A Dynamic Helical Supramolecular Peptide Framework Displaying Enantioselective Guest Adsorption
The ability to use bio-inspired building-blocks in the assembly of novel supramolecular frameworks is at the forefront of this exciting research field. Herein, we present the first helical polyproline peptide to self-assemble to form a reversibly porous, crystalline, supramolecular peptide framework (SPF). This framework is assembled from a short oligoproline, adopting the polyproline II conformation, driven by hydrogen-bonding and -interactions. Thermal activation, guest-induced dynamic porosity and enantioselective guest inclusion have been demonstrated for this novel system. The principles of the selfassembly associated with this SPF will be used as a blueprint allowing for the further development of helical, proline-based peptide linkers in the rational design of SPFs and metal-peptide frameworks.
Introduction<!>Results and discussion<!>PP4-SPF Characterization<!><!>Please do not adjust margins<!>Guest encapsulation and thermal release
<p>Peptide based porous frameworks are an emerging class of materials, which have found applications as adaptive [1][2][3] and reversibly tuneable, 4 porous materials, capable of capturing greenhouse gases, [5][6][7] and, due to their inherent chirality, have found applications in facilitating chiral events and transformations, 8,9 and separating chiral drugs 8,10 . Peptides can be prepared at scale with high purity, have canonical and noncanonical amino acids incorporated into their primary structure with high accuracy and are biocompatible. As such, the efforts to investigate this class of compounds as an alternative to the bulky, aromatic and non-adaptable ligands in the creation of extended frameworks, has seen a surge in recent years. 8,[10][11][12][13][14] While efforts to post-functionalise metal-organic frameworks (MOFs) with helical peptides, 9,15 to induce chiral selectivity, and to synthesise a broad range of metal-peptide frameworks (MPFs) have seen considerable advancements, 8,10,11,13,16 it remains a challenge to develop peptide-based extended porous networks guided by supramolecular interactions (e.g. Hbonding, halogen bonding, π-interactions and host-guest interactions). [17][18][19] Hydrogen-bond based frameworks have found applications in gas separation and storage, [20][21][22] albeit to limited degree due to the inherent weak nature of the interaction and solvent guest dependent porous behaviour. Despite these drawbacks this class of porous materials has been gaining momentum during the past decade due to processability in common organic solvents and ease of crystal growth compared to MOFs or covalent-organic frameworks (COFs). 17,23 To the best of our knowledge, the use of cyclic peptides to yield porous channels in crystalline materials and the use of self-assembling hydrophobic dipeptides, are amongst the few examples of well characterised peptide-based frameworks (i.e. Supramolecular Peptide Frameworks; SPFs). 5,7,14,24 In recognition of the immense chemical space yet to be explored in this field, we set out to exploit peptides with stable secondary structures in the construction of SPFs. In particular, we focused our efforts on the use of polyproline helices as SPF building blocks due to the accessibility to rigid yet stimuli sensitive secondary structures. 25 Polyprolines can interconvert between two different secondary conformations, polyproline II with all trans amide bonds and polyproline I with all cis amide bonds. This interconversion can be controlled as a function of the environment it is exposed to (i.e. temperature, solvent polarity and pH). [26][27][28] Polyproline helices have shown remarkable resilience to a diverse range of functionalisation and, in contrast to other helices used in supramolecular chemistry, [29][30][31] can retain their conformation even in short sequences, highlighting the stability of these helices. 32 These properties make them promising candidates for the synthesis of novel porous SPFs. Herein, we report the first reversibly porous SPF formed via the self-assembly of a helical tetraproline peptide (Figure 1). This supramolecular peptide framework is formed through the selfassembly of a short helical tetraproline via hydrogen-bonding and π-interactions. This remarkably resilient SPF is capable of reversibly hosting guest molecules within the channels through</p><!><p>Wennemer's group reported the first crystal structure of a polyproline hexamer in the polyproline II conformation providing insight into the stability of the polyproline helix, 33 followed by Hanessian, who reported the crystal structure of the tetrameric proline congener (cis-4,5-methanoproline) in the polyproline II form. 34 Previously, a crystal structure of a tetrameric oligoproline was reported by Matsuzaki, with several of the proline units deviating from the typical polyproline II conformation. 