Exploiting specific properties of squid pens for the preparation of oligochitosan hydrochloride.

Herein, we describe in first the application of squid pens for the preparation of pharmaceutical-grade oligochitosan hydrochloride with the physicochemical characteristics corresponding with the requirements of the European Pharmacopoeia. It is shown that the use of specific properties of squid pens as a source of parent chitosan allows preparing the product with a high yield at relatively moderate process conditions used for squid pens treatments and chitosan depolymerization.

Preparation of natural high-mannose-type oligosaccharides (Glc1Man9GlcNAc2) with the asparagine-glycine-threonine as consensus sequence from chicken egg yolk.

High-mannose-type glycan structure of N-glycoproteins plays important roles in the proper folding of proteins in sorting glycoprotein secretion and degradation of misfolded proteins in the endoplasmic reticulum (ER). The Glc1Man9GlcNAc2 (G1M9)-type N-glycan is one of the most important signaling molecules in the ER. However, current chemical synthesis strategies are laborious, warranting more practical approaches for G1M9-glycopeptide development. Wang et al. reported the procedure to give G1M9-Asn-Fmoc through chemical modifications and purifications from 40 chicken eggs, but only 3.3 mg of G1M9-glycopeptide was obtained. Therefore, better methods are needed to obtain more than 10 mg of G1M9-glycopeptide. In this study, we report the preparation of G1M9-glycopeptide (13.2 mg) linking Asn-Gly-Thr triad as consensus sequence from 40 chicken eggs. In this procedure, λ-carrageenan treatment followed by papain treatment was used to separate the Fc region of IgY antibody that harbors high-mannose glycans. Moreover, cotton hydrophilic interaction liquid chromatography was adapted for easy purification. The resulting G1M9-Asn(Fmoc)-Gly-Thr was identified by nuclear magnetic resonance and mass spectroscopy. G1M9-Asn(Fmoc)-Gly, G1M9-Asn(Fmoc), and G1M9-OH were also detected by mass spectroscopy. Here, our developed G1M9-tripeptide might be useful for the elucidation of glycoprotein functions as well as the specific roles of the consensus sequence.

Analogues of the pan-selectin antagonist rivipansel (GMI-1070).

The selectin family consisting of E-, P- and L-selectin plays dominant roles in atherosclerosis, ischemia-reperfusion injury, inflammatory diseases, and metastatic spreading of some cancers. An early goal in selectin-targeted drug discovery campaigns was to identify ligands binding to all three selectins, so-called pan-selectin antagonists. The physiological epitope, tetrasaccharide sialyl Lewisx (sLex, 1) binds to all selectins, albeit with very different affinities. Whereas P- and L-selectin require additional interactions contributed by sulfate groups for high binding affinity, E-selectin can functionally bind sLex-modified glycolipids and glycoproteins. Rivipansel (3) marked the first pan-selectin antagonist, which simultaneously interacted with both the sLex and the sulfate binding site. The aim of this contribution was to improve the pan-selectin affinity of rivipansel (3) by leveraging a new class of sLex mimetics in combination with an optimized linker length to the sulfate bearing group. As a result, the pan-selectin antagonist 11b exhibits an approximatively 5-fold improved affinity for E-, as well as P-selectin.

Total Synthesis of the Conjugation-Ready Tetrasaccharide Repeating Unit of Shewanella japonica Type Strain KMM 3299T.

Here we report the first total synthesis of the conjugation-ready tetrasaccharide repeating unit of Shewanella japonica type strain KMM 3299T. The presence of rare deoxyamino sugars and installation of three consecutive 1,2-cis glycosidic linkages makes the synthesis formidable. The challenging late-stage oxidation was overcome by using a galacturonate donor. The total synthesis was completed via a longest linear sequence of 22 steps in an overall yield of 3.5% starting from d-mannose.

Chemical Synthesis of Fucosylated Chondroitin Sulfate Tetrasaccharide with Fucosyl Branch at the 6-OH of GalNAc Residue.

