Seaweed-derived fucoidans and rhamnan sulfates serve as potent anti-SARS-CoV-2 agents with potential for prophylaxis.

Seaweeds represent a rich source of sulfated polysaccharides with similarity to heparan sulfate, a facilitator of myriad virus host cell attachment. For this reason, attention has been drawn to their antiviral activity, including the potential for anti-SARS-CoV-2 activity. We have identified and structurally characterized several fucoidan extracts, including those from different species of brown macroalga, and a rhamnan sulfate from a green macroalga species. A high molecular weight fucoidan extracted from Saccharina japonica (FSjRPI-27), and a rhamnan sulfate extracted from Monostroma nitidum (RSMn), showed potent competitive inhibition of spike glycoprotein receptor binding to a heparin-coated SPR chip. This inhibition was also observed in cell-based assays using hACE2 HEK-293 T cells infected by pseudotyped SARS-CoV-2 virus with IC50 values <1 µg/mL. Effectiveness was demonstrated in vivo using hACE2-transgenic mice. Intranasal administration of FSjRPI-27 showed protection when dosed 6 h prior to and at infection, and then every 2 days post-infection, with 100 % survival and no toxicity at 104 plaque-forming units per mouse vs. buffer control. At 5-fold higher virus dose, FSjRPI-27 reduced mortality and yielded reduced viral titers in bronchioalveolar fluid and lung homogenates vs. buffer control. These findings suggest the potential application of seaweed-based sulfated polysaccharides as promising anti-SARS-CoV-2 prophylactics.

Semisynthesis of new sulfated heterorhamnan derivatives obtained from green seaweed Gayralia brasiliensis and evaluation of their anticoagulant activity.

Marine green algae produce sulfated polysaccharides with diverse structures and a wide range of biological activities. This study aimed to enhance the biotechnological potential of sulfated heterorhamnan (Gb1) from Gayralia brasiliensis by chemically modifying it for improved or new biological functions. Using controlled Smith Degradation (GBS) and O-alkylation with 3-chloropropylamine, we synthesized partially water-soluble amine derivatives. GBS modification increase sulfate groups (29.3 to 37.5 %) and α-l-rhamnose units (69.9 to 81.2 mol%), reducing xylose and glucose, compared to Gb1. The backbone featured predominantly 3- and 2-linked α-l-rhamnosyl and 2,3- linked α-l-rhamnosyl units as branching points. Infrared and NMR analyses confirmed the substitution of hydroxyl groups with aminoalkyl groups. The modified compounds, GBS-AHCs and GBS-AHK, exhibited altered anticoagulant properties. GBS-AHCs showed reduced effectiveness in the APTT assay, while GBS-AHK maintained a similar anticoagulant activity level to Gb1 and GBS. Increased nitrogen content and N-alkylation in GBS-AHCs compared to GBS-AHK may explain their structural differences. The chemical modification proposed did not enhance its anticoagulant activity, possibly due to the introduction of amino groups and a positive charge to the polymer. This characteristic presents new opportunities for investigating the potential of these polysaccharides in various biological applications, such as antimicrobial and antitumoral activities.

Optimization of the Synthesis and Conjugation of the Methyl Rhamnan Tip of Pseudomonas aeruginosa A-Band Polysaccharide and Immunogenicity Evaluation for the Continued Development of a Potential Glycoconjugate Vaccine.

Pseudomonas aeruginosa is an antimicrobial-resistant bacterium that has no vaccine approved for human use. Additionally, it has been identified by the World Health Organization as a priority pathogen for novel vaccines and therapeutic development. We previously developed a synthetic mimic of the A-band polysaccharide tip that showed promise in terms of immunogenicity for use as a glycoconjugate vaccine. In this current manuscript, we improve upon the previous work to continue the development of this glycoconjugate vaccine. Herein, we report a higher-yielding synthesis of mimics containing a handle and a spacer that improved conjugation efficiency, resulting in better carbohydrate-to-protein ratios and also good immunogenicity of these conjugates in mice and rabbits. The data suggested that perhaps only a tetrasaccharide was required to induce an immune response capable of recognizing whole cells of P. aeruginosa.