35 However, reports have suggested that tetraproline can be found in the structured polyproline II helical conformation when in solution. 32,34 To the best of our knowledge, tetraproline is the shortest helical peptide (polyproline II helix) reported in the literature, therefore we decided to focus our initial efforts on this oligomer in the construction of structured SPFs. Analysis of the single crystal data in the literature for polyproline helices, allowed us to gain insight into the type of supramolecular interactions governing assembly. For example; in the case of an oligoproline hexamer a hydrogen bond interaction between the lone pair on the carbonyl oxygen of the second proline and the C terminus carboxylic acid (H---O distance of 2.05(7) Å) was present. 33 With this insight in hand, we anticipated that a terminal primary amide could act as a H-Donor (HD) to promote the formation of an extended network through H bonds with H-Acceptor (HA) groups (i.e. carbonyl groups). The N-terminus was capped with an Fmoc carbamate group to facilitate intramolecular assembly of peptides via πinteractions. With these design principles in mind, PP4 was successfully synthesized using solid phase peptide methodology on a 0.3 mmol scale and in a quantitative yield (SI 1). PP4 was characterized in solution with far-UV circular dichroism (CD) analysis of aqueous, ethanol, and propanol solutions (0.25 mM), showing that the peptide adopts a polyproline II helix conformation in all solvents (λmax at 223/229 nm and λmin at 202 nm; SI 5, Figure S6). High resolution mass spectrometry of PP4 also evidenced the presence of dimers (1277.601 m/z, [2M+Na] + ) (SI 2, Figure S2). Crystals of PP4 suitable for single crystal X-ray diffraction analysis were reproducibly obtained via slow cooling of a super-saturated hot solution of PP4 in ethanol (SI 7). The structure was solved and refined to an atomic resolution of 0.81 Å (Figure 1), revealing that the peptide adopts the polyproline II helical conformation. The characteristic dihedral angles and an analysis of the puckering of the pyrrolidine rings, have been summarized in Table S1 (SI 9.2). We found that pyramidalization of the amide carbonyls clearly indicated the presence of n-π* interactions and these interactions were more significant in prolines exhibiting the exo conformation with a larger degree of pyramidalization (SI 9.3, Table 2). 33,36</p><!><p>Analysis of the packing of PP4 showed an extended supramolecular peptide framework (PP4-SPF) with channels extending in one dimension through the network (Void volume 226.19 Å 3 , SI 9.4), which are occupied with disordered solvent molecules (i.e. ethanol; SI 9, Figure S8).</p><!><p>Please do not adjust margins</p><!><p>The formation of the extended framework is driven by a combination of hydrogen-bonding and π-interactions. The peptides are arranged in alternating anti-parallel rows, extending through the network, such that each protected Nterminus faces another Fmoc group (Figure 1d-f). Each row of peptides is offset from the next row such that the C-terminal amide can hydrogen bond with two other peptides. In particular, the amide group of each peptide interacts with different carbonyl groups on the two opposing peptides. The Cterminal amides hydrogen bond with the carbonyl group of Pro2 on peptide 1 and the Pro3 carbonyl on peptide 2 (Figure 2a, 2.1478(19) Å and 2.1930(18) Å respectively), therefore each peptide acts as both a HD and HA with another peptide, which then extends in one dimension. These 2D sheets are then stacked with weaker interlayer interactions in an alternating manner creating an extended network. The crystal structure suggests that interactions between the Fmoc groups determine the second aspect of the self-assembly process. The closest distance (2.7515(19) Å) between the Fmoc moieties is that of a proton of one fluorenyl to the aromatic region of another (SI 9, Figure S11), well within the possible distance to classify a potential interaction. [37][38][39] There is also a close contact (2.7933(15) Å) between a Cβ hydrogen, on Pro3 to the centroid of a neighbouring peptide's C24-29 aromatic ring in the Fluorenyl moiety (Figure S8, SI9), which is similar to the contact seen in Wennemer's crystal structure of the polyproline hexamer with a Pro5 Cγ hydrogen towards the p-bromobenzoyl aromatic ring (2.865(2) Å). 