Innovative direct introduction-ion mobility-mass spectrometry (DI-IM-MS) approach for fast and robust isomer-specific quantification in a complex matrix: Application to 2'-fucosyllactose (2'-FL) in breast milk.

Identification and specific quantification of isomers in a complex biological matrix by mass spectrometry alone is not an easy task due to their identical chemical formula and therefore their same mass-to-charge ratio (m/z). Here, the potential of direct introduction combined with ion mobility-mass spectrometry (DI-IM-MS) for rapid quantification of isomers as human milk oligosaccharides (HMOs) was investigated. Differences in HMO profiles between various analyzed breast milk samples were highlighted using the single ion mobility monitoring (SIM2) acquisition for high ion mobility resolution detection. Furthermore, the Se+ (secretor) or Se- (non-secretor) phenotype could be assigned to breast milk samples studied based on their HMO contents, especially on the response of 2'-fucosyllactose (2'-FL) and lacto-N-fucopentaose I (LNFP I). The possibility of quantifying a specific isomer in breast milk by DI-IM-MS was also investigated. The standard addition method allowed the determination of the 2'-FL despite the presence of other oligosaccharides, including 3-fucosyllactose (3-FL) isomer in breast milk. This proof-of-concept study demonstrated the high potential of such an approach for the rapid and convenient quantification of isomers in complex mixtures.

Isolation of Human Milk Difucosyl Nona- and Decasaccharides by Ultrahigh-Temperature Preparative PGC-HPLC and Identification of Novel Difucosylated Heptaose and Octaose Backbones by Negative-Ion ESI-MSn.

Despite their many important physiological functions, past work on the diverse sequences of human milk oligosaccharides (HMOs) has been focused mainly on the highly abundant HMOs with a relatively low degree of polymerization (DP) due to the lack of efficient methods for separation/purification and high-sensitivity sequencing of large-sized HMOs with DP ≥ 10. Here we established an ultrahigh-temperature preparative HPLC based on a porous graphitized carbon column at up to 145 °C to overcome the anomeric α/ß splitting problem and developed further the negative-ion ESI-CID-MS/MS into multistage MSn using a combined product-ion scanning of singly charged molecular ion and doubly charged fragment ion of the branching Gal and adjacent GlcNAc residues. The separation and sequencing method allows efficient separation of a neutral fraction with DP ≥ 10 into 70 components, among which 17 isomeric difucosylated nona- and decasaccharides were further purified and sequenced. As a result, novel branched difucosyl heptaose and octaose backbones were unambiguously identified in addition to the conventional linear and branched octaose backbones. The novel structures of difucosylated DF-novo-heptaose, DF-novo-LNO I, and DF-novo-LNnO I were corroborated by NMR. The various fucose-containing Lewis epitopes identified on different backbones were confirmed by oligosaccharide microarray analysis.

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.

Potential biological functions and future perspectives of sialylated milk oligosaccharides.

Sialyllactoses (SLs) primarily include sialylated human milk oligosaccharides (HMOs) and bovine milk oligosaccharides (BMOs). First, the safety assessment of 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) revealed low toxicity in various animal models and human participants. SLs constitute a unique milk component, highlighting the essential nutrients and bioactive components crucial for infant development, along with numerous associated health benefits for various diseases. This review explores the safety, biosynthesis, and potential biological effects of SLs, with a specific focus on their influence across various physiological systems, including the gastrointestinal system, immune disorders, rare genetic disorders (such as GNE myopathy), cancers, neurological disorders, cardiovascular diseases, diverse cancers, and viral infections, thus indicating their therapeutic potential.

Evaluation of Prebiotic Glycan Composition in Human Milk and Infant Formula: Profile of Galacto-Oligosaccharides and Absolute Quantification of Major Milk Oligosaccharides by UPLC-Cyclic IM-MS and UPLC-MS/MS.

Prebiotic oligosaccharides have attracted immense interest in the infant formula (IF) industry due to their unique health benefits for infants. There is a need for the reasonable supplementation of prebiotics in premium IF products. Herein, we characterized the profile of galacto-oligosaccharides (GOS) in human milk (HM) and IF using ultrahigh-performance liquid chromatography-cyclic ion mobility-mass spectrometry (UPLC-cIM-MS) technique. Additionally, we further performed a targeted quantitative analysis of five essential HM oligosaccharides (HMOs) in HM (n = 196), IF (n = 50), and raw milk of IF (n = 10) by the high-sensitivity UPLC-MS/MS method. HM exhibited a more abundant and variable HMO composition (1183.19 to 2892.91 mg/L) than IF (32.91 to 56.31 mg/L), whereas IF contained extra GOS species and non-negligible endogenous 3'-sialyllactose. This also facilitated the discovery of secretor features within the Chinese population. Our study illustrated the real disparity in the prebiotic glycome between HM and IF and provided crucial reference for formula improvement.

