molecules Article Synthesis and Gelling Abilities of Polyfunctional Cyclohexane-1,2-dicarboxylic Acid Bisamides: Influence of the Hydroxyl Groups Bernat Pi-Boleda 1 , María Campos 2 , Marta Sans 1,3 , Antonio Basavilbaso 2 , Ona Illa 1 , Vicenç Branchadell 1, *, Juan Carlos Estévez 2, * and Rosa M. Ortuño 1, * 1 Department de Química, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Barcelona, Spain; bernat@piboleda.cat (B.P.-B.); marta.sans.valls@cfel.de (M.S.); ona.illa@uab.es (O.I.) 2 CIQUS (Centro Singular de Investigación en Química Biológica y Materiales Moleculares), Departamento de Química Orgánica Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain; maria.campos.torrado@rai.usc.es (M.C.); antonio.basavilbaso.glz@gmail.com (A.B.) 3 The Hamburg Center for Ultrafast Imaging (CUI), Luruper Chaussee 149, 22761 Hamburg, Germany * Correspondence: vicenc.branchadell@uab.es (V.B.); juancarlos.estevez@usc.es (J.C.E.); rosa.ortuno@uab.es (R.M.O.); Tel.: +34-935-811-602 (R.M.O.)  Received: 8 January 2019; Accepted: 18 January 2019; Published: 19 January 2019  Abstract: New enantiomerically pure C16 -alkyl diamides derived from trihydroxy cyclohexane-1, 2-dicarboxylic acid have been synthesized from (−)-shikimic acid. The hydroxyl groups in these compounds are free or, alternatively, they present full or partial protection. Their gelling abilities towards several solvents have been tested and rationalized by means of the combined use of Hansen solubility parameters, scanning electron microscopy (SEM), and circular dichroism (CD), as well as computational calculations. All the results allowed us to account for the capability of each type of organogelator to interact with different solvents and for the main mode of aggregation. Thus, compounds with fully protected hydroxyl groups are good organogelators for methanol and ethanol. In contrast, a related compound bearing three free hydroxyl groups is insoluble in water and polar solvents including alcohols but it is able to gelate some low-polarity solvents. This last behavior can be justified by strong hydrogen bonding between molecules of organogelator, which competes advantageously with polar solvent interactions. As an intermediate case, an organogelator with two free hydroxyl groups presents an ambivalent ability to gelate both apolar and polar solvents by means of two aggregation patterns. These involve hydrogen bonding interactions of the unprotected hydroxyl groups in apolar solvents and intermolecular interactions between amide groups in polar ones. Keywords: polyfunctional cycloalkane bisamides; organogelator; self-assembly; chirality; hydrogen bonds 1. Introduction Low molecular weight organogelators (LMWOGs) are soft materials widely used at present in several fields that include products employed as lubricants, cosmetics, drug delivery systems, tissue regeneration materials, biosensors, molecular electronic devices or chiral catalysts [1–5]. Very recently, applications of supramolecular gels as materials for environmental remediation [6] or for practical and eco-friendly oil spill recovery [7,8] have been reported. The gelling ability of molecules in different solvents has been the subject of a number of studies. However, the ultimate reasons for the hierarchical self-assembling of a gelator in a specific solvent still remain incomplete [9,10]. Therefore, there is a lack of prediction tools. Rationalization of the gelling Molecules 2019, 24, 352; doi:10.3390/molecules24020352 www.mdpi.com/journal/molecules Molecules 2019, 24, x 2 of 17 Molecules 2019, 24, 352 2 of 17 gelling power of a LMWOG has been attempted based on various solubility indicators [11–15]. Amongof them, power a LMWOG Hansen has solubility parameters been attempted based on (HSPs) various[13,16] haveindicators solubility been applied [11–15].to Among elucidate the them, behaviorsolubility Hansen of LMWOGs towards parameters solvents (HSPs) andhave [13,16] to reduce the number been applied of trialsthe to elucidate usually behaviorinvolved during of LMWOGs the identification of a suitable gelator for a particular application [17–19]. towards solvents and to reduce the number of trials usually involved during the identification of Amides a suitable havefor gelator been reported application a particular as functional groups present in some of the simplest LMWOGs that [17–19]. haveAmides been described have been [20].reported We haveas previously functional developed good LMWOGs groups present in some of of peptide the simplestnature based on LMWOGs chiral β-cyclobutane amino acids [21,22], or hybrid peptides presenting that have been described [20]. We have previously developed good LMWOGs of peptide nature a cyclobutane β-amino acid joined on based in alternation with linearamino chiral β-cyclobutane residues [23].[21,22], acids In bothor cases, hybrida model peptidesto explain the self-assembly presenting a cyclobutane of the individual molecules to produce the gel was suggested by means β-amino acid joined in alternation with linear residues [23]. In both cases, a model to explain of computational calculations. More the recently, we of self-assembly reported a combined the individual experimental molecules and computational to produce study to investigate the gel was suggested by meansand of rationalize the calculations. computational gelling abilityMore of diastereomeric 1,2-disubstituted recently, we reported a combined carbocyclic experimentalcompounds (1, Figure 1) and computational [24]. The study influence and to investigate of the cis/transthe rationalize relative gellingconfiguration of the monomers ability of diastereomeric in their hierarchical 1,2-disubstituted carbocyclic compounds (1, Figure 1) [24]. The influence of the cis/trans relative configuration of thewith self-assembly was analyzed and their gelling behavior compared well that in monomers of trans-cyclohexane-1,2-diamine their hierarchical self-assemblyderivatives was analyzed described and their bygelling Hanabusa et al. [25] behavior and van compared wellEsch et that with al. [26] of suggesting that regiochemistry does not play a relevant role in the trans-cyclohexane-1,2-diamine derivatives described by Hanabusa et al. [25] and van Esch et al. [26] gelation process. It was remarkablethat suggesting thatregiochemistry chiral aggregates doeswere observed not play evenrole a relevant fromin meso molecules the gelation cis-1Itaswas process. an remarkable example of stochastic symmetry breaking induced by sonication. that chiral aggregates were observed even from meso molecules cis-1 as an example of stochastic Otherwise, symmetry breaking regarding induced by polyhydroxylated sonication. organogelators, only few results on modified carbohydrate Otherwise,or cholestane regarding derivatives have polyhydroxylated been described organogelators, only fewand theironability results modified to carbohydrate gelate organic or solvents has been interpreted by using different techniques in each case [27–30]. cholestane derivatives have been described and their ability to gelate organic solvents has been interpreted Nevertheless, as far as we by usingknow, theretechniques different is neither information in each case in the literature [27–30]. on polyhydroxylated Nevertheless, as far as we know, simple cycloalkane there is neither derivatives nor on compounds susceptible of additional hydrogen-bonding information in the literature on polyhydroxylated simple cycloalkane derivatives nor on compounds promoted by further functional groups such as amides. susceptible of additional hydrogen-bonding promoted by further functional groups such as amides. In this In thispaper, paper, we we describe describe the investigation the investigation of five of newfive new polyhydroxylated polyhydroxylated cyclohexane cyclohexane bisamides bisamides (cis- and trans-2, 3–5, Figure 2), prepared from (−)-shikimic (cis- and trans-2, 3–5, Figure 2), prepared from (−)-shikimic acid. All of them bear two long acid. All of them bear twoalkyl long alkyl chains and three hydroxyl groups with different degrees chains and three hydroxyl groups with different degrees of protection. These new polyfunctional of protection. These new polyfunctional LMWOGs have LMWOGs been compared havewithbeentheir compared with their cis- parent compounds parent compounds and trans-1 focusingcis-the and trans-1 attention on the influence of the polar functional groups at the ring, especially the free hydroxyl groups,free focusing the attention on the influence of the polar functional groups at the ring, especially the on hydroxyl their gelling groups, power on andtheir gelling mode aggregation powerinand aggregation different solvents.mode in different With this purpose, solvents. With this the complementary purpose, results the complementary obtained from severalresults obtainedtechniques experimental from several (useexperimental techniques (use of Hansen parameters, SEM ofand Hansen CD parameters, SEM and CD spectroscopy) as well as from computational spectroscopy) as well as from computational calculations have been taken into account to understand calculations have been taken into rationalize and account to the understand and rationalize