O-acetyl Groups Research Articles

Effect of O-Acetylation on the Antigenicity and Glycoconjugate Immunogenicity of the Streptococcus Pneumoniae Serotype 7F Capsular Polysaccharide.

Streptococcus pneumoniae is a bacterial pathogen causing diseases as severe as pneumonia, sepsis and meningitis. Most commercial pneumococcal conjugate vaccines contain the 7F serotype, which is epidemiologically relevant and highly invasive. This serotype contains an O-acetyl group at the internal L-rhamnose of its polysaccharide repeating unit. Herein we report on the role of the O-acetyl moiety of 7F polysaccharide in both antigen recognition and the induction of a protective antibody response against 7F. Fully and partially de-O-acetylated 7F polysaccharides were chemically prepared and compared with the O-acetylated counterpart in their antigenicity and immunogenicity of their tetanus toxoid glycoconjugates. These comparative studies showed a slight but consistent decrease in the antigenicity for the fully de-O-acetylated polysaccharide, but not for the partly de-O-acetylated variant. The glycoconjugates derived from the O-acetylated and the fully de-O-acetylated polysaccharides had similar sizes and polysaccharide-to-protein ratio, and all proved both to be immunogenic and induce opsonophagocytic responses in mice. Nevertheless, the immune response elicited by the O-acetylated glycoconjugate was better in both quantity and quality, proving that the O-acetyl group is not strictly necessary but also not irrelevant for the antigenicity and immunogenicity of the 7F serotype polysaccharide and its glycoconjugates.

Serotype 15C Streptococcus pneumoniae resistant to classical complement deposition and agglutination by polyclonal rabbit anti-capsular 15B sera

BackgroundS. pneumoniae (SP) serotypes 15B and 15C are frequent causative agents of invasive pneumococcal disease (IPD), otitis media, and nasopharyngeal colonization in the post PCV13 era. The principal difference between serotypes 15B and 15C is the presence of an O-acetyl group on the pentasaccharide repeating unit of 15B polysaccharide. It remains unclear if antibodies against SP15B polysaccharide demonstrate functional cross reaction with SP15C strains. We compared functional activity of polyclonal rabbit anti-capsular 15B sera against SP15B and SP15C isolates. MethodsUsing flow cytometry we measured complement factors C3c and C4d deposition on SP15B and 15C in the presence of polyclonal rabbit anti capsular 15B sera. We measured the binding of C3c, common to all complement pathways, and C4d, specific to classical pathway, on SP serotypes 15B and 15C when co-incubated with polyclonal rabbit anti capsular 15B sera and antibody depleted complement. Both the proportion of bacteria with complement deposition and the fluorescence intensity were measured. We also measured agglutination as the increase in forward and side scatter. ResultsPolyclonal rabbit anti-capsular 15B sera activated classical pathway resulting in deposition of C4d and C3c at high intensity on all SP15B cells but only achieved limited deposition and intensity of C4 with SP15C. Similarly, polyclonal rabbit anti-capsular 15B sera induced agglutination of SP15B strains in a dose dependent manner and limited agglutination of SP15C. ConclusionsAnti-capsular 15B sera induce limited C4d and C3c deposition, and minimal agglutination with SP15C strains, reflecting lower classical pathway activation in contrast to high C4d and C3c deposition and agglutination of SP15B. These observations support limited functional cross reactivity of anti-15B to SP15C strains and are consistent with the reduction in opsonophagocytic killing of SP15C reported following immunization with vaccines containing 15B polysaccharide.