33 It is also worth noting that a reversable single crystal to single crystal transition in the unit cell of PP4-SPF (SI 9.1-9.3, PP4-SPFFlash) is observed, whereby the Pro3 puckering switches from exo to endo (SI 9.3, Table 1). This was achieved by flash freezing crystals at 150 K, remarkably, upon returning to room temperature the crystals readopt their initial unit cell and conformation. This was not found when slowly ramping the temperature down to 150 K, with only a slight reduction of the cell volume, but no significant conformational changes. Computational calculations at [B3LYP-D3(BJ)/6-311++G(d,p)] on an Fmoc associated pair (geometry fixed to that of the PP4-SPF crystal structure) showed orbital overlap between the groups (Figure 2b and SI14 Figure S44). The electrostatic potential map showed relatively electron poor inner regions for the Fmoc groups, as expected, but no obvious points of significant electrostatic interaction between the groups (SI 14.1, Figure S44). Energy decomposition analysis (EDA) of the isolated dimer system (geometry fixed to that of the PP4-SPF crystal structure) was used to determine the relative breakdown of the intermolecular forces between the Fmoc groups in the crystal lattice. Calculations were run with two different functionals, BLYP-D3(BJ) and PBE-D, giving interaction energies of -44.2 and -32.4 kJmol -1 respectively. The breakdown suggests that dispersion interactions are the predominant interaction between the Fmoc groups with smaller contributions from electrostatic and orbital interactions (and the Pauli repulsive interaction, SI 14.2, Table S4).</p><!><p>Due to the porosity of the framework, it has the potential for activation by removal of the solvent from the pores, thereby allowing the introduction of other guests into the activated pores. To investigate the ability for thermal release of ethanol from the framework channels, 1 H-NMR analysis of PP4-SPF in methanol-d4 was carried out after activating the crystals for various timeframes under vacuum at 45 o C. These studies revealed that thermal activation could gradually reduce the ethanol content over time up to a 90 % reduction after 12 h, compared to the initial content present within the framework (SI 11, Figure S16). Subsequently, simultaneous thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) of PP4-SPF showed a crystalline melting point with an endothermic peak (150 o C, SI 6, Figure S7). Samples of the evacuated material, PP4-SPFact, were then taken for gas adsorption studies and surprisingly showed negligible gas adsorption, suggesting a lack of porosity in the de-solvated material. As such powder X-ray diffraction (PD-XRD) analyses was carried out, which revealed a change in phase upon desolvation, with significant differences to the original sample (Figure 3, SI 12, Figure S19). Due to these changes SCXRD analysis was carried out on an activated crystal to investigate the change in porosity. Although the crystal showed very poor single crystallinity after the evacuation of the solvent, and no useful reflections were observed beyond 1 Å resolution (SI 12.1-12.3), the diffraction data could be solved to give a connectivity model. This low-resolution model is presented purely as a comparative connectivity model which suggests collapse of the porous structure with the 2D hydrogen bonded layers shifted relative to one another such that the Fmoc group fills the pore space. However, the predicted powder pattern for the modelled de-solvated structure matches very well with the powder pattern of the bulk material (SI 12.1, Figure S20), suggesting the correct assignment of the collapsed structure for the material. Please do not adjust margins Please do not adjust margins Subsequent to these findings the activated sample was then reincubated in an ethanol solution to investigate if this phenomenon was reversible. Remarkably within 15 minutes the crystals showed an almost complete phase change back towards the original structure with a complete return clear after 21 h (SI 12.2, Figure S21). To rule out the possibility of partial recrystallization causing the phase change, a sample of PP4-SPFact after PD-XRD analysis, while still on the mount, was placed in a chamber with saturated ethanol vapour for 26 h, before again recording the powder diffraction, which showed a complete change from the activated phase to the original phase (Figure 3, SI 13.4). This type of behaviour, exhibiting a dynamic porosity whereby the framework is capable of reversible collapse, is more typically seen for MOFs or coordination polymers, whereby the conformational flexibility of a ligand or weak interactions between 2D layers allows for an adaptable transition dependent on the guest molecules present. 1,12,40 In this case the behaviour results due to the 2D hydrogen-bonded layers of peptide the layers shifting against one another behaving as weakly bound 2D interdigitated layers. 