Exploring the coordination chemistry of ruthenium complexes with lysozymes: structural and in-solution studies.

This study presents a comprehensive structural analysis of the adducts formed upon the reaction of two Ru(III) complexes [HIsq][trans-RuIIICl4(dmso)(Isq)] (1) and [H2Ind][trans-RuIIICl4(dmso)(HInd)] (2) (where HInd-indazole, Isq-isoquinoline, analogs of NAMI-A) and two Ru(II) complexes, cis-[RuCl2(dmso)4] (c) and trans-[RuCl2(dmso)4] (t), with hen-egg white lysozyme (HEWL). Additionally, the crystal structure of an adduct of human lysozyme (HL) with ruthenium complex, [H2Ind][trans-RuCl4(dmso)(HInd)] was solved. X-ray crystallographic data analysis revealed that all studied Ru complexes, regardless of coordination surroundings and metal center charge, coordinate to the same amino acids (His15, Arg14, and Asp101) of HEWL, losing most of their original ligands. In the case of the 2-HL adduct, two distinct metalation sites: (i) Arg107, Arg113 and (ii) Gln127, Gln129, were identified. Crystallographic data were supported by studies of the interaction of 1 and 2 with HEWL in an aqueous solution. Hydrolytic stability studies revealed that both complexes 1 and 2 liberate the N-heterocyclic ligand under crystallization-like conditions (pH 4.5) as well as under physiological pH conditions, and this process is not significantly affected by the presence of HEWL. A comparative examination of nine crystal structures of Ru complexes with lysozyme, obtained through soaking and co-crystallization experiments, together with in-solution studies of the interaction between 1 and 2 with HEWL, indicates that the hydrolytic release of the N-heterocyclic ligand is one of the critical factors in the interaction between Ru complexes and lysozyme. This understanding is crucial in shedding light on the tendency of Ru complexes to target diverse metalation sites during the formation and in the final forms of the adducts with proteins.

Structure, Mutagenesis, and QM:MM Modeling of 3-Ketosteroid Δ1-Dehydrogenase from Sterolibacterium denitrificans─The Role of a New Putative Membrane-Associated Domain and Proton-Relay System in Catalysis.

3-Ketosteroid Δ1-dehydrogenases (KstD) are important microbial flavin enzymes that initiate the metabolism of steroid ring A and find application in the synthesis of steroid drugs. We present a structure of the KstD from Sterolibacterium denitrificans (AcmB), which contains a previously uncharacterized putative membrane-associated domain and extended proton-relay system. The experimental and theoretical studies show that the steroid Δ1-dehydrogenation proceeds according to the Ping-Pong bi-bi kinetics and a two-step base-assisted elimination (E2cB) mechanism. The mechanism is validated by evaluating the experimental and theoretical kinetic isotope effect for deuterium-substituted substrates. The role of the active-site residues is quantitatively assessed by point mutations, experimental activity assays, and QM/MM MD modeling of the reductive half-reaction (RHR). The pre-steady-state kinetics also reveals that the low pH (6.5) optimum of AcmB is dictated by the oxidative half-reaction (OHR), while the RHR exhibits a slight optimum at the pH usual for the KstD family of 8.5. The modeling confirms the origin of the enantioselectivity of C2-H activation and substrate specificity for Δ4-3-ketosteroids. Finally, the cholest-4-en-3-one turns out to be the best substrate of AcmB in terms of ΔG of binding and predicted rate of dehydrogenation.

Polymer Brushes via Surface-Initiated Electrochemically Mediated ATRP: Role of a Sacrificial Initiator in Polymerization of Acrylates on Silicon Substrates.

Silicon wafers as semiconductors are essential components of integrated circuits in electronic devices. For this reason, modification of the silicon surface is an important factor in the manufacturing of new hybrid materials applied in micro- and nanoelectronics. Herein, copolymer brushes of hydrophilic poly(2-hydroxyethyl acrylate) (PHEA) and hydrophobic poly(tert-butyl acrylate) (PtBA) were grafted from silicon wafers via simplified electrochemically mediated atom transfer radical polymerization (seATRP) according to a surface-initiated approach. The syntheses of PHEA-b-PtBA copolymers were carried out with diminished catalytic complex concentration (successively 25 and 6 ppm of Cu). In order to optimize the reaction condition, the effect of the addition of a supporting electrolyte was investigated. A controlled increase in PHEA brush thickness was confirmed by atomic force microscopy (AFM). Various other parameters including contact angles and free surface energy (FSE) for the modified silicon wafer were presented. Furthermore, the effect of the presence of a sacrificial initiator in solution on the thickness of the grafted brushes was reported. Successfully fabricated inorganic-organic hybrid nanomaterials show potential application in biomedicine and microelectronics devices, e.g., biosensors.

