Targetable Mechanical Properties by Switching between Self‐Sorting and Co‐assembly with In Situ Formed Tripodal Ketoenamine Supramolecular Hydrogels

Abstract A new family of supramolecular hydrogelators are introduced in which self‐sorting and co‐assembly can be utilised in the tuneability of the mechanical properties of the materials, a property closely tied to the nanostructure of the gel network. The in situ reactivity of the components of the gelators allows for system chemistry concepts to be applied to the formation of the gels and shows that molecular properties, and not necessarily the chemical identity, determines some gel properties in these family of gels.


Technical Details
All references in this SI document refer to the reference list of the main manuscript.

Chemical list
Chemicals were obtained from the following and used as supplied.

Powder X-Ray Diffraction:
PXRD patterns were collected at room temperature using a Bruker D8 Advance powder diffractometer in reflectance mode. Lynxeye super speed detector was used with the radiation being monochromated Cu Kα1, with a characteristic wavelength of 1.541 Å. 30 minute scans over the range 5° ≤ 2θ ≤ 60° (stepsize = 0.014481°/counting time = 0.5 s/steps).

NMR:
NMR spectra were recorded on a Bruker AV 400 operating at 400.1 MHz for 1 H, 282.4 MHz for 19 F and 100.6 MHz for 13 C experiments. All spectra were recorded using 10 -15 mg of sample in 0.6 ml of solvent. Spectra recorded in D2O also feature the addition of NaOH (approximately 5 mg) to dissolve the sample unless otherwise stated.

SEM:
SEM images were produced using a Philips XL30 LaB6 ESEM equipped with an Oxford Instrument X-max 80 EDX detector at 3 kV. Wet gel samples were placed on carbon sticky tabs mounted on aluminium SEM stubs. Once mounted, samples were dried under dynamic vacuum (< 10 mBar) in a desiccator for 48 hours. These dried samples were given a thin coating of gold, to prevent charging effects during SEM imaging, by placing samples in a sputter coater for two minutes at 12 μA.

IR:
IR spectra were recorded on a Nicolet is5 instrument using 24 scans at a resolution of 1 cm -1 and data spacing of 0.964 cm -1 .

Rheology:
Rheological experiments were performed on a Bohlin nano II rheometer. A 40 mm aluminium cone at 4 o was used with an operating gap of 300 μm gap and a solvent trap. For all gels 5 ml of the solution was prepared and added to the plate with a syringe in the operating gap so the gel could form in contact with the cone.

Time sweep experiments:
Commenced immediately upon addition and dissolution of the appropriate amount of GdL to the gelator solutions. All samples were recorded over 15 h at 20 o C with a torque of 100 μNm and a frequency of 1 Hz, data was taken every 30 s for a sampling time of 3 s. Some of the time sweeps show unusual abrupt changes; this was due to syneresis and partial drying of samples, which could not be removed experimentally by varying instrumental parameters.
1.6.2 Frequency sweep experiment: Performed immediately after the time sweep at 20 o C. The torque was kept constant at 100 μNm. Data was obtained for the Frequency sweep from 0.01 Hz to 100 Hz, as a log scale. We have chosen to plot the Frequency sweeps' x axes not as log scales in this paper as the data overlapped too significantly at lower frequencies (which are always noisier) to convey clear differences in the materials' properties.

Stress sweep experiments:
Recorded after frequency sweep experiments. All samples were recorded at 20 o C and at a frequency of 1 Hz.

pH meter:
The pH meter used was a Mettler Toledo FiveEasy, a two point calibration was performed each morning.

Synthesis of 1,3,5-triformylphloroglucinol (A)
To a mixture of hexamethylenetetramine (22.2 g, 157.5 mmol, 2.2 equiv.) and phloroglucinol (9 g, 71.4 mmol, 1 equiv) trifluoroacetic acid (135 ml) was added, the solution was then heated to 100 o C and stirred under nitrogen for 2.5 h. To the reaction mixture 3 M HCl (300 ml) was slowly added with continued heating at 100 o C and stirred for 1 h. After being allowed to cool to room temperature the reaction mixture was filtered and the filtrate obtained was extracted with dichloromethane (5 × 100 ml) before being dried with MgSO4. Once dry the dichloromethane was removed by rotary evaporation yielding an orange solid. This solid was washed with hot ethanol (3 × 100 ml) to yield a \pale yellow free flowing powder. This powder was dried in the oven overnight to yield the final product I. The synthesis is a scaled up version of the synthesis presented by Chong et .al 1 .

