C-glycosidic Bond Research Articles

An Unexpected Lewis Acid-Catalyzed Cascade during the Synthesis of the DEF-Benzoxocin Ring System of Nogalamycin and Menogaril: Mechanistic Elucidation by Intermediate Trapping Experiments and Density Functional Theory Studies.

An unexpected Lewis acid-catalyzed carbohydrate rearrangement of a 1,5-bis-glycopyranoside to the product of a formal intramolecular C-aryl glycosylation reaction is reported. Mechanistic studies based mainly on intermediate trapping experiments and density functional theory (DFT) calculations reveal a cascade process involving three transient (a)cyclic oxocarbenium cations, the breaking of three single C(sp3)-O bonds, and the formation of three single bonds (i.e., exo-, endo-, and C-glycosidic bonds), leading to the 2,6-epoxybenzoxocine skeleton of bioactive natural glycoconjugates related to serjanione A and mimocaesalpin E. DFT calculations established that the generation of the pyran moiety embedded in the bridged benzoxocin ring system is likely to proceed through an unusual ring-closure of an ortho-quinone methide intermediate in which the attacking nucleophile is a carbonyl oxygen.

C-Glycoside-Metabolizing Human Gut Bacterium, Dorea sp. MRG-IFC3.

Biochemical gut metabolism of dietary bioactive compounds is of great significance in elucidating health-related issues at the molecular level. In this study, a human gut bacterium cleaving C-C glycosidic bond was screened from puerarin conversion to daidzein, and a new, gram-positive C-glycoside-deglycosylating strain, Dorea sp. MRG-IFC3, was isolated from human fecal sample under anaerobic conditions. Though MRG-IFC3 biotransformed isoflavone C-glycoside, it could not metabolize other C-glycosides, such as vitexin, bergenin, and aloin. As evident from the production of the corresponding aglycons from various 7-O-glucosides, MRG-IFC3 strain also showed 7-O-glycoside cleavage activity; however, flavone 3-O-glucoside icariside II was not metabolized. In addition, for mechanism study, C-glycosyl bond cleavage of puerarin by MRG-IFC3 strain was performed in D2O GAM medium. The complete deuterium enrichment on C-8 position of daidzein was confirmed by 1H NMR spectroscopy, and the result clearly proved for the first time that daidzein is produced from puerarin. Two possible reaction intermediates, the quinoids and 8-dehydrodaidzein anion, were proposed for the production of daidzein-8d. These results will provide the basis for the mechanism study of stable C-glycosidic bond cleavage at the molecular level.

Photoredox-catalyzed C-glycosylation of peptides with glycosyl bromides

Glycosyl radicals, produced under mild photoredox conditions, show unique utility in the preparation of C-linked glycoconjugates. We herein report the construction of C-glycosidic bonds on α,β-dehydroalanine (DHA) of peptides with easily available glycosyl bromides as glycosyl radical precursors under highly anomeric control, leading to C-glycosylation modifications of peptides. This method not only has outstanding functional group compatibility, but also is feasible in near-physiological conditions (pH ∼ 7 and temperature T ≤ 37 °C in aqueous media).

Divergent Synthesis of Stachyurin and Casuarinin Focusing on C-Glycosidic Bond Reactivity.

Stachyurin and casuarinin are ellagitannins, a class of polyphenols that exhibit various biological activities that have an impact on human health. Casuarinin is a stachyurin stereoisomer. These compounds contain the characteristic C-glycosidic bond between the open-chain d-glucose and the phenol aromatic ring. Therefore, chemical elucidation of the C-glycosidic bond reactivity is required to exploit their multiple bioactivities. This study developed a method for the divergent synthesis of stachyurin and casuarinin via the α-selective C-glycosylation as well as the β-selective introduction of the oxygen functional group, focusing on structural specificity. The proposed method applies to the syntheses of stachyurin and casuarinin analogues, thereby facilitating the utilisation of their beneficial bioactivities.

