Inter-Ligand STD NMR: An Efficient 1D NMR Approach to Probe Relative Orientation of Ligands in a Multi-Subsite Protein Binding Pocket.

In recent years, Saturation Transfer Difference NMR (STD NMR) has been proven to be a powerful and versatile ligand-based NMR technique to elucidate crucial aspects in the investigation of protein-ligand complexes. Novel STD NMR approaches relying on "multi-frequency" irradiation have enabled us to even elucidate specific ligand-amino acid interactions and explore the binding of fragments in previously unknown binding subsites. Exploring multi-subsite protein binding pockets is especially important in Fragment Based Drug Discovery (FBDD) to design leads of increased specificity and efficacy. We hereby propose a novel multi-frequency STD NMR approach based on direct irradiation of one of the ligands in a multi-ligand binding process, to probe the vicinity and explore the relative orientation of fragments in adjacent binding sub-sites, which we called Inter-Ligand STD NMR (IL-STD NMR). We proved its applicability on (i) a standard protein-ligand system commonly used for ligand-observed NMR benchmarking: Naproxen as bound to Bovine Serum Albumin, and (ii) the biologically relevant system of Cholera Toxin Subunit B and two inhibitors adjacently bound within the GM1 binding site. Relative to Inter-Ligand NOE (ILOE), the current state-of-the-art methodology to probe relative orientations of adjacent ligands, IL-STD NMR requires about one tenth of the experimental time and protein consumption, making it a competitive methodology with the potential to be applied in the pharmaceutical industries.

Host-guest interactions between cyclodextrins and surfactants with functional groups at the end of the hydrophobic tail.

The aim of this work was to investigate the influence of the incorporation of substituents at the end of the hydrophobic tail on the binding of cationic surfactants to α-, ß-, and γ-cyclodextrins. The equilibrium binding constants of the 1:1 inclusion complexes formed follow the trend K1(α-CD)>K1(ß-CD)≫K1(γ-CD), which can be explained by considering the influence of the CD cavity volume on the host-guest interactions. From the comparison of the K1 values obtained for dodecyltriethylammonium bromide, DTEAB, to those estimated for the surfactants with the substituents, it was found that the incorporation of a phenoxy group at the end of the hydrocarbon tail does not affect K1, and the inclusion of a naphthoxy group has some influence on the association process, slightly diminishing K1. This makes evident the importance of the contribution of hydrophobic interactions to the binding, the length of the hydrophobic chain being the key factor determining K1. However, the presence of the aromatic rings does influence the location of the host and the guest in the inclusion complexes. The observed NOE interactions between the aromatic protons and the CD protons indicate that the aromatic rings are partially inserted within the host cavity, with the cyclodextrin remaining close to the aromatic rings, which could be partially intercalated in the host cavity. To the authors' knowledge this is the first study on the association of cyclodextrins with monomeric surfactants incorporating substituents at the end of the hydrophobic tail.

Micellar kinetic effects in gemini micellar solutions: influence of sphere-to-rod transitions on kinetics.

The reactions 4-nitrobenzenesulfonate+Br(-) and methyl naphthalene-2-sulfonate+Br(-) were studied in various water-ethylene glycol, EG, [C(12)H(25)(CH(3))(2)N(CH(2))(s)N(CH(3))(2)C(12)H(25)]Br(2) micellar solutions (12-s-12,2Br(-) with s=3-5 methylene groups). Results showed that the observed rate constant of the two reactions varied when a sphere-to-rod transition occurs. This morphological transition is accompanied by changes in the interfacial region water content and in its polarity. The micellar ionization degree is also altered, making the counterion interfacial concentration change. Finally, variations on the molar surfactant volume, V(m), also follow the sphere-to-rod transitions. A simple pseudophase kinetic model is inadequate for quantitatively discussing the kinetic micellar effects observed, since the changes accompanying micellar growth affect the second-order rate constant in the micellar pseudophase, the equilibrium binding constant and the surfactant molar volume, neither of them remaining constant in the whole surfactant concentration range. However, this simple model can be helpful in the treatment of kinetic data for surfactant concentrations below the morphological transitions, providing some interesting, although approximate, information.