main interactions involved thein main eachinteractions case. involved in each case. Figure 1. Previously Figure 1. Previously studied studied LMWOGs LMWOGs (cis- and trans-1) (cis- and trans-1) [16] [16] and and new new LMWOGs LMWOGs investigated investigated in in this this work (2–5). work (2–5). Molecules 2019, 24, 352 3 of 17 Molecules 2019, 24, x 3 of 17 2. Results and Discussion 2.1. Synthesis of Organogelators 2–5 The synthesis of polysubstituted 1,2-cyclohexanedicarboxamides 1,2-cyclohexanedicarboxamides 2–5 2–5 was achieved from polysubstituted lactonelactone6 and6 2-nitromethylcyclohexanecarboxylic and 2-nitromethylcyclohexanecarboxylic acid methyl acid ester (9). respectively, methyl ester (9). previously obtained from commercially available ( − )-shikimic acid (Scheme 1) respectively, previously obtained from commercially available (−)-shikimic acid (Scheme 1) [31]. [31]. For the preparation of 1,2-cyclohexanedicarboxamides cis-2, 3 and 5, lactone 6 was transformed into acid 7 in 76% yield through a Nef reaction reaction conducted conducted under remarkably mild conditions, with sodium nitrite nitrite and andacetic aceticacid acidusing usingdimethylsulfoxide dimethylsulfoxide as as solvent andand solvent carefully controlling carefully the pH controlling the (not pH lower (not thanthan lower 3) in3)order to preserve in order the the to preserve acetonide protecting acetonide group protecting (Scheme group (Scheme 1).1). The resulting The resulting acid acid7 was 7 wasthen condensed then condensed with hexadecylamine, using PyBop as with hexadecylamine, coupling using agentas PyBop andcoupling diisopropylethylamine agent and (DIEA) as a base, to give diisopropylethylamine amide (DIEA) as8ain 98%toyield. base, Subsequent give amide reaction 8 in 98% yield.with a secondreaction Subsequent equivalent withofa hexadecylamine second equivalentinofthe presence of 2-hydroxypyridine hexadecylamine in the presence ofafforded diamide 3 in 2-hydroxypyridine 30% yield. afforded diamideIn turn, 3 in 3 wasyield. 30% transformed In turn, 3intowasfully protectedinto transformed derivative cis-2 by protection fully protected derivative of theby cis-2 free hydroxylofgroup protection as the free tert-butyldimethylsilyl hydroxyl ether by treatment with group as tert-butyldimethylsilyl ethertert-butyldimethylsilyl chloride and imidazole by treatment with tert-butyldimethylsilyl (74% chloride yield). and Alternatively, imidazole aqueous (74% yield). trifluoroaceticaqueous Alternatively, acid-promoted hydrolysis trifluoroacetic of the acetonide acid-promoted in 3 afforded hydrolysis of the product acetonide 5 in 42% yield. product 5 in 42% yield. 3 afforded Following a similar route, nitroester 9 was transformed into amide 11 in 41% yield, for the two intermediate afforded diamide trans-2 in 30% yield, when reacted with hexadecylamine steps. This intermediate and 2-hydroxypyridine as catalyst. The acetonide in fully protected diamide trans-2 was removed as described before giving diamide diamide 4 in 47% yield, which surprisingly preserved the silyl ether group that was inert under several conditions. Scheme 1. Synthesis of diamides 2–5. 2.2. Gelation 2.2. Gelation Studies Studies The gelling The gellingability abilityofof compounds compounds waswas 2–52–5 studied usingusing studied 14 protic or aprotic 14 protic or solvents with different aprotic solvents with polarity. differentCompounds cis- and trans-2, polarity. Compounds andtrans-2, cis- 3and 4 were soluble 3 and in 4 water were whereas soluble 5inwas insoluble; water whereasformation 5 was of hydrogels insoluble; was notof formation observed in any hydrogels wascase. Results for not observed inorganic any case. solvents Resultsarefor summarized in Table organic solvents are1 where data for summarized inrelated where datacis- Table 1compounds forand trans-1 related [24] are also compounds cis-shown for comparison. and trans-1 [24] are also shown for comparison. Molecules 2019, 24, 352 4 of 17 The gels produced were stable at room temperature but unstable at 37 ◦ C. Thus, a gel became a solution by heating with a hand. Once the solution was left to cool down to room temperature, the gel was formed again and remained unaltered for weeks (see the Experimental Section for details on the gel preparation). Compounds 2–5 presented a different behavior with respect to cis- and trans-1, as expected, clearly indicating that the additional substitution of the ring plays a significant role. Compounds cis- and trans-2, and 4 are able to gelate alcohols contrariwise to cis- and trans-1 that are insoluble as well as trihydroxylated compound 5. In addition, cis-2 also promotes the formation of gels in pentane, ethyl acetate and isopropanol although with higher mgc (minimum gelation concentration) values. Nevertheless, the differences between these diastereoisomers are not very remarkable in contrast with the behavior of isomers 1, being trans-1 a much better gelator than the cis diastereomer (Table 1) [24]. Table 1. Gelling behavior of previously known LMWOG 116 and of the new compounds 2–5 in common organic solvents a, b, c . Pentane 1,4-Dioxane Toluene Et2 O CHCl3 EtOAc THF CH2 Cl2 i PrOH Acetone EtOH MeOH CH3 CN 30 51 100 cis-116 (48) (82) (161) I S I I I I I I I I O O O 3 7 18 trans-116 I (5) (11) I (29) I I I I I I I I T T T 83 100 100 21 17 cis-2 (101) S S S S (122) S S (122) S (26) (21) S C O O O O 50 16 trans-2 S S S S S S S S S S (61) (19) S T O 51 102 3 I (72) (144) S S S S S S S S S S T T 54 61 64 70 45 22 56 70 4 (69) (78) (82) (90) S (58) S S S (28) (72) (90) S O O T O O O O O 102 82 82 5 I (193) (156) I (156) I S S I I I I I O T T aDielectric constant increases from left to right; b mgc (minimum gelation concentration) in mg mL−1 . mgc values in mM in parentheses; c I: insoluble (precipitates before formation of a gel), S: soluble, C: clear, O: opaque, T: translucent. Compound 3 is a bad organogelator because it is soluble in nearly all solvents tested, except in 1,4-dioxane and toluene, but it exhibits very high mgc values. Compound 3 is related to cis-2 but with a free hydroxyl group; comparing their behavior, it seems clear that having an unprotected alcohol disfavors the gelling ability especially in methanol and ethanol. Compound 4, bearing two free hydroxyl groups, gelates a large variety of solvents of very different dielectric constants and it is a good organogelator for acetone. Comparison with trans-2 suggests that the structural features of 4 exert a significant influence on its properties allowing it to gelate plenty more solvents. Compound 5 has all three hydroxyl groups unprotected. Its behavior is similar to that of disubstituted cyclohexanes cis- and trans-1 as it forms gels in 1,4-dioxane, toluene and chloroform and it is insoluble in most other solvents (except in THF and dichloromethane), although the mgc values are significantly higher for 5. It is noteworthy that, although this compound bears three free hydroxyl groups, it is insoluble in all the alcohols tested (methanol, ethanol and isopropanol) as well as in water. Some rational behind these observations will be discussed below. 2.3. Hansen Solubility Parameters (HSPs) Inspection of the data in Table 1 makes evident that the gelling properties observed for the studied LMWOGs do not only depend on the dielectric constant of the solvents but also other factors need to be considered for their understanding. Molecules 2019, 24, x 5 of 17 2.3. Hansen Solubility Parameters (HSPs) Inspection of the data in Table 1 makes evident that the gelling properties observed for the Molecules 2019, 24, 352 5 of 17 studied LMWOGs do not only depend on the dielectric constant of the solvents but also other factors need to be considered for their understanding. Plotting Plotting thethe HSPs HSPs of of eacheach solvent, solvent, a 3Da 3D space space is generated is generated andandthethe solvents solvents areare sorted sorted over over thethe space space depending depending onon three three parameters: parameters: δd ,δwhich d, which accounts accounts forfor dispersive dispersive interactions interactions that that dominate dominate forfor lowlow polarity polarity solvents; solvents; δp ,δwhich p, which is related is related to to polar polar interactions, interactions, andandδh δthat h that arises arises from from hydrogen hydrogen bonding bonding interactions. interactions. The representation The representation of the HSPsof thefor HSPs 4 showsfor two4 areas shows two areas of solvents of solvents corresponding to corresponding those gelated by to this those gelated by this organogelator, organogelator, which whichin are highlighted are highlighted blue, and those in in blue, redand those it in which inisred in which it is soluble (Figure 2a,b). In order to better observe the two areas, soluble (Figure 2a,b). In order to better observe the two areas, the plane δp -δd was also represented the plane δ p-δ d was also represented showing showing that there are two that thereofare clusters two solvents gelling clusters which of gelling can besolvents related towhich can be related two different to two aggregation different aggregation patterns. One area corresponds to apolar solvents patterns. One