Structural Analysis of Mammalian Sialic Acid Esterase

Sialic acid esterase (SIAE) catalyzes the removal of O-acetyl groups from sialic acids found on cell surface glycoproteins to regulate cellular processes such as B cell receptor signalling and apoptosis. Loss-of-function mutations in SIAE are associated with several common autoimmune diseases including Crohn’s, ulcerative colitis, and arthritis. To gain a better understanding of the function and regulation of this protein, we determined crystal structures of SIAE from three mammalian homologs, including an acetate bound structure. The structures reveal that the catalytic domain adopts the fold of the SGNH hydrolase superfamily. The active site is composed of a catalytic dyad, as opposed to the previously reported catalytic triad. Attempts to determine a substrate-bound structure yielded only the hydrolyzed product acetate in the active site. Rigid docking of complete substrates followed by molecular dynamics simulations revealed that the active site does not form specific interactions with substrates, rather it appears to be broadly specific to accept sialoglycans with diverse modifications. Based on the acetate bound structure, a catalytic mechanism is proposed. Structural mapping of disease mutations reveals that most are located on the surface of the enzyme and would only cause minor disruptions to the protein fold, suggesting that these mutations likely affect binding to other factors. These results improve our understanding of SIAE biology and may aid in the development of therapies for autoimmune diseases and cancer.

Structural characteristics and wound-healing effects of Bletilla striata fresh tuber polysaccharide

A homogeneous polysaccharide from Bletilla striata fresh tuber (BSPS) was prepared and extensively characterized using HP-GPC, colorimetry, FT-IR, methylation, GC–MS, NMR, Congo red experiment, SEM, and AFM. The molecular weight of BSPS was 722.90 kDa. BSPS consisted of glucose and mannose in the molar ratio of 1 : 2.5. BSPS had a linear chain structure consisting mainly of →4)-β-d-Glcp-(1→ and →4)-β-d-Manp-(1→ residues. O-acetyl group linked to C2 of →4)-β-d-Manp-(1→ residue. Its monosaccharide molar ratio, molecular weight, and O-acetyl substituted position were different from that of the polysaccharide from B. striata dried tuber reported previously. Furthermore, BSPS at concentrations of 3.125–25 μg/mL significantly promoted the viability (ca. 10%), differentiation (1.5–4 folds), migration (15%–70%), and invasion (1.84–4.65 folds) of C2C12 cells. Of note, BSPS remarkably accelerated the epidermal regeneration and wound healing in mice. This study for the first time reported the structure of polysaccharides in B. striata fresh tubers. The results demonstrated that BSPS could be explored as a novel natural wound-healing drug.

Structural characteristics and anti-tumor effect of low molecular weight Dendrobium officinale polysaccharides by reconstructing tumor microenvironment

In this study, glucanase and mannanase were selected to hydrolysis DOP to obtain low molecular weight DOP (LMWDOP), namely glucanase-hydrolysis Dendrobium officinale polysaccharides (GDOP) and mannanase-hydrolysis Dendrobium officinale polysaccharides (MDOP). The antitumor activity of LMWDOPs and its effect on tumor immune microenvironment were investigated. The growth of transplantable S180 sarcoma in mice was significantly inhibited by GDOP, and MDOP in a dose-dependent manner compared with the model group (P < 0.05, P < 0.01). This indicated that mannanase-hydrolysis Dendrobium officinale polysaccharides have stronger anti-tumor activity. The structure of MDOP was systematically characterized to explore the structure–activity relationship. The average molecular weight of MDOP was 3494 Da. MDOP had a linear chain structure consisting mainly of →6)-α-d-Glcp-(1→ and →4)-β-d-Manp-(1 → residues. O-acetyl group linked to C2 of →4)-β-d-Manp-(1 → residue. In conclusion, it has been determined that the small molecule dendrobium polysaccharides obtained from the hydrolysis of mannanase have good anti-tumor activity and can reconstruct the immune microenvironment. “→ 6-α-d-Glcp- (1 → 4)-β-d-Manp-(1→) may be the structural basis for Dendrobium officinale polysaccharides to exert anti-tumor effects.

