Fluoro-Aryl Substituted α,β2,3-Peptides in the Development of Foldameric Antiparallel β-Sheets: A Conformational Study

α,β2,3-Disteroisomeric foldamers of general formula Boc(S-Ala-β-2R,3R-Fpg)nOMe or Boc(S-Ala-β-2S,3S-Fpg)nOMe were prepared from both enantiomers of syn H-2-(2-F-Phe)-h-PheGly-OH (named β-Fpg) and S-alanine. Our peptides show two appealing features for biomedical applications: the presence of fluorine, attractive for non-covalent interactions, and aryl groups, crucial for π-stacking. A conformational study was performed, using IR, NMR and computational studies of diastereoisomeric tetra- and hexapeptides containing the β2,3-amino acid in the R,R- and S,S-stereochemistry, respectively. We found that the stability of peptide conformation is dependent on the stereochemistry of the β-amino acid. Combining S-Ala with β-2R,3R-Fpg, a stable extended β-strand conformation was obtained. Furthermore, β-2R,3R-Fpg containing hexapeptide self-assembles to form antiparallel β-sheet structure stabilized by intermolecular H-bonds and π,π-interactions. These features make peptides containing the β2,3-fluoro amino acid very appealing for the development of bioactive proteolytically stable foldameric β-sheets as modulators of protein-protein interaction (PPI).


INTRODUCTION
Amino acids are small molecules able to induce high molecular complexity, promoting discrete three-dimensional folded structures in peptides. α-Amino acid-based peptides show some drawbacks such as the low stability to proteases, and, in the case of short peptides, the lack of a stable secondary structure. The use of oligomers with unnatural backbones is a powerful strategy to overcome those issues, while maintaining the desired fragment structure.
Due to the lack of information related to α,β 2,3 -amino acids repeating sequences, here we report on the preparation and conformational studies of α,β 2,3 -peptides, containing syn H-2-(2-F-Phe)-h-PheGly-OH, named β-Fpg, and helicogenic Salanine (Figure 1). This scaffold shows two features that can be appealing for biomedical applications: the presence of a fluorine, useful for non-covalent interactions, and two aryl groups, crucial for π-stacking and hydrophobic interactions. Taking advantage of the use of fluorine-substituted β-2S,3S-Fpg (Figure 1), in combination with S-Ala or S-Arg-S-Ala, we recently prepared short peptides able to generate proteolytically stable nanotubes  and spherical aggregates (Bucci et al., 2017b) as drug delivery systems.
Our results showed that the stability of peptide conformation is dependent on the stereochemistry of the β-amino acid. Combining S-Ala with β-2R,3R-Fpg, a stable extended βstrand conformation was obtained. Moreover, it was found that hexapeptide 11 (Figure 1) self-assembles to form antiparallel βsheet structure stabilized by intermolecular H-bonds and π,πinteractions. This result is important since, to the best of our knowledge, no examples of extended α,β 2,3 -foldamers are reported. Furthermore, there is a need of the development of bioactive proteolytically stable foldameric β-sheets that might be useful as modulators of protein-protein interactions (PPI) (Hegedüs et al., 2016). The design of this secondary structure is indeed not trivial, although β-sheet interfaces frequently occurred in PPI.
Melting points were determined with a Stuart Scientific melting point apparatus in open capillary tubes and are uncorrected. Chemicals were purchased from Sigma Aldrich and were used without further purification. ESI MS were recorded on a LCQ Advantage spectrometer from Thermo Finningan and a LCQ Fleet spectrometer from Thermo Scientific. The FT-IR absorption spectra were recorded with a Perkin-Elmer 1720X spectrophotometer, nitrogen flushed, equipped with a sampleshuttle device, at 2 cm −1 nominal resolution, averaging 100 scans. Solvent (base-line) spectra were recorded under the same conditions. Cells with path lengths of 1.0 and 10 mm (with CaF 2 windows) were used. Spectrograde CDCl 3 (99.8% D) was purchased from Fluka. The NMR spectroscopic experiments were carried out either on a Varian MERCURY 200 MHz (200 and 50 MHz for 1 H and 13 C, respectively), Varian MERCURY 300 MHz (300, 75, 282 MHz for 1 H, 13 C, and 19 F, respectively), or Bruker Avance I 500 MHz spectrometers (500 and 125 MHz for 1 H and 13 C, respectively). Chemical shifts (δ) are given in ppm relative to the CHCl 3 internal standard, and the coupling constants J are reported in Hertz (Hz). Optical rotations were measured on a Perkin-Elmer 343 polarimeter at 20 • C (concentration in g/100 mL).