Formation and immunological evaluation of Moraxella catarrhalis glycoconjugates based on synthetic oligosaccharides.

Published work has shown that glycoconjugate vaccines, based on truncated detoxified lipopolysaccharides from Moraxella catarrhalis attached through their reducing end to a carrier protein, gave good protection for all three serotypes A, B, and C in mice immunisation experiments. The (from the non-reducing end) truncated LPS structures were obtained from bacterial glycosyl transferase knock-out mutants and contained the de-esterified Lipid A, two Kdo residues and five glucose moieties. This work describes the chemical synthesis of the same outer Moraxella LPS structures, spacer-equipped and further truncated from the reducing end, i.e., without the Lipid A part and containing four or five glucose moieties or four glucose moieties and one Kdo residue, and their subsequent conjugation to a carrier protein via a five­carbon bifunctional spacer to form glycoconjugates. Immunisation experiments both in mice and rabbits of these gave a good antibody response, being 2-7 times that of pre-immune sera. However, the sera produced only recognized the immunizing glycan immunogens and failed to bind to native LPS or whole bacterial cells. Comparative molecular modelling of three alternative antigens shows that an additional (2 â†’ 4)-linked Kdo residue, not present in the synthetic structures, has a significant impact on the shape and volume of the molecule, with implications for antigen binding and cross-reactivity.

Unlocking the mysteries of milk oligosaccharides: Structure, metabolism, and function.

Milk oligosaccharides (MOs), complex carbohydrates prevalent in human breast milk, play a vital role in infant nutrition. Serving as prebiotics, they inhibit pathogen adherence, modulate the immune system, and support newborn brain development. Notably, MOs demonstrate significant variations in concentration and composition, both across different species and within the same species. These characteristics of MOs lead to several compelling questions: (i) What distinct beneficial functions do MOs offer and how do the functions vary along with their structural differences? (ii) In what ways do MOs in human milk differ from those in other mammals, and what factors drive these unique profiles? (iii) What are the emerging applications of MOs, particularly in the context of their incorporation into infant formula? This review delves into the structural characteristics, quantification methods, and species-specific concentration differences of MOs. It highlights the critical role of human MOs in infant growth and their potential applications, providing substantial evidence to enhance infant health and development.

Advances in the preparation, characterization, and biological functions of chitosan oligosaccharide derivatives: A review.

Chitosan oligosaccharide (COS), which represent the positively charged basic amino oligosaccharide in nature, is the deacetylated and degraded products of chitin. COS has become the focus of intensive scientific investigation, with a growing body of practical and clinical studies highlighting its remarkable health-enhancing benefits. These effects encompass a wide range of properties, including antibacterial, antioxidant, anti-inflammatory, and anti-tumor activities. With the rapid advancements in chemical modification technology for oligosaccharides, many COS derivatives have been synthesized and investigated. These newly developed derivatives possess more stable chemical structures, improved biological activities, and find applications across a broader spectrum of fields. Given the recent interest in the chemical modification of COS, this comprehensive review seeks to consolidate knowledge regarding the preparation methods for COS derivatives, alongside discussions on their structural characterization. Additionally, various biological activities of COS derivatives have been discussed in detail. Lastly, the potential applications of COS derivatives in biomedicine have been reviewed and presented.

Preparation, characterization, antioxidant, and antifungal activity of phenyl/indolyl-acyl chitooligosaccharides.

In this study, carboxylic acids compounds were grafted onto chitooligosaccharides to prepare seven phenyl/indolyl-acyl chitooligosaccharides derivatives. The structures of the derivatives were characterized by IR spectroscopy, 13C NMR and elemental analysis. Meanwhile, antioxidant activities in vitro of the novel derivatives were analyzed. Compared to COS and carboxylic acid, the derivatives showed higher scavenging capacity for superoxide anion and DPPH radicals, with scavenging rates of 59.39% and 94.86%, respectively. The hydroxyl radical scavenging ability of the derivatives was only 18.89%. The antifungal activities of chitooligosaccharide derivatives against Diaporthe batatas and Phytophthora capsici were studied by the growth rate method. Compared with chitooligosaccharide itself, derivatives were inhibited by 97.77% and 100%. The above results showed that chitooligosaccharide derivatives have good biocompatibility and can be used in food, agriculture and medicine.

