Anomeric Triflates versus Dioxanium Ions: Different Product-Forming Intermediates from 3-Acyl Benzylidene Mannosyl and Glucosyl Donors.

Minimal structural differences in the structure of glycosyl donors can have a tremendous impact on their reactivity and the stereochemical outcome of their glycosylation reactions. Here, we used a combination of systematic glycosylation reactions, the characterization of potential reactive intermediates, and in-depth computational studies to study the disparate behavior of glycosylation systems involving benzylidene glucosyl and mannosyl donors. While these systems have been studied extensively, no satisfactory explanations are available for the differences observed between the 3-O-benzyl/benzoyl mannose and glucose donor systems. The potential energy surfaces of the different reaction pathways available for these donors provide an explanation for the contrasting behavior of seemingly very similar systems. Evidence has been provided for the intermediacy of benzylidene mannosyl 1,3-dioxanium ions, while the formation of the analogous 1,3-glucosyl dioxanium ions is thwarted by a prohibitively strong flagpole interaction of the C-2-O-benzyl group with the C-5 proton in moving toward the transition state, in which the glucose ring adopts a B2,5-conformation. This study provides an explanation for the intermediacy of 1,3-dioxanium ions in the mannosyl system and an answer to why these do not form from analogous glucosyl donors.

Detection and Characterization of Rapidly Equilibrating Glycosylation Reaction Intermediates Using Exchange NMR.

The stereoselective introduction of glycosidic bonds (glycosylation) is one of the main challenges in the chemical synthesis of carbohydrates. Glycosylation reaction mechanisms are difficult to control because, in many cases, the exact reactive species driving product formation cannot be detected and the product outcome cannot be explained by the primary reaction intermediate observed. In these cases, reactions are expected to take place via other low-abundance reaction intermediates that are in rapid equilibrium with the primary reaction intermediate via a Curtin-Hammett scenario. Despite this principle being well-known in organic synthesis, mechanistic studies investigating this model in glycosylation reactions are complicated by the challenge of detecting the extremely short-lived reactive species responsible for product formation. Herein, we report the utilization of the chemical equilibrium between low-abundance reaction intermediates and the stable, readily observed α-glycosyl triflate intermediate in order to infer the structure of the former species by employing exchange NMR. Using this technique, we enabled the detection of reaction intermediates such as ß-glycosyl triflates and glycosyl dioxanium ions. This demonstrates the power of exchange NMR to unravel reaction mechanisms as we aim to build a catalog of kinetic parameters, allowing for the understanding and eventual prediction of glycosylation reactions.

Investigation of in vitro histone H3 glycosylation using H3 tail peptides.

Posttranslational modifications (PTMs) on histone tails regulate eukaryotic gene expression by impacting the chromatin structure and by modulating interactions with other cellular proteins. One such PTM has been identified as serine and threonine glycosylation, the introduction of the ß-N-acetylglucosamine (GlcNAc) moiety on histone H3 tail at position Ser10 and Thr32. The addition of the ß-O-GlcNAc moiety on serine or threonine residues is facilitated by the O-GlcNAc transferase (OGT), and can be removed by the action of O-GlcNAcase (OGA). Conflicting reports on histone tail GlcNAc modification in vivo prompted us to investigate whether synthetic histone H3 tail peptides in conjunction with other PTMs are substrates for OGT and OGA in vitro. Our enzymatic assays with recombinantly expressed human OGT revealed that the unmodified and PTM-modified histone H3 tails are not substrates for OGT at both sites, Ser10 and Thr32. In addition, full length histone H3 was not a substrate for OGT. Conversely, our work demonstrates that synthetic peptides containing the GlcNAc functionality at Ser10 are substrates for recombinantly expressed human OGA, yielding deglycosylated histone H3 peptides. We also show that the catalytic domains of human histone lysine methyltransferases G9a, GLP and SETD7 and histone lysine acetyltransferases PCAF and GCN5 do somewhat tolerate glycosylated H3Ser10 close to lysine residues that undergo methylation and acetylation reactions, respectively. Overall, this work indicates that GlcNAcylation of histone H3 tail peptide in the presence of OGT does not occur in vitro.

Competing C-4 and C-5-Acyl Stabilization of Uronic Acid Glycosyl Cations.

