The regulation of calmodulin (CaM), a calcium ion sensing protein, is a vital translator of calcium signaling in cells. CaM, capable of binding four calcium (II) ions, regulates the calcium concentration within the cell. Once activated by Ca2+, CaM is capable of binding other proteins involved in signaling processes. These interactions are necessary for a multitude of functions in the body including nerve signaling, smooth muscle constriction, intracellular movement, metabolism, and memory. CaM antagonists, substrates capable of inhibiting CaM, are commonly used to study CaM’s effects on biological systems, whose complex binding mechanisms are still not fully understood. In conjunction with probing CaM’s structure and function, these antagonists are used to purify CaM from CaM expressing E. coli via affinity chromatography. The most reported method for purifying CaM is a Phenyl Sepharose affinity column; however, this method has a major drawback – it is incapable of purifying CaM in one purification step. It is necessary to perform back-to-back Phenyl Sepharose columns, as well as ion-exchange chromatography, to purify CaM in &gt; 95% purity. Another, but much less reported, method to purify CaM is affinity chromatography utilizing the antagonist 2-trifluoromethyl-10-aminopropylphenothiazine (TAPP). This method has been shown to only require one purification step. Despite the benefits of using TAPP Sepharose for CaM purification, it is not a commonly used method due to the long, complicated, and hazardous synthesis of TAPP reported in the literature. The classic synthesis of TAPP makes this antagonist inaccessible to most biochemists, who have limited organic synthesis experience. Herein we report an improved synthesis of TAPP with the goal of making this antagonist much more accessible to the biochemists who need it. Starting from 2-(trifluoromethyl)phenothiazine and 3-aminopropylbromide hydrobromide, TAPP was successfully synthesized in 96% yield over three simple steps. 3-Aminopropylbromide was first boc-protected and then coupled to 2-(trifluoromethyl)phenothiazine by simple SN2 chemistry in the presence of potassium hydroxide. After the amino arm was installed, the boc-protecting group was subsequently removed using trifluoroacetic acid. Once synthesized, TAPP was immobilized on Sepharose 4B and it was used to isolate CaM in &gt; 95% purity.

Improved, efficient synthesis of the calmodulin antagonist TAPP

2019 Spring National Meeting

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