Studies of the β -sheet mediated self-assembly of designed synthetic peptides of general formula PhCO-Gly-Xx-OCH 2 Ph and the possible role of aromatic π - π interactions in the self-assembly

A set of three peptides 1 - 3 of general formula PhCO-Gly-Xx-OCH 2 Ph, where Xx is Gly in peptide 1 , Ala in 2 and Aib ( α -animo isobutyric acid) in 3 has been chosen to study the self-assembly and the morphology of the solid biomaterials. FT-IR and single crystal X-ray diffraction studies reveal that the peptides 1 - 3 self-assemble to form supramolecular β -sheet structures through intermolecular hydrogen bonds and aromatic π - π interactions. Field emission scanning electron micrographs (FE-SEM) of the dried materials of the peptides 1 - 3 show the formation of flat ribbon like structures which are formed through β -sheet mediated self-assembly.


Introduction
Generally peptides are efficient building blocks for producing nanoscopic materials through selfassembly. 1,2 esigned small peptides that self-assemble into β-sheet fibrils may find useful applications in tissue engineering and fabricating biomaterials. 3Under suitable condition selfaggregating β-sheet forming peptides can create gels, 4 and provide molecular scaffolds for growing neurons and cartilage.3a, 5 It has been established that β-sheet-driven aggregation of misfolded proteins is responsible for various neurodegenerative diseases such as Alzheimer's disease, 6 Parkinson's disease, 7 and prion related diseases. 8Therefore design and construction of β-sheets is important if a better understanding of the peptide self-aggregation mechanism to be gained.
It has been observed that aromatic π-π interactions play a crucial role in many areas of chemistry and biochemistry, most notably in molecular recognition and self-assembly. 9Aromatic π-π interactions are known to provide energetic contribution as well as order and directionality in the self-assembly of amyloid structures. 10Generally, short peptides with aromatic side chains are found to have the ability to form β-sheet mediated amyloid fibrils. 10,11 n this context, we chose a set of three peptides 1-3 of general formula PhCO-Gly-Xx-OCH 2 Ph where Xx is Gly in peptide 1, Ala in 2 and Aib (α -animo isobutyric acid) in 3 to examine the role of aromatic π-π interactions in self-assembly (Fig. 1).The incorporation of Gly is expected to provide extended and semi-extended conformations in the back-bone of the peptides.The investigation would establish whether the peptides 1-3 produce the same supramolecular structure and morphologically similar materials through their self-assembly or differ significantly.Peptides 1-3 were synthesized using conventional solution phase methodology and their solid state structures were determined by FT-IR and X-ray diffraction analysis.The morphological features of the biomaterials generated from the peptides 1-3 in the solid state were examined by field emission scanning electron microscopy (FE-SEM).

FT-IR study
The preliminary information about the peptide conformation was obtained from solid state FT-IR studies.In the solid state (KBr matrix) all the peptides 1-3 show intense bands at 3286-3368 cm -1 indicating the presence of strongly hydrogen bonded NH groups (Fig. 2).Important IR data for all the peptides are listed in Table 1.The absence of a band attributed to free NH (over 3430 cm -1 ) indicates that all the NH groups in peptides 1-3 are involved in intermolecular hydrogen bonding.The CO stretching band at around 1647-1656 cm -1 (amide I), and the NH bending peak near 1530-1552 cm -1 (amide II) suggest the presence of intermolecular hydrogen bonded supramolecular β-sheet-like conformations for all the peptides in the solid state (Fig. 2). 12The bands around 1736-1752 cm -1 are due to the CO stretching frequencies of free ester groups (-CO 2 CH 2 Ph).Therefore the solid-state FT-IR data supports the hypothesis that the peptides adopt the β-sheet-like structures in the solid state.In order to obtain detailed information about the intermolecular hydrogen bonding networks, aromatic π-π interactions and self-assembly mechanisms, single crystal X-ray diffraction studies were carried out.
Wavenumbers (cm -1 )   2).The incorporation of Gly  in the sequence helps to attain a fully extended conformation, which is necessary for β-sheet formation.In the solid state the molecules are connected via intermolecular hydrogen bond between Gly(1)-NH of one molecule with Ph-CO of another (N9---O8, 2.85 Ǻ) along c-axis to create an infinite strand-like structure where the molecules are arranged in anti-parallel fashion (Fig. 4a, Table 3).Several such strand like structures are self-assembled in b direction to form two dimensional (b, c axis) layers of β-sheet through zipper-like assembly stabilized by van der Waals interactions between the phenyl rings (Fig. 4a).Again the molecules of 1 are packed in a direction through intermolecular hydrogen bond between Gly(2)-NH of one molecule with Gly(1)-CO of another (N13---O12, 2.83 Ǻ) in parallel fashion (Fig. 4b, Table 3 ).The arrangement is further stabilized by aromatic π-π interactions between the two neighbouring phenyl rings of the benzoyl groups with an average distance of 4.57 Ǻ between the eclipsed C atoms. 13 Therefore the β-sheet layers (b, c axis) of peptide 1 are stacked one on top of the other in a direction to form laminated supramolecular β-sheet assembly in the solid state.
interactions to create laminated supramolecular β-sheet assembly in the solid state (Fig. 6b).The average distance between the eclipsed C atoms of two neighbouring phenyl rings of benzoyl groups is 4.81 Ǻ and that of benzyl groups is also 4.81 Ǻ.The colourless monoclinic crystals of peptide PhCO-Gly-Aib-OCH 2 Ph (3) were obtained from ethanol by slow evaporation.The space group is P2 1 /n with one molecule in the asymmetric unit.A diagram of peptide 3 is presented in Fig. 7.In contrast to peptide 1 and 2, peptide 3 adopts a bent structure.The incorporation of helicogenic Aib 14 creates a bent structure where the backbone torsion angles within Gly are φ 1 : -168.6 o and ψ 1 : 179.8 o and within Aib they are φ 2 : 54.0 o and ψ 2 : -155.2 o (Table 2).Two neighbouring molecules of peptide 3 are interlocked by two hydrogen bonds between Aib-NH and Ph-CO (N13---O8, 2.90 Å) to create molecular duplex (Fig. 8a, Table 3).The duplexes are further self-assembled through π-π interactions between the phenyl rings of benzyl groups to create layer (a, c axis) of β-sheets (Fig. 8b).The distances between the eclipsed C atoms of two neighbouring phenyl rings of benzyl groups are approximately 4.94 Ǻ.

