Towards antibacterial hydrogels. Synthesis, structural studies and biological activity

Towards antibacterial hydrogels OBJECTIVE Towards antibacterial hydrogels. Synthesis, structural studies and biological activity INDEX 1 TUTORS APPROVAL D. Juan Carlos Estévez Cabanas, Doctor in Organic Chemistry from the University of Santiago de Compostela (1992) and D. Ramón José Estévez Cabanas, Doctor in Organic Chemistry from the University of Santiago de Compostela (1979). CERTIFY: That this Master Dissertation entitled "Towards antibacterial hydrogels. Synthesis, structural studies and biological activity" presented by María Costa de Dios to apply for the title of Master in Chemistry at the Interface with Biology and Materials Science, has been carried out under our direction in the laboratory of this research group and, considering that it has been concluded, we authorise its presentation at the University of Santiago de Compostela. For the record, we sign the present report: Sgd: Juan C. Estévez Cabanas Sgd: Ramón J. Estévez Cabanas Santiago de Compostela, 31st January 2024. Firmado por ESTEVEZ CABANAS JUAN CARLOS - ***7123** el día 30/01/2024 con un certificado emitido por AC FNMT Usuarios ESTEVEZ CABANAS RAMON J. - 76854712M Firmado digitalmente por ESTEVEZ CABANAS RAMON J. - 76854712M Fecha: 2024.01.30 20:27:41 +01'00' Towards antibacterial hydrogels. Synthesis, structural studies and biological activity INDEX 2 1. INDEX 1. INDEX ......................................................................................................................................................... 2 2. SUMMARY ................................................................................................................................................. 4 3. INTRODUCTION ....................................................................................................................................... 5 4. OBJECTIVE .............................................................................................................................................. 14 5. WORK PLANNING .................................................................................................................................. 15 6. DISCUSSION ............................................................................................................................................ 17 5.1. Synthesis of the low molecular weight gelators (LMWGs) ............................................................... 17 5.1.1 Synthesis of the β-amino ester 5 ............................................................................................. 17 5.1.2 Synthesis of the gelling agent 6 .............................................................................................. 19 5.1.3 Synthesis of the gelling agent 7 .............................................................................................. 20 5.1.4 Synthesis of the gelling agent 9 .............................................................................................. 21 5.1.5 Synthesis of the racemic mixture (rac-7) ................................................................................ 23 5.2. Gelification studies ............................................................................................................................ 25 5.2.1 Temperature-dependent gel formation .................................................................................... 25 5.2.2 pH-dependent gel formation ................................................................................................... 28 5.2.3 Racemic mixture: gelation study............................................................................................. 30 5.3. Hydrogels structural studies ............................................................................................................... 31 5.3.1 Infrared Spectroscopy (IR) ..................................................................................................... 31 5.3.2 Scanning Electron Microscopy (SEM) ................................................................................... 33 5.3.3 X-ray Spectroscopy ................................................................................................................. 34 5.4. Drug release studies ........................................................................................................................... 37 5.4.1 Release testing ........................................................................................................................ 38 5.4.2 Release kinetics....................................................................................................................... 39 5.5. Biological “in vitro” tests .................................................................................................................. 41 5.5.1 Bacteriostatic activity: Study of hypotheses ........................................................................... 43 5.5.2 GdL hydrogels: The general effect .......................................................................................... 45 5.5.3 SEM study .............................................................................................................................. 47 7. CONCLUSIONS ....................................................................................................................................... 51 8. EXPERIMENTAL WORK ........................................................................................................................ 52 7.1. Experimental Techniques ................................................................................................................... 52 Towards antibacterial hydrogels. Synthesis, structural studies and biological activity INDEX 3 7.2. Synthetic procedures .......................................................................................................................... 53 7.2.1 Synthesis of (R)-N-benzyl-1-phenylethan-1-amine (1) ........................................................... 53 7.2.2 Synthesis of (1S,2R)-2-benzyl((R)-1-phenylethyl)amino)cyclohexane-1-carboxylate methyl ester (3) .................................................................................................................................... 55 7.2.3 Synthesis of (1R,2R)-2-benzyl((R)-1-phenylethyl)amino)cyclohexane-1-carboxylate methyl ester (4) .................................................................................................................................... 57 7.2.4 Synthesis of (1R,2R)-2-aminocyclohexane-1-carboxylate methyl ester (5) ............................ 59 7.2.5 Synthesis of (1R,2R)-2-dodecanamidocyclohexane-1-carboxylate methyl ester (6) .............. 61 7.2.6 Synthesis of (1R,2R)-2-dodecanamidocyclohexane-1-carboxylic acid (7) ............................. 63 7.2.7 Synthesis of 2-((1R,2R)-2-dodecanamidocyclohexane-1-carbonyl)hydrazine-1-carboxylate tert-butyl ester (8) .................................................................................................................... 65 7.2.8 Synthesis of N-((1R,2R)-2-(hydrazinocarbonyl)cyclohexyl)dodecanamide (9) ...................... 67 7.2.9 Synthesis of the (1R,2R)-2-carbamoylcyclohexane-1-carboxylic acid ((1R,2R)-11) and the (1S,2S)-2-carbamoylcyclohexane-1-carboxylic acid ((1S,2S)-11) (rac-11) ............................. 69 7.2.10 Synthesis of the hydrochloride of (1R,2R)-2-aminocyclohexanecarboxylic acid ((1R,2R)-12) and the hydrochloride of (1S,2S)-2-aminocyclohexanecarboxylic acid ((1S,2S)-12) (rac-12) 71 7.2.11 Synthesis of the methyl (1R,2R)-2-aminocyclohexane-1-carboxylate ((1R,2R)-5) and the methyl (1S,2S)-2-aminocyclohexane-1-carboxylate ((1S,2S)-5) (rac-5) ................................. 73 7.2.12 Synthesis of the (1R,2R)-2-dodecanamidocyclohexane-1-carboxylate methyl ester ((1R,2R)-6) and the (1S,2S)-2-dodecanamidocyclohexane-1-carboxylate methyl ester ((1S,2S)-6)(rac-6) 75 7.2.13 Synthesis of the (1R,2R)-2-dodecanamidocyclohexane-1-carboxylic acid ((1R,2R)-7) and the (1S,2S)-2-dodecanamidocyclohexane-1-carboxylic acid ((1S,2S)-7) (rac-7) .......................... 77 7.2.14 Synthesis of 1,3:2,4-dibenzylidene-D-sorbitol-p,p'-dimethylester (15) .................................. 79 7.2.15 Synthesis of 1,3:2,4-dibenzylidene-D-sorbitol-p,p´-dicarboxylic acid (16) ............................ 81 7.2.16 Synthesis of tert-butyl (((9H-fluoren-9-λl)methoxy)carbonyl)leucylglycinate (19) ............... 83 7.2.17 Sythesis of N-alpha-(9-Fluorenylmethyloxycarbonyl)-L-leucinyl-glycin (20) ....................... 85 7.3. Infrared Spectra .................................................................................................................................. 87 7.4. Ampicillin calibration curve .............................................................................................................. 89 7.5. Study of gelation through pH variation for Gt12-COOH-GdL ........................................................ 90 7.6. Sample preparation protocol for SEM ............................................................................................... 90 Towards antibacterial hydrogels. Synthesis, structural studies and biological activity SUMMARY 4 2. SUMMARY The controlled release of bioactive molecules and the encapsulation of living cells are active areas of research in drug discovery. Hydrogels are attracting significant attention in the medical field, particularly in drug delivery applications, due to their unique physical and chemical properties. This study focuses on the design and study of three new low molecular weight gelators (LMWGs) derived from N-alkylamides derived from β-cyclohexanic amino acids. The study of their gel-forming properties was carried out by determining their minimum gelation concentration (mgc) in 14 solvents through a heating-cooling inversion test. Due to their potential applicability as drug delivery systems, the possibility of forming hydrogels was investigated, and it was only possible with two of the three gel-forming agents: Gt12-COOH and Gt12-NHNH2. These two hydrogels underwent structural studies using Infrared, X-ray and Scanning Electron Microscopy (SEM) to further understand their supramolecular organization and three-dimensional structures. Additionally, they were subjected to studies on the release of a model antibiotic, Ampicillin. These studies determined that the Gt12-COOH hydrogel formed with D-gluconolactone (GdL) established temporal control in drug release, prolonging its activity over time and positioning itself as a promising candidate for drug delivery applications. To conclude, the activity of both hydrogels was studied in biological cultures. A surprising inhibitory effect in the negative control of Gt12-COOH-GdL led to the consideration of various hypotheses regarding its interaction with different bacterial strains in the study. This antibacterial activity was ultimately linked to a tendency towards filamentation, observed exclusively in Gram-negative microorganisms. Towards antibacterial hydrogels. Synthesis, structural studies and biological activity INTRODUCTION 5 3. INTRODUCTION Gels have emerged as highly intriguing materials with a wide range of applications due to their exceptional properties. However, due to their great variability, it is often difficult to encapsulate their entire meaning in a single sentence. So, what is a gel? The first definition of a gel was proposed by Thomas Graham in 1861. 