Ex Situ Synthesis of gelators R1 to R9 inclusively
To a suspension of A (.250 mg, 1.19 mmol) in ethanol (50 ml) the appropriate amine was added (3.69 mmol, 3.1 molar equivalents) and the resultant suspension was refluxed for 16 hours. Once complete the reflux was cooled to room temperature then cooled to 0 o C, before the reaction mixture was filtered to obtain a yellow precipitate. The precipitate was washed with hot ethanol (5 × 50 ml) then dried overnight at 80 o C this produced the desired product at good yields and purity.

Synthesis of compound R10
To a suspension of A (.250 mg, 1.19 mmol) in ethanol (50 ml) 6-aminohexanoic acid was added (3.69 mmol, 3.1 molar equivalents) and the resulting solution was refluxed for 16 hours. Once complete the reflux was cooled to room temperature and rotary evaporation was used to remove all the ethanol resulting in a dark orange oil. After a week at 5 o C the oil had solidified to give a dark orange solid. The solid ground to a fine powder and washed with hot ethanol (5 × 50 ml) then dried overnight at 80 o C this produced the desired product at good yields (71 %) and purity.

Chemical Analysis notes
Ligands R2, R6, R7 and R8 prooved too insoluble in any solvent to obtain a 1 H spectra of the pure compounds. This resulted in proton data experiments having to be run in D2O with the addition of NaOH in order to solublise the ligand. To obtain chemical information for the ligands the spectra where recorded between 5 and 15 ppm in order to remove the H2O signal which would have otherwise suppressed the signals for the ligand 1 H nuclei. Due to deportonation and exchange, the -NHand -COOH signals were lost.
All 13 C NMR spectra with the exception of R10 were recorded in the manner described above. Due to the two distinct conformations of the ligands and the overlapping of the subsequent signals exact assignment of signals proved impossible.

Methods for hydrogel Preparation.
Nine hydrogels could be prepared with two preparation methods attributed to each individual hydrogel. The 'ex situ' preparation method describes the use of the previously prepared compounds R1 to R9 to form the corresponding hydrogel. The 'in situ' preparation method relates to the mixing and subsequent reaction of the amine and aldehyde component of the gelator in the volume of water that is to be gelled.

Setting gel using In situ gel preparation method (gels R1 to R8 inclusively):
This procedure describes the method used in a typical in situ setting of the hydrogels using a solution of 1,3,5-triformylglucinol and the appropriate aminobenzoic acid.
1,3,5-triformylglucinol (0.082g, 0.39 mmol) was suspended in water (5 ml), to this suspension sodium hydroxide (0.05 g, 1.25 mmol) was added and sonication and gentle heating was used to produce a clear pale yellow solution. To a separate portion of water (5 ml) the appropriate aminobenzoic acid was added (1.17 mmol) along with sodium hydroxide (0.016 g, 0.4 mmol) to ensure the complete dissolution of the aminobenzoic acid. These two aqueous solutions were then mixed together vigorously shaken before being allowed to react at room temperature for 4 hours. After this reaction period glucono delta-lactone (GdL) (0.08 g, 0.45 mmol) was added and the solution was shaken until the GdL had completely dissolved, this resulted in the formation of the gel after approximately 2 hours.

In situ R9 gel formation:
This procedure describes the method used in a typical in situ setting of the hydrogel R9 using a solution of 1,3,5-triformylglucinol and the 4-aminophenol.
1,3,5-triformylglucinol (0.082g, 0.39 mmol) was suspended in water (5 ml), to this suspension sodium hydroxide (0.05 g, 1.25 mmol) was added and sonication and gentle heating was used to produce a clear pale yellow solution. To a separate portion of water (5 ml) the 4-aminophenol was added (0.13 g, 1.17 mmol) along with sodium hydroxide (0.016 g, 0.4 mmol) to ensure the complete dissolution of the 4-aminophenol. These two aqueous solutions were then mixed together vigorously shaken before being allowed to react at room temperature for 4 hours. After this reaction period conc. Hydrochloric acid (0.8 ml) was rapidly added to the solution instantly producing a bright orange gel.

