# Designing and Controlling Transient Supramolecular Gels

Emma L. Bowley, Simona Bianco, Fin Hallam Stewart, Chloe M. Wallace, Rebecca E. Ginesi, Alex S. Loch, Martin Rosenthal, Andrew J. Smith, Dave J. Adams

► To cite this version:

Emma L. Bowley, Simona Bianco, Fin Hallam Stewart, Chloe M. Wallace, Rebecca E. Ginesi, et al.. Designing and Controlling Transient Supramolecular Gels. ChemSystemsChem, 2025, 7 (2), pp.e202400073. 10.1002/syst.202400073. hal-05099676

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Distributed under a Creative Commons Attribution 4.0 International License
# Designing and Controlling Transient Supramolecular Gels

Emma L. Bowley,[a] Simona Bianco,[a] Fin Hallam Stewart,[a] Chloe M. Wallace,[a] Rebecca E. Ginesi,[a] Alex S. Loch,[a] Martin Rosenthal,[b] Andrew J. Smith,[c] and Dave J. Adams*[a]

Supramolecular systems are often designed such that a steady state exists. However, the ability to design systems with pre-determined changes in state can lead to highly dynamic materials, with evolving supramolecular structures and adaptable material properties. This approach is of great interest from the perspective of designing adaptive systems as well as from a broader systems chemistry perspective. Here, we report how a transient system can be altered to access different mechanical

properties and transitions by varying the trigger and temperature. The aging of these systems is also explored, as the networks continually evolve long past the common cut-off point of analysis of one day. We therefore provide new insights into the control of transient gels, as well as an understanding as to the underpinning supramolecular structures and how they evolve with time.

## Introduction

Supramolecular transient gel systems can be used to access novel materials by mimicking natural out-of-equilibrium systems.[1,2] These systems are designed to be dynamic in nature and changes in state or properties are induced by internal or external stimuli such as changes in pH, changes in temperature, enzymatic reactions, or redox reactions.[2-6]

Boekhoven et al. reported the first transient self-assembled system fuelled by a chemical reaction for a sol-to-gel-to-sol transition.[4] Since then, there have been numerous examples of transient systems driven by altering the equilibrium of gel systems.[7,8] In most cases, a chemical fuel alters the equilibrium of the self-assembly and causes a change in the state or gel network.[6] For example, a transient system containing the urea/urease reaction and methyl formate hydrolysis reactions causes an increase and then decrease in pH over time resulting in a gel-to-sol-to-gel transition.[9,10] The urease induced hydrolysis of urea produces ammonia which initially increases the system's pH uniformly, the hydrolysis of methyl formate then becomes dominant and its production of protons decreases the pH i.e.[11] There are also examples of gel-to-gel and gel-to-gel-to-gel systems where no change in state occurs; however, there is still a significant change in the mechanical properties of the system indicating the dynamic nature of these systems Figure 1.[10,12]

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Diagram showing the pH change over time, for the 1ThNapFF transient system, with images showing the gel-to-sol-to-gel transitions.</figcaption></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

Despite the many existing examples, most of the systems reported detail a limited scope of gelator, chemical fuel, and the conditions under which the system is measured. Although it is often implied that these systems can be applied to a wide scope of gelators or conditions, the rheological studies are rarely reported meaning the mechanical properties of the system are unknown. Within this field, the properties of the "final gel" are often reported with data recorded 24 hours after the gel was triggered.[11] For example, rheological properties are often reported shortly after the system's pH appears to have plateaued.[11] However, this is not necessarily the point at which the system stops evolving.

<footnote>[a] School of Chemistry, University of Glasgow, Glasgow, UK</footnote>

<footnote>[b] ESRF - The European Synchrotron, Grenoble, France</footnote>

<footnote>[c] Diamond Light Source Ltd., Didcot, Oxfordshire, UK</footnote>

<footnote>**Correspondence:** Dave J. Adams, School of Chemistry, University of Glasgow, Glasgow G12 8QQ, UK.</footnote>

<footnote>Email: dave.adams@glasgow.ac.uk</footnote>

<footnote>Supporting information for this article is available on the WWW under https://doi.org/10.1002/syst.202400073</footnote>

<footnote>© 2024 The Authors. ChemSystemsChem published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</footnote>
<figure><img src="image_5.png" /></figure>

The gel-to-sol-to-gel transient system using urea, urease, and methyl formate, as discussed above, has been well-established within the current literature for the 1ThNapFF gelator.[10–12] Herein, we examine a wide library of low molecular weight gelators, using various pH triggers over a range of temperatures. Through this, we demonstrate how the urea/urease/methyl formate system can be fully exploited to access desired final gel properties. We examine the effect of replacing methyl formate with ethyl formate and *n*-propyl formate to affect the rate of pH decrease. We also examine the ageing process of these systems by measuring mechanical properties over a month after the start of the transient process.