Bioactive Xanthone C-glycoside Derivatives – QSAR Approach

Introduction. Xanthone glycosides have unique structures and properties. Many efforts focus on the search for C-glycoside derivatives of mangiferin with higher bioavailability. The application of the QSAR approach allows for the optimization of the search for novel xanthone derivatives with the desired characteristics.Aim. Using available descriptors of chemical structure, physical-chemical properties, and biological activity, analyze a sample set of known homologs and analogs of mangiferin to QSAR prognosis bioactivity of new xanthone C-glycosides.Materials and methods. 26 molecules of natural homologs and modified derivatives of mangiferin formed the analyzed sample set. Topological graphs of compounds were constructed using ChemicPen software. ChemicDescript software was used for the calculation of molecular descriptors, including the Balaban index. Physicochemical characteristics of molecules as well as Lipinski's rule criteria were calculated in Molinspiration. The spectrum of the most probable (Pa > 0.7) biological activity of the described compounds were predicted using Pass Online. The software Origin (OriginLab, USA) was used for the graphical representation of the results.Results and discussion. Mangiferin and its natural homologs are the most hydrophilic compounds. The hydrolysis of the C-glycosidic bond, alkylation, acylation, and the introduction of an amino substituent radical into the mangiferin structure led to the increase of its lipophilic properties. The spectrum of the most probable biological activities of the described molecules: antitumor, antioxidant, and cardioprotective effects. The results of ADMET modeling based on the substance-drug similarity criteria showed that only 4 compounds correspond to the rule of five. We proposed the validation model to predict bioactivity from lipophilicity and molecule structure described with Balaban index. The error of prediction obtained in a result of cross-validation turned out to be about less than 3 %.Conclusion. A correlation between the structure and properties of the molecules discussed has been demonstrated. The obtained results can be used for further prediction of the properties of natural and synthetic xanthone C-glycosides and directed synthesis of new active compounds.

Glycosyl Radical-Based Synthesis of C-Glycoamino Acids and C-Glycopeptides.

Radical-based reactions usually exhibit excellent functional-group compatibilities due to their mild initiation conditions. Glycosyl radical involved C-glycosylation modifications are important strategies to achieve highly regio- and chemoselective constructions of C-glycosidic bonds or C-glycoside linkages of peptides and proteins. In this Concept, we cover recent developments in glycosyl radical-based synthesis of unnatural amino acids and late-stage modification of peptides and proteins, and provide a preliminary outlook on the possible development of this direction in the future.

Structural mechanism of a dual-functional enzyme DgpA/B/C as both a C-glycoside cleaving enzyme and an O- to C-glycoside isomerase

The C-glycosidic bond that connects the sugar moiety with aglycone is difficult to be broken or made due to its inert nature. The knowledge of C-glycoside breakdown and synthesis is very limited. Recently, the enzyme DgpA/B/C cascade from a human intestinal bacterium PUE was identified to specifically cleave the C-glycosidic bond of puerarin (daidzein-8-C-glucoside). Here we investigated how puerarin is recognized and oxidized by DgpA based on crystal structures of DgpA with or without substrate and biochemical characterization. More strikingly, we found that apart from being a C-glycoside cleaving enzyme, DgpA/B/C is capable of efficiently converting O- to C-glycoside showing the activity as a structure isomerase. A possible mechanistic model was proposed dependently of the simulated complex structure of DgpB/C with 3″-oxo-daidzin and structure-based mutagenesis. Our findings not only shed light on understanding the enzyme-mediated C-glycosidic bond breakage and formation, but also may help to facilitate stereospecific C-glycoside synthesis in pharmaceutical industry.

Characterization of the Oxazinomycin Biosynthetic Pathway Revealing the Key Role of a Nonheme Iron-Dependent Mono-oxygenase.

