β-Turn Induction by a Diastereopure Azepane-Derived Quaternary Amino Acid

β-Turns are one of the most common secondary structures found in proteins. In the interest of developing novel β-turn inducers, a diastereopure azepane-derived quaternary amino acid has been incorporated into a library of simplified tetrapeptide models in order to assess the effect of the azepane position and peptide sequence on the stabilization of β-turns. The conformational analysis of these peptides by molecular modeling, NMR spectroscopy, and X-ray crystallography showed that this azepane amino acid is an effective β-turn inducer when incorporated at the i + 1 position. Moreover, the analysis of the supramolecular self-assembly of one of the β-turn-containing peptide models in the solid state reveals that it forms a supramolecular helical arrangement while maintaining the β-turn structure. The results here presented provide the basis for the use of this azepane quaternary amino acid as a strong β-turn inducer in the search for novel peptide-based bioactive molecules, catalysts, and biomaterials.


■ INTRODUCTION
Protein−protein and peptide−protein interactions mediate essential cellular processes, including antigen−antibody interactions, cell signaling networks, or programmed cell death. 1 Misregulation of these interactions is often implicated in disease states, so they constitute promising therapeutic targets via either their inhibition or stabilization. 2 Despite the large and often flat contact surfaces implicated in these interactions, a few key residues with a defined secondary structure often contribute to the majority of the binding affinity. 3,4These interactions can therefore be modulated by small-molecule mimetics or stabilizers of such secondary structure elements, as the conformation and binding capabilities of the native sequences are not retained when they lack the structural reinforcement provided by the protein environment. 5Among common elements of secondary structure found in proteins, the β-turn accounts for more than 20% of protein residues. 6,7It reverses the peptide chain direction, facilitating the formation of globular structures. 8In addition to its key role in protein folding and the stabilization of tertiary structures, β-turns are often implicated in molecular recognition. 9For example, octreotide is a synthetic octapeptide mimicking natural somatostatin and is used for the treatment of carcinoid syndrome and neuroendocrine tumors (Figure 1a). 10,11It possesses enhanced biological affinity and metabolic stability due to the β-turn stabilization provided by substitution of L-Trp by D-Trp. 12Another relevant example is the type II′ β-turn arrangement adopted by the cyclic pentapeptide c(RGDfV), which is essential for the inhibition of integrins implicated in angiogenesis and metastasis. 13A cyclic, conformationally restricted analogue of α-melanocyte-stimulating hormone (α-MSH) displaying a β-turn conformation is four orders of magnitude more active than the native hormone. 14Interestingly, the β-turn structure was found to be crucial for other applications apart from medicinal chemistry, such as peptide-based catalysis and self-assembly.Figure 1b shows an example of a β-turn-containing short peptide able to catalyze the atroposelective bromination of pharmaceutically relevant quinazolinones with high levels of enantioinduction. 15,16−21 As an example, a tetrapeptide model containing Leu, Aib (αaminoisobutyric acid), and Ser adopts a type-III′ β-turn, which self-assembles to form a supramolecular nanotube in the solid state (Figure 1c). 19he role of β-turns in the regulation of key biological processes and as useful scaffolds in peptide-based catalysts and biomaterials has prompted the development of strategies to mimic or stabilize such peptide secondary structure over the last decades.−35 Nevertheless, the most common strategy for the stabilization of β-turns is the incorporation of constrained amino acids into short peptide sequences. 36,37−52 However, in most cases, the constrained residue is used in combination with other restricted amino acids such as proline or Aib.