## Results and Discussion

We have previously reported a transient gel system, where by a gel-to-sol-to-gel transition was achieved using 1ThNapFF (structure in Figure 2). The gelator was in a H₂O/DMSO mixture with a final concentration of 2 mg/mL as the gelator system, with the addition of urea/urease and methyl formate.[2,11] A static gel formed using 1ThNapFF in H₂O/DMSO provides a suitable non-transient analogue for comparison. We have examined a range of other low molecular weight gelators (Figure 2) to understand the scope of the transient systems and the range of properties that can be achieved.

Initially, we examined whether solvent-triggered gels can be formed from this library of gelators with H₂O/DMSO at a constant ratio of 20/80 v/v. The mixtures were left for 16 hours the results of which can be seen in columns two and three of Table 1. As a guide, successful gels are formed from those gelators that were more hydrophobic, with calculated logP (clogP) values approximately greater than 2.10. For each of the molecules, the hydrophobicity was estimated from a predicted value of clogP, the partition coefficient.[13] The different gelation rates can be explained by the solubility differences in both DMSO and water.

The gelators that formed hydrogels after approximately one minute were then examined with urease, urea, methyl formate, the same conditions used in the 1ThNapFF transient system. Urea and methyl formate were added to the DMSO gelator solution and then gelation was triggered by adding urease to this mixture. Of these, 2NapFF, FmocFA, CarbFV, and 7MeO₂-NapFV all underwent a successful gel-to-sol-to-gel transition in the presence of urea, urease, and methyl formate, as shown in Figure 3. The gel formed from CF₃PhF1F did not undergo a transition into a sol state as the pH increased. The gelators that did not transition into a sol state had an apparent p*K*ₐ above the maximum pH (≈8.1) reached by the system; the pH simply does not get sufficiently high to deprotonate the carboxylic acid to induce micelle formation which is characteristic of the sol phase, evidenced by the SAXS data.[14–16] FmocLG underwent a gel-to-sol transition but it did not transition back into a gel state. The gelators that did not transition into a gel state had an apparent p*K*ₐ below the minimum pH reached by the system; the pH simply does not get sufficiently low to protonate the carboxylic acid to induce network formation. For those systems

where a complete gel-to-sol-to-gel transition occurred, the rates of the transitions were related to the apparent p*K*ₐ of the gelators, Table 1. Altogether therefore, to show a gel-to-sol-to-gel transition, the gelator needs to successfully form a H₂O/DMSO solvent switch gel, have a clogP value above 2.10, and an apparent p*K*ₐ below 8.1.

To understand the structures underpinning the gel and sol phases, the transient systems were probed over 16 hours using *in situ* small angle X-ray scattering (SAXS). Due to beamline time limitations, the H₂O/DMSO hydrogels were initially measured for 2NapFF, FmocFA, CarbFV, and 7MeO₂NapFV, and the gelators which had the most obvious scattering (FmocFA, and CarbFV) were chosen for the kinetic experiments. Static gels refer to the H₂O/DMSO solvent switch gels.

<figure><img src="image_6.png" /><figcaption>**Figure 2.** Molecular structure of the low molecular weight gelators investigated in this study, along with acronyms used.</figcaption></figure>
<figure><img src="image_7.png" /></figure>

