Glycosides Research Articles

Enzymatic advances in starch modification: Creating functional derivatives and exploring applications

Enzyme based starch modification has attracted much attention form recent years due to formation various functional derivatives with novel processing and functional properties such as resistant to digestion, thermos-reversible gel formation, etc. It has emerges as a promising method due to their high specificity, safety, and eco-friendly nature. Starch-modifying enzymes hydrolyse or form glycosidic bonds causing alteration in the amylose/amylopectin ratio, molecular weight and distribution of branch chain-length that has reduced limitation possessed by NSs i.e., low water-holding capacity, insolubility, syneresis retrogradation, etc. The review delves into the key enzymes used for modifying starch, affecting the structural and functional properties of starch. These alteration in starch properties provide guidelines in designing a new starch-based products with desired structure and properties for the food industry.

Linkage-Editing of Melibiosamine: Synthesis and Biological Evaluation of CH2- and CHF-Linked Analogs.

Melibiosamine (Gal-α(1,6)-GlcNH2), consisting of galactose and glucosamine linked by an α(1,6)-glycosidic bond, is an artificial disaccharide derivative that selectively inhibits the proliferation of K562 tumor cells relative to HUC-F2 normal cells. In this study, we employed a linkage-editing strategy to synthesize CH2- and CHF-linked melibiosamine analogs through chemo- and stereoselective hydrogenation of fluorovinyl-C-glycoside. (R)-CHF-Melibiosamine exhibited more potent antiproliferative activity than O-linked melibiosamine, while (S)-CHF-melibiosamine was less potent.

Utilization of papermaking black liquor as liquid phase for hydrothermal carbonization of corn stalks: The unique role of alkali for production of hydrochar

Papermaking black liquor (BL) contains a large amount of organic matter, alkali, and salt, which has high value in industrial and agricultural production. In the present paper, hydrothermal carbonization (HTC) technology has been employed to deal with BL and corn stalk (CS) under various conditions. A variety of analytical technologies, including Fourier Transform Infrared Spectrometer, X-Ray Diffraction, element analyzer, comprehensive thermal analyzer, scanning electron microscope, chromatography/mass spectrometry, TOC analyzer, and density functional theory (DFT), were used to characterize the physicochemical properties of hydrochar and aqueous phase product. The effects of experimental parameters (temperature, solid-liquid ratio, and time) on the HTC of BL and CS were systematically investigated. The higher energy yield and densification of hydrochar prepared by our HTC technology were obtained at 260 ℃ in 30 min with a solid-liquid ratio of 1:3. Mechanism study indicates that NaOH, which comes from the original BL, could effectively facilitate the cleavage of β–O–4 bond in lignin and β–1–4 glycosidic bond in cellulose and hemicellulose molecules. Hemicellulose and cellulose are more susceptible to degradation relevant to the lignin in the presence of NaOH. Elementary analysis shows that the dosage of BL is important for improving energy densification of hydrochar. The combustion characteristics show that hydrochar presents a thermochemical behavior, which is close to bituminous coal. Owing to hydroxyl ion in NaOH ion pair was consumed in the series of dehydrogenation reactions, pH of HTC aqueous phase product significantly decreases from 11 to 13 in original BL to 4.8–5. Such results indicate that a completely change for the components of BL could be obtained by our HTC technology, which provide a novel idea for the treatment of Papermaking BL without concentration.

Investigation of Neighboring Group Participation in 3,4-Diacetylated Glycosyl Donors in the Gas Phase.