Tunable Approach to C-Linked Analogs of Glycosamines.

The synthesis of (1R)-2-amino-2-deoxy-ß-l-gulopyranosyl benzene and the α and ß forms of 2-amino-2-deoxy-l-idopyranosyl benzene derivatives was accomplished through stereospecific addition of tributylstannyllithium to readily available (SR)- or (SS)-N-tert-butanesulfinyl-arabinofuranosylamine building blocks, followed by stereoretentive Pd-catalyzed Migita-Kosugi-Stille cross-coupling, stereoselective reduction, and an activation-cyclization strategy. Application of this methodology paves the way to new three-dimensional chemical space and preparation of unknown (non-natural) and complex 2-amino-2-deoxy sugars of biological interest.

Structural Characterization and Evaluation of an Epitope at the Tip of the A-Band Rhamnan Polysaccharide of Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces a variety of cell surface glycans. Previous studies identified a common polysaccharide (PS) antigen often termed A-band PS that was composed of a neutral d-rhamnan trisaccharide repeating unit as a relatively conserved cell surface carbohydrate. However, nuclear magnetic resonance (NMR) spectra and chemical analysis of A-PS preparations showed the presence of several additional components. Here, we report the characterization of the carbohydrate component responsible for these signals. The carbohydrate antigen consists of an immunogenic methylated rhamnan oligosaccharide at the nonreducing end of the A-band PS. Initial studies performed with the isolated antigen permitted the production of conjugates that were used to immunize mice and rabbits and generate monoclonal and polyclonal antibodies. The polyclonal antibodies were able to recognize the majority of P. aeruginosa strains in our collection, and three monoclonal antibodies were generated, one of which was able to recognize and facilitate opsonophagocytic killing of a majority of P. aeruginosa strains. This monoclonal antibody was able to recognize all P. aeruginosa strains in our collection that includes clinical and serotype strains. Synthetic oligosaccharides (mono- to pentasaccharides) representing the terminal 3-O-methyl d-rhamnan were prepared, and the trisaccharide was identified as the antigenic determinant required to effectively mimic the natural antigen recognized by the broadly cross-reactive monoclonal antibody. These data suggest that there is considerable promise in this antigen as a vaccine or therapeutic target.

Synthesis and Immunogenicity of a Methyl Rhamnan Pentasaccharide Conjugate from Pseudomonas aeruginosa A-Band Polysaccharide.

Pseudomonas aeruginosa was added to the World Health Organization's priority pathogen list for research and development of new antibiotics in 2017. Alongside the development of new antibiotics to fight antimicrobial-resistant P. aeruginosa, vaccines would be an appealing addition to the toolbox health professionals have against this bacteria, which causes life-threatening respiratory infections. Recently, the structure of a novel immunogenic terminal carbohydrate moiety on the cell surface of P. aeruginosa was elucidated, consisting of a 3-O-methyl (1→4)-α-d-rhamnan pentasaccharide. As isolating this oligosaccharide from P. aeruginosa in sufficient amounts for producing a conjugate vaccine is challenging, herein we describe the synthesis of 3-O-methyl d-rhamnose oligosaccharide. We also report the conjugation of the synthetic pentasaccharide to human serum albumin and its resulting immunogenicity.

Hypolipidemic activity and safety evaluation of a rhamnan-type sulfated polysaccharide-chromium (III) complex.