<table><caption>**Table 1.** Results of the solvent switch gelator test.</caption><thead><tr><th>Gelator</th><th>pK<sub>a</sub></th><th>Gel 1 min<br>after H<sub>2</sub>O<br>addition</th><th>Gel 16 hrs<br>after H<sub>2</sub>O<br>addition</th><th>clogP<sup>[a]</sup></th><th>T<sub>gel(°C)</sub></th></tr></thead><tbody><tr><td>1ThNapFF</td><td>6.35</td><td>✓</td><td>✓</td><td>2.74</td><td>&gt; 90</td></tr><tr><td>2NapFF</td><td>6.46</td><td>✓</td><td>✓</td><td>2.76</td><td>79.2</td></tr><tr><td>2NapIF</td><td>7.18,<br>5.42</td><td>x</td><td>✓</td><td>2.58</td><td>73.0</td></tr><tr><td>2NapFV</td><td>-</td><td>x</td><td>x</td><td>2.08</td><td>-</td></tr><tr><td>2NapVG</td><td>-</td><td>x</td><td>x</td><td>0.83</td><td>-</td></tr><tr><td>4CINapLIF</td><td>-</td><td>x</td><td>x</td><td>3.94</td><td>-</td></tr><tr><td>BrNapAA</td><td>-</td><td>x</td><td>x</td><td>0.62</td><td>-</td></tr><tr><td>6MeONapA</td><td>-</td><td>x</td><td>x</td><td>0.44</td><td>-</td></tr><tr><td>7MeO2NapFV</td><td>8.08,<br>6.91</td><td>✓</td><td>✓</td><td>2.11</td><td>35.9</td></tr><tr><td>FmocAG</td><td>7.87</td><td>x</td><td>✓</td><td>2.87</td><td>34.9</td></tr><tr><td>FmocFA</td><td>8.00,<br>5.88</td><td>✓</td><td>✓</td><td>4.11</td><td>31.7</td></tr><tr><td>FmocLG</td><td>7.62,<br>4.81</td><td>✓</td><td>✓</td><td>4.18</td><td>73.0</td></tr><tr><td>CarbFV</td><td>8.08</td><td>✓</td><td>✓</td><td>4.75</td><td>38.9</td></tr><tr><td>CF<sub>3</sub>PhFIF</td><td>8.54</td><td>✓</td><td>✓</td><td>3.19</td><td>51.4</td></tr></tbody></table>

[a] clogP calculated using an online prediction program and represent the predicted hydrophobicities.[13]

<figure><img src="image_8.png" /><figcaption>**Figure 3.** Variation of G' (black) and G'' (red) at a frequency of 50 rad/s over time and pH (orange) over time for a) 2NapFF, b) 7MeO2NapFV, c) FmocFA, and d) CarbFV in the presence of urea-urease reaction and methyl formate, with a final gelator concentration of 2 mg/mL. pH and rheology measurements were performed with two different vials under identical conditions.</figcaption></figure>

Due to weak scattering at 2 mg/mL, the concentration was increased to 5 mg/mL for the *in situ* SAXS experiments. This more concentrated system was optimised by increasing the concentrations of urea and urease to 4 M and 0.5 mg/mL respectively. The volumes of urea (4 M), urease (0.5 mg/mL), and methyl formate were kept the same. From this point, the system with a final concentration of 5 mg/mL is used.

For FmocFA, the scattering data for the initial gel in the transient system and the static gel both fit well to an elliptical cylinder model combined with a power law. From Figure 5, it is evident that the structure changes over the first 20 minutes as the transition from gel-to-sol occurs. The fibres start as elliptical cylinders combined with a power law which transition into cylinders combined with a power law during the gel-to-sol transition at approximately 8 mins. This matches with the rheological data taken of the system (Figure 4). After 2 hours, the second transition from sol-to-gel has occurred and the data fit to cylinders combined with a power law for the images taken at 2 hours and 4 hours. After this, the system fits to a power law and remains a power law for the remaining 12 hours. This is indicative of the cylinders becoming more entangled and forming a larger network that is unable to be captured on this length scale. (Figure 5)

Due to the speed of the first gel-to-sol transition, the initial gel for the CarbFV transient system was unable to be captured. Therefore, the structure is assumed to be the same as the static gel as with the previous systems. As the data for the static gel are modelled as elliptical cylinders, the SAXS data implies that during the gel-to-sol transition the system has transformed into hollow cylinders with a power law during this transition. The hollow cylinders increase in radius from 107 Å–116 Å during the sol phase and are retained through the gel-to-sol transition. However, during the sol phases the thickness of the hollow cylinders decreases from 37 Å–30 Å.