Computational Methods
The non-natural β-amino acids, capped respectively with an acetyl (Ac) and an OMe group at the N-and C-termini, were designed using MOE (Molecular Operating Environment, 2013) and submitted to a "low mode" conformational search (MMFF94x force field, Born solvation, iteration limit = 40 000, MM iteration limit = 2,500, rejection limit = 500). The two lowest energy conformations having φ, θ, and ψ dihedrals corresponding to two different secondary structures, namely the extended one and a β-turn, were selected to derive partial charges with the R.E.D.III.52 software (Dupradeau et al., 2010). Each geometry was optimized at the HF/6-31G(d) level, and two different spatial orientations were used to derive orientation-and conformation-independent RESP-A1 charges.
The tetra-(m7 and m8) and hexapeptides (m11 and m12) of general formula 8 were built with the tleap module of Amber14 (Case et al., 2014) by imposing an extended conformation (φ = ψ = ω = θ =180 • ), and solvated with an octahedral box of CHCl 3 . All the systems were consequently submitted to a preliminary equilibration with the pmemd module of Amber14 package, using the ff14SB (Maier et al., 2015) force field. In detail, the systems were relaxed by minimizing hydrogens and solvent (2,000 cycles of steepest descent and 5,000 cycles of conjugated gradient). The solvent box was equilibrated at 300 K by 100 ps of NVT and 100 ps of NPT simulation using a Langevin thermostat with a collision frequency of 2.0 ps −1 . Successively, a minimization of side chains and CHCl 3 with restraints on backbone atoms of 25 kcal/mol and a total minimization (2,500 cycles of steepest descent and 5,000 cycles of conjugated gradient) were performed. The systems were then heated up to 300 K in 6 steps of 5 ps each ( T = 50 K), where backbone restraints were reduced from 10.0 to 5 kcal/mol. Full equilibration was performed in the NVT ensemble (100 ps, backbone restraints = 5.0 kcal/mol) and in the NPT ensemble (1 step of 200 ps, backbone restraints = 5 kcal/mol; 3 steps of 100 ps each, reducing the backbone restraints from 5.0 to 1.0 kcal/mol, and 1 step 1 ns with 1.0 kcal/mol of nd backbone restraints). Finally, unrestrained production runs were run at 300 K for 15 ns. An electrostatic cutoff of 8.0 Å was applied to all the calculations. The equilibrated geometries and the average dihedral energies and potential obtained through the accurate systems equilibrations were used as starting point for accelerated MD (aMD) simulations, where the α value was set to 0.2 and 0.16, for the dihedral and potential factors, respectively.
The aMD simulations of each peptide were run until convergence, for a total of 500 ns for the tetrapeptides m7 and m8, and 800 ns for hexapeptides m11 and m12, respectively. The root mean square displacement of backbone heavy atoms from the extended conformation was used as a metric of convergence ( Figure S1). All the analyses were conducted on the last 200 ns of trajectory. Cluster analyses were performed with the cpptraj module of Amber14 by sampling one of every 8 frames, using the average-linkage algorithm and requesting ten clusters; the pairwise mass-weighted RMSD on backbone Cα atoms was used as a metric. H-bonds were computed with cpptraj by setting a donor-acceptor distance threshold of 4.0 Å and an angle cutoff of 110 • . Only H-bonds with an occupancy >20% were considered. Cpptraj was used also for computing the radius of gyration of investigated peptides.

