Stereochemical Behavior of Pyrrolo-Pyrazole Peptidomimetics Promoting Phase-Selective Supramolecular Organogels

Supramolecular gels were developed by taking advantage of an assembly of small dipeptides containing pyrrolo-pyrazole scaffolds. The dipeptides were prepared through a robust and ecofriendly synthetic approach from the commercially available starting materials of diazoalkanes and maleimides. By playing with the functionalization of the scaffold, the choice of the natural amino acid, and the stereochemistry, we were able to obtain phase-selective gels. In particular, one peptidomimetic showed gelation ability and thermoreversibility in aromatic solvents at very low concentrations. Rheology tests showed a typical viscoelastic solid profile, indicating the formation of strong gels that were stable under high mechanical deformation. NMR studies were performed, allowing us to determine the conformational and stereochemical features at the base of the supramolecular interactions.


Introduction
The self-assembly of small peptides has received great attention in nanoscience and nanotechnology [1][2][3].These molecules can be easily produced at a large scale and their spontaneous assembly can lead to the formation of ordered architectures, from nanotubes to spheres, micelles, and fibers.This hierarchical process is driven by complex, highly specific non-covalent interactions including hydrogen bonding, van der Waals and electrostatic forces, hydrophobic interactions, and the π-π stacking of aromatic rings [3].Although several functional supramolecular structures have been reported in the last few decades, their design and preparation are still a key research interest.Furthermore, the possibility of introducing both natural and non-coded amino acids in the sequence could expand the scope of possible applications [4].
A particularly intriguing topic is the development of supramolecular gels, in which semi-solid materials are formed by entrapping large amounts of solvents in ordered 3D networks formed by the assembly of small molecules and peptides [5,6].
Due to the reversible nature of non-covalent interactions, the obtained supramolecular gels are highly sensitive and dynamic in nature [7,8].When the entrapped solvent is an organic one, organogels are formed, and when 1% or less of the entrapping compound is needed for their formation, this compound is called a super-organogelator [9].One profitable feature of an organogelator is the ability to induce the phase-selective gelation (PSG) of the organic phase from non-miscible solvents with water.The first report on an organic compound able to selectively gelate the oil phase from a two-phase mixture of water and oil was published by Bhattacharya and Ghosh in 2001 [10].They exploited N-lauroyl alanine amphiphile, which selectively assembled into a fibrous network within the oil phase, leading to PSG.Afterwards, several other molecules with PSG properties were developed, of water and oil was published by Bhattacharya and Ghosh in 2001 [10].They exploited N-lauroyl alanine amphiphile, which selectively assembled into a fibrous network within the oil phase, leading to PSG.Afterwards, several other molecules with PSG properties were developed, finding various applications such dye absorption and oil-spill recovery [11].In particular, PSGs showing gelation ability in aromatic solvents have been actively investigated, as they could find application in minimizing environmental pollution [12][13][14].In this context, a challenging task is the development of PSGs enabled to work at room temperature, as the majority of them require a high-temperature trigger or the presence of an organic co-solvent to be efficient.
Only a few examples of the use of small peptides and peptidomimetics for PSG are reported in the literature.Their use is becoming of large interest due to their ability to generate well-defined secondary structures [15,16], as well as the possibility of introducing specific substituents to stabilize π,π interactions [17][18][19] or induce pH responsiveness [20].All of these features allow the provision of molecules characterized by the structural diversity necessary to finely control the physical properties of products.On the other hand, the exploitation of peptidomimetics as supramolecular gelators could be hampered by their sometimes-difficult synthesis.As a result, there is a growing interest in obtaining gelator molecules through few synthetic steps, and in using non-expensive starting materials [21,22].
Continuing our research on non-coded amino acids able to generate specific secondary structures when inserted into peptides [23] and enhancing the spectrum of fundamental materials [24], we recently synthesized 3-carboxylic-pyrrolo-pyrazole derivatives 3 (Scheme 1), which can be considered as mimetics of γ-amino acid [25].This versatile scaffold can be obtained in one step from commercially available compounds and can be easily decorated at the carboxylic function, at the-nitrogen of the imide function and of pyrazoline, affording a small library of compounds.Of relevance, their functionalization with Gly makes peptidomimetics able to form a homogeneous fibrous matrix by electrospinning.Taking advantage of the above feature of pyrrolo-pyrazole scaffold 3, here we aimed to expand the scope of its application by developing PSG containing this scaffold.Accordingly, a small library of peptidomimetics 4 (Scheme 1) characterized by a wide chemical variability was synthesized, allowing shifting from electrospun fibers to supramolecular gelators able to entrap aromatic solvents.Taking advantage of the above feature of pyrrolo-pyrazole scaffold 3, here we aimed to expand the scope of its application by developing PSG containing this scaffold.Accordingly, a small library of peptidomimetics 4 (Scheme 1) characterized by a wide chemical variability was synthesized, allowing shifting from electrospun fibers to supramolecular gelators able to entrap aromatic solvents.
