Redox potential fine tuning of ferrocene-peptide conjugates

We report the synthesis of new ferrocene (Fc)-peptide conjugates, namely, N-Fc-succinamide-Gly-Ala-OEt, N-Fc-glutaramide-Gly-Ala-OEt, N-Fc-pimelamide-Gly-Ala-OEt, and N-Fc-glutaramide-Gly-Gly-Ala-OEt. They were designed on the base of four components: (i) a Fc unit as an electroactive element; (ii) the linker -NH-CO- to adjust the oxidation potential of Fc towards the physiological conditions; (iii) spacers of different lengths between Fc and the peptide; (iv) a peptide unit to drive Fc to selected biological targets. Fc-peptide conjugates have already been explored as potential anticancer drugs. It is reported that Fc displays the anti-proliferative activity through the production of reactive oxygen species (ROS) as its redox potential (0.40 V vs. SCE) is compatible with the intracellular potential which varies from +0.40 V to −0.44 V. However, the way Fc binds to the peptide significantly influences its redox potential. So far, this issue was not deeply addressed in the literature. Therefore, in this contribution we aimed at investigating the influence of the linker, and of the length of spacer and peptide on the Fc oxidation potential. Noteworthy, we linked Fc to the remaining part of the molecule via an amide bond, but with N-end attached to Fc and not the carbonyl, as reported in the literature so far. The cyclic voltammetry measurements we performed revealed that the transition from an electron-withdrawing (Fc-CO-NH-) to an electron-donating group (Fc-NH-CO-) significantly affects the Fc redox potential. On the contrary, spacer and peptide lengths display a moderate effect. We also carried out a conformational study in the crystal state (X-ray diffraction), and in solution (2D-NMR) on three intermediate molecules. Interestingly, the tripeptide Boc-Gly-Gly-Ala-OEt adopts a β-turn structure in all environments. This finding help to explain its resistance to the enzymatic hydrolysis. Enzymatic degradation tests in human serum were performed on the other conjugates as well, highlighting that the Fc unit acts as a protector of the peptide portion.