Fucosylated chondroitin sulfate is a unique glycosaminoglycan isolated from sea cucumbers, with excellent anticoagulant activity. The fucosyl branch in FCS is generally located at the 3-OH of D-glucuronic acid but, recently, a novel structure with α-L-fucose linked to the 6-OH of N-acetyl-galactosamine has been found. Here, using functionalized monosaccharide building blocks, we prepared novel FCS tetrasaccharides with fucosyl branches both at the 6-OH of GalNAc and 3-OH of GlcA. In the synthesis, the protective group strategy of selective O-sulfation, as well as stereoselective glycosylation, was established, which enabled the efficient synthesis of the specific tetrasaccharide compounds. This research enriches knowledge on the structural types of FCS oligosaccharides and facilitates the exploration of the structure-activity relationship in the future.

Synthesis and Structural Revision of a Natural Tetrasaccharide from Starfish Asterias rollestoni Bell.

Starfish provide important saponins with diverse bioactivities as the secondary metabolites, among which 2-O-glycosylated glycosides are commonly found. Preparation of those 1,2-trans 2-O-glycosylated glycosides usually relies on 2-O-acyl participation requiring the selective installation and cleavage of 2-O-acyl groups. A convergent synthesis using 2-O-glycosylated oligosaccharide donors would be more straightforward but also pose greater challenges. Herein, we report a convergent synthesis of a distinctive tetrasaccharide isolated from starfish Asterias rollestoni Bell. Dual 2-(diphenylphosphinoyl)acetyl (DPPA) groups at O3 and O4 on galactose moiety led to high ß-selectivities (ß/α=12/1 or ß only) in the challenging [2+2] glycosylation, giving the desired tetrasaccharides in >90 % yields from the 2-O-glycosylated disaccharide donors. These synthetic studies have also unambiguously revised the structure of these natural tetrasaccharides. This work would facilitate further studies on new inhibitors of α-glucosidase as hypoglycemic drugs.

Stereoselective gold(I)-catalyzed approach to the synthesis of complex α-glycosyl phosphosaccharides.

Glycosyl phosphosaccharides represent a large and important family of complex glycans. Due to the distinct nature of these complex molecules, efficient approaches to access glycosyl phosphosaccharides are still in great demand. Here, we disclose a highly efficient and stereoselective approach to the synthesis of biologically important and complex α-glycosyl phosphosaccharides, employing direct gold(I)-catalyzed glycosylation of the weakly nucleophilic phosphoric acid acceptors. In this work, the broad substrate scope is demonstrated with more than 45 examples, including glucose, xylose, glucuronate, galactose, mannose, rhamnose, fucose, 2-N3-2-deoxymannose, 2-N3-2-deoxyglucose, 2-N3-2-deoxygalactose and unnatural carbohydrates. Here, we show the glycosyl phosphotriester prepared herein was successfully applied to the one-pot synthesis of a phosphosaccharide from Leishmania donovani, and an effective preparation of a trisaccharide diphosphate of phosphosaccharide fragments from Hansenula capsulate via iterative elongation strategy is realized.

Chemoenzymatic Synthesis of Sialyl Sulfo-Oligosaccharides as Potent Siglec-8 Ligands via Transglycosylation Catalyzed by Keratanase II.

Sialyl type-II sulfo-oligosaccharides are gaining much attention as bioactive ligands for Siglecs. In this study, we have achieved the first synthesis of sialyl type-II sulfo-oligosaccharides chemoenzymatically by utilizing the transglycosylation activity of keratanase II. The oxazoline derivative of α(2→3)-sialylated 6,6'-di-sulfo-LacNAc (3) was newly designed as the glycosyl donor for enzymatic transglycosylation. Keratanase II efficiently catalyzed the transglycosylation of 3 with two kinds of glycosyl acceptors, 6-sulfo-Lewis X and 6,6'-di-sulfo-LacNAc derivatives, providing sialyl sulfo-hexasaccharide (1) and sialyl sulfo-pentasaccharide (2) with 86 and 95% yields, respectively. The products 1 and 2 showed higher affinity to Siglec-8 with KD 70 and 25 µmol·L-1, respectively, compared to the known ligand of the α(2→3)-sialylated 6,6'-di-sulfo-Lewis X with KD 185 µmol·L-1. Thus, this study will advance not only the study of Siglec-8 biology but also the exploration of functions of sialyl sulfo-oligosaccharides having various microstructures.

Preparation, characterization, and bioactivity evaluation of oligosaccharides from Atractylodes lancea (Thunb.) DC.