For all the gelators discussed, it is evident from the SAXS data that the gel-to-sol transition enables the fibres to organise and gain more kinetic stability. This more stable network is then trapped by the sol-to-gel transition. Regarding the CarbFV gelators system, this increases the strength of the system as seen in Figure 4. Despite the superior kinetic stability of the final gel network, the mechanical properties are not improved after the transition occurs, as is shown by the FmocFA data.

The choice of the counter pH trigger is essential in controlling the final gel properties. The effect of using the hydrolysis of different formates, namely ethyl formate and *n*-propyl formate, to lower the pH was explored as these reactions occur at a slower timescale compared to methyl formate.[17] Altering the hydrolysis reaction allows the solution to remain at high pH for a longer time and therefore the system is in the sol

<figure><img src="image_9.png" /><figcaption>**Figure 4.** Variation of G' (black) and G'' (red) over time at 10 rad/s and pH (orange) over time for FmocFA (left), and CarbFV (right) in the presence of urea-urease reaction and methyl formate, with a final gelator concentration of 5 mg/mL. pH and rheology measurements were performed with two different vials under identical conditions.</figcaption></figure>
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>**Figure 5.** SAXS data for the static gel (top) and in-situ transient gels over 20 mins (middle) and 16 hours (bottom) for the FmocFA and methyl formate transient gel (left) and CarbFV and methyl formate transient gel (right). The overlaid red (top) and black (bottom) lines are the fits to the data (see Tables S2–4 and S7–9).</figcaption></figure>

phase for a longer time. This high degree of temporal control allows us to access gels with new properties. FmocFA, and CarbFV were explored, and the counter pH trigger of methyl formate was replaced with one molar equivalent of ethyl formate or *n*-propyl formate.

The effect of changing out the formate reaction on pH was immediately evident (Figure 6). Increasing the alkyl chain length on the formate increases the time that the system spends in the sol phase, although the initial increase in pH is slowed down. The effect on the pH rate change is independent on the gelator choice as both FmocFA and CarbFV show this same trend, The formate systems all reach approximately the same maximum pH before starting to decrease, due the pH needed to start the formate hydrolysis; rather it is the time that it takes to reach this pH which is delayed, as the curve follows the same general shape.

Although the effect on the pH is independent of gelator choice, the effect on the mechanical properties differs for each gelator. For FmocFA, the systems with ethyl formate have lower G' and G'' values compared to the methyl formate system and also produce a weaker final gel when comparing G' and G'' to the initial gel. For the *n*-propyl formate systems, G' and G'' are again lower than the other formates used.

All of systems are less stiff after 16 hours compared to the initial gel before the transition. Despite this, the rheological values for all three systems are still increasing and have not reached a plateau (Figure 6). Therefore, it would be inaccurate

<figure><img src="image_12.png" /><figcaption>**Figure 6.** Variation of G' and G'' over time at 10 rad/s (left) and pH over time (right) for FmocFA (top), and CarbFV (bottom) in the presence of urea-urease reaction with methyl formate (green), ethyl formate (orange), and *n*-propyl formate (red) with a final gelator concentration of 5 mg/mL. pH and rheology measurements were performed with two different vials under identical conditions.</figcaption></figure>

to draw a conclusion at this time point as the gels are clearly still evolving.

For the CarbFV systems, the final gels are stiffer than the initial gel regardless of the formate used. However, unlike the FmocFA gels, the mechanical properties seem to be independent of the rate of pH change. Each formate system forms a gel with similar mechanical properties when compared to each other that are higher than the initial gel after 16 hours.

It should also be noted that these systems can also be formed in other solvents such as ethanol. This has been tested for FmocFA (Figure S8) and transitions were observed for methyl formate and ethyl formate in an ethanol/water system.

These alternative hydrolysis systems were also measured using *in situ* SAXS to understand the underlying network (Figure 7). The increase in radius seen in the methyl formate systems was also observed in the ethyl and *n*-propyl formate systems for FmocFA and CarbFV. The *n*-propyl formate system for both gelators echoed the same fibre shape transitions seen for the methyl formate system. The ethyl formate systems stayed the same shape as the initial gel and only showed the radius increase mentioned above.