Oxazinomycin is a C-nucleoside natural product with antibacterial and antitumor activities. In addition to the characteristic C-glycosidic linkage shared with other C-nucleosides, oxazinomycin also features a structurally unusual 1,3-oxazine moiety, the biosynthesis of which had previously been unknown. Herein, complete in vitro reconstitution of the oxazinomycin biosynthetic pathway is described. Construction of the C-glycosidic bond between ribose 5-phosphate and an oxygen-labile pyridine heterocycle is catalyzed by the C-glycosidase OzmB and involves formation of an enzyme-substrate Schiff base intermediate. The DUF4243 family protein OzmD is shown to catalyze oxygen insertion and rearrangement of the pyridine C-nucleoside intermediate to generate the 1,3-oxazine moiety along with the elimination of cyanide. Spectroscopic analysis and mutagenesis studies indicate that OzmD is a novel nonheme iron-dependent enzyme in which the catalytic iron center is likely coordinated by four histidine residues. These results provide the first example of 1,3-oxazine biosynthesis catalyzed by an unprecedented iron-dependent mono-oxygenase.

A newly isolated human intestinal strain deglycosylating flavonoid C-glycosides.

Glycosidic bond of C-glycosides is difficult to be broken due to its chemical stability. Screening specific microbe from microbiota is a practical way to deglycosylate these compounds. In this study, a new strain W974-1 which is capable of cleaving C-glycosidic bonds was isolated from human gut microbiota by spread plate method. It deglycosylates flavonoid 8-C-glycosides such as orientin and vitexin to their aglycones with the enzymes secreted outside the bacterial cells. This strain was identified as Enterococcus avium by 16S rDNA sequencing, physiological and biochemical characterization.

Discovery of novel glycoside hydrolases from C-glycoside-degrading bacteria using sequence similarity network analysis.

C-Glycosides are an important type of natural product with significant bioactivities, and the C-glycosidic bonds of C-glycosides can be cleaved by several intestinal bacteria, as exemplified by the human faeces-derived puerarin-degrading bacterium Dorea strain PUE. However, glycoside hydrolases in these bacteria, which may be involved in the C-glycosidic bond cleavage of C-glycosides, remain largely unknown. In this study, the genomes of the closest phylogenetic neighbours of five puerarin-degrading intestinal bacteria (including Dorea strain PUE) were retrieved, and the protein-coding genes in the genomes were subjected to sequence similarity network (SSN) analysis. Only four clusters of genes were annotated as glycoside hydrolases and observed in the genome of D. longicatena DSM 13814T (the closest phylogenetic neighbour of Dorea strain PUE); therefore, genes from D. longicatena DSM 13814T belonging to these clusters were selected to overexpress recombinant proteins (CG1, CG2, CG3, and CG4) in Escherichia coli BL21(DE3). In vitro assays indicated that CG4 efficiently cleaved the O-glycosidic bond of daidzin and showed moderate β-D-glucosidase and β-D-xylosidase activity. CG2 showed weak activity in hydrolyzing daidzin and pNP-β-D-fucopyranoside, while CG3 was identified as a highly selective and efficient α-glycosidase. Interestingly, CG3 and CG4 could be selectively inhibited by daidzein, explaining their different performance in kinetic studies. Molecular docking studies predicted the molecular determinants of CG2, CG3, and CG4 in substrate selectivity and inhibition propensity. The present study identified three novel and distinctive glycoside hydrolases, highlighting the potential of SSN in the discovery of novel enzymes from genomic data.

Natural product C-glycosyltransferases - a scarcely characterised enzymatic activity with biotechnological potential.

Covering: up to 2020C-Glycosyltransferases are enzymes that catalyse the transfer of sugar molecules to carbon atoms in substituted aromatic rings of a variety of natural products. The resulting β-C-glycosidic bond is more stable in vivo than most O-glycosidic bonds, hence offering an attractive modulation of a variety of compounds with multiple biological activities. While C-glycosylated natural products have been known for centuries, our knowledge of corresponding C-glycosyltransferases is scarce. Here, we discuss commonalities and differences in the known C-glycosyltransferases, review attempts to leverage them as synthetic biocatalysts, and discuss current challenges and limitations in their research and application.

