Electrochemically produced local pH changes stimulating (bio)molecule release from pH-switchable electrode-immobilized avidin-biotin systems.

Immobilized avidin-biotin complexes were used to release biotinylated (bio)molecules upon producing local pH changes near an electrode surface by electrochemical reactions. The nitro-avidin complex with biotin was dissociated by increasing local pH with electrochemical O2 reduction. The avidin complex with iminobiotin was split by decreasing local pH with electrochemical oxidation of ascorbate. Both studied systems were releasing molecule cargo species in response to small electrical potentials (-0.4 V or 0.2 V for the O2 reduction or ascorbate oxidation, respectively) applied on the modified electrodes.

Boolean Logic Networks Mimicked with Chimeric Enzymes Activated/Inhibited by Several Input Signals.

The front cover artwork is provided by groups of Prof. Evgeny Katz and Prof. Artem Melman (Clarkson University, NY, USA) as well as Prof. Kirill Alexandrov (Queensland University of Technology, Brisbane, Australia). The image shows activation/inhibition of a chimeric enzyme with biomolecular signals and a corresponding logic network - the artistic vision. Read the full text of the Communication at 10.1002/cphc.201901050.

Boolean Logic Networks Mimicked with Chimeric Enzymes Activated/Inhibited by Several Input Signals.

Reactions catalyzed by artificial allosteric enzymes, chimeric proteins with fused biorecognition and catalytic units, were used to mimic multi-input Boolean logic systems. The catalytic parts of the systems were represented by pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH). Two biorecognition units, calmodulin or artificial peptide-clamp, were integrated into PQQ-GDH and locked it in the OFF or ON state respectively. The ligand-peptide binding cooperatively with Ca2+ cations to a calmodulin bioreceptor resulted in the enzyme activation, while another ligand-peptide bound to a clamp-receptor inhibited the enzyme. The enzyme activation and inhibition originated from peptide-induced allosteric transitions in the receptor units that propagated to the catalytic domain. While most of enzymes used to mimic Boolean logic gates operate with two inputs (substrate and co-substrate), the used chimeric enzymes were controlled by four inputs (glucose - substrate, dichlorophenolindophenol - electron acceptor/co-substrate, Ca2+ cations and a peptide - activating/inhibiting signals). The biocatalytic reactions controlled by four input signals were considered as logic networks composed of several concatenated logic gates. The developed approach allows potentially programming complex logic networks operating with various biomolecular inputs representing potential utility for different biomedical applications.

Selective Derivatization of Hexahistidine-Tagged Recombinant Proteins.

Covalent modification of proteins is extensively used in research and industry for biosensing, medical diagnostics, targeted drug delivery, and many other practical applications. The conventional method for production of protein conjugates has changed little in the last 20 years mostly relying on reactions of side chains of cysteine and lysine residues. Due to the presence of large numbers of similar reactive amino acid residues in proteins, common synthetic methods generally produce complex mixtures of products, which are difficult to separate. An emerging alternative technology for covalent modification of proteins involves formation of a covalent bond with a hexahistidine affinity tag present in a majority of recombinant proteins without interfering with other amino acid residues. The approach is based on formation of a ternary complex of the hexahistidine sequence with a bivalent metal cation chelated by ligand bearing an electrophilic Baylis-Hillman ester group capable of subsequent formation of a covalent bond with one of the histidine residues of the tag. The reaction proceeds under mild reaction conditions in neutral aqueous solutions under high dilutions (10-5 to 10-4 M) providing a stable covalent bond between the label and an imidazole residue in a hexahistidine tag at either C- or N-terminus. Because hexahistidine affinity tag methodology is a de-facto standard for preparation of recombinant proteins our approach can be easily implemented for selective derivatization of these proteins with fluorescent groups, alkynyl groups for "click" reactions, or biotinylation.

Investigation of Unusual N-(Triphenyl-λ5-phosphanylidene) Amide Fragmentation Observed upon MS/MS Collision-Induced Dissociation.

A mechanism of unusual tandem (MS/MS) fragmentation of protonated species of N-(triphenyl-λ5-phosphanylidene) derivatives, [M + H]+ to generate triphenylphosphine oxide (TPPO) within the mass spectrometer has been investigated and reported. Collision-induced dissociation of these molecules resulted in the generation of TPPO as a signature fragment. This fragment suggested the presence of a P-O bond in the structure which was contrary to the structure of the compound identified by nuclear magnetic resonance spectrometry (NMR) and single-crystal X-ray diffractometry (SXRD) techniques with a P═N bond rather than a P-O bond. In order to confirm the generation of the TPPO fragment within the mass spectrometer, 14 different N-(triphenyl-λ5-phosphanylidene) derivatives containing amide, 18O-labeled amide, thiamide, and nonacyl phosphazene derivatives were synthesized and their MS/MS behavior was studied by liquid chromatography-high-resolution mass spectrometry. Fragmentation of these amide derivatives generated TPPO/TPPS or their 18O-labeled analogues as the major fragment in almost all cases under similar MS conditions. Based on the outcome of these experiments, a plausible mechanism for such fragmentation, involving the intramolecular shifting of oxygen from carbon to phosphorus, has been proposed. DFT calculations for the protonated species at B3LYP-D3/6-31+G(d,p) further supported the proposed mechanism involving a four-membered ring, P-O-C-N, as the transition state. Details of this work are presented here.

Electrochemical control of the catalytic activity of immobilized enzymes.

Regulation of the catalytic activity of enzymes immobilized on carbon nanotube electrodes was achieved by changing their local pH environment using electrochemical reactions. Reduction of oxygen increased the interfacial pH while oxidation of ascorbate decreased it, thus allowing changing rates of enzymatic reactions of electrode-immobilized amyloglucosidase and trypsin enzymes over a wide activity range.

Electrochemically stimulated molecule release associated with interfacial pH changes.

A new linker with a hydrolyzable aryl ester bond was used for electrode modification. Basic pH locally produced at the electrode surface upon electrochemical reduction of O2 resulted in the hydrolytic cleavage of the aryl ester bond and release of the immobilized fluorescent dye used as a model compound.