1 He defined materials with a jelly-like appearance as colloids, describing them as an opposite mode of material aggregation compared to crystalline structures because "While the rigidity of the crystalline structure shuts out external impressions, the softness of the gelatinous colloid contributes to its fluidity, allowing the colloid to function as a medium for liquid diffusion". The first comprehensive definition that combines the microscopic and macroscopic perspectives was provided by Flory (Nobel laureate in Chemistry in 1974). According to Flory 2 , a gel is a substance that exhibits a continuous microscopic structure with macroscopic dimensions, remains stable over the time scale of an analytical experiment and behaves as a solid in terms of its rheological behaviour. Gels behave as elastic solids at low values of mechanical stress and transform into viscous liquids above this threshold. Nowadays, a gel can be defined as a meta-stable state of condensed matter in which a reticular solid structure, referred to as the continuous phase, coexists within a liquid, known as the dispersed phase. As Tanaka stated 3 , this state is only possible by the combination of these two parts, where the reticular solid prevents it from flowing like a liquid, while the liquid component prevents the collapse of the solid. This initially positioned the gel state as an intermediate between the solubilization and crystallization of the aggregate 4 , being referred to by some authors as "frustrated crystallization". 5 While it is true that both processes share an important point of convergence, as they are based on the self-assembly of molecules in a homogeneous solution, which is heated and then cooled, their packing is different (even if both are formed in the same solvent!). This statement is supported by the X-ray diffraction data obtained experimentally by Professor Adams. 6 During crystallization, molecules pack isotropically in all three spatial 1 Graham, T. X. Liquid diffusion applied to analysis. Philos. Trans. R. Soc. London. 1861, 151, 183-224. 2 Guenet, J.-M. Physical Aspects of Organogelation: A Point of View. Gels, 2021, 7 (2), 65. 3 Tanaka, T. Gels. Scientific American. 1981, 244 (1), 124-138. 4 García Velázquez, D. Gelificadores multifuncionales de bajo peso molecular. Propiedades y aplicaciones de arquitecturas inteligentes. An. Quím. 2010, 106 (4), 257–267. 5 Bradford, S. C. On the Theory of Gels. Biochemical Journal. 1921, 15 (4), 553–562.1. 6 Houton, K. A.; Morris, K. L.; Chen, L.; Schmidtmann, M.; Jones, J. T. A.; Serpell, L. C.; Lloyd, G. O.; Adams, D. J. On Crystal versus Fiber Formation in Dipeptide Hydrogelator Systems. Langmuir. 2012, 28 (25), 9797–9806. Towards antibacterial hydrogels. Synthesis, structural studies and biological activity INTRODUCTION 6 directions, forming highly ordered crystals. In contrast, gelation favours one over the other dimensions, resulting in anisotropic growth that leads to the formation of fibers (Figure 1). 7 Figure 1. Schematic representation of the equilibrium between the processes of crystallization, dissolution, and gelation. Image taken from Hanabusa (2014). 8 It is important to highlight that the definition of a gel does not solely rely on a material's common identification. In fact, this category encompasses a wide range of complex biological systems that may not exhibit the typical appearance of a gel: as cytoskeletons, blood clots, microbial colonies and inflamed cellular tissues. 9 There are several examples of gels found in nature like gelatin, collagen, agar or starch, but the search for novel materials has driven the development of synthetic gels. These gels exhibit greater variability, leading to the need for two distinct classification approaches. Based on the type of interaction that gives rise to the three-dimensional network we can come across polymeric gels - where the bonds connecting the network's monomers are covalent, resulting in irreversible gelation - or molecular gels - where the structure is maintained by weak bonds, allowing for reversibility between the gel and sol states. On the other hand, if we focus on the dispersed phase of gels, we can distinguish between organogels - when the dispersed phase is an organic solvent - and hydrogels - when it is water or a mixture of other solvent in water at concentrations below 50%. All these gels transform into xerogels when the dispersed phase is removed, while maintaining the reticular arrangement of the continuous phase. 10 7 Menger, F. M.; Caran, K. L. Anatomy of a Gel. Amino Acid Derivatives That Rigidify Water at Submillimolar Concentrations. J. Am. Chem. Soc. 2000, 122 (47), 11679–11691. 8 Hanabusa, K.; Suzuki, M. Development of low-molecular-weight gelators and polymer-based gelators. Polymer Journal. 2014, 46, 776–782. 9 Jones, C. D.; Steed, J. W. Gels with Sense: Supramolecular Materials That Respond to Heat, Light and Sound. Chem. Soc. Rev. 2016, 45 (23), 6546–6596. 10 Sangeetha, N. M., & Maitra, U. Supramolecular gels: Functions and uses. Journal of Chemical Society. 2005, 34 (10), 821830. Towards antibacterial hydrogels. Synthesis, structural studies and biological activity INTRODUCTION 7 Molecular or physical gels can be created through a wide range of substance combinations, such as clays, polymers, proteins, colloids, and specific small organic compounds serving as the components of the continuous phase. 11 In this study, our focus will be on supramolecular gels, a type of molecular gels where small organic compounds act as the network-forming component. The organic compounds responsible for forming these gels are referred to as low molecular weight gelators (LMWGs). 12 These LMWGs, with a molecular weight between 200 and 400 Da, possess the ability to self-assemble into fibrous networks through non-covalent interactions, including hydrogen bonds, van der Waals forces, π-π interactions and electrostatic interactions. All these interactions form cavities so, only a small amount of solid (typically, <1% wt/vol) is capable of immobilizing large quantities of solvent through capillary and surface tension forces, allowing the gel formation. LMWGs offer several advantages over polymeric gels due to their reversibility and higher biocompatibility. For instance, when it comes to biological applications, finding biodegradable polymeric gels remains a challenge. However, many molecular gels consist of biocompatible molecules, making them easily processable by the body. Furthermore, their reversible bond nature renders them potential functional materials capable of responding reversibly to external stimuli, including temperature, light, pH changes, ultrasound, and more. 13 The literature is witnessing an increasing number of reports on these gelators, with an expanding array of applications being documented. LMWGs have started to have a significant impact on the industry in recent years. One of the most well-known examples is 1,3:2,4Dibenzylidene-D-sorbitol (DBS), which has numerous patents in various sectors such as cosmetics, construction, food industry (food packaging), dental composites, adhesives, liquid crystal gel devices, and many more. 14 However, their unique properties such as softness, hydrophilicity, biodegradability, non-toxicity, flexibility, transparency, ability to mimic the mechanical properties of biological tissues and the ability to retain large volumes of liquid have placed them at the forefront of biomedicine and pharmacology. 15 Among the new 11 Zweep, N.; van Esch, J. H. The Design of Molecular Gelators. In Functional Molecular Gels. Escuder, B.; Miravet, J. F., Eds.; The Royal Society of Chemistry, 2013; Chapter 1, pp 1-29. 12 Terech, P.; Weiss, R. G. Low Molecular Mass Gelators of Organic Liquids and the Properties of Their Gels. Chemical Reviews, 1997, 97 (8), 3133–3160. 13 Jindrich Kopecek. Hydrogels: From soft contact lenses and implants to self-assembled nanomaterials. Journal of polymer science. 2009, 47 (22), 5929–5946. 14 Okesola, B. O.; Vieira, V. M. P.; Cornwell, D. J.; Whitelaw, N. K.; Smith, D. K. 1,3:2,4-Dibenzylidene-D-Sorbitol (DBS) and Its Derivatives – Efficient, Versatile and Industrially-Relevant Low-Molecular-Weight Gelators with over 100 Years of History and a Bright Future. Soft Matter 2015, 11 (24), 4768–4787. 15 Nafo, W. Hydrogel Biomaterials for Drug Delivery: Mechanisms, Design, and Drugs. In Hydrogels - From Tradition to Innovative Platforms With Multiple Applications; IntechOpen, 2023; Chapter 3. Towards antibacterial hydrogels. Synthesis, structural studies and biological activity INTRODUCTION 8 applications, we can find their use as tissue engineering scaffolds, 3D cell culture moulds, wound dressings, surgical adhesives and soft devices such as sensors, contact lenses or ocular treatments (ophthalmic gels). 16 Moreover, their porosity allows them to act as absorbents for contaminated water 17 , purifying it through the selective gelation of oil spills, as proposed by Professor John. 18 However, just as they can absorb, they can also release substances trapped within their structure, making them valuable for controlled drug release systems. 