Crystal Structures of the C-Glycosyltransferase UGT708C1 from Buckwheat Provide Insights into the Mechanism of C-Glycosylation.

C-Glycosyltransferases (CGTs) catalyze the formation of C-glycosidic bonds for the biosynthesis of C-glycosides, but the underlying mechanism is unclear. This process improves the solubility and bioavailability of specialized metabolites, which play important roles in plant growth and development and represent rich resources for drug discovery. Here, we performed functional and structural studies of the CGT UGT708C1 from buckwheat (Fagopyrum esculentum). Enzymatic analysis showed that UGT708C1 is capable of utilizing both UDP-galactose and UDP-glucose as sugar donors. Our structural studies of UGT708C1 complexed with UDP-glucose and UDP identified the key roles of Asp382, Gln383, Thr151, and Thr150 in recognizing the sugar moiety of the donor substrate and Phe130, Tyr102, and Phe198 in binding and stabilizing the acceptor. A systematic site-directed mutagenesis study confirmed the important roles of these residues. Further structural analysis combined with molecular dynamics simulations revealed that phloretin binds to the acceptor binding pocket in a bent state with a precise spatial disposition and complementarity. These findings provide insights into a catalytic mechanism for CGTs.

Deglycosylation of the Isoflavone C-Glucoside Puerarin by a Combination of Two Recombinant Bacterial Enzymes and 3-Oxo-Glucose.

A human intestinal bacterium strain related to Dorea species, PUE, can metabolize the isoflavone C-glucoside puerarin (daidzein 8-C-glucoside) to daidzein and glucose. We reported previously that 3″-oxo-puerarin is an essential reaction intermediate in enzymatic puerarin degradation, and we characterized a bacterial enzyme, the DgpB-DgpC complex, that cleaved the C-glycosidic bond in 3″-oxo-puerarin. However, the exact enzyme catalyzing the oxidation of the C-3″ hydroxyl in puerarin has not been identified. In this study, we demonstrated that recombinant DgpA, a Gfo/Idh/MocA family oxidoreductase, catalyzed puerarin oxidation in the presence of 3-oxo-glucose as the hydride acceptor. In the redox reaction, NAD(H) functioned as the cofactor, which bound tightly but noncovalently to DgpA. Kinetics analysis of DgpA revealed that the reaction proceeded via a ping-pong mechanism. Enzymatic C-deglycosylation of puerarin was achieved by a combination of recombinant DgpA, the DgpB-DgpC complex, and 3-oxo-glucose. In addition, the metabolite derived from the sugar moiety in the 3″-oxo-puerarin-cleaving reaction catalyzed by the DgpB-DgpC complex was characterized as 1,5-anhydro-d-erythro-hex-1-en-3-ulose, suggesting that the C-glycosidic linkage is cleaved through a β-elimination-like mechanism.IMPORTANCE One important role of the gut microbiota is to metabolize dietary nutrients and supplements such as flavonoid glycosides. Ingested glycosides are metabolized by intestinal bacteria to more-absorbable aglycones and further degradation products that show beneficial effects in humans. Although numerous glycoside hydrolases that catalyze O-deglycosylation have been reported, enzymes responsible for C-deglycosylation are still limited. In this study, we characterized enzymes involved in the C-deglycosylation of puerarin from a human intestinal bacterium, PUE. Here, we report the purification and characterization of a recombinant oxidoreductase involved in C-glucoside degradation. This study provides new insights for the elucidation of mechanisms of enzymatic C-deglycosylation.

Total Synthesis of Casuarinin.

This study involves the total synthesis of casuarinin, a naturally occurring ellagitannin, in which an open-chain glucose is esterified with two (S)-hexahydroxydiphenoyl (HHDP) groups. One HHDP group incorporates a C-glycosidic bond between its benzene ring and the glucose moiety, which was constructed with complete stereoselectivity using a benzyl oxime group that opened the glucopyranose ring and acted as a scaffold for C-glycoside production. This total synthesis enables future structure-activity relationship studies of this compound.

