High-resolution nuclear magnetic resonance spectroscopy is a powerful method for analysis of the conformations, molecular structures, purity, and stereochemistry of carbohydrates, including monosaccharide, oligosaccharide, and polysaccharide types that are of prime interest in bacterial vaccine development. Part of our function is to provide analytical NMR support as needed for current vaccine projects in the lab that involve the characterization of saccharide structure and purity. Another part includes more fundamental investigations of the application of measurements of NMR chemical shifts, nuclear spin-spin coupling constants, and nuclear Overhauser effects to the structural analysis of carbohydrates. We also characterize the molecular geometry of saccharides of interest in these studies by molecular dynamics computations followed by energy minimization by molecular mechanics.? ? DEFINITION OF THE ANGULAR DEPENDENCE OF 1H-15N COUPLING CONSTANTS IN AMINO SUGAR DERIVATIVES: Amino sugars are common components of bacterial polysaccharides. 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 recent studies of amino sugar derivatives, the NH to ND induced isotope effects on the chemical shifts of 13C nuclei surrounding the nitrogen atom have been characterized. The high sensitivity of an NMR cryoprobe allowed the rapid acquisition of well-resolved 1D and 2D NMR spectra, before significant anomerization of the N-acetyl-amino-deoxy sugars had occurred, thus facilitating their spectral assignment. ? In our current work, the 15N-1H coupling constants of various derivatives of amino sugars have been measured by the inverse-detected, heteronuclear single quantum, multiple bond correlation (HSQMBC) technique, and the values so obtained compared with those measured by the more traditional procedure of synthesizing highly enriched 15N-labeled amino sugar derivatives and analyzing their 1H NMR spectra. The HSQMBC method is highly selective, in this instance filtering out all non-15N NMR interactions, thereby yielding simplified NMR spectra. Acquisition of the HSQMBC spectra from reasonably small quantities of materials (for example, 30 mg/0.5 mL) having 15N at natural abundance was facilitated by the enhanced sensitivity of a 1H/13C/15N inverse NMR cryoprobe. ? ? A number of bicyclic amino sugar models of fixed or known geometry have been studied, including a series of methyl 2- and 3-amino-4,6-O-benzylidene-deoxy-alpha-D-hexopyranosides in chair or skew boat conformations, and methyl 2,6-anhydro-3-deoxy-3-phthalimido-alpha-D-mannopyranoside in a locked, almost classical boat conformation. The major conformational features of these molecules have been determined from 1H-1H NMR coupling constants measured by complete assignment of the 1H NMR spectra of the amino derivatives. The magnitudes of the vicinal 1H-15N coupling constants have been correlated with the geometry of the coupled nuclei in the conformations of the amino sugar models, as determined by molecular dynamics/mechanics computations. Various methods for analysis of the HSQMBC spectra have been investigated. The most convenient method was found to be """"""""NMR multiplet total width subtraction"""""""", based on the fact that the total width of first order NMR multiplets is equal to the sum of their coupling constants.? ? 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. Applications of the equation are expected in NMR analysis of the stereochemistry of amino sugar components of nitrogen-containing saccharides and aminoglycoside antibiotics.
| 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 |
