Supramolecular Self-Assembly of Engineered Polyproline Helices

The ability to rationally design biomaterials to form desired supramolecular constructs presents an ever-growing research field, with many burgeoning works within recent years providing exciting results; however, there exists a broad expanse of promising avenues of research yet to be investigated. As such we have set out to make use of the polyproline helix as a rigid, tunable, and chiral ligand for the rational design and synthesis of supramolecular constructs. In this investigation, we show how an oligoproline tetramer can be specifically designed and functionalized, allowing predictable tuning of supramolecular interactions, to engineer the formation of supramolecular peptide frameworks with varying properties and, consequently, laying the groundwork for further studies utilizing the polyproline helix, with the ability to design desired supramolecular structures containing these peptide building blocks, having tunable structural features and functionalities.


HP2H,
Peptides P3H, AcHP2H, Cis-HP2H and AcP4 were separated via RP-HPLC using a HiChrom KR100 5C18 5263 column at 40 o C on a Dionex UltiMate 3000. Gradient: 5% B for 5 minutes then from 5% B to 100% B over 20 minutes, and held at 100% B for 5 minutes. Where A is Water (0.1 % formic acid) and B is methanol (0.1 % formic acid). Flow rate is 1.0 ml/min. Wavelength: 225 nm. The flow was directed into directed into the electrospray source of a Thermo Scientific MSQ Plus Mass Detector, operating in positive ion mode, at 75 kV and mass spectra recorded from 100-2000 m/z. All other peptides, unless otherwise stated, were separated on a 2.1 x 150 mm, 3.6 um, XB-C18 Aeris Widepore column, from Phenomenex, at 30 o C on an Agilent 1100 HPLC. Gradient: 5% B for 5 minutes then from 5% B to 100% B over 25 minutes, and held at 100% B for 5 minutes. Where A is Water (0.1 % formic acid) and B is acetonitrile (0.1 % formic acid). Flow rate is 0.2 ml/min. The flow was directed into directed into the electrospray source of a Bruker micrOTOF-QII mass spectrometer, operating in positive ion mode, at 4.5 kV and mass spectra recorded from 150-3000 m/z. Data was analysed with Bruker's Compass Data Analysis software.
Peptides HP3, PHP2, P3H and HP2H were crystallised by slow evaporation from an EtOH/EtOAc solution, forming crystals (colourless planks). However, no crystals of PHP2 suitable for SCXRD analysis were found.
Peptide AcHP2H was dissolved in hot acetonitrile before slowly evaporating to form colourless needles. Sonication of a supersaturated solution instead forms an organogel, this occurred in acetonitrile, chloroform, and dichloromethane, with the strongest gelation in chloroform ( Figure S27).
Crystallisation of peptide AcP4 was attempted in MeOH, EtOH, EtOH/EtOAc, ACN, and EtOH/ACN, producing a non-crystalline glassy solid or a viscous oil. The peptide crystallised readily from slow evaporation of a CHCl3 solution to form colourless needles, however, the crystal quality was poor and unsuitable for single crystal analysis. The peptide was crystallised successfully from vapour diffusion of Et2O into a solution of the peptide in CHCl3 to produce colourless needles which were analysed via SCXRD analysis.
All crystalline samples were stable outside of their mother liquor. Crystals of peptide AcHP2H melted under a stream of room temperature N2 (290 K), likely due to the loss of ACN from the framework. Crystals of peptide Cis-HP2H, degraded under a room temperature N2 flow (300 K) also likely due to loss of encapsulated solvent within the framework pores.

