Synthesis of branched Man5 oligosaccharides and an unusual stereochemical observation.
ABSTRACT: Branched mannopentaoses were synthesized through two routes. While assembly from the nonreducing end to the reducing end was more convergent with fewer intermediate steps, two diastereomers were obtained. On the other hand, synthesis from the reducing end to the nonreducing end yielded the mannopentaose with the desired stereochemistry as a single isomer. Our results suggest that it is still challenging to reliably predict stereochemical outcome of a glycosylation reaction. It is necessary to thoroughly characterize anomeric configurations of newly formed glycosidic linkages in complex oligosaccharide synthesis.
Project description:Here we describe the concise syntheses of the 15 diastereomers and key analogs of the natural product tyroscherin. While systematic analysis of the analogs clearly demonstrated that the hydrocarbon tail is important for biological activity, structure-activity relationship studies of the complete tyroscherin diastereoarray revealed a surprisingly expansive stereochemical tolerance for the cytotoxic activity. Our results represent a departure from the tenet that biological activity is constrained to a narrow pharmacophore, and highlight the recently emerging appreciation for stereochemical flexibility in defining the essential structural elements of biologically active small molecules.
Project description:4-Hydroxy-2-nonenal (HNE) is a toxic aldehyde generated during lipid peroxidation and has been implicated in a variety of pathological states associated with oxidative stress. Glutathione S-transferase (GST) A4-4 is recognized as one of the predominant enzymes responsible for the metabolism of HNE. However, substrate and product stereoselectivity remain to be fully explored. The results from a product formation assay indicate that hGSTA4-4 exhibits a modest preference for the biotransformation of S-HNE in the presence of both enantiomers. Liquid chromatography mass spectrometry analyses using the racemic and enantioisomeric HNE substrates explicitly demonstrate that hGSTA4-4 conjugates glutathione to both HNE enantiomers in a completely stereoselective manner that is not maintained in the spontaneous reaction. Compared with other hGST isoforms, hGSTA4-4 shows the highest degree of stereoselectivity. NMR experiments in combination with simulated annealing structure determinations enabled the determination of stereochemical configurations for the GSHNE diastereomers and are consistent with an hGSTA4-4-catalyzed nucleophilic attack that produces only the S-configuration at the site of conjugation, regardless of substrate chirality. In total these results indicate that hGSTA4-4 exhibits an intriguing combination of low substrate stereoselectivity with strict product stereoselectivity. This behavior allows for the detoxification of both HNE enantiomers while generating only a select set of GSHNE diastereomers with potential stereochemical implications concerning their effects and fates in biological tissues.
Project description:A new anomeric linker has been developed that facilitates the purification of glycans prepared by chemoenzymatic approaches and can readily give compounds that are appropriately modified for microarray development or glycan derivatives with a free reducing end that are needed as standards for the development of analytical protocols.
Project description:Heparitinase I, a key lyase enzyme essential for structural analysis of heparan sulfate (HS), degrades HS domains that are undersulfated at glucuronyl residues through an elimination mechanism. Earlier studies employed viscosimetric measurements and electrophoresis to deduce the mechanism of action of heparitinase I and two other related lyases, heparitinase II and heparitinase III. However, these findings lack molecular evidence for the intermediates formed and could not distinguish whether the cleavage occurred from the reducing end or the nonreducing end. In the current study, 2-aminoacridone (2-AMAC)-labeled HS precursor oligosaccharides of various sizes were prepared to investigate the mechanism of heparitinase I-mediated depolymerization using sensitive and quantitative methodologies. Furthermore, fluorescent (2-AMAC) tagging of HS precursor oligosaccharides allowed us to distinguish fragments that result from cleavage of the substrates at various time intervals and sites farther away from the reducing and nonreducing ends of oligosaccharide substrates. This study provides the first direct molecular evidence for a predominantly random endolytic mechanism of cleavage of HS precursor oligosaccharides by heparitinase I. This robust strategy can be adapted to deduce the mechanism of action of other heparitinases and also to deduce structural information of complex HS oligosaccharides of biological importance.
Project description:Bioactivity-guided fractionation of the methanolic root bark extract of Leucophyllum frutescens led to the identification of leubethanol (1), a new serrulatane-type diterpene with activity against both multi-drug-resistant and drug-sensitive strains of virulent Mycobacterium tuberculosis. Leubethanol (1) was identified by 1D/2D NMR data, as a serrulatane closely related to erogorgiane (2), and exhibited anti-TB activity with minimum inhibitory concentrations in the range 6.25-12.50 ?g/mL. Stereochemical evidence for 1 was gleaned from 1D and 2D NOE experiments, from 1H NMR full spin analysis, and by comparison of the experimental vibrational circular dichroism (VCD) spectrum to density functional theory calculated VCD spectra of two diastereomers.
Project description:Spectra-structure relationships were investigated for estimating the anomeric configuration, residues and type of linkages of linear and branched trisaccharides using 13C-NMR chemical shifts. For this study, 119 pyranosyl trisaccharides were used that are trimers of the ? or ? anomers of D-glucose, D-galactose, D-mannose, L-fucose or L-rhamnose residues bonded through a or b glycosidic linkages of types 1?2, 1?3, 1?4, or 1?6, as well as methoxylated and/or N-acetylated amino trisaccharides. Machine learning experiments were performed for: (1) classification of the anomeric configuration of the first unit, second unit and reducing end; (2) classification of the type of first and second linkages; (3) classification of the three residues: reducing end, middle and first residue; and (4) classification of the chain type. Our previously model for predicting the structure of disaccharides was incorporated in this new model with an improvement of the predictive power. The best results were achieved using Random Forests with 204 di- and trisaccharides for the training set-it could correctly classify 83%, 90%, 88%, 85%, 85%, 75%, 79%, 68% and 94% of the test set (69 compounds) for the nine tasks, respectively, on the basis of unassigned chemical shifts.
