The use of 1H-31P GHMBC and covariance NMR to unambiguously determine phosphate ester linkages in complex polysaccharide mixtures.

Poly- and oligo-saccharides are commonly employed as antigens in many vaccines. These antigens contain phosphoester structural elements that are crucial to the antigenicity, and hence the effectiveness of the vaccine. Nuclear Magnetic Resonance (NMR) is a powerful tool for the site-specific identification of phosphoesters in saccharides. We describe here two advances in the characterization of phosphoesters in saccharides: (1) the use of (1)H-(31)P GHMBC to determine the site-specific identity of phosphoester moieties in heterogeneous mixtures and (2) the use of Unsymmetrical/Generalized Indirect Covariance (U/GIC) to calculate a carbon-phosphorus 2D spectrum. The sensitivity of the (1)H-(31)P GHMBC is far greater than the "standard" (1)H-(31)P GHSQC and allows long-range (3-5)J(HP) couplings to be readily detected. This is the first example to be reported of using U/GIC to calculate a carbon-phosphorus spectrum. The U/GIC processing affords, in many cases, a fivefold to tenfold or greater increase in signal-to-noise ratios in the calculated spectrum. When coupled with the high sensitivity of (1)H-(31)P HMBC, U/GIC processing allows the complete and unambiguous assignments of phosphoester moieties present in heterogeneous samples at levels of ~5% (or less) of the total sample, expanding the breadth of samples that NMR can be used to analyze. This new analytical technique is generally applicable to any NMR-observable phosphoester.