NMR studies of the tetrasaccharide (4-mer), octasaccharide (8-mer), and dodecasaccharide (12-mer) glycosides have been completed. The problem encountered previously with broadness of the 1H spectra of the 8-mer and 12-mer in D2O solutions has been solved by addition of acetone-d6 to the solutions, which permitted more accurate shimming of the magnetic field in the spectrometer. The best procedure found was to shim on the deuterium signal of the acetone-d6, and then lock the field-frequency ratio of the spectrometer on the D2O signal. The rationale is that the lock level based on the D2O signal is insensitive to shimming. With few exceptions, complete assignments of the 1H and 13C NMR spectra of the 5-methoxycarbonylpentyl glycosides of the 4-mer, 8-mer, and 12-mer have been obtained by 2D NMR methods. Also, almost complete sets of 1H-1H coupling constants have been measured for the three oligosaccharide glycosides. Interpretation of these coupling constants according to Karplus considerations confirmed the types of sugar residues present, and confirmed the chair conformations of the pyranose rings. The observation of exclusively large 1J(C-1,H-1) values (168.9-175.8 Hz) by 1H-coupled 2D HSQC NMR indicates the alpha anomeric configuration for all linkages in the three oligosaccharide glycosides, as synthetically designed. All of the linkage positions in the three oligosaccharide glycosides have been confirmed by the observation of specific HMBC connectivities for one or two 13C1H pairs per linkage. Proof of structure by NMR allows for preparation of protein conjugates of the oligosaccharides, which will be used for immunogenic testing of potential vaccine components against E. coli O148 and S. dysenteriae type 1. We found previously that two major components of the native antigenic material from B. burgdorferi are 1-O-palmitoyl-2-O-oleoyl-3-O-alpha-D-galactopyranosyl-sn-glycerol and 1-O-oleoyl-2-O-palmitoyl-3-O-alpha-D-galactopyranosyl-sn-glycerol, which we have synthesized and now characterized completely by high-resolution NMR. The 1H NMR spectra of these structural isomers are extremely similar, and since we required a method that would reliably distinguish these isomers, we investigated the application of 13C NMR. Resolution enhancement of the carbonyl 13C region of the 13C NMR spectra of these two diglycerides revealed characteristic differences in 13C chemical shift that can be used to distinguish the isomers and assess their chemical purity. Detailed analysis of the NMR spectra allowed complete 1H and 13C signal assignments to be made, and also the following generalizations: (a) The major 13C=O chemical shift is due to the substituent position on the glycerol moiety, (b) the minor 13C=O shift is due to the type of fatty acid, (c) the 1-sustituent C=Os resonate at lower field that the 2-substituent C=Os, (d) the palmitic C=Os are at lower field than the oleoyl C=Os.
| Pozsgay, Vince; Kubler-Kielb, Joanna; Coxon, Bruce et al. (2011) Synthesis and antigenicity of BBGL-2 glycolipids of Borrelia burgdorferi, the causative agent of Lyme disease. Carbohydr Res 346:1551-63 |
