An Artificial Miniaturized Peroxidase for Signal Amplification in Lateral Flow Immunoassays

Signal amplification strategies are widely used for improving the sensitivity of lateral flow immunoassays (LFiAs). Herein, the artificial miniaturized peroxidase Fe(III)-MimochromeVI*a (FeMC6*a), immobilized on gold nanoparticles (AuNPs), is used as a strategy to obtain catalytic signal amplification in sandwich immunoassays on lateral flow strips. The assay scheme uses AuNPs decorated with the mini-peroxidase FeMC6*a and anti-human-IgG as a detection antibody (dAb), for the detection of human-IgG, as a model analyte. Recognition of the analyte by the capture and detection antibodies is first evidenced by the appearance of a red color in the test line (TL), due to the accumulation of AuNPs. Subsequent addition of 3,3',5,5'-tetramethylbenzidine (TMB) induces an increase of the test line color, due to the TMB being converted into an insoluble colored product, catalyzed by FeMC6*a. This work shows that FeMC6*a acts as an efficient catalyst in paper, increasing the sensitivity of an LFiA up to four times with respect to a conventional LFiA. Furthermore, FeMC6*a achieves lower limits of detection that are found in control experiments where it is replaced with horseradish peroxidase (HRP), its natural counterpart. This study represents a significant proof-of-concept for the development of more sensitive LFiAs, for different analytes, based on properly designed artificial metalloenzymes

Enzymatic and Bioinspired Systems for Hydrogen Production

The extraordinary potential of hydrogen as a clean and sustainable fuel has sparked the interest of the scientific community to find environmentally friendly methods for its production. Biological catalysts are the most attractive solution, as they usually operate under mild conditions and do not produce carbon-containing byproducts. Hydrogenases promote reversible proton reduction to hydrogen in a variety of anoxic bacteria and algae, displaying unparallel catalytic performances. Attempts to use these sophisticated enzymes in scalable hydrogen production have been hampered by limitations associated with their production and stability. Inspired by nature, significant efforts have been made in the development of artificial systems able to promote the hydrogen evolution reaction, via either electrochemical or light-driven catalysis. Starting from small-molecule coordination compounds, peptide- and protein-based architectures have been constructed around the catalytic center with the aim of reproducing hydrogenase function into robust, efficient, and cost-effective catalysts. In this review, we first provide an overview of the structural and functional properties of hydrogenases, along with their integration in devices for hydrogen and energy production. Then, we describe the most recent advances in the development of homogeneous hydrogen evolution catalysts envisioned to mimic hydrogenases

Halophenol bioremediation catalyzed by an artificial peroxidase

Halophenols (HPs) have been widely used as pesticides, herbicides and wood-preserving agents. Once released into the environment, they exert toxic effects onto living systems such as plants, animals and humans.[1] Among bioremediation strategies targeting HPs, oxidative degradation is efficiently catalyzed by natural heme-enzymes, such as Horseradish Peroxidase (HRP),[2,3] in the presence of hydrogen peroxide as an oxidant. Peroxidases activate the phenol ring, by generating both phenoxy radical and carbocationic species, which further react to give coupling and/or oxidative dehalogenation products, such as chlorinated benzo-p-dioxins and quinones. The ability of these enzymes to cause phenolic coupling may allow the immobilization of toxic phenolic substances, such as HPs, limiting their bioavailability and suppressing their toxic effects. Humic acids (HA) are ubiquitous organic materials in terrestrial and aquatic ecosystems to which HPs can covalenty bind upon activation. In order to improve the chemical stability of natural peroxidases along with their catalytic efficiency, in recent years a variety of artificial biomimetic systems has been developed and evaluated to this purpose. [4] In this area, our ongoing project, focused on the design and synthesis of artificial enzymes led us to explore the activity of an artificial peroxidase, FeIII-Mimochrome VI*a (FeMC6*a), towards HPs.[5] Herein, the oxidative degradation of HPs catalyzed by FeMC6*a and its use in bioremediation strategies are reported. FeMC6*a is able to convert a variety of HPs, including 2,4,6-trichlorophenol (TCP) with 840-fold higher catalytic efficiency than natural HRP. 1. J. Huff, Chemosphere 2012, 89, 521. 2. S. Sumithran, M. Sono, G. M. Raner, J. H. Dawson, J. Inorg. Biochem. 2012, 117, 316. 3. K. Morimoto, K.Tatsumi, K-I Kuroda, Soil Biology & Biochemistry 2000, 32, 1071. 4. M. Chino, L. Leone, G. Zambrano, F. Pirro, D. D’Alonzo, V. Firpo, D. Aref, L. Lista, O. Maglio, F. Nastri, A. Lombardi, Biopolymers, 2018, e23107. 5. G. Caserta, M. Chino, V. Firpo, G. Zambrano, L. Leone, D. D’Alonzo, F. Nastri, O. Maglio, V. Pavone, A. Lombardi, ChemBioChem 2018, cbic.201800200