2-Styryl-quinoline-4-carboxylic acids: Synthesis, characterization, and investigation of their optical properties and antimicrobial activity

Styrylquinoline-based fluorophores 1–10 were synthesized via Knoevenagel and Pfitzinger reactions. The molecules were fully characterized and studied for the impact of their substitution patterns (A-D) on their optical and bioactivity properties. With exception of nitro substituted compounds 9-D and 10-D, all compounds were fluorescent in solution with a significant solvatochromic behavior measured in toluene, dioxane, tetrahydrofurane, ethyl acetate, dichloromethane, dimethylformamide, dimethylsulfoxide, acetonitrile, isopropanol, and methanol. The maximum wavelength of their emission ranged from 408 to 539 nm. Additionally, there was a significant variation in the photoluminescence (PL) quantum yield concerning different solvent polarities. The PL quantum yields ranged from small values, such as 1.7 % in methanol for compound 1-A, to good PL quantum yields, such as 44.4 % in dioxane for 7-C. For comparison, the quantum yield of 7-C in methanol was 3.3 %, a total range variation of 41.1 %. 4-B and 5-B with O-acetyl groups at ortho and at para position to the CC double bond, respectively, presented an interesting acidochromism with a redshift of about 102 nm in their emission bands along with a visible emitted color change from blue to green. In addition, the antimicrobial activity for all compounds and the theoretical mechanism of action of the more significant bioactive compounds were investigated. In inverse virtual screening studies, the enzymes Dehydrosqualene synthase and Squalene synthase were indicated as putative biological targets for 3-B and 4-B. Molecular docking indicated the amino acid residues critical for interaction in bacteria and fungi.

Development and Application of a High-Throughput Method for the Purification and Analysis of Surface Carbohydrates from Klebsiella pneumoniae.

Klebsiella pneumoniae (Kp) is a Gram-negative bacterium, and a leading cause of neonatal sepsis in low- and middle-income countries, often associated with anti-microbial resistance. Two types of polysaccharides are expressed on the Kp cell surface and have been proposed as key antigens for vaccine design: capsular polysaccharides (known as K-antigens, K-Ags) and O-antigens (O-Ags). Historically, Kp has been classified using capsule serotyping and although 186 distinct genotypes have been predicted so far based on sequence analysis, many structures are still unknown. In contrast, only 11 distinct OAg serotypes have been described. The characterization of emerging strains requires the development of a high-throughput purification method to obtain sufficient K- and O-Ag material to characterize the large collection of serotypes and gain insight on structural features and potential cross-reactivity that could allow vaccine simplification. Here, this was achieved by adapting our established method for the simple purification of O-Ags, using mild acetic acid hydrolysis performed directly on bacterial cells, followed by filtration and precipitation steps. The method was successfully applied to purify the surface carbohydrates from different Kp strains, thereby demonstrating the robustness and general applicability of the purification method developed. Further, antigen characterization showed that the purification method had no impact on the structural integrity of the polysaccharides and preserved labile substituents such as O-acetyl and pyruvyl groups. This method can be further optimized for scaling up and manufacturing to support the development of high-valency saccharide-based vaccines against Kp.

Quantum DFT studies on the drug delivery of favipiravir using pristine and functionalized chitosan nanoparticles

Considering the spread of the COVID-19 pandemic, finding new drugs along with the development of effective drug delivery methods can help in the treatment of this disease. For this reason, in this research work, the possibility of drug-delivery of Favipiravir (FP), one of the drugs approved in the treatment of COVID-19, by pristine chitosan (Chit) nanoparticles (NP), and functionalized chitosan nanoparticles with N-acylate, N-methyl, O-acetyl, and Oxazoline functional groups was studied using quantum mechanical DFT methods at B3LYP-D3(BJ)/6-311 + g(d,p) theoretical level in water medium. The QTAIM, NBO, DOS, frontier orbital, conceptual-DFT indices, and non-covalent interaction analysis were further implemented to investigate the possible interactions between FP and Chit NPs. The results show that the adsorption of FP on Chit NPs is done through the creation of hydrogen bonds, and the highest absorption energy of − 18.15 kcal/mol between pristine chitosan and FP. In the case of all functionalized Chit NPs, a decrease in the absorption energy is observed, which is more noticeable in the case of N-acylated and O-acetyl functionalize Chit NPs, and indicates the weakening of the van der Waals interactions for these cases. Considering the compatibility of Chit NPs with the human body and their non-toxicity, as well as the fact that factors such as pH, solubility, the ionic strength, and so on can be adjusted to control the release rate using the functionalized Chit NPs, it seems that the results of this work can be a comprehensive guide to design the drug delivery methods of FP drug using Chit NPs, to reduce the symptoms of COVID-19 disease.

