Stigmasterol-based novel low molecular weight/mass organic gelators.

Conjugates consisting of stigmasterol and L-phenylalanine, interconnected through short-chained dicarboxylic acyls by ester and amide bonds, respectively, were synthesized as potential low molecular weight/mass organic gelators (LMWGs/LMMGs). Their physico-chemical properties were subjected to investigation, especially their ability to form gels reversibly based on changes of the environmental conditions. Other self-assembly properties detectable by UV-VIS traces were measured in systems consisting of two miscible solvents (water/acetonitrile) with varying solvent ratios and using constant concentrations of the studied compounds. Partition and diffusion coefficients and solubility in water were calculated for the target conjugates. The conjugate 3a was the only compound from this series capable of forming a gel in 1-octanol. All three conjugates 3a-3c displayed supramolecular characteristics in the UV-VIS spectra.


Introduction
Investigations in the area of supramolecular chemistry during the last two decades have resulted in the discovery of a large number of new compounds displaying self-assembly characteristics, often assisted by solvents. Many of those novel compounds have been proved to form reversible gels, and since that time, as Professor Dastidar has stated [1]: "Gels are everywhere!", and the number of papers dealing with different aspects of this field of supramolecular chemistry has been steadily increasing. The most frequent components of self-assembly systems are heterocycles and other aromatic systems and amino acids (both aromatic and non-aromatic in nature), combined with steroid molecules, monoand oligosaccharides, fatty acyls and even hydrocarbons and organometallic compounds [1][2][3]. Steroid compounds, particularly the bile acids [4][5][6][7][8], but also other types of steroids, like phytosterols and phytoecdysteroids [2] can be a basis for designing supramolecules in combination with other small molecules, e.g., amino acids, nucleotides, heterocycles, etc. [1][2][3]. A specific group among such structures is represented by steroid-based anion and cation receptors and transporters. Ion transportation across biological membranes represents a fundamental process essential for homeostasis in multicellular life forms. Biological systems use a number of mechanisms by which hydrophilic ions pass through phospholipid bilayers possessing hydrophobic nature. Steroid compounds represent convenient natural molecules capable of assisting in transportation of ions through the hydrophobic bilayers or modifying the gelator properties [9]. In turn, several types of steroid derivatives with other small molecules of natural or synthetic origin have already been proved to become a specific part of supramolecular gelators, i.e., low molecular weight/mass organic gelators (LMWGs/LMMGs). Recently, we have published a paper dealing with the synthesis of phytosterol-amino acid conjugates and investigation of their physico-chemical characteristics as supramolecular gelators [2]. Among steroid molecules, phytosterols have not yet received adequate attention, despite the fact that these natural products can be relatively easily extracted from numerous plants. Phytosterols are biogenetic precursors of phytoecdysteroids, which are important plant products acting mainly in plant defense systems. Phytosterols are structurally close to cholesterol, and many of them display important pharmacological activities, including cholesterol lowering activity in the human body.
Amino acids represent key products for humans and other living organisms. Enantiomerically pure non-natural amino acids are often used as valuable intermediates in the preparation of pharmaceuticals [10,11]. In the other hand, both enantiomerically pure natural and non-natural amino acids may become parts of molecules of low molecular weight organic gelators (LMWGs) [2]. Amino acids bear a charge on their molecules. In combination with steroid compounds like phytosterols, the resulting conjugates may provide H-bond donor functionality combined with the lipophilic nature of phytosterol molecules [2]. Hydrogen bonds, - stacking of aromatic rings and multiple bond systems represent the key forces responsible for supramolecular self-assembly of the studied systems.
The objectives of this investigation were: (a) a synthesis of conjugates of stigmasterol and L-phenylalanine, connected by ester and amide bonds through short-chained dicarboxylic acyl compounds; and (b) a study of the physico-chemical properties of these novel potential LMWGs.