Uronic acids are carbohydrates carrying a terminal carboxylic acid and have a unique reactivity in stereoselective glycosylation reactions. Herein, the competing intramolecular stabilization of uronic acid cations by the C-5 carboxylic acid or the C-4 acetyl group was studied with infrared ion spectroscopy (IRIS). IRIS reveals that a mixture of bridged ions is formed, in which the mixture is driven towards the C-1,C-5 dioxolanium ion when the C-5,C-2-relationship is cis, and towards the formation of the C-1,C-4 dioxepanium ion when this relation is trans. Isomer-population analysis and interconversion barrier computations show that the two bridged structures are not in dynamic equilibrium and that their ratio parallels the density functional theory computed stability of the structures. These studies reveal how the intrinsic interplay of the different functional groups influences the formation of the different regioisomeric products.

Stabilization of Glucosyl Dioxolenium Ions by "Dual Participation" of the 2,2-Dimethyl-2-(ortho-nitrophenyl)acetyl (DMNPA) Protection Group for 1,2-cis-Glucosylation.

The stereoselective introduction of glycosidic bonds is of paramount importance to oligosaccharide synthesis. Among the various chemical strategies to steer stereoselectivity, participation by either neighboring or distal acyl groups is used particularly often. Recently, the use of the 2,2-dimethyl-2-(ortho-nitrophenyl)acetyl (DMNPA) protection group was shown to offer enhanced stereoselective steering compared to other acyl groups. Here, we investigate the origin of the stereoselectivity induced by the DMNPA group through systematic glycosylation reactions and infrared ion spectroscopy (IRIS) combined with techniques such as isotopic labeling of the anomeric center and isomer population analysis. Our study indicates that the origin of the DMNPA stereoselectivity does not lie in the direct participation of the nitro moiety but in the formation of a dioxolenium ion that is strongly stabilized by the nitro group.

Characterization of Elusive Reaction Intermediates Using Infrared Ion Spectroscopy: Application to the Experimental Characterization of Glycosyl Cations.

A detailed understanding of the reaction mechanism(s) leading to stereoselective product formation is crucial to understanding and predicting product formation and driving the development of new synthetic methodology. One way to improve our understanding of reaction mechanisms is to characterize the reaction intermediates involved in product formation. Because these intermediates are reactive, they are often unstable and therefore difficult to characterize using experimental techniques. For example, glycosylation reactions are critical steps in the chemical synthesis of oligosaccharides and need to be stereoselective to provide the desired α- or ß-diastereomer. It remains challenging to predict and control the stereochemical outcome of glycosylation reactions, and their reaction mechanisms remain a hotly debated topic. In most cases, glycosylation reactions take place via reaction mechanisms in the continuum between SN1- and SN2-like pathways. SN2-like pathways proceeding via the displacement of a contact ion pair are relatively well understood because the reaction intermediates involved can be characterized by low-temperature NMR spectroscopy. In contrast, the SN1-like pathways proceeding via the solvent-separated ion pair, also known as the glycosyl cation, are poorly understood. SN1-like pathways are more challenging to investigate because the glycosyl cation intermediates involved are highly reactive. The highly reactive nature of glycosyl cations complicates their characterization because they have a short lifetime and rapidly equilibrate with the corresponding contact ion pair. To overcome this hurdle and enable the study of glycosyl cation stability and structure, they can be generated in a mass spectrometer in the absence of a solvent and counterion in the gas phase. The ease of formation, stability, and fragmentation of glycosyl cations have been studied using mass spectrometry (MS). However, MS alone provides little information about the structure of glycosyl cations. By combining mass spectrometry (MS) with infrared ion spectroscopy (IRIS), the determination of the gas-phase structures of glycosyl cations has been achieved. IRIS enables the recording of gas-phase infrared spectra of glycosyl cations, which can be assigned by matching to reference spectra predicted from quantum chemically calculated vibrational spectra. Here, we review the experimental setups that enable IRIS of glycosyl cations and discuss the various glycosyl cations that have been characterized to date. The structure of glycosyl cations depends on the relative configuration and structure of the monosaccharide substituents, which can influence the structure through both steric and electronic effects. The scope and relevance of gas-phase glycosyl cation structures in relation to their corresponding condensed-phase structures are also discussed. We expect that the workflow reviewed here to study glycosyl cation structure and reactivity can be extended to many other reaction types involving difficult-to-characterize ionic intermediates.