Synthesis
Dipeptides 1 and 2 were efficiently synthesized in gram scale starting from a racemic mixture of amino acid β-Fpg, according to a known procedure . Pure dipeptides were used for the preparation of α,β 2,3 -tetra-and hexa-peptide sequences (Scheme 1).

IR Analysis
In our FTIR absorption investigation we focused on the N-H stretching (amide A) region (3,470-3,240 cm −1 ). Absorptions in this region are highly dependent on H-bond formation and, consequently, they are conformationally informative. To get insights into the modes of folding and self-association we chose as solvent CDCl 3 . It has a relatively low polarity and it is known to support ordered secondary structures in peptides.
As the peptide chain is elongated, the relative intensity of the N-H stretching band at about 3,330 cm −1 markedly increases as compared to that of the band at about 3,430 cm −1 (Figure S9). The first band is assigned to H-bonded NH groups, while the second is due to free (solvated) NH groups (Palumbo et al., 1976;Toniolo et al., 1985).
In the absence of aggregation, this behavior can be ascribed to the formation of ordered secondary structures, stabilized by intramolecular H-bonds. From our analysis we conclude that the chirality of the two stereogenic centers of the fluoro amino acid remarkably affects the peptide 3D-structure. In particular, the maximum of the bonded N-H band is located at 3,320 cm −1 for peptide 12, but at higher energy, 3,330 cm −1 , for peptide 11 (Figure 2). This latter observation implies that the H-bonds in the 11 hexapeptide are weaker. Indeed, an inspection to the behavior of the two hexapeptides at two concentrations (0.1 and 1 mM) is highly informative (Figure 2). For peptide 12 the relative intensities of the bands centered at 3,430 cm −1 (free NHs) and at 3,320 cm −1 (H-bonded NHs) do not significantly change upon diluting 10 times ( Figure 2B). Therefore, we assume that the H-bonded NHs originate mainly from intramolecular interactions in 12. Conversely, a clear dilution effect is observed for 11 (Figure 2A) highlighting an important contribution from intermolecular H-bonds. The onset of a β-sheet conformation can thus be hypothesized for 11 based on this FTIR absorption analysis.
In conclusion, this IR absorption analysis indicates that the longer oligomers adopt secondary structures strongly dependent on the chirality of the fluoro-β 2,3 -diarylamino acid.
As reported in the literature (Cheng et al., 2001;Balamurugan and Muraleedharan, 2012;March et al., 2012), J 2,3 and J NH−CHβ values help to predict the secondary structure of peptides containing β-amino acids. Large values (10-12 Hz) indicate the antiperiplanar arrangement of C α /C β substituents and of NH-CH β and are consistent with an extended conformation. Smaller values (3-4 Hz) indicate a gauche conformation corresponding to turn/helix constructs. Since we started from a syn-amino acid, an extended conformation could be expected. On the other hand, the presence of helicogenic alanine could influence the secondary conformation of the peptide. As shown in Table 1, large J 2,3 and J NH−CHβ values were detected for peptides containing β-2R,3R-Fpg amino acid indicating their extended conformation. Lower J values were detected for peptides containing β-2S,3S-Fpg amino acid, mostly for J 2,3 of amino acids at position 2 and 4 of peptide 12. On the other hand, these J values are higher with respect to those reported for helix constructs containing β-amino acids (J 3-4 Hz), suggesting that for 12 more than one preferred conformation is present (Seebach et al., 1999).
A further main difference between the two series is the Me Ala resonances. According to an extended conformation, Me Ala3 of 7 (see Figure 7A) is located between Ph β2 and Ar Fβ4 possessing the same orientation. This induces a strong shielding effect on the methyl group that resonates at higher field (δ 0.48). Similarly, 11 is characterized by two shielded methyl groups [Me Ala3 (δ 0.32); Me Ala5 (δ 0.35)], in agreement with an extended conformation ( Figure 8A). Me Ala3 (δ 0.97) in 8, as well as all three Me Ala [Me Ala1 (δ 1.36); Me Ala3 (δ 1.20); Me Ala5 (δ 1.01)] of 12, not affected by this electronic effect, resonate at lower field. Furthermore, differences of NH chemical shift are mostly found for hexapeptides 11 and 12, being the NH resonances of this last at lower field.