area corresponds to apolar solvents with low δp and high δd and the other to aprotic with low δ p and high δd and the other (acetone) or to aprotic protic (acetone) (alcohols) or solvents, polar protic (alcohols) with higherpolarδpsolvents, and alsowith higher higher δh inδthe p andcasealso of higher methanol δh in andtheethanol case of(Figure methanol2a). and These ethanol results(Figure suggest2a). thatThese 4 could results suggest interact with thethatsolvent 4 could bothinteract through with thethe solvent both unprotected through hydroxyl the unprotected groups and through hydroxyl hydrogen groups bondingandbetween through thehydrogen bondingThese amide groups. betweenfactsthe amide groups. resulting Theseoffacts in two types resulting aggregates as in two types ofbyaggregates corroborated as corroborated by SEM (see below). SEM (see below). Figure 2. (a,c) Figure HSPs 2. (a,c) representation HSPs representationandand (b,d) 2D2D (b,d) representation of δof representation p vs. δd δfor δp vs. thethe d for gelation study gelation of of study compounds compounds 4 (a,b) and 4 (a,b) 5 (c,d). and Red: 5 (c,d). Solvents Red: in in Solvents which 4 or which 5 are 4 or soluble; 5 are blue: soluble; solvents blue: gelled. solvents gelled. Despite Despitebearing three bearing unprotected three unprotected hydroxyl hydroxyl groups, groups,compound compound 5 is insoluble 5 is insolublein in polar polarsolvents, solvents, especially in alcohols and also in water. One could have expected that the interaction especially in alcohols and also in water. One could have expected that the interaction of the of the three three free hydroxyl groups of 5 should be favorable for solubility or gelation through hydrogen free hydroxyl groups of 5 should be favorable for solubility or gelation through hydrogen bonding. bonding. Instead, as shown Instead, as shownin Figure 2, compound in Figure 2, compound5 is only 5 issoluble in apolar only soluble in solvents while the apolar solvents solvents while the gelled solvents aregelled clustered in the region with the highest are clustered in the region with the δ and lowest δ in the HSPs space (Figure 2d). d highest δd and lowest δp in the HSPs space (Figure p This result 2d). suggests that the hydroxyl groups in 5 do not interact with solvents but they are This result suggests that the hydroxyl groups in 5 do not interact with solvents but they are preferably involved in inter-gelator interactions. Only few examples on the fact that gelling solvents for polyhydroxylated compounds are clustered in a region of lower δp and δh but high δd have been described in the Molecules 2019, 24, x 6 of 17 preferably involved Molecules 2019, 24, 352 in inter-gelator interactions. Only few examples on the fact that gelling solvents 6 of 17 for polyhydroxylated compounds are clustered in a region of lower δp and δh but high δd have been described in the literature [17]. These observations were fully supported by the results of literature [17]. calculations computational These observations and CD were fully supported by the results of computational calculations (see below). and CD (see below). 2.4. Scanning Electron Microscopy 2.4. Scanning Electron Microscopy SEM experiments were carried out to investigate the morphology of some of the gels produced SEM experiments were carried out to investigate the morphology of some of the gels produced at the mgc for each compound. The micrographs were taken from the corresponding xerogels and at the mgc for each compound. The micrographs were taken from the corresponding xerogels and selected examples are shown in Figure 3. selected examples are shown in Figure 3. Figure Figure 3. SEM images 3. SEM imagesofofxerogels xerogels at the at the mgcmgc of: cis-2 of: (a,b) (a,b) from cis-2methanol; from methanol; (c,d)from (c,d) trans-2 trans-2 from methanol; methanol; (e,f) 4 from acetone; (g,h) 4 from pentane, at two magnifications each (e,f) 4 from acetone; (g,h) 4 from pentane, at two magnifications each (200 and 50 µm). (200 and 50 μm). SEM images SEM images show showthatthatcompounds compounds cis-cis- andandtrans-2 present trans-2 different present morphologies different despitedespite morphologies having a similar having a gelling similar behavior in methanol. gelling behavior CompoundCompound in methanol. cis-2 forms cis-2 fibersforms of different fiberslengths, some lengths, of different of which are very some long (around of which are very200 longµm) (Figure200 (around 3a,b). μm)Its (Figures 3a and b). trans-2, diastereoisomer, also forms big Its diastereoisomer, aggregates trans-2, also but inbig forms thisaggregates case in the but shape of fibrous in this case inplatelets the shape (Figure 3c,d). platelets of fibrous Therefore, this confirms (Figures 3c andthat in this case, d). Therefore, as well this confirms that in this cis/trans-1, as for diastereomers [24] the case, as well as cis/trans stereochemistry for diastereomers plays a [24] cis/trans-1, role on thethecis/trans pattern of aggregation. stereochemistry plays a role on the pattern of aggregation. Compound 44 was studied Compound studiedin intwo twodifferent different solvents, solvents, acetone acetone(Figure (Figure3e,f)3e,f) andand pentane (Figure pentane 3g,h). (Figure The appearance 3g,h). of the micrographs The appearance is quite different; of the micrographs is quite in acetone in different; theacetone aggregates the form disorganized aggregates form shapes whileshapes disorganized in pentane while the compound in pentane forms clearforms the compound platelets. Therefore, clear platelets. the presence Therefore, of the free the presence of hydroxyl the groups also free hydroxyl influences groups the morphology also influences of aggregates the morphology in differentinsolvents, of aggregates differentwhich agrees solvents, with which the results agrees with from consideration the results of the Hansen from consideration solubility of the Hansenparameters as discussedasabove. solubility parameters discussed above. 2.5.Computational 2.5. ComputationalCalculations Calculationsand andCircular CircularDichroism Dichroism In earlier 1 H-NMR spectroscopies to gain information In earlier studies studiesofofother LMWOGs, other LMWOGs, we used we IR and IR used and 1H-NMR spectroscopies to gain about the aggregation process [21–23]. Nevertheless, in information about the aggregation process [21–23]. Nevertheless, the present case in in which the different present case groups can in which contribute different to the can groups gel formation, contributewe to thought the gel that the combined formation, resultsthat we thought from computational the calculations combined results from and CD spectroscopy would be better to provide an overall view and a more reliable computational calculations and CD spectroscopy would be better to provide an overall view and a interpretation. Indeed, more CD allowed reliable us to obtain interpretation. information Indeed, about the CD allowed us hierarchical organizationabout to obtain information of thethe molecules in the hierarchical aggregates and organization to corroborate of the molecules the predictions in the from aggregates calculations and that, the to corroborate in turn, led us tofrom predictions bettercalculations understand the structure of the gels and their formation mode. Computational calculations that, in turn, led us to better understand the structure of the gels and their formation mode. of compounds 2–5 were carried out using the M06-2X/6-31G(d) level of theory for the optimization of the geometry of Molecules 2019, 24, x 7 of 17 Molecules 2019, 24, x 7 of 17 Computational calculations of compounds 2–5 were carried out using the M06-2X/6-31G(d) level of Molecules 2019, 24, 352 7 of 17 Computational theory for the calculations optimization of compounds of the geometry 2–5 were of carried out usingand the monomer the M06-2X/6-31G(d) the tetramer andlevel using of theory for the ofoptimization minimizations the energy with of the geometry molecular of the for mechanics monomer and the the structure tetramer of the hexamer andand usingthe minimizations octamer the of the (see Supplementary monomer energy and the tetramer with Materialsmolecular and using mechanics forminimizations details). for the structure of the hexamer of the energy with molecular mechanics for and the octamer (see Supplementary By means the structure of of thecalculations hexamer andMaterials we thefoundfor that octamer details). monomers (see can aggregate Supplementary Materials according to two types of for details). By By means of calculations we found that monomers can aggregate according to two means interactions. The of calculations first one, whichwe isfound the that only monomers observed for can 2 andaggregate 3, occurs according through to the types types of formation two of interactions. The –NH···OC– hydrogen interactions. first one, The firstbonds which is the in oneisdimension one, which only observed for (1-D) involving the only observed 2 and for 2 and 3, the3, occurs amide occurs through groups through the (α-type formation aggregates). the formation of of –NH···OC– In turn, –NH ···OC– hydrogen monomers hydrogenwith bonds bonds