Chloromethyl Glycosides as Versatile Synthons to Prepare Glycosyloxymethyl-Prodrugs.

This work investigates the addition of monosaccharides to marketed drugs to improve their pharmacokinetic properties for oral absorption. To this end, a set of chloromethyl glycoside synthons were developed to prepare a variety of glycosyloxymethyl-prodrugs derived from 5-fluorouracil, thioguanine, propofol and losartan. Drug release was studied in vitro using ß-glucosidase confirming rapid conversion of the monosaccharide prodrugs to release the parent drug, formaldehyde and the monosaccharide. To showcase this prodrug approach, a glucosyloxymethyl conjugate of the tetrazole-containing drug losartan was used for in vivo experiments and showed complete release of the drug in a dog-model.

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 Cyclic N-Acyliminium Ions by Infrared Ion Spectroscopy.

N-Acyliminium ions are highly reactive intermediates that are important for creating CC-bonds adjacent to nitrogen atoms. Here we report the characterization of cyclic N-acyliminium ions in the gas phase, generated by collision induced dissociation tandem mass spectrometry followed by infrared ion spectroscopy using the FELIX infrared free electron laser. Comparison of DFT calculated spectra with the experimentally observed IR spectra provided valuable insights in the conformations of the N-acyliminium ions.

A comprehensive overview of substrate specificity of glycoside hydrolases and transporters in the small intestine : "A gut feeling".

The human body is able to process and transport a complex variety of carbohydrates, unlocking their nutritional value as energy source or as important building block. The endogenous glycosyl hydrolases (glycosidases) and glycosyl transporter proteins located in the enterocytes of the small intestine play a crucial role in this process and digest and/or transport nutritional sugars based on their structural features. It is for these reasons that glycosidases and glycosyl transporters are interesting therapeutic targets to combat sugar related diseases (such as diabetes) or to improve drug delivery. In this review we provide a detailed overview focused on the molecular structure of the substrates involved as a solid base to start from and to fuel research in the area of therapeutics and diagnostics.

Characterization of glycosyl dioxolenium ions and their role in glycosylation reactions.

Controlling the chemical glycosylation reaction remains the major challenge in the synthesis of oligosaccharides. Though 1,2-trans glycosidic linkages can be installed using neighboring group participation, the construction of 1,2-cis linkages is difficult and has no general solution. Long-range participation (LRP) by distal acyl groups may steer the stereoselectivity, but contradictory results have been reported on the role and strength of this stereoelectronic effect. It has been exceedingly difficult to study the bridging dioxolenium ion intermediates because of their high reactivity and fleeting nature. Here we report an integrated approach, using infrared ion spectroscopy, DFT computations, and a systematic series of glycosylation reactions to probe these ions in detail. Our study reveals how distal acyl groups can play a decisive role in shaping the stereochemical outcome of a glycosylation reaction, and opens new avenues to exploit these species in the assembly of oligosaccharides and glycoconjugates to fuel biological research.

A Revised Modular Approach to (-)-trans-Δ8-THC and Derivatives Through Late-Stage Suzuki-Miyaura Cross-Coupling Reactions.

A revised modular approach to various synthetic (-)-trans-Δ8-THC derivatives through late-stage Suzuki-Miyaura cross-coupling reactions is disclosed. Ten derivatives were synthesized allowing both sp2- and sp3-hybridized cross-coupling partners with minimal ß-hydride elimination. Importantly, we demonstrate that a para-bromo-substituted THC scaffold for Suzuki-Miyaura cross-coupling reactions has been initially reported incorrectly in recent literature.

Synthesis and cellular uptake of carbamoylated mannose derivatives.

A series of 3-carbamoyl- and 2,3-dicarbamoyl-mannose derivatives were synthesized, conjugated to a fluorescent dye (Cy5GE, AF 647 or NBD) and their cellular uptake in A549 and THP-1 cell lines was studied by FACS. In contrast to earlier studies on carbamoyl mannosides, the observed uptake was not related to carbamoyl group on the mannose residue but rather to the cyanine dye attached, a trend previously observed for Cy5-fructose conjugates. The NBD-conjugates however, showed a temperature and concentration dependent uptake in case of mannose conjugates. These results suggest a profound impact of the dye which should be taken into consideration when studying the uptake of small molecules by dye conjugation.

