Lignocellulosic-biomolecules conjugated systems: green-engineered complexes modified by covalent linkers.

Lignocellulosic biomass represents an abundant and eco-friendly material widely explored in recent years. The main lignocellulosic fractions include cellulose, hemicellulose, and lignin. Nonetheless, the heterogeneity and complexity of these components pose challenges in achieving the desired properties. Conversely, their attractive functional groups can covalently link with other biomolecules, facilitating the creation and enhancement of material properties. Lignocellulosic molecules can form different linkages with other biomolecules through classic and modern methods. Bioconjugation has emerged as a suitable alternative to create new nuances, empowering the linkage between lignocellulosic materials and biomolecules through linkers. These conjugates (lignocellulosic-linkers-biomolecules) attract attention from stakeholders in medicine, chemistry, biology, and agriculture. The plural formations of these biocomplexes highlight the significance of these arrangements. Therefore, this review provides an overview of the progress of lignocellulosic-biomolecule complexes and discusses different types of covalent bioconjugated systems, considering the formation of linkers, applicability, toxicity, and future challenges.

Papain Covalent Immobilization in Bacterial Cellulose Films as a Wound Dressing.

Ideally, the dressings used in the clinic have characteristics that help the wound closure process. Among several factors that affect the success of this healing process, there is debridement. It manages the wound bed components and the re-epithelialization process. Still, the property of debridement is not generally associated with dressings. Here, we show a chemically modified bacterial cellulose film conjugated to a proteolytic enzyme, papain, as a dressing with debridement properties. Bacterial cellulose films were reacted with a spacer derived from succinic acid and finally had this enzyme covalently immobilized in its structure by an amide bond. FT-IR and UV-vis showed bands typically of bioconjugated polymer. Enzymatic immobilization was very effective under the conditions applied with high yield (33% w/w), and these remained activated after the coupling reaction. The bacterial cellulose film with the enzyme papain attached to it was also very compatible with fibroblast cells, suggesting that it could be a promising wound dressing material for future research.

Liposome surface modification by phospholipid chemical reactions.

Liposomal systems are well known for playing an important role as drug carriers, presenting several therapeutic applications in different sectors, such as in drug delivery, diagnosis, and in many other academic areas. A novel class of this nanoparticle is the actively target liposome, which is constructed with the surface modified with appropriated molecules (or ligands) to actively bind a target molecule of certain cells, system, or tissue. There are many ways to functionalize these nanostructures, from non-covalent adsorption to covalent bond formation. In this review, we focus on the strategies of modifying liposomes by glycerophospholipid covalent chemical reaction. The approach used in this text summarizes the main reactions and strategies used in phospholipid modification that can be carried out by chemists and researchers from other areas. The knowledge of these methodologies is of great importance for planning new studies using this material and also for manipulating its properties.

Improvement of antimalarial activity of a 3-alkylpiridine alkaloid analog by replacing the pyridine ring to a thiazole-containing heterocycle: Mode of action, mutagenicity profile, and Caco-2 cell-based permeability.

The development of new antimalarial drugs is urgent to overcome the spread of resistance to the current treatment. Herein we synthesized the compound 3, a hit-to­lead optimization of a thiazole based on the most promising 3-alkylpyridine marine alkaloid analog. Compound 3 was tested against Plasmodium falciparum and has shown to be more potent than its precursor (IC50 values of 1.55 and 14.7 µM, respectively), with higher selectivity index (74.7) for noncancerous human cell line. This compound was not mutagenic and showed genotoxicity only at concentrations four-fold higher than its IC50. Compound 3 was tested in vivo against Plasmodium berghei NK65 strain and inhibited the development of parasite at 50 mg/kg. In silico and UV-vis approaches determined that compound 3 acts impairing hemozoin crystallization and confocal microscopy experiments corroborate these findings as the compound was capable of diminishing food vacuole acidity. The assay of uptake using human intestinal Caco-2 cell line showed that compound 3 is absorbed similarly to chloroquine, a standard antimalarial agent. Therefore, we present here compound 3 as a potent new lead antimalarial compound.

Enhancement of fibroblast growing on the mannosylated surface of cellulose membranes.

Bacterial cellulose membrane is a biomaterial with high value in the biomedical field. Many groups have been making efforts to promote chemical modifications of its structure and, consequently, add new characteristics. Recently, our group has developed a methodology to insert monoester succinic acid in bacterial cellulose membrane without disrupting the microfibril network and bind a protein on it. Considering the role of carbohydrates in the molecular recognition process in biological events, we continued these studies by inserting covalently multiples copies of aryl monosaccharide to bacterial cellulose succinylated and to study the in vitro tissue compatibility using fibroblasts. The mix of synthetical chemistry and material modification was performed to prepare aminoaryl mannoside and conjugate it, via amide bond using ultrasonic irradiation, to succinic group of bacterial cellulose. This material was characterized chemically (IR, UV-vis, 13C NMR CP-MAS) and physically (TGA and AFM). Mannosylated cellulose showed good in vitro compatibility with fibroblasts demonstrating its potential in the tissue engineering field which could provide a tissue compatible scaffold.