in in one free dimension hydroxyl one groups dimension (1-D) involving can (1-D) interactthe involving byamide the groups additional amide groups (α-type (α-typeaggregates). –OH···OH– hydrogen aggregates). In In turn,monomers bonding turn, monomers to give β-type with with free free hydroxyl aggregates. hydroxyl These groups groups can interact aggregates can interact formed by additional by two by additional kinds –OH –OH···OH– of ···OH– directional hydrogen hydrogen bonding bonding interactions to to give β-type give would β-type aggregates. aggregates. be dimers of α-ones These These aggregates aggregates (Figure 4) and formed formed byhave by two been predicted two kinds kinds of directional for gelators of directional bonding bonding4interactions and 5. interactions would be dimers of α-ones (Figure 4) and have would be dimers of α-ones (Figure 4) and have been predicted for gelators 4 and 5.been predicted for gelators 4 and 5. Figure 4. Cartoon representing the spatial disposition of monomers in α- and β-aggregates. Figure 4. Cartoon representing the spatial disposition of monomers in α- and β-aggregates. Predicted structures Predicted structuresofofthethe octamers octamers from compounds from compoundscis- and cis- trans-2, and 3 are and trans-2, andshown 3 are inshown Figurein 5. As itPredicted can Figure 5.be itstructures Asobserved, can bedue ofto the observed,the octamers presence due to of from thethe compounds TBDMS presence thecis-TBDMS group of and and thetrans-2, group and relativeand 3the arerelative shown cis cis configuration in of Figure the two5.amide As itof configuration can thebetwo groups, observed, compound due cis-2 to amide groups, the shows presence of the a significantly compound cis-2 TBDMS curved shows groupaggregate. avertical and curved significantly the relative Otherwise, cis vertical configuration compounds of the aggregate. Otherwise, two amide trans-2 andcompounds groups, 3 form a right-handed compound trans-2 andhelical cis-2 3 formaggregateshows a significantly where the a right-handed chains helical curved are placed aggregate vertical in such where the aggregate. achains way that Otherwise, maximizes are placed compounds the in such aVan way der trans-2 Waals that and 3the form Vanader interactions. maximizes right-handed helical aggregate where the Waals interactions. chains are placed in such a way that maximizes the Van der Waals interactions. Frontviews Figure 5. Front viewsof ofcentral central66molecules moleculesininoctameric octameric 1-D 1-D aggregates aggregates (amide (amide hydrogen-bonds) hydrogen-bonds) of Figure of cis- cis- and 5. and Front views trans-2, trans-2, and andof central 6 molecules Non-polarhydrogen 3. 3.Non-polar in octameric hydrogenatoms atomshave 1-D havebeen aggregates beenomitted (amide omittedfor hydrogen-bonds) forclarity. clarity. Atoms of Atoms in amide cis- andhave groups trans-2, beenand 3. Non-polar represented represented withhydrogen with red atoms red (oxygen), (oxygen), bluehave blue been omitted (nitrogen) (nitrogen) and and grey greyfor(carbon) clarity. spheres. (carbon) Atoms in amide spheres. groups have been represented with red (oxygen), blue (nitrogen) and grey (carbon) spheres. CD spectra CD forcis- spectra for andtrans-2 cis-and trans-2were were recorded recordedboth in methanol both solution in methanol and and solution as xerogels (dry gels) as xerogels (dry from methanol CD spectra(Figure for cis-6). andIn this way, trans-2 more were insight recorded could both be in obtained methanol on the transfer solution and gels) from methanol (Figure 6). In this way, more insight could be obtained on the transfer of of as chirality xerogels from (dry achirality single gels) molecule from to the methanol from a single aggregates. (Figure molecule 6).toInthethis way, more insight could be obtained on the transfer of aggregates. chirality from a single molecule to the aggregates. Molecules 2019, 24, x 8 of 17 Molecules 2019, 24, 352 8 of 17 Molecules 2019, 24, x 8 of 17 1.0 cis-2 5 (MeOH solution) 1.0 cis-2 5 (MeOH xerogel) cis-2 5 (MeOH solution) 0.5 trans-2 6 (MeOH solution) 5 (MeOH xerogel) Normalized [Φ] cis-2 trans-2 6 (MeOH xerogel) 0.5 trans-2 6 (MeOH solution) Normalized [Φ] trans-2 6 (MeOH xerogel) 0.0 0.0 -0.5 -0.5 -1.0 -1.0 200 210 220 230 240 250 260 270 280 200 210 220 Wavelength 230 240(nm)250 260 270 280 Wavelength (nm) Figure 6. Normalized CD spectra of cis-and trans-2, respectively, in methanol solution (2.4 mM for Figure 6. Normalized CD spectra of cis-and trans-2, respectively, in methanol solution (2.4 mM for cis-2 cis-2 and 2.7 Figure 6. mM for trans-2) Normalized CDand xerogel spectra at the mgc ofthe cis-and in methanol trans-2, in KBr (20 respectively, mM) at◦solution 25 °C. (2.4 mM for and 2.7 mM for trans-2) and xerogel at mgc in methanol in KBr (20inmM) methanol at 25 C. cis-2 and 2.7 mM for trans-2) and xerogel at the mgc in methanol in KBr (20 mM) at 25 °C. In Insolution, solution,cis- cis-and andtrans-2 trans-2show showaaband bandin inthe theCDCDspectrum. spectrum.cis-2 cis-2Presents Presentsaanegativenegativeband bandwith with aamaximum maximum atat222 In solution, nm 222cis-nmand and trans-2 andtrans-2 trans-2 shows show shows aapositive a band in the band positive CD band with withaamaximum spectrum. cis-2 Presents maximum atat221 nm nmand a negative 221 aasmall andband with small negative a maximum negative lobule at lobuleatat222 211 211nmnm. nm. andThe The CD trans-2 spectra shows of CD spectra of these a positive organogelators band with a maximum these organogelators in methanol in methanol solution at solution 221 nm and were also werea alsosmall computed negativegiving computed lobulepredictions giving at 211 nm.in predictions The in very veryCD good spectra good agreement of thesewith agreement the theexperimental organogelators with experimental spectra spectra(see in methanol (seeFigure solutionFigurewereS1 S1inalso in Supplementary computed giving Supplementary Materials). predictions in very good agreement with the experimental spectra (see Figure S1 in Materials). For Forthethexerogels Supplementary xerogels from frommethanol, Materials). methanol,the theshape shapeofofthe theCD CDspectra spectra is is visibly visibly different. different. ForForcis-2 as as cis-2 a xerogel a xerogel from Forfrom methanol, the xerogels methanol, a frombisignate Cotton methanol,Cotton a bisignate effect the shape was effectofwas observed the CD with spectrawith observed a positive is visibly a positive band different. at bandFor 215 at 215 and cis-2anda as a negative a xerogel negative one from one at methanol, 223223 at nmnm withawith zero bisignate crossing zero Cottonateffect crossing 218218 at nm. was nm. The band observed The band of withcompound of a positive compound trans-2 band trans-2 presents at presents 215 and a a hypsochromic a negative hypsochromic shift one atshift and 223 and it nm withhas a it haszeromaximum at crossing at 207 a maximum 207 nm. This 218 nm. The shift Thisband is associated shift of compound to is associated to the formation trans-2 of presents a the formation H-type H-typeaggregates ofhypsochromic aggregates in which shift and in which ittwo hastwoor more a maximum or more monomersat 207 are monomers nm.arranged This are shift arrangedonisthe top the of associated on topeach other, to each of the i.e., the formation other, i.e.,of stacking theH-type due stacking to aggregates amide due to amide π−π* in which interactions π−π*two is oriented or more monomers interactions in is oriented in a direction area arranged that direction that is on the roughly top of each is roughly perpendicular other, i.e.,to perpendicular the to the the molecular stacking molecular plane; dueplane;to amide the the band band is moved π−π*isinteractions moved to lower to lower is oriented wavelengths wavelengthsin a direction because because the the thatabsorption absorption is roughlyisperpendicular is more to more energetic energetic thethat than than molecular of the that of the the plane; monomer monomerband is suggesting suggesting moved a strongto a strong lower interaction interaction wavelengths between the between because monomers thethemonomers absorption [32,33]. [32,33]. is more In view energetic In viewthan of the of the CD thatCD spectra of the of monomer spectra the xerogels of thesuggesting (Figure xerogels (Figure 6) a strong and their 6)interactionpredicted structures between the and their predicted (Figure monomers structures 5), it is [32,33]. (Figure 5), important to In view of it is important remark to the that the CD spectra remark bisignate that theofbisignate spectrum the xerogels of cis-2 (Figureof6)cis-2 spectrum is and is in agreement their with predicted structures in agreement a curved with a curved (Figurevertical 5), it is vertical aggregate important aggregate whereas to remark whereas the themonosignate that the bisignate monosignate band bandfor fortrans-2 trans-2suggests spectrum of cis-2 isaahelical suggests inhelical structure agreementstructure withfor foraits aggregates. curved its vertical aggregates. This This relationship aggregate relationship whereas is consistent the monosignate is consistent with previous with previous observations bandobservations for trans-2 suggests for compounds for compoundsa helical cis- and cis-structure and trans-1,trans-1, for its [24] andand [24]aggregates. also also applies This applies for thefor other relationship the other xerogels isxerogels consistent considered with considered previous in this in this work. observations work. for compounds cis- and trans-1, [24] and Compound also applies for33the Compound also differs alsoother differs in the xerogels in the shape shapeof considered ofthethein CDCD this spectra work.ininsolution spectra solution andand in in thethe xerogel xerogel (Figure (Figure 7a). 