Oligosaccharide Structures Research Articles

NMR Analyses of the Enzymatic Degradation End-Products of Diabolican: The Secreted EPS of Vibrio diabolicus CNCM I-1629.

Diabolican, or HE800, is an exopolysaccharide secreted by the non-pathogenic Gram-negative marine bacterium Vibrio diabolicus (CNCM I-1629). This polysaccharide was enzymatically degraded by the Bacteroides cellulosilyticus WH2 hyaluronan lyase. The end products were purified by size-exclusion chromatography and their structures were analyzed in depth by nuclear magnetic resonance (NMR). The oligosaccharide structures confirmed the possible site of cleavage of the enzyme showing plasticity in the substrate recognitions. The production of glycosaminoglycan-mimetic oligosaccharides of defined molecular weight and structure opens new perspectives in the valorization of the marine polysaccharide diabolican.

Coupling an ion chromatography to high resolution mass spectrometry (IC-MS) for the discovery of potentially prebiotic oligosaccharides in Chardonnay grape marc

Oligosaccharides are carbohydrates made of three to twenty monosaccharide units linked through glycosidic bonds. Emerging research into the potential prebiotic activity of oligosaccharides is creating opportunities to use industrial byproducts as value-added products. Grape marc is a residue left after winemaking and has been shown to provide health benefits to humans. In this study, we analyzed the oligosaccharides in Chardonnay grape marc by utilizing a hyphenated platform in which an ion chromatography (IC) system is coupled to an Orbitrap mass spectrometer (MS). With this platform, we obtained a structural library including 32 oligosaccharides with unique compositions of monosaccharides and 61 oligosaccharide structures. Notably, the ion chromatographic separation provided resolution of charged isomers while maintaining separation capacity for small, neutral oligosaccharides. High-quality tandem MS also facilitated the identification of oligosaccharides with structural modifications including methylation and the presence of sugar alditols and hexuronic acids. The data acquired by the IC-MS system were also compared with previously published LC-MS data. We found that these two platforms are largely complementary and, in combination, provide a more comprehensive characterization of oligosaccharides than either platform achieves alone.

Cyclodextrin-Based Polymeric Materials Bound to Corona Protein for Theranostic Applications.

Cyclodextrins (CDs) are cyclic oligosaccharide structures that could be used for theranostic applications in personalized medicine. These compounds have been widely utilized not only for enhancing drug solubility, stability, and bioavailability but also for controlled and targeted delivery of small molecules. These compounds can be complexed with various biomolecules, such as peptides or proteins, via host-guest interactions. CDs are amphiphilic compounds with water-hating holes and water-absorbing surfaces. Architectures of CDs allow the drawing and preparation of CD-based polymers (CDbPs) with optimal pharmacokinetic and pharmacodynamic properties. These polymers can be cloaked with protein corona consisting of adsorbed plasma or extracellular proteins to improve nanoparticle biodistribution and half-life. Besides, CDs have become famous in applications ranging from biomedicine to environmental sciences. In this review, we emphasize ongoing research in biomedical fields using CD-based centered, pendant, and terminated polymers and their interactions with protein corona for theranostic applications. Overall, a perusal of information concerning this novel approach in biomedicine will help to implement this methodology based on host-guest interaction to improve therapeutic and diagnostic strategies.

Site-Selective Modification of (Oligo)Saccharides.

Oligosaccharides, either as such or as part of glycolipids, glycopeptides, or glycoproteins, are ubiquitous in nature and fulfill important roles in the living cell. Also in medicine and to some extent in materials, oligosaccharides play an important role. In order to study their function, modifying naturally occurring oligosaccharides, and building in reactive groups and reporter groups in oligosaccharides, are key strategies. The development of oligosaccharides as drugs, or vaccines, requires the introduction of subtle modifications in the structure of oligosaccharides to optimize efficacy and, in the case of antibiotics, circumvent bacterial resistance. Provided the natural oligosaccharide is available, site-selective modification is an attractive approach as total synthesis of the target is often very laborious. Researchers in catalysis areas, such as transition-metal catalysis, enzyme catalysis, organocatalysis, and photoredox catalysis, have made considerable progress in the development of site-selective and late-stage modification methods for mono- and oligosaccharides. It is foreseen that the fields of enzymatic modification of glycans and the chemical modification of (oligo)saccharides will approach and potentially meet each other, but there is a lot to learn and discover before this will be the case.

Use of Opuntia ficus-indica Fruit Peel as a Novel Source of Mucilage with Coagulant Physicochemical/Molecular Characteristics.