Chemical Synthesis of Δ-4,5 Unsaturated Heparan Sulfate Oligosaccharides for Biomarker Discovery.

A methodology is described that can provide heparan sulfate oligosaccharides having a Δ4,5-double bond, which are needed as analytical standards and biomarkers for mucopolysaccharidoses. It is based on chemical oligosaccharide synthesis followed by modification of the C-4 hydroxyl of the terminal uronic acid moiety as methanesulfonate. This leaving group is stable under conditions used to remove temporary protecting groups, O-sulfation, and hydrogenolysis. Treatment with NaOH results in elimination of the methanesulfonate and formation of a Δ4,5-double bond.

Chromatography based profiling of feruloylated arabinans and galactans.

Profiling of pectic arabinans and galactans by analysis of the released oligosaccharides after backbone cleavage provides information on the complexity of the polymer structure. In plants of the family Amaranthaceae, arabinan and galactan substitution with ferulates extends the polysaccharide complexity, changing its chemical properties. Knowledge of the ferulate environment is crucial to understand structure-function-relationships of feruloylated pectins. Here, we present an approach to separate enzymatically generated feruloylated and non-feruloylated arabino- and galactooligosaccharides, followed by deesterification and semiquantitative analysis by HPAEC-PAD using previously reported relative response factors. Application of this approach to sugar beet pectins and insoluble and soluble dietary fiber preparations of amaranth and quinoa suggests that ferulates are preferably incorporated into more complex structures, as nicely demonstrated for feruloylated galactans. Also, ferulate substitution appears to negatively affect enzymatic cleavage by using endo-enzymes. As a consequence, we were able to tentatively identify new feruloylated tri- and tetrasaccharides of galactans isolated from sugar beet pectins.

Enzymatic modular synthesis of asymmetrically branched human milk oligosaccharides.

Human milk oligosaccharides (HMOs) are intricate glycans that promote healthy growth of infants and have been incorporated into infant formula as food additives. Despite their importance, the limited availability of asymmetrically branched HMOs hinders the exploration of their structure and function relationships. Herein, we report an enzymatic modular strategy for the efficient synthesis of these HMOs. The key branching enzyme for the assembly of branched HMOs, human ß1,6-N-acetylglucosaminyltransferase 2 (GCNT2), was successfully expressed in Pichia pastoris for the first time. Then, it was integrated with six other bacterial glycosyltransferases to establish seven glycosylation modules. Each module comprises a one-pot multi-enzyme (OPME) system for in-situ generation of costly sugar nucleotide donors, combined with a glycosyltransferase for specific glycosylation. This approach enabled the synthesis of 31 branched HMOs and 13 linear HMOs in a stepwise manner with well-programmed synthetic routes. The binding details of these HMOs with related glycan-binding proteins were subsequently elucidated using glycan microarray assays to provide insights into their biological functions. This comprehensive collection of synthetic HMOs not only serves as standards for HMOs structure identification in complex biological samples but also significantly enhances the fields of HMOs glycomics, opening new avenues for biomedical applications.

Impacts of monosaccharide composition on immunomodulation by cello-pentaose, manno-pentaose, and xylo-pentaose: Unraveling the underlying molecular mechanisms.

Different types of functional oligosaccharides exhibit varying degrees of immune-enhancing effects, which might be attributable to differences in their glycosyl structures. The differences in the immunomodulatory action of three functional oligosaccharides with distinct glycosyl compositions: cello-oligosaccharides (COS), manno-oligosaccharides (MOS), and xylo-oligosaccharides (XOS), were investigated in mouse-derived macrophage RAW264.7. Moreover, the immune enhancement mechanism of oligosaccharides with diverse glycosyl compositions was investigated from a molecular interaction perspective. The TLR4-dependent immunoregulatory effect of functional oligosaccharides was shown by measuring the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in RAW264.7 cells treated with different functional oligosaccharides, both with and without Resatorvid [TAK-242] (a Toll-like receptor 4 [TLR4] inhibitor). Western blot analysis showed that binding of the three oligosaccharides to TLR4 activated the downstream signaling pathway and consequently enhanced the immune response. The fluorescence spectra and molecular docking results revealed that the main mechanisms by which these oligosaccharides attach to the TLR4 active pocket are hydrogen bonds and van der Waals forces. Functional oligosaccharides were ranked according to their affinity for TLR4, as follows: MOS > COS > XOS, indicating that oligosaccharides or polysaccharides containing mannose units may confer significant advantages for immune enhancement.

