Reversibility in the Physical Properties of Agarose Gels following an Exchange in Solvent and Non-Solvent.

Agarose forms a homogeneous thermoreversible gel in an aqueous solvent above a critical polymer concentration. Contrary to the prevailing consensus, recent confirmations indicate that agarose gels are also stable in non-solvents like acetone and ethanol. A previous study compared gel characterisations and behaviours in water and ethanol, discussing the gelation mechanism. In the current work, the ethanol gel is exchanged with water to explore the potential reversibility of the displacement of water in agarose. Initially, the structure is characterised using 1H NMR in DMSO-d6 and D2O solvents. Subsequently, a very low yield (0.04) of methyl substitution per agarobiose unit is determined. The different gels after stabilisation are characterised using rheology, and their physical properties are compared based on the solvent used. The bound water molecules, acting as plasticizers in aqueous medium, are likely removed during the exchange process with ethanol, resulting in a stronger and more fragile gel. Next, the gel obtained after the second exchange from ethanol back to water is compared with the initial gel prepared in water. This is the first time where such gel has been characterised without undergoing a phase transition when switching from a good solvent to a non-solvent, and vice versa, thereby testing the reversibility of the solvent exchange. Reversibility of this behaviour is demonstrated through swelling and rheology experiments. This study extends the application of agarose in chromatography and electrophoresis.

Synthesis of Neuraminidase-Resistant Sialyllactose Mimetics from N-Acyl Mannosamines using Metabolically Engineered Escherichia coli.

Herein, we describe the efficient gram-scale synthesis of α2,3- and α2,6-sialyllactose oligosaccharides as well as mimetics from N-acyl mannosamines and lactose in metabolically engineered bacterial cells grown at high cell density. We designed new Escherichia coli strains co-expressing sialic acid synthase and N-acylneuraminate cytidylyltransferase from Campylobacter jejuni together with the α2,3-sialyltransferase from Neisseria meningitidis or the α2,6-sialyltransferase from Photobacterium sp. JT-ISH-224. Using their mannose transporter, these new strains actively internalized N-acetylmannosamine (ManNAc) and its N-propanoyl (N-Prop), N-butanoyl (N-But) and N-phenylacetyl (N-PhAc) analogs and converted them into the corresponding sialylated oligosaccharides, with overall yields between 10 % and 39 % (200-700 mg.L-1 of culture). The three α2,6-sialyllactose analogs showed similar binding affinity for Sambucus nigra SNA-I lectin as for the natural oligosaccharide. They also proved to be stable competitive inhibitors of Vibrio cholerae neuraminidase. These N-acyl sialosides therefore hold promise for the development of anti-adhesion therapy against influenza viral infections.

Characterization of Agarose Gels in Solvent and Non-Solvent Media.

Agarose is known to form a homogeneous thermoreversible gel in an aqueous medium over a critical polymer concentration. The solid-liquid phase transitions are thermoreversible but depend on the molecular structure of the agarose sample tested. The literature has mentioned that agarose gels could remain stable in non-solvents such as acetone or ethanol. However, there has been no characterization of their behavior nor a comparison with the gels formed in a good solvent such as water. In the first step of this article, the structure was characterized using 1H and 13C NMR in both D2O and DMSO-d6 solvents. DMSO is a solvent that dissolves agarose regardless of the temperature. First, we have determined a low yield of methyl substitution on the D-galactose unit. Then, the evolution of the 1H NMR spectrum was monitored as a function of temperature during both increasing and decreasing temperature processes, ranging from 25 to 80 °C. A large thermal hysteresis was obtained and discussed, which aided in the interpretation of rheological behavior. The hysteresis of NMR signals is related to the mobility of the agarose chains, which follows the sol/gel transition depending on the chains' association with H-bonds between water and the -OH groups of agarose for tightly bound water and agarose/agarose in chain packing. In the second step of the study, the water in the agarose gel was exchanged with ethanol, which is a non-solvent for agarose. The resulting gel was stable, and its properties were characterized using rheology and compared to its behavior in aqueous media. The bound water molecules that act as plasticizers were likely removed during the exchange process, resulting in a stronger and more brittle gel in ethanol, with higher thermal stability compared to the aqueous gel. It is the first time that such gel is characterized without phase transition when passing from a good solvent to a non-solvent. This extends the domains of application of agarose.

Biotechnological production of sialylated solid lipid microparticles as inhibitors of influenza A virus infection.