Sixteen oligosaccharide monomers with the degree of polymerization 3 to 18 (DP 3 to DP 18) and three active fractions (DP 3-9, DP 8-11, and DP 11-17) were separated from Atractylodes lancea (Thunb.) DC. by optimized fast protein liquid chromatography coupled with refractive index detector (FPLC-RID) and preparation hydrophilic interaction chromatography (Pre-HILIC). Gas chromatography-mass spectrometer (GC-MS), liquid chromatography tandem mass spectrometry (LC-MS/MS), nuclear magnetic resonance (NMR) spectroscopy, and methylation analysis showed that the oligosaccharide in A. lancea was 1-kestose [ß-D-fructofuranosyl-(2 â†’ 1)-ß-D-fructofuranosyl-(2 â†’ 1)-α-D-glucopyranoside] (inulin-type fructooligosaccharides, FOS). Particularly, DP 3-9 showed the best capacity in stimulating phagocytic, NO, and cytokines production on RAW264.7 cells than any other purified oligosaccharide monomers and active fractions. It could also activate T-cells in Peyer's patch cells and enhance the production of colony stimulation factors. Besides, FPLC-RID showed a good capacity for large-scale preparation of DP 3-9 with the recovery of more than 93%. The bioactivity of sixteen FOS monomers (DP 3 to DP 18) and three FOS fractions (DP 3-9, DP 8-11, and DP 11-17) investigated in this study are beneficial for the utilization of FOS as a functional ingredient in novel product development.

Synthesis of branched ß-1,3-glucan oligosaccharide with narrow degree of polymerization by fungi co-cultivation.

The large molecular weight and poor water solubility of ß-1,3-glucan impede its potential applications. In this study, the ß-1,3-glucan producing fungi and Trichoderma harzianum capable of secreting endo-ß-1,3-glucanase were co-cultivated to produce branched ß-1,3-glucan oligosaccharides (bOßGs) by fermentation with Sclerotium rolfsii and Schizophyllum commune. The highest bOßG yields from S. rolfsii in flasks were 4.53 and 9.94 g/L in a 7 L fermenter. Structural analysis proved that bOßG from S. rolfsii had a narrow degree of polymerization of 5-12, whereas bOßG from S. commune had a degree of polymerization of 5-15. Antioxidant tests showed that both bOßGs had remarkable DPPH radical scavenging activity and hydroxyl radical scavenging activity, and the activity of bOßG from S. commune was better than that of bOßG from S. rolfsii. In addition, bOßGs could promote the secretion of NO by mouse macrophages and increase the production of TNF-α, IL-1ß, and IL-6 in RAW264.7.

Engineering of a chitosanase fused to a carbohydrate-binding module for continuous production of desirable chitooligosaccharides.

Chitooligosaccharides (CHOS) with multiple biological activities are usually produced through enzymatic hydrolysis of chitosan or chitin. However, purification and recycling of the enzyme have largely limited the advancement of CHOS bioproduction. Here, we engineered a novel enzyme by fusing the native chitosanase Csn75 with a carbohydrate-binding module (CBM) that can specifically bind to curdlan. The recombinase Csn75-CBM was successfully expressed by Pichia pastoris and allowed one-step purification and immobilization in the chitosanase immobilized curdlan packed-bed reactor (CICPR), where a maximum adsorption capacity of 39.59 mg enzyme/g curdlan was achieved. CHOS with degrees of polymerization of 2-5 (a hydrolysis yield of 97.75%), 3-6 (75.45%), and 3-7 (73.2%) were continuously produced by adjusting the ratio of enzyme and chitosan or the flow rate of chitosan. Moreover, the CICPR exhibited good stability and reusability after several cycles. The recombinase Csn75-CBM has greatly improved the efficiency of the bioproduction of CHOS.

Potential applications of alginate oligosaccharides for biomedicine - A mini review.

Extensive research on marine algae, especially on their health-promoting properties, has been conducted. Various ingredients with potential biomedical applications have been discovered and extracted from marine algae. Alginate oligosaccharides are low molecular weight alginate polysaccharides present in cell walls of brown algae. They exhibit various health benefits such as anti-inflammatory, anti-microbial, anti-oxidant, anti-tumor and immunomodulation. Their low-toxicity, non-immunogenicity, and biodegradability make them an excellent material in biomedicine. Alginate oligosaccharides can be chemically or biochemically modified to enhance their biological activity and potential in pharmaceutical applications. This paper provides a brief overview on alginate oligosaccharides characteristics, modification patterns and highlights their vital health promoting properties.