The peels obtained as a byproduct from the processing of fruits (prickly pears) of the Cactaceae family are a rich source of mucilage, a hydrocolloid biopolymer that may have potential application in water/wastewater treatment as a natural coagulant. In this study, the structural (UPLC-QTOF-MS, FTIR, Raman, NMR, XRD, and zeta potential), morphological (SEM), and thermal (DSC/TGA) characterizations of the mucilage extracted from the peels of Opuntia ficus-indica (OFI) fruits were carried out. UPLC-QTOF-MS results revealed the presence of a branched polymer with an average molecular weight of 0.44 KDa for this mucilage in aqua media. The NMR spectra of mucilage in DMSO-d6 indicated that it seemed well-suited as a coagulant with its typical oligosaccharide structure. FTIR studies confirmed the presence of hydroxyl and carboxyl functional groups in the mucilage, indicating its polyelectrolyte nature that could provide coagulating properties through binding and adsorption mechanisms. Likewise, the zeta potential of −23.63 ± 0.55 mV showed an anionic nature of the mucilage. Power XRD technique evidenced the presence of crystalline poly(glycine-β-alanine), glutamic acid, and syn-whewellite. SEM images revealed an irregular and amorphous morphology with cracks, which are suitable characteristics for adsorption mechanisms. The mucilage exhibited two endothermic transitions, with a decomposition temperature in uronic acid of 423.10 °C. These findings revealed that mucilage obtained from OFI fruit peels has molecular and physicochemical characteristics that are suited to its possible application as a natural coagulant in water/wastewater treatments.

Galactosylation of cell-surface glycoprotein required for hyphal growth and cell wall integrity in Schizosaccharomyces japonicus

Schizosaccharomyces japonicus is a dimorphic yeast, transiting between unicellular and hyphal growth. The glycoproteins of fission yeast contain, in addition to mannose (Man), a large number of galactose (Gal) residues. Previously, we reported that the cell-surface O-glycans of S.japonicus comprise mainly tri-saccharides (Gal-Man-Man) as a main component, in contrast to the tetra-saccharides observed in other Schizosaccharomyces species. Here we have investigated the function of cell-surface Gal residues in S.japonicus. Because disruption of gms1+, encoding the UDP-Gal transporter required for galactomannan synthesis, abolishes cell-surface galactosylation in Schizosaccharomyces pombe, we constructed a deletion mutant of the homologous gene in S.japonicus gms1Δ [gms1 (S.j)] and determined the N- and O-linked oligosaccharide structures present on the cell surface. Disruption of gms1 (S.j) resulted in a complete lack of Gal on the cell surface, indicating that Gms1 plays an essential role in supplying UDP-Gal from the cytoplasm to the Golgi lumen. Analytical microscopy of gms1Δ demonstrated that the lack of cell-surface Gal did not affect cell growth or morphology during vegetative growth. However, hyphal development was blocked in gms1Δ, even in the presence of the topoisomerase I inhibitor camptothecin, which is known to induce hyphal differentiation in wild-type S.japonicus. Collectively, these findings show that Gal-containing oligosaccharides are required for cell wall integrity during filamentous growth in S.japonicus.

Microwave-assisted degradation of β-D-glucan from Ganoderma lucidum and the structural and immunoregulatory properties of oligosaccharide fractions

Microwave-assisted degradation of β-(1 → 3,1 → 6)-D-glucan from Ganoderma lucidum and correlated immunoregulatory activities were investigated in this study. The optimal temperature and degradation time for microwave hydrothermal hydrolysis were 140 °C and 40 min, respectively. Under these conditions, a high yield of degradation rate (98.4 %) and abundant β-oligosaccharide products (GLOS) with different degrees of polymerization (DP 2–24) were obtained. Four fractions including F1 (DP 2–8), F2 (DP 6–19), F3 (DP 8–24) and F4 (high DPs) with different average ratios of β-(1 → 3) to β-(1 → 6)-linked glucose units were isolated from GLOS. The structures of oligosaccharides with DP (2–6) in F1 were identified as linear β-(1 → 3)-linked glucooligosaccharides without or with β-(1 → 6)-linked glucose residues based on MS/MS analysis. The immunoregulation activity of β-glucooligosaccharides was correlated with their DPs and the average ratios of β-(1 → 3) to β-(1 → 6)-linked glucose units. F4 fraction with high DPs and ratio of 3.29:1 exhibited higher immunoenhancing activity on inducing NF-κB activation through binding to dectin-1. Surface plasmon resonance (SPR) analysis indicated that β-glucooligosaccharides could bind to Dectin-1 directly and the binding affinity increased with the increase of DPs and the ratios of β-(1 → 3)-linked glucose.

