Efficient heterogeneous synthesis of nucleophilic carboxymethyl hydrazides of polysaccharides.

Nucleophilic moieties in polysaccharides (PS) with distinct higher reactivity compared with the hydroxy group are interesting for sustainable applications in chemistry, medicine, and pharmacy. An efficient heterogeneous method for the formation of such nucleophilic PS is described. Employing alcohols as slurry medium, protonated carboxymethyl (CM) PS and hydrazine hydrate are allowed to react at elevated temperatures. The CM derivatives of starch and pullulan can be transformed almost quantitatively to the corresponding hydrazides. The reaction is less efficient for CM dextrans and CM xylans. As slurry media, 2-propanol and ethanol were probed, and the results are compared with a homogeneous procedure performed in water. Overall, the heterogeneous procedure is superior compared with the homogeneous route. 2-Propanol is the best slurry medium investigated yielding PS hydrazides with the highest nitrogen content.

High-Solids, Solvent-Free Modification of Engineered Polysaccharides.

The nature-identical engineered polysaccharide α-(1,3) glucan, produced by the enzymatic polymerization of sucrose, was chemically modified by acylation with succinic anhydride. This modification reaction was initially performed at the micro scale in a TGA reactor to access a range of reaction conditions and to study the mechanism of the reaction. Subsequently, the best performing conditions were reproduced at the larger laboratory scale. The reaction products were characterized via coupled TGA/DSC analysis, FT-IR spectroscopy, solution viscosity and pH determination. The acylation path resulted in partially modifying the polysaccharide by altering its behavior in terms of thermal properties and solubility. The acylation in a solvent-free approach was found promising for the development of novel, potentially melt-processable and fully bio-based and biodegradable ester compounds.

Solubilization of poorly water-soluble bioactive molecules in neutral aqueous media by complexation with renatured ß-1,3-1,6-glucan nanoparticles.

The design of scaffolds for solubilizing/dispersing poorly water-soluble bioactive molecules in neutral aqueous media is a major challenge of functional food, pharmaceuticals, and cosmetics development, as highlighted by the plethora of corresponding solubilization/dispersion strategies. Herein, renatured ß-1,3-1,6-glucan (r-glucan) nanoparticles prepared by neutralization of alkali-denatured ß-1,3-1,6-glucan and subsequent centrifugation are used as a host to disperse water-insoluble bioactive molecules (curcumin, all-trans-retinoic acid, and rebamipide) by simple mixing of host and guest solutions. Curcumin in the r-glucan cavity is found to be stacked in the form of J-aggregates and twisted along the helix, and is demonstrated to be retained for significantly longer than curcumin in the corresponding γ-cyclodextrin (γ-CD) complex. Specifically, curcumin incorporated in γ-CD is released within 5.5 hours, whereas that in the r-glucan complex is released very slowly, with 12% of curcumin in the latter complex retained after 31-day incubation at 37°C. Thus, inclusion protocol simplicity and slow release ability make r-glucan nanoparticles a potential carrier scaffold for various applications.

Safe-by-Design of Glucan Nanoparticles: Size Matters When Assessing the Immunotoxicity.

Glucan (from Alcaligenes faecalis) is a polymer composed of ß-1,3-linked glucose residues, and it has been addressed in different medical fields, namely in nanotechnology, as a vaccine or a drug delivery system. However, due to their small size, nanomaterials may present new risks and uncertainties. Thus, this work aims to describe the production of glucan nanoparticles (NPs) with two different sizes, and to evaluate the influence of the NPs size on immunotoxicity. Results showed that, immediately after production, glucan NPs presented average sizes of 129.7 ± 2.5 and 355.4 ± 41.0 nm. Glucan NPs of 130 nm presented greater ability to decrease human peripheral blood mononuclear cells and macrophage viability and to induce reactive oxygen species production than glucan NPs of 355 nm. Both NP sizes caused hemolysis and induced a higher metabolic activity in lymphocytes, although the concentration required to observe such effect was lower for the 130 nm glucan NPs. Regarding pro-inflammatory cytokines, only the larger glucan NPs (355 nm) were able to induce the secretion of IL-6 and TNF-α, probably due to their recognition by dectin-1. This higher immunomodulatory effect of the larger NPs was also observed in its ability to stimulate the production of nitric oxide (NO) and IL-1ß. On the contrary, a small amount of Glu 130 NPs inhibited NO production. In conclusion, on the safe-by-design of glucan NPs, the size of the particles should be an important critical quality attribute to guarantee the safety and effectiveness of the nanomedicine.