A key challenge in oligosaccharide synthesis is the stereoselective installation of glycosidic bonds. Each glycosidic linkage has one of two possible stereo-chemical geometries, α/β or 1,2-cis/trans. An established approach to install 1,2-trans glycosidic bonds is neighboring group participation (NGP), mediated by a 2-O-acyl group. Extension of this intramolecular stabilization to nucleophilic groups located at more remote positions has also been suggested, but remains poorly understood. Previously, we employed infrared ion spectroscopy to characterize the molecular ions of monoacetylated sugar donors and showed how the strength of the stabilizing effect depends on the position of the participating ester group on the glycosyl donor ring as well as on its relative stereochemistry. In this work, we investigated glycosyl donors carrying two acyl groups. Using isotope labelling and isomer population analysis we were able to resolving spectra of isomeric mixtures and establish the relative contribution of individual species. We conclude that 3,4-diacetyl mannosyl donors exclusively form a dioxanium ion as a result of C-3 acyl stabilization. In contrast, the glucosyl and galactosyl cations form mixtures of C-3 and C-4 acyl participation products. Hence, the combination of isotope labeling and population analysis allows for the study of increasingly complex glycosyl cations.

Functional characterization and biotechnological applications of exopolysaccharides produced by newly isolated Enterococcus hirae MLG3-25-1.

This study investigated the potential applications of Enterococcus hirae MLG3-25-1 exopolysaccharides (EPS), with a focus on their isolation, identification, production, and functional characteristics. After the bacterial strain was cultured in De Man-Rogosa-Sharpe (MRS) medium containing 1% glucose at 37°C, the EPS was refined, and the highest yield of 0.85mg/mL was achieved at the 24-h incubation period. Enterococcus hirae MLG3-25-1 was found to be able to produce EPS. The study explored the microstructure of the EPS, which resembles polysaccharide sheets with smooth surfaces, through scanning electron microscope (SEM) analysis. Through Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance(NMR) analysis, the chemical composition, aligning with glycosidic bond characteristics, has been deciphered. Furthermore, the antimicrobial and antibiofilm activities against pathogenic bacteria, particularly Bacillus sp., demonstrated potential applications in combating antibiotic resistance. The EPS exhibited notable antioxidant activity (89.36% DPPH scavenging), along with high water-holding capacity (575%), emulsifying activity, and flocculation activity, suggesting its potential as a stabilizing agent in the food industry. Overall, this study provides a comprehensive characterization of Enterococcus hirae MLG3-25-1 EPS, emphasizing its diverse applications in antimicrobial, antioxidant, and food-related industries. These findings lay the groundwork for further exploration and utilization of this EPS in various sectors.

Photoprotective sulfated mannogalactan from heterotrophic Bacillus velezensis blocks UV-A mediated matrix metalloproteinase expression and nuclear DNA damage in human dermal fibroblast

Prolonged exposure of human dermal fibroblasts (HDF) to ultraviolet (UV) radiation triggers the production of reactive oxygen species by upregulating the expression of matrix metalloproteinases (MMPs), causing type-I collagen degradation and photoaging. A sulfated (1 → 3)/(1 → 4) mannogalactan exopolysaccharide (BVP-2) characterized as [→3)-α-Galp-{(1 → 4)-α-6-O-SO3-Manp}-(1 → 3)-α-6-O-SO3-Galp-(1→] was isolated from seaweed-associated heterotrophic bacterium Bacillus velezensis MTCC13097. Whole genome analysis of B. velezensis MTCC13097 (Accession number JAKYLL000000000) revealed saccharine biosynthetic gene clusters for exopolysaccharide production. BVP-2 administered cells showed noteworthy reduction in mitochondrial superoxide (∼85 %, p < 0.05) and ROS production (62 %) than those exhibited by UV-A irradiated HDF cells. Oxidative imbalance in HDF cells (after UV-A exposure) was recovered with BVP-2 treatment by significantly downregulating nitric oxide (NO) production (98.6 μM/mL, 1.9-fold) and DNA damage (⁓67 %) in comparison with UV-A induced cells (191.8 μM/mL and 98.7 %, respectively). UV-irradiated HDF cells showed a ∼30-50 % downregulation in the expression of MMPs (1, 2, and 9) following treatment with BVP-2. Considerable amount of sulfation (18 %) along with (1 → 3)/(1 → 4) glycosidic linkages in BVP-2 could be pivotal factors for down-regulation of the intracellular MMP-1, which was further supported by molecular docking and structure-activity studies. The (1 → 3)/(1 → 4)-linked bacterial exopolysaccharide (BVP-2) might be used as prospective natural lead to attenuate and mitigate UV-A-induced photoaging.