Morphological studies
Recently several reports show that supramolecular β-sheet forming small peptides promote amyloid-like fibrillation in the solid state. 15Flat laminated ribbon-like materials have been fabricated through β-sheet mediated self-assembly of designed peptide incorporating Pro-Pro segment at the middle. 16In amyloidogenic peptide calcitonin ribbon-like structures twist back upon themselves to afford hollow tube-like assemblies. 17Various attempts to fabricate flat ribbon-like structures using amyloid proteins, peptides, and viruses have been reported. 18It is believed that the quasi-crystalline structures of flat non-twisted morphology may be useful for specific nano-applications.Therefore, we become interested in exploring the possibility of formation of materials of specific morphology with peptides 1-3.Field emission scanning electron microscopic (FE-SEM) images of the dried fibrous materials of peptides 1-3 grown slowly from CHCl 3 -petroleum ether mixture clearly demonstrate that the aggregates in the solid state are bunches of flat ribbon like structures (Fig. 9).Interestingly peptide 1 generates a net-like structure of ribbons.All peptides exhibit higher ordered self-assembly to create ribbon like materials.The hierarchical assembly of the peptides initially create layers of β-sheets.Subsequently the β-sheet layers stack one on top of the other to produce flat ribbon like structures.

Synthesis of peptides
The peptides 1-3 were synthesized by conventional solution phase methods. 19The benzoyl group was used for N-terminal protection, and the C-terminal was protected as a benzyl ester.Couplings were mediated by dicyclohexylcarbodiimide (DCC).All intermediates were characterized by thin layer chromatography on silica gel and used without further purification.Final peptides were purified by column chromatography using silica gel (100-200 mesh) as the stationary phase and ethyl acetate and petroleum ether mixture as the eluent.The peptides 1-3 were characterized by X-ray crystallography, NMR and IR.

Synthesis of the peptide 1, PhCO-Gly-Gly-OCH 2 Ph
PhCO-Gly-OH (2.0 g, 11.2 mmol) was dissolved in DMF (10 ml).Gly-OCH 2 Ph obtained from its p-toluenesulphonic acid salt (7.5 g, 22.3 mmol) was added to it, followed by DCC (3.45 g, 16.7 mmol).The reaction mixture was stirred at room temperature for 1 day.The precipitated dicyclohexylurea (DCU) was filtered and to the filtrate 20 ml of ethyl acetate was added.The organic layer was washed with 1 N HCl (3 x 30 mL), 1 M Na 2 CO 3 solution (3 x 30 mL) and water.The solvent was then dried over anhydrous Na 2 SO 4 and evaporated in vacuo, giving a white solid.Yield: 3.1 g (85.1 %).Purification was done using silica gel as stationary phase and ethyl acetate-petroleum ether mixture as the eluent.Single crystals were grown from chloroform and petroleum ether mixture (1:3)

Field emission scanning electron microscopic study
The morphologies of the fibrous materials of peptides 1-3 were investigated using field emission scanning electron microscopy (FE-SEM).For the FE-SEM study, fibrous materials of the peptides (grown slowly from chloroform-petroleum ether) were dried and platinum coated.The micrographs were taken in a FE-SEM apparatus (JEOL JSM-6700F).

Figure 4 .
Figure 4. (a) Packing diagram of peptide 1 showing the formation of two dimensional (b, c axis) layer of β-sheet through zipper-like self-assembly driven by intermolecular hydrogen bond (between N9--08) and van der Waals interactions; (b) Packing of peptide 1 in a direction through intermolecular hydrogen bond (between N13---O12) and π-π interactions.Intermolecular hydrogen bonds are shown as dotted lines.

Figure 6 .
Figure 6.(a) Packing diagram of peptide 2 showing the formation of two dimensional (a, c axis) layer of β-sheet through intermolecular hydrogen bond (between N9--08) and van der Waals interactions; (b) The molecules of peptide 2 are packed in b direction through intermolecular hydrogen bond (between N13---O12) and π-π interactions.Intermolecular hydrogen bonds are shown as dotted lines.

Figure 8 .
Figure 8.(a) Showing two molecules of peptide 3 are locked by intermolecular hydrogen bonds to create molecular duplex; (b) Packing diagram of peptide 3 showing the formation of two dimensional (a, c axis) layer of β-sheet through intermolecular hydrogen bonds and π-π interactions between the phenyl rings.Intermolecular hydrogen bonds are shown as dotted lines.

Peptide 1 2 3 Figure 9 .
PeptidePeptide The FE-SEM images of the fibrous materials of the peptides showing the formation of flat tape-like morphology.The fibrous materials of the peptides grown slowly from chloroformpetroleum ether are dried and platinum for imaging.