The temperature dependence experiments of the NH proton chemical shifts variation, giving insights on NH inaccessibility or on NH-bond network (Augspurger et al., 1995) were performed for peptides 7, 8, and 12 (NHs proximity to aromatic protons prevented a clear overview of their behavior in peptide 11).
Very high δ/ T values (>-9.4 ppb K −1 ; this value is not detectable for NH β2 , overlapped signal) were found for 7 and 8, indicating absence of H-bonds. As exception, a medium value (−4.3 ppb K −1 ) was detected for NH Ala1 of 8, indicating an equilibrium between a H-bonded and non-bonded status or a shielded environmental (Figure 3).
These results agree with IR data indicating, for both peptides, a low intensity of the bond at about 3,320 cm −1 and a lower area for 7 with respect to 8 (Figure 2).
Smaller δ/ T values were found for NHs of 12 with respect to 8, mostly for NH Ala1 , NH β6 , and NH Ala3 (−2.9, −3.01, −4.4 ppb K −1 , respectively; Figure 4). These data are consistent with  IR analyses (Figure 2B) indicating the tendency of 12 to give strong/medium intramolecular C=O···H-N H-bonds. A complete set of CH α /NH β (i, i+1) and H β -2/NH α (i,i+1) NOEs characterizes peptides 7 (Figure S2), 8 (Figure S3), and 12 ( Figure S8). A peculiar NOE for 8 is between H-5 of Ar β4 (δ 7.11) with Ph β2 (δ 7.31, Figure 5). The resonances of Ph protons in both Ph-3 β2 and Ph-3 β4 range in δ 7.31-7.22 region. On the other hand, the antiperiplanar position of Ph with Ar F of β-4 amino acid (J 2,3 10.3 Hz) excludes that the detected spatial proximity between Ar F and Ph derives from the aromatic moieties of the same amino acid. The formation of a turn in β-2/Ala3/β-4 region is suggested, as confirmed by computational data (Figure 7C), that is consistent with the deviation of J NH−CHα values characterizing the extended peptides (Table 1).
Considering peptide 12, apart the above-mentioned NOEs, only a weak spatial proximity was detected between NH-2 and NH-3 ( Figure S8). Detection of possible NOEs between nonsequential Ph/Ar protons, as shown for 8, were prevented because of the presence of several overlapped aryl protons.
Focusing on 11, only some CH α /NH β (i, i+1) and H β -2/NH α (i,i+1) NOEs were detected, because several signals are overlapped preventing certain NOEs. Interestingly, the formation of an antiparallel pleated sheet arrangement in which the C-terminus of one strand is faced on the N-terminus of a second strand is supported by Noesy/Roesy experiments. Different inter-strand spatial proximities are present that are Me Ala1 with both Me Ala5 (m) and OMe (vw) and Boc with both CH β6 -2(m) and OMe(w) (Figure 6). 1 H NMR studies at variable concentration (1.15-9.10 mM; Figure S32) showed concentration dependence chemical shift changes indicating aggregation. Noesy/Roesy experiments confirmed this tendency also at very low concentration (1.5 mM in CDCl 3 , Figure S5). DMSO-solvent titration of 11 was performed that was matched with Tocsy experiments (0%, 8% and 20% v/v of DMSO in CDCl 3 ) to ensure the correct correlation between NH with the corresponding amino acid. A strong NH downshift was detected for NH β6 , NH β4 , and NH Ala−3 ( δ > 1, 0.89, and 0.54, respectively) indicating their solvent exposure. A mediumweak H-bond ( δ 0.35) was detected for NH-2. Instead, NH-1 and NH-5 are strongly involved in a H-bond ( δ < 0.05) ( Figure S7). As a result, our hypothesis is that this antiparallel sheet is stabilized by intermolecular H-bond.