A biomimetic metalloporphyrin catalyzes indole oxidation with high selectivity

Indole is one of the most common heterocyclic scaffolds available in nature. It occurs in several natural compounds, including alkaloids, plant hormones, flower scents and dyes.1 Despite the structural simplicity of this molecule, indole oxidation commonly results in the formation of a large number of products, including the 2- or 3-oxygenated compounds, di-oxygenated and more complex molecules. For this reason, indole oxidation has become a widespread model reaction to test the efficacy of both biological catalysts2,3 and their synthetic analogues.4,5 Most of the catalysts examined so far gave poor selectivity toward any of the oxidation products.2-5 Here we present the results concerning oxidation of indole and its derivatives catalyzed by Mn-Mimochrome VI*a (Mn-MC6*a). Mn-MC6*a is a synthetic peptide-porphyrin conjugate conceived to act as a miniaturized heme-protein model.6 Mn-MC6*a is able to oxidize indole under unprecedented site-selective conditions, yielding to 3-oxindolenine as single product. Additionally, the reaction selectivity is dramatically altered when 1- or 3-methyl-substituted indoles are used as substrates. The formation and isolation of the reactive 3-oxindolenine is highly important, since it is believed to represent a useful synthon in organic synthesis. Accordingly, the exploitation of its reactivity with nucleophiles, in order to provide one pot transformations, is currently ongoing, with the aim to further increase the synthetic potential of our catalyst. 1. Burton, T.C. in Heterocyclic scaffolds II: Reactions and applications of indoles; Gribble, G.W., Ed.; Springer-Verlag Berlin Heidelberg, 2011. 2. Kuo, H. H. and Mauk, A. G.; Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 13966–13971. 3. Barrios, D. A. et al. J. Am. Chem. Soc. 2014, 136, 7914-7925. 4. Linhares, M. et al. Appl. Catal. A. 2014, 470, 427–433. 5. Poon L. C.-H. et al. J. Am. Chem. Soc. 2011, 133, 1877–1884. 6. Caserta, G. et al. ChemBioChem 2018 (doi: 10.1002/cbic.201800200

Synthesis of temporin L hydroxamate-based peptides and evaluation of their coordination properties with iron (III)

Ferric iron is an essential nutrient for bacterial growth. Pathogenic bacteria synthesize iron-chelating entities known as siderophores to sequestrate ferric iron from host organisms in order to colonize and replicate. The development of antimicrobial peptides (AMPs) conjugated to iron chelators represents a promising strategy for reducing iron availability, inducing bacterial death, and enhancing simultaneously the efficacy of AMPs. Here we designed, synthesized, and characterized three hydroxamate-based peptides Pep-cyc1, Pep-cyc2, and Pep-cyc3, derived from a cyclic temporin L peptide (Pep-cyc) developed previously by some of us. The Fe3+ complex formation of each ligand was characterized by UVvisible spectroscopy, mass spectrometry, IR, and NMR spectroscopies. In addition, the effect of Fe3+ on the stabilization of -helix conformation of hydroxamate-based peptides and the cotton effect were examined by CD spectroscopy. Moreover, the antimicrobial results obtained in vitro on some Gram-negative strains (K. Pneumoniae and E. coli) showed the ability of each peptide to chelate efficaciously Fe3+ obtaining a reduction of MIC values in comparison to their parent peptide Pepcyc. Our results demonstrated that siderophore conjugation could increase the efficacy and selectivity of AMPs used for the treatment of infectious diseases caused by Gram-negative pathogens

Site-selective indole oxidation catalyzed by a Mn-containing artificial metalloenzyme