Surface Density of Ligands Controls In-Plane and Aggregative Modes of Multivalent Glycovesicle-Lectin Recognitions.

Glycovesicles are ideal tools to delineate finer mechanisms of the interactions at the biological cell membranes. Multivalency forms the basis which, in turn, should surpass more than one mechanism in order to maintain multiple roles that the ligand-lectin interactions encounter. Ligand densities hold a prime control to attenuate the interactions. In the present study, mannose trisaccharide interacting with a cognate receptor, namely, Con A, is assessed at the vesicle surface. Synthetic (1→3)(1→6)-branched mannose trisaccharides tethered with a diacetylene monomer and glycovesicles of varying sugar densities were prepared. The polydiacetylene vesicles were prepared by maintaining uniform lipid concentrations. The interactions of the glycovesicles with the lectin were probed through dynamic light scattering and UV-Vis spectroscopy techniques. Binding efficacies were assessed by surface plasmon resonance. Aggregative and in-plane modes of interactions show ligand-density dependence at the vesicle surface. Vesicles with sparsely populated ligands engage lectin in an aggregative mode (trans-), leading to a cross-linked complex formation. Whereas glycovesicles embedded with dense ligands engage lectin interaction in an in-plane mode intramolecularly (cis-). Sub-nanomolar dissociation constants govern the intramolecular interaction occurring within the plane of the vesicle, and are more efficacious than the aggregative intermolecular interactions.

Synthesis of 3-O-Sulfated Heparan Sulfate Oligosaccharides Using 3-O-Sulfotransferase Isoform 4.

Heparan sulfate (HS) 3-O-sulfotransferase isoform 4 (3-OST-4) is a specialized carbohydrate sulfotransferase participating in the biosynthesis of heparan sulfate. Here, we report the expression and purification of the recombinant 3-OST-4 enzyme and use it for the synthesis of a library of 3-O-sulfated hexasaccharides and 3-O-sulfated octasaccharides. The unique structural feature of the library is that each oligosaccharide contains a disaccharide domain with a 2-O-sulfated glucuronic acid (GlcA2S) and 3-O-sulfated glucosamine (GlcNS3S). By rearranging the order of the enzymatic modification steps, we demonstrate the synthesis of oligosaccharides with different saccharide sequences. The structural characterization was completed by electrospray ionization mass spectrometry and NMR. These 3-O-sulfated oligosaccharides show weak to very weak anti-Factor Xa activity, a measurement of anticoagulant activity. We discovered that HSoligo 7 (HS oligosaccharide 7), a 3-O-sulfated octasaccharide, binds to high mobility group box 1 protein (HMGB1) and tau protein, both believed to be involved in the process of inflammation. Access to the recombinant 3-OST-4 expands the capability of the chemoenzymatic method to synthesize novel 3-O-sulfated oligosaccharides. The oligosaccharides will become valuable reagents to probe the biological functions of 3-O-sulfated HS and to develop HS-based therapeutic agents.

Agarose oligosaccharide- silver nanoparticle- antimicrobial peptide- composite for wound dressing.

Marine polysaccharides or oligosaccharides have potential to promote wound healing due to their biocompatibility and physicochemical properties. However, microbial infection delays wound healing process, and novel antimicrobial wound dressings are urgently needed. Here, agarose oligosaccharides (AGO) obtained from marine red algae were used as a reducing and stabilizer for green synthesis of silver nanoparticles (AgNPs), and further successfully connected with odorranain A (OA), one of antimicrobial peptides (AMPs), to obtain a novel composite nanomaterial (AGO-AgNPs-OA). Transmission electron microscopy (TEM) and Malvern particle size analyzer showed that AGO-AgNPs-OA was spherical or elliptic with average size of about 100 nm. Circular dichroism (CD) spectroscopy showed that AGO-AgNPs stabilized the α-helical structure of OA. AGO-AgNPs-OA showed stronger anti-bacterial activities than AGO-AgNPs, and had good biocompatibility and significant promoting effect on wound healing. Our data suggest that AMPs conjugated marine oligosaccharides and AgNPs may be effective and safe antibacterial materials for wound therapy.

