Glycosylation Conditions Research Articles

Highly Reactive Glycosylation with 1-O-(Methylthio)thiocarbonyl Glycosyl Donors under Acidic to Neutral Reaction Conditions.

In this study, we have successfully developed a glycosylation method using 1-O-(methylthio)thiocarbonyl-glycoses as donors. Such xanthate donors are easily accessible and shelf-stable. The glycosylation reaction could be promoted by cations (acidic to neutral conditions) under mild conditions, exhibiting a reactivity intermediate between that with glycosyl trichloroacetimidate as the donor and that with thioglycoside as the donor. This methodology tolerates both "armed" and "disarmed" glycosyl donors, as well as various sugar acceptors, and affords the corresponding glycosides in good to excellent yields. Based on the relative higher reactivity of such xanthate donors than thioglycoside donors under the same glycosylation conditions, a trisaccharide was further synthesized in a one-pot glycosylation strategy.

Exploring the Dual Functions of Distal Acyl Group Direction in Various Nucleophilic Environments.

A universal glycosylation strategy could significantly simplify glycoside synthesis. One approach to achieving this goal is through acyl group direction for the corresponding 1,2-, 1,3-, 1,4-, or 1,6-trans glycosylation; however, this approach has been challenging for glycosidic bonds that require distal equatorial-acyl group direction. We developed an approach in weakly nucleophilic environments for selective 1,4-trans glycosylation directed by the equatorial-4-O-acyl group. Here, we explored this condition in other distal acyl groups and found that, besides 1,n-trans direction, acyl groups also mediated hydrogen bonding between acyl groups and alcohols. The latter showed a diverse effect and classified the acyl group direction into axial and equatorial categories. Corresponding glycosylation conditions were distinguished as guidance for acyl group direction from either category. Hence, acyl group direction may serve as a general glycosylation strategy.

Structural and functional characterization of mung bean protein-peach gum conjugate through the Maillard reaction as a novel encapsulation agent

This study used peach gum (PG) and mung bean protein isolate (MBPI) as an encapsulation agent. This study used peach gum (PG) and mung bean protein isolate (MBPI) as an encapsulation agent. The present investigation was undertaken to examine and refine the conjugation process of MBPI-PG, while concurrently evaluating the impact of Maillard modification on functional properties through the wet heat method. Through the analyses of SDS-PAGE, glycation degree, SEM micrographs, and FT-IR spectroscopy, it was ascertained that optimal glycosylation conditions were achieved at a pH of 8.5 and a temperature of 90 °C for a duration of 1 h. Conjugation resulted in the enhanced of solubility, emulsifying properties, and DPPH radical scavenging activity of MBPI. In comparison to their un-conjugated one, the observed improvements in thermal stability and solubility of proteins were attributed to the process of glycation. Collectively, the findings imply that the conjugation with PG imparts additional functional attributes to MBPI, thereby potentially enhancing its applicability in the food industries as a novel encapsulation agent.

Convergent Synthesis of the Octasaccharide Repeating Unit of the K55 Capsular Polysaccharide of Acinetobacter baumannii BAL_204 Strain

AbstractSynthesis of the octasaccharide repeating unit of the K55 capsular polysaccharide of Acinetobacter baumannii BAL_204 strain has been achieved in very good yield using a convergent [5+3] block glycosylation strategy. The pentasaccharide and trisaccharide components were synthesized using sequential stereoselective glycosylations. The p-methoxybenzyl (PMB) group was used as temporary alkyl protecting group, which was removed under the thiophilic glycosylation condition by raising the temperature. A late-stage TEMPO-mediated selective oxidation of primary hydroxyl group into carboxylic acid allowed getting the d-glucuronic acid moiety in the octasaccharide. A combination of N-iodosuccinimide (NIS) and perchloric acid supported over silica (HClO4­-SiO2) was used as a thiophilic promoter for the activation of thioglycosides. HClO4-SiO2 was also used as a solid acid activator for glycosyl trichloroacetimidate derivative.

Accelerated Solid Phase Glycan Synthesis: ASGS.