Pyrrolo-pyrazole-containing gels were fully characterized by studying their molecular and supramolecular structures, morphology, and chemical-physical behavior.Rheological experiments were performed to assess the mechanical and thermal stability of organogels formulated with different organic solvents.NMR studies were performed to understand the conformational and stereochemical features at the base of supramolecular interactions.
Transmission and scanning electron microscopy were used to emphasize the presence of certain conformations.
Finally, the gelators' efficiency at inducing the selective gelation of the organic phase into an aqueous/organic mixture was evaluated.Interestingly, compound 5b (Scheme 1) underwent gelation at room temperature without changes in the water phase amount.This is an added value of our gelators, since one of the main limitations of some PSGs described in the literature is related to the need for heating and cooling cycles to induce the gelation, making this protocol troublesome for the removal of high flammability in most oil phases [17,26].
All compounds were characterized by NMR.Specifically, diastereoisomers 5/5 ′ (CDCl 3 , 400 MHz) showed significant differences: the ∆δ values of H 3a /H 6a were found to be ≥1 for 5 and <1 for the 5 ′ series.NH Ala resonates to a higher field for 5 with respect to 5 ′ .A different trend was detected for the Me group of the Ala side chain.

Gelation Studies
The gelation phenomenon is the equilibrium state between dissolution and phase separation.In a gel system, the intermolecular forces are balanced.None of the compounds formed gels in water, while different results were obtained in organic solvents depending on the substitution patterns on the scaffold.The gelation results in different solvents at room temperature are reported in the Supporting Information: compound 4 (Table S1), diastereoisomers 5a,c,d/5 ′ a,c,d (Table S2), 5b (Table S3), and 5 ′ b (Table S4).Compounds 4a-d, all containing Gly, moiety failed in giving a gel (Table S1).On the other hand, diastereoisomers 5/5 ′ containing Ala afforded promising results.The most interesting compound was 5b, containing the Fmoc group on Ala, the tBu ester, and the N-phenyl as substituents on the pyrrolo-pyrazole core, whose gelation ability was deeply investigated (Table S3).Concerning aliphatic solvents, gels were formed only in mixtures of hexane/AcOEt (6:4) or tBuOMe (2% w/v), while 5b in Et 2 O (1% w/v) provided a weak gel not suitable for any purpose.When the amount of 5b increased, a partial dissolution was observed.On the other hand, all the tested aromatic solvents provided gels except for chloro-benzene, where 5b was not fully soluble at the tested concentrations.The best results were achieved in toluene/dioxane (10:1), benzene, and m-xylene, using a substrate concentration of 2% w/v that led to a gel.Curiously, the diastereoisomer 5 ′ b was soluble in all the tested conditions except for hexane (Table S4), without giving a gel, indicating that the stereochemistry of the bicyclic ring plays a crucial role in gel formation.

Rheology Experiments and Thermo-Reversibility
Rheology experiments were performed on 5b-based supramolecular gels obtained from toluene/dioxane 10:1, xylene, and benzene (2% w/v of substrate for all solvents; Figure 1).investigated (Table S3).Concerning aliphatic solvents, gels were formed only in mixtures of hexane/AcOEt (6:4) or tBuOMe (2% w/v), while 5b in Et2O (1% w/v) provided a weak gel not suitable for any purpose.When the amount of 5b increased, a partial dissolution was observed.On the other hand, all the tested aromatic solvents provided gels except for chloro-benzene, where 5b was not fully soluble at the tested concentrations.The best results were achieved in toluene/dioxane (10:1), benzene, and m-xylene, using a substrate concentration of 2% w/v that led to a gel.
Curiously, the diastereoisomer 5′b was soluble in all the tested conditions except for hexane (Table S4), without giving a gel, indicating that the stereochemistry of the bicyclic ring plays a crucial role in gel formation.

