NMR case study of ropinirole: Concentration-dependent effects of nonexchangeable proton resonances

NMR is an established technique that is commonly used for struc-ture characterization and determination of purity of small molecules.It is generally accepted that nonexchangeable protons of chemicalstructures show a characteristic chemical shift and signal multiplicitydependingonthelocalgeometry,such as binding partners, bondlengths and binding angle.[1,2]The chemical shifts of nonexchangeableprotons, unlike for exchangeable protons, are typically independentfrom the sample concentration. However, significant changes inchemical shifts of nonexchangeable protons have been previouslyreported for a range of scaffolds including quinolones, indoles,benzofurans, naphthalenes and other related structures.[3–7]Detailsabout the concentration of a sample are generally not reported forNMR experiments; however, unidentified as well as unreportedconcentration-dependent NMR effects complicate the reproducibil-ity and comparison of spectral data from different experiments andpotentially lead to false interpretation of results.More recently, NMR techniques alsofind their application forthe screening of ligands or fragments to identify pharmaceuti-cally relevant interactions with target proteins and enzymes. Itis probably not surprising that concentration-dependent changesin chemical shifts affect the outcome of such screening cam-paigns. It has previously been shown that compound aggrega-tion of small molecules can lead to promiscuous results in NMRscreening experiments and various other assays.[8–12]In otherwords, it is very important that scientists are more mindful ofconcentration-dependent NMR effects of nonexchangeable pro-tons and the potential consequences for structural characteriza-tion and pharmacological evaluations