Preparation and Structure Characterization of High-Value Laminaria digitata Oligosaccharides.

Algae-derived marine oligosaccharides have been reported to be promising bioactive compounds because of their various properties with health benefits and potential significance in numerous applications in industrial biotechnology. In this study, laminaran oligosaccharides (LOs) with varying degrees of polymerization were obtained through partial acid hydrolysis of laminaran derived from Laminaria digitata. Based on response surface methodology, the optimum LOs yield was obtained for acid hydrolysis laminaran at a hydrolysis time of 55 min, temperature of 71°C, and acid concentration of 1.00 mol/L. The size-exclusion resin Bio-Gel P-2 was considered to be a better option for LOs purification. The structure of the purified oligosaccharides was analyzed through mass spectrometry and nuclear magnetic resonance. They demonstrated the main oligosaccharide structure corresponding to the connection of glucose with β-D-Glcp-(1→3)-β-D-Glcp, which was identified as laminaribiose (DP2), laminaritriose (DP3), laminaritetrose (DP4), and laminaripentaose (DP5). LOs demonstrate excellent antioxidant activities, as evidenced from their reactions with oxidizing free radicals, 1, 1-diphenyl-2-picryl-hydrazyl, and 2, 2′-azino-bis (3-etilbenzotiazoline-6-sulfonic acid) radicals. LOs exhibited a prebiotic effect on the growth of Bifidobacterium adolescentis and Lactobacillus plantarum. Therefore, we propose the development of LOs as natural antioxidants and prebiotics in the functional food and pharmaceutical industries.

Cranberry Arabino-Xyloglucan and Pectic Oligosaccharides Induce Lactobacillus Growth and Short-Chain Fatty Acid Production.

Numerous health benefits have been reported from the consumption of cranberry-derived products, and recent studies have identified bioactive polysaccharides and oligosaccharides from cranberry pomace. This study aimed to further characterize xyloglucan and pectic oligosaccharide structures from pectinase-treated cranberry pomace and measure the growth and short-chain fatty acid production of 86 Lactobacillus strains using a cranberry oligosaccharide fraction as the carbon source. In addition to arabino-xyloglucan structures, cranberry oligosaccharides included pectic rhamnogalacturonan I which was methyl-esterified, acetylated and contained arabino-galacto-oligosaccharide side chains and a 4,5-unsaturated function at the non-reducing end. When grown on cranberry oligosaccharides, ten Lactobacillus strains reached a final culture density (ΔOD) ≥ 0.50 after 24 h incubation at 32 °C, which was comparable to L. plantarum ATCC BAA 793. All strains produced lactic, acetic, and propionic acids, and all but three strains produced butyric acid. This study demonstrated that the ability to metabolize cranberry oligosaccharides is Lactobacillus strain specific, with some strains having the potential to be probiotics, and for the first time showed these ten strains were capable of growth on this carbon source. The novel cranberry pectic and arabino-xyloglucan oligosaccharide structures reported here combined with the Lactobacillus strains that can metabolize cranberry oligosaccharides and produce short-chain fatty acids, have excellent potential as health-promoting synbiotics.

Unusual free oligosaccharides in human bovine and caprine milk

Free oligosaccharides are abundant macronutrients in milk and involved in prebiotic functions and antiadhesive binding of viruses and pathogenic bacteria to colonocytes. Despite the importance of these oligosaccharides, structural determination of oligosaccharides is challenging, and milk oligosaccharide biosynthetic pathways remain unclear. Oligosaccharide structures are conventionally determined using a combination of chemical reactions, exoglycosidase digestion, nuclear magnetic resonance spectroscopy, and mass spectrometry. Most reported free oligosaccharides are highly abundant and have lactose at the reducing end, and current oligosaccharide biosynthetic pathways in human milk are proposed based on these oligosaccharides. In this study, a new mass spectrometry technique, which can identify linkages, anomericities, and stereoisomers, was applied to determine the structures of free oligosaccharides in human, bovine, and caprine milk. Oligosaccharides that do not follow the current biosynthetic pathways and are not synthesized by any discovered enzymes were found, indicating the existence of undiscovered biosynthetic pathways and enzymes.