Rheology Experiments and Thermo-Reversibility
Rheology experiments were performed on 5b-based supramolecular gels obtained from toluene/dioxane 10:1, xylene, and benzene (2% w/v of substrate for all solvents; Figure 1).To elucidate the self-assembly mechanism of 5b responsible for gelation, the organogel was observed by transmission electron microscopy (TEM).A two-fold dilution was carried out to facilitate the visualization.Figure 2 shows the formation of selfassembled interconnected fibrillar networks.The variable thickness of the fibrils was determined from 15 to 50 nm with variable length.Similar nanotubular bundles were observed for 5b organogels in benzene (Figure 2A,B) and in toluene/dioxane (Figure 2C,D).To elucidate the self-assembly mechanism of 5b responsible for gelation, the organogel was observed by transmission electron microscopy (TEM).A two-fold dilution was carried out to facilitate the visualization.Figure 2 shows the formation of self-assembled interconnected fibrillar networks.The variable thickness of the fibrils was determined from 15 to 50 nm with variable length.Similar nanotubular bundles were observed for 5b organogels in benzene (Figure 2A,B) and in toluene/dioxane (Figure 2C,D).SEM images (Figure 3) of the dried organogels revealed a closely knit network of fibers, each exhibiting different thicknesses owing to the self-assembly ability of 5b.Both TEM and SEM analyses underscored the gelation process of 5b, demonstrating its ability, mediated by the pyrrole-pyrazole scaffold, to form fiber bundles with varying  Amplitude sweeps are generally used to measure the linear viscoelastic region of a sample (LVER).In the LVER, the storage and loss moduli are independent of the applied deformation.The tested samples confirmed the gel-like behavior, since the storage modulus (G') referring to the elastic properties of the material was always higher than the loss modulus (G″) correlated to the viscous characteristics.
The amplitude sweep for 5b showed that the viscoelastic region was between 0.1 and Amplitude sweeps are generally used to measure the linear viscoelastic region of a sample (LVER).In the LVER, the storage and loss moduli are independent of the applied deformation.The tested samples confirmed the gel-like behavior, since the storage modulus (G ′ ) referring to the elastic properties of the material was always higher than the loss modulus (G ′′ ) correlated to the viscous characteristics.
The amplitude sweep for 5b showed that the viscoelastic region was between 0.1 and 1 Pa stress and 0.001 and 0.5% strain (Figure 4).Strain/stress from within the linear region was used for all subsequent dynamic tests.Considering 5b-based organogels, their mechanical strength was evaluated by comparing the values of G ′ in the LVER.All the samples were gel since G ′ differs from G ′′ by one order of magnitude.The results were influenced by the organic solvent used.Organogels with benzene (x) had higher G ′ values compared to 5b dissolved in xylene ( ) and in the mixture of toluene/dioxane (∆), indicating a higher mechanical strength.Figure 4B-D show that with a gradual increase in strain, the values of both G ′ and G ′′ are unchanged and get deviated from the linearity beyond a certain point (from 0.5 to 2% strain).The presence of a cross-point of G ′ and G ′′ at high strain indicates that the organogel is broken.Close cross-points were observed for 5b-based gels in xylene and toluene/dioxane, with 4-6% and 7-12% strains, respectively.Higher cross-points were observed for organogel in benzene (28-37% strains).A high cross-point is commonly observed for other supramolecular gels in the literature [28,29], which is attributed to the highly dynamic nature of these self-assembled systems.24, 10, x FOR PEER REVIEW 7 mechanical strength was evaluated by comparing the values of G' in the LVER.A samples were gel since G' differs from G″ by one order of magnitude.The results influenced by the organic solvent used.Organogels with benzene (x) had higher G' v compared to 5b dissolved in xylene (◯) and in the mixture of toluene/dioxane indicating a higher mechanical strength.Figure 4B-D show that with a gradual inc in strain, the values of both G' and G" are unchanged and get deviated from the line beyond a certain point (from 0.5 to 2% strain).The presence of a cross-point of G' an at high strain indicates that the organogel is broken.Close cross-points were observe 5b-based gels in xylene and toluene/dioxane, with 4-6% and 7-12% strains, respecti Higher cross-points were observed for organogel in benzene (28-37% strains).A cross-point is commonly observed for other supramolecular gels in the literature [2 which is attributed to the highly dynamic nature of these self-assembled systems.The oscillation frequency sweep test involves oscillating the sample at fixed s (0.5 Pa) while varying the frequency.Since this test is performed in the linear viscoe region, where structural integrity is not affected, a frequency sweep is considere fingerprint spectrum of the material and is a very characteristic measurement of a sam Depending on the type of material, three typical frequency responses can be reve visco-elastic solid, gel-like, and visco-elastic liquid [30].As observed in Figure 5 storage modulus for 5b was always higher than the loss modulus for all the investig solvents, denoting that the elastic character is always dominant when a load is app Considering all this evidence, 5b shows a gel-like behavior.This type of sample ca The oscillation frequency sweep test involves oscillating the sample at fixed stress (0.5 Pa) while varying the frequency.Since this test is performed in the linear viscoelastic region, where structural integrity is not affected, a frequency sweep is considered the fingerprint spectrum of the material and is a very characteristic measurement of a sample.Depending on the type of material, three typical frequency responses can be revealed: viscoelastic solid, gel-like, and visco-elastic liquid [30].As observed in Figure 5, the storage modulus for 5b was always higher than the loss modulus for all the investigated solvents, denoting that the elastic character is always dominant when a load is applied.Considering all this evidence, 5b shows a gel-like behavior.This type of sample can be classified as strong or weak depending on the relationship between the elastic modulus and the applied frequency [31].A low dependence of elastic modulus on frequency was found for all the gels.This trend is commonly observed for strong gels that manifest the typical behavior of viscoelastic solids that rupture rather than flow above a critical deformation value [32].This was also confirmed by the visual examination of the gels; at large deformations, 5bbased organogels ruptured and failed without becoming liquid, and they were not able to recover.As observed before, 5b-based organogels derived from benzene showed the highest strength with G ′ values higher than 140 kPa, regardless of the frequency.