The NMR analysis of 11 is also performed in DMSOd 6 (10 mM), that is known to be a dissociating solvent. A similar 1 H NMR spectrum was detected concerning protons multiplicity, indicating an extended architecture, except for the NH resonances that are at lower field (Table TS4). This allowed to detect a complete set of CH α /NH β and H β -2/NH α (i,i+1) spatial proximities. As expected, inter-strand spatial proximities were absent in the ROESY experiment ( Figure S6).

Computational Studies
To gain additional insight on the conformational behavior of both tetra (m7 and m8) and hexapeptides (m11 and m12), we performed accelerated molecular dynamics (aMD) simulations in explicit CHCl 3 solvent (Pierce et al., 2012).
Concerning the tetrapeptides, both clustering and hydrogen bond (H-bond) analyses suggested a significantly different conformational behavior for m7 and m8. The analyses of the aMD trajectories of both peptides only evidenced two potential H-bonds, one between the acetyl carbonyl and NH β2 , the other between C=O β2 and NH β4 . However, the occupancy is about 20% higher in the case of m8 ( Table 2), suggesting that m8 can adopt a more compact conformation. This behavior is also confirmed by the analysis of the φ, ψ, and θ dihedrals of the representative structures of the most populated clusters (Table 3).
Indeed, m7 prefers an almost completely extended conformation (Figure 7A), except for a γ-turn involving the acetyl cap at the N-terminus. This conformation is consistent with the high chemical shift observed for Me Ala3 (see above). At a minor extent, a geometry characterized by two γ-turns, the first involving the acetyl cap and the NH β2 , the second between the C=O β2 carbonyl and NH β4 , is also observed as the secondary cluster ( Figure 7B). This conformation is possibly stabilized by a π-π interaction between the Ph β2 and the Ar Fβ4 . However, the low H-bond population, the relatively long donor-acceptor distances and narrow donor-H···angles (Table 2) suggest that these γ-turns are not stable. Overall, these data suggest that m7 can switch from a completely extended conformation to a partially folded conformation. However, this last is only marginally stabilized by π-π interactions between the two β-amino acids and by γ-turn H-bonds. The equilibrium is consequently shifted toward the extended conformation, coherently with NMR observations. Conversely, m8 tetrapeptide showed an opposite behavior. A highly populated primary cluster (pop = 87.7%; Figure 7C) is indeed characterized by a relatively compact geometry, showing a γ-turn between β-2 and β-4 and a π-π interaction between the Ph β2 and Ar Fβ4 . This interaction is consistent with Noesy experiments (see above). A poorly sampled fully extended conformation (pop = 9.1%) was also found as a secondary cluster ( Figure 7D).
The analysis of the aMD simulations performed on m11 and m12 hexapeptides led to similar conclusions, compared to the tetrapeptide series. Indeed, m11 most populated cluster (pop = 43.3%) corresponds to an extended conformation, as showed by the φ, ψ, and θ dihedrals of the corresponding representative structure (Table 4 and Figure 8A). The second most populated cluster (pop = 29.5%) represents conformations showing π-π interactions involving the Ar F of both β-2 and β-4, while the C-terminus remains extended (Table 4 and Figure 8B). Only the third cluster (pop = 19.8%) presented the Ar F /Ph π-π interactions between the aryl groups of β-2/β-4 and of β-4/β-6, similarly to what observed in m7. We could also observe γ-turns between the backbone C=O of Ac capping group with NH β2 , and carbonyls of β-2 and β-4 with NH β4 and NH β6 ( Table 5).
The top two clusters of the m12 hexapeptide are relatively low populated (pop = 27.4 and 27.0%, respectively). However, they both show a folded conformation, with π-π interactions between the aromatic rings of the β-amino acids, and the above described γ-turns (Figures 8C,D; Table 4). This behavior resembles what previously observed for the tetrapeptides series, where m8 principal geometry was more compact, compared to m7. A similar behavior between the hexa-and tetrapeptide series was also observed in terms of H-bonds. Indeed, the occupancies of the H-bonds defining the γ-turns are about 15% higher in m12, compared to m11 (Table 5). In the hexapeptide series, also a relatively populated third cluster (pop = 16.4%) showed a partially folded representative structure. Indeed, computed φ, ψ, and θ dihedrals (ψ3, φ4, and θ4, in particular), assume values that are compatible with interactions between the Ph β2 and the Ar Fβ4 , as well as between the Ph β4 and Ar Fβ6 .
