Fibril formation through self-assembly of a simple glycine derivative and X-ray diffraction study.

N-(N-benzoyl glycinyl)-N,N′-dicyclohexylurea was synthesised by conjugating N-benzoyl glycine and dicyclohexylcarbodiimide (DCC) using triethylamine as base catalyst. A single crystal X-ray diffraction study reveals that the compound self-assembles into a supramolecular sheet structure by intermolecular N–H · · · O, C–H · · · O hydrogen bonding and non-bonding van der Waals interactions. A high resolution transmission electronic microscopic (HR-TEM) image of the compound exhibits formation of fibrils in the solid state.

Keywords: fibrils; glycine derivative; self-assembly; supramolecular sheet

In nature, various biological systems are the result of molecular self-assembly [[1]], [[2]], [[3]], [[4]]. Supramolecular assemblies are often stabilised by hydrogen bonding, hydrophobic, hydrophilic interactions, π–π stacking etc [[5]], [[6]]. The design and synthesis of suitable molecular building blocks that self-assemble into desired supramolecular achitectures is an active area of current research. Micro and nanoscale soft materials derived from amino acid based molecules are important for their versatile functionality. Construction of supramolecular sheet structures through self-assembly of small building blocks has attracted considerable attention for their diverse applications in the field of drug delivery, biosensing, 3D cell culture etc [[7]], [[8]], [[9]], [[10]]. Several examples are available, where small peptide molecules are acting as suitable molecular building blocks for β-sheet self-assembly [[11]], [[12]], [[13]], [[14]], [[15]]. Very few reports are available where supramolecular structures and morphological properties of small capped amino acid based molecules have been discussed. For example, m-aminobenzoic (non-coded amino acid) acid protected with Boc and N, N′-dicyclohexylurea was reported to adopt a supramolecular double helical motif in the solid state [[16]]. With these in mind, we wanted to explore the self-assembly and morphology of a small molecule derived from glycine. We have chosen glycine because it is a profuse biomonomer in nature as well as the smallest coded amino acid.

In this report, we prepared a compound, namely, N-(N-benzoyl glycinyl)-N, N′-dicyclohexylurea 1 (Figure 1) and examined the formation of its supramolecular motif. Here, the N-terminal of glycine is intentionally protected by a benzoyl group to get an extra benefit to find order and directionality in the self-assembly process. Again two cyclohexyl groups in the C-terminal of glycine may provide hydrophobic interactions to promote the desired supramolecular structure. The title compound was synthesised by conjugating N-benzoyl glycine and dicyclohexylcarbodiimide (DCC) using triethylamine as base catalyst. The solid state structure of compound 1 was determined by single crystal X-ray diffraction. High resolution transmission electronic microscopy (HR-TEM) has also been employed to examine the morphological properties of the molecule.

Graph: Fig. 1: Structure of compound 1.

PhCO-Gly-OH (1.0 g, 5.6 mmol) was dissolved in 10 mL DMF, followed by dicyclohexylcarbodiimide (2.3 g, 8.4 mmol) and two drops of triethylamine were added and the reaction mixture was stirred at room temperature for 12 h. The reaction mixture was filtered and to the filtrate 20 mL of ethylacetate was added. The organic layer was washed with 1 N HCl (3×30 mL), 1 M Na2CO3 solution (3×30 mL) and brine, respectively. The solvent was then dried over anhydrous Na2SO4 and evaporated in vacuo, giving a white solid. Yield: 3.8 g (88%). Purification was done using silica gel as stationary phase and an ethyl acetate-petroleum ether mixture as the eluent. Single crystals were grown from methanol by slow evaporation and were stable at room temperature. Mp=165–167°C; 1H NMR 500 MHz (DMSO-d6, δ ppm): 8.65 (GlyNH, 1H, t, J=5.5 Hz), 8.41 (Substituted urea NH, 1H, d, J=8 Hz), 7.87–7.46 (Phenyl ring protons, 5H, m), 4.08 (CαHs of Gly, 2H, d, J=5.5 Hz), 3.95 (1H, m), 3.50 (1H, m), 1.83–1.69 (8H, m), 1.57–1.47 (4H, m), 1.00–1.32 (8H, m); 13C NMR 75 MHz (DMSO-d6, δ ppm): 167.02, 166.90, 153.43, 134.49, 131.80, 128.76, 127.75, 53.89, 50.11, 42.13, 32.19, 30.71, 25.90, 25.59, 25.56, 24.85; DEPT-135: 42.13, 32.19, 30.71, 25.90, 25.60, 25.56, 24.85 (Negative).