Characterization of a GH20 ß-N-Acetylhexosaminidase from Flavobacterium algicola Suitable to Synthesize Lacto-N-triose II.

ß-N-Acetylhexosaminidases have attracted much attention in the enzymatic synthesis of lacto-N-triose II (LNT2) as a backbone precursor of human milk oligosaccharides (HMOs). In this study, a novel glycoside hydrolase (GH) 20 family ß-N-acetylhexosaminidase, FlaNag2353, from Flavobacterium algicola was biochemically characterized and applied to synthesize LNT2. FlaNag2353 displayed optimal activity to p-nitrophenyl N-acetyl-ß-d-glucosaminide (pNP-GlcNAc) at 40 °C and pH 8.0. In addition to its excellent hydrolysis activity toward pNP-GlcNAc and chitooligosaccharides, FlaNag2353 showed trans-glycosylation activity. Under conditions of pH 9.0 and 55 °C for 2 h and utilizing 200 mM lactose and 10 mM pNP-GlcNAc, FlaNag2353 synthesized LNT2 with a conversion ratio of 4.15% calculated from pNP-GlcNAc. Moreover, when applied to LNT2 synthesis with 10 mM pNP-GlcNAc and 9.7% (w/v) industrial waste whey powder, FlaNag2353 achieved a conversion ratio of 2.39%. This study has significant implications for broadening the applications of GH20 ß-N-acetylhexosaminidases and promoting the high-value utilization of whey powder.

Effect of alkali treatment on enzymatic hydrolysis of p-toluenesulfonic acid pretreated bamboo substrates.

The comprehensive separation and utilization of whole components of lignocellulosic materials has received extensive attention in present research. This study focused on the efficacy of alkali treatment for enzymatic saccharification of cellulose based on p-toluenesulfonic acid (p-TsOH) pretreated bamboo substrate. The results showed that the cellulose to glucose conversion yield was 94.69 % under optimized conditions of 0.4 g NaOH/g, 160 °C and 4 h (soaked), which after only 6 h enzymatic hydrolysis time. Alkali lignin recovery was 88.51 %, with potential for conversion to lignin derivatives. The yield of hemicellulose in the pretreated filtrate was 51.85 % after the 4th cycling reuse of p-TsOH. This work has borrowed the advantages of p-TsOH pretreatment of depolymerized hemicellulose from bamboo, combined with a low-priced weak alkali secondary treatment method, which can be effectively applied to the co-production of lignin, xylooligosaccharide, xylose and glucose, and the whole process is green and economically sustainable.

Gram-scale chemical synthesis of galactosyllactoses and their impact on infant gut microbiota in vitro.

Galactooligosaccharides (GOS) are widely used as a supplement in infant nutrition to mimic the beneficial effects found in prebiotic human milk oligosaccharides (HMOs). However, the complexity of the GOS mixture makes it challenging to ascertain which of the GOS components contribute most to their health benefits. Galactosyllactoses (GLs) are lactose-based trisaccharides containing a ß-galactopyranosyl residue at the 3'-position (3'galactosyllactose, 3'-GL), 4'-position (4'-galactosyllactose, 4'-GL), or the 6'-position (6'-galactosyllactose, 6'-GL). These GLs are of particular interest as they are present in both GOS mixtures and human milk at early stages of lactation. However, research on the potential health benefits of these individual GLs has been limited. Gram quantities are needed to assess their health benefits but these GLs are not readily available at this scale. In this study, we report the gram-scale chemical synthesis of 3'-GL, 4'-GL, and 6'-GL. All three galactosyllactoses were obtained on a gram scale in good purity from cheap and commercially available lactose. Furthermore, in vitro incubation of GLs with infant faecal microbiota demonstrates that the GLs were able to increase the abundance of Bifidobacterium and stimulate short chain fatty acid production.