Lipopolysaccharide of Legionella pneumophila Serogroup 1 Facilitates Interaction with Host Cells.

Legionella pneumophila is the primary causative agent of Legionnaires' disease. The mutant-type strain interrupted in the ORF7 gene region responsible for the lipopolysaccharide biosynthesis of the L. pneumophila strain Heysham-1, lacking the O-acetyl groups attached to the rhamnose of the core part, showed a higher surface polarity compared with the wild-type strain. The measurement of excitation energy transfer between fluorophores located on the surface of bacteria and eukaryotic cells showed that, at an early stage of interaction with host cells, the mutant exhibited weaker interactions with Acanthamoeba castellanii cells and THP-1-derived macrophages. The mutant displayed reduced adherence to macrophages but enhanced adherence to A. castellanii, suggesting that the O-acetyl group of the LPS core region plays a crucial role in facilitating interaction with macrophages. The lack of core rhamnose O-acetyl groups made it easier for the bacteria to multiply in amoebae and macrophages. The mutant induced TNF-α production more strongly compared with the wild-type strain. The mutant synthesized twice as many ceramides Cer(t34:0) and Cer(t38:0) than the wild-type strain. The study showed that the internal sugars of the LPS core region of L. pneumophila sg 1 can interact with eukaryotic cell surface receptors and mediate in contacting and attaching bacteria to host cells as well as modulating the immune response to infection.

Chemical Synthesis of Peptides and Proteins Bearing Base-Labile Post-Translational Modifications: Evolution of the Methods in Four Decades.

The S-palmitoylation on Cys residue and O-acetylation on Ser/Thr residues are two types of base-labile post-translational modifications (PTMs) in cells. The lability of these PTMs to bases and nucleophiles makes the peptides/proteins bearing S-palmitoyl or O-acetyl groups challenging synthetic targets, which cannot be prepared via the standard Fmoc-SPPS and native chemical ligation. In this review, we summarized the efforts towards their preparation in the past 40 years, with the focus on the evolution of synthetic methods.

The polysaccharides from Auricularia auricula alleviate non-alcoholic fatty liver disease via modulating gut microbiota and bile acids metabolism

The polysaccharides from Auricularia auricula (AAPs), containing a large number of O-acetyl groups that are related to the physiological and biological properties, seem to be potential prebiotics like other edible fungus polysaccharides. In the present study, therefore, the alleviating effects of AAPs and deacetylated AAPs (DAAPs, prepared from AAPs by alkaline treatment) on nonalcoholic fatty liver disease (NAFLD) induced by high-fat and high-cholesterol diet combined with carbon tetrachloride were investigated. The results revealed that both AAPs and DAAPs could effectively relieve liver injury, inflammation and fibrosis, and maintain intestinal barrier function. Both AAPs and DAAPs could modulate the disorder of gut microbiota and altered the composition of gut microbiota with enrichment of Odoribacter, Lactobacillus, Dorea and Bifidobacterium. Further, the alteration of gut microbiota, especially enhancement of Lactobacillus and Bifidobacterium, was contributed to the changes of bile acids (BAs) profile with increased deoxycholic acid (DCA). Farnesoid X receptor could be activated by DCA and other unconjugated BAs, which participated the BAs metabolism and alleviated the cholestasis, then protected against hepatitis in NAFLD mice. Interestingly, it was found that the deacetylation of AAPs negatively affected the anti-inflammation, thereby reducing the health benefits of A. auricula-derived polysaccharides.

