Eurico J. Cabrita

The use of agar-based biomaterials for the development of emerging areas, such as tissue engineering or ‘smart materials’ production has recently gained great interest. Understanding how these gel-forming polysaccharides self-organise in aqueous media and how these associations can be tuned to meet the specific needs of each application is thus of great relevance. As an extension of previous pioneering research concerning the application of the microwave-assisted extraction (MAE) technique in the recovery of native (NA) and alkali-modified (AA) agars, this article focuses on the different molecular assemblies assumed by these novel NA and AA when using different MAE routes. The molecular architectures in dilute (5, 10, 50 and 100 mg mL
1) and concentrated (1.5% (w/w)) aqueous media were imaged by AFM and cryoSEM, respectively. Relevant structural and physicochemical properties were investigated to support the microscopic data. Different extraction routes led to polysaccharides with unique properties, which in turn resulted in different molecular assemblies. Even at 5 mg mL
1, AFM images included individual fibers, cyclic segments, aggregates and local networks. At higher polymer concentrations, the structures further aggregated forming multilayer polymeric networks for AA. The more compact and denser 3D networks of AA, imaged by cryoSEM, and their higher resistance to large deformations matched the 2D-shapes observed by AFM. Depending on the nature of the AA chains, homogeneous or heterogeneous growth of assemblies was seen during network formation. The obtained results support well the view of double helix formation followed by intensive double helix association proposed for agar gelation.

Non-catalytic cellulosomal carbohydrate-binding modules (CBMs) are responsible for increasing the catalytic efficiency of cellulosic enzymes by selectively putting the substrate (a wide range of poly- and oligosaccharides) and enzyme into close contact. In the present work we carried out an atomistic rationalization of the molecular determinants of ligand specificity of a family 11 CBM from thermophilic C. thermocellum (CtCBM11), based on a NMR and molecular modeling approach. We have determined the NMR solution structure of CtCBM11 at 25 and 50 ºC and derived information on the residues of the protein involved in ligand recognition and on the influence of the length of the saccharide chain on binding. We obtained models of the CtCBM11/cellohexaose and CtCBM11/cellotetraose complexes by docking in accordance with the NMR experimental data. Specific ligand/protein CH-π and Van der Waals interactions were found to be determinant for the stability of the complexes and for defining specificity. Using the order parameters derived from backbone dynamics analysis in the presence and absence of ligand and at 25 and 50 ºC, we determined that the protein’s backbone conformational entropy is slightly positive. This data in combination with the negative binding entropy calculated from ITC studies supports a selection mechanism where a rigid protein selects a defined oligosaccharide conformation.

A novel porous polymer-ionic liquid composite with poly(2-hydroxyethyl methacrylate) (PHEMA) and 1-butyl-3-methylimidazolium hexafluorophosphate (BMIPF6) has been synthesized by γ-irradiation without heat or chemical initiators. The products can be reversibly converted into organogels. The composites are potential candidates for electrochemical applications. The use of γ-radiation can be a simple and versatile alternative way to obtain these materials.

Pulsed field gradient spin echo high resolution magic angle spinning nuclear magnetic resonance spectroscopy is a powerful technique to characterize confined biosystems. We used this approach to assess the diffusion of solvent and reaction species within sol–gel matrices differing in enzyme loading.

Molybdenum and tungsten enzymes require specific chaperones for folding and cofactor insertion. PaoD is the chaperone of the periplasmic aldehyde oxidoreductase PaoABC. It is the last gene in the paoABCD operon in Escherichia coli and its presence is crucial for obtaining mature enzyme. PaoD is an unstable, 35 kDa, protein. Our biochemical studies showed that it is a dimer in solution with a tendency to form large aggregates, especially after freezing/thawing cycles. In order to improve stability, PaoD was thawed in the presence of two ionic liquids [C4mim]Cl and [C2OHmim]PF6 and no protein precipitation was observed. This allowed protein concentration and crystallization using polyethylene glycol or ammonium sulfate as precipitating agents. Saturation transfer difference – nuclear magnetic resonance (STD-NMR) experiments have also been performed in order to investigate the effect of the ionic liquids in the stabilization process, showing a clear interaction between the acidic ring protons of the cation and, most likely, negatively charged residues at the protein surface. DLS assays also show a reduction of the overall size of the protein aggregates in presence of ionic liquids. Furthermore, cofactor binding studies on PaoD showed that the protein is able to discriminate between molybdenum and tungsten bound to the molybdenum cofactor, since only a Mo-MPT form of the cofactor remained bound to PaoD.

The human macrophage galactose-type lectin (hMGL) is a key physiological receptor for the carcinoma-associated Tn antigen (GalNAc-α-1-O-Ser/Thr) in mucins. We herein report NMR- and modeling-based data on the molecular recognition features of synthetic Tn-bearing glycopeptides by hMGL. Cognate epitopes on the sugar and matching key amino acids involved in the interaction have been identified by saturation transfer difference (STD) NMR spectroscopy. Only the amino acids close to the glycosylation site in the peptides are involved in lectin contact. Moreover, control experiments with non-glycosylated MUC1 peptides unequivocally showed that the sugar residue is essential for hMGL binding, as is Ca2+. The dissociation constants (Kd) have been estimated by STD titrations and/or STD competition experiments and show that Gal was a poor binder for hMGL, with a Kd in the mM range, while GalNAc and MUC1 Tn-glycopetides reached Kd values in the lower μM range. STD-based results suggested a distinct interacting epitope for the two monosaccharides. NMR data have been complemented with molecular dynamics simulations and Corcema- ST to establish a 3D view on the molecular recognition process between Gal, GalNAc and the Tn-presenting glycopeptides and hMGL. Gal and GalNAc have a dual binding mode with opposite trend of the main interaction pattern and the differences in affinity can be explained by additional hydrogen bonds and CH-π contacts involving exclusively the NHAc moiety.

