Antifreeze Protein Mimetic Metallohelices with Potent Ice Recrystallization Inhibition Activity

Antifreeze proteins are produced by extremophile species to control ice formation and growth, and they have potential applications in many fields. There are few examples of synthetic materials which can reproduce their potent ice recrystallization inhibition property. We report that self-assembled enantiomerically pure, amphipathic metallohelicies inhibited ice growth at just 20 μM. Structure–property relationships and calculations support the hypothesis that amphipathicity is the key motif for activity. This opens up a new field of metallo-organic antifreeze protein mimetics and provides insight into the origins of ice-growth inhibition.


General Synthetic Details S2
Physical & Analytical Details S2 Syntheses S3 Ice recrystallisation inhibition (splat) assay. S6 Molecular structure of Zn(II) perchlorate analogue of 6 S7 Location of counter-ions and solvent in crystal structures S9 Hydrophobicity calculations S13

Synthetic details
All solvents and chemicals purchased from commercial sources (Sigma-Aldrich, Acros, Fisher Scientific or Alfa Aesar) were used without further purification unless otherwise stated.
Deuterated solvents were purchased from Sigma-Aldrich and Cambridge Isotope Laboratories.

Physical and Analytical Details
NMR spectra were recorded on Bruker Spectrospin 300/400/500 MHz spectrometers. Routine NMR assignments were confirmed by 1
Thermogravimetric and microanalytical data are consistent with the presence of an octahydrate.

Ice Recrystallisation Inhibition Assay
Ice recrystallisation inhibition was measured using a modified splay assay. 3  Southend on sea, UK), equipped with a Canon DSLR 500D digital camera. Images were taken of the initial wafer (to ensure that a polycrystalline sample had been obtained) and after 30 minutes. Image processing was conducted using Image J, which is freely available. 4 In brief, ten of the largest ice crystals in the field of view were measured and the single largest length in any axis recorded. This was repeated for at least three wafers and the average (mean) value was calculated to find the largest grain dimension along any axis. The average of this value from three individual wafers was calculated to give the mean largest grain size (MLGS). This average value was then compared to that of a PBS buffer negative control.

Molecular structure of Zn(II) perchlorate analogue of 6
The water-soluble compounds such as 6 [see (a) below] resist formation of sufficiently large single crystals for X-ray diffraction so we turned to the isostructural Zn(II) perchlorate analogue (b) (see ref 1 for synthesis and characterisation of this compound). Single crystals were grown from acetonitrile/ethyl acetate. A suitable crystal was selected at the UK National Crystallographic Service 5 and mounted using a MiTeGen loop using Fomblin on a 3-circle AFC11 with a Rigaku Saturn944+ (2x2 bin mode) CCD.
The crystal was kept at 100.15 K during data collection. Using Olex2, 6

S8
The asymmetric unit contains the complex, one molecule of ethyl acetate, two fully occupied and one half occupied acetonitrile molecules, three fully occupied perchlorate anions and two half-occupied perchlorates. The acetonitrile N300-C302 was refined isotropically at half occupancy. Perchlorate Cl30 was modeled as disordered over two positions related by rotation around the Cl30-O31 bond. The occupancies were fixed at 50:50 and the disordered components refined isotropically. Perchlorate Cl40 sits just off the cell wall and was refined under a Part -1 instruction at 50% occupancy. Perchlorate Cl50 sits on a 2 fold axis and was refined at half occupancy. Four positions were located for the oxygen atoms (O51-O54 refined isotropically at 50% occupancy) which under the action of the two fold axis form a perchlorate disordered over two positions around Cl50. Some additional electron density was modeled as a water molecule disordered over three very closely related positions (too close to be bonded), each position was refined isotropically at a third occupancy. No hydrogens were located for the disordered water.
Many SIMU, DELU and DFIX restraints were used to give the disordered components chemically sensible bond lengths, angles and thermal parameters.
The refinement gave values of Hooft y: 0.080(5) (Olex2) and Flack x: 0.038(8) (Shelxl 2016). Since the Flack parameter deviates from zero by slightly greater than 3s, the refinement was completed with TWIN/BASF instruction as an inversion twin. The synthesis was undertaken with starting material of a known handedness and the error on the Flack parameter is not unexpected for data from weakly diffracting crystals. The same view of the cation in spacefill mode is shown below.

Location of counter-ions and solvent in crystal structures
In contrast, this view perpendicular to the remaining pendant arene p-stack shows that it is relatively free of polar contacts. The same view of the cation in spacefill is shown below.
The following examples are re-analysis of all relevant previously published data.

Monometallic Co complex
CCDC code DAJHAZ, Figure 14  From the "rear" of the complex, while the coordinated iminopyridine units are surrounded by perchlorate and acetonitrile the packing is less dense than the Co structure above. This difference probably arises because the charge on the complex is 2+ rather than 3+ for the Co structure.

Hydrophobicity calculations
Calculations were performed using the Firefly QC package, 9 which is partially based on the GAMESS (US) source code. 10 The computed structure 1 of the Zn(II) analogue of the cationic metallohelix unit in L-1 was used as the basis for this study since suitable Fe parameters were not available. The Zn(II) and Fe(II) compounds are isostructural. 11 A polar coordinates system (defining one metal as the origin, the primary axis as the two metal atoms, and one coordinating nitrogen N3 as the third point of reference) was used to define the position of the oxygen atom of a water molecule relative to the frozen atom positions of the tetra-cation D-1. In each calculation the position of the water oxygen was defined with a fixed azimuthal angle, (0° to 350° in 10° steps) relative to nitrogen atom 3, and a fixed polar angle (170° to 50° in 10° steps) and a freely floating radial distance from the origin (metal 1) starting at 10 Å. The position of the two water hydrogen atoms were defined relative to the water oxygen atom (initially with standard bond lengths and angle) and allowed to move freely relative to the oxygen, including torsional angle relative to the origin (metal 1). An energy minimisation (AM1 semi-empirical basis set, with Fe changed to Zn due to lack of Fe parameters) was then performed keeping all atoms in complex cation D-1 fixed as well as the water S14 oxygen position defining azimuthal and radial angles, while origin (metal 1) oxygen distance and the positons of the water hydrogen atoms were allowed to vary. Thus 468 calculations were performed for all combinations of 36 azimuthal angles and 13 polar angles. Another 468 calculations were then performed defining the origin as the other metal (metal 2, with the primary axis as the two metal atoms, and one coordinating nitrogen N3 as the third point of reference) to complete the other half of the solvation "sphere" of the complex cation D-1. The results from two of these calculations (polar angle 50 azimuthal, angle 190, and polar angle 50, azimuthal angle 310) were not included in the final results as they did not minimize to the reasonable energy gradient. Polar coordinates of the water oxygen atoms for each of the 934 (468x2 -2) calculations were then converted into xyz coordinates and plotted in MATLAB2016 using the scatter3 function, with the minimised energy as the colour scale. The difference between the highest and lowest energy in 25.5 kJmol -1 .