Reagent controlled stereoselective synthesis of teichoic acid α-(1,2)-glucans.

The stereoselective construction of 1,2-cis-glycosidic linkages is key in the assembly of biologically relevant glycans, but remains a synthetic challenge. Reagent-controlled glycosylation methodologies, in which external nucleophiles are employed to modulate the reactivity of the glycosylation system, have become powerful means for the construction of 1,2-cis-glycosidic linkages. Here we establish that nucleophilic additives can support the construction of α-1,2-glucans, and apply our findings in the construction of a d-alanine kojibiose functionalized glycerol phosphate teichoic acid fragment. This latter molecule can be found in the cell wall of the opportunistic Gram-positive bacterium, Enterococcus faecalis and represents a structural element that can possibly be used in the development of therapeutic vaccines and diagnostic tools.

Comparative Studies on Regioselectivity of α- and ß-Linked Glucan Tosylation.

Alpha- and beta-linked 1,3-glucans have been subjected to conversion with p-toluenesulfonic acid (tosyl) chloride and triethylamine under homogeneous reaction conditions in N,N-dimethyl acetamide/LiCl. Samples with a degree of substitution of tosyl groups (DSTs) of up to 1.91 were prepared by applying 5 mol reagent per mole repeating unit. Hence, the reactivity of α-1,3-glucan is comparable with cellulose and starch, while the ß-1,3-linked glucan curdlan is less reactive. The samples dissolve in aprotic dipolar media independent of the DSTs and possess a solubility in less polar solvents that depends on the DSTs. NMR studies on the tosyl glucans and of the peracylated derivatives showed a preferred tosylation of position 2 of the repeating unit. However, the selectivity is less pronounced compared with starch. It could be concluded that the α-configurated glycosidic bond directs tosyl groups towards position 2.

Reagent Controlled Stereoselective Synthesis of α-Glucans.

The development of a general glycosylation method that allows for the stereoselective construction of glycosidic linkages is a tremendous challenge. Because of the differences in steric and electronic properties of the building blocks used, the outcome of a glycosylation reaction can vary greatly when switching form one glycosyl donor-acceptor pair to another. We here report a strategy to install cis-glucosidic linkages in a fully stereoselective fashion that is under direct control of the reagents used to activate a single type of donor building block. The activating reagents are tuned to the intrinsic reactivity of the acceptor alcohol to match the reactivity of the glycosylating agent with the reactivity of the incoming nucleophile. A protecting group strategy is introduced that is based on the sole use of benzyl-ether type protecting groups to circumvent changes in reactivity as a result of the protecting groups. For the stereoselective construction of the α-glucosyl linkages to a secondary alcohol, a per-benzylated glusosyl imidate donor is activated with a combination of trimethylsilyltriflate and DMF, while activation of the same imidate donor with trimethylsilyl iodide in the presence of triphenylphosphine oxide allows for the stereoselective cis-glucosylation of primary alcohols. The effectiveness of the strategy is illustrated in the modular synthesis of a Mycobacterium tuberculosis nonasaccharide, composed of an α-(1-4)-oligoglucose backbone bearing different α-glucosyl branches.

Formulation, Characterization, and Optimization of Captopril Fast-Dissolving Oral Films.