Results and Discussion
Hemiesters of stigmasterol 2a-2c were prepared by reacting the sterol 1 with the corresponding acid anhydride in either dry pyridine (in the case 2a and 2b) or in dry dichloromethane (for 2c) in the presence of 4-dimethylaminopyridine (DMAP). Using pyridine in the synthesis of 2c resulted in only a 13% yield. The subsequent amide bond formation between the amino group of methyl L-phenylalanine and the corresponding hemiesters 2a-2c was carried out in dichloromethane in the presence of dicyclohexylcarbodiimide (DCC) as an auxiliary condensation reagent and small quantity of DMAP as reaction promoter, and resulted in the target conjugates 3a-3c (Scheme 1).
3a, X-X = CH 2 CH 2 3b, X-X = CH 2 CH 2 CH 2 3c, X-X = CH 2 C(CH 3  The obtained conjugates 3a-3c were subjected to a screening of their supramolecular properties. Their UV-VIS spectra were recorded in solvent systems consisting of water and an organic solvent miscible with water (acetonitrile in our experiments). The concentration of the studied compound was constant in all solutions, and the ratio of both solvents was changed stepwise in 5-25% steps. Under the described conditions, the UV-VIS spectra of the compounds able to show self-assembly properties, display irregularities indicating formation of supramolecular structures in the studied systems consisting of two solvents. Figure 1 shows several examples of those results and irregularities. When the compound forms no supramolecular structures in the solutions in two-solvent-based systems, then the absorbance intensity follows the sequence of solvent ratio change. However, when the compound is able to form supramolecular structures, then the absorbance intensity is responsive to structural changes due to hydrogen bond formation (supramolecular polymer) or due to the - stacking of aromatic rings. Formation of both types of van der Waals forces may be accompanied by absorbance maximum shift in different solvent ratio solutions. Supramolecular structures are formed with different rates, and the structure of supramolecular systems can change during the longer time, as shown in Figure 1. It follows from this figure that the conjugates 3a and 3b display supramolecular characteristics in their UV-VIS spectra. The supplementary material contains the whole sequence of long-term UV-VIS measurement traces, supporting this finding.   Based on the UV-VIS measurement results, several solvents were tested to determine if the studied compounds would display the ability to form gels. Thus, benzene, toluene, dichloromethane, methanol and 1-octanol were tested as solvents in gelation ability screening experiments for the new compounds. Table 1 shows the results of this screening. Only compound 3a formed a reversible gel in 1-octanol (Figure 2), while the compounds 3b and 3c formed solutions in this solvent. All tested compounds formed precipitates in methanol, and solutions in all other tested solvents. Compound 3a formed the gel in 1-octanol upon cooling in a refrigerator. The gel was transformed into a solution by slowly increasing the temperature to the laboratory temperature, and the gel reversibly formed by repeated cooling in the refrigerator.

General
The 1 H-NMR and the 13 C-NMR spectra were recorded on a Bruker AVANCE 600 MHz spectrometer at 600.13 MHz and 150.90 MHz in deuteriochloroform using tetramethylsilane ( = 0.0) as internal reference. 1 H-NMR data are presented in the following order: chemical shift () expressed in ppm, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet), coupling constants in Hertz, number of protons. Infrared spectra were measured with a Nicolet 205 FT-IR spectrometer. Mass spectra were measured with a Waters ZMD mass spectrometer in a positive ESI mode. TLC was carried out on silica gel plates (Merck 60F254) and the visualization was performed both, by UV detection and by spraying with the methanolic solution of phosphomolybdic acid (5%) followed by heating. Elemental analyses were performed on a Perkin Elmer 2400, series II CHNS/O analyzer (USA). Melting points were determined on a Kofler MHK melting point apparatus (Franz Küstner Nacht, KG, Dresden, Germany) and are uncorrected. All chemicals and solvents were purchased from regular commercial sources in analytical grade and the solvents were purified by general methods before use. For column chromatography, silica gel 60 (0.063-0.200 mm) from Merck was used. ACD/Labs software, ACD/logD DB, version 12.01, was used for calculation of solubility, partition coefficient (log P) and distribution coefficient (log D) of the prepared compounds.