7a). It is bisignate Compound It is bisignate in solution 3 alsowhile in solution while differs it shows in the ashape it shows a band for band of the for the CD the xerogel, spectra xerogel, which which in solution is in agreement and in with is in agreement the xerogel with the (Figure the predicted predicted 7a). It isstructure structure ofbisignate ofinitssolution its aggregates aggregates while that suggests that suggests it ashows a helical helicala torsion band for torsion asthe as in shown xerogel, shown which5. Figure inisFigure 5. in agreement with the predicted structure of its aggregates that suggests a helical torsion as shown in Figure 5. a) b) c) 1.0a) 7G xerogel xerogel 1.0b) 8 xerogel pentane c) 1.0 3 4 8 solution methanol 5 9 xerogel 7G solution solution 8 xerogel acetone 9 solution Normalized [φ] 0.5 1.0 0.5 1.0 Normalized [φ] 8 xerogel pentane Normalized [Φ] 0.5 1.0 7G xerogel xerogel Δε 3 4 8 solution methanol 5 9 xerogel 7G solution solution 8 xerogel acetone 9 solution Normalized 0.0 0.5 0.0 0.5 Normalized [φ] Normalized [φ] 0.0 Normalized [Φ] 0.5 Normalized Δε -0.5 -0.5 0.0 -0.5 0.0 0.0 -1.0 -1.0 -0.5 -1.0 -0.5 -0.5 200 210 220 230 240 250 260 200 210 220 230 240 250 260 270 200 210 220 230 240 250 -1.0 -1.0 -1.0 Wavelength (nm) Wavelength (nm) Wavelength (nm) 200 210 220 230 240 250 260 200 210 220 230 240 250 260 270 200 210 220 230 240 250 (a)Normalized Figure7.7. (a) Normalized CDspectra spectraofofmethanol methanolWavelength solution(nm) (2.47 mM) and xerogel atWavelength mgc(nm) themgc from Figure Wavelength (nm) CD solution (2.47 mM) and xerogel at the from toluene in KBr (20 mM) of 3 at 25 ◦ C; (b) Normalized CD spectra of methanol solution (2.56 mM) and toluene Figure 7. (a) Normalized CD spectra of methanol solution (2.47 mM) and xerogel at the mgcand in KBr (20 mM) of 3 at 25 °C; (b) Normalized CD spectra of methanol solution (2.56 mM) from xerogels at xerogels at the mgc inin acetone andand pentane, respectively, in KBrin(20KBrmM) 4 at 25of◦ C;4 (c) of mM) Normalized toluene in KBr (20 mM) of 3 at 25 °C; (b) Normalized CD spectra of methanol solution (2.56 mM)(c)and the mgc acetone pentane, respectively, (20 at 25 °C; CD spectra of Normalized CD5 in methanol spectra solution (2.17 mM) and xerogel mM)atand the xerogel mgc from toluene mgcin KBr toluene (20 mM)inat xerogels at the mgc of in5 acetone in methanol and solution pentane,(2.17 respectively, in KBr at(20themM) from of 4 at 25 °C; (c) 25 ◦ C. KBrNormalized (20 mM) at CD 25 °C.spectra of 5 in methanol solution (2.17 mM) and xerogel at the mgc from toluene in KBr (20 mM) at 25 °C. Molecules 2019, 24, x 9 of 17 Molecules 2019, 24, 352 9 of 17 Compounds 4 and 5 were also computed. As was hypothesized when the HSPs were analyzed (see above), these compounds could present two different aggregation patterns depending on the Compounds 4 and 5 were also computed. As was hypothesized when the HSPs were analyzed predominant interactions in apolar or polar solvents. In α-type aggregates from 4, the molecules are (see above), these compounds could present two different aggregation patterns depending on the placed in a zig-zag disposition because of the intermolecular interactions between amide groups (see predominant interactions in apolar or polar solvents. In α-type aggregates from 4, the molecules are Figure S3). This type of interactions would be favored in polar solvents and, indeed, the CD placed in a zig-zag disposition because of the intermolecular interactions between amide groups (see spectrum of the xerogel from acetone (prepared at the mgc) is bisignate (Figure 7b). Figure S3). This type of interactions would be favored in polar solvents and, indeed, the CD spectrum On the other hand, the predicted β-type structure (Figure S3) shows that compound 4 can of the xerogel from acetone (prepared at the mgc) is bisignate (Figure 7b). self-assemble through hydrogen bonding interactions of the unprotected hydroxyl groups, and then On the other hand, the predicted β-type structure (Figure S3) shows that compound 4 can forms an aggregate which shows a torsion that will produce some helicity (right-handed helix). This self-assemble through hydrogen bonding interactions of the unprotected hydroxyl groups, and then type of interaction is expected to be predominant in apolar solvents as confirmed by the forms an aggregate which shows a torsion that will produce some helicity (right-handed helix). monosignate CD spectrum of 4 as a xerogel from pentane (Figure 7b). This type of interaction is expected to be predominant in apolar solvents as confirmed by the Therefore, the results from the consideration of HSPs for organogelator 4 are consistent with the monosignate CD spectrum of 4 as a xerogel from pentane (Figure 7b). CD spectra of the xerogels from polar or apolar solvents and can be explained by the predictions Therefore, the results from the consideration of HSPs for organogelator 4 are consistent with the from computational calculations. CD spectra of the xerogels from polar or apolar solvents and can be explained by the predictions from In a similar manner, compound 5 can aggregate in two different ways. Figure 8 shows the computational calculations. α−type and the β−type structure, where two α−aggregates are interacting with each other through In a similar manner, compound 5 can aggregate in two different ways. Figure 8 shows the α-type hydrogen bond interactions between the unprotected hydroxyl groups. In this way, the free and the β-type structure, where two α-aggregates are interacting with each other through hydrogen hydroxyl groups of the molecule would be used to form aggregates avoiding interactions with the bond interactions between the unprotected hydroxyl groups. In this way, the free hydroxyl groups of solvent. This could explain the insolubility of 5 in alcohols. Moreover, if the polar head of this the molecule would be used to form aggregates avoiding interactions with the solvent. This could organogelator was strongly interacting with another polar head, the parts of the molecule available explain the insolubility of 5 in alcohols. Moreover, if the polar head of this organogelator was strongly for aggregation would only be the amide groups and the long alkyl chains, which would explain interacting with another polar head, the parts of the molecule available for aggregation would only be why 5 behaves in a very similar manner to the non-substituted cyclohexane-based compounds cis- the amide groups and the long alkyl chains, which would explain why 5 behaves in a very similar and trans-1 [24] (see Table 1). manner to the non-substituted cyclohexane-based compounds cis- and trans-1 [24] (see Table 1). 8. (a) Figure 8. Figure (a) side sideview viewofofcentral central6 molecules 6 moleculesin in octameric 1-D1-D octameric aggregate (amide aggregate hydrogen-bonds) (amide 5-α. hydrogen-bonds) (b) side views of octameric 2-D aggregates (amide and hydroxyl hydrogen-bonds) 5-α. (b) side views of octameric 2-D aggregates (amide and hydroxyl hydrogen-bonds) 5-β. 5-β. Non-polar hydrogen atoms have been omitted for clarity. Atoms in amide groups have been represented with red Non-polar hydrogen atoms have been omitted for clarity. Atoms in amide groups have been (oxygen), blue (nitrogen) and grey (carbon) spheres. represented with red (oxygen), blue (nitrogen) and grey (carbon) spheres. Although compound 5 is rather insoluble in methanol, the low concentration required for CD Although compound 5 is rather insoluble in methanol, the low concentration required for CD spectroscopy, 2.17 mM in this case, allowed the spectrum to be recorded in this solvent. Thus, spectroscopy, 2.17 mM in this case, allowed the spectrum to be recorded in this solvent. Thus, the the normalized CD spectra (Figure 7c) show that while compound 5 presents a band in methanol normalized CD spectra (Figure 7c) show that while compound 5 presents a band in methanol solution, the spectrum for the xerogel is bisignate with zero crossing at 205 nm, which is consistent solution, the spectrum for the xerogel is bisignate with zero crossing at 205 nm, which is consistent with the formation of a curved vertical aggregate. However, both predicted α- and β-type structures with the formation of a curved vertical aggregate. However, both predicted α- and β-type structures Molecules 2019, 24, 352 10 of 17 Molecules 2019, 24, x 10 of 17 for compound compound 5 would be compatible compatible with with these results and the CD data does not provide enough evidence to distinguish between them. These structural features were also supported by the aggregation energy of the formation of the dimeric totothethe octameric octameric aggregates, aggregates, whichwhich was calculated was calculated for compoundsfor compounds 2–5 (∆Eagg ).2–5 The(ΔE agg). The aggregation aggregation energy per energy per molecule (∆Emolecule agg /n) (ΔE was also agg /n) was also considered considered (see Figure (see S2). Figure Figure S2). 