Project description:Oligosaccharide synthesis is hindered by the need for multiple steps as well as numerous selective protections and deprotections. Herein we report a highly efficient de novo route to various oligosaccharide motifs, of use for biological and medicinal structure activity studies. The key to the overall efficiency is the judicious use of asymmetric catalysis and synthetic design. These green principles include the bidirectional use of highly stereoselective catalysis (Pd(0)-catalyzed glycosylation/post-glycosylation). In addition, the chemoselective use of C-C and C-O ?-bond functionality, as atom-less protecting groups as well as an anomeric directing group (via a Pd-?-allyl), highlights the atom-economical aspects of the route to a divergent set of natural and unnatural oligosaccharides (i.e., various d-/l-diastereomers of oligosaccharides as well as deoxysugars which lack C-2 anomeric directing groups). For example, in only 12 steps, the construction of a highly branched heptasaccharide with 35 stereocenters was accomplished from an achiral acylfuran.
Project description:The indium-mediated allylation reaction has been applied to melibiose, a disaccharidic substrate. This elongation methodology allows for a short, efficient and diastereoselective approach towards complex glycosylated carbohydrate structures. The stereochemical outcome of the key intermediates, allylated disaccharides, has been determined by X-ray analysis. Ozonolysis of the introduced double bond yielded the unprotected elongated disaccharides in the equilibrium of the pyranoid as well as furanoid isomers in both anomeric forms, respectively. Per-O-acetylation has been performed to facilitate separation of the isomeric mixture for structural identification. The main product revealed to adopt a β-pyranoid form of the elongated unit at the reducing end of the disaccharide.
Project description:The stereochemical course of the reaction catalysed by endo-1,3-1,4-beta-D-glucan 4-glucanohydrolase (EC 18.104.22.168) has been determined by 1H n.m.r. The enzyme-catalysed hydrolysis of barley beta-glucan proceeds with overall retention of the anomeric configuration, indicating that the enzyme operates through a double-displacement mechanism. The structures of the final oligosaccharide products, 3-beta-O-cellobiosyl D-glucopyranoside and 3-beta-O-cellotriosyl D-glucopyranoside, have been completely assigned by 1H- and 13C-n.m.r. spectroscopy.
Project description:In this study, we characterized the mode of action of reducing-end xylose-releasing exoxylanase (Rex), which belongs to the glycoside hydrolase family 30-7 (GH30-7). GH30-7 Rex, isolated from the cellulolytic fungus Talaromyces cellulolyticus (Xyn30A), exists as a dimer. The purified Xyn30A released xylose from linear xylooligosaccharides (XOSs) 3 to 6 xylose units in length with similar kinetic constants. Hydrolysis of branched, borohydride-reduced, and p-nitrophenyl XOSs clarified that Xyn30A possesses a Rex activity. 1H nuclear magnetic resonance (1H NMR) analysis of xylotriose hydrolysate indicated that Xyn30A degraded XOSs via a retaining mechanism and without recognizing an anomeric structure at the reducing end. Hydrolysis of xylan by Xyn30A revealed that the enzyme continuously liberated both xylose and two types of acidic XOSs: 22-(4-O-methyl-?-d-glucuronyl)-xylotriose (MeGlcA2Xyl3) and 22-(MeGlcA)-xylobiose (MeGlcA2Xyl2). These acidic products were also detected during hydrolysis using a mixture of MeGlcA2Xyl n (n?=?2 to 14) as the substrate. This indicates that Xyn30A can release MeGlcA2Xyl n (n?=?2 and 3) in an exo manner. Comparison of subsites in Xyn30A and GH30-7 glucuronoxylanase using homology modeling suggested that the binding of the reducing-end residue at subsite +2 was partially prevented by a Gln residue conserved in GH30-7 Rex; additionally, the Arg residue at subsite -2b, which is conserved in glucuronoxylanase, was not found in Xyn30A. Our results lead us to propose that GH30-7 Rex plays a complementary role in hydrolysis of xylan by fungal cellulolytic systems.IMPORTANCE Endo- and exo-type xylanases depolymerize xylan and play crucial roles in the assimilation of xylan in bacteria and fungi. Exoxylanases release xylose from the reducing or nonreducing ends of xylooligosaccharides; this is generated by the activity of endoxylanases. ?-Xylosidase, which hydrolyzes xylose residues on the nonreducing end of a substrate, is well studied. However, the function of reducing-end xylose-releasing exoxylanases (Rex), especially in fungal cellulolytic systems, remains unclear. This study revealed the mode of xylan hydrolysis by Rex from the cellulolytic fungus Talaromyces cellulolyticus (Xyn30A), which belongs to the glycoside hydrolase family 30-7 (GH30-7). A conserved residue related to Rex activity is found in the substrate-binding site of Xyn30A. These findings will enhance our understanding of the function of GH30-7 Rex in the cooperative hydrolysis of xylan by fungal enzymes.