Setting gel using the ex situ prepared compound (gels R1 to R8 inclusively):
This procedure describes the method used in a typical setting of the hydrogel at 2 wt% using the previously prepared synthesised compound. The gel ligand (0.1 g) was suspended in water, to this suspension sodium hydroxide was added (0.05 g, 1.25 mmol). Sonication and gentle heating was used to ensure the ligand had completely dissolved giving a solution with a pH of 9 -10. To this solution glucono deltalactone (GdL) (0.08 g, 0.45 mmol) was added and the solution was shaken until the GdL had completely dissolved, this resulted in the formation of the gel after approximately 2 hours.

Setting gel R9 using the ex situ prepared compound:
This procedure describes the method used in a typical setting of the hydrogel R9 at 2 wt% using the previously prepared synthesised compound R9.
The synthesised compound R9 (0.1 g, 0.21 mmol) was suspended in water (5 ml), to this suspension sodium hydroxide was added (0.05 g, 1.25 mmol). Sonication and gentle heating was used to ensure the ligand had completely dissolved giving a solution with a pH of 9 -10. To this solution conc. Hydrochloric acid (0.4 ml) was rapidly added instantly producing a bright orange gel.

Critical gel concentration (CGC):
Critical gel concentration was determined using the inverted vial method in 2 ml sample vials using 0.5 ml of water. This method can often give lower CGC values than are determined using a rheometer due to the low volume of solution and the high surface tension within the vials.

Morphology determination of single amine Gels
The morphology of the gels could be determined using SEM images. The gels were prepared by both the ex situ and in situ in vials before being mounted on carbon covered SEM stubs. The gels were then dried under vacuum for 72 hours before being gold coated. Figure S12. SEM images of gel R1 ex situ preparation method (left) in situ preparation method (right). Figure S13. SEM images of gel R2 ex situ preparation method (left) in situ preparation method (right). Figure S14. SEM images of gel R3 ex situ preparation method (left) in situ preparation method (right).

Gel R4
Figure S15. SEM images of gel R4 ex situ preparation method (left) in situ preparation method (right).

Gel R5
Figure S16. SEM images of gel R5 ex situ preparation method (left) in situ preparation method (right). Figure S17. SEM images of gel R6 ex situ preparation method (left) in situ preparation method (right).

Gel R7
Figure S18. SEM images of gel R7 ex situ preparation method (left) in situ preparation method (right).

Gel R8
Figure S19 SEM images of gel R8 ex situ preparation method (left) in situ preparation method (right).                        The conversion of the initial solution to each of the gels could be observed by monitoring the growth of G over time after the addition of GdL. The results of these time sweep experiments make it possible to determine the Avrami constants for the formation of the gels. The Avrami constants for gels R1 to R9 were calculated to be 1.7, 1.6, 1.9, 1.7, 1.8, 1.7, 2.0, 1.6, respectively.

Rheological characterisation of in situ and
By preforming a time sweep, the data acquired allowed determination of the Avrami constant for gelation sample. The Avrami constant is a useful method of quantifying the kinetics of a phase change. 22 More recently it has been used to describe the sol to gel transition for supramolecular gelation systems. 22 The use of the Avrami equation, which is used to determine the Avrami constant, relies on the general sigmoidal profile for the extent of transformation from one phase to another.