We investigated imidazolium-based ionic liquid (IL) interactions with human serum albumin (HSA) to discern the level of cation interactions towards protein stability. STD-NMR spectroscopy was used to observe the imidazolium IL protons involved in direct binding and to identify the interactions responsible for changes in Tm as accessed by differential scanning calorimetry (DSC). Cations influence protein stability less than anions but still significantly. It was found that longer alkyl side chains of imidazolium- based ILs (more hydrophobic) are associated with a higher destabilisation effect on HSA than short-alkyl groups (less hydrophobic). The reason for such destabilisation lies on the increased surface contact area of the cation with the protein, particularly on the hydrophobic contacts promoted by the terminus of the alkyl chain. The relevance of the hydrophobic contacts is clearly demonstrated by the introduction of a polar moiety in the alkyl chain: a methoxy or alcohol group. Such structural modification reduces the degree of hydrophobic contacts with HSA explaining the lesser extent of protein destabilisation when compared to longer alkyl side chain groups: above [C2mim]+. Competition STD-NMR experiments using [C2mim]+, [C4mim]+ and [C2OHmim]+ also validate the importance of the hydrophobic interactions. The combined effect of cation and anion interactions was explored using 35Cl NMR. Such experiments show that the nature of the cation has no influence on the anion–protein contacts, still the nature of the anion modulates the cation–protein interaction. Herein we propose that more destabilising anions are likely to be a result of a partial contribution from the cation as a direct consequence of the different levels of interaction (cation–anion pair and cation–protein).

The lipoxygenase (LOX) products have been identified as mediators of a series of inflammatory diseases, namely rheumatoid arthritis, inflammatory bowel disease, psoriasis, allergic rhinitis, atherosclerosis and certain types of cancer. Hence, LOX inhibitors are of interest for the modulation of these phenomena and resolution of the inflammatory processes. During LOX activity, peroxyl radical complexes are part of the reaction and may function as sources of free radicals. Thus antioxidants, such as flavonoids, capable of inhibiting lipid peroxidation and scavenging free radicals, may act as LOX inhibitors. The aim of this work was to assess the structure–activity relationship among a series of flavonoids concerning 5-LOX inhibition, through a systematic study of the inhibition of the formation of LTB4 in human neutrophils. The type of inhibition of the flavonoids was further studied using soybean LOX, type I, and Saturation Transfer Difference 1H NMR (STD-1H NMR) was used to characterize the binding epitopes of the compounds to LOX-1. The obtained results reinforce flavonoids as effective inhibitors of LTB4 production in human neutrophils. It was also possible to establish a structure/activity relationship for the inhibitory activity and the type of inhibition.

The thermal behavior and transport properties of several ion jellys (IJs), a composite that results from the combination of gelatin with an ionic liquid (IL), were investigated by dielectric relaxation spectroscopy (DRS), differential scanning calorimetry (DSC), and pulsed field gradient nuclear magnetic resonance spectroscopy (PFG NMR). Four different ILs containing the dicyanamide anion were used: 1-butyl-3-methylimidazolium dicyanamide (BMIMDCA), 1-ethyl-3-methylimidazolium dicyanamide (EMIMDCA), 1-butyl-1-methylpyrrolidinium dicyanamide (BMPyrDCA), and 1-butylpyridinium dicyanamide (BPyDCA); the bulk ILs were also investigated for comparison. A glass transition was detected by DSC for all materials, ILs and IJs, allowing them to be classified as glass formers. Additionally, an increase in the glass transition temperature upon dehydration was observed with a greater extent for IJs, attributed to a greater hindrance imposed by the gelatin matrix after water removal, rendering the IL less mobile. While crystallization is observed for some ILs with negligible water content, it was never detected for any IJ upon thermal cycling, which persist always as fully amorphous materials. From DRS measurements, conductivity and diffusion coefficients for both cations (D+) and anions (D–) were extracted. D+ values obtained by DRS reveal excellent agreement with those obtained from PFG NMR direct measurements, obeying the same VFTH equation over a large temperature range (ΔT ≈ 150 K) within which D+ varies around 10 decades. At temperatures close to room temperature, the IJs exhibit D values comparable to the most hydrated (9%) ILs. The IJ derived from EMIMDCA possesses the highest conductivity and diffusion coefficient, respectively, 10–2 S·cm–1 and 10–10 m2·s–1. For BMPyrDCA the relaxational behavior was analyzed through the complex permittivity and modulus formalism allowing the assignment of the detected secondary relaxation to a Johari–Goldstein process. Besides the relevant information on the more fundamental nature providing physicochemical details on ILs behavior, new doorways are opened for practical applications by using IJ as a strategy to produce novel and stable electrolytes for different electrochemical devices.

Non-steroidal anti-inflammatory drugs exert their pharmacological activity through inhibition of cyclooxygenase 1 and 2 (COX-1 and COX-2). Recent research suggests that a balanced inhibition of both COX-1 and COX-2 is the key to reduce the side-effects exhibited by COX inhibitors. We developed new benzimidazole-based compounds that showed a balanced COX inhibition, supported by molecular docking screening. The human whole blood assays demonstrated that the ester derivatives were potent inhibitors. Competitive saturation transfer difference (STD)-NMR experiments, in the presence of COX-2, using naproxen and diclofenac demonstrated that ester derivatives do not compete with diclofenac for the same binding site, but compete with the allosteric inhibitor naproxen. Combination of NMR spectroscopy with molecular docking has permitted us to detect a new naproxen-like inhibitor, which could be used for future drug development.