Antitumour Activity of Different Bioactive Compounds from Hemolymph and Mucus of Mollusca against Human Urinary Bladder Cancer Cell Lines

In this study, we isolated different bioactive compounds and fractions from Rapana venosa (RvH), Helix aspersa (HaH) and Helix lucorum (HlH) and then we evaluated their in vitro antitumour activity on CAL-29 and T- 24 cell lines (transitional cell carcinoma). Also, we performed fluorescence staining with propidium iodide to assess nuclear changes caused by molluscan extracts. Briefly, our results have demonstrated antineoplastic activity of: subunit βc-HaH of H. aspersa hemocyanins to CAL-29 cell line, subunit βc-HaH and subunit RvH II of R. venosa hemocyanin against T-24 cells. Also, fractions from R. venosa hemolymph exerted significant cytotoxic activity against CAL- 29 cells. Furthermore, fluorescence microscopy results demonstrated chromatin condensation and nuclear morphological changes which are typical for apoptosis induction. We assume that the observed death of T-24 and CAL-29 cells is due to the interaction between the oligosaccharide structures of the proteins on the tumour cell membranes and the hemolymph from R. venosa, active components isolated from hemolymph of R. venosa, H. aspersa and H. lucorum, as well as H. aspersa mucus.

Selective oligosaccharide recognition with phenylboronic acid through Cu(I)-catalysed click imprinted surfaces

With the emerging role of glycans as potential diagnostic markers of disease, highly selective diagnostic platforms must be developed to realise the clinical value of irregular glycan expression patterns. Overcoming chemical and structural homogeneity between oligosaccharide structures however remains a significant challenge, which largely rests on the capability to develop materials embedding precise carbohydrate recognition. Herein we report a surface imprinting strategy that takes advantage of the high yield Cu(I)-catalysed azide-alkyne cycloaddition (CuAAC) click reaction to achieve oligosaccharide imprinted cavities featuring precisely organized phenylboronic acid (PBA) motifs. Central to the construction of the imprinted surface was the choice of a foundation alkyne-terminated monolayer of a long alkyl chain to impart a high surface click efficiency of the PBA motif. By pre-forming and clicking an oligosaccharide:PBA complex onto the alkyne-terminated monolayer and capping the free space, selective oligosaccharide imprinted cavities were formed with low millimolar dissociation constants.

Ulva (Enteromorpha) Polysaccharides and Oligosaccharides: A Potential Functional Food Source from Green-Tide-Forming Macroalgae.

The high-valued utilization of Ulva (previously known as Enteromorpha) bioresources has drawn increasing attention due to the periodic blooms of world-wide green tide. The polysaccharide is the main functional component of Ulva and exhibits various physiological activities. The Ulva oligosaccharide as the degradation product of polysaccharide not only possesses some obvious activities, but also possesses excellent solubility and bioavailability. Both Ulva polysaccharides and oligosaccharides hold promising potential in the food industry as new functional foods or food additives. Studies on Ulva polysaccharides and oligosaccharides are increasing and have been the focus of the marine bioresources field. However, the comprehensive review of this topic is still rare and do not cover the recent advances of the structure, isolation, preparation, activity and applications of Ulva polysaccharides and oligosaccharides. This review systematically summarizes and discusses the recent advances of chemical composition, extraction, purification, structure, and activity of Ulva polysaccharides as well as oligosaccharides. In addition, the potential applications as new functional food and food additives have also been considered, and these will definitely expand the applications of Ulva oligosaccharides in the food and medical fields.

Pd nanoparticles decorated thiol-functionalized MOF as an efficient matrix for differentiation and quantitation of oligosaccharide isomers by laser desorption/ionization mass spectrometry

Oligosaccharides play a key role in many biological functions, and the accurate identification of oligosaccharide structures is an important prerequisite for a comprehensive understanding of the biological functions of oligosaccharides. MALDI-TOF/TOF tandem mass spectrometry has been considered as a potential technique for the structural characterization of oligosaccharides. In this work, palladium nanoparticles decorated thiol-functionalized metal organic framework nanocomposite (UiO-66-(SH)2@Pd NPs) was fabricated as an efficient matrix to assist laser desorption/ionization mass spectrometry (LDI-MS) for oligosaccharides analysis. The ionization efficiency of oligosaccharides was significantly improved owning to the synergistic effect of MOF and Pd nanoparticles, which is favorable for further oligosaccharide structure identification. By combining LDI-LIFT-TOF/TOF, 24 oligosaccharide isomers including disaccharides, trisaccharides and tetrasaccharides, were effectively distinguished. In addition, the relative quantification curves for isomeric oligosaccharides were established with good linear correlations. The method was successfully applied to the identification and quantification of sucrose and maltose in three batches of Asian ginseng and American ginseng respectively, showing potentiality of MOF materials and metal nanomaterials assisted structural analysis of oligosaccharide isomers.

