A series of dimeric through octameric (1→5) amide-linked
sialooligomers were prepared using solid-phase peptide methods on Rink resin with Fmoc protecting group
chemistry. The oligomers were
conjugated to ε-amino caproic acid in order to model membrane-bound
conformations. The secondary
structure of the oliogomers was probed with NH/ND exchange rates
determined by NMR, and with
circular dichroism. The combined structural studies show that a
tetramer is required for ordered
secondary structure, and that secondary structure is stabilized upon
elongation to pentameric and
hexameric species. Interestingly, the heptamer shows rapid NH/ND
exchange rates; however,
ordered secondary structure is restored in the octamer. These
studies provide the first evidence
that oligomers composed of constrained carbohydrate-derived amino acids
form stable secondary
structures in water.
A molecular dynamics study and solution-phase 1 H and 13 C chemical shift anisotropy determination of a symmetric cryogenic disaccharide, R,R-trehalose, has been performed in a temperature range between 264 and 350 K. Negligible temperature dependence of proton-carbon couplings of the asymmetrically [1-13 C]-labeled trehalose suggest that the averaged conformation of the interglycosidic linkage is centered around dihedral angles φ ) ψ ) -41°with (5°uncertainty. Homonuclear NOE-s in the labeled trehalose support the dominance of similar conformation. Close to the slow and fast motional regime, R,R-trehalose can be considered as a spherical top, and global correlation times can be determined easily at extreme temperatures. This allows the construction of an Arrhenius plot for the whole range of temperature. The approach, which we call "trouble-free", yields E a ) 28.2 kJ/mol for the activation energy of molecular reorientation. The model-free analysis of 13 C T 1 , T 2 , and NOE data showed a local maximum of the generalized order parameters with S 2 ) 0.9 around 273 K. Monte-Carlo error analysis corroborated that this effect could be real; however, effective correlation times have relatively high error limits. Thermodynamically, the S 2 data can be interpreted in terms of changes in the Gibbs free energy due to increased or diminished spatial restriction of rapid CH fluctuations. Liquid state 1 H and 13 C chemical shift anisotropies were determined from the interference of dipole-dipole and chemical shift anisotropy relaxation. In solution, chemical shift anisotropies cannot be separated from an inherent geometrical factor, so a combined CSA g factor was used. Cross-correlated spectral densities could be well fitted for the C-1,H-1 vector over the entire temperature range with the "trouble-free" global correlation times. The resulting numerical values for CSA g were smaller compared to the model-free evaluation, due to the omission of internal fluctuations. The measured shift anisotropies were found to be independent from the selection of isotropic or anisotropic dynamical models. Apparent CSA g factors were nearly constant in the entire temperature range except C-3, H-2, and H-3. Comparison with deuterium labeled [2,4,6-2 H]-trehalose proved that temperature-induced changes of the ABX-type strong coupling pattern (caused by the change of differential chemical shift of vicinal H-2 and H-3 protons) interfere with asymmetric multiplet relaxation and potentially lead to misinterpretation of CSA/DD relaxation rates.
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