40 Thus negligible conformational change of the peptide units occurs and the strong hydrogen bond interactions are retained along two axes, allowing for minimal disruption of a significant portion of the framework (SI This exemplifies the exceptional rigidity of the polyproline helix compared to other biomolecules, 41 highlighting its applicability as a structural unit in constructing supramolecular frameworks. Due to the finding that the framework exhibits dynamic porosity, and specifically a sponge-like behaviour towards ethanol, 42 attempts were made to see if this behaviour would apply with other guests. Initially PP4-SPFact was incubated in hexane and showed no change in phase on the powder pattern. As such hexane was used as the diluent for other guest molecules, with the framework incubated in a 5 % solution of various guests in hexane as previously (Table 1, further details SI 13). Subsequent to incubation in the guest solution the SPF was washed thoroughly with fresh hexane, and PDXRD analysis was carried out to determine whether a change in phase had occurred (i.e. reflation) for each guest (SI 13.4). The amount of guest retained within the pores was analysed via 1 H NMR, after dissolving the framework in methanol-d4 releasing the guest molecules (Table S3, SI13 & 13.3). All the guests analysed (Acetone, THF, 1-bromohexane, ethyl acetate, toluene, (±) 1-phenylethanol, and iodine), elicited a change in phase from the de-solvated structure, after analysis of the solid SPF after incubation, suggesting they all act to reflate the structure (SI 13.4). However, differences in the powder patterns of the incubated samples suggests there may be a degree of adaptation of the framework to accommodate different guests when compared to the original ethanol guest. Although, differences in peak intensities and loss of some low angle peaks are likely attributable to both loss of crystal quality through the processes of activation and incubation. It should be noted that the activated material loses crystallinity when mechanically ground (SI Fig. S19), and so the measured diffraction patterns for the (unground) materials also exhibit some preferred orientation effects. The encapsulation of the guests was supported by 1 H NMR analysis of the dissolved SPF (washed and dried), thus releasing any encapsulated guest molecules, allowing for the analysis of the mol% of encapsulated guest (Table 1, SI 13.3). This data showed significantly higher proportions of hexane than for the SPF incubated alone in hexane (Table S3, SI 13), which suggests that once porosity is recovered hexane is adsorbed into the framework. However, the proportion of guest alters significantly (e.g. from 5 % v/v), favouring adsorption of guest molecules over the hexane diluent (SI 13). As the SPF building blocks are inherently chiral, with the specific helicity introduced by the polyproline II conformation, we decided to investigate the framework's ability for enantioselective adsorption. With this in mind, and with the successful encapsulation of the chiral molecule (±) 1-Phenylethanol (PhEtOH) within the framework (Table 1, SI 13), chiral HPLC was utilized to investigate whether any enantioselectivity was exhibited towards this encapsulated guest. The de-solvated solid SPF after incubation in a racemic PhEtOH solution, was dissolved in propan-2-ol to release the encapsulated guest before precipitating the peptide in hexane and filtering, this filtrate was then analysed on a chiral HPLC (SI 13.5). These results showed a clear shift in the enantiomeric ratio from the reference racemic (±) PhEtOH after exposure to PP4-SPF (≈24 % ee, S-1-Phenylethanol) (Figure 4, SI 13.5). No  Please do not adjust margins enrichment of either enantiomer was observed with amorphous peptide when used in the same method (SI 13.5), highlighting the role of the SPF in the enantioselectivity.</p><p>In an attempt to observe the crystal structure of the reflated framework hosting a guest molecule, activated single crystals were exposed to a saturated solution of iodine in hexane. Due to the high electron density of iodine, compared to the other organic guest molecules, it made an ideal candidate for visualization within the pores through single crystal diffraction, even at lower guest loadings. By visual inspection of the crystals, a color change was evident with the crystals taking on a yellow-orange hue. SC-XRD analysis of PP4-SPF@I2 was performed which showed that the incubated SPF readopted the initial solvated structural geometry (Figure 5) and was able to adsorb iodine with a chemical occupancy for I2 of ≈12.3 % (SI 13.1). The interatomic distances (2.813(16) Å) between iodine and the Pro1 carbonyl oxygens were indicative of halogen bond formation (Figure 5). To investigate the ability to perform reversible guest absorption with iodine, the same single crystal of PP4-SPF@I2 was thermally treated, while mounted on the stage, using a heated flow of N2 at 50 o C for 3 hours, prior to repeating SC-XRD analysis. The crystal was mounted without the use of any inert oil, to facilitate guest loss. Upon analysis of the thermally treated crystal, the chemical occupancy of I2 was reduced by 50 % while crystallinity of the sample was retained (SI 13.1). This experiment again demonstrates the potential of PP4-SPF to perform thermally responsive guest release. Finally, a batch of PP4-SPF@I2, kept at room temperature for four weeks, showed an I2 occupancy of 10 % (initial occupancy 12.3 %) from SC-XRD analysis, which exemplifies the necessity for thermal treatment to remove the guest I2.</p>