OR PEER REVIEW 8 of 20 benzene showed the highest strength with G' values higher than 140 kPa, regardless of the frequency.We then investigated the thermoreversibility of the gels obtained by 5b (2% w/v; Table 1).Different melting temperatures and behaviors were observed depending on the solvent.Gel in benzene (entry 1) becomes a solution at 96 °C.Likewise, gels in m-xylene at 136 start to change to a viscous liquid (entry 2); neither are thermoreversible.Operating in toluene/dioxane (entry 3), a transition from gel to solution was observed at 60 °C.As the system cooled to room temperature, the solution became a gel within 10 min.Accordingly, compound 5b is able to give a thermoreversible gel upon heating and returns to gel upon cooling.Rheological changes in 5b gels in toluene/dioxane (10:1; 2%v/w) across a temperature ramp were evaluated by a temperature sweep test (Figure 6).Temperature was increased from RT to 70 °C at a rate of 2 °C/min, and a constant frequency of 1 Hz and a strain percentage of 0.01% were used.As shown in Figure 5, the 5b organogel in toluene/dioxane underwent gel-sol transition at 65-68 °C.We then investigated the thermoreversibility of the gels obtained by 5b (2% w/v; Table 1).Different melting temperatures and behaviors were observed depending on the solvent.Gel in benzene (entry 1) becomes a solution at 96 • C. Likewise, gels in mxylene at 136 start to change to a viscous liquid (entry 2); neither are thermoreversible.Operating in toluene/dioxane (entry 3), a transition from gel to solution was observed at 60 • C. As the system cooled to room temperature, the solution became a gel within 10 min.Accordingly, compound 5b is able to give a thermoreversible gel upon heating and returns to gel upon cooling.Rheological changes in 5b gels in toluene/dioxane (10:1; 2% v/w) across a temperature ramp were evaluated by a temperature sweep test (Figure 6).Temperature was increased from RT to 70 • C at a rate of 2 • C/min, and a constant frequency of 1 Hz and a strain percentage of 0.01% were used.As shown in Figure 5, the 5b organogel in toluene/dioxane underwent gel-sol transition at 65-68 • C.

Phase-Selective Gelation (PSG)
An ideal phase-selective gelator (PSG) should exhibit organogelator skill at room temperature, water insolubility, and high mechanical rigidity in a gelled organic solvent.The gelation features of 5b in various aromatic solvents motivated us to investigate whether it is a good candidate as a PSG for realistic applications in the separation and recovery of aromatic solvents.The PSG ability of compound 5b was investigated by adding different percentages of compound 5b to a mixture of organic solvent/water with (Method A) or without (Method B) shaking, and then keeping the mixture at 25 °C overnight (Table 2).At the organic-to-water volume ratio of 1:1, weak gel was formed at the interface of the water/organic layers.A residual solvent was observed and measured (Table 2, entries 1, 2, and 6) and the content of the organic phase in the gel was higher than 50% (v/v).The obtained organogel showed different morphological features compared to the organogel prepared in pure organic solvent.However, a complete PSG was achieved by adding 10 mg of 5b in a biphasic mixture of benzene/water and m-xylene/water using a volume ratio of 1:5 (v/v) (Table 2, entries 5 and 8).No alteration in the water phase was observed.It can be speculated that the presence of water can interfere with gel formation and that a higher concentration of 5b was needed.This mismatch has already been observed elsewhere in the literature, albeit with inconsistent findings [20].