Genome-Wide Identification of GH17s Family Genes and Biological Function Analysis of SlA6 in Tomato.

Glycoside hydrolases (GHs), enzymes that break down glycosidic bonds in carbohydrates and between carbohydrates and non-carbohydrates, are prevalent in plants, animals, microorganisms, and other organisms. The tomato is a significant crop that contains the GH17 gene family. However, its role in tomatoes has yet to be fully investigated. In this study, we identified 43 GH17 genes from the tomato genome, distributed unevenly across 12 chromosomes. We further analyzed their gene structure, phylogenetic relationships, promoter elements, and expression patterns. The promoter element analysis indicated their potential roles in response to biotic and abiotic stresses as well as phytohormone effects on growth and development. The expression studies across different tomato tissues revealed that 10 genes were specifically expressed in floral organs, with SlA6 prominently expressed early during bud formation. By using CRISPR/Cas9 gene-editing technology, SlA6 knockout plants were generated. Phenotypic characterization showed that pollen viability, pollen tube germination, fruit weight, and seed number were significantly reduced in the Sla6 mutant, but the soluble solids content (TSS) was significantly higher in the Sla6 mutant, suggesting that SlA6 affects pollen development and fruit quality.

Protonation-Induced Tautomerization Lowers the Activation Barriers for N-Glycosidic Bond Cleavage of Thymidine and 5-Methyluridine

Synergistic infrared multiple photon dissociation (IRMPD) action spectroscopy experiments and electronic structure calculations revealed that both 2,4-dihydroxy tautomers and O2 protonated conformers of protonated thymidine, [dThd+H]+, and protonated 5-methyluridine, [Thd+H]+ coexist in the gas phase with the 2,4-dihydroxy tautomers dominating the population. In the current study, the kinetic energy dependence of the collision-induced dissociation behavior of [dThd+H]+ and [Thd+H]+ are examined in a guided ion beam tandem mass spectrometer. The primary dissociation pathways observed involve N-glycosidic bond cleavage leading to competitive elimination of either protonated or neutral thymine. The potential energy surfaces (PESs) for these N-glycosidic bond cleavage pathways are mapped via electronic structure calculations for the mixture of 2,4-dihydroxy tautomers and O2 protonated conformers of [dThd+H]+ and [Thd+H]+ populated in the IRMPD experiments. The activation energies and heats of reaction predicted for N-glycosidic bond cleavage at the B3LYP and MP2(full) levels of theory are compared to the measured values. The agreement between experiment and theory indicates that B3LYP provides better estimates of the energetics of the species along the PESs for N-glycosidic bond cleavage of [dThd+H]+ and [Thd+H]+ than MP2, and that the 2,4-dihydroxy tautomers, which are stabilized by strong hydrogen-bonding interactions, control the threshold dissociation behavior of [dThd+H]+ and [Thd+H]+.

Identification and characterization of Lacticaseibacillus rhamnosus HP-B1083-derived β-glucuronidase and its application for baicalin biotransformation

Baicalein, showing higher bioavailability and stronger pharmacological activity, can be obtained via a β-glucuronidase (GUS)-catalyzed transformation of baicalein 7-O-β-D-glucuronide (baicalin). Recently, we have found that the fermentation broth of Lacticaseibacillus rhamnosus HP-B1083 can efficiently convert baicalin to baicalein. In this study, the L. rhamnosus HP-B1083-derived enzyme involved in baicalin biotransformation was identified and characterized. First, the LruidA gene, encoding the responsible enzyme, was cloned and sequenced. Sequence analysis revealed that the deduced enzyme (designated as LrUidA) belonged to the glycosyl hydrolase family 2. The recombinant LrUidA was expressed and purified for characterization. LrUidA had a molecular weight of 70 kDa, with an optimal temperature of 50 °C and pH 4.5. Although LrUidA was susceptible to temperature, it possessed a relative pH stability. Its Michaelis–Menten constant, maximum reaction velocity and catalytic constant values were 9.710 mM, 13.08 mM/min/mg, and 14.95 s−1, respectively. Site-directed mutagenesis experiment results demonstrated that the enzyme reaction uses side chains of E509 and E415 to hydrolyze the glycosidic bond of baicalin and involves three negatively charged residues, E450, D451, and D452, respectively. Surprisingly, biotransformation was performed under optimized reaction conditions by incubating the purified enzyme with 0.1 % baicalin for 4 h, resulting in a considerable conversion ratio of 99 %. Altogether, our findings provide insights into the properties of L. rhamnosus HP-B1083-derived enzyme and expand our understanding regarding using GUS for the industrial production of baicalein.

