Synthesis, crystal structure, and Hirshfeld analysis of an ultrashort hybrid peptide

2.1 Materials and methods

Commercially available reagents and chemicals sourced from Merck and Spectrochem were utilized to synthesize the hybrid peptide. All solvents used in the reaction were obtained commercially and used directly without additional purification steps. IR spectra were recorded on Perkin-Elmer L120-00A spectrometer (ν_max in cm⁻¹) using KBr pellet. ¹H NMR and ¹³C NMR spectra were registered on a Bruker NMR spectrometer (400 MHz and 100 MHz, respectively) in CDCl₃.

2.2 Crystal structure determination

A single crystal suitable for X-ray diffraction was loaded on a Bruker D8 QUEST diffractometer configured with PHOTON 100 detector device, and the diffraction data were collected using a monochromatic Mo-target rotating-anode X-ray source and graphite monochromator (Mo-Kα, λ = 0.71073 Å) with the ω and φ scan technique. The crystal was refined as an inversion twin. The unit cell was determined using SMART, ²⁷ the diffraction data were integrated with Bruker SAINT System ²⁷ and the data were corrected for absorption using SADABS. ²⁷ The structure was solved using SHELXS 97 ²⁸ by direct method and was refined by full matrix least squares based on F² using SHELXL-2018/3. ²⁹ All non-hydrogen atoms were refined anisotropically. Most of the H atoms were included at calculated positions as riding atoms with C (sp²)–H distances of 0.95 Å and C(sp³)–H distances of 0.99 Å for CH₂ units and 0.98 Å for CH₃ units and others being located from respective Q picks. Some most disagreeable reflections were excluded from the refinement. ORTEP-plot and packing diagram were generated with ORTEP-3 for Windows. ³⁰ WinGX ³⁰ was used to prepare the material for publication. CCDC 2428616 contains Supplementary crystallographic data for this paper.

2.3 Hirshfeld surface analysis

The CRYSTAL EXPLORER 17.5 software program ^{31 , 32} was used to compute the Hirshfeld surfaces and the associated 2D fingerprint plots across the constituent ionic and molecular geometries. The properties such as normalized contact distance, shape index, curvedness, and fragment patch were mapped over the Hirshfeld surface and plotted with the appropriate colour scale. The 2D fingerprint plots were presented as d_e versus d_i.

3.1 Synthesis of hybrid peptide

The t-butyloxycarbonyl and methyl ester groups were used for amino and carboxyl protections, respectively. Couplings were mediated by dicyclohexylcarbodiimide/1-hydroxybenzotriazole (DCC/HOBT). All intermediates were characterized by thin layer chromatography on silica gel and used without further purification. Final peptide was purified by column chromatography using silica gel (100–200 mesh) as the stationary phase and an ethyl acetate and petroleum ether (60:40, v/v) mixture as the eluent. The reported peptide was fully characterized by FT IR, NMR and X-ray crystallography.

Boc-L-Pro-5-AIA-(OMe) ₂ (hybrid peptide), 1: Boc-L-Pro-OH (1.0 g, 4.7 mmol) was dissolved in DCM (15 ml). Free amine of 5-AIA-(OMe)₂ was extracted with ethyl acetate by treating the aqueous solution of 5-AIA-(OMe)₂.HCl (2.3 g, 9.3 mmol) with excess NaHCO₃, and added to the reaction mixture, followed by DCC (1.4 g, 7.0 mmol). The reaction mixture was stirred at room temperature for 1 day. The precipitated dicyclohexylurea (DCU) was filtered off and the filtrate was diluted with ethyl acetate. The organic layer was washed with excess of water, 1M HCl (3 × 30 ml), 1M Na₂CO₃ solution (3 × 30 ml), and again with water. The solvent was then dried over anhydrous Na₂SO₄ and evaporated in vacuo, giving a light yellow solid, which was purified by column chromatography using silica gel (100–200 mesh) as the stationary phase and an ethyl acetate and petroleum ether (60:40, v/v) mixture as the eluent.