We have previously described the stereoselective synthesis of a quaternary azepane amino acid from commercial ornithine derivatives (Scheme 1). 53The key intermediate is the diastereopure β-lactam II synthesized via stereoselective intramolecular cyclization of the corresponding chloropropionyl derivative I.The spontaneous intramolecular β-lactam ring opening reaction, after deprotection of the ornithine side chain, leads to the formation of the 2-oxoazepane amino acid III.Selective reduction of the 2-oxoazepane ring yields diastereomerically pure (3R,4S)-4-amino-4-carboxy-3-methylazepane (Aze) 1.This quaternary amino acid displays a set of properties that make it especially suited for its application as peptide secondary structure inducer.There are not many studies on the ability of seven-membered quaternary amino acids to constrain peptide conformations on their own, i.e., without the synergistic effect of additional restricted residues. 48,49,54,55The azepane heterocycle in 1 also enables the incorporation of structural diversity and functionality in the constrained peptides through the Alloc-protected nitrogen, for example, fluorescent dyes or affinity labels for visualization and immobilization.In addition, it contains two chiral centers, which can be easily defined in the synthesis step opening the possibility of studying the effect of chirality in the induction of secondary structure elements.In fact, we have reported the synthesis of the four possible diastereoisomers of the 2oxoazepane derivative III. 53In a previous report, we have also demonstrated that the azepane 1 is an effective inducer of 3 10 helices in short alanine-based pentapeptide models. 56In this article, we report the induction of single β-turns by this quaternary amino acid in the shortest possible peptide model.We present a study of the key factors governing the folding of the peptide chain into a β-turn, such as the position of the constrained amino acid in the sequence and the compatibility with proteinogenic amino acids in the remaining central position of the turn.

■ RESULTS AND DISCUSSION
The conformational preferences of tetrapeptide models incorporating the azepane amino acid 1 were first analyzed using molecular dynamics (MD) simulations with the Amber The Journal of Organic Chemistry 10 suite of programs. 57Simplified tetrapeptide systems were used in which the N-terminal amino acid (i) was replaced by an acetyl group and the C-terminal (i + 3) by a Nmethylamide.The β-turn induction was evaluated through its characteristic topographic parameters, i.e., H-bond distance CO i −NH i+3 and the backbone dihedral angles of the central residues.We first studied the influence of the position of the constrained amino acid within the central residues of alaninebased tetrapeptide models.−60 When the azepane residue was incorporated at the i + 1 position, molecular modeling results showed that the 100% of the conformers within a 3 kcal•mol −1 window from the global minimum stabilize type I or III βturns.At the i + 2 position, the global minimum also displays a β-turn, but there is another family of conformers within a 3 kcal•mol −1 window in which the characteristic β-turn H-bond is not present (see the Supporting Information for details).Thus, MD studies suggest that the incorporation of 1 at the i + 1 position is preferred over that at the i + 2 position, with a higher degree of β-turn induction.This is in agreement with the preferred position for other constrained amino acids, as the restriction in the dihedral angles imposed by these residues at such position facilitates the β-turn nucleation. 61,62ith the azepane fixed at the i + 1 position, we next evaluated the effect of the amino acid side chain at position i + 2, again using MD simulations.Calculations were performed in tetrapeptide models incorporating the azepane residue at the i + 1 position and the 20 proteinogenic amino acids at the i + 2 position.MD studies suggested that the azepane derivative is a strong β-turn inducer, except when it is combined with Gln, Arg, and Asp at the i + 2 position.In these cases, less than 50% of the conformers within a 3 kcal•mol −1 window from the global minimum display a β-turn structure.The presence of polar residues tends to destabilize the β-turn due to competing H-bond formation between the side chain and the peptide backbone (see the Supporting Information for details).Taking these results into account, we selected a representative set of amino acids to prepare a library of Boc-Aze(Alloc)-Xaa-NHMe tetrapeptide models, where Xaa was chosen to be alanine, valine, leucine, or phenylalanine (residues with a high tendency to adopt β-turns) as well as serine, lysine, or glycine (with a medium tendency toward β-turns).This small library covers from hydrophobic aliphatic and aromatic residues to polar ones, displaying different side chain branching patterns.Additionally, we also prepared the corresponding alaninebased tetrapeptide model Boc-Ala-Aze(Alloc)-NHMe to experimentally assess the influence of the azepane position in the reverse turn induction.