Enhanced saccharification levels of corn starch using as a strategy a novel amylolytic complex (AmyHb) from the thermophilic fungus Humicola brevis var. thermoidea in association with commercial enzyme.

Amylases represent a versatile group of catalysts that are used for the saccharification of starch because they can hydrolyze the glycosidic bonds of starch molecules to release glucose, maltose, and short-chain oligosaccharides. The amylolytic complex of the thermophilic filamentous fungus Humicola brevis var. thermoidea (AmyHb) was produced, biochemically characterized, and compared with the commercial amylase Termamyl. In addition, the biotechnological application of AmyHb in starch saccharification was investigated. The highest production was achieved using a wheat bran medium at 50°C for 5-6days in solid-state fermentation (849.6 ± 18.2U·g-1) without the addition of inducers. Optimum amylolytic activity occurred at pH 5.0 at 60°C, and stability was maintained between pH 5.0 and 6.0, with thermal stability at 50-60°C, especially in the presence of Ca2+. These results were superior to those found with Termamyl. Both enzymes were strongly inhibited by Hg2+, Cu2+, and Ag+; however, AmyHb displayed increased activity in the presence of Mn2+ and Na+. In addition, AmyHb showed greater tolerance to a wide range of ethanol concentrations. AmyHb appears to be a complex consisting of glucoamylase and α-amylase, based on its substrate specificity and TLC. The hydrolysis tests on cornstarch flour showed that the cocktail of AmyHb50% + Termamyl50% significantly increased the release of glucose and total reducing sugars (36.6%) when compared to the enzymes alone. AmyHb exhibited promising physicochemical properties and good performance with commercial amylase; therefore, this complex is a biotechnological alternative candidate for the bioprocessing of starch sources.

Facilely tuning the triazine-heptazine ratios in crystalline g-C3N4 for efficient photo-degradation of tylosin

Intrinsically, g-C3N4 has high-density defects, insufficient efficiencies of photogenerated charge carrier separation and transfer, and inadequate photocatalytic ability against antibiotics. A facile crystallinity engineering on g-C3N4 assisted by molten-salts and simultaneously fine-tuning the triazine-heptazine ratio led to forming type-II homojunctions, without introducing foreign atoms and being metal-free. It was successfully achieved through modulating the calcination temperatures and using KCl-LiCl. The salts accelerated deamination reaction kinetics, tuned the unit ratio, and improved the degree of carbon nitride polymerization. At a triazine-heptazine ratio of 74:26 obtained at 450 °C, 10 mg/L of tylosin was completely mitigated within 9 min by the catalyst. Active radicals including O2− and OH were boosted from charge carriers under visible light, and the recombination of photogenerated excitons was relieved on the interfaces of the homojunctions. Breaking glycosidic bonds and dropping off deoxyglycosyl moieties are considered the main degradation mechanism. The cytotoxicity and acute toxicity of the degradation product are classified as harmless. In addition, 3D-molecular docking between the transformation products and target proteins (L4 and L22) in ribosome confirms toxicity reduction. This work paves a way for efficient and harmless photocatalytic degradation of tylosin by facilely modulating the unit ratio and enhancing the crystallinity of carbon nitride without introducing foreign atoms.