SI 3.1 Single-crystal XRD data:
Single crystal XRD data for all peptides were collected on a Rigaku Oxford Diffraction SuperNova AS2 single crystal diffractometer using Cu Kα (λ = 1.54184) radiation, AcHP2H and AcP4 used Mo Kα (λ = 0.71073). The crystals were mounted on a Mitegen micromount in Paratone immersion oil and temperature controlled using an Oxford Cryosystems 800-series Cryostream.
Using the software Olex2, 2 the structures were solved with the ShelXT structure solution program using intrinsic phasing and refined with the ShelXL refinement package using least squares minimization. [3][4][5] CCDC-2127751, 1 2234312, 2238152, 2238155, 2238160-1, 2238180, 2238252 and 2264145 contain the supplementary crystallographic data for this paper, including structure factors and refinement instructions, and can be obtained free of charge from The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK (e-mail: depos-it@ccdc.cam.ac.uk), or via https://www.ccdc.cam.ac.uk/getstructures.   1 for ease of comparison to other crystal structures, in both this section 3.1 and section 3.2, the collapsed structure after thermal activation is available from the above reference and CCDC-2156434. The crystal structure data was obtained from plank-shaped colourless crystals, crystallised by dissolving P4 in hot EtOH (60 o C, ≈25 mgml -1 ) and allowing to slowly cool to room temperature forming crystals overnight within the solution. The crystals were stable outside of solution at room temperature showing no signs of deterioration over the timeframe of the experiment. To model the disordered solvent, the ethanol molecule was split into two components with a total occupancy of 1. The distances between the oxygen-carbon and carbon-carbon atoms were set to the expected values of 1.43 Å and 1.51 Å respectively. Upon refinement, these distances were then fixed to the same value between the two components and the anisotropic displacement parameters were set to be equivalent within the molecule.  P4 (PP4-SPF) 1  The crystal structure data was obtained from colourless plank crystals, the crystal quality was relatively poor, the crystal was mounted on a Mitegen micromount in Paratone immersion oil and cooled to 150 K using an Oxford Cryosystems 800-series Cryostream. The crystal structure was obtained with some regions of significant disorder. The linker to the fluorenyl group was significantly disordered as such the atom C13-14 were split and EADP applied. Split SAME was also carried out on O2. The Pro1 pyrrolidine ring was disordered, as such C16-19 were split and EADP applied, split SAME was applied to O8. The pro3 ring was also disordered as such C26-28 were split and DELU applied.
Only small voids are present (40.80 Å 3 , 1.3 %-unit cell volume, Probe radius 1.2 Å, grid spacing 0.5 Å) within the structure that do not extend through the framework, forming a non-porous structure. The fmoc region is disordered and this extends to the neighbouring peptide's Pro3 sidechain, while the Cterminus is well ordered.  Crystal data and structure refinement for HP3 Analysing the other positions on the P4 backbone, the Pro2 residue on the P4 backbone is aligned with adjacent peptides' aromatic groups and the closest hydrogen bond acceptor (HA) is 4.5 Å from the C4 position, which suggests the current crystal structure is unlikely to satisfy the hydrogen bond donation of a new hydroxyl at this position (SI 3.1.3, Figure S32).

