{'fraction_non_alphanumeric': 0.048314259201979584, 'fraction_numerical': 0.017816269718527684, 'mean_word_length': 4.874273255813954, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Bioinspired Reductionistic Peptide Engineering for Exceptional Mechanical Properties

A simple solution-processing and self-assembly approach that exploits the synergistic interactions between multiple hydrogen bonded networks and aromatic interactions was utilized to synthesize molecular crystals of cyclic dipeptides (CDPs), whose molecular weights (~0.2 kDa) are nearly three orders of magnitude smaller than that of natural structural proteins (50–300 kDa). Mechanical properties of these materials, measured using the nanoindentation technique, indicate that the stiffness and strength are comparable and sometimes better than those of natural fibres. The measured mechanical responses were rationalized by recourse to the crystallographic structural analysis and intermolecular interactions in the self-assembled single crystals. With this work we highlight the significance of developing small molecule based bioinspired design strategies to emulate biomechanical properties. A particular advantage of the successfully demonstrated reductionistic strategy of the present work is its amenability for realistic industrial scale manufacturing of designer biomaterials with desired mechanical properties.

Naturally occurring biological materials and structures in general are highly optimized to serve multifunctional purposes while being lightweight 1-3 . A key difference between these and man-made high performance structures are that the former are synthesized through 'bottom-up' approach using the combination of solvo-chemical and self-assembly routes [4][5][6] . Further, their remarkable mechanical performance is due to the ingenious use of weak noncovalent interactions [7][8][9] . Specifically, the mechanical properties of natural materials like cocoon and spider silks, and amyloid fibers can all be attributed to the exploitation of hydrogen bonding interactions. For example, theoretically predicted elastic modulus, E, of hydrogen bonded assemblies is ~10 GPa, which is effectively achieved in spider dragline silks 8 . It is in this context, we envision that it may be possible to synthesize biomolecular materials with desired mechanical properties by exploiting the crystal engineering principles. For this, we employ the simplest forms of peptide, viz. CDPs, which can be tailored into hydrogen bonded one-dimensional (1D) molecular chains or two-dimensional (2D) molecular layers depending on the nature of the side chains [10][11][12][13][14][15][16] . Such materials offer the following important advantages: (i) rigid structural and self-complementary (multiple hydrogen bonding) motifs, (ii) functional tailorability through the introduction of α -substituents to impart additional functionalities or noncovalent interactions, (iii) configurational variability, (iv) biocompatibility, (v) solution processability that entails easy synthesis and scalability.
In the current work, we employ CDPs of alanine (LL-Ala and LD-Ala) to exploit multiple hydrogen bonding interactions. Further, CDPs of an unnatural amino acid phenylglycine (LL-Phg and LD-Phg), with aromatic substituents were utilized to introduce additional noncovalent (aromatic) interactions ( Fig. 1a-d) [17][18][19][20] . Here, LL and LD represent the stereochemistry of the two amino acids in CDP. This allows control over the spatial orientation of the amino acid side chain (α -substituent) and in turn modulation of the self-assembly of CDPs. It is important to note that the molecular weights of the CDPs utilized in the current study are only about 0.2 kDa, whereas the proteins in natural biomaterials such as silk and amyloid fibers are of few tens to few hundreds of kDa 21 . Such a reductionistic strategy was intentionally employed so as to simplify the molecular material's design, which in turn would pave way for realistic industrial scale manufacturing, if the designed materials are found to have the desired properties.

