Branched Porphyrins as Functional Scaffolds for Multi-Site Bioconjugation

Bioconjugation is a rapidly expanding field, due to the numerous potential applications of bioconjugate materials. We explored the usefulness of branched porphyrins as rigid scaffolds, bearing multiple sites for bioconjugation. To this end, we first selected the tetrakis(p-(aminomethyl) phenyl) porphyrin (TAMPP) macrocycle, and we developed a straightforward synthetic protocol, able to provide the desired tetraphenylporphyrin, carrying four functional amino groups. The partially protection of the amino groups by tert-butoxy-carbonyl (t-Boc) allowed the selective and specific decoration of the porphyrin with different peptide sequences. To explore the utility of the macrocycle as molecular scaffold for bioconjugation, we selected peptide sequences able to function as thrombin inhibitors. In particular, two peptide sequences, named CS3 and ES7, able to interact respectively with the thrombin catalytic site and with the fibrinogen recognition exosite, were joined onto the porphyrin macrocycle, providing the multi-site directed inhibitor CS3-TAMPP-ES7. This multi-site inhibitor and its MnIII-complex are able to inhibit α-thrombin catalyzed hydrolysis of Tos-Gly-Pro-Arg-nitroanilide with inhibition constants in the micromolar range, as well as the hydrolysis of the natural substrate fibrinogen. The inhibitor is resistant against enzymatic degradation by thrombin and is highly selective. The MnIII-complex is capable of interacting with clot-bound thrombin, and partially inhibit clot-growth in the presence of fibrinogen. The results herein reported are very promising, suggesting the potential of the newly developed conjugate as new imaging agents for clot detection.