This work aimed to using optimization study to formulate a patient-friendly captopril fast-dissolving oral film with satisfactory disintegration time. Films were made with pullulan and hydroxypropyl methyl cellulose (HPMC) by using the solvent-casting method. Cellulose nanofiber (CNF) was used as a compatibilizer and glycerine was used as a plasticizer. In order to find an optimum formulation, a response surface methodology and a central composite design were employed. The concentration percentages of pullulan and glycerine were considered to be the design factors. Disintegration time, tensile strength, percent elongation at break, and folding endurance were considered to be the responses. The results showed that CNF improved the compatibility and tensile strength of the pullulan and HPMC blend. Also, the rigid nature of CNF reduced the film elongation but the addition of glycerine improved its flexibility. All formulations showed an acceptable uniformity content and dissolution rate. Complete dissolution for all formulations occurred within 2 min. Films with 26% pullulan, 74% HPMC, 1% CNF, and 5% glycerine were reported to be optimum formulations for captopril fast-dissolving oral films, with 95% confidence levels. The in vivo comparison of optimized formulation with a conventional captopril sublingual tablet exhibited significant increase in AUC (~ 62%) and Cmax (~ 52%) and a major decrease in Tmax (~ 33%). The overall results showed that the captopril FDF is a promising candidate for enhanced in vivo orotransmucosal absorption.

Chemical Synthesis of Sulfated Yeast (Saccharomyces cerevisiae) Glucans and Their In Vivo Antioxidant Activity.

The effects of sulfation of yeast glucans was optimized using response surface methodology. The degree of sulfation was evaluated from 0.11 to 0.75 using ion-chromatography. The structural characteristics of SYG (sulfation of yeast glucans) with a DS = 0.75 were determined using high-performance liquid chromatography/gel-permeation chromatography and finally by Fourier transform infrared spectrometry. The SYG had lower viscosity and greater solubility than the native yeast glucans, suggesting that the conformation of the SYG had significantly changed. The results also showed that SYG had a significantly greater antioxidant activity in vivo compared to native yeast glucans.

Automated glycan assembly of xyloglucan oligosaccharides.

We report the automated glycan assembly of oligosaccharide fragments related to the hemicellulose xyloglucan (XG). Iterative addition of monosaccharide and disaccharide building blocks to a solid support provided seven cellulose and xyloglucan fragments including XXGG- and XXXG-type oligosaccharides.

Hydrogen bond mediated aglycone delivery: synthesis of linear and branched α-glucans.

A Hydrogen bond mediated aglycone delivery (HAD) method was applied to the synthesis of α-glucans, which are abundant in nature, but as targets represent a notable challenge to chemists. The synthesis of linear oligosaccharide sequences was accomplished in complete stereoselectivity in all glycosylations. The efficacy of HAD may diminish with the increased bulk of the glycosyl acceptor, and may be an important factor for the syntheses of oligomers beyond pentasaccharides. The synthesis of a branched structure proved more challenging, particularly with bulky trisaccharide acceptors.

In vitro synthesis of hyperbranched α-glucans using a biomimetic enzymatic toolbox.

Glycogen biosynthesis requires the coordinated action of elongating and branching enzymes, of which the synergetic action is still not clearly understood. We have designed an experimental plan to develop and fully exploit a biomimetic system reproducing in vitro the activities involved in the formation of α(1,4) and α(1,6) glycosidic linkages during glycogen biosynthesis. This method is based on the use of two bacterial transglucosidases, the amylosucrase from Neisseria polysaccharea and the branching enzyme from Rhodothermus obamensis . The α-glucans synthesized from sucrose, a low cost agroresource, by the tandem action of the two enzymes, have been characterized by using complementary enzymatic, chromatographic, and imaging techniques. In a single step, linear and branched α-glucans were obtained, whose proportions, morphology, molar mass, and branching degree depended on both the initial sucrose concentration and the ratio between elongating and branching enzymes. In particular, spherical hyperbranched α-glucans with a controlled mean diameter (ranging from 10 to 150 nm), branching degree (from 10 to 13%), and weight-average molar mass (3.7 × 10(6) to 4.4 × 10(7) g.mol(-1)) were synthesized. Despite their structure, which is similar to that of natural glycogens, the mechanisms involved in their in vitro synthesis appeared to be different from those involved in the biosynthesis of native hyperbranched α-glucans.

Determination of glucan phosphorylation using heteronuclear 1H, 13C double and 1H, 13C, 31P triple-resonance NMR spectra.