Effects of the Lactobacillus fermentation on Dendrobium officinale polysaccharide: Structural, physicochemical properties and in vitro bioactivities

Structural modification of polysaccharides using microbial fermentation is of great interest due to its specificity and environmental friendliness. The aim of this research was to compare structure, physicochemical properties and in vitro bioactivities of the Dendrobium officinale polysaccharides before and after fermentation using Lactobacillus paracasei and Lactobacillus acidophilus. The main fractions of crude polysaccharides from unfermented Dendrobium officinale (DOP-1A) and fermented Dendrobium officinale (FDOP-1A) were obtained by column chromatography purification. Compare to DOP-1A, the molecular weight of FDOP-1A was significantly reduced (from 412.566 to 25.161 kDa), smaller particle size (from 21.05 to 19.35 μm) and lower apparent viscosity. Although the microscopic surface morphologies of DOP-1A and FDOP-1A were significantly different, neither formed a triple-helix structure. Additionally, FDOP-1A contained more t-Manp and less 4-Manp, and interestingly the (1 → 4)-β-D-Glcp glycosidic bond site between the repeat units →4)-2-O-acetyl-β-D-Manp-(1 → 4)-β-D-Glcp-(1→ and →4)-β-D-Manp-(1 → 4)-β-D-Glcp-(1→ was cleaved. Moreover, FDOP-1A exhibited excellent scavenging capacity for ABTS+, DPPH, hydroxyl radicals and inhibited Nitric Oxide production by RAW 264.7 macrophages, thereby demonstrating effective antioxidant and anti-inflammatory effects. In conclusion, Lactobacillus fermentation could improve physicochemical properties and enhance biological activities of Dendrobium officinale polysaccharide, which has potential prospects for development and application in dietary supplements.

Bimodal substrate binding in the active site of the glycosidase BcX.

Bacillus circulans xylanase (BcX) from the glycoside hydrolase family 11 degrades xylan through a retaining, double-displacement mechanism. The enzyme is thought to hydrolyze glycosidic bonds in a processive manner and has a large, active site cleft, with six subsites allowing the binding of six xylose units. Such an active site architecture suggests that oligomeric xylose substrates can bind in multiple ways. In the crystal structure of the catalytically inactive variant BcX E78Q, the substrate xylotriose is observed in the active site, as well as bound to the known secondary binding site and a third site on the protein surface. Nuclear magnetic resonance (NMR) titrations with xylose oligomers of different lengths yield nonlinear chemical shift trajectories for active site nuclei resonances, indicative of multiple binding orientations for these substrates for which binding and dissociation are in fast exchange on the NMR timescale, exchanging on the micro- to millisecond timescale. Active site binding can be modeled with a 2 : 1 model with dissociation constants in the low and high millimolar range. Extensive mutagenesis of active site residues indicates that tight binding occurs in the glycon binding site and is stabilized by Trp9 and the thumb region. Mutations F125A and W71A lead to large structural rearrangements. Binding at the glycon site is sensed throughout the active site, whereas the weak binding mostly affects the aglycon site. The interactions with the two active site locations are largely independent of each other and of binding at the secondary binding site.

Volatile-char interactions during biomass pyrolysis: Effects of functionalized graphitized carbon nanotubes on volatile distribution of cellulose and its model compounds