BACKGROUND: Hyperlipidaemia is a chronic disorder characterized by imbalance of energy metabolism and high blood lipid level. The rhamnan-type sulfated polysaccharide is an excellent metal-ion chelating ligands. In this study, hypolipidemic activity and safety evaluation of a rhamnan-type sulfated polysaccharide-chromium (III) complex (RSPC) were studied. METHODS: Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to characterize the structure of RSPC. The effects of the RSPC on lipid metabolism in hyperlipidemic mice were evaluated by lipid contents, histopathological observation, immunofluorescent analysis, and adipocytokine levels. Moreover, sub-acute toxicity evaluation of RSPC was carried out on ICR mice. RESULTS: SEM and AFM further demonstrated formation of the polysaccharide-chromium (III) complex and revealed the intertwined network of RSPC. The RSPC significantly (p < 0.05) regulated lipid levels in the mice. The RSPC inhibited over-growth of adipocytes and reduced inflammatory infiltration induced by hyperlipidemia. The RSPC promoted differentiation of white adipose tissue into beige adipocytes and increased expression of uncoupling protein 1 (UCP1), thereby eliminating fat accumulation. Moreover, RSPC (5 mg/kg for mice; equivalent to 924 µg/d for adults) promoted secretion of adiponectin and suppressed resistin, leptin, and tumor necrosis factor alpha. Sub-acute toxicity evaluation showed that 1500 mg/kg of RSPC exhibited no apparent adverse effects on the mice. CONCLUSION: These results indicated that RSPC could be safely used to prevent hyperlipidemia and inflammation and may provide a new idea for the prevention of hyperlipidaemia and the related metabolic disorders.

Anti-Inflammatory Activity of Orally Administered Monostroma nitidum Rhamnan Sulfate against Lipopolysaccharide-Induced Damage to Mouse Organs and Vascular Endothelium.

We previously reported that rhamnan sulfate (RS) purified from Monostroma nitidum significantly suppressed lipopolysaccharide (LPS)-induced inflammation in cultured human vascular endothelial cells. Here, we analyzed the effect of orally administered RS on LPS-induced damage to mouse organs and vascular endothelium. RS (1 mg) was orally administered daily to BALB/c mice, 50 µg of LPS was intraperitoneally administered on day 8, and Evans blue was injected into the tail vein 6 h later. After 30 min, LPS-treated mice showed pulmonary Evans blue leakage and elevated plasma levels of liver damage markers, whereas this reaction was suppressed in LPS + RS-treated mice. Immunohistochemical and Western blot analysis of mouse organs 24 h after LPS treatment showed significant neutrophil infiltration into the lung, liver, and jejunum tissues of LPS-treated mice and high expression levels of inflammation-related factors in these tissues. Expression levels of these factors were significantly suppressed in LPS + RS-treated mice. Analysis of lung glycocalyx showed a significant reduction in glycocalyx in LPS-treated mice but not in LPS + RS-treated mice. Levels of syndecan-4, one of the glycocalyx components, decreased in LPS-treated mice and increased in LPS + RS-treated mice. The current results suggest that orally administered RS protects organs and vascular endothelium from LPS-induced inflammation and maintains blood circulation.

4,5-Dioxo-imidazolinium Cation-Promoted α-Selective Dehydrative Glycosylation of 2-Deoxy- and 2,6-Dideoxy Sugars.

Herein, we report the great potential of the 4,5-dioxo-imidazolinium cation activation strategy for dehydrative glycosylation reactions employing the readily available and economical geminal dichloroimidazolidinediones (DCIDs) that promotes the glycosylation between 2-deoxy- and 2,6-dideoxy-sugar hemiacetals with various acceptors in good yields and high α-selectivity. This research not only provides a mild and efficient alternative approach for stereoselective dehydrative glycosylation but also extends the dichloroimidazolidinedione as a novel promoter in the field of glycoscience.

Deoxy sugars. General methods for carbohydrate deoxygenation and glycosidation.

Deoxy sugars represent an important class of carbohydrates, present in a large number of biomolecules involved in multiple biological processes. In various antibiotics, antimicrobials, and therapeutic agents the presence of deoxygenated units has been recognized as responsible for biological roles, such as adhesion or great affinity to receptors, or improved efficacy. The characterization of glycosidases and glycosyltranferases requires substrates, inhibitors and analogous compounds. Deoxygenated sugars are useful for carrying out specific studies for these enzymes. Deoxy sugars, analogs of natural substrates, may behave as substrates or inhibitors, or may not interact with the enzyme. They are also important for glycodiversification studies of bioactive natural products and glycobiological processes, which could contribute to discovering new therapeutic agents with greater efficacy by modification or replacement of sugar units. Deoxygenation of carbohydrates is, thus, of great interest and numerous efforts have been dedicated to the development of methods for the reduction of sugar hydroxyl groups. Given that carbohydrates are the most important renewable chemicals and are more oxidized than fossil raw materials, it is also important to have methods to selectively remove oxygen from certain atoms of these renewable raw materials. The different methods for removal of OH groups of carbohydrates and representative or recent applications of them are presented in this chapter. Glycosidic bonds in general, and 2-deoxy glycosidic linkages, are included. It is not the scope of this survey to cover all reports for each specific technique.