9 Figure shows 9 the shows the computed computed values and values and suggests thatsuggests that the the aggregation of aggregation all compounds of isallfavorable compounds is favorable because because the aggregation the energy aggregation becomes more energy becomes negative as themore negative number as the number of monomers of monomers increases. increases. 50 5cis-3 cis-2 6trans-3 trans-2 0 743 8-α 5−α 4-α -50 8-β 5−β 4-β (kcal/mol) 9-α 6−α 5-α -100 9-β 6−β 5-β agg -150 ΔE -200 -250 -300 2 4 6 8 Number of monomers Figure 9. Aggregation energies of compounds 2–5. Figure 9. Aggregation energies of compounds 2–5. Moreover, for compounds that have two different aggregation patterns (4 and 5) these data Moreover, for compounds that have two different aggregation patterns (4 and 5) these data provide more information. Although the formation of a dimer with β-type structure is in both provide more information. Although the formation of a dimer with β−type structure is in both cases cases not favorable because the aggregation energy of the formation is positive, the formation of not favorable because the aggregation energy of the formation is positive, the formation of the the tetrameric, the hexameric and the octameric aggregates becomes favorable. This fact probably tetrameric, the hexameric and the octameric aggregates becomes favorable. This fact probably means means that, first of all, the monomer self-assembles by hydrogen bonding through the amide groups that, first of all, the monomer self-assembles by hydrogen bonding through the amide groups and and then, in a second step, the formation of the β−aggregate is favorable. In addition, it is shown then, in a second step, the formation of the β−aggregate is favorable. In addition, it is shown that the that the aggregation energy to form the octamer with the predicted β-type structures for 4 and 5 is aggregation energy to form the octamer with the predicted β−type structures for 4 and 5 is more more favorable than the formation of the octamers with the predicted α-type structures, allowing the favorable than the formation of the octamers with the predicted α−type structures, allowing the possibility of the coexistence of both of them. For the case of organogelator 5, the network formed possibility of the coexistence of both of them. For the case of organogelator 5, the network formed by by inter- and intramolecular hydrogen bonding between the unprotected hydroxyl groups seems inter- and intramolecular hydrogen bonding between the unprotected hydroxyl groups seems to be to be much stronger than the predicted β-type structure of compound 4. The β-type aggregate much stronger than the predicted β−type structure of compound 4. The β−type aggregate predominantly formed in polar media would explain the insolubility of 5, because the free hydroxyl predominantly formed in polar media would explain the insolubility of 5, because the free hydroxyl groups are not able to interact with the solvent. groups are not able to interact with the solvent. 3. Materials and Methods 3. Materials and Methods 3.1. General Procedures 3.1. General Procedures Melting points were determined using a Kofler Thermogerate apparatus (Reichert, Wien, Austria) Melting and are points Specific uncorrected. were determined using rotations were a Kofler recorded on aThermogerate DIP-370 opticalapparatus polarimeter(Reichert, (JASCO, Wien, Tokio, Austria) and arespectra Japan). Infrared uncorrected. Specificon were recorded rotations a UATRwere recorded Spectrum on a DIP-370 two (Perkin optical polarimeter Elmer, Waltham, MA, USA, (JASCO, films on Tokio, NaCl). Japan). NuclearInfrared magneticspectra were spectra resonance recordedwereon arecorded UATR Spectrum on a Mercurytwo (Perkin Elmer, 300 apparatus Waltham, MA, (Varian, Palo USA, Alto, CA,films USA).on NaCl). Mass Nuclear spectra magneticonresonance were obtained a MS 50 TC spectra were recorded mass spectrometer on a (Kratos, Mercury Manchester, UK). Thin layer chromatography (tlc) was performed using GF-254 type 60 silica TC 300 apparatus (Varian, Palo Alto, CA, USA). Mass spectra were obtained on a MS 50 gel mass spectrometer (Merck, Kenilworth,(Kratos, Manchester, NJ, USA) UK). Thin mixtures and EtOAc/hexane layer chromatography as eluents; the(tlc) was performed tlc spots using were visualized GF-254 type 60 silica with a Hanessian staingel (Merck, (dipping Kenilworth, into NJ, a solution of USA) 12.5 g of and (NHEtOAc/hexane 4 )4 Mo7 O24 ·4H2mixtures as eluents; O, 5 g of Ce(SO 4 )2 ·4Hthe 2O tlc spots were visualized with a Hanessian stain (dipping into a solution of 12.5 g of (NH4)4Mo7O24.4H2O, 5 g of Ce(SO4)2·4H2O and 50 mL of H2SO4 in 450 mL of H2O, and warming). Column chromatography was carried out using Merck type 9385 silica gel. Molecules 2019, 24, 352 11 of 17 and 50 mL of H2 SO4 in 450 mL of H2 O, and warming). Column chromatography was carried out using Merck type 9385 silica gel. 3.2. Experimental Section Synthesis of (3aS,4R,7S,8S,8aR)-2,2-dimethyl-6-oxohexahydro-4,7-methano[1,3]dioxolo[4–c]oxepine-8-carboxylic acid (7): Sodium nitrite (0.48 g, 7.00 mmol) and acetic acid (1.33 mL, 23.3 mmol) were added over a solution of lactone 6, prepared according to reference 31, (0.6 g, 2.33 mmol) in DMSO (7.0 mL), under argon. The resulting mixture was stirred at 35 ◦ C for 72 h, 25 mL of HCl aqueous 3 M solution were then added and the reaction allowed to stir at rt for 15 min. H2 O (50 mL) were added over the reaction, the resulting mixture extracted with Et2 O (3 × 25 mL), the organic layers dried with anhydrous sodium sulfate, filtered, and evaporated to dryness to give an oil that, purified by flash column chromatography (CH2 Cl2 /MeOH 95:5), gave compound 7 (0.43 g, 76% yield) as a yellow oil. Rf = 0.2 (CH2 Cl2 /MeOH 95:5); [α]D 1 20 −5.8 (c = 2.1 in CHCl3 ); H-NMR (300 MHz, CDCl3 ) δ = 1.39 (s, 3H, CH3 ); 1.56 (s, 3H, CH3 ); 2.18–2.44 (m, 2H, CH2 ); 3.32 (dd, 1H, J = 2.0 Hz, J = 4.3 Hz, H7); 3.40–3.49 (m, 1H, H8); 4.43 (dd, 1H, J = 4.0 Hz, J = 7.6 Hz, H3a); 4.53 (dd, 1H, J = 4.2 Hz, J = 8.0 Hz, H8a); 4.78 (td, 1H, J = 1.5 Hz, J = 1.7 Hz, J = 4.0 Hz, H4); 5.13 (bs, 1H, CO2 H) ppm; 13 C-NMR (75 MHz, CDCl3 ) δ = 23.4 (CH2 ); 24.5 (CH3 ); 26.0 (CH3 ); 34.0 (CH); 44.6 (CH); 72.8 (CH); 76.9 (CH); 114.4 (C); 173.2 (C); 176.4 (C) ppm; IR: ν = 3452, 1766, 1701, 1210, 1078 cm−1 ; HRMS (ESI+ ) calcd m/z for C11 H14 NaO6 [M + Na]+ : 265.0688, found: 265.0684. Synthesis of (3aS,4R,7S,8S,8aR)-N-hexadecyl-2,2-dimethyl-6-oxohexahydro-4,7-methano[1,3]-dioxolo[4–c] oxepin-8-carboxamide (8): PyBOP (0.78 g, 0.15 mmol) and DIEA (0.11 mL, 0.63 mmol) were added over a solution of acid 7 (40 mg, 0.10 mmol), in anhydrous CH2 Cl2 (1.4 mL) and the resulting mixture was stirred for 15 min at rt, under argon. Hexadecylamine (0.124 g, 0.52 mmol) in anhydrous CH2 Cl2 (1.4 mL) and anhydrous DMF (0.5 mL) was then added and the reaction stirred for 2 h at rt. The mixture was then evaporated to dryness, the resulting residue dissolved in CH2 Cl2 (10 mL), extracted with saturated aqueous solution of citric acid (3 × 5 mL), saturated aqueous solution of NaHCO3 (3 × 5 mL) and H2 O (10 mL). The organic solution was dried with anhydrous sodium sulfate and evaporated to give a solid, which was purified by flash column chromatography (CH2 Cl2 /MeOH 95:5) to afford compound 8 (50 mg, 98% yield) as a yellow solid. Rf = 0.3 (CH2 Cl2 /MeOH 95:5); m.p. 209–211 ◦ C (MeOH); [α]D 1 20 +15.2 (c = 1.0 in CHCl3 ); H-NMR (300 MHz, CDCl3 ) δ = 0.86 (t, 3H, J = 6.6 Hz, CH3 ); 1.22–1.29 (m, 26H, 13 × CH2 ), 1.37 (s, 3H, CH3 ); 1.44–1.49 (m, 2H, CH2 ); 1.54 (s, 3H, CH3 ); 2.21–2.27 (m, 2H, CH2 ); 3.03 (dd, 1H, J = 4.1 Hz, J = 1.9 Hz, H8); 3.10 (ddd, 1H, J = 9.9 Hz, J = 6.6 Hz, J = 2.0 Hz, H7); 3.22 (q, 2H, J = 6.7 Hz, -CH2 CO); 4.41 (dd, 1H, J = 8.1 Hz, J = 4.2 Hz, H8a); 4.48 (dd, 1H, J = 8.0 Hz, J = 4.1 Hz, H3a); 4.75 (td, 1H, J = 3.9 Hz, J = 1.9 Hz, H4); 5.69 (t, 1H, J = 5.7 Hz, NH) ppm; 13 C-NMR (75 MHz, CDCl3 ) δ = 14.2 (CH3 ); 22.8 (CH2 ); 23.0 (CH2 ); 24.4 (CH3 ); 25.8 (CH3 ); 27.0 (CH2 ); 29.4 (CH2 ); 29.5 (CH2 ); 29.6 (CH2 ); 29.7 (CH2 ); 29.8 (CH2 ); 29.8 (CH2 ); 32.0 (CH2 ); 34.5 (CH); 40.1 (CH2 ); 43.9 (CH); 71.8 (CH); 71.9 (CH); 75.0 (CH); 113.3 (C); 171.0 (C); 172.3 (C) ppm; IR: ν = 1766, 1645, 1209, 1077 cm−1 ; HRMS (ESI+ ) calcd m/z for C27 H48 N2 O5 [M + H]+ : 466.3527, found: 466.3537. Synthesis of (3aR,4S,5S,7R,7aS)-N,N0 -dihexadecyl-7-hydroxy-2,2-dimethylhexahydrobenzo[d][1,3]dioxole-4, 5-dicarboxamide (3): 2-Hydroxypyridine (5.8 mg, 0.06 mmol) and hexadecylamine (0.27 g, 1.11 mmol) were added over a solution of carboxamide 8 (47.2 mg, 0.10 mmol) in dry THF (1 mL), under argon. The resulting solution was stirred for 2 h at rt, evaporated to dryness and the amorphous solid obtained purified by flash column chromatography (CH2 Cl2 /MeOH, 97:3), to