Definition Of The Stereochemical Dependence Of 1H-15N Coupling Constants In Amino Sugar Derivatives:? ? Amino sugars are common components of bacterial polysaccharides, either as their N-acetylated forms (for example, Neisseria meningitidis group A, Shigella dysenteriae type 1) or as sugars having a free amino group (Shigella sonnei). As part of a program of vaccine development based on such polysaccharides, we are interested in extending the use of nitrogen NMR parameters for the structural, stereochemical, and conformational analysis of these materials.? In our previous work, the 15N-1H coupling constants of a suite of organic-soluble amino sugar derivatives were measured by the inverse-detected, heteronuclear single quantum, multiple bond correlation (HSQMBC) technique. These derivatives were selected for the initial study because their expected long relaxation times appeared to offer the greatest chance of success for application of the multi-pulse HSQMBC technique, and because the derivatives available contained 15N and 1H nuclei in several different stereochemical orientations.? ? The magnitudes of the vicinal 1H-15N coupling constants were correlated with the geometry of the coupled nuclei in the conformations of the amino sugar models, as determined by molecular dynamics/mechanics computations. Non-linear regression of the vicinal 1H-15N coupling constants to the HCCN dihedral angles determined by molecular dynamics led to definition of a new Karplus equation: 3JHCCN = 3.1 cos2 phi = 0.6 cos phi + 0.4, which describes the dependence of the vicinal, 3JHCCN coupling constant on the HCCN dihedral angle phi in amino sugars derivatives.? ? During the past year, these measurements have been extended to the common amino sugars in aqueous solution, conditions that correspond more closely to physiological conditions than the organic solvents used in our initial work. These common amino sugars are precisely those which occur commonly in bacterial polysaccharides, including N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, and 3-acetamido-3-deoxy-D-glucose, and the free amino sugars glucosamine, mannosamine, and galactosamine, studied as their stable hydrochloride salts. The recently published CPMG-HSQMBC NMR technique (Kover et al., 2006) was adopted for these measurements. This technique involves repetitive refocusing of nuclear magnetization by the Carr-Purcell-Meiboom-Gill procedure to suppress evolution of proton magnetization, thus affording anti-phase, HSQMBC NMR spectra of higher quality than the older method.? ? A number of polar and semi-polar solvent systems have been tested, including deuterium oxide, 10% deuterium oxide:90% water, methylsulfoxide-d6, and 107:1 w/v methylsulfoxide-d6:trifluoroacetic acid. At the commencement of this study, it was not known whether dipolar relaxation of the 15N nucleus by an attached proton would be required for application of the HSQMBC method, and, therefore, whether deuterium oxide could be used as a solvent at all. In deuterium oxide, all of the OH and NH protons are expected to undergo deuterium exchange, thus removing the strong dipolar relaxation mechanism provided by these protons. However, the results of the study have revealed that dipolar relaxation by 1H directly bonded to 15N is not required for success of the experiment, and therefore, that the use of deuterium oxide is entirely feasible.? ? In our previous work, we found that the determination of vicinal 1H15N coupling constants from the HSQMBC NMR spectra is most facile if the inverse-detected 1H multiplets have minimum multiplicity, a situation that pertains when the sugar hydroxyl protons are in rapid chemical exchange, or have been exchanged with deuterium. In the present work, we observed that the use of deuterium oxide as solvent fulfils both of these conditions. The use of semi-polar methylsulfoxide-d6 was also investigated. We found that the use of a solvent containing 1 part of trifluoroacetic acid in 107 parts of methylsulfoxide-d6 catalyzed rapid hydroxyl proton exchange, thus removing the hydroxyl proton coupling constants from the multiplets, and facilitating interpretation of the spectra.? ? Interpretation of the HSQMBC NMR spectra requires complete 1H and 13C assignments for the amino sugars. Therefore, part of the project involves analyzing the 1D 1H and 13C NMR spectra of mixtures of the anomers of the amino sugars, with 2D COSY and 2D HSQC spectra used to obtain definitive 1H and 13C assignments, respectively, for the pyranose anomers.? ? Analysis of the NMR data is underway, and the results are expected to provide a clearer understanding of the effects of the orientation of electronegative substituents on the 1H-15N coupling constants, as well as additional information on their dihedral angle dependence.
| Coxon, Bruce (2007) A Karplus equation for (3)J(HCCN) in amino sugar derivatives. Carbohydr Res 342:1044-54 |
| Coxon, Bruce (2005) Deuterium isotope effects in carbohydrates revisited. Cryoprobe studies of the anomerization and NH to ND deuterium isotope induced 13C NMR chemical shifts of acetamidodeoxy and aminodeoxy sugars. Carbohydr Res 340:1714-21 |
