Eurico J. Cabrita

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.

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.

The close structural similarity between the two cyclooxygenase (COXs) isoforms and the absence of selective inhibitors without side effects continues to stimulate the development of novel approaches towards selective anti-inflammatory drugs. In the present study a small library of new indolic compounds involving two different substitutions patterns at the indole scaffold was synthesized. In order to establish a relation between the spatial distribution of known functional groups related with inhibitory activity, two substitution patterns were explored: one with substituents at N-1, C-3, C-5 positions and another at C-2, C-3 and C5 positions. Accordingly, indole positions C-5, C-3 and N-1 were substituted with: sulfonamide or methylsulfone at C-5, p-halo-benzyl group at C-3, and an alkyl chain with a trifluoromethyl group at N-1. Alternatively, a p-halo-benzyl group was introduced at C-2, leaving the indolic nitrogen free. Inhibitory studies were performed and the activity results obtained against both COXs isoforms were rationalized based on docking and NMR studies. Docking studies show that dialkyation at C-2 and C-3 favors a binding with an orientation similar to that of the known selective inhibitor SC-558. From the tested compounds, this substitution pattern is correlated with the highest inhibitory activity and selectivity: 70% COX-2 inhibition at 50 M, and low COX-1 inhibition (18±9%). Additionally, Saturation Transfer Difference NMR experiments reveal different interaction patterns with both COXs isoforms that may be related with different orientations of the sulfonamide group in the binding pocket. Despite the moderated inhibitory activities found, this study represents an innovative approach towards COXs inhibitory activity rationalization and to the design of anti-inflammatory drugs.

Native agars from Gracilaria vermiculophylla produced in sustainable aquaculture systems (IMTA) were extracted under conventional (TWE) and microwave (MAE) heating. The optimal extracts from both processes were compared in terms of their properties. The agars’ structure was further investigated through Fourier transform infrared and NMR spectroscopy. Both samples showed a regular structure with an identical backbone, β-D-galactose (G) and 3,6-anhydro-α-L-galactose (LA) units; a considerable degree of methylation was found at C6 of the G units and, to a lesser extent, at C2 of the LA residues. The methylation degree in the G units was lower for MAEopt agar; the sulfate content was also reduced. MAE led to higher agar recoveries with drastic extraction time and solvent volume reductions. Two times lower values of [η] and Mv obtained for the MAEopt sample indicate substantial depolymerization of the polysaccharide backbone; this was reflected in its gelling properties; yet it was clearly appropriate for commercial application in soft-texture food products.