Solid phase synthesis is the most dominant approach for the preparation of biological oligomers as it enables the introduction of monomers iteratively. Accelerated solid phase synthesis of biological oligomers is crucial for chemical biology, but its application to the synthesis of oligosaccharides is not trivial. Solid-phase oligosaccharide assembly is a slow process performed in a variety of conditions and temperatures, requires an inert gas atmosphere, and demands high excess of glycosyl donors. The process is done in special synthesizers and poor mixing of the solid support increases the risk of diffusion-independent hydrolysis of the activated donors. High shear stirring is a new way to accelerate solid phase synthesis. The efficient mixing ensures that reactive intermediates can diffuse faster to the solid support thereby increasing the kinetics of the reactions. We report here a stirring-based accelerated solid-phase oligosaccharide synthesis. We harnessed high shear mixing to perform diffusion-dependent glycosylation in a short reaction time. We minimized the use of glycosyl donors and the need to use an inert atmosphere. We showed that by tailoring the deprotection and glycosylation conditions to the same temperature, assembly steps are performed continuously, and full glycosylation cycles are completed in minutes.

Concise Synthesis of the Hexasaccharide Repeating Unit of the Capsular Polysaccharide of Klebsiella K19 Strain

AbstractA convergent [4+2] stereoselective block glycosylation strategy has been developed for the synthesis of the hexasaccharide repeating unit of the capsular polysaccharide of Klebsiella K19 strain in very good yield. The p-methoxybenzyl (PMB) group was used as a temporary alkyl protecting group, which was removed by tuning the glycosylation conditions. A thioglycoside was used as a glycosyl acceptor in an orthogonal glycosylation reaction. A late-stage TEMPO-mediated selective oxidation of a primary hydroxyl group into carboxylic acid allowed incorporation of the d-glucuronic acid moiety in the hexasaccharide. A combination of N-iodosuccinimide (NIS) and perchloric acid supported over silica (HClO4–SiO2) was used as a thiophilic promoter for the activation of thioglycosides. HClO4–SiO2 was also used as a solid acid activator for a glycosyl trichloroacetimidate derivative.

The synthesis, biological activities and applications of protein–polysaccharide conjugates in food system: a review

AbstractProteins are important ingredients in the food industry for their excellent nutritional, functional and biological properties, while the application is limited by their sensitivity to environmental stresses such as high temperature, pH changes and ionic strength. The Maillard reaction and transglutaminase-catalyzed glycosylation are the two main effective glycosylation ways to improve the stability and biological properties of proteins by mainly attaching monosaccharides or disaccharides to proteins, while protein–polysaccharide conjugates with superior functional properties are rarely discussed. Therefore, the present study reviews current state of the synthesis, biological activities and applications in the food system of protein–polysaccharide conjugates formed via the Maillard reaction and transglutaminase-catalyzed glycosylation. The biological activities including antioxidant, antimicrobial and immunomodulatory activities of proteins are improved after glycosylation, and the formed protein–polysaccharide conjugates can be applied to stabilize emulsions or deliver bioactive compounds in foods. The bioactivities produced under precisely controlled glycosylation conditions would make protein–polysaccharide conjugates a promising application in foods with healthy properties.

Changes in partial properties of glycosylated egg white powder during storage.

Glycosylation is an effective method to modify protein. However, there is a lack of research on the property changes of glycosylated protein during storage. In the present study, the changes in the physicochemical, functional, and structural properties of xylo-oligosaccharide (XOS) glycosylated egg white powder (EWP) (XOS-EWP conjugates) prepared with different glycosylation conditions (XOS/EWP ratio and reaction time) were investigated when stored at 25 °C and 60% relative humidity. In the 12 weeks of storage, the degree of grafting, browning, and the formation of Maillard reaction products of XOS-EWP conjugates increased. The increase in XOS/EWP ratio and reaction time led to an increase in protein aggregation, though a decrease in solubility, due to increased degree of glycosylation and structural changes. Furthermore, improved gel hardness of XOS-EWP conjugates deteriorated, while improved emulsification ability was kept stable during storage. For the sample with a lower XOS/EWP ratio and reaction time, the gel hardness and emulsifying properties underwent little or no deterioration even improving during storage. The results could be attributed to the limited degree of glycosylation, further unfolding of the protein structure, increased surface hydrophobicity of protein, and improved thermal characteristics. During storage, the Maillard reaction would continue to occur in the glycosylated EWP, further affecting the performance of modified EWP. Modified EWP prepared under different glycosylation conditions performed differently during storage. Modified EWP with a larger XOS/EWP ratio and reaction time meant it was harder to maintain good performance. Modified EWP with a smaller XOS/EWP ratio and reaction time changed significantly to better performances. © 2022 Society of Chemical Industry.