19 They greatly improve the administration of poorly soluble drugs, ensuring spatial and temporal control over the release process while being easily degradable. Due to its great potential and still being in the development stage, this is the chosen application towards which we will focus our study. Figure 2. Schematic depiction of the process of supramolecular gel formation. Image taken from Krieg (2009). 20 The standard process for forming these gels involves preparing a suspension of the LMWG in a suitable solvent and heating it until complete dissolution occurs, resulting in a supersaturated isotropic solution -where more solute has been dissolved than the maximum amount. As the solution cools down, a hierarchical assembly process of their individual molecules takes place, in which several levels of organization can be distinguished through 16 Sánchez-Fernández, J.A. Structural Strategies for Supramolecular Hydrogels and Their Applications. Polymers. 2023, 15, 1365. 17 Giuri, D.; D’Agostino, S.; Ravarino, P.; Faccio, D.; Falini, G.; Tomasini, C. Water Remediation from Pollutant Agents by the Use of an Environmentally Friendly Supramolecular Hydrogel. ChemNanoMat. 2022, 8 (4), e202200093. 18 Jadhav, S. R.; Vemula, P. K.; Kumar, R.; Raghavan, S. R.; John, G. Sugar-Derived Phase-Selective Molecular Gelators as Model Solidifiers for Oil Spills. Angew. Chem. Int. Ed. 2010, 49 (42), 7695–7698. 19 Vigata, M., Meinert, C., Hutmacher, D. W., & Bock, N. Hydrogels as Drug Delivery Systems: A Review of Current Characterization and Evaluation Techniques. Pharmaceutics. 2020, 12 (12), 1188. 20 Krieg, E.; Shirman, E.; Weissman, H.; Shimoni, E.; Wolf, S. G.; Pinkas, I.; Rybtchinski, B. Supramolecular Gel Based on a Perylene Diimide Dye: Multiple Stimuli Responsiveness, Robustness, and Photofunction. J. Am. Chem. Soc. 2009, 131, 14365–14373. Towards antibacterial hydrogels. Synthesis, structural studies and biological activity WORK PLANNING 15 5. WORK PLANNING As indicated in the 'Objective' section, this work can be differentiated into three sections: the synthesis of new gelling agents, the study of their gelling capacity and the study of the drug release ability of the obtained hydrogels. The synthesis of our molecular gelling agents will be carried out from the beta-amino acid 5, which will be obtained from the methyl ester of cyclohexanecarboxylate 2, as shown in Scheme 1. From there, we will introduce as a variable the nature of the apolar group as an alkyl chain of twelve carbon atoms in the amino functionality, and we will modify the polar part by introducing three groups of increasing polarity in the carboxyl functionality: ester, carboxylic acid and hydrazide. Scheme 1. Retrosynthetic scheme The main transformations we face in this synthesis are: • The synthesis of compound 3 by Michel addition of amine 1 amidure over methyl cyclohex-1-ene-1-carboxylate 2. • The epimerization of compound 3 with sodium methoxide to obtain pure compound 4 in their trans conformation. Towards antibacterial hydrogels. Synthesis, structural studies and biological activity WORK PLANNING 16 • The hydrolysis of the dibenzylamino group of compound 4 to give beta-amino acid 5. • The peptide coupling between compound 5 and dodecylcarboxylic acid. • And finally, the transformations of the ester moiety of compound 6 into carboxylic acid 7 and hydrazide 9. Once synthesized our three candidates 6, 7 and 9, we will study their gelation properties in fourteen solvents with different dielectric constants: hexane, dioxane, toluene, diethyl ether, chloroform, ethyl acetate, tetrahydrofuran, dichloromethane, isopropanol, acetone, ethanol, methanol, acetonitrile and water. This study will be carried out through a heating-cooling vial inversion test. This way, various factors can be determined, such as the appearance of the formed gel - which is related to its strength -, the minimum gelation concentration (mgc), solubility in each of the solvents, etc. Special attention will be paid to its behaviour in water, as it is the most biocompatible solvent and therefore the most interesting one for the development of the main objective of achieving new drug delivery systems. Therefore, efforts will be made to minimize its mgc, water will be the chosen solvent for structural studies as well as for following release tests. Finally, the drug release capacity of the hydrogels exhibiting the highest potential will be studied. For their evaluation in a liquid environment, a gelator-drug cogel will be brought into contact with a known volume of distilled water, where the antibiotic - retained in its structure - will be released towards it. The amount released will be measured using ultravioletvisible spectroscopy (UV-VIS) tests. Additionally, the release kinetics of the gel will be also determined, concluding with its final assessment in a more realistic environment through in vitro tests on bacterial cultures. Towards antibacterial hydrogels. Synthesis, structural studies and biological activity DISCUSSION 17 6. DISCUSSION 5.1. Synthesis of the low molecular weight gelators (LMWGs) As mentioned in the introduction, an amphiphilic structure is often key for certain organic molecules to exhibit gelling capacity. To obtain this specific structure from derivatives of cyclohexanic β-amino acids, we have designed a synthetic route that allowed us to obtain new low molecular weight organic gelators to be studied. 5.1.1 Synthesis of the β-amino ester 5 According to the established work plan, the synthetic route began with a four-step transformation from two commercial products, (R)-1-phenylethan-1-amine and benzaldehyde, to yield the target β-amino ester 5. The first transformation corresponds to a reductive amination to obtain Davies' amine 1. This chiral amine was subsequently treated with n-BuLi to obtain (R)-N-benzyl-N-(αmethylbenzyl)lithium amide, which reacts with the commercial methyl cyclohex-1-ene-1carboxylate 2 through a Michael addition to yield a mixture of the major product 3 (where the amino and carboxyl groups are in a cis arrangement) and traces of product 4 (where they are in a trans arrangement). Scheme 2 Towards antibacterial hydrogels. Synthesis, structural studies and biological activity DISCUSSION 18 The reaction was confirmed by comparing their 1H-NMR spectrum with those present in the literature 37 , as it is a previously characterized molecule. 38 In which, a multiplet at 7.127.44 ppm -corresponding to the ten aromatic protons of the two phenyls that are part of the amine reagentcan be observed, as well as the signals of the two CH3 groups present in the molecule, the signal of the methyl ester at 3.61 ppm and the one of the amine at 1.30 ppm. Also, the characteristic signals of the CH groups absent in compound 2 appear, such as the proton in the α position of the amine, which appears as a doublet of triplets at 2.66-2.74 ppm, and the one in the α position of the ester as a quartet at 2.52 ppm. This diastereomeric mixture 3+4 was submitted, without further purification, to epimerization for its complete transformation into the compound with trans configuration 4. The mixture was treated with 2M sodium methoxide as a strong base and refluxed in methanol for 48 hours, obtaining the tertiary amine 4 as the only product with a final yield of 85%. Finally, in order to obtain the β-amino ester 5, compound 4 was subjected to catalytic hydrogenation in methanol, with 5% Pd(OH)2/C as the catalyst. This resulted in the reduction of the tertiary amine to the primary one 5 (87%). Scheme 3 The compound 5 was characterized by comparing its 1H-NMR spectrum with those already reported 39 , wherein the disappearance of signals from the ten aromatic protons of the substituents of the starting amine 4 was indeed observed. Additionally, the appearance of two signals corresponding to the CH protons in the α position of both the amine (doublet of doublets at 2.91-2.98 ppm) and the methyl ester (doublet of triplets at 2.39-2.49 ppm). 37 Marcin, L. R.; Higgins, M. A. Antiamyloid Phenylsulfonamides: N-Cycloalkylcarboxamides Derivatives. U.S. Patent US 2005/0113442 A1, 2005. 38 Ait Said, L.; El Hammoumi, M. M.; El Haimer, C.; El Bachiri, A.; Khoukhi, M. Synthesis of limonene β-amino alcohol from (R)-(+)-α-methylbenzylamine and (+)-limonene 1,2-epoxide. J. Mol. Struct. 2021, 1241, 130691. 39 Vogensen, S. B.; Jørgensen, L.; Madsen, K. K.; Jurik, A.; Borkar, N.; Rosatelli, E.; Nielsen, B.; Ecker, G. F.; Schousboe, A.; Clausen, R. P. Structure-Activity Relationship of Selective GABA Uptake Inhibitors. Bioorg. Med. Chem. 2015, 23 (10), 2480-2488. Towards antibacterial hydrogels. Synthesis, structural studies and biological activity DISCUSSION 19 5.1.2 Synthesis of the gelling agent 6 Once precursor 5 was obtained pure and with good yields, we proceeded with the synthesis of first of the gelling agent that we had proposed as the object of study in this work, the (1R,2R)-2-dodecanamidocyclohexane-1-carboxylate methyl ester (6). Scheme 4 A peptide coupling was performed between the primary amine present in the synthesized precursor 5 and commercial dodecanoic acid. Following the procedure outlined in Scheme 4, the coupling was induced using PyBop as the acid activator, DIEA as the base, and dry DCM as the solvent. Under these conditions, a white amorphous solid was obtained with an 80% yield. The spectroscopic properties allowed us to confirm that this solid is compound 6. In its 1H-NMR spectrum, a high-intensity signal appeared at 1.22-1.30 ppm, corresponding to the sixteen CH2 protons of the acid chain, as well as a triplet at 0.77-1.00 ppm characteristic of a terminal CH3 in the hydrocarbon chain. Additionally, we identified signals for the α-CH in the ester (at 2.20-2.38 ppm) and the formed amide (at 3.89-4.09 ppm), along with a singlet integrating for three at 3.65 ppm, corresponding to the ester methyl. In its 13C-NMR spectrum, the characteristic signals of its two carbonyl carbons stand out at 172.80 and 174.20 ppm. Finally, in its high-resolution mass spectrum, a signal with a ratio m/z = 340.2846 (theoretical m/z = 340.2843) corresponding to the [M+Na]+ ion was observed, allowing us to confirm its molecular formula as C20H38NO3. Towards antibacterial hydrogels. Synthesis, structural studies and biological activity DISCUSSION 20 5.1.3 Synthesis of the gelling agent 7 To obtain the (1R,2R)-2-dodecanamidocyclohexane-1-carboxylic acid (7), we started with ester 6 