On the influence of solvent on the stereoselectivity of glycosylation reactions.

Methodology development in carbohydrate chemistry entails the stereoselective formation of C-O bonds as a key step in the synthesis of oligo- and polysaccharides. The anomeric selectivity of a glycosylation reaction is affected by a multitude of parameters, such as the nature of the donor and acceptor, activator/promotor system, temperature and solvent. The influence of different solvents on the stereoselective outcome of glycosylation reactions employing thioglucopyranosides as glycosyl donors with a non-participating protecting group at position 2 has been studied. A large change in selectivity as a function of solvent was observed and a correlation between selectivity and the Kamlet-Taft solvent parameter π* was found. Furthermore, molecular modeling using density functional theory methodology was conducted to decipher the role of the solvent and possible reaction pathways were investigated.

Complete 1H and 13C NMR chemical shift assignments of mono-to tetrasaccharides as basis for NMR chemical shift predictions of oligo- and polysaccharides using the computer program CASPER.

Carbohydrate structure can be elucidated or confirmed by using NMR spectroscopy as the prime technique. Prediction of 1H and 13C NMR chemical shifts by computational approaches makes this assignment process more efficient and the program CASPER can perform this task rapidly. It does so by relying on chemical shift data of mono-, di-, and trisaccharides. In order to improve accuracy and quality of these predictions we have assigned 1H and 13C NMR chemical shifts of 30 monosaccharides, 17 disaccharides, 10 trisaccharides and one tetrasaccharide; in total 58 compounds. Due to different rotamers, ring forms, α- and ß-anomeric forms and pD conditions this resulted in 74 1H and 13C NMR chemical shift data sets, all of which were refined using total line-shape analysis for the 1H resonances in order to obtain accurate chemical shifts. Subsequent NMR chemical shift predictions for three sialic acid-containing oligosaccharides, viz., GD1a, a disialyl-LNnT hexasaccharide and a polysialic acid-lactose decasaccharide, and NMR-based structural elucidations of two O-antigen polysaccharides from E. coli O174 were performed by the CASPER program (http://www.casper.organ.su.se/casper/) resulting in very good to excellent agreement between experimental and predicted data thereby demonstrating its utility for carbohydrate compounds that have been chemically or enzymatically synthesized, structurally modified or isolated from nature.

A Lead-Based Fragment Library Screening of the Glycosyltransferase WaaG from Escherichia coli.

Glucosyl transferase I (WaaG) in E. coli catalyzes the transfer of an α-d-glucosyl group to the inner core of the lipopolysaccharide (LPS) and plays an important role in the biogenesis of the outer membrane. If its activity could be inhibited, the integrity of the outer membrane would be compromised and the bacterium would be susceptible to antibiotics that are normally prevented from entering the cell. Herein, three libraries of molecules (A, B and C) were docked in the binding pocket of WaaG, utilizing the docking binding affinity as a filter to select fragment-based compounds for further investigations. From the results of the docking procedure, a selection of compounds was investigated by molecular dynamics (MD) simulations to obtain binding free energy (BFE) and KD values for ligands as an evaluation for the binding to WaaG. Derivatives of 1,3-thiazoles (A7 and A4) from library A and 1,3,4-thiadiazole (B33) from library B displayed a promising profile of BFE, with KD < mM, viz., 0.11, 0.62 and 0.04 mM, respectively. Further root-mean-square-deviation (RMSD), electrostatic/van der Waals contribution to the binding and H-bond interactions displayed a favorable profile for ligands A4 and B33. Mannose and/or heptose-containing disaccharides C1-C4, representing sub-structures of the inner core of the LPS, were also investigated by MD simulations, and compound C42- showed a calculated KD = 0.4 µM. In the presence of UDP-Glc2-, the best-docked pose of disaccharide C42- is proximate to the glucose-binding site of WaaG. A study of the variation in angle and distance was performed on the different portions of WaaG (N-, the C- domains and the hinge region). The Spearman correlation coefficient between the two variables was close to unity, where both variables increase in the same way, suggesting a conformational rearrangement of the protein during the MD simulation, revealing molecular motions of the enzyme that may be part of the catalytic cycle. Selected compounds were also analyzed by Saturation Transfer Difference (STD) NMR experiments. STD effects were notable for the 1,3-thiazole derivatives A4, A8 and A15 with the apo form of the protein as well as in the presence of UDP for A4.

Exploiting 13C/14N solid-state NMR distance measurements to assign dihedral angles and locate neighboring molecules.

The RESPDOR NMR method rapidly provides multiple 13C/14N distance measurements in natural abundance solids. In this study, 13C/14N RESPDOR information is shown, for the first time, to provide accurate molecular conformation and to locate non-bonded neighboring molecules.

Conformational Dynamics and Exchange Kinetics of N-Formyl and N-Acetyl Groups Substituting 3-Amino-3,6-dideoxy-α-d-galactopyranose, a Sugar Found in Bacterial O-Antigen Polysaccharides.

Three dimensional shape and conformation of carbohydrates are important factors in molecular recognition events and the N-acetyl group of a monosaccharide residue can function as a conformational gatekeeper whereby it influences the overall shape of the oligosaccharide. NMR spectroscopy and quantum mechanics (QM) calculations are used herein to investigate both the conformational preferences and the dynamic behavior of N-acetyl and N-formyl substituents of 3-amino-3,6-dideoxy-α-d-galactopyranose, a sugar and substitution pattern found in bacterial O-antigen polysaccharides. QM calculations suggest that the amide oxygen can be involved in hydrogen bonding with the axial OH4 group primarily but also with the equatorial OH2 group. However, an NMR J coupling analysis indicates that the θ1 torsion angle, adjacent to the sugar ring, prefers an ap conformation where conformations <180° also are accessible, but does not allow for intramolecular hydrogen bonding. In the formyl-substituted compound 4JHH coupling constants to the exo-cyclic group were detected and analyzed. A van't Hoff analysis revealed that the trans conformation at the amide bond is favored by ΔG° ≈ - 0.8 kcal·mol-1 in the formyl-containing compound and with ΔG° ≈ - 2.5 kcal·mol-1 when the N-acetyl group is the substituent. In both cases the enthalpic term dominates to the free energy, irrespective of water or DMSO as solvent, with only a small contribution from the entropic term. The cis-trans isomerization of the θ2 torsion angle, centered at the amide bond, was also investigated by employing 1H NMR line shape analysis and 13C NMR saturation transfer experiments. The extracted transition rate constants were utilized to calculate transition energy barriers that were found to be about 20 kcal·mol-1 in both DMSO-d6 and D2O. Enthalpy had a higher contribution to the energy barriers in DMSO-d6 compared to in D2O, where entropy compensated for the loss of enthalpy.

Synthesis of ß-(1→2)-Linked 6-Deoxy-l-altropyranose Oligosaccharides via Gold(I)-Catalyzed Glycosylation of an ortho-Hexynylbenzoate Donor.

The ß-(1→2)-linked 6-deoxy-l-altropyranose di- to pentasaccharides 2-5, relevant to the O-antigen of the infectious Yersinia enterocolitica O:3, were synthesized for the first time. The challenging 1,2-cis-altropyranosyl linkage was assembled effectively via glycosylation with 2-O-benzyl-3,4-di-O-benzoyl-6-deoxy-l-altropyranosyl ortho-hexynylbenzoate (7) under the catalysis of PPh3AuNTf2. NMR and molecular modeling studies showed that the pentasaccharide (5) adopted a left-handed helical conformation.