NMR structure analysis of uniformly 13C-labeled carbohydrates

Abstract

In this study, a set of nuclear magnetic resonance experiments, some of them commonly used in the study of (13)C-labeled proteins and/or nucleic acids, is applied for the structure determination of uniformly (13)C-enriched carbohydrates. Two model substances were employed: one compound of low molecular weight [(UL-(13)C)-sucrose, 342 Da] and one compound of medium molecular weight ((13)C-enriched O-antigenic polysaccharide isolated from Escherichia coli O142, ~10 kDa). The first step in this approach involves the assignment of the carbon resonances in each monosaccharide spin system using the anomeric carbon signal as the starting point. The (13)C resonances are traced using (13)C-(13)C correlations from homonuclear experiments, such as (H)CC-CT-COSY, (H)CC-NOESY, CC-CT-TOCSY and/or virtually decoupled (H)CC-TOCSY. Based on the assignment of the (13)C resonances, the (1)H chemical shifts are derived in a straightforward manner using one-bond (1)H-(13)C correlations from heteronuclear experiments (HC-CT-HSQC). In order to avoid the (1) J CC splitting of the (13)C resonances and to improve the resolution, either constant-time (CT) in the indirect dimension or virtual decoupling in the direct dimension were used. The monosaccharide sequence and linkage positions in oligosaccharides were determined using either (13)C or (1)H detected experiments, namely CC-CT-COSY, band-selective (H)CC-TOCSY, HC-CT-HSQC-NOESY or long-range HC-CT-HSQC. However, due to the short T2 relaxation time associated with larger polysaccharides, the sequential information in the O-antigen polysaccharide from E. coli O142 could only be elucidated using the (1)H-detected experiments. Exchanging protons of hydroxyl groups and N-acetyl amides in the (13)C-enriched polysaccharide were assigned by using HC-H2BC spectra. The assignment of the N-acetyl groups with (15)N at natural abundance was completed by using HN-SOFAST-HMQC, HNCA, HNCO and (13)C-detected (H)CACO spectra.

Exploiting Uniformly 13C-Labeled Carbohydrates for Probing Carbohydrate–Protein Interactions by NMR Spectroscopy

“…In particular, the insertion of constant-time periods (CT) in the indirect dimension or virtual decoupling in the direct dimension were implemented to avoid the splittings from 13 C- 13 C couplings. 2 Furthermore, using uniformly 13 C-labeled carbohydrates permits access to the larger spectral dispersion of 13 C, compared to 1 H, and opens up the possibility to perform 3D or higher order NMR experiments from which information can be extracted more easily. Efforts are also under way to improve accessibility to 13 C-labeled material either employing production of complex 13 C-labeled oligosaccharides through overexpression using engineered yeast strains 4 and/or chemoenzymatic strategies.…”

Insights from NMR Spectroscopy into the Conformational Properties of Man‐9 and Its Recognition by Two HIV Binding Proteins

“…However, making unambiguous assignments of the chemical shifts needed for 3 D structure determination can be challenging due to the abundance of overlapping 1 H and, to a lesser degree, 13 C resonances. Overlapping signals can be resolved by conducting 13 C‐edited or 13 C‐coupled NMR experiments, but limitations in sensitivity generally mean that uniform isotopic labeling is required . In contrast to proteins, the lack of templated biosynthesis of and the presence of heterogeneity in naturally occurring glycans has limited the production of uniformly isotopically labeled sugars to those present on the outer membrane of Escherichia coli or to oligomannosides produced in an engineered yeast strain .…”

Recent Advances in NMR Studies of Carbohydrates

“…For all compounds, the proton, carbon, and (if applicable) phosphorus spectra were first assigned using a set of homonuclear and heteronuclear techniques (DQF COSY, 13 C HMQC, 13 C HMQC-TOCSY, 13 C HMQC-COSY, 13 C DEPT-HMQC, 13 C HMBC, and 31 P HMBC). Recently, Fontana et al [41] presented a set of NMR experiments for the structure determination of uniformly 13 Cenriched carbohydrates. They present an approach to the analysis of lowand medium-size 13 C-enriched oligosaccharides by 1D 13 C experiment to assign an anomeric carbon.…”