Yield: 1.7 g (72 %). Mp = 148–150 °C; IR (KBr): 3,433, 3,295, 2,981, 1,729, 1,662, 1,561, 1,423 cm⁻¹; ¹H NMR 400 MHz (CDCl₃, δ ppm): 9.87 (5-AIA-NH, 1H, s); 8.32 (5-AIA-H, 3H, m); 4.51(C^αH of Pro, 1H, m); 3.91 (–OCH₃, 6H, s); 3.44 (Pro-H, 2H, m); 2.45 (Pro-H, 1H, m); 1.93 (Pro-H, 3H, m); 1.51 (Boc-CH₃s, 9H, s); ¹³C NMR 100 MHz (CDCl₃, δ ppm): 170.6, 165.9, 139.1, 131.2, 125.9, 125.8, 124.5, 81.2, 60.5, 52.4, 47.3, 28.4, 27.5, 24.6.

3.2 Single crystal X-ray and supramolecular assembly

The hybrid peptide 1 crystallizes by slow evaporation of its methanolic solution. The obtained colourless monoclinic crystals exhibit the space group C 2. Crystal data, data collection, and structure refinement details are summarized in Table 1. The asymmetric unit is composed of four peptide molecules and water. The L-Pro at the N-terminus generates a turn, which is evident from the backbone torsion angles in the solid state conformation (Figure 2). The molecular conformations are similar in all four molecules in the asymmetric unit, which is reflected from the backbone torsion angles with L-Pro. φ and Ψ values at L-Pro are ranging from −56.4(8)° to −78.4(6)° and 137.5(5)° to 148.3(5)° respectively (Figure 2, Table 2). L-Proline’s torsion angles are often visualized in the Ramachandran plot with φ value around −65° and Ψ clusters around 150° (β region), comparable with this hybrid peptide. Two peptide molecules are interlocked in antiparallel criss-cross fashion, through two N–H⋯O hydrogen bonding interaction, involving 5-AIA-NH⋯O=C-Βοc (N3–H3⋯O27 and N7–H7⋯O13; N1–H1⋯O20 and N5–H5⋯O6) with donor–acceptor distances ranging from 1.97 Å to 2.22 Å (Figure 2b, Table 3) forming a robust dimer. Here, proline-induced bent molecular conformation is responsible for a unique criss-cross twin formation. The dimeric assemblage is further strengthened by π−stacking in a parallel manner between the aromatic rings of 5-AIA with Cg–Cg distance 3.756(4) and 3.651(4) Å (Figure 3, Table S1). One water molecule holds two asymmetric units via hydrogen bonding with the ester carbonyl of 5-AIA (O29–H29⋯O3) with donor–acceptor distances of 2.14 Å (Table 3). In the self-assembly process, two termini of peptide molecule, i.e., Boc-CH₃ at N-terminus and ester-CH₃ at C-terminus are participate in C–H⋯π interaction with C–H⋯Cg distance ranging from 2.82 to 2.93 Å (Figure 4, Table S2). Moreover, many non-classical C–H⋯O interactions reinforce the supramolecular arrangement involving C–H of proline ring, aromatic ring, and Boc group with O-atom of amide carbonyl, ester group, and oxycarbonyl group (Table 3). As a result, self-assembly of the ultrashort hybrid peptide unveils a unique water-mediated supramolecular sheet architecture (Figure 5). Notably, in this supramolecular arrangement, 5-AIA plays a substantial role in π-stacking and water-mediated hydrogen bonding interaction.

Figure 2:

The single crystal stucture of the hybrid peptide 1. (a) ORTEP diagram of the single molecule of 1 drawn of 30 % probability, (b) two molecules are arranged in anti-parallel criss-cross pattern (H atoms are omitted for clarity) forming a robust dimer, and (c) asymmetric unit with ellipsoid drawn of 30 % probability consist of two dimers and a water molecule.