Structure of an unprecedent glucuronoxylogalactoglucomannan from fruit bodies of Auricularia auricula-judae (black woody ear)

An unprecedent glucuronoxylogalactoglucomannan (GXG′G″M), ME-2 (Mw, 2.60 × 105 g/mol; O-acetyl % = 16.7 %), was isolated and purified from water extracts of Auricularia auricula-judae (black woody ear). Firstly, due to much higher O-acetyl contents, we prepared its fully deacetylated products (dME-2; Mw, 2.13 × 105 g/mol) for convenient structure survey. The repeating structure-unit of dME-2 was readily proposed based on Mw determination, monosaccharide compositions, methylation analysis, free-radical degradation and 1/2D NMR spectroscopy. The dME-2 was identified as a highly branched polysaccharide with an average of 10 branches per 10 sugar backbone units. The backbone was only repeating →3)-α-Manp-(1→ residues, substituted at the C-2, C-6 and C-2,6 positions. The side chains included β-GlcAp-(1→, β-Xylp-(1→, α-Manp-(1→, α-Galp-(1→ and β-Glcp-(1→. Secondly, the complex substituted positions of O-acetyl groups in ME-2 were determined to be at C-2, C-4, C-6 and C-4,6 in the backbone and at C-2 and C-2,3 in some side chains. Finally, the anti-inflammatory activity of ME-2 was preliminarily explored on LPS-stimulated THP-1 cells. The above date not only provided the first example for structural studies of GXG′G″M type polysaccharides, but also facilitated development and application of black woody ear polysaccharides as medicinal agents or functional dietary supplements.

Structural properties of Bletilla striata polysaccharide and the synergistic gelation of polysaccharide and xanthan gum

Bletilla strata polysaccharide (BSP) has been attracting increasing attention because of its numerous bioactive components. In this study, BSP was obtained by water-extraction and ethanol-precipitation, and its structure and synergistic interaction with xanthan gum (XG) were then investigated. BSP was identified as a highly linear polymer with a weight average molecular weight (Mw) of 323.7 kDa, whose ratio of mannose to glucose was 2.4:1.0. BSP had the backbone of →4)-β-Manp-(1→, →4)-β-Glcp-(1→, as well as a small amount of →3)-β-Manp-(1→, terminated by T-Manp residues. Moreover, it was also substituted by a small number of O-acetyl groups at C-3 position of →4)-Manp-(1→ residue. Despite the poor gelling ability of BSP itself, it could form a firm and thixotropic gel when mixing with xanthan gum (XG). Upon cooling, irrespective of the mixing ratio, all BSP/XG mixtures exhibited a steep increase in G′ at ∼60 °C, which was defined as the onset temperature of the synergistic gelation. On heating, all gels melted at 60 °C, without detectable thermal hysteresis. The most pronounced synergistic interaction occurred at BSP/XG ratio = 5:5. Overall, this study provides new knowledge about the structure of BSP and its synergistic interaction with XG. Especially, the thixotropy of the mixed gels may find usefulness in formulating easy-to-swallowing food products.

