Branched porphyrins as functional scaffolds for multisite bioconjugation

Abstract

Bioconjugation is a rapidly expanding field because of 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 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 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 the fibrinogen recognition exosite, were joined onto the porphyrin macrocycle, providing the multisite-directed inhibitor CS3-TAMPP-ES7. This multisite inhibitor and its Mn(III) 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 Mn(III) complex is capable of interacting with clot-bound thrombin and partially inhibits 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.

Porphyrinoid biohybrid materials as an emerging toolbox for biomedical light management

“…Alternatively, Engelen et al synthesized a conjugate composed of a Por and two different peptides, CS3 and ES7, which act as thrombin inhibitors. 103 In particular, the authors prepared tetrakis( p-[aminomethyl]phenyl) H 2 Por, which carries four amino groups in meso positions, allowing the incorporation of the different peptide fragments through an adequate protection/ deprotection strategy. The obtained PS hybrids could be used for sensing and imaging the interaction between different molecules and thrombin.…”

Toward Intracellular Bioconjugation Using Transition-Metal-Free Techniques

“…To illustrate this, many examples involving direct functionalization of a target protein by a fluorescent dye or through nonpeptide biomolecules, as linkers for bioconjugation, exist in the literature. One such instance is elucidated in a work involving tetrakis­( p -[aminomethyl] phenyl)­porphyrin (TAMPP) synthesized from tetraphenylporphyrins, to act as a rigid macrocycle with multiple amine groups for further conjugation with different peptide sequences . To validate the macrocyclic scaffold as a linker for bioconjugation techniques, known thrombin inhibitors CS3 and ES7 were conjugated to TAMPP.…”

“…One such instance is elucidated in a work involving tetrakis(p-[aminomethyl] phenyl)porphyrin (TAMPP) synthesized from tetraphenylporphyrins, to act as a rigid macrocycle with multiple amine groups for further conjugation with different peptide sequences. 125 To validate the macrocyclic scaffold as a linker for bioconjugation techniques, known thrombin inhibitors CS3 and ES7 were conjugated to TAMPP. The conjugated novel porphyrin system, shown in Figure 2, was found to bind and inhibit clot-bound thrombin, apart from resisting enzymatic degradation over long periods of time, being the first of its kind.…”

Indolizine-Based Scaffolds as Efficient and Versatile Tools: Application to the Synthesis of Biotin-Tagged Antiangiogenic Drugs

“…and their use for bioconjugations. 3 − 7 So far, there are a few heterocyclic platforms, 8 − 10 and none with intrinsic luminescent properties. In a previous work, we considered the use of pyridinium ylide-alkyne cycloaddition forming indolizine as fluorogenic coupling methodology.…”

“…During the last decades, the development of orthogonal chemistries , opened the way to the conception of molecular scaffolds decorated with orthogonal reactive groups (azide, alkyne, alkene, carbonyl, etc.) and their use for bioconjugations. − So far, there are a few heterocyclic platforms, − and none with intrinsic luminescent properties. In a previous work, we considered the use of pyridinium ylide-alkyne cycloaddition forming indolizine as fluorogenic coupling methodology .…”