Low-Molecular-Weight Organogelators Based on N-dodecanoyl-L-amino Acids—Energy Frameworks and Supramolecular Synthons

Lauric acid was used to synthesize the low-molecular-weight organogelators (LMOGs), derivatives of two endogenous (L)-alanine, (L)-leucine, and three exogenous (L)-valine, (L)-phenylalanine, and (L)-proline amino acids. The nature of processes responsible for the gel formation both in polar and in apolar solvents of such compounds is still under investigation. Knowing that the organization of surfactant molecules affects the properties of nano scale materials and gels, we decided to elucidate this problem using crystallographic diffraction and energy frameworks analysis. The single crystals of the mentioned compounds were produced successfully from heptane/tBuOMe mixture. The compounds form lamellar self-assemblies in crystals. The energetic landscapes of single crystals of a series of studied amphiphilic gelators have been analyzed to explore the gelling properties. The presented results may be used as model systems to understand which supramolecular interactions observed in the solid state and what energy contributions are desired in the designing of new low-molecular-weight organic gelators.


Introduction
Pharmaceutical gels are willingly applied in pharmaceutical formulations where active pharmaceutical ingredients (API) are combined to yield a final medicinal product of well-defined properties. The stability of the obtained formulation is a crucial issue in the pharmaceutical and cosmetics industries [1]. Stability, in the case of pharmaceutical products, is an ability to maintain the original form and properties (to ensure that the product maintains its intended quality, safety and efficacy as well as functionality) under the influence of various factors, such as time, humidity, temperature, etc. when stored under appropriate conditions throughout the shelf life [2]. The problem of stability is especially visible in the cases of semiliquid formulations, gels, and nanoparticle-based drugs [3][4][5][6]. The latter offer the unique possibility to overcome cellular barriers in order to improve the delivery of active ingredients. Such a gel form may improve a medicine's efficacy and decrease side effects. Moreover, the drug distribution to target sites via passive and active targeting may be modulated [7][8][9]. A hydrophilic nanoparticle can be an effective carrier for insoluble drugs of low bioavailability [10]. The properties of the nanoparticle-based drugs depend on the particle size. On the one hand, the small particles have an extremely large specific surface, and therefore large specific API capacity. On the another hand, the size of nanoparticles modifies the ability of the drug to pass from the blood vessels to the targeted site destination [11,12]. It helps to reach the extracellular fluid of the tissue by

General Chemistry
Melting points were recorded in open capillaries and are uncorrected. 1 H NMR (500 MHz) and 13 C NMR (125 MHz) spectra were taken on a Bruker AVANCE 500 MHz spectrometer using CDCl 3 as a solvent (Figures S1-S5).
Compounds 1-5 were prepared according to our previously reported protocols [48] and their spectral and other physicochemical properties were consistent with the literature data [29,49].

Preparation of the Gels
The dry glass vial equipped with the stirrer magnet was charged with 10 mL of proper solvent and 1 g of 1-Ala. The vial was closed, and the reaction mixture was heated in an oil bath at 20 • C above the boiling point of the solvent used, maintaining continuous stirring for 5 min. Next the vial was cooled down in an ice bath without the stirring to 5-0 • C. After the cooling was completed the stable at ambient (and lower) temperature gels in toluene, water, t-BuOMe and petroleum ether were formed as presented in Figure 1. The gel obtained with CHCl 3 melts above 19 • C. The gelation in alcohols, DMF, DMSO was unsuccessful; however, gel obtained with 40% v/v aqueous ethyl alcohol was stable for a few days at ambient temperature.
Here, we present the study of crystal packing and energy frameworks of a series of amphiphilic gelators based on N-dodecanoyl-L-amino acid derivatives for which we managed to obtain single crystals by recrystallization from heptane/ t BuOMe mixture to explore if the solid state supramolecular synthons and the energy frameworks may be an indicator for desirable gelling properties. Obtained gelators were proved to be suitable for organogels and hydrogels formation.

General Chemistry
Melting points were recorded in open capillaries and are uncorrected. 1 H NMR (500 MHz) and 13 C NMR (125 MHz) spectra were taken on a Bruker AVANCE 500 MHz spectrometer using CDCl3 as a solvent ( Figures S1-S5).
Compounds 1-5 were prepared according to our previously reported protocols [48] and their spectral and other physicochemical properties were consistent with the literature data [29,49].