The Glycosylation Mechanisms of 6,3-Uronic Acid Lactones.

Uronic acids are important constituents of polysaccharides found on the cell membranes of different organisms. To prepare uronic-acid-containing oligosaccharides, uronic acid 6,3-lactones can be employed as they display a fixed conformation and a unique reactivity and stereoselectivity. Herein, we report a highly ß-selective and efficient mannosyl donor based on C-4 acetyl mannuronic acid 6,3-lactone donors. The mechanism of glycosylation is established using a combination of techniques, including infrared ion spectroscopy combined with quantum-chemical calculations and variable-temperature nuclear magnetic resonance (VT NMR) spectroscopy. The role of these intermediates in glycosylation is assayed by varying the activation protocol and acceptor nucleophilicity. The observed trends are analogous to the well-studied 4,6-benzylidene glycosides and may be used to guide the development of next-generation stereoselective glycosyl donors.

Modular synthesis and immunological evaluation of suspected allergenic galactooligosaccharides.

Galactooligosaccharides (GOS) are widely used in the food industry as prebiotics and in very rare cases, can lead to an allergic reaction. Due to the microheterogeneity of GOS it is very difficult to extract pure and well defined oligosaccharides to establish which component is responsible for the observed allergenicity. Herein, we report the chemical synthesis of a suspected allergen 4PX and three closely related oligosaccharides based on a modular approach. The fact that synthesized 4PX and a regioisomer did not cause basophil activation in subjects with confirmed GOS-allergy excludes both tetrasaccharides as key-epitopes in GOS-allergenicity in Singapore.

Potent Metabolic Sialylation Inhibitors Based on C-5-Modified Fluorinated Sialic Acids.

Sialic acid sugars on mammalian cells regulate numerous biological processes, while aberrant expression of sialic acid is associated with diseases such as cancer and pathogenic infection. Inhibition of the sialic acid biosynthesis may therefore hold considerable therapeutic potential. To effectively decrease the sialic acid expression, we synthesized C-5-modified 3-fluoro sialic acid sialyltransferase inhibitors. We found that C-5 carbamates significantly enhanced and prolonged the inhibitory activity in multiple mouse and human cell lines. As an underlying mechanism, we have identified that carbamate-modified 3-fluoro sialic acid inhibitors are more efficiently metabolized to their active cytidine monophosphate analogues, reaching higher effective inhibitor concentrations inside cells.

Direct Experimental Characterization of Glycosyl Cations by Infrared Ion Spectroscopy.

Glycosyl cations are crucial intermediates formed during enzymatic and chemical glycosylation. The intrinsic high reactivity and short lifetime of these reaction intermediates make them very challenging to characterize using spectroscopic techniques. Herein, we report the use of collision induced dissociation tandem mass spectrometry to generate glycosyl cations in the gas phase followed by infrared ion spectroscopy using the FELIX infrared free electron laser. The experimentally observed IR spectra were compared to DFT calculated spectra enabling the detailed structural elucidation of elusive glycosyl oxocarbenium and dioxolenium ions.

Stereoselective ß-Mannosylation by Neighboring-Group Participation.

The stereoselective synthesis of glycosidic bonds is the main challenge of oligosaccharide synthesis. Neighboring-group participation (NGP) of C2 acyl substituents can be used to provide 1,2-trans-glycosides. Recently, the application of NGP has been extended to the preparation of 1,2-cis-glycosides with the advent of C2 chiral auxiliaries. However, this methodology has been strictly limited to the synthesis of 1,2-cis-gluco-type sugars. Reported herein is the design and synthesis of novel mannosyl donors which provide 1,2-cis-mannosides by NGP of thioether auxiliaries. A key element in the design is the use of (1) C4 locked mannuronic acid lactones to enable NGP of the C2 auxiliary. In addition to C2 participation a new mode of remote participation of the C4 benzyl group was identified and provides 1,2-cis-mannosides.