Protonation equilibria of Hoechst 33258 in aqueous solution

The bis-benzimidazole derivative Hoechst 33258 (2'-(4-hydroxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bi-1H-benzimidazole) binds to the minor groove of DNA duplexes and is widely used as fluorescent cytological stain for DNA. The neutral compound, 1, is amphiphilic with four basic and three acidic sites. We have determined all seven acidity constants by spectrophotometric titration to define the pH-dependent distribution of species, from the fully protonated tetracation 1(4+) to the fully deprotonated trianion 1(3-), in aqueous solution. The structures of the intermediate protonation states were assigned with the aid of density-functional calculations. Electrostatic interaction free energies were calculated to adjust the acidity constants of the molecular subunits of 1 to their environment in the species 1(4+) to 1(3-). The experimental and theoretical pK(a) values agree well, but they differ substantially from previous estimates given in the literature.