Anti-SARS-CoV-2 Activity of Rhamnan Sulfate from Monostroma nitidum.

The COVID-19 pandemic is a major human health concern. The pathogen responsible for COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), invades its host through the interaction of its spike (S) protein with a host cell receptor, angiotensin-converting enzyme 2 (ACE2). In addition to ACE2, heparan sulfate (HS) on the surface of host cells also plays a significant role as a co-receptor. Our previous studies demonstrated that sulfated glycans, such as heparin and fucoidans, show anti-COVID-19 activities. In the current study, rhamnan sulfate (RS), a polysaccharide with a rhamnose backbone from a green seaweed, Monostroma nitidum, was evaluated for binding to the S-protein from SARS-CoV-2 and inhibition of viral infectivity in vitro. The structural characteristics of RS were investigated by determining its monosaccharide composition and performing two-dimensional nuclear magnetic resonance. RS inhibition of the interaction of heparin, a highly sulfated HS, with the SARS-CoV-2 spike protein (from wild type and different mutant variants) was studied using surface plasmon resonance (SPR). In competitive binding studies, the IC50 of RS against the S-protein receptor binding domain (RBD) binding to immobilized heparin was 1.6 ng/mL, which is much lower than the IC50 for heparin (~750 ng/mL). RS showed stronger inhibition than heparin on the S-protein RBD or pseudoviral particles binding to immobilized heparin. Finally, in an in vitro cell-based assay, RS showed strong antiviral activities against wild type SARS-CoV-2 and the delta variant.

Direct access to various C3-substituted sialyl glycal derivatives from 3-iodo-sialyl glycals.

A new and efficient method was developed for the synthesis of C3-substituted sialyl glycals that are useful for novel sialidase inhibitor discovery. This method was based on the cross-coupling reactions of 3-iodo-sialyl glycal methyl ester with boronic acids, alkenes and alkynes to directly introduce various functional groups to the sialyl glycal C3-position. A series of C3-aryl, alkyl, alkenyl, and alkynyl derivatives of sialyl glycal were efficiently and conveniently synthesized for the first time by this method, which has demonstrated its wide application scope.

Biochemical investigation of an N-acetyltransferase from Helicobacter pullorum.

N-acetylated sugars are often found, for example, on the lipopolysaccharides of Gram-negative bacteria, on the S-layers of Gram-positive bacteria, and on the capsular polysaccharides. Key enzymes involved in their biosynthesis are the sugar N-acetyltransferases. Here, we describe a structural and functional analysis of one such enzyme from Helicobacter pullorum, an emerging pathogen that may be associated with gastroenteritis and gallbladder and liver diseases. For this analysis, the gene BA919-RS02330 putatively encoding an N-acetyltransferase was cloned, and the corresponding protein was expressed and purified. A kinetic analysis demonstrated that the enzyme utilizes dTDP-3-amino-3,6-dideoxy-d-glucose as a substrate as well as dTDP-3-amino-3,6-dideoxy-d-galactose, albeit at a reduced rate. In addition to this kinetic analysis, a similar enzyme from Helicobacter bilis was cloned and expressed, and its kinetic parameters were determined. Seven X-ray crystallographic structures of various complexes of the H. pullorum wild-type enzyme (or the C80T variant) were determined to resolutions of 1.7 Å or higher. The overall molecular architecture of the H. pullorum N-acetyltransferase places it into the Class II left-handed-ß-helix superfamily (LßH). Taken together, the data presented herein suggest that 3-acetamido-3,6-dideoxy-d-glucose (or the galactose derivative) is found on either the H. pullorum O-antigen or in another of its complex glycoconjugates. A BLAST search suggests that more than 50 non-pylori Helicobacter spp. have genes encoding N-acetyltransferases. Given that there is little information concerning the complex glycans in non-pylori Helicobacter spp. and considering their zoonotic potential, our results provide new biochemical insight into these pathogens.

