SYNTHESIS AND ANALYSIS OF THE ENANTIOMERS OF CALMIDAZOLIUM, AND A H-1-NMR DEMONSTRATION OF A CHIRAL INTERACTION WITH CALMODULIN

Calmidazolium {R24571, [(2,4-dichlorophenyl)methoxy]ethyl]-1H-imidazol ium chloride} is a potent calmodulin inhibitor. This paper describes t he synthesis and properties of the enantiomers of calmidazolium from t he enantiomers of miconazole 4-dichlorobenzyloxy)-2-(2,4-dichloropheny l))-ethyl imidazole}, prepared from the racemate by chiral preparative scale high performance liquid chromatography. Overlap between ligand and protein resonances in the aromatic region of the H-1 NMR spectrum of the calmidazolium-calmodulin complexes has been obviated by prepara tion of the protein with all of its nine phenylalanine rings deuterate d (Phe-d(5) calmodulin). This has been accomplished by the overexpress ion of calmodulin derived from Trypanosoma brucei rhodiesiense in E. c oli in a medium supplemented with ring-deuterated phenylalanine. The k inetics of binding of each enantiomer are slow on the H-1 NMR time sca le as judged by the behaviour of the H2 resonance of Histidine-107, wh ich is clearly visible under the sample conditions used. The aromatic spectral regions of the protein-bound (+) and (-) enantiomers contrast strikingly, reflecting differences in bound environment and/or confor mation. (C) 1997 Wiley-Liss, Inc.