(equation 1.)
The work of Avrami is generally thought of as relating to the formation of bulk crystals, it describes how phase and ordered growth kinetics relate to physical properties such as temperature and concentration. This also means that different morphologies can arise from different growth rates. 22 Liu used the foundations laid by Avrami to develop a method where by the growth of the fibres involved in the formation of the three-dimensional supramolecular gel networks could be characterised in situ. The work of Liu describes the Avrami constant as the fractal dimension, however, throughout this work the term Avrami constant will be used. 22 The general form of the Avrami constant (equation 1.) where k = constant, t = time and Xcr = the systems crystallinity which is equal to φ(t)/φ(∞), where φ(t) is the volume fraction of crystal materials t, and φ(∞) is φ(t) at t → ∞. D describes the dimensions in which the bulk crystal grows, with rod-like growth described by D = 1, plate like crystals described by D = 2 and spherulite growth described by D = 3. By considering the relationship proposed by Einstein between relative viscosity and volume concentration of suspension of spheres 22 it is possible to relate Xcr to specific viscosity ηsp. Equation 2 describes specific viscosity of a system in terms of the complex viscosity η*, and the viscosity of the medium in which the phase change is taking place, η0.
It is possible to define the crystallinity of the solution in terms of η* and η0 with respect to time as can be seen in equation 3.

(equation 3.)
With equation 3 it possible to observe how the viscosity of the solution as a function of time can be used to determine Xcr. Viscosity is a rheological property, with this in mind it is possible to use other rheological material properties to determine Xcr. Although gels do exhibit a relatively high degree of long-range order, they are not strictly crystalline, in most cases. With this in mind, from now on Xcr will be discussed as X, which represents gelation as opposed to crystallinity. With a supramolecular gel's rheological characterisation the elastic modulus G' is representative of the materials elastic, solid-like behaviour. X can be replaced with equation 4, K represents a constant that is related to temperature, t is time and D is the Avrami constant itself. The in situ reaction system discussed here relies on the addition of GdL to set the gel meaning that the term K does not have to be considered. A plot of ln(ln(1/1-X)) against ln(t) will result in a straight line with a gradient equal to D.

Apparent pKas for synthesised gelators R1 -R9
In order to determine the apparent pKa of gelators R1 to R9 a series of titration experiments were performed. 1 mmol of gelator was dispersed in 15 ml of water and dissolved with the addition of NaOH. Further NaOH was added until the pH of the solution had been adjusted to approximately 10. Before aliquots of HCl(aq) were added and the resulting pH recorded.              The apparent pKa is defined as the plateau region where the change in the pH of the solution is minimal. As can be seen for the R1 : R6 mixtures there is a gradual and constant reduction in the apparent pKa from the value of 100% R1 to that of 100% R6. This shows that both gelators influence the apparent pKa region collectively allowing co-assembly to occur given the correct gelation conditions.

NMR of R1 : R6 gelator co-assembly process
A solution was created by dissolving 25 µmol of R1 and 25 µmol of R6 in D2O with the addition of NaOH and a spectra was obtained. 10 µmol of GdL was then added and a spectra was recorded every five minutes for a total of two hours and over a range of 6 -9 ppm. Examination of the integrations of peaks (determined against the internal standard of 4-aminobenzoic acid) that relate to the aromatic protons of both R1 and R6 allowed the plot shown in Figure S64 to be produced. The initial measurement at time zero (before the addition of GdL) was used as the reference with which to normalise the data.   Step in the plateau of R1 and R10 (50 : 50) mix shows a stepwise self-sorting co-assembly process.

NMR of R1 : R10 gelator self-sorting process
A solution was created by dissolving 25 µmol of R1 and 25 µmol of R10 in D2O with the addition of NaOH and a spectra was obtained. 10 µmol of GdL was then added and a spectra was recorded every five minutes. The same procedure described for the R1 and R6 mixed samples, section 7.5, was utilised. Figure S68. Self-sorting experiment of gelators R1 and R10 over time as determined by 1 H NMR. The two compounds are not disappearing through self-assembly at the same rate as the pH is changing, nor have they had the same initiation point (5 mins vs 15mins).  . All possible reaction products for the in situ R1 + R10 + II, including the known molecules R1 and R10, and products composed of mixed amines R1 2 ,10 1 and R1 1 ,10 2 . Chemical analyses highlight a chemical self-sorting of these compounds resulting in only the pure R10 and R1 species during in situ reactions. R11 variants would be the methyl derivatives of the four compounds shown above.