid 2, which includes an N-terminal Ns1npe residue, at varied temperatures in 5 mM citrate-phosphate buffer, pH 7