Volatile-char interactions are prevalent in biomass pyrolysis processes. To gain deeper insights into their impact on biomass pyrolysis volatile distribution, interactions between volatile compounds and char during flash pyrolysis of glucose, cellobiose, and cellulose were investigated using a pyrolysis–gas chromatography/mass spectrometry detection technique, by modifying both the chain lengths of the raw materials and the functional groups of graphitized multi-walled carbon nanotubes (CNTs). The results reveal that volatile-char interactions markedly influence the distribution of the final pyrolysis products derived from glucose-based compounds. In the presence of any type of CNTs, dehydrated saccharides in the primary products, particularly levoglucosan (LG), exhibit the highest sensitivity to the interactions. Consequently, primary products such as LG and 5-hydroxymethylfurfural (HMF) undergo secondary pyrolysis, yielding compounds such as levoglucosenone (LGO) and 5-methyl-2-furancarboxaldehyde (FCM). Additionally, CNTs without functional groups are advantageous for the production of expensive LGO (The relative abundance reached 9.65%), whereas the relative abundance of FCM increases significantly with hydroxyl-, carboxyl-, and amino-modified carbon nanotubes. Particularly, the relative abundance of FCM is highest in the presence of amino-modified carbon nanotubes, regardless of whether the feedstock is glucose, cellobiose, or cellulose, with values of 25.14%, 23.32%, and 4.23%, respectively. Furthermore, consistent changes in product distribution trends among different glucose-based compounds under identical CNT conditions suggest that glycosidic bonds have minimal impact on volatile-char interactions.

Research progress in biosynthesis of arbutin

Arbutin, a glycosylated compound of hydroquinone, exists in two forms of β-arbutin and α-arbutin based on the configuration of the glycosidic bond. As a safe and stable whitening agent, arbutin is widely used in cosmetics, and it has antioxidant, antimicrobial, anti-inflammatory, and anti-tumor activities. The production of arbutin by plant extraction faces challenges such as long plant growth periods, complex extraction processes, and low yields. The chemical synthesis of arbutin suffers from harsh reaction conditions, poor stereo-selectivity, and low yields. In recent years, biosynthesis emerges as the most popular method to produce arbutin because of the simple and mild reaction conditions, low costs, and environmental friendliness. This review summarizes the research progress in four biosynthetic strategies for arbutin, including plant conversion, enzyme catalysis, whole-cell catalysis, and microbial fermentation. The advantages and limitations of these biosynthetic strategies are discussed, and future research directions are proposed.

The dissolution of galactomannans in type II/III deep eutectic solvents: Effect of polysaccharide structure and interaction mechanism

Deep eutectic solvents (DESs) have garnered increasing attention as sustainable solvents for natural polysaccharides. Galactomannans (especially guar and locust bean gums) are abundant natural compounds used in food, medicine, agriculture and daily chemical products, whereas their high molecular weight and randomly distributed non-linear structures remain a huge challenge in solubilization and high value-added utilization. In this study, the dissolution of galactomannans in type II/III DESs was comprehensively investigated using density functional theory (DFT) calculation, experimental results, and molecular dynamics simulation (MD). The results indicated that immense anionic and neutral H-bonds supported by type II DESs (consisting of a quaternary ammonium salt and a metal chloride hydrate) could break and rebuild galactomannan H-bond networks, thereby exhibiting remarkable physical solubilization. A large number of active acid sites in type III DESs (consisting of a quaternary ammonium salt and a hydrogen bond donor) greatly improved solubility and cleaved the glycosidic bond, enabling the recovery of polysaccharides with a target molecular weight. Furthermore, significant correlations between solubility and the structure of galactomannans had been established, which meant that high molecular weight and rich galactose side-chains produced repulsive interactions with DESs, directly hindering dissolution. Overall, this work not only offered a framework for enhancing the dissolution of polysaccharides, but also proposed a promising strategy for development of task specific DESs promoting green chemistry and the use of sustainable bioresources.

Purinyl N-directed aroylation of 6-arylpurine ribo- and 2'-deoxyribonucleosides, and mechanistic insights.

The purinyl ring contains four embedded nitrogen atoms of varying basicities. Selective utilization of these ring nitrogen atoms can lead to relatively facile remote functionalization, yielding modified purinyl motifs that are otherwise not easily obtained. Herein, we report previously undescribed N-directed aroylation of 6-arylpurine ribo and the more labile 2'-deoxyribonucleosides. Kinetic isotope analysis as well as reaction with a well-defined dimeric, palladated 9-benzyl 6-arylpurine provided evidence for N-directed cyclometallation as a key step, with a plausible rate-limiting C-H bond cleavage. Radical inhibition experiments indicate the likely intermediacy of aroyl radicals. The chemistry surmounts difficulties often posed in the functionalization of polynitrogenated and polyoxygenated nucleosidic structures that possess complex reactivities and a labile glycosidic bond that is more sensitive in the 2'-deoxy substrates.