Synthesis of 4'-C-(Aminoethyl)thymidine and 4'-C-[(N-Methyl)aminoethyl] Thymidine Nucleosides to Enhance DNA Stability.

Antisense oligonucleotide (ASO) therapeutics target the pathogenic mRNA directly and modulate protein expression. Novel chemical modifications help to improve the action of ASOs with better thermal stability and resistance against nucleases. Oligodeoxynucleotides (ODNs) containing 4'-C-(aminoethyl)thymidine modifications exhibit efficient and stable hybridization with complementary DNA as well as RNA strands showing remarkably improved resistance against nucleolytic hydrolysis, which makes them promising candidates for antisense therapeutics. This article describes the synthesis of a novel nucleoside analog, 4'-C-[(N-methyl)aminoethyl]-thymidine (4'-MAE-T), 3, and previously reported 4'-C-aminoethyl-thymidine (4'-AE-T), 2, through a newly designed synthetic route to obtain a high overall yield. This has been established by changing the starting material from thymidine to diacetone-D-glucofuranose and synthesizing the known 4-C-hydroxyethyl pentofuranose. Conversion of the hydroxy group to an azide functional group through Mitsunobu azidation and performing acetolysis, provide the common intermediate 4-C-(2-azidoethyl)-ribofuranose. Subsequent coupling of the thymine nucleobase with the common intermediate under Vorbrüggen glycosylation conditions provides the corresponding modified nucleoside in high yield. It was subjected for conversion of the azide to an amine by Staudinger reaction and 2'-deoxygenation using Barton-McCombie conditions. Debenzylation with Lewis acid and mono-dimethoxytritylation of the 5'-OH afforded a fully protected 3'-OH intermediate for phosphitylation to give the corresponding phosphoramidites. In the case of 4'-MAE-T, benzyloxymethyl protection of the N3 -position and methylation were carried out prior to debenzylation. These phosphoramidite monomers were suitable with conventional oligonucleotide synthesis, and imparted ameliorated nuclease resistance, and competent RNase H activity, suggesting its potential utilization in ASO drugs. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of 4-C-(2-azidoethyl)-ribofuranose (6) Basic Protocol 2: Synthesis of 4'-C-aminoethyl thymidine phosphoramidite (15) Basic Protocol 3: Synthesis of 4'-C-(N-methyl)aminoethyl thymidine phosphoramidite (20).

Synthesis of new imidazole-triazole-glycoside hybrids as anti-breast cancer candidates