Phase-Selective Gelation (PSG)
An ideal phase-selective gelator (PSG) should exhibit organogelator skill at room temperature, water insolubility, and high mechanical rigidity in a gelled organic solvent.The gelation features of 5b in various aromatic solvents motivated us to investigate whether it is a good candidate as a PSG for realistic applications in the separation and recovery of aromatic solvents.The PSG ability of compound 5b was investigated by adding different percentages of compound 5b to a mixture of organic solvent/water with (Method A) or without (Method B) shaking, and then keeping the mixture at 25 • C overnight (Table 2).At the organic-to-water volume ratio of 1:1, weak gel was formed at the interface of the water/organic layers.A residual solvent was observed and measured (Table 2, entries 1, 2, and 6) and the content of the organic phase in the gel was higher than 50% (v/v).The obtained organogel showed different morphological features compared to the organogel prepared in pure organic solvent.However, a complete PSG was achieved by adding 10 mg of 5b in a biphasic mixture of benzene/water and m-xylene/water using a volume ratio of 1:5 (v/v) (Table 2, entries 5 and 8).No alteration in the water phase was observed.It can be speculated that the presence of water can interfere with gel formation and that a higher concentration of 5b was needed.This mismatch has already been observed elsewhere in the literature, albeit with inconsistent findings [20].
To visualize the PSG, 5b (40 mg) was added to benzene (400 µL) and water (2000 µL).The gelation of the organic solvent compared to a biphasic system of water and organic solvents is shown in Figure 7. Interestingly, without shaking, 5b underwent gelation at RT, forming a more compact organogel at RT without any residual organic solvent (Figure 7A).To visualize the PSG, 5b (40 mg) was added to benzene (400 μL) and water (2000 μL).The gelation of the organic solvent compared to a biphasic system of water and organic solvents is shown in Figure 7. Interestingly, without shaking, 5b underwent gelation at RT, forming a more compact organogel at RT without any residual organic solvent (Figure 7A).
As shown in Table 3, a strong up-shift in all proton resonances was found by changing the solvent at 298 K from CDCl 3 to a CDCl 3 /C 6 D 6 mixture and C 6 D 6 alone, except for CH Ala and CH 2Fmoc , which were down-shifted.Of relevance, it was observed that H-1 and H-8 of the symmetric fluorenyl scaffold were split in C 6 D 6 (Figure S1), but not in CDCl 3 , indicating a different environment for these two protons.Furthermore, Ph-protons were also strongly up-shifted in benzene.These results suggest that a different conformation is adopted by 5b in the two solvents.Indeed, two possible conformers can be present in solution due to the presence of a H-bond between NH and the nitrogen of C=N in the pyrazoline ring (six-member ring, conformer I, Figure 6) or the C=O of imide (δ-turn, conformer II, Figure 8).Interestingly, by changing the concentration of 5b from 1% to 2% w/v in C 6 D 6 (298 K), an up-shift of all protons was detected.This result, together with temperature-dependence experiments (see below), is of relevance when studying the driving force of the sol-gel process [34].
The NMR of 5b in the toluene d8 /dioxane d8 (10:1; 1%) mixture showed resonances in between those observed in CDCl 3 and C 6 D 6 , except for a strong down-shift in NH Ala (Table 3).No significant changes were detected by increasing the concentration of 5b (2%; Table 3).
Variable-temperature NMR experiments were then performed to investigate the potential NH involvement in H-bonds [35].Low ∆δ/∆T values (1% CDCl 3 ; 273-328 K; Figure S2) were detected for the NH Ala of isomer 5b (−2.1 ppb/K), suggesting the formation of an intramolecular H-bond.Focusing on the spectrum at 273 K, it should be pointed out that only a trace amount of a second conformer was detectable, indicating the formation of a stable conformer.
Considering the possibility of 5b forming a gel in C 6 D 6 at 2%, we focused on a systematic temperature-dependent NMR study, since it is well known that H-bonding is one of the major driving forces in organogelation for low-molecular-weight gelators [14].In the first experiment, a clear solution of the fresh prepared sample of 5b (2%) was recorded (Figure S3).A single conformer was detectable at all temperatures.No significant changes in chemical shifts were detected at the different temperatures.Clear broad signals were those of aromatic protons at 298 K that became sharp after heating.A similar behavior was observed for aliphatic protons, except for CH Ala , which was broad at all temperatures.The very low ∆δ/∆T value for NH Ala (−0.67 ppb/K) has to be pointed out, indicating the formation of a strong intramolecular H-bond.Focusing on S,S-5′b, similar results to 5b were observed: the change in the solvent from CDCl3 to C6D6 strongly affects the chemical shifts, as reported in Table S6.Furthermore, differently from 5b in the same solvent, two main conformers were detected in C6D6.In the second experiment, the sample was prepared the day before to ensure the formation of a gel.The spectra of 5b in C 6 D 6 were recorded from 298 to 348 K (Figure S4, Table S5), where the dissolution of the gel was observed.Then, the sample was cooled from 348 to 283 K, where the gel was obtained again (Figure S5, Table S5).Finally, the 1 H NMR of this last sample was recorded after 24 h at 298 K (Figure S6).