Results
Single-crystals of CDPs were obtained by self-assembly process in a solvent mixture comprising 50:50 (v/v) ratios of dichloromethane and methanol. LL-Ala and LD-Ala were obtained in the form of thin single-crystalline sheets that are 100 to 500 μ m thick with 1 to 5 mm width and > 10 mm length (Fig. 1e,f). LL-Phg was also obtained as single-crystal sheets with lateral dimensions of ~1 mm (Fig. 1g). LD-Phg formed rhombohedral shaped crystals that are several mm in size (Fig. 1h). All of these were subjected to single-crystal X-ray diffraction (XRD) studies in order to understand the molecular organization in them. LL-Ala sheets crystallized in the triclinic P1 space group with the cyclic ring possessing non-planar conformation. Each LL-Ala molecule comprises two hydrogen bond donor (N-H) and acceptor (C = O) functionalities on either side of the CDP ring, which facilitate the formation of strong N-H···O hydrogen bond dimers with the two neighbouring molecules. These linkages, in turn, result in a 1D molecular chain that extends over the crystallographic a axis (Fig. 1i). The molecular organization in the LD-Ala crystals (monoclinic, P21/n space group), which were obtained by incorporating only one modification in the stereochemistry of the amino acid side chain of LL-Ala, are significantly different with a nearly-planar cyclic ring with the methyl functionalities on its either sides (Fig. 1b). This stereochemical modification facilitates the formation of four strong N-H···O hydrogen bonds with the neighbouring molecules for each LD-Ala molecule, which in turn results in the formation of 2D molecular layers as shown in Fig. 1j.
In the crystals of LL-Phg that crystallize into triclinic P1 space group, each molecule binds to two neighboring molecules by means of strong N-H···O hydrogen bond dimers, which ultimately results in the formation of 1D molecular chains along the crystallographic a axis (Fig. 1k). In addition, the phenyl functionalities of LL-Phg were involved in π -π as well as CH-π interactions with the phenyl functionalities of neighbouring molecules along the crystallographic a axis (Supplementary Figure 1). LD-Phg was found to crystallize in orthorhombic Pbca space group to form rhombohedral architectures. Herein, each LD-Phg facilitates strong N-H···O hydrogen bonds as well as CH-π interactions with the four neighbouring molecules, which result in 2D molecular layers ( Fig. 1l and Supplementary Figure  1). In summary, the LL stereochemistry of LL-Ala and LL-Phg favour the formation of 1D molecular chains, while the LD stereochemistry favours 2D molecular layers, as in LD-Ala and LD-Phg. In addition to the above discussed strong N-H···O hydrogen bonds, all four CDPs comprises of relatively weaker C-H···O interactions ( Fig. 2 Table 4). LL-Ala showed the presence of 2D network of C-H···O interactions along the ac plane, while LD-Ala showed the presence of 1D chains ( Fig. 2a-d).

