Determination of the Cholecalciferol-Lipidcomplex Using a Combination of Comparative Modelling and NMR Spectroscopy

Abstract

Exploration of the systemic disposition of macromolecules in relation to their physicochemical properties, could be a strategy for designing targeting system. This work deals with the investigation of the Vitamin D3 conformationls in the phospholipid bilayer l ,2 in order to define a possible preferred binding site at the C=O-or POz-phosp,holipid moiety (structure-function studies) to be exploited into drug discovery efforts (forthcomingpaper). NMR analysis ID-n.O.e. data were compliant to a s-trans conformation of the diene moiety with the proton 14 and Mel8 respectively bent towards H9 and the diene moiety (Fig 1). The presence of equilibrating conformers, ex. and p (Fig 2), having the OH group respectively in equatorial and axial orientations, already well documented 3 , is also in agreement with the observation of almost equivalent interproton dipolar interactions between the proton HI9E and the protons H la and Hlb, almost equivalent intern.O.e.s H6-H4a and H6-~b were observed. Moreover, the observation of a dipolar interaction between H3 and H la is an indication of the fact that such protons are in a diaxial arrangement as in conformation ex., whereas the detection of a clear n.Q.e. between the methyl protons Mel8 andHl9z, aside from the complete absence of n.O.e. between HI9z and HIS belonging to the D ring, reveals the presence of a conformer in which the unsaturated group is on the same side of the Me18, outside the diene plane, as in conformation p. l3e T I measurements revealed that the hypothesis of isotropic overall motion is nearly satisfied, indeed the methylene I3C TIs (0.55 s) of Vitamin D3, belonging to the A and CD rings were similar each other and correlated to the 13C TIs of methinecarbons (0.99 s). Thus, the samere-orientational time can be attributed to all the molecules and the ratios ofthe different interproton cross-relaxation rates sij' determined by proton selective relaxation rates measurements 2 , can be simply correlated to the ratios of the internuclear distances (syiSik = (rwJrij)6, eq. 1), thus allowing a more precise definition of the stereochemistry. Hence, we determined the cross-relaxation rate S19Z-19E 325 Molecular Modeling and Prediction of Bioactivity, edited by Gundertofte and J~rgensen.