A Kinase and a Glycosylase Catabolize Pseudouridine in the Peroxisome to Prevent Toxic Pseudouridine Monophosphate Accumulation.

Pseudouridine (Ψ) is a frequent nucleoside modification that occurs in both noncoding RNAs and mRNAs. In pseudouridine, C5 of uracil is attached to the Rib via an unusual C-glycosidic bond. This RNA modification is introduced on the RNA by site-specific transglycosylation of uridine (U), a process mediated by pseudouridine synthases. RNA is subject to constant turnover, releasing free pseudouridine, but the metabolic fate of pseudouridine in eukaryotes is unclear. Here, we show that in Arabidopsis (Arabidopsis thaliana), pseudouridine is catabolized in the peroxisome by (1) a pseudouridine kinase (PUKI) from the PfkB family that generates 5'-pseudouridine monophosphate (5'-ΨMP) and (2) a ΨMP glycosylase (PUMY) that hydrolyzes ΨMP to uracil and ribose-5-phosphate. Compromising pseudouridine catabolism leads to strong pseudouridine accumulation and increased ΨMP content. ΨMP is toxic, causing delayed germination and growth inhibition, but compromising pseudouridine catabolism does not affect the Ψ/U ratios in RNA. The bipartite peroxisomal PUKI and PUMY are conserved in plants and algae, whereas some fungi and most animals (except mammals) possess a PUMY-PUKI fusion protein, likely in mitochondria. We propose that vacuolar turnover of ribosomal RNA produces most of the pseudouridine pool via 3'-ΨMP, which is imported through the cytosol into the peroxisomes for degradation by PUKI and PUMY, a process involving a toxic 5'-ΨMP intermediate.

Unveiling the Dynamical and Structural Features That Determine the Orientation of the Acceptor Substrate in the Landomycin Glycosyltransferase LanGT2 and Its Variant with C-Glycosylation Activity.

Many bioactive compounds are O-glycosylated metabolites; however, the hydrolytic sensitivity of O-glycosidic linkage limits their therapeutic applications. Enzymatically and chemically stable C-glycosidic bonds are thought of as a potential solution to overcome this problem, although the insufficient information about the structural preferences and interactions that distinguish the C- from the O-glycosylation reactions has hindered the development of enzyme engineering strategies. Thus, in this work, the O-glycosyltransferase LanGT2 (O-LanGT2) and its engineered C-C bond-forming variant (C-LanGT2), which catalyze the initial glycosylation step in the biosynthesis of the antibiotic landomycin A, were studied by means of all-atom Molecular Dynamics simulations. Our results indicate that precise positioning of the acceptor substrate tetrangulol (TET) seems to be determined by the flexibility of the loop 51-62, which gives rise to slightly different secondary structural elements that modulate the interactions between this loop and TET. In O-LanGT2, the most notable interactions between TET and the loop 51-62 involve R59 and A62, whereas in C-LanGT2 they involve A8, I58, and I62. Although A8 is not part of the loop 51-62, it turns out to be key to the binding mode exhibited by TET in C-LanGT2. Thus, the TET-A62 (O-LanGT2) and TET-A8 (C-LanGT2) interactions appear to be critical to accomplish the O- and the C-glycosidic bond specificity, respectively. Finally, all results together shed light on the molecular basis governing the O- and C-bond specificity, revealing that the underlying molecular mechanism that tunes the orientation of TET at its binding pocket involves hydrophobic interactions.