give compound 3 (21.2 mg, 30% yield) as a crystalline white solid. Rf = 0.3 (CH2 Cl2 /MeOH 97:3); m.p. 150–151 ◦ C; (MeOH); [α]D 20 +51.3 (c = 1.0 in CHCl3 ); 1 H-NMR (300 MHz, CDCl3 ) δ = 0.85–0.89 (m, 6H, 2 × CH3 ); 1.24–1.31 (m, 52H, 26 × CH2 ); 1.38–1.51 (m, 10H, 2 × CH3 + 2 × CH2 ); 1.96 (dt, 1H, J = 15.1 Hz, J = 5.4 Hz, H6); 2.12 (dt, 1H, J = 15.1 Hz, J = 2.6 Hz, H60 ); 2.59 (dd, 1H, J = 10.1 Hz, J = 4.3 Hz, H5); 3.03–3.37 (m, 5H, H4 + 2 × CH2 ); 4.08–4.13 (m, 1H); 4.32–4.41 (m, 2H); 6.44 (d, 1H, J = 10.4 Hz, OH); 6.96 (t, 1H, J = 5.7 Hz, NH); 7.33 (t, 1H, J = 5.6 Hz, NH) ppm; 13 C-NMR (75 MHz, CDCl3 ) δ = 14.3 (CH3 ); 22.8 (CH2 ); 26.2 (CH3 ); Molecules 2019, 24, 352 12 of 17 27.0 (CH2 ); 27.1 (CH2 ); 28.6 (CH3 ); 29.4 (CH2 ); 29.5 (CH2 ); 29.6 (CH2 ); 29.7 (CH2 ); 29.8 (CH2 ); 29.8 (CH2 ); 30.9 (CH2 ); 32.1 (CH2 ); 39.7 (CH2 ); 39.9 (CH2 ); 40.5 (CH); 46.5 (CH); 66.1 (CH); 72.6 (CH); 80.4 (CH); 108.5 (C); 172.4 (C); 174.7 (C) ppm; IR: ν = 3327, 1652, 1550, 1468, 1221, 1068 cm−1 ; HRMS (ESI+ ) calcd m/z for C43 H83 N2 O5 [M + H]+ : 707.6302, found: 707.6291. Synthesis of (3aR,4S,5S,7R,7aR)-7-((tert-butyldimethylsilyl)oxy)-N,N-dihexadecyl-2,2-dimethyl-hexahydrobenzo [d][1,3]dioxole-4,5-dicarboxamide (cis-2): Imidazole (0.13 g, 1.88 mmol) and TBDMSCl (0.15 g, 0.96 mmol) were added over a stirred solution of amide 3 (0.3 g, 0.42 mmol) in dry DMF (17 mL), under argon. The resulting mixture was stirred for 16 h at rt, quenched with brine (20 mL) and then extracted with EtOAc (32 × 20 mL). The organic layers were dried with anhydrous sodium sulfate, filtered and evaporated. The crude product obtained was purified by flash column chromatography (EtOAc/hexane 1:3) to give compound cis-2 (0.26 g, 74% yield) as a white solid. Rf = 0.3 (EtOAc/hexane 1:3); m.p. 82–83 ◦ C (EtOAc); [α]D 1 20 −16.1 (c = 2.2 in CHCl3 ); H-NMR (300 MHz, CDCl3 ) δ = 0.05 (s, 3H, CH3 ), 0.09 (s, 3H, CH3 ), 0.85–0.90 (m, 15H, (CH3 )3 + 2 × CH3 ), 1.25 (bs, 52H, 26 × CH2 ), 1.34 (s, 3H, CH3 ), 1.39–1.48 (m, 7H, CH3 + 2 × CH2 ), 1.37–1.94 (m, 2H, H6 + H60 ), 2.92 (t, 1H, J = 5.8 Hz, H4), 3.04 (td, 1H, J = 7.2 Hz, J = 4.9 Hz, H5), 3.17 (ddt, 4H, J = 10.5 Hz, J = 7.5 Hz, J = 4.8 Hz, 22 × NCH2 ), 3.73 (dt, 1H, J = 10.3 Hz, J = 6.8 Hz), 4.19 (t, 1H, J = 6.9 Hz), 4.52 (t, 1H, J = 6.4 Hz), 5.94 (t, 1H, J = 5.4 Hz, NH), 6.58 (t, 1H, J = 5.7 Hz, NH) ppm; 13 C-NMR (75 MHz, CDCl3 ) δ = −4.7 (CH3 ), −4.2 (CH3 ), 14.3 (CH3 ), 18.2 (C), 22.8 (CH2 ), 25.8 (CH3 ), 26.0 (CH3 ), 27.1 (CH2 ), 28.0 (CH3 ), 29.5 (CH2 ), 29.6 (CH2 ), 29.7 (CH2 ), 29.8 (CH2 ), 30.9 (CH2 ), 32.1 (CH2 ), 39.6 (CH2 ), 39.8 (CH), 39.9 (CH2 ), 45.4 (CH), 72.2 (CH), 74.3 (CH), 80.4 (CH), 108.4 (C), 171.4 (C), 173.4 (C) ppm; IR: ν = 3287, 1652, 1560, 1112, 837 cm−1 ; HRMS (ESI+ ) calcd m/z for C49 H97 N2 O5 Si [M + H]+ : 821.7161, found: 821.7129. Synthesis of (1S,2S,3R,4S,5R)-N,N-dihexadecyl-3,4,5-trihydroxycyclohexane-1,2-dicarboxamide (5): H2 O (9.0 mL) and TFA (18.0 mL) were added over a stirred solution of diamide 3 (0.3 g, 0.42 mmol) in MeOH (10 mL) and the obtained mixture was stirred for 12 h at rt. The solvent was removed under vacuum, the residue coevaporated with toluene (3 × 10 mL) and the crude obtained purified by flash column chromatography (CH2 Cl2 /MeOH 93:7) to give dicarboxamide 5 (0.13 g, 42% yield) as a white solid. Rf = 0.3 (CH2 Cl2 /MeOH 93:7); m.p. 166.0–166.6 ◦ C (MeOH); [α]D 20 −27.3 (c = 2.0 in CHCl3 ); 1 H-NMR (300 MHz, THF-d8) δ = 0.88 (t, 6H, J = 6.6 Hz, 2 × CH ), 1.25–1.51 (m, 56H, 28 × CH ), 3 2 1.79–1.96 (m, 2H, H6 + H60 ), 2.83–3.17 (m, 6H, 2 × NCH2 + H1 + H2), 3.34–4.39 (m, 6H, H3 + H4 + H5 + 3 × OH), 7.33 (bs, 1H, NH), 7.69 (t, 1H, J = 5.6 Hz, NH) ppm; 13 C-NMR (75 MHz, THF-d8, main rotamer) δ = 14.6 (CH3 ), 23.7 (CH2 ), 28.1 (CH2 ), 28.2 (CH2 ), 30.3 (CH2 ), 30.5 (CH2 ), 30.6 (CH2 ), 30.7 (CH2 ), 30.8 (CH2 ), 30.8 (CH2 ), 33.0 (CH2 ), 40.1 (CH2 ), 40.5 (CH2 ), 42.8 (CH), 46.4 (CH), 69.4 (CH), 70.9 (CH), 75.3 (CH), 172.7 (C), 175.8 (C) ppm; IR: ν = 3328, 1649, 1542, 1068 cm−1 ; HRMS (ESI+ ) calcd m/z for C40 H79 N2 O5 [M + H]+ : 667.5984, found: 667.5976. Synthesis of (3aR,4S,5R,7R,7aR)-7-((tert-butyldimethylsilyl)oxy)-5-(methoxycarbonyl)-2,2-dimethyl-hexa- hydrobenzo[d][1,3]dioxole-4-carboxylic acid (10): Sodium nitrite (26 mg, 0.37 mmol) and acetic acid (71 µL, 1.24 mmol) were added over a solution of nitroester 9 [20] (50 mg, 0.12 mmol) in DMSO (0.6 mL), under argon. The resulting mixture was stirred at 35 ◦ C for 72 h, 15 mL of H2 O and deactivated Dowex-50 resin were then added until pH = 3 and the reaction allowed to stir for 15 min at rt. H2 O (20 mL) were added over the reaction, the resulting mixture extracted with Et2 O (3 × 10 mL), the organic layers dried with anhydrous sodium sulfate, filtered, and evaporated to dryness to give an oil that, purified by flash column chromatography (CH2 Cl2 /MeOH 95:5), gave compound 10 (30 mg, 55% yield) as a clear oil. Rf = 0.3 (CH2 Cl2 /MeOH 95:5); [α]D 1 20 +4.1 (c = 1.2 in CHCl3 ): H-NMR (300 MHz, CDCl3 ) δ = 0.08 (bs, 6H, 2 × CH3 ); 0.88 (bs, 9H, (CH3 )3 ); 1.35 (s, 3H, CH3 ); 1.52 (s, 3H, CH3 ); 1.85–1.89 (m, 2H, CH2 ); 2.86 (dd, 1H, J = 10.7 Hz, J = 8.2 Hz), 2.94–3.13 (m, 1H, H5), 3.68 (s, 3H, OCH3 ); 3.99 (dd, 1H, J = 5.0 Hz, J = 2.9 Hz), 4.21 (q, 1H, J = 3.2 Hz), 4.40 (dd, 1H, J = 5.0 Hz, J = 8.3 Hz); 9.32 (bs, 1H, OH) ppm; 13 C-NMR (75 MHz, CDCl3 ) δ = −4.9 (CH3 ); −4.8 (CH3 ); 18.1 (C); 25.8 (CH3 ); 26.2 (CH3 ); 28.2 (CH3 ); 30.9 (CH2 ); 37.0 (CH); 47.4 (CH); 52.3 (CH3 ); 67.1 (CH); 74.6 (CH); 76.9 (CH); 109.7 (C); 174.6 (C); Molecules 2019, 24, 352 13 of 17 178.9 (C) ppm; IR: ν = 3375, 1735, 1258, 1100, 810 cm−1 ; HRMS (ESI+ ) calcd m/z for C18 H32 NaO7 Si (M + Na)+ : 411.1815, found: 411.1809. Synthesis of Methyl (3aR,4S,5R,7R,7aR)-7-((tert-butyldimethylsilyl)oxy)-4-(hexadecyl-carbamoyl)-2,2- dimethylhexahydrobenzo[d][1,3]dioxole-5-carboxylate (11): A mixture containing a solution of acid 10 (50 mg, 0.13 mmol), PyBOP (0.10 g, 0.19 mmol) and DIEA (0.14 mL, 0.80 mmol) in anhydrous CH2 Cl2 (1.8 mL) was stirred for 15 min at rt, under argon. Hexadecylamine (0.15 g, 0.52 mmol) in anhydrous CH2 Cl2 (1.8 mL) and anhydrous DMF (0.5 mL) were then added. The reaction was stirred for 2 h at rt, evaporated to dryness, the resulting residue dissolved in CH2 Cl2 (10 mL), extracted with saturated aqueous solution of citric acid (3 × 5 mL), saturated aqueous solution of NaHCO3 (3 × 5 mL) and water (10 mL). The organic layer was then dried with anhydrous sodium sulfate and evaporated to give a yellow solid, which on purification by flash column chromatography (CH2 Cl2 /MeOH 99:1) afforded compound 11 (60 mg, 74% yield) as a clear oil. Rf = 0.3 (CH2 Cl2 /MeOH 99:1); [α]D 20 +48.3 (c = 1.0 in CHCl3 ); 1 H-NMR (300 MHz, CDCl3 ) δ = 0.07 (s, 3H, CH3 ); 0.08 (s, 3H, CH3 ); 0.84–0.89 (m, 12H, -(CH3 )3 + CH3 ); 1.24–1.29 (m, 26H, 13 × CH2 ); 1.36 (s, 3H, CH3 ); 1.42–1.52 (m, 2H, CH2 ); 1.56 (s, 3H, CH3 ); 1.77–1.83 (m, 2H, H6 + H60 ); 2.58 (dd, 1H, J = 11.9 Hz, J = 9.8 Hz, H4); 3.00 (ddd, 1H, J = 12.4 Hz, J = 8.9 Hz, J = 6.7 Hz, H5); 3.21 (q, 2H, J = 6.8 Hz, N-CH2 ); 3.68 (s, 3H, OCH3 ); 3.98 (dd, 1H, J = 4.9 Hz, J = 2.6 Hz); 4.20–4.25 (m, 2H); 6.19 (t, 1H, J = 5.8 Hz, NH) ppm; 13 C-NMR (75 MHz, CDCl3 ) δ = −4.9 (CH3 ); −4.7 (CH3 ); 14.2 (CH3 ); 18.1 (C); 22.8 (CH2 ); 25.8 (CH3 ); 26.5 (CH3 ); 27.0 (CH2 ); 28.7 (CH3 ); 29.4 (CH2 ); 29.5 (CH2 ); 29.7 (CH2 ); 29.8 (CH2 ); 29.8 (CH2 ); 32.1 (CH2 ); 32.3 (CH2 ); 36.3 (CH); 39.6 (CH2 ); 48.3 (CH); 52.0 (CH3 ); 67.3 (CH); 75.1 (CH); 77.3 (CH); 109.4 (C); 172.5 (C); 176.0 (C) ppm; IR: ν = 3322, 1740, 1652, 1249, 1070, 837 cm−1 ; HRMS (ESI+ ) calcd m/z for C34 H66 NO6 Si (M + H)+ : 612.4659, Found: 612.4654. Synthesis of (3aR,4S,5R,7R,7aR)-7-((tert-butyldimethylsilyl)oxy)-N,N-dihexadecyl-2,2-dimethyl-hexahydro- benzo[d][1,3]dioxole-4,5-dicarboxamide (trans-2): 2-Hydroxipyridine (78 mg, 0.49 mmol) and hexadecylamine (2.17 g, 8.99 mmol) were added over a solution of amide 11 (0.5 g, 0.82 mmol), in dry THF (10.2 mL), under argon. The reaction mixture was refluxed for 12 h, the solvent removed under vacuum and the obtained residue extracted with CH2 Cl2 (3 × 50 mL). The organic layers were dried with anhydrous sodium sulfate, filtered and evaporated in vacuo to give a residue, which on purification by flash column chromathography (CH2 Cl2 /MeOH 99:1) gave compound trans-2 (0.2 g, 30% yield) as a white solid. Rf = 0.4 (CH2 Cl2 /MeOH 99:1); m.p. 63–64 ◦ C (MeOH); [α]D 20 +31.0 (c = 1.0 in CHCl3 ); 1 H-NMR (300 MHz, CDCl3 ) δ = 0.07 (s, 3H, CH3 ), 0.08 (s, 3H, (CH3 )3 ) 0.84–0.89 (m, 15H, (CH3 )3 + 2 × CH3 ), 1.24 (bs, 52H, 26 × CH2 ), 1.34 (s, 3H, CH3 ), 1.41–1.47 (m, 4H, 2 × CH2 ), 1.53 (s, 3H, CH3 ), 1.63 (dt, 1H, J = 14.0 Hz, J = 3.8 Hz, H6), 2.00 (ddd, 1H, J = 14.1 Hz, J = 11.3 Hz, J = 2.7 Hz, H60 ), 2.45 (dd, 1H, J = 11.7 Hz, J = 9.3 Hz, H4), 2.70 (td, 1H, J = 11.5 Hz, J = 3.5 Hz, H5), 3.15–3.19 (m, 4H, 2 × N-CH2 ), 3.98 (dd, 1H, J = 5.2 Hz, J = 2.9 Hz, H7), 4.25 (q, 1H, J = 3.0 Hz, H3a), 4.32 (dd, 1H, J = 9.3 Hz, J = 5.1 Hz, H7a), 5.76 (t, 1H, J = 5.8 Hz, NH), 5.93 (t, 1H, J = 5.7 Hz, NH) ppm; 13 C-NMR (75 MHz, CDCl3 ) δ = −4.8 (CH3 ), −4.7 (CH3 ), 14.2 (CH3 ), 18.2 (C), 22.8 (CH2 ), 25.9 (CH3 ), 26.3 (CH3 ), 27.0 (CH2 ), 28.6 (CH3 ), 29.5 (CH2 ), 