synthesized in the previous step, which underwent a basic hydrolysis reaction with LiOH to transform the ester group into a carboxylic acid. As indicated in Scheme 5, this hydrolysis took place in a solution of MeOH/THF/H2O (2.5:1:1), wherein both reagents were dissolved and refluxed at 70°C. Under these conditions, a lithium salt was obtained, which during the purification steps was protonated using 1M HCl, allowing the compound to be isolated in its acidic form as a white amorphous solid. This acid 7 was obtained with a good yield of 70%. Scheme 5 We confirmed it was the sought-after compound through its spectroscopic properties. In its 1H-NMR spectrum, the disappearance of the signal at 3.65 ppm from the ester methyl group was observed. Furthermore, it continued to exhibit all other signals, including those from the CH2 groups belonging to the hydrocarbon chain (1.22-1.40 ppm), the CH groups in the αposition to the acid (at 2.29 ppm), and amide (at 3.91 ppm). Additionally, it was characterized through its 13C-NMR spectrum, highlighting the signals of the carbonyls, this time at 174.02 ppm for the amide and 176.44 ppm for the acid. A single CH3 signal at 13.01 ppm corresponding to the end of the hydrocarbon chain appeared, confirming the disappearance of the ester methyl group. Its high-resolution mass spectrum showed a signal with m/z = 326.2690 (theoretical m/z = 326.2687) corresponding to the [M+Na]+ ion, confirming its molecular formula as C19H36NO3. Towards antibacterial hydrogels. Synthesis, structural studies and biological activity DISCUSSION 21 5.1.4 Synthesis of the gelling agent 9 Continuing with our synthetic plan, once acid 7 was obtained, two transformations were carried out to reach N-((1R,2R)-2-(hydrazinocarbonyl)cyclohexyl)dodecanamide (9), the third and last of our synthetic objectives. Scheme 6 At first, a peptide coupling were done between the acid group of compound 7 and the amine group of tert-butyl carbazate. This reaction takes place using PyBop as the acid activator, DIEA as the base and dry DCM as the solvent. A colorless oil was obtained, corresponding to 2-((1R,2R)-2-dodecanamidocyclohexane-1-carbonyl)tert-butyl hydrazine-1carboxylate (8), with a yield of 63%. Scheme 7 The structure of this product was confirmed through its spectroscopic properties. In its 1H-NMR spectrum, characteristic proton signals at 6.96 and 9.13 ppm corresponding to the two nitrogen-bound protons of the hydrazide were observed. The presence of the Boc group, lacking protons, can only be justified by the 13C-NMR spectrum, where the quaternary carbon appears at 81.12 ppm and the signal corresponding to the three methyl groups at 28.21 ppm. The corresponding high-resolution mass spectrum shows a signal at m/z=440.3483 (theoretical m/z=440.3487) corresponding to the [M+Na]+ ion, confirming its molecular formula C24H46N3O4. The second step of this transformation involved deprotection, removing the Boc group by acidifying the medium with trifluoroacetic acid (TFA). As it is a good leaving group, the Towards antibacterial hydrogels. Synthesis, structural studies and biological activity DISCUSSION 22 N-((1R,2R)-2-(hydrazinocarbonyl)cyclohexyl)dodecanamide (9) was obtained at a yield of 75% in a short time and at room temperature. Scheme 8 The formation of the final compound 9 was confirmed through spectroscopic studies. Its 1H-NMR spectrum presented characteristic signals of the newly introduced hydrazide group, showing a doublet integrating for two protons at 3.62-4.04 ppm, corresponding to the NH2 group, and a very low intensity signal at 7.73 ppm, related to the NH from the same region of the molecule. The NH of the amide group attached to the hydrocarbon chain appeared at 6.65 ppm. Furthermore, the 13C-NMR spectrum showed the disappearance of signals related to the removed Boc group, highlighting the two carbonyl groups at 171.74 and 173.63 ppm. Its high-resolution mass spectrum shows a signal at m/z=340.2959 (theoretical m/z=340.2958) corresponding to the [M+Na]+ ion, confirming its molecular formula C19H38N3O2. Towards antibacterial hydrogels. Synthesis, structural studies and biological activity DISCUSSION 23 5.1.5 Synthesis of the racemic mixture (rac-7) Additionally, it was decided to study the gelling capacity of the racemic mixture of compound 7, in an attempt to reduce production costs of this potential gelling agent in a potential industrial application. Scheme 9 For this purpose, a 5-step synthetic route was designed to obtain the racemic mixture, as shown in Scheme 9. The first step involved the opening of the commercial (3aR,7aS)- hexahydroisobenzofuran-λ3-dione 10 with a solution of ammonia in methanol (Scheme 10). The formation of the methoxide facilitated the opening of the furan ring, where the ester underwent aminolysis, resulting in the formation of the racemic mixture of monoamide 11 (rac-11). Scheme 10 Next, rac-11 underwent the Hofmann-Löffler-Freytag reaction. Using PhI(OAc)2 as the oxidizing agent, the formation of an iodoamine intermediate was achieved, and through intramolecular rearrangement, the racemic mixture of hydrochloride salt rac-12 was obtained. Towards antibacterial hydrogels. Synthesis, structural studies and biological activity DISCUSSION 24 The quaternary ammonium salt was transformed into its primary amine form through dealkylation with concentrate sulfuric acid and under reflux conditions (Scheme 11). Scheme 11 Fourthly, the peptide coupling of dodecanoic acid with the primary amine took place - using DIEA as a base and PyBop as a coupling agentresulting in the racemic mixture rac-6. The latter was subjected to basic hydrolysis with lithium hydroxide, converting it from an ester to a carboxylic acid, yielding rac-7. The conditions for these last two steps are outlined in the Scheme 12. Scheme 12 All racemates were characterized as shown in the experimental part and the success of the synthetic sequence demonstrated by comparing the spectroscopic data of the final compounds rac-6 and rac-7, which turned out to be identical to compounds 6 and 7 with the exception of their optical rotations (+5.9º and +5.4º for 6 and 7 respectively and 0 for rac-6 and rac-7). Towards antibacterial hydrogels. Synthesis, structural studies and biological activity DISCUSSION 31 In summary, two hydrogels were successfully obtained from the gelators Gt12-COOH and Gt12-NHNH2, with mgc of 0.7 and 3.5 mg/mL, respectively. For the third gelling molecule, Gt12-COOMe, it was impossible to achieve its solubilization in water or in possible H2O/EtOH mixtures; therefore, hydrogel formation was not achieved. Furthermore, the gelation capacity of the racemic mixture of compound 7 (rGt12-COOH) was studied to compare its ordering capacity and reduce the production costs of the gelator. As it proved to be a poorer hydrogelator than its pure trans-form, both the racemic mixture and Gt12COOMe will be discarded from the future antibiotic release tests proposed. 5.3. Hydrogels structural studies To understand the hierarchical self-assembly process of molecular gels, structural studies have been conducted from the microscopic to macroscopic scale using spectroscopic (FTIR), microscopic (SEM), and X-ray diffraction studies. Due to the initially set objective, all these studies were carried out in water, as the applicability of hydrogels is the main focus, so the Gt12-COOMe is excluded. 5.3.1 Infrared Spectroscopy (IR) Infrared spectroscopy can be used to obtain information about the self-assembly process, allowing determination of the non-covalent interactions responsible for gelation, particularly effective in characterizing hydrogen bonding interactions. In amide groups, there are three characteristic vibration bands: the N-H bond stretching band at around 3500 cm-1, another one corresponding to the amide carbonyl stretching at 1600-1700 cm-1 and the last one between 1510-1580 cm-1, associated with the C-N bending. The displacement of these bands towards higher or lower values can be indicative of the establishment of weak interactions between the amide groups of different monomers. To study this, at least two IR measurements need to be taken: one of the gelator in solution under mgc and another of the xerogel. To do this, we prepared the Gt12-COOH hydrogels - formed with GdL and HCl - as well as the Gt12-NHNH2 hydrogel in their mgc and they were vacuum-dried to remove the solvent. This Towards antibacterial hydrogels. Synthesis, structural studies and biological activity DISCUSSION 32 resulted in the formation of xerogels, which correspond to the collapse of the threedimensional structure formed but still retain the appearance of the constituent fibers, providing insights into the arrangement of the monomers. The solid sample was placed onto an ATR accessory of an FTIR spectrometer and was measured. The characteristic peak wavelengths of the main groups are listed in the Table 2. Table 2. IR data for the NH and CO amide bands of the two compounds Gt12-COOH and Gt12-NHNH2; xerogel and solution. Compound N-H (cm-1) C=O (cm-1) Xerogel Solution Xerogel Solution Gt12-COOMe - - - - Gt12-COOH GdL 3290,48 3297,92 1646,66 1648,93 HCl 3288,88 1646,07 Gt12-NHNH2 3290,10 3287,43 1646,94 1648,74 In the case of the Gt12-COOH hydrogel, the N-H and C=O stretching bands in solution appear at 3297.92 and 1648.93 cm-1, respectively. While their counterparts in the xerogel appear shifted towards lower values, at 3290.48 and 3288.88 cm-1 for the NH stretching and 1646.66 and 1646.07 cm-1 for the C=O stretching, in the hydrogel formed with GdL and with HCl, respectively. This clear bathochromic effect in both stretching signals is related to the presence of hydrogen bonds between the amide groups of different gelating agents, identifying them as the main driving force in hydrogel formation. However, for Gt12-NHNH2, this effect is only shared by the C=O stretching band, which shifts from 1648.74 to 1646.94 cm-1 upon gelation, while the N-H band shifts from 3267.53 to 3290.10 cm-1. The latter shifts towards higher values, which is known as a hypsochromic effect and is not associated with hydrogen bond formation. In the case of hydrogels, water competes significantly in the formation of these hydrogen bonds, so in this instance, the hydrogel might primarily be sustained by Van der Waals forces instead hydrogen bonds. This would justify its tendency to form micelles, something not observed with the previous gelating agent, and which we will revisit later in the release studies. Towards antibacterial hydrogels. Synthesis, structural studies and biological activity DISCUSSION 33 5.3.2 Scanning Electron Microscopy (SEM) Due to the characteristic structural features of fibers and their network, which typically fall within the range of 5–1000 nm, it is necessary toemploy appropriate direct imaging microscopy techniques for studying the structure of hydrogels at such extended length scales. Over time, a variety of transmission electron microscopy (TEM) and scanning electron microscopy (SEM) techniques have been used for that purpose. 