Doxorubicin (DOX) is a potent anti-tumoral agent widely used for cancer therapy. Despite numerous studies, the fluorescence properties of DOX, usually exploited for the characterization of the interaction with biological media, have until now led to controversial interpretations, mainly due to self-association of the drug in aqueous solution. We present here the first femtosecond study of DOX based on measurements with the fluorescence up-conversion technique in combination with time-correlated single photon counting using the same laser source. We provide evidence that fluorescence signals of DOX stem from monomers and dimers. DOX dimerization induces a dramatic decrease in the fluorescence quantum yield from 3.9 × 10⁻² to 10⁻⁵ associated with the red shift of the fluorescence spectrum by ∼25 nm. While the fluorescence lifetime of the monomer is 1 ns, the dimer fluorescence is found to decay with a lifetime of about 2 ps. In contrast to monomers, the fluorescence anisotropy of dimers is found to be negative. These experimental observations are consistent with an ultrafast internal conversion (<200 fs) between two exciton states, possibly followed by a charge separation process.

We report a comparative study of the isomerization reaction in native and denatured photoactive yellow protein (PYP) and in various chromophore analogues in their trans deprotonated form. The excited-state relaxation dynamics was followed by subpicosecond transient absorption and gain spectroscopy. The free p-hydroxycinnamate (pCA²⁻) and its amide analogue (pCM⁻) are found to display a quite different transient spectroscopy from that of PYP. The excited-state deactivation leads to the formation of the ground-state cis isomer without any detectable intermediate with a mechanism comparable to trans-stilbene photoisomerization. On the contrary, the early stage of the excited-state deactivation of the free thiophenyl-p-hydroxycinnamate (pCT⁻) and of the denatured PYP is similar to that of the native protein. It involves the formation of an intermediate absorbing in the spectral region located between the bleaching and gain bands in less than 2 ps. However, in these two cases, the formation of the cis isomer has not been proved yet. This difference with pCA⁻ and pCM⁻ might result from the fact that, in the thioester substituted chromophore, simultaneous population of two quasi-degenerate excited states occurs upon excitation. This comparative study highlights the determining role of the chromophore structure and of its intrinsic properties in the primary molecular events in native PYP.