Influenza viruses bind to their target through a multivalent interaction of their hemagglutinins (HAs) with sialosides at the host cell surface. To fight the virus, one therapeutic approach consists in developing sialylated multivalent structures that can saturate the virus HAs and prevent the binding to host cells. We describe herein the biotechnological production of sialylated solid lipid microparticles (SSLMs) in 3 steps: (i) a microbiological step leading to the large-scale production of sialylated maltodextrins by metabolic engineering of an Escherichia coli strain, (ii) a new in vitro glycosylation process using the amylomaltase MalQ, based on the transglycosylation of the terminal sialoside ligand of the sialylated maltodextrin onto a long-chain alkyl glucoside, and (iii) the formulation of the final SSLMs presenting a multivalent sialic acid. We also describe the morphology and structure of the SSLMs and demonstrate their very promising properties as influenza virus inhibitors using hemagglutination inhibition and microneutralization assays on the human A/H1N1 pdm09 virus.

Structure of the Polysaccharide Secreted by Vibrio alginolyticus CNCM I-5035 (Epidermist 4.0TM).

Vibrio alginolyticus (CNCM I-5035) secretes an exopolysaccharide used as ingredient in cosmetic industry under the trademark Epidermist 4.0TM. It is appreciated for its ability to improve the physical and chemical barrier functions of the skin by notably increasing the keratinocyte differentiation and epidermal renewal. Composition analyses and in depth characterization of the polysaccharides as well as oligosaccharides obtained by mild acid hydrolyses revealed that it was composed of a repetition unit of three residues: d-galactose (d-Gal), d-N-acetylglucosamine (GlcNAc) and l-N-acetylguluronic acid, of which 30% (M/M) was acetylated in position 3. The complete structure of the polysaccharide was resolved giving the repetition unit: [→3)-α-d-Gal-(1→4)-α-l-GulNAcA/α-l-3OAc-GulNAcA-(1→4)-ß-d-GlcNAc-(1→].

Boronic acid and diol-containing polymers: how to choose the correct couple to form "strong" hydrogels at physiological pH.

Dynamic covalent hydrogels crosslinked by boronate ester bonds are promising materials for biomedical applications. However, little is known about the impact of the crosslink structure on the mechanical behaviour of the resulting network. Herein, we provide a mechanistic study on boronate ester crosslinking upon mixing hyaluronic acid (HA) backbones modified, on the one hand, with two different arylboronic acids, and on the other hand, with three different saccharide units. Combining rheology, NMR and computational analysis, we demonstrate that carefully selecting the arylboronic-polyol couple allows for tuning the thermodynamics and molecular exchange kinetics of the boronate ester bond, thereby controlling the rheological properties of the gel. In particular, we report the formation of "strong" gels (i.e. featuring slow relaxation dynamics) through the formation of original complex structures (tridentate or bidentate complexes). These findings offer new prospects for the rational design of hydrogel scaffolds with tailored mechanical response.

Injectable Self-Healing Hydrogels Based on Boronate Ester Formation between Hyaluronic Acid Partners Modified with Benzoxaborin Derivatives and Saccharides.

We demonstrate here, for the first time, formation of injectable dynamic covalent hydrogels at physiological pH using benzoxaborin-saccharide complexation as a reversible cross-linking method. The gels were prepared by simply mixing hyaluronic acid modified with an original boronic acid derivative, 3,4-dihydro-2H-benzo[e][1,2]oxaborinin-2-ol (1,2-ABORIN), and HA functionalized with 1-amino-1-deoxy-d-fructose. Dynamic rheological experiments confirmed the gel-like behavior (storage modulus (G') > loss modulus (G″) in the frequency window explored) for the designed HA-1,2-ABORIN/HA-fructose network. Furthermore, this hydrogel exhibited excellent self-healing and injectability behaviors in aqueous conditions and was found to be responsive to pH. Additionally, fibroblast cells encapsulated in the HA network showed high viability (>80% after 7 days of cell culture), as monitored by Live/Dead staining. Taken together, this new class of boronate ester cross-linked hydrogel provides promising future for diverse biomedical applications.

Structure of the Exopolysaccharide Secreted by a Marine Strain Vibrio alginolyticus.