Compared to the freshly prepared sample, in which the chemical shift of H Ala , NH Ala , and H 6a were well defined (δ 5.24-5.26region; Figure S3), a different behavior was detected when starting from the gel (Figures S4 and S5).In this last case, NH and H 6a signals were very close, resonating in the δ 5.16-5.06region.The H Ala signal was isolated at 298 K but overlapped with the other signals when heating.Even if it is impossible to exactly define the NH resonance at 348 K, overall, the ∆δ/∆T value cannot be more than 1.4 ppb/K, indicating the formation of a strong intramolecular H-bond.The presence of intermolecular H-bonds is excluded since the example in the literature showed values higher than 5 ppb/K when intermolecular H-bonds were at the base of the formation of the gel [11,14].
As for the signal shape, a loss of resolution was found when lowering the temperature, as shown in Figures S5 and S6, but mostly in Figure S7.Focusing on the aromatic region when heating, not only was a shift in aromatic protons detected (Table S5), but so was a strong change in their resolution, mostly for the Fmoc group.All the above data indicate the transition of the gel-sol process, confirming the trend reported in the literature for gels [14,33,34].
Focusing on the experiment in toluene d8 /dioxane d8 at variable temperatures (from 298 to 363 K; Figure S7) of a sample of 5b recorded 24 h after its preparation, as expected, a change in the state of the sample and an increase in the resolution of the signals, as well as their resonances, were detected.A ∆δ/∆T value of −7.5 ppb/K was detected, excluding the formation of an intramolecular H-bond.On the other hand, the formation of an intermolecular H-bond cannot be excluded.
In a second step, NOESY analyses were performed, helping us to obtain information on the conformation of compound 5b according to the intramolecular H-bond formation detected with the experiment at variable temperatures in different solvents, and to define the absolute stereochemistry at C 3a and C 6a (Figures S8-S12).The complete set of chemical shifts and NOEs for 5b in the different solvents is reported in Figures 6 and S1.Operating in CDCl 3 (1%, 293 K; Figure S8), the following spatial proximities were found: (i) Me Ala with both NHAla and HAla, and (ii) H 3a with both tBu and H 6a , and those in between the protons of the Fmoc group.NH Ala showed a weak NOE with aromatic H Fmoc (δ 7.62).In a recent work, recording the spectrum of compound 5e (CDCl 3 ; [25] Figure 5), an analogous form of 5b, we found that NH Ala showed spatial proximity with the ethyl ester (not detected for 5b, in which a tBu-ester is present).Accordingly, we propose the formation of a six-member ring due to a H-bond of NH with C=N in the triazoline ring (conformer I, Figure 5).The involvement of C=N in the isooxazoline ring as a H-bond acceptor has already been observed [34].Similar NOEs were found for compound 5b in 1% CDCl 3 /C 6 D 6 (1:1; Figure S9).Performing the NOESY experiment in C 6 D 6 (1%; Figure S10A), similar results were achieved as in CDCl 3 , except for the spatial proximity between Me Ala and H 6a (Figure S10B).This last result is consistent with the formation of a δ-turn stabilized by a H-bond between NH and the carbonyl of imide (conformer II, Figure 6).Of relevance, this NOE allowed us to define the absolute R,R-stereochemistry for diastereomer 5b, first eluted in column chromatography (EtOAc/hexane).
The NOESY experiment in toluene/dioxane gave similar results as in CDCl 3 (Figure S10A), except for the medium spatial proximity of both H 6a and H 3a with aromatic protons in the 7.07 region (Figure S10B).Since there is no intramolecular spatial proximity between H 6a and H 3a and the Ph ring, resonating in the above region, it must be assumed that intermolecular NOEs occurred with toluene or the Ph-group of a second molecule of 5b.These spatial proximities were not observed in CDCl 3 and C 6 D 6 , confirming again the different behavior of 5b in the two solvents.
Focusing on S,S-5 ′ b, similar results to 5b were observed: the change in the solvent from CDCl 3 to C 6 D 6 strongly affects the chemical shifts, as reported in Table S6.Furthermore, differently from 5b in the same solvent, two main conformers were detected in C 6 D 6 .