The efficient Δ1-dehydrogenation of a wide spectrum of 3-ketosteroids in a broad pH range by 3-ketosteroid dehydrogenase from Sterolibacterium denitrificans.

Cholest-4-en-3-one Δ1-dehydrogenase (AcmB) from Sterolibacterium denitrificans, a key enzyme of the central degradation pathway of cholesterol, is a protein catalyzing Δ1-dehydrogenation of a wide range of 3-ketosteroids. In this study, we demonstrate the application of AcmB in the synthesis of 1-dehydro-3-ketosteroids and investigate the influence of reaction conditions on the catalytic performance of the enzyme. The recombinant AcmB expressed in E. coli BL21(DE3)Magic exhibits a broad pH optimum and pH stability in the range of 6.5 to 9.0. The activity-based pH optimum of AcmB reaction depends on the type of electron acceptor (2,6-dichloroindophenol - DCPIP, phenazine methosulfate - PMS or potassium hexacyanoferrate - K3[Fe(CN)6]) used in the biocatalytic process yielding the best kinetic properties for the reaction with a DCPIP/PMS mixture (kcat/Km = 1.4·105 s-1·M-1 at pH 9.0) followed by DCPIP (kcat/Km = 1.0·105 s-1·M-1 at pH = 6.5) and K3[Fe(CN)6] (kcat/Km = 0.5·102 s-1·M-1 at pH = 8.0). The unique feature of AcmB is its capability to convert both testosterone derivatives (C20-C22) as well as steroids substituted at C17 (C27-C30) such as cholest-4-en-3-one or (25R)-spirost-4-en-3-one (diosgenone). Apparent steady-state kinetic parameters were determined for both groups of AcmB substrates. In a batch reactor synthesis, the solubility of water-insoluble steroids was facilitated by the addition of a solubilizer, 2-hydroxypropyl-ß-cyclodextrin, and organic co-solvent, 2-methoxyethanol. Catalytic properties characterization of AcmB was tested in fed-batch reactor set-ups, using 0.81 µM of isolated enzyme, PMS and aerobic atmosphere resulting in >99% conversion of the C17-C20 3-ketosteroids within 2 h. Finally, the whole cell E. coli system with recombinant enzyme was demonstrated as an efficient biocatalyst in the synthesis of 1-dehydro-3-ketosteroids.

Surface Modifications of Poly(Ether Ether Ketone) via Polymerization Methods-Current Status and Future Prospects.

Surface modification of poly(ether ether ketone) (PEEK) aimed at applying it as a bone implant material aroused the unflagging interest of the research community. In view of the development of implantology and the growing demand for new biomaterials, increasing biocompatibility and improving osseointegration are becoming the primary goals of PEEK surface modifications. The main aim of this review is to summarize the use of polymerization methods and various monomers applied for surface modification of PEEK to increase its bioactivity, which is a critical factor for successful applications of biomedical materials. In addition, the future directions of PEEK surface modifications are suggested, pointing to low-ppm surface-initiated atom transfer radical polymerization (SI-ATRP) as a method with unexplored capacity for flat surface modifications.

Surface-Initiated Atom Transfer Radical Polymerization for the Preparation of Well-Defined Organic-Inorganic Hybrid Nanomaterials.

Surface-initiated atom transfer radical polymerization (SI-ATRP) is a powerful tool that allows for the synthesis of organic-inorganic hybrid nanomaterials with high potential applications in many disciplines. This review presents synthetic achievements and modifications of nanoparticles via SI-ATRP described in literature last decade. The work mainly focuses on the research development of silica, gold and iron polymer-grafted nanoparticles as well as nature-based materials like nanocellulose. Moreover, typical single examples of nanoparticles modification, i.e., ZnO, are presented. The organic-inorganic hybrid systems received according to the reversible deactivation radical polymerization (RDRP) approach with drastically reduced catalyst complex concentration indicate a wide range of applications of materials including biomedicine and microelectronic devices.