Consecutive Glycosylation Research Articles

Multi-omics analysis reveals promiscuous O-glycosyltransferases involved in the diversity of flavonoid glycosides in Periploca forrestii (Apocynaceae)

Flavonoid glycosides are widespread in plants, and are of great interest owing to their diverse biological activities and effectiveness in preventing chronic diseases. Periploca forrestii, a renowned medicinal plant of the Apocynaceae family, contains diverse flavonoid glycosides and is clinically used to treat rheumatoid arthritis and traumatic injuries. However, the mechanisms underlying the biosynthesis of these flavonoid glycosides have not yet been elucidated. In this study, we used widely targeted metabolomics and full-length transcriptome sequencing to identify flavonoid diversity and biosynthetic genes in P. forrestii. A total of 120 flavonoid glycosides, including 21 C-, 96 O-, and 3 C/O-glycosides, were identified and annotated. Based on 24,123 full-length coding sequences, 99 uridine diphosphate sugar-utilizing glycosyltransferases (UGTs) were identified and classified into 14 groups. Biochemical assays revealed that four UGTs exhibited O-glycosyltransferase activity toward apigenin and luteolin. Among them, PfUGT74B4 and PfUGT92A8 were highly promiscuous and exhibited multisite O-glycosylation or consecutive glycosylation activities toward various flavonoid aglycones. These four glycosyltransferases may significantly contribute to the diversity of flavonoid glycosides in P. forrestii. Our findings provide a valuable genetic resource for further studies on P. forrestii and insights into the metabolic engineering of bioactive flavonoid glycosides.

Synthesis and in vitro synergistic antifungal activity of analogues of Panax stipulcanatus saponin against fluconazole-resistant Candida albicans

Oleanolic acid 3-O-β-d-glucopyranosyl-(1 → 3)-β-d-glucopyranoside (1) and oleanolic acid 3-O-β-d-glucopyranosyl-(1 → 3)-[α-l-arabinofuranosyl-(1 → 4)]-β-d-glucopyranoside (2), novel Panax stipulcanatus saponin analogues, were synthesized for the first time starting from commercially available oleanolic acid, d-glucose, and L-(+)-arabinose. Glycosyl N-phenyltrifluoroacetimidates as donors and two-step consecutive glycosylation reactions are crucial in the synthesis. In vitro antifungal activity results indicated that analogue 2 combined with fluconazole showed synergistic antifungal activity against fluconazole-resistant Candida albicans, with MIC50 values 31.80 μg/mL and FICI values 0.32. We also found that intermediate compounds 16 and 17 revealed synergistic antifungal activity against susceptible Candida albicans when combined with fluconazole, with MIC50 values 1.43 μg/mL and 1.59 μg/mL, FICI values 0.29 and 0.32, respectively.

Unravelling the consecutive glycosylation and methylation of flavonols in peach in response to UV‐B irradiation

Flavonol glycosides are bioactive compounds important for plant defence and human nutrition. Glycosylation and methylation play an important role in enriching the diversity of flavonols in response to the environment. Peach flowers and fruit are rich in flavonol diglycosides such as isorhamnetin 3-O-rutinoside (I3Rut), kaempferol 3-O-rutinoside and quercetin 3-O-rutinoside, and flavonol monoglycosides such as I 3-O-glucoside and Q 3-O-galactoside. UV-B irradiation of fruit significantly induced accumulation of all these flavonol glycosides. Candidate biosynthetic genes induced by UV-B were identified by genome homology searches and the in vitro catalytic activities of purified recombinant proteins determined. PpUGT78T3 and PpUGT78A2 were identified as flavonol 3-O-glucosyltransferase and 3-O-galactosyltransferase, respectively. PpUGT91AK6 was identified as flavonol 1,6-rhamnosyl trasferase catalysing the formation of flavonol rutinosides and PpFOMT1 was identified as a flavonol O-methyltransferase that methylated Q at the 3'-OH-OH to form isorhamnetin derivatives. Transient expression in Nicotiana benthamiana confirmed the specificity of PpUGT78T3 as a flavonol 3-O-glucosyltransferase, PpUGT78A2 as a 3-O-galactosyltransferase, PpUGT91AK6 as a 1,6-rhamnosyltrasferase and PpFOMT1 as an O-methyltransferase. This study provides new insights into the mechanisms of glycosylation and methylation of flavonols, especially the formation of flavonol diglycosides such as I3Rut, and will also be useful for future potential metabolic engineering of complex flavonols.

