Sanela Martić

Reversible Intramolecular C−C Bond Formation/Breaking and Color Switching Mediated by a N,C-Chelate in (2-ph-py)BMes2 and (5-BMes2-2-ph-py)BMes2

A diboron compound with both 3-coordinate boron and 4-coordinate boron centers, (5-BMes2-2-ph-py)BMes2 (1) and its monoboron analogue, (2-ph-py)BMes2 (2) have been synthesized. Both compounds are luminescent but have a high sensitivity toward light. UV and ambient light cause both compounds to isomerize to 1a and 2a, respectively, via the formation of a C-C bond between a mesityl and the phenyl group, accompanied by a drastic color change from yellow or colorless to dark olive green or dark blue. The structures of 1a and 2a were established by 2D NMR experiments and geometry optimization by DFT calculations. Both 1a and 2a can thermally reverse back to 1 and 2 via the breaking of a C-C bond, with the activation barrier being 107 and 110 kJ/mol, respectively. The N,C-chelate ligands in 1 and 2 were found to play a key role in promoting this unusual and reversible photo-thermal isomerization process on a tetrahedral boron center. Reactions with oxygen molecules convert 1a and 2a to 5-BMes2-2-[(2-Mes)-ph]-pyridine (1b) and 2-(2-Mes)-ph-pyridine (2b), respectively.

Ferrocene-peptido conjugates: From synthesis to sensory applications

The field of chemical and biological sensing is increasingly dependent on the availability of new functional materials that enhance the ability of the system to respond to chemical interactions. Organometallic bioconjugates derived from amino acids, peptides, proteins, peptide nucleic acids, and dendrimers have had a profound effect in this area and have endowed modern sensory systems with a superior performance. Owing to their fairly high stability, solubility in various solvents, and excellent redox properties, ferrocene and ferrocenyl conjugates have emerged as one of the most important classes of materials that enable direct observation of molecular interactions and as electron mediators. The low potential, reversible redox behavior of the ferrocene/ferrocenium couple is a unique property that finds widespread application in the design of sensory platforms. Currently, there is significant drive to exploit new organometallic systems, in which the presence of ferrocene acting as a redox center is critical and allows the design of highly sensitive electrochemical sensors for the sensing and recognition of a vast array of analytes.

Structure-activity relationships of targeted RuII(η6-p-cymene) anticancer complexes with flavonol-derived ligands.

RuII(arene) complexes have been shown to be promising anticancer agents, capable of overcoming major drawbacks of currently used chemotherapeutics. We have synthesized RuII(η6-arene) compounds carrying bioactive flavonol ligands with the aim to obtain multitargeted anticancer agents. To validate this concept, studies on the mode of action of the complexes were conducted which indicated that they form covalent bonds to DNA, have only minor impact on the cell cycle, but inhibit CDK2 and topoisomerase IIα in vitro. The cytotoxic activity was determined in human cancer cell lines, resulting in very low IC50 values as compared to other RuII(arene) complexes and showing a structure-activity relationship dependent on the substitution pattern of the flavonol ligand. Furthermore, the inhibition of cell growth correlates well with the topoisomerase inhibitory activity. Compared to the flavonol ligands, the RuII(η6-p-cymene) complexes are more potent antiproliferative agents, which can be explained by potential multitargeted properties.

Versatile Strategy for Biochemical, Electrochemical and Immunoarray Detection of Protein Phosphorylations

Protein kinases catalyze the phosphorylation of cellular proteins involved in the regulation of many cellular processes and have emerged as promising targets for the treatment of several diseases. Conventional assays to monitor protein kinase activity are limited because they typically rely on transfer of radioactive phosphate or phospho-specific antibodies that recognize specific substrates or sequence motifs. To overcome the limitations of conventional assays, we have developed a versatile approach based on transfer of ferrocene-phosphate that can be readily monitored using electrochemical detection or detection with antiferrocene antibodies in an immunoarray format. This assay is readily adapted to multiplex arrays and can be employed for monitoring kinase activity in complex mixtures and for kinase inhibitor profiling.

Electrochemical analysis of HIV-1 reverse transcriptase serum level: Exploiting protein binding to a functionalized nanostructured surface

This manuscript describes an electrochemical approach to the detection of the reverse transcriptase of the human immunodeficiency virus type-1 (HIV-1 RT) in serum exploiting an organometallic peptide conjugate that is chemically linked to a nanostructured gold surface. The assay format is based on the formation of a thin film of a ferrocene-labeled lipoic acid (Fc-LA) onto a gold nanoparticles-functionalized screen-printed carbon electrode (GNPs-SPCE). Time-of-Flight secondary ion mass spectrometry (TOF-SIMS) and X-ray photoelectron spectroscopy were employed to confirm the binding of the Fc-LA to the electrode surface via formation of a gold-thiol bond. The RT biosensor was developed by covalent attachment of the peptide VEAIIRILQQLLFIH to the carboxylic acid group of Fc-LA. Square wave voltammetry offered a two-dimensional measurement of RT based on the anodic shift and reduction of current density of the Fc redox signal upon binding of RT to its specific peptide. This allowed a linear quantification of the target RT in the range of 1-500 pg mL(-1) equivalent to 0.9-427 fM, with a detection limit of 0.8 pg mL(-1) (0.7 fM) with a short response time.