43 However, it is important to note that these techniques do not capture images of gel fibers in their natural state, leading to the potential occurrence of artifacts stemming from the sample preparation process. Scanning electron microscopy experiments were conducted on the obtained hydrogels to gain a closer understanding of their microscopic structure. For this purpose, the corresponding hydrogels of Gt12-COOH and Gt12-NHNH2 were prepared at a concentration of 2 mgc. The one containing the hydrazide group was formed in a H2O/EtOH 30% solution following the previously described procedure, while the carboxylic acid group's hydrogel was studied in two variations: acidifying with GdL and with HCl. All three were vacuum-dried to remove the solvent, resulting in the formation of the xerogels. They correspond to the collapse of the threedimensional structure formed but still retain the appearance of the constituent fibers, providing insights into the arrangement of the monomers. Figure 7. SEM image of the compound Gt12-NHNH2 (15K X). 43 Zweep, N.; van Esch, J. H. The Design of Molecular Gelators. In Functional Molecular Gels. Escuder, B.; Miravet, J. F., Eds.; The Royal Society of Chemistry, 2013; Chapter 1, pp 1-29. Towards antibacterial hydrogels. Synthesis, structural studies and biological activity DISCUSSION 34 Figure 8. SEM images of the compound A) Gt12-COOH-GdL (10K X) and B) Gt12-COOH-HCl (10K X). Comparing the images, we can see that all three hydrogels are structured into fibers. In the first case, Gt12-NHNH2, we find a higher organization into finer and branched fibrils, with the presence of some "nest" type structures where the fibers are positioned in a spherical manner (Figure 7, boxed details). However, in the case of the Gt12-COOH’s hydrogels we have an image from each gelification process. However, both share a plate-like aggregation pattern, resulting in wide and flat structures of larger dimensions and less branched (Figure 8). 5.3.3 X-ray Spectroscopy Once it seems clear that our studied gelling agents are capable of aggregating with hierarchical and directional stacking in water, in order to understand this process at a molecular level, X-ray experiments were conducted on our hydrogels. However, to confidently affirm the orientation of the molecules in their supramolecular structure, we should be able to crystallize our systems. Faced with the impossibility of obtaining such single crystals, this technique was used to investigate the degree of similarity between the two formed Gt12COOH hydrogels. The samples of both hydrogels, the one formed with D-gluconolactone (Gt12-COOH-GdL) and the one formed with hydrochloric acid (Gt12-COOH-HCl), were prepared in 2 mm diameter capillaries. Measurements were performed on the Bruker D8 Towards antibacterial hydrogels. Synthesis, structural studies and biological activity DISCUSSION 35 VENTURE PHOTON-III diffractometer. The X-ray images of the hydrogels revealed two regions of different density within the formed gels, so both were studied. For the Gt12-COOH-GdL hydrogel, two measurements were taken at different points of the capillary: one at low density, GdL_a, and another at high density, GdL_b (Figure 9A). Once the diffractograms were obtained, the background - the capillary without the sample - was removed and they were compared alongside the diffractogram of the xerogel (Figure 9B). The appearance of four bands - not peaks, as they are not single crystals - provided information about the interactions that are preserved or not when comparing both states. Two broad bands appeared at 2.2 and 3.2 Å, which do not correspond to any signal from the xerogel. At 4.0 and 4.2 Å, we can see two more defined peaks, which are present in the xerogel, and a band at a low angle, the exact position of which cannot be determined due to the diffractometer's geometry (it cannot be positioned at values greater than 20 Å). b Figure 9. A) Images of the Gt12-COOH-GdL hydrogel in the capillary, areas of low and high density. B)Analysis of X-ray Diffraction (PXRD) of the Gt12-COOH-GdL in water. A B Towards antibacterial hydrogels. Synthesis, structural studies and biological activity DISCUSSION 36 For the Gt12-COOH-HCl hydrogel, 4 measurements were taken, two at low density (HCl_a and HCl_b), and two at high density (HCl_c and HCl_d), as shown in the Figure 10A. The treatment of the diffractograms was the same as in the previous case, and they were compared again with the peaks of the xerogel (Figure 10B). In this case, we observed the appearance of a band at 2.2 Å and another, larger one, at 3.2 Å that do not correspond to any signal from the xerogel. However, the one they do share with the xerogel, as well as with its analog formed with GdL, is a small band at 4.0-4.2 Å. Figure 10. A) Images of the Gt12-COOH-HCl hydrogel in the capillary, areas of low and high density. B)Analysis of X-ray Diffraction (PXRD) of the Gt12-COOH-HCl in water. A B Towards antibacterial hydrogels. Synthesis, structural studies and biological activity DISCUSSION 37 The appearance of bands in the study of gels compared to the characteristic peaks in the X-ray study is due to the flexible/dynamic organization of these systems, as opposed to the rigidity of crystals. The comparative study proposed will help us understand the distance between planes, between chains of Gt12-COOH molecules, which interact through hydrogen bonds, and see if it is shared by both hydrogels. If we compare both hydrogels, we can assert that there are two arrangements/two characteristic distances at the beginning of packing, which are shared by both hydrogels at both densities. Thus, the contacts at 2.2 and 3.2 Å would be responsible for the network of contacts in the gel system. These two appear as broad bands, which is proportional to the number of times a characteristic distance is repeated. However, we must highlight that the Gt12-COOH-GdL hydrogel shows a higher degree of crystallinity (synonymous with organization) due to the appearance of a signal at 4.2 Å and the band at a low angle, characteristic of long-range contacts. This signal at 4.2 Å is shared by Gt12COOH-HCl but only at high densities. From this study, we conclude that both hydrogels form a similar structure, with a higher degree of ordering for Gt12-COOH-GdL and samples at high opacity. 5.4. Drug release studies Considering the possible future biomedical applications of these gels, the potential use as drug release systems for the two N-alkyl amides that proved to be effective gelation agents for polar solvents, particularly water, was investigated. To this end, Ampicillin (AMP), a wellknown bactericidal antibiotic (Figure 11), was chosen as drug-model to conduct proof-ofconcept tests to evaluate the properties of our hydrogels as controlled release systems. Figure 11. Structure of the two gelling agents under study and the antibiotic used, Ampicillin. Towards antibacterial hydrogels. Synthesis, structural studies and biological activity DISCUSSION 38 5.4.1 Release testing In order to prepare the co-gel for release testing, the suitable amount of gelling agent was weighed in a 5 mL vial to achieve the mgc or 2 mgc of each compound in a 2 mL volume. One milliliter of this volume corresponds to H2O/NaOH solution for Gt12-COOH or H2O/EtOH 30% solution for Gt12-NHNH2. The remaining 1 mL is for the AMP solution at the corresponding concentration (500, 1000, or 2000 μg/mL). In the case of Gt12-COOHAMP, after obtaining a fully transparent solution with both components, D-gluconolactone is added and immediately stirred until complete dissolution to form a homogeneous gel. After a few minutes of resting at room temperature, the gel formation can be visually observed as the transparent solution turns whitish and eventually becomes an opaque gel. Regarding Gt12NHNH2-AMP, complete dissolution of the gelling agent is achieved by heating the 2 mL in a sand bath at 120 °C, followed by immersion in an ice-water bath to induce gelation by thermal shock. In both cases, no precipitation of the antibiotic within the gel was observed; it remained dissolved in the liquid component of the gel structure. Figure 12. Representative scheme of the antibiotic release test by the hydrogel. Once each co-gel (AMP-gelator) was formed a known volume of distilled water (3 mL) was added onto the co-gel as the supernatant and left undisturbed for 48 hours (Figure 12). After this time, it is understood that the system has reached equilibrium and an aliquot of the supernatant is taken to measure the concentration of AMP using UV-Vis Spectroscopy, employing a previously obtained calibration curve for AMP in water (Experimental Work section, Figure 27). With these measurements and the standard curve, the percentage of release is calculated for each initial Ampicillin concentration that was used, resulting in the values presented in Table 3. Towards antibacterial hydrogels. Synthesis, structural studies and biological activity DISCUSSION 39 Table 3. Percentages of Ampicillin release for both hydrogels. [AMP] μg mL-1 Gt12-COOH Gt12-NHNH2 mgc 2 mgc mgc 2 mgc 500 70% 75% 87% 89% 1000 57% 58% 46% 48% 2000 51% 52% 41% 41% Note: mgc (minimum gelation concentration), 2 mgc (two times the minimum gelation concentration). The values were calculated based on the 268 nm band of the UV-VIS spectrum of Ampicillin. The obtained results establish both hydrogels as promising carriers for this drug. For both Gt12-COOH and Gt12-NHNH2, it was observed that the amount of Ampicillin released is remarkably similar, regardless of the gelator concentration (mgc vs 2 mgc). Furthermore, this percentage decreases as the concentration of Ampicillin in the co-gel increases. This phenomenon can be easily explained if we perceive the release process as an equilibrium process. With a higher amount of AMP within the gel, a lesser amount is needed to be released in order to "saturate" the supernatant. The only difference was that Gt12-NHNH2 showed higher release percentages at 500 μg/mL of AMP co-gels. This could be attributed to the tendency of Gt12 NHNH2 to form vesicles, resulting in a less compact three-dimensional network, that facilitates greater exchange of the antibiotic between the "trapped" water and the supernatant. 