Preparation of the Gels
The dry glass vial equipped with the stirrer magnet was charged with 10 mL of proper solvent and 1 g of 1-Ala. The vial was closed, and the reaction mixture was heated in an oil bath at 20 °C above the boiling point of the solvent used, maintaining continuous stirring for 5 min. Next the vial was cooled down in an ice bath without the stirring to 5-0 °C. After the cooling was completed the stable at ambient (and lower) temperature gels in toluene, water, t-BuOMe and petroleum ether were formed as presented in Figure 1. The gel obtained with CHCl3 melts above 19 °C. The gelation in alcohols, DMF, DMSO was unsuccessful; however, gel obtained with 40% v/v aqueous ethyl alcohol was stable for a few days at ambient temperature.  toluene (transparent  gel), water, t-BuOMe, petroleum ether (120-140 °C), CHCl3 (opaque gels), no gelation in alcohols C2H5OH and n-C5H11OH, and finally a gel in a mixture of water/ethyl alcohol (6/4 v/v).

X-Ray Diffraction
The crystal structures of the analyzed compounds 1-Ala, 2-Pro, 3-Phe, 4-Leu, and 5-Val were determined based on the X-ray diffraction experiment performed on single crystals at 120 K on SuperNova X-ray diffractometer, CuKα radiation (λ = 1.54184 Å). The structural models were obtained by direct methods using the SHELXS-97 program [50] and refined by the full-matrix least-squares method on F 2 using the SHELXL-97 program implemented on Olex2.refine package [51]. Crystal structure of 3-Phe was redetermined. The first report was published by Chen et al. with final R1 = 10.96% [30]. The propan-2-yl fragment of the valine derivative in 5-Val was refined as disordered over two positions with site occupation factors being of 0.72 and 0.28. The compounds crystallized in noncentrosymmetric space groups. The absolute configuration was established by reference to the unchanging chiral center in the synthetic route because the Flack test results were ambiguous (no elements heavier than O in the crystal structure). Crystal

X-ray Diffraction
The crystal structures of the analyzed compounds 1-Ala, 2-Pro, 3-Phe, 4-Leu, and 5-Val were determined based on the X-ray diffraction experiment performed on single crystals at 120 K on SuperNova X-ray diffractometer, CuKα radiation (λ = 1.54184 Å). The structural models were obtained by direct methods using the SHELXS-97 program [50] and refined by the full-matrix least-squares method on F 2 using the SHELXL-97 program implemented on Olex2.refine package [51]. Crystal structure of 3-Phe was redetermined. The first report was published by Chen et al. with final R 1 = 10.96% [30]. The propan-2-yl fragment of the valine derivative in 5-Val was refined as disordered over two positions with site occupation factors being of 0.72 and 0.28. The compounds crystallized in non-centrosymmetric space groups. The absolute configuration was established by reference to the unchanging chiral center in the synthetic route because the Flack test results were ambiguous (no elements heavier than O in the crystal structure).
Crystal Data for 1-Ala, C 15

Energy Framework Calculations
Hirshfeld analysis is a well-known method for exploring crystal data to gain deeper insight into the characteristics of intermolecular interactions in a solid state [52][53][54][55]. For each crystal, a cluster of the closest molecules was generated and the unique pair energies have been calculated using CrystalExplorer [56][57][58] with the CE-B3LYP model derived from wavefunctions computed by Gaussian09 [59]. The energies were scaled by factors 1.057, 0.74, 0.871, and 0.618 for coulombic, polarization, dispersion and repulsion energy terms, respectively, according to Mackenzie et al. [37]. The initial geometry for the calculations was taken from the single crystal diffraction experiments. In the case of 1-Ala with 2 independent molecules in the unit cell, the lattice energy is given as an average of the lattice sums for each of them. In 5-Val, the two disordered orientations of the molecule with the partial occupancies were included in calculations