Understanding the structure and composition of recalcitrant oligosaccharides in hydrolysate using high-throughput biotin-based glycome profiling and mass spectrometry

Novel Immunological and Mass Spectrometry Methods for Comprehensive Analysis of Recalcitrant Oligosaccharides in AFEX Pretreated Corn Stover. Lignocellulosic biomass is a sustainable alternative to fossil fuel and is extensively used for developing bio-based technologies to produce products such as food, feed, fuel, and chemicals. The key to these technologies is to develop cost competitive processes to convert complex carbohydrates present in plant cell wall to simple sugars such as glucose, xylose, and arabinose. Since lignocellulosic biomass is highly recalcitrant, it must undergo a combination of thermochemical treatment such as Ammonia Fiber Expansion (AFEX), dilute acid (DA), Ionic Liquid (IL) and biological treatment such as enzyme hydrolysis and microbial fermentation to produce desired products. However, when using commercial fungal enzymes during hydrolysis, only 75–85% of the soluble sugars generated are monomeric sugars, while the remaining 15–25% are soluble recalcitrant oligosaccharides that cannot be easily utilized by microorganisms. Previously, we successfully separated and purified the soluble recalcitrant oligosaccharides using a combination of charcoal and celite-based separation followed by size exclusion chromatography and studies their inhibitory properties on enzymes. We discovered that the oligosaccharides with higher degree of polymerization (DP) containing methylated uronic acid substitutions were more recalcitrant towards commercial enzyme mixtures than lower DP and neutral oligosaccharides. Here, we report the use of several complementary techniques that include glycome profiling using plant biomass glycan specific monoclonal antibodies (mAbs) to characterize sugar linkages in plant cell walls and enzymatic hydrolysate, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) using structurally-informative diagnostic peaks offered by negative ion post-secondary decay spectra, gas chromatography followed by mass spectrometry (GC–MS) to characterize oligosaccharide sugar linkages with and without derivatization. Since oligosaccharides (DP 4–20) are small, it is challenging to mobilize these molecules for mAbs binding and characterization. To overcome this problem, we have applied a new biotin-coupling based oligosaccharide immobilization method that successfully tagged most of the low DP soluble oligosaccharides on to a micro-plate surface followed by specific linkage analysis using mAbs in a high-throughput system. This new approach will help develop more advanced versions of future high throughput glycome profiling methods that can be used to separate and characterize oligosaccharides present in biomarkers for diagnostic applications.

Resolving Heparan Sulfate Oligosaccharide Positional Isomers Using Hydrophilic Interaction Liquid Chromatography-Cyclic Ion Mobility Mass Spectrometry.

Heparan sulfate (HS) is a linear polysaccharide covalently attached to proteoglycans on cell surfaces and within extracellular matrices in all animal tissues. Many biological processes are triggered by the interactions among HS binding proteins and short structural motifs in HS chains. The determination of HS oligosaccharide structures using liquid chromatography-mass spectrometry (LC-MS) is made challenging by the existence of positional sulfation and acetylation isomers. The determination of uronic acid epimer positions is even more challenging. While hydrophilic interaction liquid chromatography (HILIC) separates HS saccharides based on their composition, there is a very limited resolution of positional isomers. This lack of resolution places a burden on the tandem mass spectrometry step for assigning saccharide isomers. In this work, we explored the use of the ion mobility dimension to separate HS saccharide isomers based on molecular shape in the gas phase. We showed that the combination of HILIC and cyclic ion mobility mass spectrometry (cIM-MS) was extremely useful for resolving HS positional isomers including uronic acid epimers and sulfate positions. Furthermore, HILIC-cIM-MS differentiated multicomponent HS isomeric saccharide mixtures. In summary, HILIC-cIM-MS provided high-quality data for analysis of HS oligosaccharide isomeric mixtures that may prove useful in the discovery of new structural motifs for HS binding proteins and for the targeted quality control analysis of commercial HS products.

Ion Mobility Mass Spectrometry Reveals Rare Sialylated Glycosphingolipid Structures in Human Cerebrospinal Fluid.

