Relayed HOHAHA, a useful method for extracting subspectra of individual components of sugar chains

Abstract

The ma in difficulty encountered in the analysis of NMR spectra of sugar chains is the overlap of most sugar proton resonances except for reporter groups such as anomerit proton resonances ( 1). Therefore, NMR methods for extracting the proton resonances of each sugar component from an overlapping region are very useful. In previous studies, we applied mu ltiple relayed COSY (2-5) and homonuclear Hartmann-Hahn spectroscopy (HOHAHA) (6, 7) to extract the subspectrum of each sugar component of glycolipids (8, 9)) utilizing magnetization transfer from wellresolved anomeric proton resonances. In contrast to homonuclear mu ltiple relay experiments, the HOHAHA experiment does not generate large amounts of mu ltiplequantum coherence during the m ixing period and it redistributes the entire integrated intensity of one proton among all N protons in the same spin system. Hence, besides relaxation during the m ixing time, the sensitivity of a ID HOHAHA spectrum is reduced only by a factor N relative to a conventional one-dimensional ‘H spectrum. Magnetization transfer in the HOHAHA experiment is especially efficient when all couplings are of a similar order of magn itude. For example, for sugar residues such as G lc and G lcNAc which have couplings of 6-9 Hz all a round the ring, complete subspectra can be obtained very efficiently. If one of the couplings around the sugar ring is very small, it essentially blocks the Hartmann-Hahn flow of magnetization. For example, in Ga l, Ga lNAc, and Fuc residues, magnetization is rapidly transferred among protons HI through H4, but because of the typically very small coupling constant between H4 and H5 protons ( l1.5 Hz), HOHAHA transfer to H5 is not very efficient. Moreover, any magnetization transferred to H5 is rapidly “diluted” because of further HOHAHA transfer to the usually fast relaxing H6 protons. Here we demonstrate that a combination of HOHAHA and the conventional ‘H‘H relay mechanism can be used effectively to circumvent this problem. The pulse sequence of 1 D-relayed HOHAHA is shown in F ig. 1, where magnetization is first transferred from Hl to H4 via H2 and H3 by HOHAHA using MLEV-