Automated, Multistep Continuous-Flow Synthesis of 2,6-Dideoxy and 3-Amino-2,3,6-trideoxy Monosaccharide Building Blocks.

An automated continuous flow system capable of producing protected deoxy-sugar donors from commercial material is described. Four 2,6-dideoxy and two 3-amino-2,3,6-trideoxy sugars with orthogonal protecting groups were synthesized in 11-32 % overall yields in 74-131.5 minutes of total reaction time. Several of the reactions were able to be concatenated into a continuous process, avoiding the need for chromatographic purification of intermediates. The modular nature of the experimental setup allowed for reaction streams to be split into different lines for the parallel synthesis of multiple donors. Further, the continuous flow processes were fully automated and described through the design of an open-source Python-controlled automation platform.

Acinetobacter baumannii K106 and K112: Two Structurally and Genetically Related 6-Deoxy-l-talose-Containing Capsular Polysaccharides.

Whole genome sequences of two Acinetobacter baumannii clinical isolates, 48-1789 and MAR24, revealed that they carry the KL106 and KL112 capsular polysaccharide (CPS) biosynthesis gene clusters, respectively, at the chromosomal K locus. The KL106 and KL112 gene clusters are related to the previously described KL11 and KL83 gene clusters, sharing genes for the synthesis of l-rhamnose (l-Rhap) and 6-deoxy-l-talose (l-6dTalp). CPS material isolated from 48-1789 and MAR24 was studied by sugar analysis and Smith degradation along with one- and two-dimensional 1H and 13C NMR spectroscopy. The structures of K106 and K112 oligosaccharide repeats (K units) l-6dTalp-(1→3)-D-GlcpNAc tetrasaccharide fragment share the responsible genes in the respective gene clusters. The K106 and K83 CPSs also have the same linkage between K units. The KL112 cluster includes an additional glycosyltransferase gene, Gtr183, and the K112 unit includes α l-Rhap side chain that is not found in the K106 structure. K112 further differs in the linkage between K units formed by the Wzy polymerase, and a different wzy gene is found in KL112. However, though both KL106 and KL112 share the atr8 acetyltransferase gene with KL83, only K83 is acetylated.

Halogen addition to some 1-C-substituted pyranoid glycals.

Addition of bromine and chlorine to O-peracylated 1-CN-, COOMe- and CONH2-substituted glycals was studied under ionic and radical conditions. The main or exclusive products were the corresponding 2,3-trans-diaxial (3-bromo-3-deoxy-α-d-heptopyranosylbromide)onic acid derivatives. Bromination of the O-peracetylated d-lyxo-hept-2-enopyranosononitrile and all chlorinations proved selective towards the 2-axial-3-equatorial (3-halogeno-3-deoxy-α-d-heptopyranosylhalide)onic acid derivatives. Silver triflate promoted glycosylation of methanol was successful with each 2,3-trans-diaxial (3-bromo-3-deoxy-α-d-heptopyranosylbromide)onic acid derivative, however, several attempted nucleophilic substitution and elimination reactions gave the parent glycal only.

D-Rhamnan and Pyruvate-Containing Teichuronic Acid from the Cell Wall of Rathayibacter sp. VKM Ac-2759.