Vibrio alginolyticus (CNCM I-4151) secretes an exopolysaccharide whose carbohydrate backbone is decorated with amino acids, likely conferring its properties that are appreciated in cosmetics. Here, the secreted polysaccharide of another strain of V. alginolyticus (CNCM I-5034) was characterized by chromatography and one- and two-dimensional NMR spectroscopy experiments. The structure was resolved and shows that the carbohydrate backbone is made of four residues: D-galactose (Gal), D-galacturonic acid (GalA) D-N-acetylglucosamine (GlcNAc) and D-glucuronic acid (GlcA), forming a tetrasaccharide repetition unit [→4)-ß-d-GlcA-(1→3)-α-d-Gal-(1→3)-α-d-GalA-(1→3)-ß-GlcNAc(1→]. GlcA is derivatized with a lactate group giving 'nosturonic acid', and GalA is decorated with the amino acid alanine.

Structure of an Amino Acid-Decorated Exopolysaccharide Secreted by a Vibrio alginolyticus Strain.

Vibrio alginolyticus (CNCM I-4994) secretes an exopolysaccharide that can be used as an ingredient in cosmetic applications. The structure was resolved using chromatography and one- and two-dimensional NMR spectroscopy experiments. The results show that the carbohydrate backbone is made of two residues: d-galacturonic acid and N-acetyl-d-glucosamine (GlcNac), which together constitute a tetrasaccharide repetition unit: [→3)-α-d-GalA-(1→4)-α-d-GalA-(1→3)-α-d-GalA-(1→3)-ß-GlcNAc(1→]. Two amino acids, alanine and serine, are linked to GalA residues via amido linkages. The position and the distribution of the amino acids were characterized by two-dimensional NMR spectroscopy. To our knowledge, this is the first description of a structure for a marine exopolysaccharide decorated with an amino acid.

Synthesis of a library of allyl alpha-L-arabinofuranosyl-alpha- or beta-D-xylopyranosides; route to higher oligomers.

Six isomeric disaccharides allyl 2,3,5-tri-O-benzoyl-alpha-l-arabinofuranosyl-alpha-d-xylopyranosides and beta-d-xylopyranosides were synthetized by the stereoselective glycosylation of pure allyl alpha- or beta-d-xylopyranosides with 1-O-acetyl-2,3,5-tri-O-benzoyl-l-arabinofuranose as donor, catalyzed with BF(3).Et(2)O in DCM. Regio- and stereoselective glycosylation with excess of donor furnished almost exclusively the trisaccharides allyl 2,3-di-O-(2,3,5-tri-O-benzoyl-alpha-l-arabinofuranosyl)-alpha- or beta-d-xylopyranosides. Extension of the reaction to the triol beta-d-xylopyranosyl-(1-->4)-1,2,3-tri-O-acetyl-alpha-d-xylopyranose, obtained from the 4-hydroxyl penta-O-acetyl-alpha-xylobiose, gave in the same manner the tetrasaccharide [2,3-di-O-(2,3,5-tri-O-benzoyl-alpha-l-arabinofuranosyl)-beta-d-xylopyranosyl]-(1-->4)-1,2,3-tri-O-acetyl-alpha-d-xylopyranose. The protocol described herein should offer the possibility to produce branched oligosaccharides with a 2,3-di-O-(alpha-l-Ara(f))-beta-d-Xyl(p) block unit at the terminal non-reducing end.

Plant chemical defense induced by a seed-eating pollinator mutualist.

Plant-seed parasite pollination mutualisms involve a specific pollinator whose larvae develop by consuming a fraction of the host plant seeds. These mutualisms are stable only if the plant can control seed destruction by the larvae. Here, we studied the chemical response of the European globeflower Trollius europaeus to infestation by an increasing number of Chiastocheta fly larvae. We used liquid chromatographic analysis to compare the content of phenolic compounds in unparasitized and parasitized fruits collected in two natural populations of the French Alps, and mass spectrometry and nuclear magnetic resonance to elucidate the structure of adonivernith, a C-glycosyl-flavone. This compound is present in many of the organs of T. europaeus, but not found in other Trollius species. Furthermore, it is overproduced in the carpel walls of parasitized fruits, and this induced response to infestation by fly larvae is density-dependent (increases with larval number), and site-dependent (more pronounced in the high-altitude site). Mechanical damage did not induce adonivernith production. This tissue-specific and density-dependent response of T. europaeus to infestation by Chiastocheta larvae might be an efficient regulation mechanism of seed-predator mutualist population growth if it decreases survival or growth of the larvae.