One of the leading causes of death worldwide among women is breast cancer. Thus, discovering new multi-target anti-breast cancer drugs is mandatory to stimulate nitric oxide (NO) and cyclooxygenase-2 (COX-2) as a cross-talking strategy for killing breast cancerous cells. 1-(3-(4-bromophenyl)-5-methyl-2-thioxo-2,3-dihydro-1H -imidazol-4-yl)ethan-1-one 1 was prepared and allowed to react with various alkyl halide propargyl bromide; 1,2-dibromoethane; and 1,3-dichloropropane to afford the corresponding alkyl imidazolo derivatives 2, and 14a,b respectively; Compound 2 allowed to react undergoes click reaction condition afforded the corresponding 1,2,3-triazole-N-acetylated glycosides 4 and 5 which reacted with methanolic ammonia solution to obtain free hydroxyl 1,2,3-triazole N-glycosides 6 and 7; respectively. Also, Compounds 14a,b reacted with sodium azide to give the azido derivatives 15,16; which reacted under click reaction condition to produce 1,2,3-triazole-C-glycosides compounds 18-21, respectively. Meanwhile, the previous compounds can be deacetylated to afford the free hydroxyl thioglycosides 22-25. Moreover, the thioglycoside derivatives of compound 1 were synthesized and underwent glycosylation conditions utilizing acetylated glycosyl bromides to give S-glycoside analogs 8-10 which proceeded to undergo deacetylated condition to obtain free hydroxyl imidazolo- S-glycosides 11-13; respectively. All newly synthesized compounds were tested against human breast MCF-7 and MDA-MB-231 cancerous cell lines. Cytotoxicity using MTT assay was performed as well as IC50 was measured for all candidates. Then, nitric oxide (NO) levels and cyclooxygenase-2 (COX-2) enzyme activity were investigated in the proposed cancerous cells. We resulted that the prepared compounds were more cytotoxic against MDA-MB-231 cells than MCF-7 cells. MCF-7 breast cancerous cells were resistant to doxorubicin in addition to the applied compounds, while MDA-MB-231 cells were sensitive. It was reported that compounds 22, 12, and 6 recorded the highest cytotoxic effects against both cancer cell lines. The signaling crosstalk between NO and COX-2 in cancer cells achieved that compounds 12 and 22 induced the significant elevation of NO levels in MCF-7 cells which in turn inhibited the COX-2 enzyme activity. In conclusion, the newly prepared compounds containing an imidazole nucleus have anti-breast cancer potential through modulation of NO and COX-2 signaling track.

Chemical Synthesis of the Highly Sterically Hindered Core Undecasaccharide of Helicobacter pylori Lipopolysaccharide for Antigenicity Evaluation with Human Serum.

Helicobacter pylori, listed as a human carcinogen by the Department of Health and Human Services, colonizes the gastric mucosa of more than half of the world's population. The individuals infected with H. pylori have a high risk to develop chronic gastritis, peptic ulcers, and even gastric cancer. The conserved core structure of H. pylori lipopolysaccharide (LPS) has been regarded as a promising candidate structure for development of a glycoconjugate vaccine targeting multiple serotypes. Here, we report a total synthesis of the core undecasaccharide of H. pylori LPS and its subunit antigens. The match and mismatch between the glycosyl donor and acceptor caused by the inert hydroxyl groups were addressed by a judicious choice of orthogonal protection strategies and glycosylation conditions. A combination of acyl remote participation and solvent effects has been applied for selective formation of the five 1,2-cis-glucosidic bonds. The high steric hindrance induced by the high carbon sugars and trinacriform architecture required that the core undecasaccharide was synthesized through a finely tuned linear assembly [2 + (1 + (3 + (1 + (1 + 3))))] rather than convergent strategies. An antigenicity evaluation using glycan microarrays showed that an α-(1 → 6)-glucan trisaccharide is recognized by IgG antibodies in sera of H. pylori-infected patients. The phosphate group of the inner core trisaccharide key epitope is very important for IgG recognition. These findings are an important step toward designing carbohydrate-based vaccines against H. pylori.

Preparation, characterization and bioavailability studies of Tegillarca granosa hemoglobin and its glycosylated products

Iron deficiency anemia (IDA) is a common micronutrient deficiency. Tegillarca granosa (T. granosa) is a good source of iron due to its high content of hemoglobin. The present study aimed to determine the effects of glycosylation on structure, physicochemical characteristics and iron bioavailability of hemoglobin. Using Box-Behnken design and response surface methodology, the optimal conditions for hemoglobin-chitosan glycosylation were obtained: 61.8 °C, pH 6.3, hemoglobin/chitosan mass ratio of 4.3 and reaction time of 15 min. The formation of hemoglobin-chitosan conjugates was verified by SDS-PAGE and fluorescence spectroscopy. The surface hydrophobicity of hemoglobin was reduced by 20.90–65.05 % after glycosylation, along with the observations of elevated water-holding capacity, likely owing to the introduction of hydrophilic groups. Antioxidant capacity of glycosylated products (0.41–0.66 μM Trolox/mg protein) was markedly greater than that of original protein (0.06 μM Trolox/mg protein) due to the formation of brown polymers with antioxidant activity. In addition, glycosylation improved in vitro digestibility of hemoglobin by 41.15–69.09 %, which could be attributed to less β-sheet in secondary structures. Moreover, hemoglobin (324.38 ng/mg) exhibited better iron absorption than FeSO4 (121.63 ng/mg), with the value being further enhanced by glycosylation (442.73 ng/mg), which may be due to the improved protein digestibility and iron-chelating capacity.