5.4.2 Release kinetics Once confirmed the retention-release capacity of the selected drug by the two hydrogels under study, we were interested in knowing its release curve. We selected the Gt12-COOHGdL hydrogel as our study target since it is visually more homogeneous and has a lower mgc. To do this, we prepared a vial with a concentration of 2 mgc and 1000 µg/mL of AMP. The utility of this hydrogel in providing controlled and prolonged release over time needed to be validated through a study of its release kinetic. To achieve this, we monitored the release of Ampicillin using UV-VIS Spectroscopy by taking aliquots of the supernatant at controlled time intervals over 24 hours (Figure 13). Towards antibacterial hydrogels. Synthesis, structural studies and biological activity DISCUSSION 40 Figure 13. UV-Vis spectra of Ampicillin release from Gt12-COOH-GdL gel at t = 0–24 h. We collected all absorbance measurements at 268 nm, the wavelength used for the Ampicillin standard curve, and calculated the released percentage in relation to the initial concentration in the gel. The Figure 14 shows the percentage of Ampicillin release in percentage for the hydrogel Gt12-COOH-GdL. As can be observed, 26% of the encapsulated Ampicillin is released within the first hour and this percentage increases to reach 39% at 9 hours. The next day, after 24 hours, a new measurement indicated that 45% of the initial Ampicillin had been released. In the previous section, a maximum release for the Gt12COOHGdL hydrogel of 58% at 48 hours was stated, with saturation of the supernatant being the maximum to achieve. This test confirms that the administration of the antibiotic by the hydrogel is carried out in a controlled and progressive manner, enhancing its activity in the damaged area in which it is located. Figure 14. Kinetics of Ampicillin release for Gt12-COOH-GdL hydrogel. Towards antibacterial hydrogels. Synthesis, structural studies and biological activity DISCUSSION 47 5.5.3 SEM study At first, it was thought that the effect of D-gluconolactone on bacterial viability was related to a charge issue. D-gluconolactone undergoes hydrolysis when dissolved in water, resulting in gluconic acid, which donates protons to our gelling agent, leaving mostly as gluconate. This gluconate has a net negative charge that opposes the also negative charge of bacterial walls and membranes on their outer part. Therefore, it was initially suggested that bacteria might be experiencing a repulsion phenomenon or even some damage to the structure of their wall, preventing the viability of the strains. To clarify this, it was decided to carry out a scanning electron microscopy study, through which we could closely observe the bacterial wall and the presence or absence of structural damage or morphological changes. Cultures were conducted following the protocol described in LB/agar plates. Small sections of the medium were isolated and placed on autoclaved glass coverslips. Subsequently, a fixation protocol with Glutaraldehyde and Osmium Tetroxide, followed by dehydrations with a series of EtOH/H2O solutions, was followed (Experimental Work). The obtained image with E.coli are collected in Figure 19. Figure 19. SEM images of E. coli cultures; comparison between negative control (first row) and post 24h contact with Gt12COOH-GdL (second row). Towards antibacterial hydrogels. Synthesis, structural studies and biological activity DISCUSSION 48 What can be observed in the images after being in contact with the hydrogel is the appearance of a new phenotype compared to the one observed in the negative control. This phenotype is much more elongated, without the presence of constriction in the bacterial wall or any apparent structural damage. The cell density in the halo zone is much lower, with a few scattered viable cells and this new phenotype, resulting from a process known as bacterial filamentation. The filamentation of bacteria will lead to their collapse, among other things, due to the diffusion of nutrients. Bacteria are 'designed' in such a way that their surface-to-volume ratio is suitable for nutrient uptake from the external environment. When division is inhibited, the volume increases significantly compared to a decreasing surface area (less contact with the external environment), making nutrient uptake difficult and resulting in the death of the organism. This mode of antibacterial action is the modus operandi of FtsI-specific antibiotics such as cephalexin (10 mg/ml), piperacillin (2 mg/ml), and furazlocillin (1 mg/ml). 47 This filamentation effect was also studied in a model Gram-positive microorganism, Staphylococcus aureus. As can be seen in Figure 20, it was not shared by this microorganism, which exhibits a completely normal and viable phenotype without any variation in morphology. This test was repeated with different microorganisms, both Gram-negative and Gram-positive, to establish that it was a general effect (Figure 21). In this way, we could determine that the extremely elongated morphology was shared by all Gram-negative bacilli. However, when introducing a Gram-positive bacillus like Bacillus circulans, the absence of any effect was observed, similar to the case of cocci. 47 Weiss, D. S.; Chen, J. C.; Ghigo, J.-M.; Boyd, D.; Beckwith, J. Localization of FtsI (PBP3) to the Septal Ring Requires Its Membrane Anchor, the Z Ring, FtsA, FtsQ, and FtsL. J. Bacteriol. 1999, 181 (2), 508–520. Figure 20. SEM images of S. aureus cultures; A) negative control, B) and C) post 24h contact with Gt12-COOH-GdL. A B C Towards antibacterial hydrogels. Synthesis, structural studies and biological activity DISCUSSION 49 Figure 21. Comparative SEM images between negative control (left) vs post 24h contact with Gt12-COOH-GdL (right) of A) Acinetobacter baumannii, B) Aeromonas hydrophila, and C) Bacillus circulans cultures. Through this test, it was concluded that gluconate should permeate inside these prokaryotes due to its small size. Once inside, it would interact with some of the proteins involved in the formation of the septum of the bacterial cell wall, which are only present in Gram-negative bacteria. This interaction could be preventing the proper organization of the tubulin-analogue FtsZ and its membrane anchor FtsA (proto-ring formation) or the activity of A B C Towards antibacterial hydrogels. Synthesis, structural studies and biological activity DISCUSSION 50 proteins related to the septal peptidoglycan synthesis, as glycosyltransferase FtsW and transpeptidase FtsI. 48 One future step to determine at which stage of the septum formation the failure in the cell division machinery occurs would be through confocal microscopy, labelling one of the proto-ring proteins with green fluorescent protein (GFP), for example, to see if they manage to position themselves in the internuclear zone. If the FtsZ-ring were to form, we should label one of the final stages involved in the constriction process. 48 Attaibi,M.; den Blaauwen, T. An Updated Model of the Divisome: Regulation of the Septal Peptidoglycan Synthesis Machinery by the Divisome. Int. J. Mol. Sci. 2022, 23, 3537. Towards antibacterial hydrogels. Synthesis, structural studies and biological activity CONCLUSIONS 51 7. CONCLUSIONS - The objective of synthesising new LMWOGs β-amino acids derivatives that can be studied was achieved. Following the synthetic route initially proposed, the three desired gelling agents were obtained: methyl (1R,2R)-2-dodecanamidocyclohexane-1carboxylate (6), (1R,2R)-2-dodecanamidocyclohexane-1-carboxylic acid (7) and N- ((1R,2R)-2-(hydrazinecarbonyl)cyclohexyl)dodecanamide (9). - The respective gelation tests were carried out for the three compounds, in each of the 14 solvents, determining those in which they exhibit gelling activity and classifying their gelling potential by mgc values. It was observed that compound 7 (Gt12-COOH) is the best of the gelling compounds as it forms gels in 11 of the 14 solvents and presents lower mgc values, highlighting its ability to form gels with water at mgc= 0.666 mg/mL. - Given this very promising mgc, the gelling capacity of its racemic counterpart (rac-7) was studied in an attempt to reduce costs. It turned out to be a less effective gelling agent than its enantiomerically pure analog. - Structural studies were conducted -IR, X-rays, SEM microscopy - on the hydrogels obtained with Gt12-COOH and for Gt12-NHNH2. In the first case, the two hydrogels obtained were compared by varying the pH with D-glucolactone and with HCL, confirming that both shared the same structure. - The releasing capacity of both hydrogels in a liquid medium was studied by calculating their respective release percentages of a model antibiotic, Ampicillin. Additionally, the release kinetics curve of Gt12-COOH-GdL was plotted, demonstrating controlled release over time. - Finally, we studied their releasing capacity in an environment closer to reality, observing their bactericidal effect on bacterial cultures. Surprisingly, one of the negative controls (Gt12-COOH-GdL) exhibited an unexpected inhibitory effect. This effect was studied and linked to the presence of gluconate resulting from the acidification process for gelation. Through SEM microscopy, its inhibitory effect could be associated with bacterial filamentation. Towards antibacterial hydrogels. Synthesis, structural studies and biological activity EXPERIMENTAL WORK 52 8. EXPERIMENTAL WORK 7.1. Experimental Techniques The NMR spectra were recorded on a Bruker DPX-250 spectrometer (250 MHz for 1H and 62.5 MHz for 13C) and Varian 300 (300 MHz for 1H and 75 MHz for 13C) in CDCl3, unless otherwise specified. TMS was used as an internal reference, and chemical shifts are reported in the δ scale (ppm). 