Mechanism of dansylation of the polyamine pentaazapentacosane 5 HCl

Abstract

Pentaazapentacosane pentahydrochloride (PAPC-HCl) is a synthetically produced aliphatic pentaamine that is being investigated for use as an anticancer agent. As part of this research project a rapid high-performance liquid chromatographic method for determination of the dansyl derivative of PAPC was developed. The chromatographic system uses a reverse phase C-8 column, a mobile phase of acetic acid buffer and acetonitrile and UV detection. The dansylation conditions were optimized with a pH of 11.0 and a 20 fold dansyl chloride excess. The yield of dansyl PAPC increased 10 fold as the reaction pH was changed from 9.5 to 10.5. An investigation of the products formed in the dansylation reaction revealed that, even under conditions of pH and dansyl chloride concentration most likely to produce partially dansylated products, only perdansyl PAPC is present. This unexpected finding is explained by a mechanism whereby (1) only completely unionized amine molecules will dansylate and (2) the ratio of unionized molecules to ionized molecules increases as dansylation proceeds. The proposed mechanism is verified by comparing the dansylation vs. pH profile of PAPC to that of a reference monoamine (piperidine ·HCl). After 4 hours at room temperature and pH 9.5, 100% of piperidine is dansylated while under the same conditions only 10% of PAPC is derivatized. A pH greater than 10.5 is required to completely dansylate PAPC. This difference is significantly greater than would be predicted from the pKₐ values but it is consistent with the proposed mechanism.