A21 ROLE OF SIALIC ACID O-ACETYLATION IN MAINTAINING MUCUS INTEGRITY AND HOMEOSTASIS OF THE COLONIC MUCOSA

Abstract Background The mucus network provides innate immune defense to protect our gastrointestinal tract from pathogens, and promote homeostasis with our resident microbiota. This network is constituted by the mucin MUC2 (Muc2 in mouse), which is ~80% complex O-linked glycans by weight. Sialic acid (Sia) is a key capping monosaccharide on complex O-glycans which has recently been linked to preserving mucus integrity. Sia can undergo enzymatic modifications including the addition of O-acetyl groups. The 9-O-acetyltransferase CasD1 is responsible for the 9-OAc Sia variants. Functionally, the OAc-modification is known to inhibit microbial sialidase activities which may preserve Sia’s protective roles on mucins. However, the extent of these OAc modifications in human and murine Mucin-2, and how they influence mucus function is unclear. Purpose: To determine whether and how Sia O-acetylation on colonic mucus regulates mucus integrity, host-microbe interactions, and colitis susceptibility. Method We used viral-derived probes that target specific OAc-Sia analogues on mucus on sections from human feces and mouse feces and colon tissues to visualize their spatial arrangement and microbial interaction in situ. For glycomics, OAc-Sia analogues were quantitated on purified human MUC2 and mouse Muc2 by HPLC-MS after derivatization with 4,5-dimethyl-1,2-diaminobenzamine (DMBA). O-glycans were released via non-reductive ammonia-catalyzed β-elimination and analyzed by mass spectrometry. For in vivo work, we generated intestinal epithelial cell-specific Casd1 KO mice (Casd1flox/flox;VillinCre or IEC Casd1-/- mice) and analyzed their mucins. Sialidase activities were quantified in the supernatants of colon fecal materials from WT and IEC Casd1-/- mutants mice using a fluorogenic substrate 4-MU-NeuNAc. Colitis susceptibility was monitored using 1.5% w/v Dextran Sodium Sulfate (DSS). Result(s) We found Sias on both human MUC2 and murine Muc2 were heavily O-acetylated, with ~75% and ~45% of Sias having 9-OAc-based modification in humans and mice respectively, and were distributed throughout the niche and barrier layers of mucus in situ. IEC Casd1-/- mice were viable and healthy with knockdown confirmed by 9-OAc staining, western blot of protein lysates and mucins, and sialylomics. The mucus encapsulation appeared overall intact regardless of OAc status. However, IEC Casd1-/- mice showed heightened susceptibility to 1.5% DSS colitis, linked to thinning of the mucus in IEC Casd1-/- vs WT littermates after challenge. Consistent with the role of OAc Sia in sialidase inhibition, loss of OAc Sia was associated with increased sialidase activities as assessed by heightened 4 MU signal in fecal supernatants in WT vs littermate IEC Casd1-/- mice. O-glycomics also showed reduction in the number of sialylated O-glycan structures upon loss of 9-OAc Sia. Conclusion(s) Sia O-acetylation appears important in maintaining key aspects of Sia-dependent mucus function and protecting from inflammatory insult. Please acknowledge all funding agencies by checking the applicable boxes below: CCC Disclosure of Interest None Declared

Phase-Transfer Catalyzed Microfluidic Glycosylation: A Small Change in Concentration Results in a Dramatic Increase in Stereoselectivity

Phase-transfer catalysis (PTC) is widely used in glycochemistry for the preparation of aryl glycosides by the glycosylation reaction. While investigating the possibility of synthesis of 4-(3-chloropropoxy)phenyl sialoside (Neu5Ac-OCPP) from N-acetylsialyl chloride with O-acetyl groups (1), we have recently discovered a strong dependence of the PTC glycosylation outcome on the mixing mode: under batch conditions, only α-anomer of Neu5Ac-OCPP was obtained, albeit in low yield (13%), while under microfluidic conditions the yield of Neu5Ac-OCPP increased to 36%, although stereoselectivity decreased (α/β ≤ 6.2). Here, we report that the outcome of this reaction, performed under microfluidic conditions using a Comet X-01 micromixer (at 2 μL/min flow rate), non-linearly depends on the concentration of N-acetylsialyl chloride 1 (5–200 mmol/L). The target Neu5Ac-OCPP was obtained in a noticeably higher yield (up to 66%) accompanied by enhanced stereoselectivity (α/β = 17:1–32:1) in the high concentration range (C &gt; 50 mmol/L), whereas the yield (10–36%) and especially, stereoselectivity (α/β = 0.9:1–6.2:1) were lower in the low concentration range (C ≤ 50 mmol/L). This dramatic stepwise increase in stereoselectivity above critical concentration (50 mmol/L) is apparently related to the changes in the presentation of molecules on the surface of supramers of glycosyl donor, which exist in different concentration ranges.

