Rational Inhibitor Design, Synthesis and NMR Spectroscopic Study by Transferred Nuclear Overhauser Spectroscopy of Novel Inhibitors of Cinnamyl Alcohol Dehydrogenase, a Critical Enzyme in Lignification

Abstract Cinnamyl alcohol dehydrogenase is one of the enzymes controlling the first two committed steps of lignification. Using a 3-dimensional similarity model of this enzyme, a series of novel phos-phonates (1-5) was designed as potential inhibitors. Phosphonates 1-5 were synthesized in good yield by reaction of the corresponding cinnamaldehydes with tetraethylmethylene diphos-phonate. Monophosphonic acids 6 and 7 were obtained by basic hydrolysis of the corresponding phosphonates while phosphonamidate 8 was synthesized by reacting benzylamine with the iminium salt intermediate of the monophosphonic acid. Using recombinant cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) the inhibitory activity of these compounds was evaluated and compared with that of the carbonyl analogues. Inhibition kinetic studies showed compounds 2 and 3 to be mixed type linear inhibitors while compound 4 was uncompetitive. H NMR studies of inhibitor 2, for which K1 and K1 were 20 and 86 pM, respectively, in the presence of CAD based on selective line-broadening showed an increased interaction of the 3-Ome group of the aromatic ring of the inhibitor with the active site of the CAD. A transferred nuclear overhauser effect spectroscopy (TRNOESY) experiment for inhibitor 2 with CAD was used to determine the conformation of this compound bound to CAD. These results were found to be consistent with the 3-dimensional structural model of the enzyme