The NOESY experiment of S,S-5 ′ b in CDCl 3 (293 K; Figure S15) showed similar results to those observed for 5b, but with stronger spatial proximities between the fluorenyl group and both CH Ala and NH Ala (Figure S15A).Furthermore, a very weak NOE between the tBu group and CH Ala was detected (Figure S15B), confirming the formation of the NH-bond with C=N, i.e., conformer I in Figure S12, as well as the behavior of these compounds in CDCl 3 in forming the six-pseudo ring by H-bond stabilization.
A more complex NOE set was found in 6 D 6 (293 K; Figure S16) due to the presence of a couple of conformers, also at 293 K, and several overlapped signals.Similar spatial proximities observed for II-5b were detected for the main conformer II-5 ′ b.As expected, no spatial proximity was found between Me Ala and H 6a .Due to the similar chemical shift of H Ala/ H 6a , we cannot define possible spatial proximities.The second isomer is probably consistent with I-5 ′ b.Experiments at 283 K were also performed, giving a better definition of the two conformers (see COSY, Figure S17).Unfortunately, the NOESY experiment showed an equilibrium between the two conformers, preventing information on the spatial proximity between the aliphatic protons (Figure S18).
In summary, the conformation of peptidomimetics 5 and 5 ′ is strongly affected by the solvent, with the stability of conformer I confirmed for both isomers R,R-5b and S,S-5 ′ b in CD 3 Cl.On the other hand, conformer II is stable in C 6 D 6 for R,R-5b, but an equilibrium between I and II for S,S-5 ′ b was observed in the same solvent.

Conclusions
A small library of peptidomimetics 4 and 5/5 ′ , containing a pyrrolo-pyrazole scaffold and, respectively, Gly and Ala natural amino acids, was prepared in two steps from easily available compounds.Compound 5b was found to be a low-molecular-weight organogelator, giving good performances mostly in aromatic solvents, and being a thermoreversible gel in toluene/dioxane.It was also able to induce phase-selective gelation.The gelation process reflects the self-assembly ability of the peptidomimetics into fibers, forming a network where the organic solvent is entrapped.
The rheological properties of 5b in different solvents were analyzed.An amplitude sweep test was carried out to determine the LVER of the organogels, as well as to evaluate the gels' strengths.Frequency sweep tests confirmed that the strength of the organogels was affected by the organic solvent.
Moreover, 5b organogels showed a typical viscoelastic solid profile and they were found to be strong gels since, upon high mechanical deformation, they collapsed without becoming a liquid.Based on the organic solvent used for the gelation, a different stress point was revealed: gel in benzene breaks at the highest strain percentage (28-37% strain), indicating a highly dynamic self-assembly.Gels made in toluene/dioxane lose their structure at 7-12% strain, showing a gel-sol transition at 65-70 • C, as confirmed by rheological measurements.
NMR studies supported the mechanism of gel formation in aromatic solvents through supramolecular interactions, as the resonances shift by changing the concentration of the sample, as well as broadening and decreasing the intensity of aromatic peaks at low temperature.Several considerations can be made on the effect of the functionalization pattern of the scaffold toward gel formation.First, it was revealed that the presence of Ala, as well as the R,R stereochemistry of the pyrrolo-pyrazole scaffold in combination with aromatic solvents, are important for gel formation.The contribution of the aromatic groups, giving π-π interactions, is fundamental to trigger self-assembly.It was indeed found that the Ph group is better than the benzyl one, probably due to its coplanarity with the pyrrolidindione system, as well as the Fmoc group, with respect to Boc.Furthermore, bulky lipophilic alkyl substituents (tBu vs. Et and Me Ala vs. H Gly ) were favorable in the formation of the gels.
The aromatic solvent plays a key role in the formation of the gels: a stable δ-turn structure, due to the formation of a very strong intramolecular H-bond in benzene affording conformer II (Figure 6), is formed.This formation allows the orientation of the lipophilic groups (alkyl group of the ester and Me Ala ) involved in the Van der Waals interactions on the same side and the aromatic ones (Fmoc and imide substituent) on the opposite side.On the other hand, no intramolecular H-bonds were detected for 5b in toluene/dioxane, but intermolecular interactions of the bicyclic ring with aromatic moieties were detected.
Finally, 5b was demonstrated as an organogelator, and it could be of potential interest in several applications, such as environmental ones [36,37].