The evolving world of milk oligosaccharides: Biochemical diversity understood by computational advances.

Milk oligosaccharides, complex carbohydrates unique to mammalian milk, play crucial roles in infant nutrition and immune development. This review explores their biochemical diversity, tracing the evolutionary paths that have led to their variation across different species. We highlight the intersection of nutrition, biology, and chemistry in understanding these compounds. Additionally, we discuss the latest computational methods and analytical techniques that have revolutionized the study of milk oligosaccharides, offering insights into their structural complexity and functional roles. This brief but essential review not only aims to provide a deeper understanding of milk oligosaccharides but also discuss the road toward their potential applications.

Crystal structure of mango α1,3/α1,4-fucosyltransferase elucidates unique elements that regulate Lewis A-dominant oligosaccharide assembly.

In various organisms, α1,3/α1,4-fucosyltransferases (CAZy GT10 family enzymes) mediate the assembly of type I (Galß1,3GlcNAc) and/or type II (Galß1,4GlcNAc)-based Lewis structures that are widely distributed in glycoconjugates. Unlike enzymes of other species, plant orthologues show little fucosyltransferase activity for type II-based glycans and predominantly catalyze the assembly of the Lewis A structure [Galß1,3(Fucα1,4)GlcNAc] on the type I disaccharide unit of their substrates. However, the structural basis underlying this unique substrate selectivity remains elusive. In this study, we investigated the structure-function relationship of MiFUT13A, a mango α1,3/α1,4-fucosyltransferase. The prepared MiFUT13A displayed distinct α1,4-fucosyltransferase activity. Consistent with the enzymatic properties of this molecule, X-ray crystallography revealed that this enzyme has a typical GT-B fold-type structure containing a set of residues that are responsible for its SN2-like catalysis. Site-directed mutagenesis and molecular docking analyses proposed a rational binding mechanism for type I oligosaccharides. Within the catalytic cleft, the pocket surrounding Trp121 serves as a binding site, anchoring the non-reducing terminal ß1,3-galactose that belongs to the type I disaccharide unit. Furthermore, Glu177 was postulated to function as a general base catalyst through its interaction with the 4-hydroxy group of the acceptor N-acetylglucosamine residue. Adjacent residues, specifically Thr120, Thr157 and Asp175 were speculated to assist in binding of the reducing terminal residues. Intriguingly, these structural elements were not fully conserved in mammalian orthologue which also shows predominant α1,4-fucosyltransferase activity. In conclusion, we have proposed that MiFUT13A generates the Lewis A structure on type I glycans through a distinct mechanism, divergent from that of mammalian enzymes.

Separation, purification and structural characterization of marine oligosaccharides: A comprehensive and systematic review of chromatographic methods.

Marine oligosaccharides have now been applied in a wide range of industry due to various kinds of physiological activities. However, the oligosaccharides with different polymeric degrees (Dps) differed in physiological activities and applicable fields. So it is promising and essential to separate, purify and structurally characterize these oligosaccharides for understanding their structure-function relationship. This review will summarize the lasted developments in the separation, purification and structural characterization of marine oligosaccharides, including the alginate oligosaccharides, carrageenan oligosaccharides, agar oligosaccharides, chitin oligosaccharides and chitosan oligosaccharides, emphasizing the successful examples of methods for separation and purification. Furthermore, an outlook for preparation of functional oligosaccharides in food biotechnology and agriculture fields is also included. This comprehensive review could definitely promote the utilization of marine functional polysaccharides for food and agriculture.

Total Synthesis of Vibrio Cholerae O43 Tetrasaccharide Repeating Unit.

Vibrio cholerae is a pathogen responsible for the deadly pandemic - cholera. The glycans present on the surface of various strains of V. cholerae are considered as potential vaccine candidates. The tetrasaccharide repeating unit (RU) of V. cholerae O43 is decorated with less-explored rare deoxy amino sugars like d-quinosamine and d-viosamine, along with a rare amino acid, N-acetyl-l-allothreonine. Herein, we report a detailed account of the total synthesis of V. cholerae O43 tetrasaccharide RU. In our earlier attempt, while a one-pot assembly of trisaccharide was successful, the final coupling with a fully functionalized d-viosamine donor was low yielding. The successful route involved employing the Fmoc-protected d-viosamine building block as a donor and a late-stage amide bond formation of the tetrasaccharide.