Artificial Heme Enzymes for the Construction of Gold-Based Biomaterials.

Many efforts are continuously devoted to the construction of hybrid biomaterials for specific applications, by immobilizing enzymes on different types of surfaces and/or nanomaterials. In addition, advances in computational, molecular and structural biology have led to a variety of strategies for designing and engineering artificial enzymes with defined catalytic properties. Here, we report the conjugation of an artificial heme enzyme (MIMO) with lipoic acid (LA) as a building block for the development of gold-based biomaterials. We show that the artificial MIMO@LA can be successfully conjugated to gold nanoparticles or immobilized onto gold electrode surfaces, displaying quasi-reversible redox properties and peroxidase activity. The results of this work open interesting perspectives toward the development of new totally-synthetic catalytic biomaterials for application in biotechnology and biomedicine, expanding the range of the biomolecular component aside from traditional native enzymes.

A Quartz Crystal Microbalance Immunosensor for Stem Cell Selection and Extraction.

A cost-effective immunosensor for the detection and isolation of dental pulp stem cells (DPSCs) based on a quartz crystal microbalance (QCM) has been developed. The recognition mechanism relies on anti-CD34 antibodies, DPSC-specific monoclonal antibodies that are anchored on the surface of the quartz crystals. Due to its high specificity, real time detection, and low cost, the proposed technology has a promising potential in the field of cell biology, for the simultaneous detection and sorting of stem cells from heterogeneous cell samples. The QCM surface was properly tailored through a biotinylated self-assembled monolayer (SAM). The biotin-avidin interaction was used to immobilize the biotinylated anti-CD34 antibody on the gold-coated quartz crystal. After antibody immobilization, a cellular pellet, with a mixed cell population, was analyzed; the results indicated that the developed QCM immunosensor is highly specific, being able to detect and sort only CD34+ cells. Our study suggests that the proposed technology can detect and efficiently sort any kind of cell from samples with high complexity, being simple, selective, and providing for more convenient and time-saving operations.

Branched porphyrins as functional scaffolds for multisite bioconjugation.

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.

Exploring the role of unnatural amino acids in antimicrobial peptides.

Cationic antimicrobial peptides (CAMPs) are a promising alternative to treat multidrug-resistant bacteria, which have developed resistance to all the commonly used antimicrobial, and therefore represent a serious threat to human health. One of the major drawbacks of CAMPs is their sensitivity to proteases, which drastically limits their half-life. Here we describe the design and synthesis of three nine-residue CAMPs, which showed high stability in serum and broad spectrum antimicrobial activity. As for all peptides a very low selectivity between bacterial and eukaryotic cells was observed, we performed a detailed biophysical characterization of the interaction of one of these peptides with liposomes mimicking bacterial and eukaryotic membranes. Our results show a surface binding on the DPPC/DPPG vesicles, coupled with lipid domain formation, and, above a threshold concentration, a deep insertion into the bilayer hydrophobic core. On the contrary, mainly surface binding of the peptide on the DPPC bilayer was observed. These observed differences in the peptide interaction with the two model membranes suggest a divergence in the mechanisms responsible for the antimicrobial activity and for the observed high toxicity toward mammalian cell lines. These results could represent an important contribution to unravel some open and unresolved issues in the development of synthetic CAMPs.

Artificial heme-proteins: determination of axial ligand orientations through paramagnetic NMR shifts.

An empirical equation, describing the relationship between the porphyrin methyl hyperfine shifts and the position of the axial ligand(s), has been applied to an artificial heme-protein in order to obtain insight into the active site properties of heme-protein models.