General
NMR spectroscopic experiments were carried out, either on Varian MERCURY 300 MHz (300 and 75 MHz for 1 H and 13 C, respectively) or Bruker Avance I 400 MHz spectrometers (400 and 101 MHz for 1 H and 13 C, respectively).Chemical shifts (δ) are given in ppm relative to the CHCl 3 internal standard, and the coupling constants J are reported in Hertz (Hz).Mass spectra were recorded on an LCQESI MS, on a LCQ Advantage spectrometer from Thermo Finningan, and a LCQ Fleet spectrometer from Thermo Scientific.Optical rotations were measured on a PerkinElmer 343 polarimeter (concentration in g/100 mL).Diazoalkanes 1a-c, maleimides 2a,b, and protected amino acids are commercially available compounds.

Gelation Test
Peptide powders were first weighed into a screw-capped glass vial (4 cm length and 1 cm diameter), as described in the literature [17].An aliquot (0.5 mL) of different solvents was directly added onto the peptide powder to reach the desired concentration listed in Table 1 and Tables S1-S4 (Supporting Information).The mixture was vortexed for 2 min, sonicated for 20 min, and then heated in order to obtain a homogenous solution, before being left overnight at 25 • C. If not otherwise stated, the inversion test was carried out.The as-prepared material was preliminary classified as a "gel" if it did not exhibit gravitational flow upon turning the vial upside-down at room temperature.This state was further confirmed by oscillatory rheological measurements.
The morphological study of the gels was investigated by transmission electron microscopy at an accelerating voltage of 200 kV (JEOL-JEM-1200-EXII). Gel samples were diluted 2-fold, and they were deposited onto a TEM grid and then dried before imaging.First, 5b-based gels were prepared using the method described above with benzene, mxylene, and the toluene/dioxane mixture.To gain a visual insight into the gels' structures, high-resolution SEM analysis (Mira3 Tescan, Czech Republic) was carried out.Small samples of gels were dried at high temperatures of 80-85 • C under vacuum.Samples were then mounted on the support and coated with graphite, and images were taken at a low voltage of 5 kV.
The gel prepared from 5b (20 mg) in the solvent (1 mL) was loaded (around half the amount) with a spatula with a plate-plate geometry of PU20:PL61 (20 mm upper-plate diameter) and a gap of 1 mm.A solvent trap was used to minimize solvent evaporation during all tests.Data processing was recorded with rSpace software version 1.50 (Malvern Panalytical, Monza, Italy)..The viscoelastic properties were assessed by measuring the storage modulus (G ′ ) and loss modulus (G ′′ ), representing the elastic and viscous behaviors of the material, respectively.The amplitude sweep test was carried out at a constant frequency of 1 Hz with a shear strain (%) from 0.001 to 100% at 25 • C to determine if the material had a linear viscoelastic region, and for which stress values the structure began to break down.
Frequency sweep tests were performed to measure the viscoelastic responses of the samples at 25 • C in an oscillation mode (10-0.1 Hz) at constant stress (0.5 Pa).The results show the viscoelastic responses of the samples at different frequencies.
All tests were performed in triplicate, and mean values are reported in logarithmic graphs (sd < 5%).T gel values were usually determined by the inverse flow method.A sealed vial containing the organogel was soaked into an oil bath, which was heated up at 2 • C/min.Tgel was defined as the temperature at which the gel started to break.Each measurement was made in duplicate and the average values are reported.The thermal gel-to-sol transition was evaluated by rheological analysis (PU20:PL61; 20 mm upper-plate diameter with a gap of 1 mm).Then, the 5b organogel (0.5 mL) was transferred with a spatula onto the lower plate at RT.The temperature was increased from 25 to 70 • C at 2 • C/min.Oscillation frequency and strain percentage were kept constant at 1 Hz and 0.01% strain (within the LVER).The mean values of G ′ and G ′′ are reported in logarithmic graphs against the temperature (sd < 5%).

Phase-Selective Gelation (PSG)
A weighed amount of the corresponding peptide (20 mg) was added to a biphasic mixture of water and an appropriate organic solvent, changing the concentration according to Table 3.The selective gelation of the organic phase was tested in situ with shaking (Method A) or without shaking (Method B).The presence of residual aromatic solvent not undergoing gelation was assessed by pipetting the liquid, and the relative volume was measured.Then, gelation was verified by the complete absence of gravitational flow upon turning the vial upside-down.
Organic phase: gelled Water phase: solution H2O (500 μL) a Method A: without shaking; b Method B: with shaking.c Organic solvent completely trapped in the gel.

Table 2 .
PSG studies of 5b in water with different aromatic solvents and concentrations.

Table 2 .
PSG studies of 5b in water with different aromatic solvents and concentrations.
a Method A: without shaking; b Method B: with shaking.c Organic solvent completely trapped in the gel.