[show abstract][hide abstract] ABSTRACT: Determination of the structure of heparin-derived oligosaccharides by (1)H NMR is challenging because resonances for all but the anomeric protons cover less than 2 ppm. By taking advantage of increased dispersion of resonances for the anomeric H(1) protons at low pD and the superior resolution of band-selective, homonuclear-decoupled (BASHD) two-dimensional (1)H NMR, the primary structure of the heparin-derived octasaccharide ∆UA(2S)-[(1 → 4)-GlcNS(6S)-(1 → 4)-IdoA(2S)-](3)-(1 → 4)-GlcNS(6S) has been determined, where ∆UA(2S) is 2-O-sulfated ∆(4,5)-unsaturated uronic acid, GlcNS(6S) is 6-O-sulfated, N-sulfated β-D: -glucosamine and IdoA(2S) is 2-O-sulfated α-L: -iduronic acid. The spectrum was assigned, and the sites of N- and O-sulfation and the conformation of each uronic acid residue were established, with chemical shift data obtained from BASHD-TOCSY spectra, while the sequence of the monosaccharide residues in the octasaccharide was determined from inter-residue NOEs in BASHD-NOESY spectra. Acid dissociation constants were determined for each carboxylic acid group of the octasaccharide, as well as for related tetra- and hexasaccharides, from chemical shift-pD titration curves. Chemical shift-pD titration curves were obtained for each carboxylic acid group from sub-spectra taken from BASHD-TOCSY spectra that were measured as a function of pD. The pK (A)s of the carboxylic acid groups of the ∆UA(2S) residues are less than those of the IdoA(2S) residues, and the pK (A)s of the carboxylic acid groups of the IdoA(2S) residues for a given oligosaccharide are similar in magnitude. Relative acidities of the carboxylic acid groups of each oligosaccharide were calculated from chemical shift data by a pH-independent method.
Analytical and Bioanalytical Chemistry 10/2010; 399(2):663-71. · 3.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: The secondary amide peptide bonds that comprise up to one-third of the bonds of peptide or protein backbones can exist as cis and trans isomers, with the trans isomer being highly favored. However, there is little quantitative data on the kinetics and equilibria of cis-trans isomerization of secondary amide peptide bonds due to the difficulty of detecting the very small population of cis isomers. Knowledge of factors that influence the kinetics and equilibria of cis-trans isomerization of secondary amide peptide bonds will contribute to a more complete understanding of the structural and dynamic behavior of the backbones of peptides and unfolded proteins and of complex protein folding kinetics. We have characterized the kinetics and equilibria of cis-trans isomerization of the Xaa-Yaa secondary amide peptide bonds of the linear dithiol and cyclic disulfide forms of the peptides Ac-Cys-Xaa-Yaa-Cys-His-NH(2), where Xaa-Yaa is Ala-Phe, Phe-Ala, Ala-Tyr, and Tyr-Ala, by (1)H NMR. Resolved resonances were observed for the Ala-methyl protons of the trans and the much less abundant cis isomers due to differential shielding of the Ala-methyl protons of the trans and cis isomers by ring current effects from the Phe and Tyr side chains. The population of the cis isomers was determined from the relative intensities of the Ala-methyl resonances for the trans and cis isomers, and rate constants for cis-to-trans and trans-to-cis isomerization were determined by the magnetization transfer NMR method. The population of the cis isomers ranges from 0.07 to 0.12%, and the rate constants indicate that, when there is a trans-to-cis interchange, it is rapidly followed by a cis-to-trans interchange back to the more stable trans conformation. Although cyclization by disulfide bond formation imposes conformational constraints on the peptide backbones, cyclization is found to have relatively small affects on the dynamics of cis-trans isomerization.
The Journal of Physical Chemistry B 02/2010; 114(9):3387-92. · 3.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: We explore strategies to enhance conformational ordering of N-substituted glycine peptoid oligomers. Peptoids bearing bulky N-alkyl side chains have previously been studied as important examples of biomimetic "foldamer" compounds, as they exhibit a capacity to populate helical structures featuring repeating cis-amide bonds. Substantial cis/trans amide bond isomerization, however, gives rise to conformational heterogeneity. Here, we report the use of N-aryl side chains as a tool to enforce the presence of trans-amide bonds, thereby engendering structural stability. Aniline derivatives and bromoacetic acid are used in the facile solid-phase synthesis of a diverse family of sequence-specific N-aryl glycine oligomers. Quantum mechanics calculations yield a detailed energy profile of the folding landscape and substantiate the hypothesis that the presence of anilide groups establishes a strong energetic preference for trans-amide bonds. X-ray crystallographic analysis and solution NMR studies verify this preference. Molecular modeling indicates that the linear oligomers can adopt helical structures resembling a polyproline type II helix. High resolution structures of macrocyclic oligomers incorporating both N-alkyl and N-aryl glycine units confirm the ability to direct the presence of trans-amide bonds specifically at N-aryl positions. These results are an important step in developing strategies for the rational de novo design of new structural motifs in biomimetic oligopeptoid systems.
Journal of the American Chemical Society 01/2009; 130(49):16622-32. · 10.68 Impact Factor