As the transitions are delayed for the *n*-propyl formate compared to the methyl formate system, the initial gel and early transitions could be captured by SAXS for CarbFV. The initial gel showed cylindrical fibres and not elliptical cylinders as expected from the data for the solvent-triggered gel. The scattering from the *n*-propyl formate system was weaker than the methyl formate system. Therefore, for the CarbFV system after the initial image, the scattering fit to a power law. However, like with FmocFA this could be indicative of larger network entanglement.
<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /><figcaption>**Figure 7.** SAXS data for the in-situ transient gels over 16 hours with ethyl formate (left) and *n*-propyl formate (right) for FmocFA (top), and CarbFV (bottom). The overlaid black lines are the fits to the data (see Tables S5–6 and S9–10).</figcaption></figure>

To further understand the morphology of the systems before and after the transition, images of the FmocFA systems were taken with a confocal microscope. From these images (Figure 8) it is evident that the addition of the formates reduces the size of the spherulites in the network after the transition. Images of the systems without urea were also taken as an analogue of the of the systems before the transitions (Figure S7). These images show that large spherulites are formed prior to the transition which are similar to those found in the DMSO/H₂O solvent switch gels.

<figure><img src="image_15.png" /><figcaption>**Figure 8.** Confocal images of FmocFA DMSO/H₂O gels, FmocFA methyl formate transient gels, FmocFA ethyl formate transient gels, and FmocFA *n*-propyl formate transient gels, all imaged 1 day after gelation was triggered.</figcaption></figure>

As noted above, at 16 hours, the systems are still evolving. The rheological properties were therefore measured after 1 day, 3 days, 7 days, 14 days, and 28 days (Figure 9). It is clear that, broadly, the systems do not stop evolving until 7 days after the start of the system. All of the CarbFV systems also underwent gradual syneresis after 3 days, with no further shrinking occurring after 7 days; at this point, the mechanical properties stabilise (see Figures S9–S11).

If these types of materials are to be used in practical applications, then it is vital that the properties and materials are observed over a longer period. This means that for practical applications, it is vital that testing of these materials takes place over long timescales.

These systems can be further manipulated by varying the temperature of the system as it is undergoing the state transition. Methyl formate transient systems containing Fmoc-FA, and CarbFV were investigated by maintaining the systems at a range of temperatures from 10–35 °C (Figure 10).

The temperature dramatically alters the kinetics of the urea/urease reaction and the methyl formate hydrolysis used which allows the gel-to-sol-to-gel system to be accurately controlled. The range of the temperature control is between 10 °C and 35 °C, although there are no gel-to-sol-to-gel transitions at these temperatures as the pH peak is lower than the $pK_a$ of the gelators. CarbFV had a different range, however, and only transitions between 15 °C and 35 °C as this gelator has the highest $pK_a$. The temperature range of a system can be predicted from the $pK_a$ of the gelator. As the temperature is increased the strength of the gel also increases as the time spent in the sol phase is longer.

The rheological properties were again measured after 1 day, 3 days, 7 days, 14 days, and 28 days (Figure 11). After the 28 days and syneresis has occurred for CarbFV, the gels all show similar mechanical properties at all three temperatures. The difference is in the rate of increase of the mechanical properties. This probably due to a plateau in the pH of the system that occurs faster in the higher temperature systems. Increasing the temperature speeds up the rate of the methyl formate hydrolysis and so the system stabilises faster as there is no longer any stimulus affecting the system.

<figure><img src="image_16.png" /><figcaption>**Figure 9.** Evolution of G' and G'' over a period of 28 days for FmocFA (left), and CarbFV (right) in the presence of urea-urease reaction with methyl formate (green), ethyl formate (orange), and *n*-propyl formate (red). Values of G' and G'' were taken at a strain of 0.5% from strain sweeps which were performed over 0.01%-1000% strain at a frequency of 10 rad/s. Strain sweeps were performed in triplicate and the standard deviations of G' and G'' were taken from these measurements.</figcaption></figure>
<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /><figcaption>**Figure 10.** Variation of G', G'' and pH with time at 10 rad/s for FmocFA (top), and CarbFV (left) in the presence of urea-urease reaction with methyl formate at 10 °C (purple), 15 °C (blue), 20 °C (teal), 25 °C (green), 30 °C (orange), and 35 °C (red) with a final gelator concentration of 5 mg/mL. pH and rheology measurements were performed with two different vials under identical conditions.</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>**Figure 11.** Evolution of G' and G'' over a period of 28 days for FmocFA (left), and CarbFV (right) at 15 °C (blue), 25 °C (green), and 35 °C (red). Values of G' and G'' were taken at a strain of 0.501% from strain sweeps which were performed over 0.01%-1000% strain at a frequency of 10 rad/s. Strain sweeps were performed in triplicate and the standard deviations of G' and G'' were taken from these measurements.</figcaption></figure>