and Supplementary
On the other hand, both LL-Phg and LD-Phg possess 2D networks of C-H···O interactions as shown in Fig. 2e-h. Therefore, with respect to C-H···O interactions, it is found that only LD-Ala possesses 1D chains whereas LL-Ala, LL-Phg and LD-Phg comprises of 2D networks.
Nanoindentation, which has been successfully employed to measure mechanical properties of organic and metal-organic framework crystals in the recent past, was utilized to evaluate the mechanical properties of the synthesized LL-Ala, LD-Ala, LL-Phg and LD-Phg crystals [22][23][24][25][26][27][28][29][30][31]    probed 23 . In the current context, they were ~4.10 nm, which is about five times d 010 (= 7.69 Å), suggesting that the pop-ins occur due to collective sliding of multiple (010) planes during indentation. Pop-ins were also noted on the loading segments of the P-h curves obtained on LL-Phg, but their number was much smaller. In this case, the average h pop-in was ~4.75 nm, which again is close to five times the d 010 (= 9.77 Å).
The P-h curves were analysed using the Oliver-Pharr method to extract elastic modulus, E, and hardness, H, of the crystals, which are listed in Table 1 23 . It is seen that the crystals with 2D hydrogen bond network (LD-Ala and LD-Phg) have far superior mechanical properties as compared to those with 1D hydrogen bonded chains (LL-Ala and LL-Phg). Amongst the four materials examined, LD-Phg is mechanically the most robust, with highest values of E and H. Notably, it is nearly-ten times stiffer and five times harder than LL-Phg. Likewise, E and H of LD-Ala are both more than double the respective values of LL-Ala. The measured E value of LD-Ala, which only contains hydrogen bonded assemblies, is in the range of 10-20 GPa predicted for such structures 8 . The synergistic interactions between hydrogen bonded networks and the aromatic interactions in LD-Phg lead to doubling of E in it vis-á-vis that of LD-Ala.
Structural reasons for such large differences in mechanical responses of the CDPs were sought through the examination of crystal packings (Fig. 4). In case of LL-Ala, strong N-H···O [D = distance between N and O in N-H···O; d = distance between H and O in N-H···O; θ = bond angle of N-H···O: 2.89 Å; 1.89 Å; 170°] hydrogen bonded 1D chains run parallel to the (010) indentation plane (Fig. 4b). These chains, which are aligned perpendicular to the indentation axis, are interlinked to each other by weak van der Waals interactions along the (010) plane (Fig. 4c). In contrast, LD-Ala comprises of strong N-H···O (2.88 Å; 1.93 Å; 157°) hydrogen bonded 2D layers that are oriented parallel to the indentation direction as shown in Fig. 4d,e. Herein, it should also be noted that LL-Ala and LD-Ala consists of 2D network and1D chains of C-H···O interactions, respectively. Thus, the interlocked and corrugated 2D molecular packing of strong N-H···O interactions in LD-Ala imparts structural rigidity to the crystal, which in turn manifests in terms of high E and H. In comparison, LL-Ala, which consists of only stacks of 1D chains of N-H···O interactions, is relatively compliant and softer. Therefore, for higher E and H, it is the nature and the strength of N-H···O interactions which contributes greatly over that of weaker C-H···O interactions, as observed in case of LL-Ala and LD-Ala. While the crystal structure of LL-Phg also contains strong N-H···O (2.88 Å; 1.87 Å; 172°) hydrogen bonded 1D chains (Fig. 4f,g), they facilitate additional π -π and C-H···π interactions that are oblique to the indentation direction. Similarly, the (11-1) indentation face of LD-Phg encompasses strong N-H···O (2.95 Å; 2.00 Å; 150°) hydrogen bonded 2D networks that are oriented skew to both the indentation plane and the direction of indentation (Fig. 4h,i). The presence of strong hydrogen bonded 2D networks as well as additional synergistic contributions from intermolecular C-H···π interactions make LD-Phg stiffer and stronger than the alanine derivatives (LL-Ala and LD-Ala).
The relatively lower H of LL-Phg, vis-á-vis LD-Phg can be ascribed to the presence of molecular slip planes along a-axis that shear slide relatively easily and smoothly during indentation. Thus, LD-Phg and LL-Phg respectively represent the synergistic and non-synergistic contributions of additional aromatic interactions that modulate E and H of hydrogen bonded organic materials. Note that both LL-Ala and LL-Phg contain relatively stronger hydrogen bonds (θ = ~170°) in comparison to their LD counterparts (θ of LD-Ala = 157°, θ of LD-Phg = 150°). Yet, the LD derivatives exhibit superior mechanical properties as compared to their LL counterparts. This observation suggests that the mechanical behavior of these biomolecular materials depend strongly on the hydrogen bond networks as well as synergistic contributions from other noncovalent interactions (as in case of LD-Phg) and is not exclusively dependent on the strength of N-H···O interactions.

