SYNTHESIS AND BINDING-AFFINITY OF BIDENTATE PHENOTHIAZINES WITH 2 DIFFERENT PHOTOACTIVE GROUPS

The development of targeted, bidentate photoaffinity reagents for mapp ing the interacting domains of calmodulin (CaM) with the enzymes that it regulates required the synthesis and evaluation of the binding affi nity of various phenothiazines. These photoaffinity reagents would pos sess a photoactive 3-azidophenothiazine group for cross-linking the hy drophobic binding domain of CaM, a second photoactive benzophenone gro up that would be activated at a different wavelength than the 3-azidop henothiazine group, and a suitable radiolabel. Difficulties were encou ntered in identifying those structural features that would be compatib le with the introduction of a benzophenone group, with the solubility of these benzophenone-substituted phenothiazines, and with the ability of these phenothiazines to inhibit the calmodulin-mediated activation of phosphodiesterase. Solutions to this problem involved the preparat ion of phenothiazines possessing a quaternary ammonium salt, a zwltter ionic amino acid, or a carbohydrate moiety. The phenothiazines that po ssessed photoactive 3-azido and benzophenone groups and in which one o f the piperazine nitrogens in the side chain was converted to a quater nary, N-methylammonium iodide inhibited the calmodulin-mediated activa tion of phosphodiesterase at a level comparable to that of chlorpromaz ine.