## Conclusions

In conclusion, many gelator molecules could be applied to the urea/urease and formate system to produce a successful transient gel. We have shown how a system could then be modified to access specific properties with a high degree of control. By varying the formate hydrolysis reaction, the timing of the transitions can be tuned to increase or decrease the time a system sends in the sol state. This can also be achieved by varying the temperature, although this also allows control over if the system undergoes a gel-to-sol-to-gel or a gel-to-gel-to-gel transition. Both methods affect the final mechanical properties, therefore, desired gel properties can be easily accessed. However, the results of the modifications are not always as predictable as is often claimed as evidenced by the difference in effect on the FmocFA and CarbFV systems. Due to the longer

time in the sol state these systems have potential applications in 3D printing and in "molding and casting" of hydrogels.

We have also shown that the aging of these systems is extremely important. Our transient gel systems are still evolving for many days after they have been formed and it is logical to conclude that this also applies to other transient systems. Therefore, it is essential to consider this when introducing transient systems.

## Experimental

### Materials

The gelators were synthesised as described previously.[18–20] CF₃PhFIF was synthesised following our established methods (see Supporting Information). Methyl formate (anhydrous, 99%), and Urease (U4002-100KU, Jack Beans, 100,000 units/g solid) were purchased from Sigma-Aldrich. *n*-Propyl formate (97%) was purchased from Thermo Scientific. Dimethyl sulfoxide (>= 99%) was purchased from Fischer Scientific. Urea (ultrapure 99%) and ethyl formate (97%) were purchased from Alfa Aesar. Deionised water was employed in all experiments.

### Preparation of Solutions

To prepare the stock solution of the gelator, the gelator was dissolved in DMSO and shaken to dissolve to give a concentration of 10 mg/mL or 25 mg/mL as required. Stock solutions of urea (2 M), urease (0.253 mg/mL), and urease (0.5 mg/mL) were dissolved in H₂O and shaken. For the urease stock solution, the concentration was determined by taking the mass of the enzyme powder (in mg) dissolved in a known volume of H₂O.

### Preparation of Hydrogels

The DMSO/H₂O hydrogels were prepared by transferring 1.6 mL of H₂O into 0.4 mL of the gelator solution (10 mg/mL or 25 mg/mL) in DMSO. The final concentration of the gelator was therefore either 2 mg/mL or 5 mg/mL in a 20% DMSO, 80% H₂O solution.

To induce the 2 mg/mL gel-to-sol-to-gel transitions, the gels were prepared in the presence of urea (2 M) and either methyl formate, ethyl formate, or *n*-propyl formate, the water was replaced with the urease (0.254 mg/mL) stock solutions. In a 7 mL sterilin vial, 10 µL of urea and 0.4 mL of the gelator solution were combined. The formate was added to this mixture, 100 µL for methyl formate, 133 µL for ethyl formate, and 158 µL for *n*-propyl formate. 1.59 mL of urease was then transferred into this solution. If not specified, then the system was left undisturbed for 16 hours.

To induce the 5 mg/mL gel-to-sol-to-gel transitions, the gels were prepared in the presence of urea (4 M) and either methyl formate, ethyl formate, or *n*-propyl formate, the water was
<figure><img src="image_20.png" /></figure>

replaced with the urease (0.5 mg/mL) stock solution. In a 7 mL
sterilin vial, 10 µL of urea and 0.4 mL of the gelator solution
were combined. The formate was added to this mixture, 100 µL
for methyl formate, 133 µL for ethyl formate, and 158 µL for n-
propyl formate. 1.59 mL of urease was then transferred into this
solution. If not specified, then the system was left undisturbed
for 16 hours.