Elucidation of the O-antigen structure of Escherichia coli O93 and characterization of its biosynthetic genes.

The structure of the O-antigen from the international reference strain Escherichia coli O93:-:H16 has been determined. A nonrandom modal chain-length distribution was observed for the lipopolysaccharide, a pattern which is typical when long O-specific polysaccharides are expressed. By a combination of (i) bioinformatics information on the gene cluster related to O-antigen synthesis including putative function on glycosyl transferases, (ii) the magnitude of NMR coupling constants of anomeric protons, and (iii) unassigned 2D 1H, 13C-HSQC, and 1H,1H-TOCSY NMR spectra it was possible to efficiently elucidate the structure of the carbohydrate polymer in an automated fashion using the computer program CASPER. The polysaccharide also carries O-acetyl groups and their locations were determined by 2D NMR experiments showing that ~½ of the population was 2,6-di-O-acetylated, ~¼ was 2-O-acetylated, whereas ~¼ did not carry O-acetyl group(s) in the 3-O-substituted mannosyl residue of the repeating unit. The structure of the tetrasaccharide repeating unit of the O-antigen is given by: →2)-β-d-Manp-(1→3)-β-d-Manp2Ac6Ac-(1→4)-β-d-GlcpA-(1→3)-α-d-GlcpNAc-(1→, which should also be the biological repeating unit and it shares structural elements with capsular polysaccharides from E. coli K84 and K50. The structure of the acidic O-specific polysaccharide from Cellulophaga baltica strain NN015840T differs to that of the O-antigen from E. coli O93 by lacking the O-acetyl group at O6 of the O-acetylated mannosyl residue.

Reversible tissue sticker inspired by chemistry in plant-pathogen relationship

Plants release phenolic molecules to protect against invading pathogens. In plant-microorganism relationships, phenolics bind to surface oligosaccharides, inactivating microorganism activities. Inspired by phenol-saccharide interactions in plant defense systems, we designed an adhesive sealant. By screening 16 different saccharides, the O-acetyl group, rich in glucomannan (GM), exhibited rapid, robust binding with the galloyl moiety of a model phenolic molecule, tannic acid (TA). Furthermore, the interaction showed both pH and temperature (upper critical solution temperature) sensitivities. Utilizing O-acetyl-galloyl interactions, materials of all dimensions from beads (0D) to strings (1D), films (2D), and objects (3D) could be prepared, as a suitable platform for printing techniques. GMTA films are elastic, adhesive, water-resistant, and effectively sealed perforations, as demonstrated by (1) a lung incision followed by an air inflation model and (2) a thoracic diaphragm model. Statement of significanceIn nature, phenolic molecules are 'nearly always' physically bound with polysaccharides, indicating that the phenolics widen the functions of polysaccharides. An example includes that phenolic-polysaccharide interactions are key defense mechanisms against microbial infection in plants whereas polysaccharide alone functions poorly. Despite the ubiquitous biochemistry of polysaccharide-phenolic interactions, efforts on understanding binding chemistry focusing on phenol/polysaccharide interactions is little. This study is important because we found for the first time that O-acetyl group is the moiety in polysaccharides to which phenolic cis-diol and/or cis-triol is spontaneously bound. The phenol-polysaccharide interaction is non-covalent yet robust, kinetically fast, and reversible. Inspired by the interaction chemistry, a simple mixture of phenolic molecules and O-acetyl group containing polysaccharides such as glucomannan opens a promising fabrication strategy toward functional polysaccharide-based material.