Phosphorylation and dephosphorylation of starch and glycogen are important for their physicochemical properties and also their physiological functions. It is therefore desirable to reliably determine the phosphorylation sites. Heteronuclear multidimensional NMR-spectroscopy is in principle a straightforward analytical approach even for complex carbohydrate molecules. With heterogeneous samples from natural sources, however, the task becomes more difficult because a full assignment of the resonances of the carbohydrates is impossible to obtain. Here, we show that the combination of heteronuclear (1) H,(13) C and (1) H,(13) C,(31) P techniques and information derived from spectra of a set of reference compounds can lead to an unambiguous determination of the phosphorylation sites even in heterogeneous samples.

Structure-activity relationship study of WSS25 derivatives with anti-angiogenesis effects.

WGEW, an α(1-4) linked glucan with an α(1-4) linked branch attached to C-6, was isolated from the rhizoma of Gastrodia elata Bl. WSS25, a sulfated derivative of WGEW, was reported to inhibit angiogenesis by disrupting BMP2/Smad/Id1 signaling pathway. However, the structure-activity relationship (SAR) for WSS25 is not known. To study the SAR, seven sulfated saccharides derived from WGEW degradation products, six sulfated polysaccharides with varying degrees of substitution, and four aminopropylated, carboxymethylated, phosphorylated, and acetylated derivatives of WGEW were prepared. A sulfated, unbranched product of polysaccharide was also obtained. The structural features of these derivatives were characterized by infrared spectroscopy and nuclear magnetic resonance spectroscopy. An HMEC-1 cell tube formation assay was employed to measure the antiangiogenic effect of the derivatives. The results indicated that only sulfated polysaccharides with molecular weights of more than 41,000 Da could inhibit HMEC-1 cell tube formation. The inhibition effect was dependent on the presence of a sulfate group, since the tube formation was not blocked by aminopropylated, carboxymethylated, phosphorylated, or acetylated WGEW. A higher degree of sulfate substitution on the polysaccharide led to a stronger inhibitory effect, and the degree of sulfate substitution between 0.173 and 0.194 was found to be optimal. Interestingly, the WGEW side chain was not required for anti-tube formation activity. All these preliminary results may provide a clue for further modification of the core structure of WSS25 to discover polysaccharide derivatives as novel anti-angiogenic inhibitors.

[Synthesis of low molecular weight mimetics of heparin].

Natural hexosaminoglycan heparin remains the most commonly prescribed anticoagulant in hospitalized patients. However its administration could induce side clinical events, including thrombocytopenia and bleeding. This explaines the need of development of alternative anticoagulant drugs based on modified heparin and polyanionic oligo- and polysaccharide derivatives, such as sulfated glucans, phosphomannans and fucoidans. Here we review the works on the synthesis of oligosaccharides related to low molecular weight hepain fragments and their derivatives, as well as oligosaccharides, which imitate parts of heparin chain responsible for biological activity. These works were aimed to develop the pharmaceutical preparations lacking ofheparin disadvantages.

ZnI2-Directed Stereocontrolled α-Glucosylation.

Here we report a glucosylation strategy mediated by ZnI2, a cheap and mild Lewis acid, for the highly stereoselective construction of 1,2-cis-O-glycosidic linkages using easily accessible and common 4,6-O-tethered glucosyl donors. The versatility and effectiveness of the α-glucosylation strategy were demonstrated successfully with various acceptors, including complex alcohols. This approach demonstrates the feasibility of the modular synthesis of various α-glucans with both linear and branched backbone structures.

A novel maltoheptaose-based sugar ester having excellent emulsifying properties and optimization of its lipase-catalyzed synthesis.

A novel maltoheptaose-palmitate ester (G7-PA) was synthesized and investigated for emulsion properties. First of all, the optimal conditions for lipase-catalyzed G7-PA synthesis, which were 0.2 of the G7/PA molar ratio, 33.5 U of immobilized CALB per 1 g of PA in 10% DMSO, were determined by response surface methodology. G7-PA was compared with the commercial sucrose-PA (S-PA) in terms of emulsion-forming ability and stability at extreme conditions. At the 0.1% surfactant concentration, G7-PA emulsion exhibited a droplet distribution similar to the 0.2% surfactant condition, while S-PA emulsion was quickly destabilized. G7-PA showed better emulsifying properties than the S-PA at the acidic condition (pH 3). Flocculation and phase separation was observed at the S-PA emulsion, but the G7-PA emulsion was stable for 7-day. In thermostability tests, G7-PA and S-PA both were stable up to the boiling temperature. Conclusively, G7-PA exhibits excellent properties as a biosurfactant in O/W emulsion compared with S-PA.