Rheological Measurements

Rheological experiments were carried out on an Anton Paar
Physica MCR 301 rheometer. The measurements were taken
with a cup and vane geometry (ST10-4 V-8.8/97.5- SN42404) at
a measuring distance of 2.1 mm. Unless specified, the measure-
ments were taken at a constant temperature of 25 °C. Strain
sweeps were performed over 0.01%-1000% strain at a
frequency of 10 rad/s. Frequency sweeps were taken at 0.5%
strain while increasing the frequency from 1 rad/s-100 rad/s.
Time sweeps were performed at a strain of 0.5% and frequency
of 50 rad/s over 16 hours for the transient systems with a final
concentration of 2 mg/mL. Time sweeps were performed at a
strain of 0.5% and frequency of 10 rad/s over 16 hours for the
transient systems with a final concentration of 5 mg/mL. All gel-
to-sol-to-gel samples were prepared immediately before per-
forming the time sweep measurement.

T<sub>gels</sub> were measured using a heat-cool cycle. Gels were made
in 7 mL aluminium cups 1 day before the measurements.
Measurements were taken with an aluminium cup and vane
geometry (ST10-4V-8.8/97.5- SN42404) and were performed at
a strain of 0.5% and frequency of 50 rad/s. The temperature
was started at 25 °C and was increased by 2 °C every minute up
to 90 °C at which point the temperature was decreased by 2 °C
every minute until 25 °C.

Small-Angle X-ray Scattering

Small-angle X-ray scattering (SAXS) measurements were per-
formed using the X-ray beam of the I22 beamline (Diamond
Light Source, Oxfordshire, UK) during experiment SM32866 and
the X-ray beam of the BM26 DUBBLE beamline (ESRF, Grenoble,
France) during SC5410. The BM26 DUBBLE beamline operates at
an energy of 12 KeV and a camera length of 5.41 m, resulting in
a Q range of 0.0036–0.129 Å⁻¹. The I22 beamline operates at an
energy of 12.4 keV and a camera length of 7.721 m, resulting in
a Q range of 0.0027–0.329 Å⁻¹.

The solutions were prepared in vials as described in the
previous sections prior to the beamtime and loaded in glass
capillaries using a 1 mL syringe with a 21G needle. The raw data
was processed using the Dawn Science software (version
2.27).[21] As part of the processing, the backgrounds were
subtracted from the raw 2D SAXS data and a full azimuthal
integration was performed to reduce the data to an 1 vs q plot.
The plots were then fitted to structural models in the SasView
software 5.0.5.[22] To calculate the X-ray scattering length
densities (SLDs) for all samples, the NIST neutron activation and

scattering calculator was used (https://www.ncnr.nist.gov/re-
sources/activation/), assuming a density of 1.55 g/cm³ for the
low molecular weight gelators.[23]

pH Measurements

pH measurements were performed using a HANNA FC200 pH
probe with a 6 mm×10 mm conical tip. Unless specified the
measurements were taken at 25 °C. The accuracy of the pH
values was ±0.1. To monitor the pH, change of the system with
urea, urease, and methyl formate, the reaction mixture was
prepared as above immediately before measurement and
measured for 16 hours. The temperature was controlled using a
circulating water bath.

The pKₐ values of the gelator systems were determined by titration via the addition of 2.5 µL aliquots of 0.1 M HCl solution to the DMSO/H₂O hydrogels with 1 molar equivalence of 0.1 M NaOH at 25 °C. The system was stirred in between recording the pH values and during HCl addition. The system was left to equilibrate overnight before the titration.

Confocal Microscopy

For imaging, nile blue A dye (0.1 wt% aqueous solution, 2 µL
per mL of gel) was incorporated into gels to allow for
observation by confocal fluorescence microscopy using a Zeiss
LSM 710 confocal microscope fitted with Zeiss N-Achroplan
10x, LD Plan- NEUFLUAR 20x (0.4 Korr Ph 2), and LD EC Epiplan
NEUFLUAR 50x (0.55 DIC) objectives. Gels were formed within a
25 mL syringe with the top removed. They were then placed
imaged onto a glass microscope slide to be imaged.

Acknowledgements

E.B. and D.J.A. thank the EPSRC for funding (EP/T517896/1). We
thank Connor McDonald for collecting SAXS data.

Conflict of Interests

The authors declare no conflict of interest.

Data Availability Statement

The data that support the findings of this study are available in
the supplementary material of this article.

Keywords: Gel • Transient • Rheology • SAXS • Dynamic

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Manuscript received: September 11, 2024

Accepted manuscript online: November 5, 2024

Version of record online: November 21, 2024

[Correction added on 13 December 2024, after first online publication: The molecular structure of compound CarbFV has been corrected.]