1 The Interaction of La 3+ Complexes of DOTA/DTPA-Glycoconjugates with the RCA 120 lectin: A Saturation Transfer Difference (STD) NMR Spectroscopic Study João M. C. Teixeira, [a] David M. Dias, [a] F. Javier Cañada, [b] José A. Martins, [c] João P. André, [c] Jesús Jiménez-Barbero, [b] and Carlos F. G. C. Geraldes * [a] Abstract: The study of ligand-receptor interactions using high resolution NMR techniques, namely the Saturation Transfer Difference (STD), is presented for the recognition process between La(III) complexes of DOTA mono(amide) and DTPA bis(amide) glycoconjugates and the galactose specific lectin Ricinus Communis agglutinin (RCA 120 ). This new class of Gd(III)-based potential targeted MRI contrast agents (CAs), bearing one or two terminal sugar (galactosyl or lactosyl) moieties, has been designed for in vivo binding to ASGPR (the asialoglycoprotein receptor), which is specifically expressed at the surface of liver hepatocytes, with the aim of leading to a new possible diagnosis of liver pathologies. The in vitro affinity constants of the divalent La(III)- glycoconjugate complexes to RCA 120 , used as a simple, water soluble receptor model, were higher than those of the monovalent analogues. The combination of the experimental data obtained from the STD NMR experiments with molecular modelling protocols (Autodock 4.1) allowed us to predict the binding mode of mono and divalent forms of these CAs to the galactose 1 binding sites of RCA 120 . The atomic details of the molecular interactions allowed corroborating and supporting the interaction of both the sugar moieties and the linkers with the surface of the protein and thus, their contribution to the observed interaction stabilities. Keywords: ligand-receptor binding • glycoconjugates • STD-NMR spectroscopy • MRI contrast agents • protein-ligand interaction • compound docking Introduction Molecular recognition events are of paramount importance in chemistry, biology and biomedicine. A large variety of techniques allow the elucidation of binding events between a ligand and its receptor. As key examples, ELISA (Enzyme Linked Immunosorbent Assay(1), Immunoblotting, RIA (Radioimmuno-assay),(2) affinity chromatography(3) or Surface Plasmon Resonance experiments (Biacore)(4) are nowadays commonly employed for this task. In recent years, NMR- based techniques(5) have become increasingly popular when filling in the existing gap for characterization of molecular binding processes at high resolution. Transferred NOESY,(6) NOE pumping (7) and WaterLOGSY (8,9) are particular and powerful examples of such approaches. Among them, the Saturation Transfer Difference (STD) NMR technique is probably one of the most popular and robust methods.(5, 10-14) This technique allows characterizing ligand binding through intermolecular saturation transfer and, moreover, allows screening of ligand libraries,(11) as well as [a] João M. C. Teixeira, D.M. Dias, C.F.G.C. Geraldes () Department of Life Sciences and Center of Neurosciences and Cell Biology, Faculty of Science and Technology University of Coimbra, P.O. Box 3046, 3001-401 Coimbra, Portugal Tel: (+) 351239853608; Fax: (+)351239853607 e-mail: geraldes@bioq.uc.pt [b] F. J. Cañada, J. Jiménez-Barbero Department of Chemical and Physical Biology, CIB-CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain [c] J.A. Martins, J.P. André Centro de Química, Campus de Gualtar, Universidade do Minho, Braga, Portugal