Pullulan films loading saffron extract encapsulated in nanoliposomes; preparation and characterization.

Nanoencapsulation of saffron extract (SE) components into the rapeseed lecithin nanoliposomes were performed by sonication of their aqueous dispersions as a green process. Dynamic light scattering (DLS) results exhibited that empty and SE loaded nanoliposomes (SENL) had average sizes in range of 118-138 nm, negative zeta potentials (-32.0 to -46.8 mV) and polydispersity index (PDI) less than 0.3 during storage for 28 days at 4 °C. Encapsulation efficiency of crocin was approximately 30%. The 70% of crocin released from SENLs within 5 h in PBS solution. Pullulan-based films were fabricated by incorporation of empty and SE loaded nanoliposomes into pullulan solution through casting method. The mechanical resistance and thermal stability of the films reduced by addition of nanoliposomes. FTIR and thermal characterizations indicated that SE was successfully encapsulated in the nanoliposomes and film matrix with high thermal stability. Incorporation of nanoliposomes enhanced the oxygen barrier properties of the films, while it didn't significantly affect the water vapor permeability (WVP) of the films. The obtained edible films or coatings can provide additional benefits due to unique flavor and color of saffron. In addition, the utilization of SE, can provide benefits for health-allegation from SE antioxidant capacity.

Chitotriazolan (poly(ß(1-4)-2-(1H-1,2,3-triazol-1-yl)-2-deoxy-d-glucose)) derivatives: Synthesis, characterization, and evaluation of antibacterial activity.

Here we describe the first synthesis of a new type of polysaccharides derived from chitosan. In these structures, the 2-amino group on the pyranose ring was quantitively replaced by an aromatic 1,2,3-triazole moiety. The 2-amino group of chitosan and di-TBDMS chitosan was converted into an azide by diazo transfer reaction. The chitosan azide and TBDMS-chitosan azide were poorly soluble but could be fully converted to triazoles by "copper-catalysed Huisgen cycloaddition" in DMF or DMSO. The reaction could be done with different alkynes but derivatives lacking cationic or anionic groups were poorly soluble or insoluble in tested aqueous and organic solvents. Derivatives with N,N-dimethylaminomethyl, N,N,N-trimethylammoniummethyl, sulfonmethyl, and phosphomethyl groups linked to the 4-position of the triazole moiety were soluble in water at neutral or basic conditions and could be analyzed by 1H, 13C APT, COSY, and HSQC NMR. The quaternized cationic chitotriazolan's had high activity against S. aureus and E. coli, whereas the anionic chitotriazolan's lacked activity.

Etherified pullulan-polyethylenimine based nanoscaffolds improved chemosensitivity of erlotinib on hypoxic cancer cells.

The current research endeavor aimed to accomplish hypoxia-responsive polyethyleneimine-conjugated carboxymethyl pullulan-based co-polymer (CMP-HA-NI-PEI-NBA) bearing nitroaromatic subunits to efficiently deliver erlotinib (ERL) to reverse its hypoxia-induced resistance in cancer cells. As compared to a control co-polymer (CMP-HA-MI-PEI-BA) devoid of hypoxia-sensitive moieties, this scaffold demonstrated a hypochromic shift in the UV spectra and rapid dismantling of its self-assembled architecture upon exposure to simulated hypoxic condition. The hypoxia-responsive co-polymer encapsulated ERL with desirable loading capacity (DEE, 63.05 ± 2.59%), causing attenuated drug crystallinity. The drug release rate of the scaffold under reducing condition was faster relative to that of non-reducing environment. Their cellular uptake occurred through an energy-dependent endocytic process, which could exploit its caveolae/lipid raft-mediated internalization pathway. The ERL-loaded scaffolds more efficiently induced apoptosis and suppressed the proliferation of drug-resistant hypoxic HeLa cells than the pristine ERL. Hence, this study presented a promising drug delivery nanoplatform to overcome hypoxia-evoked ERL resistance.