Simulation Modeling of Reduced Glycosylation Effects on Potassium Channels of Mouse Cardiomyocytes.

Dilated cardiomyopathy (DCM) is the third most common cause of heart failure and the primary reason for heart transplantation; upward of 70% of DCM cases are considered idiopathic. Our in-vitro experiments showed that reduced hybrid/complex N-glycosylation in mouse cardiomyocytes is linked with DCM. Further, we observed direct effects of reduced N-glycosylation on Kv gating. However, it is difficult to rigorously determine the effects of glycosylation on Kv activity, because there are multiple Kv isoforms in cardiomyocytes contributing to the cardiac excitation. Due to complex functions of Kv isoforms, only the sum of K+ currents (IKsum) can be recorded experimentally and decomposed later using exponential fitting to estimate component currents, such as IKto, IKslow, and IKss. However, such estimation cannot adequately describe glycosylation effects and Kv mechanisms. Here, we propose a framework of simulation modeling of Kv kinetics in mouse ventricular myocytes and model calibration using the in-vitro data under normal and reduced glycosylation conditions through ablation of the Mgat1 gene (i.e., Mgat1KO). Calibrated models facilitate the prediction of Kv characteristics at different voltages that are not directly observed in the in-vitro experiments. A model calibration procedure is developed based on the genetic algorithm. Experimental results show that, in the Mgat1KO group, both IKto and IKslow densities are shown to be significantly reduced and the rate of IKslow inactivation is much slower. The proposed approach has strong potential to couple simulation models with experimental data for gaining a better understanding of glycosylation effects on Kv kinetics.

Translating solution to solid phase glycosylation conditions.

Optimization of glycosylation conditions for automated glycan assembly is highly challenging, demands wasteful use of precious building blocks, and relies on nontrivial analyses. We developed a semi-quantitative method for automated optimization of glycosylation temperature that utilized minute quantities of donors and translated those conditions to solid-phase glycan assembly.

Characterization of Mannosyl Dioxanium Ions in Solution Using Chemical Exchange Saturation Transfer NMR Spectroscopy.

The stereoselective introduction of the glycosidic bond remains one of the main challenges in carbohydrate synthesis. Characterizing the reactive intermediates of this reaction is key to develop stereoselective glycosylation reactions. Herein we report the characterization of low‐populated, rapidly equilibrating mannosyl dioxanium ions that arise from participation of a C‐3 acyl group using chemical exchange saturation transfer (CEST) NMR spectroscopy. Dioxanium ion structure and equilibration kinetics were measured under relevant glycosylation conditions and highly α‐selective couplings were observed suggesting glycosylation took place via this elusive intermediate.

Characterization of Mannosyl Dioxanium Ions in Solution Using Chemical Exchange Saturation Transfer NMR Spectroscopy

AbstractThe stereoselective introduction of the glycosidic bond remains one of the main challenges in carbohydrate synthesis. Characterizing the reactive intermediates of this reaction is key to develop stereoselective glycosylation reactions. Herein we report the characterization of low‐populated, rapidly equilibrating mannosyl dioxanium ions that arise from participation of a C‐3 acyl group using chemical exchange saturation transfer (CEST) NMR spectroscopy. Dioxanium ion structure and equilibration kinetics were measured under relevant glycosylation conditions and highly α‐selective couplings were observed suggesting glycosylation took place via this elusive intermediate.

Synthesis of 6-Aza-2-Hydroxyimino-5-Methylpyrimidine Nucleosides for Antiviral Evaluation.