Overall, the computational analysis shows a coherent influence of the stereochemical configuration of α and β carbons on the investigated β-amino acids inserted in model  Ac(S-Ala-β-Fpg) n OMe peptides. Indeed, our observation suggest that β-2R,3R-Fpg helps to stabilize an extended conformation. Conversely, β-2S,3S-Fpg seems to induce more compact conformations, possibly stabilized by inter-residue π-π interactions and/or by γ-turns. This behavior is also supported by J NH−CHβ and J 2,3 coupling constants obtained by 1 H NMR experiments. Indeed, larger J values were obtained for both 7 and 11, compared to 8 and 12, suggesting a more folded conformation for these latter peptides (Li et al., 2015). To provide additional evidences of this behavior, we compared the frequency of the radius of gyrations (RoG) sampled in the last 200 ns of simulation (Figure 9). For both tetra-and hexapeptides, it can be observed that a larger RoG is sampled more frequently for peptides containing β-2R,3R-Fpg, compared to peptides containing β-2S,3S-Fpg. Figure 9 also shows that two peaks are found for m11, centered at 6.5 and 8.0 Å. This is coherent with the results of the clustering analysis, which evidence the possibility of a "bending" of the extended geometry at the Ala3/β4 level (Figure 8). m7 instead shows a "spiked" peak centered at 5.0 Å, corresponding to the representative geometry shown in Figure 7A. The two "spikes" at 4.8 and 5.4 Å correspond to the breaking of the N-terminal γ-turn (involving the acetyl cap) and to the forming of the additional γ-turn (involving β-2 and β-4), respectively. Conversely, both m8 and m12 show a main peak (sharper, for m8) that is centered onto a RoG value that is significantly lower to that computed for the corresponding peptides containing β-2R,3R-Fpg.

CONCLUSIONS
Taken together these data, we can conclude that the architecture of the above peptides depends on the balance between the features of the selected amino acids, i.e., the syn β-amino acid, favoring an extended conformation, and alanine, inducing turns. The chirality of the two stereogenic centers of β-amino acid remarkably affects the peptide 3D-structures. A preferred extended conformation was found matching the β-R,R-amino acid with S-alanine. This is confirmed by J values of both tetra-and hexapeptides containing β-2R,3R-Fpg as well as by computational analysis. The extended conformation of this peptide induces the formation of an antiparallel β-sheet, also at low concentration, as documented by NMR data of 11. Our hypothesis is that the formation of this pleated sheet is favored by π,π-interactions of aromatic moieties and is stabilized by H-bonds. IR analysis of this peptide indicates the involvement of NHs in H-bonds, confirmed by the titration experiment (NH-1 and NH-6 involvement). The NH-network proposed by computational experiments does not fit with NMR data. On the other hand, computational data are consistent with a 11 single strand and not with pleated sheet architecture. In this case the formation of H-bonds is driven by γturn formation. β-S,S-amino acid with S-alanine gives more compact folded structures that are probably driven by alanine moieties. In fact, peptides containing β-2S,3S-Fpg display a higher stability and a larger content of an ordered secondary structure as compared to the peptides containing β-2R,3R-Fpg. Both NMR and computational data support the formation of a turn for 8 stabilized by π-π interactions between Ph β2 and Ar Fβ4 . IR absorption analysis and experiments at variable temperature confirm that the longer oligomer 12 is stabilized by intramolecular C=O···H-N bonds. On the other hand, J values indicate that an equilibrium occurred between multiple compact conformations.

AUTHOR CONTRIBUTIONS
MG conceived the research. RB and SP carried out change with RB, EB, and SP carried out the synthesis. MG, FC, and FF carried out the spectroscopic analyses. AC and IM carried out the computational studies. MG and AC supervised the work. MG, AC, and RB wrote the paper. All the authors revised the manuscript.