Protonation Equilibria of Hoechst 33258 in Aqueous Solution

Abstract

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 14+ to the fully deprotonated trianion 13−, 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 14+ to 13−. The experimental and theoretical pKa values agree well, but they differ substantially from previous estimates given in the literature.

UV-induced Spectral Shift and Protonation of DNA Fluorescent Dye Hoechst 33258

“…Also the data reported here are consistent with the view that the predominant form of Hoechst 33258 in a neutral pH is the single-protonated form, while in a low pH environment it is the triple-protonated form [3], and at pH 4.5 Hoechst 33258 exists in a double-protonated form. Hoechst 33258 fluorescence emission spectra, excited by 365 nm, recorded at four different pH values (7, 4.5, 1.5 and 11) were reported before [5].…”

Calorimetric and Spectroscopic Studies of Hoechst 33258: Self-association and Binding to Non-cognate DNA

“…We conducted H33258 dilution experiments under conditions where H33258 is monoprotonated. 50 The enthalpy of H33258 self-interaction in Mops and TEA buffers (Table 1) is essentially equal, within error. Mops and TEA have deprotonation enthalpies of 21.8 kJ mol − 1 and 33.6 kJ mol − 1 , respectively, 51 hence the fact that self-aggregation enthalpy of H33258 is the same in the two different buffers indicates that aggregation is not accompanied by (de)protonation.…”

Positive and negative ion mode ESI-MS and MS/MS for studying drug–DNA complexes

“…This behavior can again be explained with the proton affinities of the drugs. In the literature, Hoechsts 33258 and 33342 are usually represented with one positive charge; while DAPI and Netropsin are usually represented with two positive charges, and this corresponds to the preferred form in solution at pH 7 [50]. In Fig.…”