Isotopic Control over Self-Assembly in Supramolecular Gels

It is common to switch between H2O and D2O when examining peptide-based systems, with the assumption being that there are no effects from this change. Here, we describe the effect of changing from H2O to D2O in a number of low-molecular-weight dipeptide-based gels. Gels are formed by decreasing the pH. In most cases, there is little difference in the structures formed at high pH, but this is not universally true. On lowering the pH, the kinetics of gelation are affected and, in some cases, the structures underpinning the gel network are different. Where there are differences in the self-assembled structures, the resulting gel properties are different. We, therefore, show that isotopic control over gel properties is possible.

Rheology. Rheological measurements were performed using an Anton Paar Physica MCR101 or MCR301 rheometers. For the frequency sweeps, measurements were carried out directly on gels formed in the Sterlin vials. Frequency sweeps were performed using a cup and vane system under a strain of 0.5 % using a scan range of 1 rad/s to 100 rad/s. Time sweeps were performed using a PP25/S plate with a measuring gap of 1 mm. An angular frequency of 10 rad/s and a strain of 0.5 % were used. To prevent the sample drying out the rim of the plate was sealed with mineral oil (Alfa Aesar).
Cryo-TEM. Cryogenic TEM imaging was performed using a FEI Tecnai 12 TWIN Transmission Electron Microscope, operating at 100 kV. Gels were immediately diluted five times with water to reduce their viscosity and 6 μL of sample solution was placed on a holey carbon film supported on a TEM copper grid (Electron Microscopy Services, Hatfield, PA). All the TEM grids used for cryo-TEM imaging were treated with plasma air to render the lacey carbon film hydrophilic. A thin film of the sample solution was produced using the Vitrobot with a controlled humidity chamber (FEI). After loading of the sample solution, the lacey carbon grid was blotted using preset parameters and plunged instantly into a liquid ethane reservoir precooled by liquid nitrogen. The vitrified samples were then transferred to a cryo-holder and cryo-transfer stage, which was cooled by liquid nitrogen. To prevent sublimation of vitreous water, the cryo-holder temperature was maintained below −170 °C during the imaging process. All images were recorded by a SIS Megaview III wide-angle CCD camera. Small Angle X-Ray Scattering (SAXS). SAXS was obtained at beamline B21 at the Diamond Light Source. This beamline operates at a fixed energy of 12.4 keV and a camera length of 4.014 m, resulting in a Q range of 0.004 -0.44 Å -1 . The samples were manually loaded into borosilicate glass capillaries (Capillary Tube Supplies UK). Measurement times were 15 s for each sample. 14 frames were acquired and averaged. Data were processed using DAWN 3 and a water background was subtracted as a 2D image within the processing pipeline.
Additional data were collected on a SAXSLAB Ganesha 300XL instrument. 70 μL of the solutions as prepared above in H 2 O were transferred to a borosilicate glass capillary (Capillary Tube Supplies UK), sealed, and measured for 3600 s in a Q range of 0.007 -0.25 Å -1 . Gels were prepared by addition of GdL to the solution in 10 mL glass vials before transfer to the capillary, sealed using a UV curable adhesive (Norland) and allowed to gel overnight. Data were corrected for transmission, absolute intensity and capillary width.
All data were fitted using SASView 4.0. 4 As the length of the scattering objects falls outside of the Q range for the experiment, an arbitrarily long length was introduced into the model and not allowed to refine.

Details of models used for SAXS data fitting:
All data were fitted in SASView 4.0 without further modification to the models. The details of the form factor, P(q) for a cylinder, a cylinder with an elliptical cross section, and a hollow cylinder are outlined below. Detailed information as to the model for a flexible cylinder is given in the references below.

a) Cylinder
The 2D scattering intensity for a cylinder is described by Guinier 5 and is outlined below. where α is the angle between the cylinder axis and , and is the particle volume, R = 2 is the particle radius, L is the length, ∆ρ is the scattering length density difference between the scatterer and the solvent, and J 1 is the first order Bessel function.
Therefore, the output for scattering in 1D from randomly oriented cylinders is given by: 5 Modelling the scattering function for a worm-like chain (WLC) cannot be done analytically; instead quantitative structural information is found by Monte Carlo simulations of the Kratky and Porod WLC model, taking into account excluded volume effects. The model used within the SASView software has been extensively detailed by Pedersen and Schurtenberger 6 as well as Chen et al. 7

c) Flexible Elliptical Cylinder
To take into account the scattering from a non-uniform elliptical cross section of a cylinder, the following applies: Where ( , , ) = 2 1 ( )sin

And
: Ψ is the orientation of the major axis with respect to , and α is the angle between the cylinder axis and .
For randomly oriented cylinders in 1D, the form factor is averaged over all possible orientations and normalised by volume.
The scattering intensity for a hollow cylinder is described in Guinier, 1955 and is outlined below.
Scale is a scale factor, H = L/2, and J 1 is the first order Bessel function.
All data had an H 2 O or D 2 O background subtracted as appropriate. Data for 1 in both H 2 O and D 2 O in solution fitted as shown previously to a flexible cylinder model with a low Chi squared value, and with values for the radius commensurate with our previous work. As shown previously the length is outside of the Q range for the experiment and was therefore fixed at an arbitrarily high value and not allowed to refine. The gels fitted well to a flexible elliptical cylinder model as has been shown before.
For 2 and 3 in both H 2 O and D 2 O in solution, we selected a hollow cylinder model based on the TEM images. For 2, a power law was included to fit the data effectively. In both cases, here the length was allowed to refine and falls into the Q range of the experiment. Comparing this fitted length to the TEM images gives rise to a discrepancy -the TEM data indicates that these hollow tubes are longer than can be probed by this Q range. We postulate that this length is likely to be representative of the Kuhn length, and have observed this behaviour in our previous work. 8 In the gel of 2, a good fit was found with a flexible elliptical cylinder model. In H 2 O, gels of 3 were best described again by a hollow cylinder model where a Schulz polydispersity was required to improve the fit. The length here should be assumed as before to be the Kuhn length of the cylinder rather than its overall length. In D 2 O the data fitted best to a flexible elliptical cylinder, showing a clear difference in the gel phase of 3 between H 2 O and D 2 O. Data for 4 in H 2 O was fitted as a flexible elliptical cylinder with an arbitrarily long length. In D 2 O a good fit was found fitting to a flexible cylinder. In the gel phase in H 2 O, again, a flexible elliptical cylinder best describes the data. However, in D 2 O, a cylinder plus a power law was found to be the most appropriate model to describe the gel.    The error bars represent the average of three different measurements on unique gels. Note that there is a weak frequency dependence at low frequency in all cases, as expected for such low molecular weight gels. At higher frequency for 3 and 4, there is an increase in G′ and G″. This is not due to slip since we collected the data using a vane and cup but may be a flow artefact. We note that similar effects are seen using nanoindentation for related gels. 9 S12 Additional Cryo-TEM data.

(H 2 O) (D 2 O) 2 (H 2 O) 2 (D 2 O) 3 (H 2 O) 3 (D 2 O) 4 (H 2 O) 4 (D 2 O)
To collect these data, a thin film has to be prepared from the gel phase. This is most commonly carried out by pressing the grid on or into the gel and hoping that the structures that adhere represent those forming the network. This can be problematic for the gel phase. Here, we find that the broken structures are observed, with no evidence of a network. This may be due to the structures that adhere to the grid being not part of the network or perhaps structures that break on adherence to the grid of during the blotting process. As such, whilst we provide example cryo-TEM images in Figure S5, we treat them with caution.