29.5 (CH2 ), 29.6 (CH2 ), 29.7 (CH2 ), 29.8 (CH2 ), 29.8 (CH2 ), 32.1 (CH2 ), 38.8 (CH), 39.7 (CH2 ), 39.8 (CH), 50.1 (CH2 ), 67.6 (CH), 75.6 (CH), 77.3 (CH), 109.2 (C), 172.7 (C), 174.1 (C) ppm; IR: ν = 1653, 1117, 1065, 838 cm−1 ; HRMS (ESI+ ) calcd m/z for C49 H97 N2 O5 Si (M + H)+ : 821.7167, found: 821.7169. Synthesis of (1R,2S,3R,4R,5R)-5-((tert-butyldimethylsilyl)oxy)-N,N-dihexadecyl-3,4-dihydroxy-cyclohexane-1, 2-dicarboxamide (4): Trifluoroacetic acid (7.4 mL) was added over a solution of diamide trans-2 (0.8 g, 0.09 mmol) in a mixture of MeOH (3.7 mL) and H2 O (3.7 mL) and the resulting mixture was stirred for 12 h at rt. Solvent was removed under vacuum, coevaporated with toluene (3 × 5 mL) and the crude obtained purified by flash column chromatography (CH2 Cl2 /MeOH 9:1) to give compound 4 (0.35 g, 47% yield) as a yellow solid. Rf = 0.4 (CH2 Cl2 /MeOH 90:10); m.p. 96–97 ◦ C (MeOH); [α]D 20 +7.0 (c = 1.7 in CHCl3 ); 1 H-NMR (300 MHz, CDCl3 ) δ = 0.04 (s, 3H, CH3 ), 0.05 (s, 3H, CH3 ), 0.85–0.89 (m, 15H, (CH3 )3 + 2 × CH3 ), 1.17–1.52 (m, 56H, 28 × CH2 ), 2.14 (t, 1H, J = 13.1 Hz, H6), 2.71–2.81 Molecules 2019, 24, 352 14 of 17 (m, 1H, H60 ), 2.89–3.26 (m, 6H, H4 + H5 + 2 × N-CH2 ), 3.87 (t, 1H, J = 3.3 Hz), 4.08 (q, 1H, J = 2.8 Hz), 4.20 (dd, 1H, J = 10.5 Hz, J = 2.8 Hz), 5.00 (bs, 2H, 2 × OH), 6.11 (t, 1H, J = 5.3 Hz, NH), 7.18 (t, 1H, J = 5.2 Hz, NH) ppm; 13 C-NMR (75 MHz, CDCl3 ) δ = −4.8 (CH3 ), 14.3 (CH3 ), 18.1 (C), 22.8 (CH2 ), 25.9 (CH3 ), 27.1 (CH2 ), 27.2 (CH2 ), 29.5 (CH2 ), 29.6 (CH2 ), 29.7 (CH2 ), 29.8 (CH2 ), 29.9 (CH2 ), 30.6 (CH2 ), 32.1 (CH2 ), 39.9 (CH2 ), 41.8 (CH), 48.2 (CH), 69.5 (CH), 70.3 (CH), 72.5 (CH), 173.1 (C), 174.5 (C) ppm; IR: ν = 3297, 1645, 1119, 1076, 837 cm−1 ; HRMS (ESI+ ) calcd m/z for C46 H93 N2 O5 Si (M + H)+ : 781.6854, found: 781.6855. Procedure for gel preparation: A small amount (5.0 ± 0.1 mg) of bisamide is weighted in a 2 mL transparent-glass vial with septum screw-on cap. When the 5 mg amount of bisamide is soluble in a specific solvent, a new vial containing 10 ± 0.1 mg is prepared and the solubility-gelation checked again. In a second step, a certain volume of solvent to be tested is added and the vial closed. The minimum volume added is 0.05 mL. Then the mixture is heated under the boiling point of the solvent using a balloon system in order to avoid solvent pressure and, once a solution is obtained the mixture is sonicated for 1 to 5 min. For high concentrations and also in some solvents, previous sonication is needed for a good solubilization during heating and sonication time is usually shorter than for diluted gels. Then, the mixture is left to stabilize and to reach room temperature. To state that the mixture is a gel the tube inversion test is done just by turning the vial upside down. If the sample is a gel it does not drop and if it drops a little it can be classified as gel-like mixture. We can also state the mixtures as solutions or insoluble systems. In order to determine the mgc, a new volume of solvent is added to the gel and the process is repeated until no gel is formed: the last volume added determines the mgc. Gels were stable at room temperature and, moreover, they were reversible at the body temperature. Thus, the gel became a solution by heating with a hand. Once the solution was left to cool down at room temperature, the gel was formed again. SEM measurements: SEM images were acquired with Quanta ESEM FEG apparatus equipped with a field emission gun. Wet gels were disposed on a carbon-film-coated copper grid and dried by standing for 30 min on the grid. The resulting xerogels (dry gels) were then introduced into the microscope working at 10 kV and under a pressure of 50 Pa, in most cases (Figure 3a–h), and 29 Pa (Figure 3f). Computational details: A conformational search of each system was carried out using a mixed low mode/torsional sampling [34] with the OPLS-2005 [35] force field implemented in the MacroModel [36] program in order to find and select an approximation of the most stable conformers. The geometries of the lowest energy conformers of each system were optimized using DFT calculations with the Gaussian09 [37] program with the M06-2X [38] functional with the 6-31G(d) basis set. This is a hybrid meta-GGA functional that includes a 54% of exact e × CHange and that was shown to describe correctly non covalent interactions [39]. For all the studied molecules, once the geometry in gas phase is obtained, the dimer is built up and the procedure is repeated. And then, the procedure is repeated again with the tetramer. Once the optimized DFT structure of the tetramer is obtained, the internal dimer of the aggregate is selected to be the model of aggregation to build up the hexamer and the octamer. A minimization of the energy of the hexamer and the octamer is carried out in toluene. Finally, a single point energy calculation in gas phase is carried out to get the energy of each system. Circular dichroism spectra were calculated by taking the optimized structure of the monomer in methanol solution and calculating different excited states with Gaussian09. Representation of the predicted circular dichroism spectra were done using GaussSum software [40]. 4. Conclusions Chiral pentasubstituted cyclohexane derivatives 2–5 show different gelling behavior depending on the degree of protection of the hydroxyl groups and, to some extent, depending on the relative cis/trans stereochemistry. Compounds cis- and trans-2 differ only in the relative configuration of the two amide groups and both compounds are good gelators for methanol and ethanol but they are very Molecules 2019, 24, 352 15 of 17 soluble in most of the other solvents tested. Nevertheless, their SEM images reveal the formation of fibers in the case of cis-2 and of platelets in the case of trans-2. Computational calculations predict the formation of a curved aggregate without a helicity trend for cis-2, whereas for trans-2 a right handed helical aggregate is predicted. These results are confirmed by the CD spectra that show of a bisignate spectrum for cis-2 and a monosignate positive band for trans-2, both as xerogels from methanol. Regarding LMWOGs 3–5, compound 3, which is polar but with low hydrogen bonding ability, is a bad organogelator for apolar solvents and it is soluble in polar ones; the CD spectrum of its toluene xerogel is in agreement with the computed structure that suggests the formation of a right handed helical aggregate. LMWOG 4, bearing two hydroxyl groups, presents ambivalent ability since two types of aggregation are predicted by calculations depending on the polarity of solvents: α-type, which is described as a vertical aggregation promoted by intermolecular amide hydrogen bonding, in polar solvents; and β-type, where two molecules are facing head-to-head and then piled vertically, in apolar ones. This solvent-mediated ambivalence is corroborate by the different morphologies shown by SEM images of xerogels form acetone and pentane, respectively, and supported by Hansen parameters and CD. Finally, LMWOG 5 with three hydroxyl groups does not interact with very polar solvents due to the formation of intra and intermolecular hydrogen bonds and, consequently, it is insoluble in alcohols and water; nevertheless, it is able to gelate low polarity solvents by means of non-hydrogen bonding interactions. Supplementary Materials: The following are available online: 1 H-NMR and 13 C-NMR spectra for compounds 7, 8, 3, cis-2, 5, 10, 11, trans-2 and 4; Figure S1. Predicted CD spectra of compounds cis- and trans-2; Figure S2. Calculated aggregation energies per monomer of compounds 2–5; Figure S3. Predicted structures for aggregates 4-α and 4-β; Total energies of aggregates of compounds 2–5; Cartesian coordinates of aggregates of compounds 2–5. Author Contributions: Conceptualization, R.M.O.; Funding acquisition, O.I., J.C.E. and R.M.O.; Investigation, B.P.-B., M.C., M.S., A.B., O.I., V.B., J.C.E. and R.M.O.; Methodology, B.P.-B., O.I., V.B. and J.C.E.; Supervision, O.I., V.B., J.C.E. and R.M.O.; Writing—original draft, R.M.O.; Writing—review & editing, O.I., V.B. and J.C.E. Funding: Financial support from Spanish Ministry of Science and Innovation (Project CTQ2009-08490) and MINECO (grant CTQ2016-77978-R), the Xunta de Galicia (Project CN2011/037 and Project GRC2014/040), Galchimia S.A., the Xunta de Galicia (Centro singular de investigación de Galicia accreditation 2016–2019, ED431G/09) and the European Union (European Regional Development Fund-ERDF). Acknowledgments: Time allocated in CSUC (Consorci de Serveis Universitaris de Catalunya) is gratefully acknowledged. Conflicts of Interest: There are not conflict to declare. References 1. Van Esch, J.H.; Feringa, B.L. New Functional Materials Based on Self-Assembling Organogels: From Serendipity towards Design. Angew. Chem. Int. Ed. 2000, 39, 2263–2266. [CrossRef] 2. Steed, W. Supramolecular gel chemistry: Developments over the last decade. Chem. Commun. 2011, 47, 1379–1383. [CrossRef] [PubMed] 3. 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[CrossRef] Sample Availability: Samples of the compounds are not available from the authors. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).