Nicotinic receptors are regulated by protein kinase C activated via a nicotinic receptors-mediated signaling pathway

Previously we reported than BO-IMI, a reagent which contains a BODIPY fluorophore linked to an imidazole group, can be used to covalently label a phosphomonoester in a single step under aqueous conditions [P. Wang, R.W. Giese, Anal. Chem. 65 (1993) 3518]. The reaction was conducted in the presence of a water-soluble carbodiimide 1-ethyl-3-(3'-N,N'-dimethylaminopropyl) carbodiimide [EDC] to activate the phosphomonoester, and the coupling took place onto both the N1 and N3 imidazole nitrogens of BO-IMI. Whether the two BO-IMI-phosphomonoester regioisomers migrated separately or together during capillary electrophoresis depended on the pH, due to a difference in their pKa values. Since then, we have studied the reaction in more detail leading to the information reported here. First, we have learned that the regioisomer ratio changes during the course of the reaction, and found that the mechanism involves both spontaneous and BO-IMI-catalyzed hydrolysis of the less stable isomer. Second, there is a background reaction in which BO-IMI becomes attached to EDC. Third, the BO-IMI-phosphomonoester product (a mixture of two isomers), that is observed by capillary electrophoresis at an alkaline pH, is found to no longer contain the two fluorine atoms present in the starting BO-IMI reagent. This is because they are placed by hydroxy groups at high pH. Finally, an event was discovered which complicates the detection of less than about 60 fmol of a phosphomonoester with BO-IMI: hydrolysis of a tiny fraction of the BO-IMI takes place during the coupling reaction, which leads to chemical noise in the capillary electropherogram.