Scheme 2 shows the synthetic pathway for the preparation of the designed small peptide library via classical solution peptide synthesis.Compounds 2-8 were obtained through peptide coupling between the azepane residue 1 56 and H-Xaa-NHMe, with Xaa being Gly, Ala, Val, Leu, Phe, Ser(Bn) and Lys(Z).Alternatively, peptide coupling between azepane 1 and MeNH 2 yielded derivative 9 in a good yield.The removal of the Boc group followed by amide coupling with Boc-Ala-OH provided dipeptide 10.
The conformational preferences in solution of tetrapeptide models 2−8 and 10 were evaluated by variable-temperature NMR experiments.β-Turns are usually stabilized by an intramolecular H-bond between the carbonyl oxygen of residue i and the NH proton of residue i + 3. The existence of this Hbond in compounds 2−8 and 10 was assessed by the variation of the amide proton chemical shift with temperature.In DMSO-d 6 , it has been established that the values of temperature coefficients (Δδ/ΔT) for amide protons equal to or lower than 3 ppb•K −1 (in absolute value) are indicative of such NHs participating in an intramolecular H-bond. 63,64In contrast, solvent-exposed amide protons typically display values over 4 ppb•K −1 , while values within 3−4 ppb•K −1 are not conclusive.Figure 2 shows the amide proton region of the 1 H NMR spectra for compound 2 at different temperatures, as well as the plot of the chemical shift variation with temperature (Δδ/ΔT).The observed linear correlation was used to calculate the temperature coefficient for all the amide protons.For compound 2, only NH i+3 has a temperature coefficient compatible with the presence of an intramolecular H-bond, suggesting the adoption of a β-turn in solution.It is worth pointing out that the splitting of the NH signal corresponding to the i + 1 residue at the lowest temperature is likely due to rotamers of the N-Alloc group, which coalesce when the temperature increases.
Following the same protocol as that for compound 2, the temperature coefficients for all the amide protons in derivatives 3−8 and 10 were extracted from VT-NMR experiments (Table 1 and Figures S2−S9 The Journal of Organic Chemistry the NH i+2 . 65Thus, this result demonstrates that branching at the β-position of the side chain seems to destabilize the β-turn conformation.
For compound 10 with the azepane residue at the i + 2 position, both NH i+2 and NH i+3 have temperature coefficients in the uncertainty range, so there is no strong evidence of the formation of either a γor β-turn.Comparing the NMR data of 3 and 10, the incorporation of the constrained amino acid at the i + 1 position seems to be more favorable for the stabilization of the β-turn, in line with the data obtained from the molecular modeling studies.
To gain further insight into the 3D structures of these tetrapeptide models, we attempted the crystallization of all of the prepared peptides.We obtained suitable crystals for X-ray diffraction for compounds 2 and 3. Figure 3a and b shows the X-ray structure of a representative molecule from the asymmetric unit.In both compounds, there is an intramolecular H-bond between the amide group of the Nmethylamide moiety (residue i + 3) and the Boc carbonyl oxygen (residue i), characteristic of a β-turn.Both β-turn structures are quite similar as observed when the two crystal structures are superimposed (Figure 3c).Deeper analysis of the asymmetric unit for both crystals allowed us to extract the topographic parameters of the observed β-turns.In particular, we measured the backbone dihedral angles of the central residues for all of the conformers (Table 2).In all cases, they are consistent with the formation of a type I β-turn.The solidstate structures of 2 and 3 are therefore in agreement with the NMR and molecular modeling data.These results confirm the ability of the azepane residue to induce β-turns in short peptides.