An Immunological Polysaccharide from Tremella fuciformis: Essential Role of Acetylation in Immunomodulation.

The edible fungus Tremella fuciformis was shown to have a high molecular weight (1.87 × 103 kDa) bioactive polysaccharide, denoted as TFP-F1. Monosaccharide composition and NMR analysis of the polysaccharide and its derivatives indicated it contained fucose (Fucp), xylose (Xylp), mannose (Manp), and glucuronic acid (GlcAp) in a ratio of 0.9:1.0:3.2:1.2. Using IR, NMR, and GC-MS spectroscopic data, the structure of TFP-F1 was elucidated as {→3)-[β-D-GlcAp-(1→2)]-α-D-Manp-(1→3)-α-D-Manp-(1→3)-[α-L-Fucp-(1→2)-β-D-Xylp-(1→2)]-α-D-Manp-(1→}n, with partial acetylation of C6-OH in mannoses. Furthermore, at a concentration of 1 μg/mL, TFP-F1 was found to stimulate the secretion of TNF-α and IL-6 in J774A.1 macrophage cells in vitro via interaction with toll-like receptor 4 (TLR4). The removal of O-acetyl groups led to the loss of immunomodulatory activities, demonstrating that O-acetyl groups play an essential role in enhancing the production of pro-inflammatory cytokines.

O-Acetyl Migration within the Sialic Acid Side Chain: A Mechanistic Study Using the Ab Initio Nanoreactor.

Many disease-causing viruses target sialic acids on the surface of host cells. Some viruses bind preferentially to sialic acids with O-acetyl modification at the hydroxyl group of C7, C8, or C9 on the glycerol-like side chain. Studies of proteins binding to sialosides containing O-acetylated sialic acids are crucial in understanding the related diseases but experimentally difficult due to the lability of the ester group. We recently showed that O-acetyl migration among hydroxyl groups of C7, C8, and C9 in sialic acids occurs in all directions in a pH-dependent manner. In the current study, we elucidate a full mechanistic pathway for the migration of O-acetyl among C7, C8, and C9. We used an ab initio nanoreactor to explore potential reaction pathways and density functional theory, pKa calculations, and umbrella sampling to investigate elementary steps of interest. We found that when a base is present, migration is easy in any direction and involves three key steps: deprotonation of the hydroxyl group, cyclization between the two carbons, and the migration of the O-acetyl group. This dynamic equilibrium may play a defensive role against pathogens that evolve to gain entry to the cell by binding selectively to one acetylation state.

High-Performance Anion-Exchange chromatography with conductivity detection method for simultaneous determination of nitrogen and phosphorus in polysaccharides

Capsular polysaccharides of Streptococcus pneumoniae contain a characteristic mix of monosaccharides in their structure resulting in immunologically distinct serotypes. Pneumococcal capsular polysaccharides include sugars such as hexoses, uronic acids, hexosamines, methyl pentoses, other functional groups are attached to the sugars are N and O-acetyl groups, nitrogen and phosphorus. Most of these components can be quantified using different colorimetric methods. However, available methods for quantifying nitrogen and phosphorus are not sensitive enough and laborious. We report a highly sensitive high-performance anion-exchange chromatography-conductivity detector (HPAEC-CD) method for quantifying nitrogen and phosphorus present in pneumococcal capsular polysaccharides. The method is reliable, robust and reproducible with no interference. The LOQ for nitrogen and phosphorus of 3.125 and 62.5 ng/mL, respectively, is highly critical for estimating low levels of total nitrogen and total phosphorus. We have implemented this method to quantify total nitrogen in Typhoid Vi polysaccharide and phosphorus in Haemophilus influenzae type-b polysaccharide. This method has greater application for quantification of nitrogen and phosphorus present in low concentrations in polysaccharide vaccines/biologicals.