NMR studies of electrostatic fields around charged monosaccharides and related molecules

A new and simple technique has been developed to experimentally measure ele ctrostatic fields around charged molecules in solution. Using a series of t hree charged or neutral [4-hydroxy (or carboxy or amino)-2,2,6,6-tetramethy l-1-piperidinyloxy (TEMPO)] spin probes, the apparent rate constants (k(+), k(o), and k(-)) for a particular nucleus can be calculated as the slope of 1/T-1, (i.e., the spin-lattice relaxation rate) vs, increasing radical con centration. Protons residing within a particular charged region suffer a de crease in their apparent rate constants due to repulsive interactions when the nitroxide spin probe bears the same charge-type as that of the local el ectrostatic environment. Accordingly, electrostatic forces of attraction in crease the apparent rate constants for these same protons when the nitroxid e spin probe bears the opposite charge to that present in the local electro static field. The apparent rate constants of the neutral nitroxide spin pro be are an indication of the solvent accessibility to that particular site i n the molecule being investigated. Ratios of these apparent relaxation rate constants (k(+)/k(-) > 1 or k(-)/k(+) > 1, etc.) thus provide a measuremen t of the sign and magnitude of the local electrostatic fields surrounding a particular proton in the target molecule. Three charged hexosamines [D-glu cosamine.HCl (4), D-galactosamine.HCl (5), and D-mannosamine.HCl (6)1, a gl ucuronide metabolite of paracetamol [p-acetamidophenyl-beta -D-glucuronide sodium salt (7)1, and a mononucleotide [thymidine-3 ' -phosphate sodium sal t,(8)1, were investigated in this study. Positively charged environments ar ound protons in the hexosamines are clearly seen by k(o)/k(+) ratios greate r than unity, while negatively charged surroundings about protons in the gl ucuronide and nucleotide are markedly evident from larger than unity k(+)/k (-) and k(o)/k(-) ratios. In the case of small charged target molecules suc h as 6-8, the electrostatic field seems to envelop the entire molecule. In general, a lack of statistically significant differences in the k(attractio n)/k(repulsion) or k(neutral)/k(repulsion) ratios as a function of proton d istance from the charged functionality was observed. This finding (with the possible exception of the methyl protons in 7) suggests that the present v ersion of this method is not sensitive enough to map subtle differences in charged environments around small and conformationally flexible or dynamic target molecules.