1H, 13C, and DEPT NMR spectra included were processed using MestreNova software. Optical rotations were measured on a JASCO P-2000 digital polarimeter, using a Na lamp (589 nm) and a 5 cm cell. The optical rotation value is the average of 10 readings taken over 15 seconds each. Sample concentrations are given in g/100 mL. Low-resolution mass spectra were obtained on a Hewlett Packard® 5980a quadrupolar spectrometer via direct insertion, using the chemical ionization technique. High-resolution mass spectra were obtained on a VG Autospect 20-250 spectrometer via direct insertion, using the electrospray technique. IR spectra were recorded on a PerkinElmer® FT-IR spectrophotometer. The electron microscopy images were obtained using the ZEISS FESEM GEMININI-500 with EDX, employing iridium sputtering for xerogels and gold sputtering for biological samples. Column chromatography was performed on Merck® silica gel 60 (230-400 mesh, flash, 0.040.063) and TLC was performed on Merck® silica gel 60 GF254. Revealing agents included a UV lamp (λ: 254-360 nm) and Hanessian, Ninhydrin, and Potassium Permanganate mixtures. Filtration on Celite® was carried out using Fluka Celite 545 Coarse. Reactions conducted under argon atmosphere were performed in flasks flame-dried with a Bunsen burner while purged with argon. Both reduced pressure and high vacuum concentrations utilized a Büchi rotary evaporator. All biological tests were conducted on Petri dishes containing 25 mL of LB Agar with an overlay of 12 mL of soft LB Agar (0.7% agar), inoculated with 200 μL of a liquid log-phase culture in LB medium of the bacteria to be tested (OD600 = 0.5). Towards antibacterial hydrogels. Synthesis, structural studies and biological activity EXPERIMENTAL WORK 53 7.2. Synthetic procedures 7.2.1 Synthesis of (R)-N-benzyl-1-phenylethan-1-amine (1) In a 500 mL round-bottom flask of dissolve (R)-1-phenylethan-1-amine (15 g, 123.78 mmol) in 180 mL of ethanol, benzaldehyde (13.14 g, 123.78 mmol) was added. The mixture was left stirring under reflux at 85 ֯C. After 4 hours, the solution was cooled to 0 ֯C in an ice-water bath for 30 minutes and NaBH4 (4.65 g, 123.78 mmol) was introduced. Subsequently, we let the reaction reach room temperature and stirring for 48 hours. Thereafter, the solution was concentrated to dryness. 180 mL of water were used to redissolve the product, which was extracted with chloroform (3 x 160 mL). The combined organic phases were dried together with anhydrous magnesium sulfate, gravity-filtered and concentrated to dryness. We obtained a colourless oil, the (R)-N-benzyl-1-phenylethan-1-amine 1 (24.36 g, 94% yield). 1H-NMR (300 MHz, CDCl3, ppm) 1.27 (d, 3H, -CH3); 3.47 – 3.59 (m, 2H, -CH2); 3.67 - 3.74 (m, 1H, -CHCH3); 7.15 – 7.27 (m, 10H, 2 x Ph). 13C-NMR (75 MHz, CDCl3, ppm) 24.46 (-CH3); 51.68 (-CH2); 57.52 (-CH); 126.72 (Ph); 126.84 (Ph); 126.93 (Ph); 126.99 (Ph); 128.13 (Ph); 128.37 (Ph); 128.47 (Ph), 128.57 (Ph); 140.69 (-CCH2), 145.61 (-CCHCH3). Towards antibacterial hydrogels. Synthesis, structural studies and biological activity EXPERIMENTAL WORK 54 1 Towards antibacterial hydrogels. Synthesis, structural studies and biological activity EXPERIMENTAL WORK 55 7.2.2 Synthesis of (1S,2R)-2-benzyl((R)-1-phenylethyl)amino)cyclohexane-1-carboxylate methyl ester (3) On a solution of (R)-Methyldibenzylamine in dry THF cooled to -78 °C, 28 mL of nBuLi were added dropwise through an addition funnel. After 30 minutes, the ester 1 dissolved in 44 mL of dry THF was added to the amine solution at -78 °C. The resulting mixture was stirred at - 78 °C for 3 hours. After 3 hours, the reaction was quenched by adding 125 mL of a saturated solution of NH4Cl, and the mixture was allowed to reach room temperature. The appearance of the reaction product was checked by TLC (Ether/Hex 1:10) at Rf=0.50 for the cis and Rf=0.40 for the trans conformation. The resulting yellow-colored solution was extracted three times with ether, and the organic phase was dried with anhydrous sodium sulfate, filtered by gravity, and concentrated to dryness. The obtained residue was purified by column chromatography (Ether/Hex 1:20), yielding a yellow oil corresponding to the product (1S,2R)- 2-benzyl((R)-1-phenylethyl)amino)cyclohexane-1-carboxylate methyl ester (3) (6.579 g, 61%) and a clear oil, (1R,2R)-2-benzyl((R)-1-phenylethyl)amino)cyclohexane-1-carboxylate methyl ester (4) (0.621 g, 5%). 1H-NMR (300 MHz, CDCl3, ppm): 1.14-1.25 (dd, 2H, -CH2CH2CHN); 1.29-1.32 (m, 4H, - CHCH3, -CHa2CHN); 1.58 – 1.74 (m, 3H, -CH2CH2CH2, -CHb2CHN); 1.78-1.87 (dd, 1H, - CH2CHCOO); 2.11 – 2.25 (m, 1H, -CH2CHCOO); 2.52 (m, 1H, -CHCOO); 2.66-2.74 (dt, 1H, -CHN); 3.61 (s, 3H, -COOCH3); 3.74-3.80 (d, 1H, -CHCH3); 3.91-4.00 (m, 2H, -CH2Ph); 7.12 – 7.44 (m, 10H, 2 x Ph). 13C-NMR (75 MHz, CDCl3, ppm): 13.49 (-CH3); 20.97 (-CH2); 26.37 (-CH2); 26.48 (-CH2); 29.06 (-CH2); 44.87 (-CH); 51.02 (-CH3); 51.05 (-CH2); 56.40 (-CH); 58.17 (-CH); 126.42, 126.47, 127.85, 128.10, 128.21, 142.36, 144.47, 176.06. Towards antibacterial hydrogels. Synthesis, structural studies and biological activity EXPERIMENTAL WORK 56 3 Towards antibacterial hydrogels. Synthesis, structural studies and biological activity EXPERIMENTAL WORK 63 7.2.6 Synthesis of (1R,2R)-2-dodecanamidocyclohexane-1-carboxylic acid (7) The starting ester 6 (165.7 mg, 0.585 mmol) was dissolved in 30 mL of a mixture of MeOH/THF/H2O (2.5:1:1). Then, LiOH·2H2O (248.4 mg, 5.851 mmol) was added at room temperature and the solution was refluxed at 70 °C with stirring. After 2 hours, by TLC (CH2Cl2/MeOH 5%), the complete disappearance of the starting material (Rf=0.4) and the appearance of a more polar reaction product (Rf=0.3) were observed. The reaction was allowed to cool to room temperature and concentrated to dryness. Inside the flask, a dense opaque gel formed, which was subjected to stirring in 1M HCl (6 mL). The obtained solution was extracted with CHCl3: iPrOH 5:1 (3x25 mL) and the combined organic phases were dried with anhydrous magnesium sulfate, filtered by gravity and concentrated to dryness, yielding a white solid corresponding to (1R,2R)-2-dodecanamidocyclohexane-1-carboxylic acid (7) (110.3 mg, 67%). [α]D23: +5.4° (c 1 mg/mL, CH2Cl2). 1H-NMR (CD3OD, 250 MHz, ppm): 0.82-0.99 (t, 3H, -CH3); 1.22-1.40 (m, 18H, 9x-CH2); 1.37-1.62 (m, 4H, 2x-CH2); 1.66-1.96 (m, 4H, CH2); 2.09 (t, 2H, -CH2CON); 2.29 (m, 1H, - CHCOO); 3.91 (m, 1H, -CHN). 13C-NMR (CD3OD, 62.5 MHz, ppm): 13.01 (CH3); 22.31 (CH2); 24.42 (2xCH2); 25.75 (2xCH2); 28.81 (2xCH2); 29.05 (4xCH2); 29.34 (CH2); 31.82 (CH2); 35.92 (CH2); 48.88 (CH); 49.71 (CH); 174.02 (C=ONH); 176.44 (C=OOH). EM-ESI+ (m/z, %): calculated for C19H36NO3 [M+H]+: 326.2687. Found: 326.2690. Towards antibacterial hydrogels. Synthesis, structural studies and biological activity EXPERIMENTAL WORK 64 7 Towards antibacterial hydrogels. Synthesis, structural studies and biological activity EXPERIMENTAL WORK 65 7.2.7 Synthesis of 2-((1R,2R)-2-dodecanamidocyclohexane-1-carbonyl)hydrazine-1carboxylate tert-butyl ester (8) In a round-bottom flask, the starting acid 7 (75 mg, 0.279 mmol) was dissolved in dry DCM (3.3 mL) and DIEA (116.5 μL) under an inert argon atmosphere at room temperature. Next, PyBOP (217.5 mg, 0.418 mmol) was added, and the mixture was stirred magnetically for 30 minutes. To this mixture, the Boc hydrazide (51.55 mg, 0.390 mmol) was added, and the stirring continued at room temperature for 18 hours. After this time, the disappearance of the starting material (Rf=0.3) and the appearance of the reaction product (Rf=0.4) were observed by TLC (CH2Cl2/MeOH 5%). DCM (10 mL) was added to the reaction solution, and it was washed with a saturated aqueous solution of NaHCO3 (15 mL), a saturated aqueous solution of NaCl (15 mL), a 10% aqueous solution of citric acid (15 mL), and again a saturated aqueous solution of NaCl (15 mL). The organic phase was dried with anhydrous magnesium sulfate, filtered, and concentrated to dryness. Subsequently, it was purified by column chromatography eluting with a mixture (CH2Cl2/MeOH 3%), yielding 2-((1R,2R)-2propionamidocyclohexane-1-carbonyl)hydrazine-1-carboxylate tert-butyl ester (8) as a colorless oil (77 mg, 63%). [α]D23: -14.4° (c 1 mg/mL, CH2Cl2). 1H-NMR (CDCl3, 250 MHz, ppm): 0.87 (t, 3H, -CH3); 1.18-1.30 (m, 18H, 9x-CH2); 1.43 (s, 9H, 3x-CH3); 1.53-1.98 (m, 8H, 4x-CH2); 2.15-2.26 (t, 2H, -CH2CO); 2.44-2.62 (m, 1H, - CHCO); 3.89-4.05 (d, 1H, -CHNH); 6.96 (s, 1H, -CONH); 9.13 (s, 1H, -NHCOO). 13C-NMR (CDCl3, 62.5 MHz, ppm): 14.06 (CH3); 22.65 (CH2); 24.30 (CH2); 24.85 (CH2); 25,08 (CH2); 25.81 (CH2); 28.21 (3XCH3); 29.34 (CH2); 29.44 (CH2); 29.50 (CH2); 29.63 (3xCH2); 31.89 (CH2); 32.79 (CH2); 36.68 (CH2); 48.54 (CH); 49.84 (CH); 81.12 (C); 155.40 (COO); 173.76 (CONHNH); 173.94 (CONH). EM-ESI+ (m/z, %): calculated for C24H46N3O4 [M+H]+: 440.3487. Found: 440.3483. Towards antibacterial hydrogels. Synthesis, structural studies and biological activity EXPERIMENTAL WORK 66 8 Towards antibacterial hydrogels. Synthesis, structural studies and biological activity EXPERIMENTAL WORK 67 7.2.8 Synthesis of N-((1R,2R)-2-(hydrazinocarbonyl)cyclohexyl)dodecanamide (9) In a round-bottom flask containing starting hydrazide 8 (112.8 mg, 0.256 mmol), a mixture of TFA/DCM 1:1 (1.28 mL) was added. The reaction mixture was stirred at room temperature for 3 hours, after which, by TLC (CH2Cl2/MeOH 5%), the disappearance of the starting material (Rf=0.3) and the appearance of a reaction product (Rf=0.1) were observed. The reaction solution was concentrated, placed on ice to precipitate the product, and triturated with ether/hexane 1:1. The resulting solid was then dissolved in a saturated aqueous solution of NaHCO3 (10 mL) and extracted with DCM (3x10 mL). The organic phases were combined, dried with anhydrous sodium sulfate, and concentrated to dryness. Purification was achieved through column chromatography (CH2Cl2/MeOH 5%), yielding the product N-((1R,2R)-2- (hydrazinocarbonyl)cyclohexyl)dodecanamide (9) as a white solid (74 mg, 85%). [α]D22: +6.6° (c 1 mg/mL, CH2Cl2). 1H-NMR (CDCl3, 250 MHz, ppm): 0.84-0.91 (t, 3H, -CH3); 0.99-1.16 (m, 2H, -CH2); 1.221.30 (m, 16H, 8x-CH2); 1.47-2.03 (m, 8H, 4x-CH2); 2.08-2.22 (t, 2H, -CH2CO); 2.34-2.56 (m, 1H, -CHCO); 3.20-3.44 (m, 1H, -CHN); 3.62-4.04 (d, 2H, -NH2); 6.65 (s, 1H, -CONHC); 7.73 (s, 1H, -CONHN). 