The growing interest in peptide-based supramolecular materials encouraged us to analyze the molecular packing of the obtained crystal structures.For the glycine analogue 2, the  The Journal of Organic Chemistry crystal belongs to the P2 1 space group, with a dimeric columnar structure stabilized by a bifurcated intermolecular Hbond between CO i+1 and both the NH i+1 and NH i+2 of different molecules (see the Supporting Information for details).In contrast, the supramolecular arrangement observed in the crystal structure of peptide derivative 3 belongs to the tetragonal system.It forms supramolecular right-handed helical assemblies with small internal pores (Figure 4, top).−20 In this case, it is composed by four dimers stabilized by H-bonds between the CO (Alloc) and CO i+1 of molecule A and the NH i+1 and NH i+2 of molecule B, respectively (Figure 4, bottom).In addition, the structure is stabilized by a set of interdimer H-bonds.For example, in the case of dimers A1-B1, the molecule A1 participates in one Hbond between the CO i+2 and the NH i+2 of A2, a second Hbond between the NH i+2 and the CO i+2 of A4, and a third Hbond between the NH i+1 and the CO i+2 of molecule B4.In addition, there is an interdimer H-bond between the CO i+2 of molecule B1 and NH i+1 of A2.Hydrophobic interactions drive the stacking of the allyl groups and packing between adjacent helices.The analysis of the crystal packing for 2 and 3 suggests that subtle changes in the molecular structure led to significant differences in the observed supramolecular assemblies in the solid state.Considering that both compounds display a nearly identical β-turn structure in the solid state and that both crystals were grown in the same solvent under the same conditions, the changes in the intermolecular H-bond pattern are likely to be determined by the replacement of one hydrogen for a methyl group.The fact that the self-assembly properties of these peptide models could be modulated by only varying the side chain substituents opens the way to expand the present study and determine the key structural features driving the self-assembly of β-turn containing peptides.Understanding the relationship between chemical structure and self-assembly in these models could provide hints not only to study how more complex proteins assemble but also to design novel peptide-based materials for drug delivery, tissue engineering or catalysis. 72

■ CONCLUSIONS
The stabilization of the peptide secondary structure elements is of interest to achieve a fundamental understanding of complex protein−protein interactions in the search of novel therapeutic agents.In particular, β-turns are some of the most common secondary structures found in proteins.They play a key role in protein folding and stabilization as well as in protein−protein and peptide−protein recognition.Moreover, they are an attractive scaffold with relevant applications in peptide-based catalysis and in supramolecular materials.The use of constrained quaternary amino acids is one of the most common strategies used to induce reverse turns in short peptide sequences.In this context, we have previously reported the stereoselective synthesis of a novel azepane-derived quaternary amino acid. 53Here, we present a systematic study of its ability to stabilize β-turns in the shortest possible peptide models.The effect of the position of the azepane and peptide sequence has been assessed using molecular modeling and NMR and X-ray crystallography.We have demonstrated that the azepane amino acid is an effective β-turn inducer when incorporated preferentially at the i + 1 position of tetrapeptide models.The same effect was observed with other amino acids at the i + 2 position, such as alanine, leucine, and serine.Only branching at the β-position of the i + 2 side chain (valine)  The Journal of Organic Chemistry seems to largely disrupt the β-turn.We have also paid attention to the supramolecular self-assembly behavior of two β-turncontaining peptide models in the solid state.When alanine is incorporated at the i + 2 residue, the tetrapeptide model selfassembles into a supramolecular right-handed helical structure, stabilized by a defined network of intermolecular H-bonds along with the characteristic β-turn intramolecular H-bond.The results here presented therefore provide the basis for the use of azepane quaternary amino acids as effective β-turn inducers with widespread potential applications in the development of novel bioactive molecules, catalysts, and biomaterials.
Compound 3 [Boc-Aze(Alloc)-Ala-NHMe]. Pure compound 3 (5 mg) was dissolved in MeOH (5 mL) and the mixture was put in a crystallizing dish, resulting in spontaneous crystallization after 20 days at 4 °C in a closed jar.X-ray diffraction was performed in a Bruker MicroStar 2.7 kW, with a four-circle goniometer, with κ-geometry, and Bruker CCD detector, using CuKα radiation.

Data Availability Statement
The data underlying this study are available in the published article and its Supporting Information.

* sı Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.joc.3c01689.General experimental details; molecular modeling methods; synthesis and characterization of compounds, including copies of 1 H, 13 C and 2D NMR spectra; VT NMR experimental details; and X-ray crystallography data for compounds 2 and 3 (PDF) Accession Codes CCDC 2281546 and 2281591 contain the supplementary crystallographic data for this paper.These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.