The importance of fluorine in medicinal chemistry is well‐recognized. An increasing number of drugs on the market contain fluorine, the presence of which is often of major importance to activity [ NMR applied to proteins, see Gerig [
NMR spectroscopy has been made possible by technical developments that have resulted in an improvement in sensitivity of the detection of fluorine signals [
transverse relaxation T
chemical shift anisotropy and chemical exchange contributions to the transverse relaxation of the
signals in the presence of the biological target. In this chapter we focus on the application of
NMR spectroscopy to simple and complex biochemical assays for the detection of inhibitors or agonists of enzymatic reactions. The chapter is organized in several sections. An initial section introduces basic notions of enzymology required for the interpretation of the NMR results. The principles at the basis of the methodology are then presented and analyzed. Application of the technique starts with the simplest biological system of one substrate and one enzyme, followed by more complex biological systems, such as several substrates in the presence of one or more enzymes, which are relevant in systems biology. A section comparing the NMR methodology with other more established biophysical techniques often used for biochemical screening is then presented. Finally, protocols for performing the assays and for measuring the inhibition strength of the identified NMR hits along with NMR procedures for the identification of false positives are presented.