Oligosaccharides Display Both Rigidity and High Flexibility in Water as Determined by13C NMR Relaxation and1H,1H NOE Spectroscopy: Evidence ofanti-φ andanti-ψ Torsions in the Same Glycosidic Linkage

Abstract

The trisaccharide beta-D-Glcp-(1-->2)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-OMe has been investigated by molecular dynamics (MD) simulations and NMR experiments in water. 13C spin-lattice (T1) and spin-spin (T2) relaxation times, together with 1H,13C NOE data were measured at two magnetic field strengths (9.4 and 14.1 T) in a 277 K D2O solution. Relaxation data interpreted by means of the model-free formalism revealed a rigid (S2 approximately 0.9) oligosaccharide tumbling in solution. 1H,1H Cross-relaxation rates were determined at 600 MHz by 1D DPFGSE NOESY and T-ROESY experiments, which provided high quality data and subsequently proton-proton distances within the trisaccharide. The presence of anti conformers at both torsions of a glycosidic linkage is demonstrated for the first time. MD simulations were carried out to facilitate analysis of the NOE data. In total, 15 simulations-starting from five different conformational states--were performed, with production runs of up to 10 ns, resulting in 83 ns of oligosaccharide dynamics in water. anti Conformers were populated to different degrees in the simulations, especially at the phi2 torsion angle. By combining the results from the NOE experiments and the MD simulations, the anti conformers at the (1-->2)-linkage were quantified as 7% anti-phi2 and 2% anti-psi2, revealing a highly flexible trisaccharide in which large conformational changes occur. From the MD simulations, interresidue hydrogen bonding, from HO2" to O2 or O3, was significantly populated (approximately 40%) in both of the anti conformational states. The contentious issue over rigidity versus flexibility in oligosaccharides has thus been thoroughly examined, showing that the dynamics should be taken into account for a relevant description of the molecular system.

Conformational properties of glucose-based disaccharides investigated using molecular dynamics simulations with local elevation umbrella sampling

“…The (1?6)-linked disaccharides are characterized by two significantly populated states (B and A for I; C and D for G; the population of the former one being in both cases about twice that of the latter one; A and C:x in gt; B and D:x in gg), along with two alternative states with marginal populations for G only. In the literature, 210,211 the most stable states are sometimes referred to as syn-/ and (Table 1). They were also selected to illustrate the main intramolecular H-bonds present in this state (Table 5; (except for I and G) syn-w conformations (for I and G: syn-/(gt) and syn-/(gg)), while the alternative (metastable) states with different values of / or w are referred to as anti-/ or anti-w, respectively.…”

“…marginally populated (up to 1.3% at most) for the D-glucpyranose disaccharides in aqueous solution at 300 K and 1 bar. Experimentally, population estimates ranging from a fraction of a percent up to 92 13% have been proposed for these metastable states in disaccharide derivatives and trisaccharides (J-coupling 92,106 or/ and NOE-based 93,212,213 mixed NMR/MD approaches 210,211 ). Note that some of these estimates have subsequently been questioned.…”

“…Note that some of these estimates have subsequently been questioned. 212,106 Higher occurrences have been suggested for C-and N-linked (rather than O-linked) disaccharides, 213,214 longer oligosaccharides, 210,215 as well of glycosides bound to proteins. 216 However, to our knowledge, no experimental study to date provides a reliable estimate in the context of unfunctionalized disaccharides in aqueous solution, against which the present simulation results could be (in)validated.…”

A Conformational Study of the Trisaccharideβ-D-Glcp-(1→2)[β-D-Glcp-(1→3)]α-D-Glcp-OMe by NMR NOESY and TROESY Experiments, Computer Simulations, and X-Ray Crystal Structure Analysis

“…In a related study of another trisaccharide the intraresidue distance also showed good agreement to simulations, but in this case the model-free approach resulted in a slightly shorter distance when NOE data were used and a somewhat longer distance when TROE data were employed. [42] Subsequently, when ISPA is used (vide infra), a reverse relationship for r NOE vs. r TROE is obtained for the short trans-glycosidic distances. Therefore, the experimental differences should represent the accuracy of the data.…”

A perspective on the primary and three-dimensional structures of carbohydrates

“…59 However, it was instead highly flexible and further studies of the trisaccharide showed that both anti-φ and anti-ψ conformational states were populated as determined by NMR spectroscopy and interpreted using molecular dynamics (MD) simulations. 60 In conformational analysis of oligosaccharides 1 H, 1 H NOEs play an important role but the decimate the contribution from a third proton to the desired interaction is to chemically exchange 1 H for 2 H atoms, which effectively eliminates contributions via the undesired relaxation pathway. 63,64 Thus, if spectral appearance permits selective irradiation, it is the method of choice, otherwise a chemical substitution approach is needed.…”