Amylosucrase from Deinococcus geothermalis can be modulated under different reaction conditions to produce novel quercetin 4′-O-α-d-isomaltoside

Amylosucrase (ASase, EC.4.2.1.4) is well-known for its distinguishable property of transglycosylation of many flavonoids and phenolics. Quercetin has diverse biological functions, however, its use is limited due to poor solubility and bioavailability. ASase derived from Deinococcus geothermalis (DGAS) showed conditional preference for producing unusual quercetin glucosides (QGs). DGAS produced a variety of QGs including quercetin monoglucosides (QG1), diglucosides (QG2 and QG2′), and triglucoside from quercetin and sucrose. The newly synthesized QG2′ was recognized as a novel quercetin isomaltoside with an α-1,6 linkage branched at the −OH of C4′ in quercetin by mass and nuclear magnetic resonance spectra. With a higher conversion yield from quercetin to QGs (60–92%), the optimum conditions for producing QG2′ were examined under various pH and sucrose concentrations by response surface methodology. QG2′ was predominantly produced under acidic conditions (pH 5.0) and at high sucrose concentrations (1000–1500 mM). In contrast, QG1 was generated as an intermediate of consecutive glycosylation. Kinetic evaluations indicated that considerable differences of transglycosylation velocities were caused by the pH and buffer salts of the reaction, which had a 3.9-fold higher overall performance (kcat/K′m) of generating QG2′ at pH 5 compared to at pH 7. A rationale of unusual transglycosylations was demonstrated with a molecular docking simulation. Taken together, our study demonstrated that ASase can be used to synthesize unusually branched flavonoid glycosides from flavonol aglycones with clear patterns by modulating reaction conditions.

Discovery of UDP-Glycosyltransferases and BAHD-Acyltransferases Involved in the Biosynthesis of the Antidiabetic Plant Metabolite Montbretin A.

Plant specialized metabolism serves as a rich resource of biologically active molecules for drug discovery. The acylated flavonol glycoside montbretin A (MbA) and its precursor myricetin 3-O-(6'-O-caffeoyl)-glucosyl rhamnoside (mini-MbA) are potent inhibitors of human pancreatic α-amylase and are being developed as drug candidates to treat type-2 diabetes. MbA occurs in corms of the ornamental plant montbretia (Crocosmia x crocosmiiflora), but a system for large-scale MbA production is currently unavailable. Biosynthesis of MbA from the flavonol myricetin and MbA accumulation occur during early stages of corm development. We established myricetin 3-O-rhamnoside (MR), myricetin 3-O-glucosyl rhamnoside (MRG), and mini-MbA as the first three intermediates of MbA biosynthesis. Contrasting the transcriptomes of young and old corms revealed differentially expressed UDP-sugar-dependent glycosyltransferases (UGTs) and BAHD-acyltransferases (BAHD-ATs). UGT77B2 and UGT709G2 catalyze the consecutive glycosylation of myricetin to produce MR and of MR to give MRG, respectively. In addition, two BAHD-ATs, CcAT1 and CcAT2, catalyze the acylation of MRG to complete the formation of mini-MbA. Transcript profiles of UGT77B2, UGT709G2, CcAT1, and CcAT2 during corm development matched the metabolite profile of MbA accumulation. Expression of these enzymes in wild tobacco (Nicotiana benthamiana) resulted in the formation of a surrogate mini-MbA, validating the potential for metabolic engineering of mini-MbA in a heterologous plant system.