Fucosylated inhibitors of recently identified bangle lectin from Photorhabdus asymbiotica

A recently described bangle lectin (PHL) from the bacterium Photorhabdus asymbiotica was identified as a mainly fucose-binding protein that could play an important role in the host-pathogen interaction and in the modulation of host immune response. Structural studies showed that PHL is a homo-dimer that contains up to seven l-fucose-specific binding sites per monomer. For these reasons, potential ligands of the PHL lectin: α-l-fucopyranosyl-containing mono-, di-, tetra-, hexa- and dodecavalent ligands were tested. Two types of polyvalent structures were investigated – calix[4]arenes and dendrimers. The shared feature of all these structures was a C-glycosidic bond instead of the more common but physiologically unstable O-glycosidic bond. The inhibition potential of the tested structures was assessed using different techniques – hemagglutination, surface plasmon resonance, isothermal titration calorimetry, and cell cross-linking. All the ligands proved to be better than free l-fucose. The most active hexavalent dendrimer exhibited affinity three orders of magnitude higher than that of standard l-fucose. To determine the binding mode of some ligands, crystal complex PHL/fucosides 2 – 4 were prepared and studied using X-ray crystallography. The electron density in complexes proved the presence of the compounds in 6 out of 7 fucose-binding sites.

A newly isolated human intestinal bacterium strain capable of deglycosylating flavone C-glycosides and its functional properties

BackgroundFlavone C-glycosides are difficult to be deglycosylated using traditional chemical methods due to their solid carbon–carbon bond between sugar moieties and aglycones; however, some bacteria may easily cleave this bond because they generate various specific enzymes.ResultsA bacterial strain, named W12-1, capable of deglycosylating orientin, vitexin, and isovitexin to their aglycones, was isolated from human intestinal bacteria in this study and identified as Enterococcus faecalis based on morphological examination, physiological and biochemical identification, and 16S rDNA sequencing. The strain was shown to preferentially deglycosylate the flavone C-glycosides on condition that the culture medium was short of carbon nutrition sources such as glucose and starch, and its deglycosylation efficiency was negatively correlated with the content of the latter two substances.ConclusionThis study provided a new bacterial resource for the cleavage of C-glycosidic bond of flavone C-glycosides and reported the carbon nutrition sources reduction induced deglycosylation for the first time.

Expression and Characterization of the Human Intestinal Bacterial Enzyme Which Cleaves the C-Glycosidic Bond in 3″-Oxo-puerarin.

Puerarin (daidzein 8-C-glucoside) is an isoflavone C-glucoside contained in the roots of Pueraria lobata OHWI. We have previously isolated the human intestinal bacterium, strain PUE, which metabolizes puerarin to daidzein, though the enzyme which cleaves C-glycosidic bond has not been clarified. Here, we identified one of the intermediates of enzymatic puerarin C-deglycosylation reaction as 3″-oxo-puerarin (1): C-3 in the glucose moiety connecting to hydroxyl is oxidized to ketone group. 1 was easily isomerized to the mixture of 1, 2″-oxo-puerarin (2a) and cyclic acetal (2b) of 2a in non-enzymatic condition. We identified the putative puerarin-metabolizing operon of strain PUE composed of 8 genes (dgpA-H). Among them, DgpB-C complex was expressed in Escherichia coli, which cleaved the C-glycosidic bond in 1 but not puerarin. These results suggested that the puerarin C-deglycosylation reaction is a two-step enzymatic reaction, including the oxidation reaction at C-3″ in puerarin to give 1, and the subsequent C-deglycosylation of 1 to provide daidzein.

Construction of the DEF-Benzoxocin Ring System of Nogalamycin and Menogaril via a Reductive Heck Cyclization.

Construction of the DEF-ring system of nogalamycin and menogaril has been achieved with a novel reductive Heck cyclization approach. Our strategy exploited the stereoelectronic preferences dictated by the anomeric effect for introduction of an O-glycosidic bond in order to direct the introduction of a bridging C-glycosidic bond with the desired stereochemistry. Our strategy relied upon a stereoselective O-aryl glycosylation reaction and a highly efficient selenoxide elimination to provide the key substrate for study of the reductive Heck cyclization reaction. The cyclization proceeded smoothly under the optimized conditions, in a yield comparable to that achieved in our previously reported model study.