Discussion
The properties obtained on the bioorganic crystals synthesized in this work are put in perspective through a comparison of nanoindentation data available on various organic crystals (Supplementary Table 1). It indicates that LD-Phg is by far the stiffest and hardest organic crystal amongst those that have been examined hitherto (Fig. 5a). Further, its E is comparable in fact slightly higher than the E of 19 GPa that was estimated through computational studies for diphenylalanine based nanotubes 32 . Moreover, LD-Phg possesses very high yield strength, σ y (estimated using the relation σ y = H/3) of 388 MPa 33 . In a broader context, it is worth noting that LD-Phg's specific properties (ratios of E and σ y to density) are comparable to the respective values of structural metals (Supplementary Table 2) as it has low density of ~1.3 gcm −3 (Fig. 5b,c).
In conclusion, the work presented in this paper demonstrates that it is possible to design peptide-based organic materials that are as strong and stiff as some of the best known natural fibres. This bioinspired design strategy employs a reductionistic method and exploits the synergistic interactions between hydrogen bonded networks and aromatic interactions in the self-assembled molecular architectures. Further, the substantial differences in the mechanical responses of the different CDP crystals demonstrate that it is possible to design bioinspired organic materials with tuneable mechanical properties, on the basis of molecular crystal engineering principles. Additionally, such low-density and high-strength biomolecular materials offer the advantages of biocompatibility, solution processability and large-scalability, and thus are promising in the contexts of biomaterial applications.

Methods
Materials. All the solvents and reagents were obtained from Sigma-Aldrich and used as received unless otherwise mentioned.

NMR Spectroscopy, Mass Spectrometry (HRMS), and Elemental Analysis. 1 H and 13 C NMR
were recorded on a Bruker AV-400 spectrometer with chemical shifts reported as ppm (in CDCl 3 with tetramethylsilane as internal standard). High resolution mass spectra (HRMS) were obtained on Agilent Technologies 6538 UHD Accurate-Mass Q-TOF LC/MS spectrometer. Elemental analysis was carried out on ThermoScientific FLASH 2000 Organic Element Analyzer.  2 mmol) was added and the reaction mixture was stirred at ice temperature for 1 h and then at room temperature for 5 h. The reaction progress was monitored by thin layer chromatography (TLC). Reaction mixture was evaporated to dryness and extracted from dichloromethane, washed with water, dried over anhydrous sodium sulphate. The solvent was evaporated to obtain Fmoc-L-Ala-L-Ala-OMe in quantitative yield. The Fmoc-deprotection of Fmoc-L-Ala-L-Ala-OMe dipeptide in 15% piperidine/ dichloromethane resulted in intramolecular cyclization to give LL-Ala, which was filtered, washed with dichloromethane, methanol and the crude white solid material was further recrystallized to obtain  Synthesis of LD-Ala. 9-Fluorenylmethoxycarbonyl protected L-alanine (Fmoc-L-Ala-OH) and D-alanine methylester (D-Ala-OMe) were prepared by using standard protection protocols. Fmoc-L-Ala-OH (2.24 g, 7.2 mmol) dissolved in dichloromethane was added with D-Ala-OMe (1 g, 7.2 mmol), 1-ethyl-3-(3 -dimethylaminopropyl)carbodiimide (EDC.HCl, 1.65 g, 8.64 mmol) and 1-hydroxybenzotriazole (HOBt, 1.17 g, 8.64 mmol). The solution was maintained at ice cold temperature. Diisopropylethylamine (DIPEA, 3.26 g, 25.2 mmol) was added and the reaction mixture was stirred at ice temperature for 1 h and then at room temperature for 5 h. The reaction progress was monitored by thin layer chromatography (TLC). Reaction mixture was evaporated to dryness and extracted from dichloromethane, washed with water, dried over anhydrous sodium sulphate. The solvent was evaporated to obtain Fmoc-L-Ala-D-Ala-OMe in quantitative yield. The Fmoc-deprotection of Fmoc-L-Ala-D-Ala-OMe dipeptide in 15% piperidine/ dichloromethane resulted in intramolecular cyclization to give LD-Ala, which was filtered, washed with dichloromethane, methanol and the white solid material was further recrystallized to obtain LD-Ala. 1

Synthesis of LL-Phg & LD-Phg.
These were synthesized as per our earlier report 12 .