Combined physicochemical and transcriptomic analyses reveal the effect of the OsGA20ox1 gene on the starch properties of germinated brown rice

Genes play a pivotal role in regulating the germination of cereal grains; however, there is limited research on the impact of germination genes on the physicochemical properties of germinated cereal starch. We investigated the effects of the OsGA20ox1 gene on the multiscale structural features and adhesion behavior of germinated brown rice starch. Compared to the knockout lines group, the wild type exhibited a decrease in double-helix content (62.74 %), relative crystallinity (47.39 %), and short-range molecular ordering (2.47 %), accompanied by enhanced erosion on the surface of starch granules. The damage to glycosidic bonds at the double-helix level and the heightened structural amorphization (90.95 %) led to reduced entanglement and interaction among starch molecules, ultimately resulting in reduced characteristic viscosity. Further transcriptomic analysis revealed that OsGA20ox1 could regulate the expression of starch-related enzyme genes in the starch metabolism pathway during germination of brown rice. This study contributes to understanding the role of germination genes in promoting the physicochemical properties of starch in germinated grains, thereby opening up new avenues for the improvement of plant-based starch, and paving the way for further research in this field.

A Multimodal System for Lipid A Structural Analysis from a Single Colony.

Structural elucidation of Gram-negative bacterial lipid A traditionally requires chemical extraction followed by tandem MS data in the negative ion mode. Previously, we reported FLAT and FLATn as methods to rapidly determine the structure of lipid A without chromatographic techniques. In this work, we extend the capability and effectiveness of these techniques to elucidate the chemical structure in a de novo manner by including the use of positive ion mode (FLAT+ and FLATn+) spectral approaches. Advantages of positive mode analysis of lipid A include the generation of more interpretable and informative fragmentation patterns that include the identification of diagnostic fragments, including selective dissociation of a glycosidic bond between two glucosamine units and the selective dissociation at the secondary acyl chain in 2'-N, allowing for the determination of the composition of fatty acids. As a proof of principle, we present here two previously uncharacterized structures of lipid A from Roseomonas mucosa (R. mucosa) and Moraxella canis (M. canis). In R. mucosa, we determined the lipid A structure with a nonconventional backbone of-β-1,6 linked 2,3-dideoxy-2,3-diamno-d-glucopyranose further modified with galacturonic acid in the place of typical 1-phosphate, and in M. canis, we assigned a single discrete structure using the specific fragmentation patterns of terminal phosphate groups present in lipid A. Therefore, FLATn+, in combination with FLAT and FLATn, provides a multimodal structural platform for rapid structure characterization of unusual and complex lipid A structures from a single colony.

Influence of the backbone chemistry and ionic functional groups of five pairs of oppositely charged polyelectrolytes on complex coacervation

Complex coacervation plays an important role in various fields. Here, the influences of the backbone chemistry and ionic functional groups of five pairs of oppositely charged polyelectrolytes on complex coacervation were investigated. These pairs include synthetic polymers with aliphatic hydrocarbon backbones, peptides with amide bonds, and carbohydrates with glycosidic linkages. Despite sharing identical charged groups, specific pairs displayed distinct liquid/liquid and liquid/solid phase separations depending on the polyelectrolyte mixing ratio, buffer, and ionic strength. The coacervate phase boundary broadened in the orders: glycosidic linkages > amide backbone > aliphatic hydrocarbon backbone, and Tris-phosphate > Tris-acetate > Tris-chloride buffers. Coacervates prepared from polyelectrolytes with lower solubilities in water resisted disassembly at high salt concentrations, and their merge rate was slow. These observations suggest that the hydrophobic segments in polyelectrolytes interfere with the formation of complex coacervates; however, following coacervate formation, the hydrophobic segments render the coacervates stable and elastic.