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.

2-Diazo-1,2-naphthoquinone (1) and its derivatives are the photoactive components in Novolak photoresists. A femtosecond infrared study has established that the photoreaction of 1 proceeds largely by a concerted Wolff rearrangement yielding the ketene 1H-inden-1-ylidene-methanone (3) within 300 fs after excitation, but earlier trapping studies gave evidence for a minor reaction path via a carbene intermediate 1-oxo-2(1H)-naphthalenylidene (2) with a lifetime of about 10 ps. Here, we provide a quantitative assessment of the stepwise pathway by Stern-Volmer analysis of the trapping of 2 by methanol to yield 2-methoxy-1-naphthol (4). We conclude that the lifetime of the carbene 2 is at least 200 ps. Moreover, [3 + 2]cycloaddition of 2 and acetonitrile yielding 2-methylnaphth[2,1-d]oxazole (5) was observed. A comparison of the yields of 5 formed upon photolysis and upon thermolysis of 1 in acetonitrile provides evidence that a substantial part of the hot nascent carbene 2 formed photolytically rearranges to the ketene 3 during its vibrational relaxation (hot ground-state reaction).