Polyvalent C-glycomimetics based on l-fucose or d-mannose as potent DC-SIGN antagonists.

The C-type lectin DC-SIGN expressed on immature dendritic cells is a promising target for antiviral drug development. Previously, we have demonstrated that mono- and divalent C-glycosides based on d-manno and l-fuco configurations are promising DC-SIGN ligands. Here, we described the convergent synthesis of C-glycoside dendrimers decorated with 4, 6, 9, and 12 α-l-fucopyranosyl units and with 9 and 12 α-d-mannopyranosyl units. Their affinity against DC-SIGN was assessed by surface plasmon resonance (SPR) assays. For comparison, parent O-glycosidic dendrimers were synthesized and tested, as well. A clear increase of both affinity and multivalency effect was observed for C-glycomimetics of both types (mannose and fucose). However, when dodecavalent C-glycosidic dendrimers were compared, there was no difference in affinity regarding the sugar unit (l-fuco, IC50 17 µM; d-manno, IC50 12 µM). For the rest of glycodendrimers with l-fucose or d-mannose attached by the O- or C-glycosidic linkage, C-glycosidic dendrimers were significantly more active. These results show that in addition to the expected physiological stability, the biological activity of C-glycoside mimetics is higher in comparison to the corresponding O-glycosides and therefore these glycomimetic multivalent systems represent potentially promising candidates for targeting DC-SIGN.

Target-Guided Synthesis and Antiplasmodial Evaluation of a New Fluorinated 3-Alkylpyridine Marine Alkaloid Analog.

The need to develop new alternatives for antimalarial treatment is urgent. Herein, we report the synthesis and antimalarial evaluation of a small library of synthetic 3-alkylpyridine marine alkaloid (3-APA) analogs. First, the compounds were evaluated in vitro against Plasmodium falciparum. The most active compound 5c was selected for optimization of its antimalarial properties. An in silico approach was used based on pure ab initio electronic structure prediction, and the results indicated that a substitution of the hydroxyl group by a fluorine atom could favor a more stable complex with heme at a molecular ratio of 2:1 (heme/3-APA halogenated). A new fluorinated 3-APA analog was synthesized (compound 7), and its antimalarial activity was re-evaluated. Compound 7 exhibited optimized antimalarial properties (P. falciparum IC50 = 2.5 µM), low genotoxicity, capacity to form a more stable heme/3-APA complex at a molecular ratio of 2:1, and conformity to RO5. The new compound, therefore, has great potential as a new lead antimalarial agent.

Antiviral activity of self-assembled glycodendro[60]fullerene monoadducts.

A series of amphiphilic glycodendro[60]fullerene monoadducts were efficiently synthesized using the CuAAC "click chemistry" approach. These glycodendrofullerenes can self-assemble in aqueous media, in a process favoured through π-π interactions between the [60]fullerene moieties. This aggregation process leads to big and well-defined compact micelles with a uniform size and spherical-shape. The supramolecular aggregates were characterized using electronic microscopy (SEM and TEM), light scattering methods (DLS) and X-ray methodologies (SAXS and XRD). The antiviral efficiency of these aggregates has been tested in an experimental infection assay using Ebola virus glycoprotein (EboGP) pseudotyped viral particles on Jurkat cells overexpressing DC-SIGN and an improvement in the IC50 value with respect to other systems endowed with a higher number of carbohydrate ligands is observed.

Preparation of succinylated cellulose membranes for functionalization purposes.

The anhydroglucose chains of cellulose possess hydroxyls that facilitate different chemical modification strategies to expand on, or provide new applications for membranes produced by the bacteria Gluconacetobacter xylinus. Conjugation with biomolecules such as proteins, especially by the amine groups, is of great value and interest for the production of biomaterial derivatives from bacterial cellulose. To assist in these modifications, cellulose was succinylated in order to prevent steric hindrance and to create an attachment point for conjugation. Bacterial cellulose membranes were first treated in dichloromethane and reacted with succinic anhydride through a series of conditions. The membrane structure remained intact after these first processes and the product was confirmed by Infra-Red spectroscopy and solid state nuclear magnetic resonance and characterized by X-ray diffraction, thermogravimetry and atomic force microscopy. Hydrolyzed collagen was used as a model protein of interest to be conjugated to these membranes, which furnished a biomaterial functionalized over its surface.

Revealing the Binding Process of New 3-Alkylpyridine Marine Alkaloid Analogue Antimalarials and the Heme Group: An Experimental and Theoretical Investigation.