SI 3.1.4 P2HP SC-XRD:
The crystal structure data was obtained from colourless plank crystals, the crystal was mounted on a Mitegen micromount in Paratone immersion oil and cooled to 150 K using an Oxford Cryosystems 800series Cryostream. P2HP crystal structure was isostructural to P4, and two water molecules were modelled within the voids of the framework. One of the water molecules was disordered as such split SAME was applied to O9. EADP was applied O2-C15. This reduced pore size is too small to accommodate EtOH molecules, as such the framework is selective towards H2O molecules in the wet solvent during crystallisation, which can be clearly modelled in the crystal structure ( Figure S33).   The crystal structure data was obtained from colourless plank crystals, the crystal was mounted on a Mitegen micromount in Paratone immersion oil and cooled to 150 K using an Oxford Cryosystems 800series Cryostream. The structure was resolved with two peptides comprising the asymmetric unit (Z' = 2), joined by the typical C-terminal amide NH2 hydrogen bonds. Each of these peptides was modelled with a disordered EtOH molecules within the pores with partial occupancies (0.5). The oxygen atoms of each were disordered as such split SAME was applied to each (O8-O8X). While a model of the atomic positions of this disordered solvent is included it is likely that an atomistic model is not fully appropriate for the disordered electron density inside the pore.  The crystal structure data was obtained from colourless plank crystals crystallised via slow cooling of a hot supersaturated ethanol solution of peptides P4 and P2HP in equimolar concentrations. The crystal was mounted on a Mitegen micromount in Paratone immersion oil and kept at 290 K using an Oxford Cryosystems 800-series Cryostream. The mixed peptides crystal structure was isostructural to P2HP and P4. Disordered EtOH molecules were modelled in the pores with a partial occupancy of 0.75. Split same was applied to the EtOH atoms C36-37. H atoms were not assigned to the EtOH molecules due to the high level of disorder. Interestingly this framework adopted the endo conformation similarly to P4 alone, having significantly less impact on the channel volume (Volume 233.02 Å 3 , 12.8 % / unit cell, Probe r = 1.2 Å, Grid spacing 0.4 Å), and thus contained EtOH within the pores rather than being selective towards H2O ( Figure S37 and S38).   The crystal structure data was obtained from colourless plank crystals, crystallised via slow evaporation of an ethanol solution of peptide HP2H. The crystal was mounted on a Mitegen micromount in Paratone immersion oil and kept at 295 K using an Oxford Cryosystems 800-series Cryostream. The first hydroxyproline residue was disordered on hydroxyl group and at the Cγ position (C18), likely to due to the presence of endo and exo ring puckering within the structure. The crystal structure is nonporous containing no significant void space. Both hydrogen bonds present in the HP3 and P3H crystal structure are present within the crystal structure between the same atoms, extending along the b-axis.   The crystal structure data was obtained from colourless plank crystals, crystallised via slow evaporation of an ethanol/acetonitrile solution of peptide Cis-HP2H. The crystal was mounted on a Mitegen micromount in Paratone immersion oil and kept at 150 K using an Oxford Cryosystems 800-series Cryostream. The crystal structure obtained shows the presence of channels within the structure, filled with disordered solvent (volume 628.9 Å 3 , 17.1 % / unit cell, Figure S42) and no suitable model could be obtained for the disordered solvent (EtOH and acetonitrile) within the pores of the structure and so a solvent masking routine was used. The electron density was therefore accounted for using a solvent mask within Olex2, 2 giving a solvent accessible volume of 167 Å 3 and containing 36 electrons asymmetric unit. Screening of multiple crystallites suitable for single crystal analysis gave the same crystal structure. The extended structure of Cis-HP2H differs from the other Fmoc peptide frameworks as the Fmoc moieties of adjacent peptides no longer face one another, resulting in staggered rather than linear H-bonded layers of the peptides (Figure 4e). The last hydroxyproline's (C-terminus) hydroxyl group is aligned into the pore space, clearly hydrogen bonding to a solvent molecule, with significant electron density adjacent to this group. Therefore, the only intermolecular hydrogen-bonding between peptides is the typical C-terminal NH2 amide bonding present in all the structures seen previously. This highlights how small changes in the placement of functional groups can be used to affect the assembly process, with control over even the flexible pyrrolidine ring endo/exo conformations possible by use of 4S versus 4R functional groups, while the polyproline II helix remains as a rigid ligand for placement of these functional groups.   The crystal structure data was obtained from colourless needle crystals, crystallised via slow evaporation of an acetonitrile solution of peptide AcHP2H. The crystals obtained formed packed fibrous assemblies that did not diffract well due to low crystal volumes requiring long exposure times. The crystal was mounted on a Mitegen micromount in Paratone immersion oil and at 294. The crystal structure obtained showed a porous structure (Volume 415.06 Å 3 , 15.6 % / unit cell, Probe r = 1.2 Å, Grid spacing 0.4 Å), in the orthorhombic P212121 spacegroup, with channels extending along the a-axis containing ordered acetonitrile molecules with no apparent strong interactions between the solvent and the peptide within the structure. The crystal was weakly diffracting and no data above 2 I/sig was observed above 1.1 angstroms. Consequently, the data were truncated at this resolution.  Crystal data and structure refinement for AcHP2H The crystal structure data was obtained from colourless crystalline needles, crystallised via vapour diffusion of Et2O into a solution of AcP4 in CHCl3. The crystal was mounted on a Mitegen micromount in Paratone immersion oil and kept at 150 K using an Oxford Cryosystems 800-series Cryostream. The crystal structure obtained showed a porous structure (Volume 572.37 Å 3 , 36.1 % / unit cell, Probe r = 1.2 Å, Grid spacing 0.4 Å), with in the monoclinic P21 spacegroup, with channels extending in a 2D layer along the b-and a-axis containing ordered chloroform molecules, with two solvent molecules per asymmetric unit, hydrogen bonding to the Pro1 and Pro4 carbonyl groups.   Crystal data and structure refinement for AcP4

P3H PD-XRD:
Peptide P3H did not crystallise well as once precipitated from solution an insoluble white solid formed preventing redissolution, as such a suitable powder pattern could not be obtained, despite the crystallisation of some single crystals suitable for SCXRD, sufficient crystalline material was not obtained for PDXRD analysis.    The experimental spectra for Cis-HP2H differs significantly from the simulated powder pattern ( Figure  S58), from the crystal structure obtained, suggesting the bulk material differs from the singly crystalline material. Screening of crystals for alternate crystal structures was unsuccessful, all crystals suitable for single crystal analysis exhibited the previously obtained structure.