Figure 3:

Non-classical π−π interactions (H atoms are omitted for clarity), Cg2, Cg4, Cg6 and Cg8 are the centroids of C1–C6, C21–C26, C41–C46 and C61–C66 rings respectively.

Figure 4:

Non-classical C–H⋯π interactions, Cg4, Cg6 and Cg8 are the centroids of C21–C26, C41–C46 and C61–C66 rings respectively; Symmetry Codes: (ii) x, −1 + y, z; (vi) x, 1 + y, z.

Figure 5:

Water-mediated supramolecular sheet arrangement of the hybrid peptide 1.

3.3 Hirshfeld surface analysis

Hirshfeld surface (HS) analysis (d_norm, shape index, curvedness, and fragment patch) was conducted to explore the weak non-covalent intermolecular interactions inside the molecule. This experiment intimates the quantitative ratios of short contacts between atoms, indicating the potential for weak hydrogen bond intermolecular interactions and their location. Colour coding was used to determine intermolecular interactions. The red, white, and blue patches in d_norm indicate intermolecular interactions with distances less than, equal to, and greater than van der Waals radii (Figure 6a). From Figure 6a, it is observed that the molecule has strong interactions, which are evident by the obvious variation in red and blue patches in the picture.

Figure 6:

Hirshfeld surface of crystal mapped over (a) d_norm, (b) shape index, (c) curvedness, and (d) fragment patch.

Useful curvature factors like shape-index and curvedness provide further chemical insight into molecular shaping. Dark-blue borders in shape index (Figure 6b) with “bumps and hollows” belonging to blue and red, respectively, to show flatness of the surface, highlight a high degree of curvature. Large green areas that are generally flat and are bordered by dark blue margins are typically indicated by the curvedness (Figure 6c). The H-bond interaction is evident by the red spots located close to the O-atom of the carbonyl group of Boc-protection at the N-terminus and the H-atom of the amide –NH, which is also reflected in the 2D fingerprint plot (Figure 8b). In addition to hydrogen bonding interactions, HS analysis also reveal weak interactions involving phenyl rings and the aliphatic protons.

Additionally, 2D fingerprint (FP) plots were generated to emphasize the quantitative information regarding the types and nature of intermolecular interactions in the crystal packing (Figure 7). 2D FP plots were produced for each interatomic contact, as well as for all interactions. The reciprocal contact of each interatomic connection was also taken into account when calculating the individual interatomic interactions. The 2D FP plots for the hybrid peptide, displayed in Figure 7, demonstrate that H atom has the maximum interactions (78.1 %) with the other atoms outside the Hirshfeld surface, followed by the O-atom (14.6 %). Their corresponding d_norm plots are also highlighted with blue patches in Figure 7d and e.

Figure 7:

2D fingerprint plots of the hybrid peptide 1 showing (a) all interactions, (b) H-atom interaction with all other atoms outside the Hirshfeld surface, (c) O-atom interaction with all other atoms outside the Hirshfeld surface, (d) d_norm plot for 7b, and (e) d_norm plot for 7c.

From the atom-to-atom interaction study (Figure 8), it is found that H–H interaction has the largest contribution to the total Hirshfeld surface which is account for 56.5 % in the range of d_e + d_i ≈ 2.2 Å, followed by the O–H interaction (28.0 %) in the range of d_e + d_i ≈ 1.9 Å. The d_norm plot of O–H interaction is provided in Figure 8d with blue patches. Other weak interactions are given in Figure 9.

Figure 8:

2D fingerprint plots of the hybrid peptide 1 showing all major interactions (a) H–H interactions, (b) H–O interaction, (c) C–H interaction with all other atoms outside the Hirshfeld surface, (d) d_norm plot for 8b.

Figure 9:

2D fingerprint plots of the hybrid peptide 1 showing weak interactions.