Synthesis as an Expanding Resource in Human Milk Science.

Few classes of natural products rival the structural audacity of oligosaccharides. Their complexity, however, has stood as an immense roadblock to translational research, as access to homogeneous material from nature is challenging. Thus, while carbohydrates are critical to the myriad functional and structural aspects of the biological sciences, their behavior is largely descriptive. This challenge presents an attractive opportunity for synthetic chemistry, particularly in the area of human milk science. First, there is an inordinate need for synthesizing homogeneous human milk oligosaccharides (HMOs). Superimposed on this goal is the mission of conducting syntheses at scale to enable animal studies. Herein, we present a personalized rumination of our involvement, and that of our colleagues, which has led to the synthesis and characterization of HMOs and mechanistic probes. Along the way, we highlight chemical, chemoenzymatic, and synthetic biology based approaches. We close with a discussion on emergent challenges and opportunities for synthesis, broadly defined, in human milk science.

Insights into structure, affinity, specificity, and function of GAG-protein interactions through the chemoenzymatic preparation of defined sulfated oligohyaluronans.

High amounts of glycosaminoglycans (GAG) such as hyaluronan (HA) occur in connective tissues. There is nowadays increasing evidence that a "sulfation code" exists which mediates numerous GAG functions. High molecular weight and inhomogeneity of GAG, however, aggravated detailed studies. Thus, synthetic oligosaccharides were urgently required. We will review here chemoenzymatic and analytic strategies to provide defined sulfated and anomerically modified GAG oligosaccharides of the HA type. Representative studies of protein/GAG interactions by (bio)chemical and biophysical methods are reported yielding novel insights into GAG-protein binding. Finally, the biological conclusions and in vivo applications of defined sulfated GAG oligosaccharides will be discussed.

Total Synthesis of the All-Rare Sugar-Containing Pentasaccharide Repeating Unit of the O-Polysaccharide of Plesiomonas shigelloides Strain 302-73 (Serotype O1).

First total synthesis of the conjugation-ready pentasaccharide repeating unit of Plesiomonas shigelloides strain 302-73 (serotype O1) is reported. The complex target pentasaccharide is composed of all-rare amino sugars such as orthogonally functionalized d-bacillosamine, l-fucosamine, and l-pneumosamine linked through four consecutive α-linkages. The poor nucleophilicity of axial 4-OH of l-fucosamine and stereoselective glycosylations are the key challenges in the total synthesis, which was completed via a longest linear sequence of 27 steps in 3% overall yield.

Improvement of Fucosylated Oligosaccharides Synthesis by α-L-Fucosidase from Thermotoga maritima in Water-Organic Cosolvent Reaction System.

The effects of water activity (aw), pH, and temperature on transglycosylation activity of α-L-fucosidase from Thermotoga maritima in the synthesis of fucosylated oligosaccharides were evaluated using different water-organic cosolvent reaction systems. The optimum conditions of transglycosylation reaction were the pH range between 7 and 10 and temperature 90-95 °C. The addition of organic cosolvent decreased α-L-fucosidase transglycosylation activity in the following order: acetone > dimethyl sulfoxide (DMSO) > acetonitrile (0.51 > 0.42 > 0.18 mM/h). However, the presence of DMSO and acetone enhanced enzyme-catalyzed transglycosylation over hydrolysis as demonstrated by the obtained transglycosylation/hydrolysis rate (rT/H) values of 1.21 and 1.43, respectively. The lowest rT/H was calculated for acetonitrile (0.59), though all cosolvents tested improved the transglycosylation rate in comparison to a control assay (0.39). Overall, the study allowed the production of fucosylated oligosaccharides in water-organic cosolvent reaction media using α-L-fucosidase from T. maritima as biocatalyst.

A Concise Synthesis of a BODIPY-Labeled Tetrasaccharide Related to the Antitumor PI-88.

A convergent synthetic route to a tetrasaccharide related to PI-88, which allows the incorporation of a fluorescent BODIPY-label at the reducing-end, has been developed. The strategy, which features the use of 1,2-methyl orthoesters (MeOEs) as glycosyl donors, illustrates the usefulness of suitably-designed BODIPY dyes as glycosyl labels in synthetic strategies towards fluorescently-tagged oligosaccharides.