ChemInform Abstract: Silver Trifluoromethanesulfonate and Metallic Copper Mediated Syntheses of 1,2,3‐Triazole‐O‐ and Triazolyl Glycoconjugates: Consecutive Glycosylation and Cyclization under One‐Pot Condition.

AbstractThe title reaction involves consecutive glycosidation and azide alkyne cyclization under one‐pot conditions using a combination of AgOTf and Cu(0) catalyst.

Silver trifluoromethanesulfonate and metallic copper mediated syntheses of 1,2,3-triazole-O- and triazolyl glycoconjugates: consecutive glycosylation and cyclization under one-pot condition

A new protocol for synthesis of 1,2,3-triazole glycoconjugates has been described. The reaction involves consecutive glycosylation and azide alkyne cyclization under one-pot condition using a combination of AgOTf and Cu(0) catalyst. Here, AgOTf acts as a promoter for the glycosylation reaction and then as a co-catalyst for the subsequent click reaction.

A Facile Synthesis of the Tetrasaccharide Repeating Unit of the O-Antigen from Cronobacter turicensis

GRAPHICAL ABSTRACTThe para-methoxyphenyl glycoside of a tetrasaccharide repeating unit, β-D-GlcNAc-(1→3)-β-D-GlcNAc-(1→3)-[α-D-Glc-(1→2)]-α-L-Rha-O-(C6H4-p-OMe), of the polysaccharide O-antigen from Cronobacter turicensis was synthesized by an efficiently and linear assembly strategy. Consecutive glycosylation of the 3-O- and 2-O-positions of an L-rhamnopyranoside derivative with free 2,3-hydroxyl groups using a D-glucosaminyl and a D-glucosyl donor, respectively, accomplished a branched trisaccharide rapidly. Selective 3′-O-deacetylation of the trisaccharide followed by glucosaminylation afforded the fully protected tetrasaccharide. Multistep protecting group manipulations of the fully protected tetrasaccharide eventually completed the synthesis of the target molecule.

Exploring Glycosylation Reactions under Continuous-Flow Conditions

The industrial development of carbohydrate-based drugs is greatly thwarted by the typical challenges inherent in oligosaccharide synthesis. The practical advantages of continuous-flow synthesis in microreactors (high reproducibility, easy scalability, and fast reaction optimization) may offer an effective support to make carbohydrates more attractive targets for drug-discovery processes. Here we report a systematic exploration of the glycosylation reaction carried out under microfluidic conditions. Trichloroacetimidates and thioglycosides have been investigated as glycosyl donors, using both primary and secondary acceptors. Each microfluidic glycosylation has been compared with the corresponding batch reaction, in order to highlight advantages and drawbacks of microreactors technology. As a significant example of multistep continuous-flow synthesis, we also describe the preparation of a trisaccharide by means of two consecutive glycosylations performed in interconnected microreactors.

ChemInform Abstract: Regioselective Glycosylation of Unprotected Phenyl 1‐Thioglycopyranosides with Phenylboronic Acid as a Transient Masking Group.

AbstractThe presented method for the title reaction can be also used for two consecutive glycosylations in one‐pot manner.

Regioselective Glycosylation of Unprotected Phenyl 1‐Thioglycopyranosides with Phenylboronic Acid as a Transient Masking Group

AbstractA useful protocol is described for the regioselective glycosylation of the secondary alcohols in unprotected glycosyl acceptors. Phenyl 1‐thioglycopyranosides derived from D‐glucose, D‐galactose, D‐glucosamine, L‐rhamnose, and L‐fucose were treated with phenylboronic acid to install a temporary boronic ester, and then submitted to a Koenigs–Knorr glycosylation with perbenzoylated glucopyranosyl or galactopyranosyl bromides. Good yields for coupling to the 3‐position in glucosides and galactosides were achieved, but lower yields were obtained with the other acceptors. With phenyl 1‐thio‐β‐D‐glucopyranoside, the coupling could also be achieved with a superarmed thiogalactoside donor. The product from a glucose–glucose coupling could be subjected to a second regioselective glycosylation at the 6‐position. In the galactose series, a β(1→3)‐linked galactotriose could be prepared in good yield by two consecutive glycosylations in one pot, where a free 2‐hydroxy group ensures neighboring group participation in the second coupling.