Synthetic 3-alkylpyridine marine alkaloid (3-APA) analogues have shown good antimalarial activity against Plasmodium falciparum. However, despite their structural originality, their molecular target was unknown. Herein, we report a proposal for the antimalarial mechanism of action of 3-APA analogues through interference with the process of hemozoin (Hz) formation. The interaction between 3-APA analogues and heme groups was investigated employing an in silico approach and biophysical techniques such as ultraviolet-visible light (UV-vis) titration and electrospray ionization-mass spectrometry (ESI-MS). The in silico approach was performed based on pure ab initio electronic structure methods in order to obtain insights at the molecular level concerning the binding process of antimalarial drugs at their target site, the heme group. In silico results showed that the formation of heme:3-APA complexes at a molecular ratio of 2:1 are more stable than 1:1 complexes. These results were further confirmed by experimental techniques, such as UV-vis and high-resolution mass spectrometry (ESI-TOF), for two of the most active 3-APA analogues.

Investigation of glycofullerene dynamics by NMR spectroscopy.

Glycofullerenes, in which carbohydrate molecules are attached via a linker to a [60]fullerene core, facilitate spherical presentation of glyco-based epitopes. We herein investigate the dynamics of two glycofullerenes, having 12 and 36 mannose residues at their periphery, by NMR translational diffusion and quantitative (13)C relaxation studies employing a model-free approach for their interpretation. The sugar residues are shown to be highly flexible entities with S(2) < 0.2 in both compounds. Notably, the larger glycofullerene with longer linkers shows faster internal dynamics and higher flexibility than its smaller counterpart. The dynamics and flexibility as well as the slower translational diffusion of the larger glycofullerene, thereby favoring rebinding to a receptor, may together with its spatial extension explain why it is better than the smaller one at blocking the DC-SIGN receptor and inhibiting the infection by pseudotyped Ebola virus particles.

Human dendritic cell activation induced by a permannosylated dendron containing an antigenic GM3-lactone mimetic.

Vaccination strategies based on dendritic cells (DCs) armed with specific tumor antigens have been widely exploited due the properties of these immune cells in coordinating an innate and adaptive response. Here, we describe the convergent synthesis of the bifunctional multivalent glycodendron 5, which contains nine residues of mannose for DC targeting and one residue of an immunogenic mimetic of a carbohydrate melanoma associated antigen. The immunological assays demonstrated that the glycodendron 5 is able to induce human immature DC activation in terms of a phenotype expression of co-stimulatory molecules expression and MHCII. Furthermore, DCs activated by the glycodendron 5 stimulate T lymphocytes to proliferate in a mixed lymphocytes reaction (MLR).

A multivalent inhibitor of the DC-SIGN dependent uptake of HIV-1 and Dengue virus.

DC-SIGN is a C-type lectin receptor on antigen presenting cells (dendritic cells) which has an important role in some viral infection, notably by HIV and Dengue virus (DV). Multivalent presentation of carbohydrates on dendrimeric scaffolds has been shown to inhibit DC-SIGN binding to HIV envelope glycoprotein gp120, thus blocking viral entry. This approach has interesting potential applications for infection prophylaxis. In an effort to develop high affinity inhibitors of DC-SIGN mediated viral entry, we have synthesized a group of glycodendrimers of different valency that bear different carbohydrates or glycomimetic DC-SIGN ligands and have studied their DC-SIGN binding activity and antiviral properties both in an HIV and a Dengue infection model. Surface Plasmon Resonance (SPR) competition studies have demonstrated that the materials obtained bind efficiently to DC-SIGN with IC50s in the µm range, which depend on the nature of the ligand and on the valency of the scaffold. In particular, a hexavalent presentation of the DC-SIGN selective antagonist 4 displayed high potency, as well as improved accessibility and chemical stability relative to previously reported dendrimers. At low µm concentration the material was shown to block both DC-SIGN mediated uptake of DV by Raji cells and HIV trans-infection of T cells.

Structure of a glycomimetic ligand in the carbohydrate recognition domain of C-type lectin DC-SIGN. Structural requirements for selectivity and ligand design.