The syntheses of a series of novel 6-aza-2-hydroxyimino-5-methylpyrimidine and related nucleosides are described. A suitably protected 2-methylthiopyrimidine nucleoside was selected as the precursor for installing a hydroxyimino moiety at the C-2 position. The starting nucleobase 6-aza-5-methyl-2-thiouracil is prepared in two steps from thiosemicarbazone and ethyl pyruvate. This is subjected to coupling with 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose under Vorbrüggen glycosylation conditions to provide the corresponding nucleoside in high yield. Activation of the nucleoside to the corresponding 2-methylthio derivative followed by treatment with hydroxylamine hydrochloride in pyridine provides the corresponding 2-hydroxyimino derivative in high yield. Finally, the synthesis of five free modified nucleoside analogs is described. The newly synthesized nucleosides have been evaluated against an RNA viral panel and moderate activity was observed against hepatitis C virus, Zika virus, and human respiratory syncytial virus. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of 6-aza-5-methyl-2-thiouracil Basic Protocol 2: Preparation of 6-aza-5-methyl-2-thiouridine and 6-aza-5-methyluridine Basic Protocol 3: Preparation of 6-aza-2-hydroxyimino-5-methyluridine Basic Protocol 4: Preparation of 6-aza-2-hydroxyimino-5-methyl-4-thiouridine and 6-aza-2-hydroxyimino-5-methylcytosine.

Whole-cell catalytic synthesis of 2-O-α-glucopyranosyl-l-ascorbic acid by sucrose phosphorylase from Bifidobacterium breve via a batch-feeding strategy

l-ascorbic acid 2-O-α-d-glucoside (AA-2G), a highly stabilized vitamin C derivative, has been widely applied in cosmetics, foods, and pharmaceuticals. Sucrose phosphorylase (SPase) was recently reported to be effective for producing AA-2G. In this study, SPase from Bifidobacterium breve (BbrSPase) was identified as a new AA-glycosylating tool with high activity and good thermostability. The optimal conditions for AA glycosylation are pH 5.5 and 55 °C. The Km and kcat when sucrose was applied as a glycosyl donor were 2.72 ± 0.49 mM and 9.70 ± 0.10 s−1, respectively. The transglycosylation activity was seriously inhibited by fructose, which caused a sharp decline in the glycosylation rate but did not necessarily stop the yield of AA-2G due to the high affinity to sucrose. Therefore, to attenuate the inhibition of fructose, a new sucrose batch-feeding strategy was developed for the effective production of AA-2G. The maximum titer was 185 g/L within 72 h. The molar conversion rate of l-AA reached 50.2 %, which is the highest conversion rate to our knowledge.

Synthesis of a cyclic tetramer of 3-amino-3-deoxyallose with axially oriented amino groups

A linear tetramer of β-(1 → 6)-linked 3-azido-3-deoxy-d-allose containing glycosyl donor and glycosyl acceptor functions in the terminal monosaccharide units was prepared starting from 3-azido-3-deoxy-1,2:5,6-di-O-isopropylidene-α-d-allofuranose. Cyclization of the linear tetramer under glycosylation conditions afforded the corresponding cyclic tetrasaccharide in 77% yield; its deprotection and reduction of the azido groups resulted in the formation of the cyclic tetramer of 3-amino-3-deoxy-d-allose with axial amino groups, a potential scaffold for the synthesis of tetravalent functional clusters.

Optimization of Hemoglobin Chitosan Glycosylation Conditions and Structural Characteristics and Functions of Glycosylated Hemoglobin after an In vitro Digestion

ABSTRACT The relation of glycosylation and function in Tegillarca Granosa hemoglobin was explored in this study. The manuscript firstly discusses the conditions of glycosylation between chitosan and hemoglobin, as well as the properties and functions of glycosylated hemoglobin. The results showed that optimal conditions for glycosylation of chitosan and hemoglobin were mass fraction ratio of hemoglobin and chitosan of 4:1, pH 5.5, reaction time of 15 min, reaction temperature of 55°C. After glycosylation, surface hydrophobicity, antioxidant, iron bioavailability of hemoglobin increased by 179.78%, 11.06%, 22.96% respectively; while water holding capacity, protein digestibility decreased by 24.80%, 30.49% respectively. Moreover, glycosylation increased structural stability of hemoglobin due to FT/IR absorption peaks without significant changes before and after In vitro digestion, meaning that glycosylated hemoglobin was more resistant to degradation. Therefore, glycosylation could make hemoglobin structure stable and enhance its antioxidation and iron supplement, which provides theoretical basis for application of glycosylation in food.