Results
The synthesis of the five N-dodecanoyl-L-amino acid derivatives was based on the optimization of well-defined approaches which usually allow obtaining the amino acids of high purity in acceptable yields [48]. Thus, the lauric acid was converted into the lauroyl chloride in the reaction with thionyl chloride (Scheme 1). The synthesis was carried out in toluene solution and the obtained product has the satisfied quality to be used without additional purification. Such obtained lauroyl chloride was subjected the Schotten-Baumann with in situ prepared sodium salts of five selected natural amino acids: endogenous sodium (L)-alaninate, (L)-leucinate, and exogenous (L)-valinate, (L)phenylalaninate, and (L)-prolinate (Scheme 1). The syntheses were carried out in 50% water-dioxane solution solubilizing the substrates and products, while the acidifying the postreaction mixture with aqueous hydrochloric acid effected in separation of mineral phase containing the majority of impurities. The additional purification of the desirable products of high purity was achieved by a single crystallization from heptane/ t BuOMe mixture. The alternative approaches tested to prepare the targeted fatty acid amides were less efficient [27,29,60,61]. sodium (L)-alaninate, (L)-leucinate, and exogenous (L)-valinate, (L)-phenylalaninate, and (L)-prolinate (Scheme 1). The syntheses were carried out in 50% water-dioxane solution solubilizing the substrates and products, while the acidifying the postreaction mixture with aqueous hydrochloric acid effected in separation of mineral phase containing the majority of impurities. The additional purification of the desirable products of high purity was achieved by a single crystallization from heptane/ t BuOMe mixture. The alternative approaches tested to prepare the targeted fatty acid amides were less efficient [27,29,60,61]. The enriched with gold nanoparticles N-lauroyl-L-alanine (1-Ala)-based hydrogel was tested as a supramolecular drug carrier [62]. The gelation properties of 1-Ala, 3-Phe, 4-Leu, and 5-Val were reported by MacLachlan and Hum et al. in the inversion test [30]. The example of gelation by 1-Ala is presented in Figure 1. The studied compounds exhibit the ability to form gels both in polar and in apolar solvents. In toluene (apolar) the gel is transparent, whereas in water (polar) it is opaque. Other tests were performed on 9% gels of 1-Ala in different solvents: the gels obtained with t-BuOMe and petroleum ether were stable at ambient temperature. The gel obtained with CHCl3 melts above 19 °C. The gelation of alcohols was not successful; however, gel obtained with 40% v/v aqueous ethyl alcohol was stable for a few days at ambient temperature, and it changed slowly from nanofiber (gel) to a crystalline form by dissolving-crystallisation process. The last observation proved that gels could be obtained in solvent mixtures, where the solubility of the gelator in at least one of the solvents used is low. The stability of gels is higher in the cases, where a possibility of dissolving of nanofiber of gelators and its recrystallization is limited. On the other hand, solvents in which the gelator is well soluble (CHCl3, EtOH, DMF, DMSO) might not be gelated in a pure form.

Crystal Structure and Energetic Landscape Analysis
As pure enantiomers all studied compounds crystallized in chiral space groups (P1, P21, P212121 and P22121, Table S1)-Ala has two symmetrically independent molecules in the unit cell-Val exhibited positional disorder of the propan-2-yl moiety. The heteroatoms formed bonds within the expected range of bond lengths (Table S2).
The conformation of the molecules (especially the alkyl chain) seems not to determine the crystal packing mode. The 3-Phe, 4-Leu and 5-Val which form similar folded layers show the conformations of the aliphatic chain from synclinal (5-Val) through anticlinal (3-Phe) up to antiperiplanar (4-Leu) ones (Figures 1 and 2, Table S2). The enriched with gold nanoparticles N-lauroyl-L-alanine (1-Ala)-based hydrogel was tested as a supramolecular drug carrier [62]. The gelation properties of 1-Ala, 3-Phe, 4-Leu, and 5-Val were reported by MacLachlan and Hum et al. in the inversion test [30]. The example of gelation by 1-Ala is presented in Figure 1. The studied compounds exhibit the ability to form gels both in polar and in apolar solvents. In toluene (apolar) the gel is transparent, whereas in water (polar) it is opaque. Other tests were performed on 9% gels of 1-Ala in different solvents: the gels obtained with t-BuOMe and petroleum ether were stable at ambient temperature. The gel obtained with CHCl 3 melts above 19 • C. The gelation of alcohols was not successful; however, gel obtained with 40% v/v aqueous ethyl alcohol was stable for a few days at ambient temperature, and it changed slowly from nanofiber (gel) to a crystalline form by dissolving-crystallisation process. The last observation proved that gels could be obtained in solvent mixtures, where the solubility of the gelator in at least one of the solvents used is low. The stability of gels is higher in the cases, where a possibility of dissolving of nanofiber of gelators and its recrystallization is limited. On the other hand, solvents in which the gelator is well soluble (CHCl 3 , EtOH, DMF, DMSO) might not be gelated in a pure form.