In genital mucosa, different fates are described for HIV according to the subtype of dendritic cells (DCs) involved in its recognition. This notably depends on the C-type lectin receptor, langerin or DC-SIGN, involved in gp120 interaction. Langerin blocks HIV transmission by its internalization in specific organelles of Langerhans cells. On the contrary, DC-SIGN enhances HIV trans-infection of T lymphocytes. Thus, approaches aiming to inhibit DC-SIGN, without blocking langerin, represent attractive anti-HIV strategies. We previously demonstrated that dendrons bearing multiple copies of glycomimetic compounds were able to block DC-SIGN-dependent HIV infection in cervical explant models. Optimization of such ligand requires detailed characterization of its binding mode. In the present work, we determined the first high-resolution structure of a glycomimetic/DC-SIGN complex by X-ray crystallography. This glycomimetic, pseudo-1,2-mannobioside, shares shape and conformational properties with Manα1-2Man, its natural counterpart. However, it uses the binding epitope previously described for Lewis X, a ligand specific for DC-SIGN among the C-type lectin family. Thus, selectivity gain for DC-SIGN versus langerin is observed with pseudo-1,2-mannobioside as shown by surface plasmon resonance analysis. In parallel, ligand binding was also analyzed by TR-NOESY and STD NMR experiments, combined with the CORCEMA-ST protocol. These studies demonstrate that the complex, defined by X-ray crystallography, represents the unique binding mode of this ligand as opposed to the several binding orientations described for the natural ligand. This exclusive binding mode and its selective interaction properties position this glycomimetic as a good lead compound for rational improvement based on a structurally driven approach.

Glycofullerenes inhibit viral infection.

Water-soluble glycofullerenes based on a hexakis-adduct of [60]fullerene with an octahedral addition pattern are very attractive compounds providing a spherical presentation of carbohydrates. These tools have been recently described and they have been used to interact with lectins in a multivalent manner. Here, we present the use of these glycofullerenes, including new members with 36 mannoses, as compounds able to inhibit a DC-SIGN-dependent cell infection by pseudotyped viral particles. The results obtained in these experiments demonstrate for the first time that these glycoconjugates are adequate to inhibit efficiently an infection process, and therefore, they can be considered as very promising and interesting tools to interfere in biological events where lectins such as DC-SIGN are involved.

Virus-like glycodendrinanoparticles displaying quasi-equivalent nested polyvalency upon glycoprotein platforms potently block viral infection.

Ligand polyvalency is a powerful modulator of protein-receptor interactions. Host-pathogen infection interactions are often mediated by glycan ligand-protein interactions, yet its interrogation with very high copy number ligands has been limited to heterogenous systems. Here we report that through the use of nested layers of multivalency we are able to assemble the most highly valent glycodendrimeric constructs yet seen (bearing up to 1,620 glycans). These constructs are pure and well-defined single entities that at diameters of up to 32 nm are capable of mimicking pathogens both in size and in their highly glycosylated surfaces. Through this mimicry these glyco-dendri-protein-nano-particles are capable of blocking (at picomolar concentrations) a model of the infection of T-lymphocytes and human dendritic cells by Ebola virus. The high associated polyvalency effects (ß>10(6), ß/N ~10(2)-10(3)) displayed on an unprecedented surface area by precise clusters suggest a general strategy for modulation of such interactions.

Targeting mannose-binding lectin confers long-lasting protection with a surprisingly wide therapeutic window in cerebral ischemia.

BACKGROUND: The involvement of the complement system in brain injury has been scarcely investigated. Here, we document the pivotal role of mannose-binding lectin (MBL), one of the recognition molecules of the lectin complement pathway, in brain ischemic injury. METHODS AND RESULTS: Focal cerebral ischemia was induced in mice (by permanent or transient middle cerebral artery occlusion) and rats (by 3-vessel occlusion). We first observed that MBL is deposited on ischemic vessels up to 48 hours after injury and that functional MBL/MBL-associated serine protease 2 complexes are increased. Next, we demonstrated that (1) MBL(-/-) mice are protected from both transient and permanent ischemic injury; (2) Polyman2, the newly synthesized mannosylated molecule selected for its binding to MBL, improves neurological deficits and infarct volume when given up to 24 hours after ischemia in mice; (3) anti-MBL-A antibody improves neurological deficits and infarct volume when given up to 18 hours after ischemia, as assessed after 28 days in rats. CONCLUSIONS: Our data show an important role for MBL in the pathogenesis of brain ischemic injury and provide a strong support to the concept that MBL inhibition may be a relevant therapeutic target in humans, one with a wide therapeutic window of application.

BODIPY-labeled DC-SIGN-targeting glycodendrons efficiently internalize and route to lysosomes in human dendritic cells.

Glycodendrons bearing nine copies of mannoses or fucoses have been prepared by an efficient convergent strategy based on Cu(I) catalyzed azide-alkyne cycloaddition (CuAAC). These glycodendrons present a well-defined structure and have an adequate size and shape to interact efficiently with the C-type lectin DC-SIGN. We have selected a BODIPY derivative to label these glycodendrons due to its interesting physical and chemical properties as chromophore. These BODIPY-labeled glycodendrons were internalized into dendritic cells by mean of DC-SIGN. The internalized mannosylated and fucosylated dendrons are colocalized with LAMP1, which suggests routing to lysosomes. The interaction of these glycodendrons with DC-SIGN at the surface of dendritic cells did not induce maturation of the cells. Signaling analysis by checking different cytokines indicated also the lack of induction the expression of inflammatory and noninflammatory cytokines by these second generation glycodendrons.