Metalloenzymes have become attractive tools for application in oxidation catalysis, since a complex protein environment exerts a highly specific control on the reactivity of the metal center.1 Compared to synthetic catalysts, enzymes cover only a limited repertoire of reactions and substrates. The development of hybrid catalysts, obtained by anchoring catalytic metal complexes to native or artificial biomolecular scaffolds, is aimed at merging the advantages of both systems while overcoming the drawbacks.2,3 In this area, our research is devoted to the development of peptide-porphyrin conjugates resembling natural heme-proteins, called “Mimochromes”.3,4 Among them, Mimochrome VIa (MC6a) is the most promising catalyst, thanks to its robust but flexible scaffold. MC6a, in its MnIII complex, (Mn-MC6a) is an efficient catalyst with enzyme-like properties, because fast and chemoselective reactions with a peroxygenase-like mechanism were found in the oxidation of thioethers. Even more remarkably, Mn-MC6a selectively exhibits either peroxygenase- or catalase-like activity depending on the reaction conditions. Here we present the oxidation of indole and its derivatives catalyzed by Mn-MC6a, with the aim of exploiting the catalytic properties of this artificial enzyme in reactions with potential synthetic applications. Indole is one of the most common heterocyclic scaffolds available in nature. It occurs in several natural compounds (such as alkaloids and plant hormones) and is part of many pharmaceuticals.5-8 Despite the structural simplicity of this molecule, indole oxidation leads to a large number of products, including mono- and di-oxygenated compounds. Indole oxidation has been studied with both biological5,6 and synthetic7,8 catalysts. In all the approaches described so far, no or weak selectivity toward any of the oxidation products has been reported.5-8 Conversely, Mn-MC6a is able to oxidize indole under unprecedented site-selective conditions, yielding to 3-oxindolenine as single product. Additionally, the reaction selectivity is dramatically altered when 1- or 3-methyl-substituted indoles are used as substrates. A detailed mechanistic analysis will help to rationalize the outstanding selectivity of the catalyst. References: 1. Sheldon, R. A. and Woodley, J. M. Chem. Rev. 2018, 118, 801–838. 2. Schwizer, F. et al. Chem. Rev. 2018, 118, 142-231. 3. Chino, M. et al. Biopolymers 2018 (doi: 10.1002/bip.23107). 4. Nastri, F. et al. Chem. Soc. Rev., 2016, 45, 5020-5054. 5. Kuo, H. H. and Mauk, A. G.; Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 13966–13971. 6. Barrios, D. A. et al. J. Am. Chem. Soc. 2014, 136, 7914-7925. 7. Linhares, M. et al. Appl. Catal. A. 2014, 470, 427–433. 8. Poon L. C.-H. et al. J. Am. Chem. Soc. 2011, 133, 1877–1884

Sistemi porfirino-peptidici modelli di emoproteine

Dottorato di ricerca in scienze chimiche. 8. ciclo. A.a. 1992-95. Tutori V. Pavone e C. Pedone. Relatore A. De RenziConsiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome; Biblioteca Nazionale Centrale - P.za Cavalleggeri, 1, Florence / CNR - Consiglio Nazionale delle RichercheSIGLEITItal

Structural and functional aspects of metal binding sites in natural and designed metalloproteins

This book chapter discusses the main properties of metal ions in proteins. First, it describes the amino acids that act as ligands and their possible binding modes. The most representative metal ions in biological systems are briefly outlined, mainly regarding their preferred geometry and their functions. Finally, the chapter focuses on two classes of iron-containing metalloproteins (heme proteins and carboxylate-bridged diiron proteins) in order to illustrate how the same metal cofactor can be engaged in a number of different roles. The various first and second ligation sphere interactions, which finely tune the cofactor properties, thus effecting such different functions, will be highlighted. The models developed by us for these two classes of metalloproteins will be also described, summarizing principles and methods for designing artificial metalloproteins

Peptide-tethered monodentate and chelating histidylidene metal complexes: synthesis and application in catalytic hydrosilylation

The Nδ,Nε-dimethylated histidinium salt (His*) was tethered to oligopeptides and metallated to form Ir(III) and Rh(I) NHC complexes. Peptide-based histidylidene complexes containing only alanine, Ala–Ala–His*–[M] and Ala–Ala–Ala–His*–[M] were synthesised ([M] = Rh(cod)Cl, Ir(Cp*)Cl2), as well as oligopeptide complexes featuring a potentially chelating methionine and tyrosine residue, Met–Ala–Ala–His*–Rh(cod)Cl and Tyr–Ala–Ala–His*–Rh(cod)Cl. Chelation of the methionine-containing histidylidene ligand was induced by halide abstraction from the rhodium centre, while tyrosine remained non-coordinating under identical conditions. High catalytic activities in hydrosilylation were achieved with all peptide-based rhodium complexes. The cationic SMet,CHis*-bidentate peptide rhodium catalyst outperformed the monodentate neutral peptide complexes and constitutes one of the most efficient rhodium carbene catalysts for hydrosilylation, providing new opportunities for the use of peptides as N-heterocyclic carbene ligands in catalysis