Synthesis and DNA interaction studies of Ru(II) cell penetrating peptide (CPP) bioconjugates

The synthesis of the first bioconjugates of a set of ruthenium(II) dipyridophenazine complexes with two different cell penetrating peptides (CPPs) is described. The CPPs, an arginine rich TAT-9 (RKKRRQRRR) sequence and the Xentry peptide (LCLRPVG), were synthesized using standard SPPS protocols, and the bioconjugates were obtained by the microwave-assisted coupling of the HOBt/TBTU preactivated metal complexes with the respective peptides on Wang resin. The racemic metal complexes were obtained by modified literature procedures. The bioconjugates were cleaved from the resin, purified by semi-preparative HPLC and characterized by analytical HPLC, high resolution mass spectrometry (HR-MS), and NMR spectroscopy. Despite the bioconjugation of the peptides to the dppz ligand, DNA intercalation was observed by CD spectroscopy, viscometry and the characteristic switch-on fluorescence of this class of compounds. Furthermore, the cellular uptake of the Xentry bioconjugates was confirmed by live cell imaging. Like the parent metal complexes, the bioconjugates show low in vitro cytotoxicity (IC₅₀ > 80 μM), which is similar to the respective metal complexes alone.