Rathayibacter sp. VKM Ac-2759 (family Microbacteriaceae, class Actinobacteria) contains two glycopolymers in the cell wall. The main chain of rhamnan, glycopolymer 1, is built from the repeating tetrasaccharide units carrying terminal arabinofuranose residues at the non-reducing end, →3)-α-[α-D-Araf-(1→2)]-D-Rhap-(1→2)-α-D-Rhap-(1→3)-α-D-Rhap-(1→2)-α-D-Rhap-(1→. Similar to other described Rathayibacter species, rhamnose in the neutral glycopolymer of the VKM Ac-2759 strain is present in the D-configuration. Acetalated with pyruvic acid teichuronic acid, glycopolymer 2, is composed of the repeating tetrasaccharide units, →4)-ß-D-GlcpA-(1→4)-ß-D-Galp-(1→4)-ß-D-Glcp-(1→3)-ß-[4,6-S-Pyr]-D-Manp-(1→. Glycopolymers 1 and 2 were identified in prokaryotic microorganisms for the first time and their structures were established by chemical analysis and NMR spectroscopy. The obtained data can be used in taxonomic research, as well as for elucidating the mechanisms of plant colonization and infection by bacteria of the Rathayibacter genus.

Functional identification of the 4-deoxy-L-erythro-5-hexoseulose uronate reductase from a brown alga, Saccharina japonica.

We previously reported the alginate lyase, SjAly, from a brown alga, Saccharina japonica, providing the first experimental evidence for a functional alginate-degradation enzyme in brown algae. 4-deoxy-L-erythro-5-hexoseulose uronate (DEHU), derived from an unsaturated monosaccharide, was identified as the minimum degradation product produced by SjAly-mediated lysis of alginate. DEHU was hitherto reported to be reduced to 2-keto-3-deoxy-gluconate (KDG) by a DEHU-specific reductase with NAD(P)H in alginate-assimilating organisms and its metabolism in alginate-producing organisms is unknown. Here, we report the functional identification of a DEHU reductase, SjRed, in S. japonica. Among the 14 tested compounds, only DEHU was used as a substrate and was converted to KDG in the presence of NADPH. Optimum temperature, pH, and KCl concentration required for SjRed activity were determined to be 25 °C, 7.2, and 100 mM, respectively. SjRed consists of 341 amino acid residues and is proposed to be a member of the aldo-keto reductase superfamily. Sequencing of SjRed revealed that it is composed of at least three exons. These results indicate the existence of an enzyme that reduces DEHU to KDG in S. japonica. This is the first report on the functional identification of a DEHU-reductase in alginate-producing organisms.

D-rhamnan and teichuronic acid from the cell wall of Rathayibacter caricis VKM Ac-1799T.

The cell wall of Rathayibacter caricis VKM Ac-1799T (family Microbacteriaceae, class Actinobacteria) was found to contain both neutral and acidic glycopolymers. The first one is D-rhamnopyranan with main chain →2)-α-D-Rhap-(1 â†’ 3)-α-D-Rhap-(1→, where a part of 2-substituted residues bears as a side-chain at position 3 α-D-Manp residues or disaccharides α-D-Araf-(1→2)-α-D-Manp-(1 â†’ . The second polymer is a teichuronic acid with a branched repeating units composed of seven monosaccharides →4)-α-[ß-D-Manp-(1 â†’ 3)]-D-Glcp-(1 â†’ 4)-ß-D-GlcpA-(1 â†’ 2)-ß-[4,6Pyr]-D-Manp-(1 â†’ 4)-ß-L-Rhap-(1 â†’ 4)-ß-D-Glcp-(1 â†’ 4)-ß-D-Glcp-(1 â†’ . The structures of the polymers were determined by chemical and NMR spectroscopic methods.

Deciphering the Biosynthesis of TDP-ß-l-oleandrose in Avermectin.

Avermectin (AVM) refers to eight macrolides containing a common l-oleandrosyl disaccharide chain indispensable to their antiparasitic bioactivities. We delineated the biosynthetic pathway of TDP-ß-l-oleandrose (1), the sugar donor of AVM, by characterizing AveBVIII, AveBV, and AveBVII as TDP-sugar 3-ketoreductase, 5-epimerase, and 3-O-methyltransferase, respectively. On the basis of this pathway, we successfully reconstituted the biosynthesis of 1 in Escherichia coli. Our work completes the biosynthetic pathway of AVM and lays a solid foundation for further studies.