Crystal Structure and Energetic Landscape Analysis
As pure enantiomers all studied compounds crystallized in chiral space groups (P1, P2 1 , P2 1 2 1 2 1 and P22 1 2 1 , Table S1)-Ala has two symmetrically independent molecules in the unit cell-Val exhibited positional disorder of the propan-2-yl moiety. The heteroatoms formed bonds within the expected range of bond lengths (Table S2).
The conformation of the molecules (especially the alkyl chain) seems not to determine the crystal packing mode. The 3-Phe, 4-Leu and 5-Val which form similar folded layers show the conformations of the aliphatic chain from synclinal (5-Val) through anticlinal (3-Phe) up to antiperiplanar (4-Leu) ones (Figures 1 and 2, Table S2).
The separation of hydrophilic and lipophilic regions (Figure 3) which may favor the gelation process occurs in a special way. The connection between gelling ability of some amino acid surfactants and 2-dimensional packing type was considered previously by Bastiat and Leroux [22]. To study such potential relationships, in the crystal packing analysis we decided to complement the study by the energy framework calculations. Table 1 contains the pairwise interaction energies for the analyzed crystals calculated using the CrystalExplorer at CE-B3LYP/6-31G(d,p) level in kJ/mol. All of the values were scaled by factors 1.057, 0.74, 0.871, and 0.618 for coulombic, polarization, dispersion, and repulsion energy terms, respectively, according to Mackenzie et al. [37]. The separation of hydrophilic and lipophilic regions (Figure 3) which may fa gelation process occurs in a special way. The connection between gelling ability o amino acid surfactants and 2-dimensional packing type was considered previou Bastiat and Leroux [22]. To study such potential relationships, in the crystal packin ysis we decided to complement the study by the energy framework calculations.  The separation of hydrophilic and lipophilic regions ( Figure 3) which may fav gelation process occurs in a special way. The connection between gelling ability o amino acid surfactants and 2-dimensional packing type was considered previou Bastiat and Leroux [22]. To study such potential relationships, in the crystal packin ysis we decided to complement the study by the energy framework calculations.  Table 1 contains the pairwise interaction energies for the analyzed crystals calc using the CrystalExplorer at CE-B3LYP/6-31G(d,p) level in kJ/mol. All of the value scaled by factors 1.057, 0.74, 0.871, and 0.618 for coulombic, polarization, dispersio repulsion energy terms, respectively, according to Mackenzie et al. [37]. The energy frameworks indicate the dominance of coulombic and dispersion energies in the crystal nets. Figures 4-6 show selected energy frameworks viewed along a and c directions. Various colors represent various energy terms: red-coulombic, yellowpolarization, green-dispersion, and blue-total energy. The scale for tube size was 150, the energy cut-off 5 kJ/mol. polarization, green-dispersion, and blue-total energy. The scale for tube size was 150, the energy cut-off 5 kJ/mol.    In crystal packing, there is an evident division for hydrophilic and lipophilic sections ( Figure 3). Within the hydrophilic regions the amide and carboxylic groups participate in various intermolecular interactions. The found supramolecular motifs formed by the stronger hydrogen bonds are presented in Figure 7. To ease the analysis of supramolecular synthons the graph set notation was used [63,64].
In the case of 1-Ala, the supramolecular primary level motif is a C(4) chain formed between amide groups (E_tot = −55.6 kJ/mol), which is assisted by dispersion interactions between parallelly arranged amphiphilic molecules. However, it is not the strongest interaction in this crystal. The carboxylic acid fragments preserve the usual cyclic R 2 2 (8) motif resulting in high total energy of ca. −116 kJ/mol. At the higher level of the graph set theory both of the mentioned motifs link into a R 4 4 (24) ring. The final patterning in 1-Ala may be considered to be parallel tapes between which dispersion forces are present. Such arrangement does not seem to ease a possible formation of gel, since the hydrophilic parts are strongly bonded and the rearrangement of the molecules to accommodate solvent would demand a large energy input.
2-Pro exhibits only a simple C(7) herring-bone pattern via O-H . . . O_amide contact (E_tot = −42 kJ/mol). The maximum total energy of pairwise interaction is smaller than in the other structures (−46 kJ/mol) and comes mainly from dispersion term. This may indicate possible formation of polymorphs. However, the blocked N atom limits the prospects of this compound to interact freely with solvent molecules. In crystal packing, there is an evident division for hydrophilic and lipophilic sections ( Figure 3). Within the hydrophilic regions the amide and carboxylic groups participate in various intermolecular interactions. The found supramolecular motifs formed by the stronger hydrogen bonds are presented in Figure 7. To ease the analysis of supramolecular synthons the graph set notation was used [63,64]. Comparing the crystal packing of 3-Phe, 4-Leu, and 5-Val, the main noticeable feature is the common second level graph set R 4 4 (20) ring with four donors and four hydrogen bond acceptors, in which the same functional groups are involved. These crystals are not isostructural, since the alkyl parts of each are arranged differently. However, some degree of similarity among these lamellar structures is observed, which is also in accordance with the energy frameworks.  In the case of 1-Ala, the supramolecular primary level motif is a C(4) chain formed between amide groups (E_tot = −55.6 kJ/mol), which is assisted by dispersion interactions between parallelly arranged amphiphilic molecules. However, it is not the strongest interaction in this crystal. The carboxylic acid fragments preserve the usual cyclic R 2 2(8) motif resulting in high total energy of ca. −116 kJ/mol. At the higher level of the graph set theory both of the mentioned motifs link into a R 4 4(24) ring. The final patterning in 1-Ala may be considered to be parallel tapes between which dispersion forces are present. Such arrangement does not seem to ease a possible formation of gel, since the hydrophilic parts are strongly bonded and the rearrangement of the molecules to accommodate solvent would demand a large energy input.
2-Pro exhibits only a simple C(7) herring-bone pattern via O-H…O_amide contact (E_tot = −42 kJ/mol). The maximum total energy of pairwise interaction is smaller than in the other structures (−46 kJ/mol) and comes mainly from dispersion term. This may indicate possible formation of polymorphs. However, the blocked N atom limits the prospects of this compound to interact freely with solvent molecules.
Comparing the crystal packing of 3-Phe, 4-Leu, and 5-Val, the main noticeable feature is the common second level graph set R 4 4(20) ring with four donors and four hydrogen bond acceptors, in which the same functional groups are involved. These crystals are not isostructural, since the alkyl parts of each are arranged differently. However, some degree of similarity among these lamellar structures is observed, which is also in accordance with the energy frameworks.
The 3-Phe structure is dominated by two interactions of very similar total energies (−68.7 and −62.7 kJ/mol); however, their origin is entirely different. One of the interactions is a strong O-H…O_amide hydrogen bond with the highest contribution of coulombic factor (−87.1 kJ/mol). The other one is N-H…O_acid with moderate coulombic (−36.6 kJ/mol) and quite high dispersion (−60.3 kJ/mol) inputs.
The strongest interaction in 4-Leu comes from O-H…O_amide hydrogen bond with high coulombic contribution (−77 kJ/mol) has a total energy of −54.5 kJ/mol. However, another pairwise total energy nearly equally strong is an effect of a combination of a weaker N-H…O_acid hydrogen bond (E_Coul = −31.5 kJ/mol) supported by high dispersion term (−52.6 kJ/mol) associated with a parallel alignment of the alkyl chains. The strongest interaction in 4-Leu comes from O-H . . . O_amide hydrogen bond with high coulombic contribution (−77 kJ/mol) has a total energy of −54.5 kJ/mol. However, another pairwise total energy nearly equally strong is an effect of a combination of a weaker N-H . . . O_acid hydrogen bond (E_Coul = −31.5 kJ/mol) supported by high dispersion term (−52.6 kJ/mol) associated with a parallel alignment of the alkyl chains.
Structure of 5-Val is stabilized by similar interactions, but the much higher coulombic energetic term of ca. −92 kJ/mol in the case of O-H . . . O_amide h-bond is a consequence of an additional contribution of an accompanying C-H . . . O_acid contact which closes the R 2 2 (8) ring. Additionally, the amide N-H . . . O_acid interaction is assisted by a weaker C-H . . . O_amide one (motif R 2 2 (11)) giving the contribution to the electrostatic energy.