Electrochemical Investigations of Tau Protein Phosphorylations and Interactions with Pin1

Phosphorylation of Tau by the protein kinase GSK‐3β was monitored by electrochemical impedance spectroscopy of immobilized Tau on gold surfaces. As a result of Tau phosphorylation, the film resistance decreases significantly due to conformational changes and reorganization of the immobilized phosphorylated Tau (pTau) protein, which in turn enables the interactions of pTau with the peptidyl‐prolyl cis/trans isomerase, Pin1. Interactions are specific to phospho‐Ser (pSer) and phospho‐Thr (pThr) residues of pTau. Impedance changes occurred as a function of pTauPin1 interactions and are related to the amount of Pin1 bound, which resulted in an increase of the charge‐transfer resistance, RCT. Our results clearly indicate that the isomerase Pin1 interacts favorably with pSer/pThr‐Pro residues in Tau, but does not bind non‐phosphorylated Tau or phospho‐Tyr residues in Tau films. Our study demonstrates the utility of electrochemical impedance studies to probe protein modifications and biomolecular interactions.

Chemical biology toolkit for exploring protein kinase catalyzed phosphorylation reactions

Current interests in biochemical transformations based on protein kinase-catalyzed phosphorylations drive the identification and characterization of biological targets and potential inhibitors of protein kinase activity. A simple transfer of a phosphate group from adenosine triphosphate (ATP) to the Ser/Thr/Tyr residues of target proteins drives cellular processes, including cell expression, growth, and death. Currently, three major experimental approaches towards kinome analysis are available (a) genetic engineering of protein kinases, (b) modifications of target substrates, and (c) derivatization of ATP co-substrate. Each approach offers advantages but also has disadvantages, which are discussed in this perspective, alongside with a rationale for designing and developing biological tools for kinome study.

Electrochemical screening of the indole/quinolone derivatives as potential protein kinase CK2 inhibitors

An electrochemical method based on the bioorganometallic Fc-ATP cosubstrate for kinase-catalyzed phosphorylation reactions was used for monitoring casein kinase 2 (CK2) phosphorylations in the absence and presence of five indole/quinolone-based potential inhibitors. Fc-phosphorylation of immobilized peptide RRRDDDSDDD on Au surfaces resulted in a current density at approximately 460 ± 10 mV. An electrochemical redox signal was significantly decreased in the presence of inhibitors. In addition, the electrochemical signal was concentration dependent with respect to the potential inhibitors 1 to 5, which proved to be viable CK2 drug targets with estimated IC₅₀ values in the nanomolar range.

Electrochemical "signal-on" reporter for amyloid aggregates.

The synthesis and characterization of four new Ferrocene (Fc) bioconjugates, bearing a podant (Lys)-Leu-Val-Phe-Phe motif, namely the hydrophobic sequence of amyloid-β-peptides (Aβ), is reported. The Fc-peptide conjugates are characterized by a reversible redox activity and the ability to undergo hydrophobic and hydrogen bonding interactions. Biomolecular interactions between Fc-bioconjugates with Aβ(12-28) fragments were studied by circular dichroism (CD), transmission electron microscopy (TEM), and electrochemistry. All four Fc-peptides interacted favourable with Aβ(12-28) and prevented fibril formation, the extent of which depended on the length of the peptide and the nature of the C-terminal group. The aggregates obtained for the Aβ(12-28)/Fc-peptide mixtures range from short fibrils to spherical aggregates. We demonstrated that in solution the peptide sequence and peptide charge affect the biomolecular interactions. Fc-peptide interactions with immobilized Aβ(12-28)-Cys films on Au surfaces were detected by measuring the current response of the Fc redox process. The formal redox potential, E(0), at ~440 (10) mV and i(pc)/i(pa) at 0.9 were observed characteristic for the monosubstituted Fc-derivative undergoing a one-electron redox process. On the surface, methyl ester-protected Fc-peptides (1 and 3) interacted only weakly with Aβ(12-28)-Cys films, giving rise to minimal redox activity. In contrast, charged Fc-peptides (2 and 4) gave a significant electrochemical readout following the interaction with Aβ(12-28)-Cys films. Interestingly, the Fc-peptide charge dictates the surface-assisted interactions, while hydrophobic and ionic effects contribute to the overall solution behaviour of the Fc-bioconjugates with Aβ(12-28).

On chip electrochemical detection of sarcoma protein kinase and HIV-1 reverse transcriptase.

In this study, we report a new multiplexed microchip platform exploiting a peptide-modified gold surface and a labeled electrochemical approach. The significance of the presented methodology lies in its ability to test related analytes, such as protein kinases and human immunodeficiency virus (HIV) proteins, that operate under separate mechanisms using a single device without interference. The technology is based on an electrochemical dual sensing mode that can be tuned towards monitoring separately two biochemical events, a biochemical reaction and a direct analyte-receptor binding. The first recognition process is illustrated by a sarcoma-related (Src) protein kinase which catalyzes phosphorylation transfer of a ferrocenoyl-phosphoryl group, from the ferrocene-labeled adenosine triphosphate (Fc-ATP) co-substrate, to the surface-bound target peptide and induces a current response. On the other hand, HIV-1 reverse transcriptase (RT) protein binding to the surface-immobilized ferrocene-labeled target peptide is characterized by a modulation in the current intensity and peak potential. This proof-of-principle study is based on two different biosensing components and serves as a new platform for monitoring multiple analytes of interest. This allowed detection limits of 0.1 μg mL(-1) and 50 pg mL(-1) for Src kinase and HIV-1 RT, respectively. The miniaturization of the electrochemical protein assay will have an impact in disease detection and treatment.