13C-NMR (DMSO, 62.5 MHz, ppm): 14.40 (CH3); 22.55 (CH2); 24.75 (CH2); 25.70 (3xCH2); 29.19 (2xCH2); 29.45 (4xCH2); 31.75 (CH2); 32.97 (CH2); 36.05 (CH2); 46.94 (CHCONHNH2); 48.95 (CHNHCO); 171.74 (CONHNH2); 173.63 (CONH). EM-ESI+ (m/z, %): calculated for C19H38N3O2 [M+H]+: 340.2958. Found: 340.2959. Towards antibacterial hydrogels. Synthesis, structural studies and biological activity EXPERIMENTAL WORK 68 9 Towards antibacterial hydrogels. Synthesis, structural studies and biological activity EXPERIMENTAL WORK 69 7.2.9 Synthesis of the (1R,2R)-2-carbamoylcyclohexane-1-carboxylic acid ((1R,2R)-11) and the (1S,2S)-2-carbamoylcyclohexane-1-carboxylic acid ((1S,2S)-11) (rac-11) In a 10 mL round-bottom flask, 3 mL of a 2M NH3 solution in MeOH was introduced. Onto this, the commercial compound 10, named (3aR,7aS)-hexahydroisobenzofuran-λ3-dione (2 g, 12.973 mmol), was added. The reaction was allowed to proceed with stirring at room temperature for 18 hours. Subsequently, it was acidified with a 12M aqueous HCl solution until the appearance of a white precipitate was observed. The precipitate was filtered under vacuum, washing with small volumes of distilled water. Thus, a white solid (1.558 g, 72%) was obtained, identified by magnetic resonance as 2-carbamoylcyclohexane-1-carboxylic acid (11). 1H-NMR (CD3COD, 250 MHz, ppm): 1.18-1.47 (m, 2H, -CH2CHa2CH2); 1.50-1.68 (m, 3H, -CH2CHb2CH2, -CH2CHCOOH); 1.76-2.00 (m, 2H, -CH2CHCONH2); 2.42-2.67 (m, 2H, 2xCH); 6.67 (s, 1H, -NH2); 7.08 (s, 1H, -NH2); 11.91 (s, 1H, -OH). Towards antibacterial hydrogels. Synthesis, structural studies and biological activity EXPERIMENTAL WORK 70 rac-11 rac-11 Towards antibacterial hydrogels. Synthesis, structural studies and biological activity EXPERIMENTAL WORK 71 7.2.10 Synthesis of the hydrochloride of (1R,2R)-2-aminocyclohexanecarboxylic acid ((1R,2R)-12) and the hydrochloride of (1S,2S)-2-aminocyclohexanecarboxylic acid ((1S,2S)-12) (rac-12) A volume of 32 mL of a MeCN/H2O (1:1) solution was used while stirring magnetically to add 2.95 g (9.17 mmol) of phenyl-λ3-iodanediyl diacetate. Once completely dissolved, compound 11 (1.57 g, 9.17 mmol) was added, and the reaction was allowed to proceed at room temperature for 14 hours. Initially, it was a yellowish solution that became more turbid as the reaction progressed. After the mentioned time, it was diluted with 70 mL of distilled water, and the total volume was acidified with 12 M HCl. Extraction with Et2O was performed, followed by washing with a 10% aqueous HCl solution. The aqueous phases were combined and concentrated to dryness using a high-vacuum rotary evaporator. The resulting solid was redissolved in the minimum volume of ethanol, to which Et2O was added. This mixture was sonicated, inducing the precipitation of a white solid, the hydrochloride of 2aminocyclohexanecarboxylic acid (12), obtained with a low yield. 1H-NMR (CD3COD, 250 MHz, ppm): 1.27-1.63 (m, 4H, 2x -CH2CH2CH2); 1.69-1.86 (m, 2H, -CH2CHCO); 1.82-1.89 (m, 2H, -CH2CHNH); 2.07-2.22 (m, 1H, CHCO); 2.87-2.95 (m, 1H, CHNH). Towards antibacterial hydrogels. Synthesis, structural studies and biological activity EXPERIMENTAL WORK 72 rac-12 Towards antibacterial hydrogels. Synthesis, structural studies and biological activity EXPERIMENTAL WORK 79 7.2.14 Synthesis of 1,3:2,4-dibenzylidene-D-sorbitol-p,p'-dimethylester (15) D-sorbitol (14) (110.9 mg, 0.609 mmol) was dissolved in 20 mL of cyclohexane and 5 mL of methanol at 50 ºC. Simultaneously, a solution of 4-methylformybenzoate (13) (200 mg, 1.218 mmol) and p-toluenesulfonic acid (231 mg, 0.609 mmol) in 10 mL of methanol was prepared. Both solutions were stirred for 20 minutes. Next, the second solution was added to the first one, and the mixture was kept at 70 ºC for 2 hours, ensuring that the reaction did not dry out due to methanol evaporation. After that time, it was allowed to cool until the formation of a precipitate in the cyclohexane was observed. Washes were performed with small volumes of hexane to promote precipitation, and the supernatant was decanted. The paste at the bottom was concentrated to dryness to encourage the appearance of a white powder. This was washed with cold water and cold DCM, and the formation of 1,3:2,4-dibenzylidene-D-sorbitol-p,p'- dimethylester (15) (190 mg, 71%) was confirmed by NMR. 1H-NMR (DMSO, 500 MHz, ppm): 3.49-3.54 (m, 1H, -CHa2OH); 3.64-3.68 (m, 1H, - CHb2OH); 3.87 (s, 6H, 2x -CH3); 3.90-3.95 (m, 1H, -CHOH); 3.98-4.01 (s, 1H, - CHCH(OR)CHO); 4.23 (s, 4H, -CH2CH(OR)CH); 5.75 (s, 2H, -RO-CHR-OR); 7.95-8.00 (d, 4H, 4x COC(CH)2); 7.59-7.64 (t. 4H, -CHC(CH)2). 13C-NMR (DMSO, 62.5 MHz, ppm): 52.39 (2x-CH3); 63.00 (-CH2); 68.34 (-CH); 69.18 (- CH); 69.89 (-CH); 70.80 (-CH); 78.47 (-CH); 99.19 (-CH); 99.26 (-CH); 126.93 (-CH); 129.34 (-CH); 129.49 (-CH); 130.49 (-C); 130.57 (-C); 143.60 (-C); 143.85 (-C); 166.53 (-C). Towards antibacterial hydrogels. Synthesis, structural studies and biological activity EXPERIMENTAL WORK 80 15 Towards antibacterial hydrogels. Synthesis, structural studies and biological activity EXPERIMENTAL WORK 81 7.2.15 Synthesis of 1,3:2,4-dibenzylidene-D-sorbitol-p,p´-dicarboxylic acid (16) In a round-bottom flask, 20 mL of a MeOH:NaOH solution (1:1) was added to DBSCOOMe (190 mg, 0.4 mmol). The mixture was refluxed at 70 ºC for 6 hours until it became completely transparent. After this time, it was concentrated under vacuum, resulting in a gel. It was dissolved in an aqueous HCl solution to reach a pH of 3-2, inducing the appearance of a precipitate. This solid was washed with acidic water, very sensitive to pH, and allowed to dry under vacuum. Thus, 1,3:2,4-dibenzylidene-D-sorbitol-p,p´-dicarboxylic acid was obtained (132.46 mg, 74%). 1H-NMR (DMSO, 500 MHz, ppm): 3.56-4.52 (m, 9H, D-sorbitol); 4.87 (s, 1H, -OHCHC); 5.75 (s, 2H, 2x -RO-CHR-OR); 7.60 (s, 4H, COC(CH)2); 7.99 (s. 4H, -CHC(CH)2); 12.98 (s, 2H, 2x -COOH). 13C-NMR (DMSO, 62.5 MHz, ppm): 63.08 (-CH2); 68.13 (-CH); 69.00 (-CH); 69.80 (-CH2); 70.67 (-CH); 78.09 (-CH); 99.18 (-CH); 126.78 (-CH); 129.62 (-CH); 131.43 (-CH); 143.19 (-C); 143.37 (-C); 167.56 (-C). Towards antibacterial hydrogels. Synthesis, structural studies and biological activity EXPERIMENTAL WORK 82 16 Towards antibacterial hydrogels. Synthesis, structural studies and biological activity EXPERIMENTAL WORK 83 7.2.16 Synthesis of tert-butyl (((9H-fluoren-9-λl)methoxy)carbonyl)leucylglycinate (19) In a 50 mL round-bottom flask, Fmoc-Leu 18 (687.5 mg, 1.945 mmol) and PyBop (1.52 g, 2.918 mmol) were dissolved in 10 mL of DCM with DIEA (340 μL, 1.945 mmol). The mixture was stirred magnetically for 30 minutes to activate the acid. Simultaneously, a solution of 17 (255.2 mg, 1.945 mmol) in 10 mL of DCM with DIEA (340 μL, 1.945 mmol) was prepared. After half an hour, the second solution was combined with the first using a cannula and left to stir under an inert atmosphere of argon for 20 hours. The progress was monitored using the Kaiser test. Liquid-liquid extraction was performed with 15 mL of 1M HCl, brine, and a saturated NaHCO3 solution. The organic phase was dried with anhydrous magnesium sulfate, gravity-filtered, and concentrated under vacuum. The crude product was purified by flash chromatography using DCM/MeOH 5%. This yielded a flake-like solid, identified by NMR as tert-butyl (((9H-fluoren-9-λl)methoxy)carbonyl)leucylglycinate 19 (726.15 mg, 80%). 1H-NMR (CDCL3, 500 MHz, ppm): 0.79-1.02 (m, 2H, -CH2CH(CH3)2); 1.20-1.36 (m, 5H, - CH(CH3)2, ethanol); 1.43-1.48 (d, 3H, -CH(CH3)2); 2.04 (s, 9H, -(CH3)3); 4.02-4.15 (m, 6H, - CHCH2OCO, -RCHCONHCH2); 7.25-7.41 (m, 4H, -CHar); 7.56-7.77 (m, 4H, -CHar). Towards antibacterial hydrogels. Synthesis, structural studies and biological activity EXPERIMENTAL WORK 84 19 Towards antibacterial hydrogels. Synthesis, structural studies and biological activity EXPERIMENTAL WORK 85 7.2.17 Sythesis of N-alpha-(9-Fluorenylmethyloxycarbonyl)-L-leucinyl-glycin (20) The dipeptide 19 was dissolved in 10 mL of DCM with 20% TFA and left overnight. Subsequently, TFA was co-evaporated with small volumes of toluene (x5) in a high vacuum rotary evaporator. The crude product was purified by flash chromatography using DCM/MeOH 2%. This resulted in the isolation of N-alpha-(9-Fluorenylmethyloxycarbonyl)- L-leucinyl-glycin 20, whose purity was confirmed by NMR and HPLC-MS. 1H-NMR (DMSO, 500 MHz, ppm): 0.83-0.91 (m, 6H, -CH(CH3)2); 1.42-1.53 (m, 2H, - CH2CH(CH3)2); 1.59-1.70 (m, 1H, -CH2CH(CH3)2); 3.66-3.82 (m, 2H, -CH2COOH ); 4.014.12 (m, 1H, -CHCH2CH(CH3)2); 4.18-4.35 (m, 3H, -CHCH2OCO); 7.32-7.51 (m, 5H, -CHar, -OCONH); 7.66-7.92 (m, 4H, -CHar); 8.06-8.13 (m, 1H, -CONHCH2). 13C-NMR (DMSO, 62.5 MHz, ppm): 21.84 (-CH3); 23.54 (-CH3); 24.63 (-CH); 41.24 (- CH2); 47.20 (-CH); 53.43 (-CH); 66.04 (-CH2); 120.54 (-CHar); 125.79 (-CHar); 127.51 (- CHar); 128.09(-CHar); 141.19 (2x -Car); 144.42 (2x -CCH); 156.40 (-OCONH); 170.61 (- RCHCONH); 173.11 (-COOH). EM-ESI+ (m/z, %): calculated for C23H26N2O5 [M+H]+: 410.18. Found: 411.15. Towards antibacterial hydrogels. Synthesis, structural studies and biological activity EXPERIMENTAL WORK 86 20 Towards antibacterial hydrogels. Synthesis, structural studies and biological activity EXPERIMENTAL WORK 87 7.3. Infrared Spectra Figure 22. FTIR spectrum of Gt12-COOH-GdL xerogel. Figure 23. FTIR spectrum of Gt12-COOH-HCl xerogel. Nombre Gt12-COOH-GdL 4000 10003500 3000 2500 2000 1500 100 55 60 65 70 75 80 85 90 95 cm-1 %T 3290.49cm-1; 57.72%T 1646.65cm-1; 60.14%T 2916.04cm-1; 68.51%T 1551.56cm-1; 68.69%T 1697.47cm-1; 69.11%T 2848.97cm-1; 74.07%T 1412.6cm-170.352%T 1727.2cm-174.245%T Nombre Gt12-COOH-HCl 4000 10003500 3000 2500 2000 1500 101 48 50 55 60 65 70 75 80 85 90 95 100 cm-1 %T 3288.88cm-1; 57.70%T 1646.07cm-1; 63.58%T 2915.97cm-1; 68.43%T 1696.08cm-1; 68.95%T 1724.36cm-1; 72.23%T 2848.99cm-1; 73.52%T Towards antibacterial hydrogels. Synthesis, structural studies and biological activity EXPERIMENTAL WORK 88 Figure 24. FTIR spectrum of Gt12-COOH solution in dH2O. Figure 25. FTIR spectrum of Gt12-NHNH2 xerogel. Gt12-COOH-sol Nombre Muestra 003 Por JCE-RE_P2L2 Fecha lunes, enero 08 2024 Descripción 4000 10003500 3000 2500 2000 1500 101 77 78 80 82 84 86 88 90 92 94 96 98 100 cm-1 %T 2924.23cm -1; 85.65% T 1643.93cm-1; 87.67%T 2853.54cm-1; 91.79%T 3297.92cm-1; 96.44%T 8 Nombre Gt12-COOH-sol Nombre Gt12-NHNH2 4000 4503500 3000 2500 2000 1500 1000 101 32 35 40 45 50 55 60 65 70 75 80 85 90 95 cm-1 %T 3290.10cm-1; 54.34%T 1726.59cm-1; 60.11%T 1646.94cm-1; 69.35%T 2916.11cm-1; 71.23%T 2849.09cm-1; 75.65%T