Gangliosides (GGs) represent an important class of biomolecules associated with the central nervous system (CNS). In view of their special role at a CNS level, GGs are valuable diagnostic markers and prospective therapeutic agents. By ion mobility separation mass spectrometry (IMS MS), recently implemented by us in the investigation of human CNS gangliosidome, we previously discovered a similarity between GG profiles in CSF and the brain. Based on these findings, we developed IMS tandem MS (MS/MS) to characterize rare human CSF glycoforms, with a potential biomarker role. To investigate the oligosaccharide and ceramide structures, the ions detected following IMS MS separation were submitted to structural analysis by collision-induced dissociation (CID) MS/MS in the transfer cell. The IMS evidence on only one mobility feature, together with the diagnostic fragment ions, allowed the unequivocal identification of isomers in the CSF. Hence, by IMS MS/MS, GalNAc-GD1c(d18:1/18:1) and GalNAc-GD1c(d18:1/18:0) having both Neu5Ac residues and GalNAc attached to the external galactose were for the first time discovered and structurally characterized. The present results demonstrate the high potential of IMS MS/MS for biomarker discovery and characterization in body fluids, and the perspectives of method implementation in clinical analyses targeting the early diagnosis of CNS diseases through molecular fingerprints.

The Microstructure, Antibacterial and Antitumor Activities of Chitosan Oligosaccharides and Derivatives.

Chitosan obtained from abundant marine resources has been proven to have a variety of biological activities. However, due to its poor water solubility, chitosan application is limited, and the degradation products of chitosan oligosaccharides are better than chitosan regarding performance. Chitosan oligosaccharides have two kinds of active groups, amino and hydroxyl groups, which can form a variety of derivatives, and the properties of these derivatives can be further improved. In this review, the key structures of chitosan oligosaccharides and recent studies on chitosan oligosaccharide derivatives, including their synthesis methods, are described. Finally, the antimicrobial and antitumor applications of chitosan oligosaccharides and their derivatives are discussed.

Preparation, structure and α-glucosidase inhibitory of oligosaccharides by enzymatic hydrolysis from Annona squamosa polysaccharide

Annona squamosa (AS) has relatively high content of polysaccharides which had effective hypoglycemic activity. The oligosaccharides have advantages in the digestion and absorption. However, there were few reports on oligosaccharides from AS. In the manuscript, pectinase was utilized to degrade Annona squamosa polysaccharide (ASP). The product was acetylated, purified by silica gel column and deacetylated to obtain a novel oligosaccharide Z. The structure of Z was identified with NMR, mass spectrometry and monosaccharide composition analysis. The results demonstrated that the oligosaccharide Z was a trisaccharide with molecular weight of 518 Da. The structure of oligosaccharide Z was linear skeleton composed of β-D-Glc-(1→2)-β-D-Glc-(1→3)-α-D-GalA. The oligosaccharide Z can effectively inhibit the activity of α-glucosidase with reversible type and mixed-type inhibition. The oligosaccharide Z had effective binding effect with α-glucosidase from molecular docking experiments. The results revealed that the oligosaccharide Z has the potential to be utilized as α-glucosidase inhibitor. The manuscript provided theoretical basis for the new applications of AS in food industry and pharmaceutical industry. It also provided valuable clues for finding new potential hypoglycemic drugs from natural sources.

Optimizing the Semisynthesis towards glycosylated interferon-β-polypeptide by utilizing bacterial protein expression and chemical modification.

The synthesis of a sufficient amount of homogeneous glycoprotein is of great interest because natural glycoproteins show considerable heterogeneity in oligosaccharide structures, making the studies on glycan structure-function relationship difficult. Herein, we report optimized methods that can accelerate the semisynthesis of homogeneous glycoproteins based on recombinant expression and chemical conversion. Peptide thioesters and peptides with Cys residues at their N-terminals are necessary intermediates to perform native chemical ligation. We successfully performed thioesterification for a peptide prepared in E. coli via Cys-cyanylation at its C-terminal followed by hydrazinolysis and acidic thiolysis. These optimized conditions could tolerate an acid labile Thz protected Cys at the N-terminal of a peptide-hydrazide and specific cyanylation of the C-terminal Cys to yield a peptide thioester. To reduce the amount of precious oligosaccharide that is required in the conventional SPPS method, an improved liquid phase glycopeptide coupling was also optimized in a good yield (46% over four steps). Lastly, chemoselective protection of the internal cysteines and activation of the N-terminal cysteine were optimized toward a long peptide prepared in E. coli. By using these strategies, a full-length interferon-β glycosyl polypeptide as a model was successfully obtained.