Conclusions
The crystal structures of a series of N-dodecanoyl-L-amino acid derivatives have been successfully determined. Energy frameworks analysis was applied for the first time to study the gelling behavior of a series of low-molecular-weight gelators, suitable for the formation of both organo-and hydrogels. The presence of carboxylic O-H and amide N-H groups impacted the most the crystal net energetics giving high coulombic contributions to the total energy in each of the studied structures. Three of the analyzed crystals (3-Phe, 4-Leu, and 5-Val) had the same cyclic supramolecular synthon R 4 4 (20), stabilized by hydrogen bonds. The different alignment of the aliphatic chains had a minor contribution to the total energy in the crystals, but the alkyl chains exhibited wide range of conformations from synclinal (5-Val) through anticlinal (3-Phe) up to antiperiplanar (4-Leu) ones. This information may be useful when predicting the tangling ability of the lipophilic parts of the gelator molecules.
Spontaneous formation of self-assembled fibrillary network (SAFiN), at least in the case of apolar solvents, seems to have its origin in two phenomena: (1) the hydrophilic parts of amphiphilic organogelators interact to each other influencing the stability of the gels as well as the energy necessary to initiate the gelling process; (2) the hydrophobic fibers may tangle up trapping in voids lipophilic solvent molecules.
The combination of crystallographic studies and energy frameworks analysis may be a useful help in understanding the gelling behavior and in designing new low-molecularweight organogelator (LMOG) materials.