Synthesis and Use of a Biotinylated 3-Azidophenothiazine to Photolabel Both Amino- and Carboxyl-Terminal Sites in Calmodulin

Abstract

The biotinylated probe, 3-azido-10-(4-(4-biotinyl-1-piperazinyl)butyl)phenothiazine, was used to examine the phenothiazine binding domains in calmodulin (CaM) by photolabeling. This phenothiazine, synthesized from 3-azido-10-(4-(1-piperazinyl)butyl)phenothiazine and d-biotinyl tosylate, inhibited the CaM-mediated activation of phosphodiesterase (PDE) with an I50 of 12.5 (+/- 2.8) microM. Photolabeling of CaM produced covalent adducts in excellent yield (32%) in a light- and Ca2+-dependent manner. Studies performed over a range of drug concentrations suggested a 2:1 stoichiometry for the binding of the phenothiazine probe to CaM. Limited trypsin digestion and purification of the resulting fragments by either SDS-PAGE or HPLC provided two principal phenothiazine-containing peptides. Amino acid composition and sequence analyses performed on these two peptides established that both the N- and C-terminal domains in CaM, particularly the regions amino terminal to Ca2+-binding loops 1 and 3, were modified by the photoactivated phenothiazine derivative. These data, particularly for the C-terminal domain, are in excellent agreement with the X-ray structure analysis of a 1:1 CaM-trifluoperazine complex.

Surface-enhanced Raman encoded polymer stabilized gold nanoparticles: Demonstration of potential for use in bioassays

“…Attempted tosylation in anhydrous pyridine resulted in a mixture of the tosylate 11a (19 %) and chloride 11b (16%) (35 % combined yield). This appearance of the chloro species 11b in the reaction of biotinol with sulfonyl chlorides has been previously observed by Islam et al 28 In this report the treatment of biotinol with methanesulfonyl chloride gave both the mesylate and chloride. Use of excess pyridine in the tosylation reaction, in our case by its use as solvent, can facilitate formation of chloro species by reaction of the pyridinium chloride by-product with the newly formed tosylate.…”

“…These data were found to have some discrepancy compared with those reported in literature. 27,28 It is suggested that some of the NMR data may have been misreported as the product reported in literature was not purified after the Soxhlet extraction.…”

Molecular Recognition Studies on Naphthyridine Derivatives

“…4S-[(3aS,6aR)-5-Iodopentyl]tetrahydro- 1 H - thieno[3,4-d]imidazol-2(3H)-one ( 3 ) [ 15 ]. This compound was prepared from biotin tosylate as reported [ 22 ]. The biotin tosylate (280 mg, 0.75 mmol) and NaI (216 mg, 0.15 mmol) were stirred in acetone (20 mL) for 24 h. The solvent was removed under reduced pressure and the residue was dissolved in CH 2 Cl 2 and the organic layer was successively washed with sodium thiosulfate (1.0 N) and water, dried over anhydrous Na 2 SO 4 , and concentrated under vacuum.…”

Ca2+-bound calmodulin forms a compact globular structure on binding four trifluoperazine molecules in solution

“…Gel-filtration experiments indicated that chlorpromazine, which is structurally similar to TFP, binds at not four but five binding sites [19]. Photolabelling of CaM with a reactive phenothiazine derivative indicated two binding sites, one in each globular domain [20]. "H-, %$Caand ""$Cd-NMR studies indicated the presence of two distinct binding sites for TFP in which the affinities differ by one or two orders of magnitude [21][22][23].…”