Use of iodine for efficient and chemoselective glycosylation with glycosyl ortho-alkynylbenzoates as donor in presence of thioglycosides

A novel and high yielding glycosylation protocol with glycosyl ortho-alkynylbenzoates as donors and iodine as promoter is described. The donors are stable and can be chemoselectively activated in the presence of thioethyl and thiophenyl glycosides. The application of this methodology in one-pot consecutive glycosylation reaction is described.

Oligosaccharide Analogues of Polysaccharides

The α- and γ-CD analogues 6 and 18, which possess a hexa-2,5-diyne-1,6-dioxy unit, were synthesised by intramolecular coupling of the bis-O-propargylated maltohexaoside 4, or the analogous maltooctaoside 16, followed by deprotection. The dialkynylated linear oligosaccharides were obtained by glycosidation of propargyl alcohol with the thioglycosides 1 and 13, reductive cleavage of the benzylidene acetal, and propargylation of the terminal HO−C(4) group, respectively. The β-CD analogues 23 and 25, which possess a penta-1,3-diyn-1-yl-5-oxy unit, were similarly obtained by intramolecular oxidative coupling of 20 and 21, respectively. The linear dialkynylated oligosaccharides 20 and 21 were obtained by two consecutive glycosylations, first with the maltohexaosyl-S-glycoside 1 as donor, and then by glycosylation of the resulting propargyl maltohexoside with the C(4)-ethynylated donor 19. The proximity of the terminal units of maltooligosaccharides allowed a facile intramolecular cycloaddition of the azido alkyne 29 to the isomeric triazoles 30 and 31, which were deprotected to 32 and 33, respectively. Analysis of the intramolecular H-bonds in 6, 23, 25, 32, and 33 showed that insertion of a noncarbohydrate link interrupts a single flip-flop H-bond.

Glycosylation of lactose: synthesis of branched oligosaccharides involved in the biosynthesis of glycolipids having blood-group I activity

Methyl 4- O-(2- O-benzoyl-4,6- O-benzylidene-β- d-galactopyranos)-2,3,6-tri- O-benzoyl-β- d-glucopyranoside ( 2) was [re[ared in four steps from methyl β-lactoside. Crystalline 2 was a convenient substrate for the synthesis of branched oligosaccharides derived from lactose. High-yield glycosylations were performed first at the 3′-position and then, after removal of the benzylidene group, at the 6′-position, using trichloroacetimidates of N-phthaloylamino sugars. 3,4,6-Tri- O-acetyl-2-deoxy-2-phthalimido-β- d-glucopyranosyl trichloroacetimidate was thus used in two consecutive glycosylations, and also in a simultaneous disubstitution of the triol 9, leading in each sequence to the branched tetrasaccharide, β- d-Glc p-NAc-(1→3)-[β- d-Glc pNAc-(1→6)]-β- d-Gal p-(1→4-β- d-GlcOME. Similar glycosylations performed with 3,6-di- O-acetyl-2-deoxy-2-phthalimido-4- O-(2,3,4,6- tetra- O-acetyl-β- d-galactopyranosyl)-β- d-g;ucopyranosyl trichloroacetimidate afforded the branched hexasaccharide β- d-Gal p-(1→4)-β- d-Glc pNAc-(1→3)-[β- d-Gal p-(1→4)-β- d-Glc pNAc-(1→6)]- β- d-Glc pNAc-(1→6)-β- d-Gal p-(1→4)-β- d-GlcOMe, which corresponds to the human-milk oligasaccharide lacto- N- neohexaose and has a strong blood-group activity.