A single crystal suitable for X-ray diffraction of 1 was loaded on a Bruker AXS D8 QEST ECO diffractometer and the diffraction data was collected using 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 unit cell was determined using SMART [[17]], the diffraction data was integrated with the Bruker SAINT system [[17]] and the data was corrected for absorption using SADABS [[17]]. The structure was solved using SHELXS 97 [[18]] by Direct Methods and was refined by full matrix least squares based on F2 using SHELXL-2018/3 [[19]]. All non-hydrogen atoms were refined anisotropically and the H atoms were included at calculated positions as riding atoms with C(sp2)–H distances of 0.93 Å and C(sp3)–H distances of 0.96 Å. Some low-angle reflections were excluded from the refinement as those were probably obscured by the beam stop. An ORTEP-plot and a packing diagram were generated with ORTEP-3 for Windows [[20]]. WinGX [[20]] was used to prepare the material for publication. CCDC 1906687 contains supplementary crystallographic data for this paper.

The morphology of our compound was investigated using high resolution transmission electron microscopy (HRTEM). A methanol solution of compound 1 (2 mM) was incubated over night at room temperature. TEM studies of the peptides were done using a small amount of the solution of the corresponding compounds on carbon coated copper grid (300 mesh) by slow evaporation and allowed to dry in vacuum at room temperature. An image was taken by JEOL JEM-2100.

Compound 1 crystallizes in the orthorhombic space group P 212121 with one molecule in crystallographic asymmetric unit (Figure 2, Table 1). The molecule adopts an extended conformation characterized by the backbone torsion angle ϕ=79.9(7) (C1–N1–C8–C9) and ψ=−161.9(5) (N1–C8–C9–N3) at Gly (Figure 2, Table 2). There is an intramolecular C–H···O interaction [C(11)–H(11)···O(2), 2.34 Å] between the H atom of the cyclohexyl moiety and the O atom of the CO group of glycine (Figure 3, Table 3). Both cyclohexyl units are in chair conformation and attached to the respective N atoms of the amide groups through their equatorial bond. These building blocks are self-assembled along the crystallographic a axis through two intermolecular H-bonds, one [N(1)–H(1)···O(3), 2.04 Å] between NH of glycine and C=O of the substituted urea part and another [N(2)–H(2)···O(1), 2.03 Å] between NH of the substituted urea part and C=O of the benzoyl group in head to tail fashion to form an antiparallel β-sheet assembly. There is also an intermolecular C–H···O interaction [C(7)–H(7)···O(3), 2.55 Å] between one H atom of the benzoyl group of one molecule and the carbonyl group of the substituted urea part of the neighbouring molecule (Figure 3, Table 3) to form pillars which are again stacked by van der Waals interactions along the crystallographic b and c axes resulting supramolecular β-sheet structure (Figure 4).

Graph: Fig. 2: ORTEP Diagram of compound 1 with thermal displacement ellipsoids shown at 25% probability.

Tab. 1: Crystal data collection and structure refinement for 1.

Tab. 2: Selected bond lengths (Å), bond angles (°) and torsion angles (°) for 1.

Graph: Fig. 3: (a) Molecular packing diagram and (b) Intermolecular and intramolecular hydrogen bonding of compound 1.

Tab. 3: Hydrogen bonded geometries in 1.

1 Symmetry code: (i) 1/2+x, 1/2−y, 1−z.

Graph: Fig. 4: (a) An antiparallel β-sheet assembly along the crystallographic a axis, (b) van der Waals interactions viewed along the crystallographic b axis, (c) A tentative model representing the supramolecular arrangement, (d) HR-TEM image, of compound 1.

Several reports show that sheet forming small peptide molecules promote fibrillation in the solid state [[11]], [[12]], [[13]], [[14]], [[15]]. In fact, nanofibers are very important for their versatile applications in life science, tissue engineering and industry. We became interested in exploring the possibility of formation of materials of specific morphology with compound 1. To understand the aggregation behaviour, high resolution transmission electronic microscopy (HR-TEM) was employed. The HR-TEM image revealed that, compound 1 is self-associated to form highly organised bunches of fibrills which were found to be 80–100 nm in width (Figure 4).

In conclusion, we have demonstrated the conformation and morphology of easily synthesized N-(N-benzoyl glycinyl)-N, N′-dicyclohexylurea. Single crystal XRD analysis reveals that the compound self-assembled through an anti-parallel β-sheet structure which was stabilised by intermolecular N–H···O, C–H···O hydrogen bonding and van der Waals interactions. Self association of this glycine-based molecule leading to β-sheet aggregation indicates the mimicry of many naturally occurring macromolecules. A HR-TEM image indicates a bunch of fibrils which is a result of the antiparallel β-sheet assemblage. We believe that this study may hold future promise for potential applications as new functional biomaterial as well as for understunding self-assembly processes towards tunable supramolecular arrays.

AD acknowledges laboratory facilities at R. B. C. Evening College, Naihati. PD is grateful to SERB (DST), India for a fellowship [No.TAR/2018/000228].