Stochastic searches and NMR experiments on four Lewis A analogues: NMR experiments support some flexibility around the fucosidic bond.

We have compared the conformational behavior of three Le(a) analogues with that of Le(a) using stochastic searches (MOE2005) and selective ROESY experiments. In the analogues either or both the ß-d-Gal and α-l-Fuc residues were replaced by ß-d-Glc and α-l-Rha units, respectively. All compounds showed similar behavior and even though four conformational families were identified, the calculations and NMR experiments support that the 'stacked conformation' known for Le(a) is predominant for all analogues. Interestingly, ROESY showed a correlation between H-1 Fuc/Rha and H-3 GlcNAc which, although small, could be seen in all analogues. For two compounds, the corresponding distance was measured and found to be shorter (∼3.7Å) than that found in the global minimum (4.5Å). While one published study suggests some motion around the fucosidic bond, this constitutes the first experimental evidence supporting such flexibility. Our MD simulation (Amber10/Glycam06) on Le(a) was in full agreement with previous studies which described a rigid conformation for this branched trisaccharide. Thus, NMR seems to indicate that these dynamic studies are underestimating flexibility around the fucosidic bond.

How the substituent at O-3 of N-acetylglucosamine impacts glycosylation at O-4: a comparative study.

An assessment of the relative reactivities of the 4-OH of N-acetylglucosamine acceptors bearing simple protecting groups, beta-linked or alpha-linked D or L sugars at O-3 is presented, using a per-O-acetylated alpha-D-glucosyl trichloroacetimidate donor under activation by BF(3) x OEt(2). The presence of either an acyl or carbonate protecting group at O-3 did not impact the reactivity at O-4 with all glycosylations proceeding successfully. On the other hand, the presence of peracetylated sugars at O-3 of N-acetylglucosamine acceptors did impact the reactivity of the 4-OH. The acceptors with an alpha-D-Man, beta-D-Gal, or beta-D-Glc at O-3 reacted promptly. In comparison, the acceptors bearing a beta-L-Fuc, alpha-L-Fuc, or alpha-L-Rha underwent glucosylation slowly, and unreacted acceptor was recovered from the reaction mixtures. Systematic searches carried out on the disaccharide acceptors and trisaccharide products carrying either a peracetylated beta-D-Gal or beta-L-Fuc at O-3 of the glucosamine residue suggest that, for these two acceptors, a conformational reorientation necessary around the fucosidic linkage contributes to the lower reactivity of the beta-fucosylated acceptor. The acceptors bearing a beta-linked D-Gal, D-Glc, or L-Fuc residue at O-3 each gave trisaccharide products that were mostly stable in the reaction conditions. In contrast, the alpha-linked residues at O-3 were rather unstable in these reaction conditions and the degradation of either the acceptors or trisaccharide products led to low glycosylation yields. In these later reactions, it was impossible to clearly assess which of the acceptor or product underwent degradation as comigration and detection issues prevented us from following these glycosylations by TLC or RP-HPLC. In contrast, the glycosylation of an acceptor carrying an alpha-linked perbenzylated L-Fuc residue at O-3 could be easily monitored by RP-HPLC. The data obtained when monitoring this glycosylation showed that the acceptor underwent prompt glycosylation but a decrease in the absorbance peak corresponding to the trisaccharide along with the appearance of a peak corresponding to a perbenzylated fucose hemiacetal indicated that the trisaccharide product was unstable in the reaction conditions.

The flexibility of the Le(a)Le(x) tumor associated antigen central fragment studied by systematic and stochastic searches as well as dynamic simulations.

The solution conformational behavior of the Tumor-Associated Carbohydrate Antigen Le(a)Le(x) central fragment: methyl alpha-L-fucopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranosyl-(1-->3)-beta-D-galactopyranoside was studied using three computational methods: a rigid systematic search as implemented in Sybyl, a stochastic search as implemented in MOE2004, and dynamics simulations using the SANDER module of AMBER9. Our results illustrate the complementarity of these methods to identify energetically relevant conformations and flexible linkages. In particular, the beta-GlcNAc-(1-->3)-Gal linkage was shown to be extremely flexible adopting a wide range of orientations around two energy minima. The modeling results were validated by comparison of theoretical distances, derived from the simulations, with experimental measurements obtained from 1D selective ROESY buildup curves on the synthetic fragment.

Evidence for two populated conformations for the dimeric Le(x) and Le(a)Le(x) tumor-associated carbohydrate antigens.

The conformational behavior of tumor-associated carbohydrate antigens (TACAs) dimLe(x) and Le(a)Le(x) was studied using a combination of NMR experiments and molecular dynamics simulations. It is shown that within the hexasaccharides, the Le(x) and Le(a) branched trisaccharide fragments adopt the rigid "stacked" conformation known for the isolated trisaccharide antigens. In contrast, the ß-D-GlcNAc-(1→3)-D-Gal glycosidic bond that connects the two Le(x) trisaccharides in dimLe(x), and the Le(a) trisaccharide to the Le(x) trisaccharide in Le(a)Le(x), was found to be very flexible in both hexasaccharides. Our results show that two distinct conformations, differing by the value of the Ψ angle for this glycosidic bond, are populated in solution. While the relative proportions of the two conformations in solution could not be determined accurately, experimental measurements indicate that both conformations are populated in significant amounts.