Agnès Anne

Optimizing electrode-attached redox-peptide systems for kinetic characterization of protease action on immobilized substrates. Observation of dissimilar behavior of trypsin and thrombin enzymes.

In this work, we experimentally address the issue of optimizing gold electrode attached ferrocene (Fc)-peptide systems for kinetic measurements of protease action. Considering human α-thrombin and bovine trypsin as proteases of interest, we show that the recurring problem of incomplete cleavage of the peptide layer by these enzymes can be solved by using ultraflat template-stripped gold, instead of polished polycrystalline gold, as the Fc-peptide bearing electrode material. We describe how these fragile surfaces can be mounted in a rotating disk configuration so that enzyme mass transfer no longer limits the overall measured cleavage kinetics. Finally, we demonstrate that, once the system has been optimized, in situ real-time cyclic voltammetry monitoring of the protease action can yield high-quality kinetic data, showing no sign of interfering effects. The cleavage progress curves then closely match the Langmuirian variation expected for a kinetically controlled surface process. Global fit of the progress curves yield accurate values of the peptide cleavage rate for both trypsin and thrombin. It is shown that, whereas trypsin action on the surface-attached peptide closely follows Michaelis-Menten kinetics, thrombin displays a specific and unexpected behavior characterized by a nearly enzyme-concentration-independent cleavage rate in the subnanomolar enzyme concentration range. The reason for this behavior has still to be clarified, but its occurrence may limit the sensitivity of thrombin sensors based on Fc-peptide layers.

Electrochemical Atomic Force Microscopy Imaging of Redox-Immunomarked Proteins on Native Potyviruses: From Subparticle to Single-Protein Resolution

We show herein that electrochemical atomic force microscopy (AFM-SECM), operated in molecule touching (Mt) mode and combined with redox immunomarking, enables the in situ mapping of the distribution of proteins on individual virus particles and makes localization of individual viral proteins possible. Acquisition of a topography image allows isolated virus particles to be identified and structurally characterized, while simultaneous acquisition of a current image allows the sought after protein, marked by redox antibodies, to be selectively located. We concomitantly show that Mt/AFM-SECM, due to its single-particle resolution, can also uniquely reveal the way redox functionalization endowed to viral particles is distributed both statistically among the viruses and spatially over individual virus particles. This possibility makes Mt/AFM-SECM a unique tool for viral nanotechnology.

New anthracyclines synthesis: Electrical reduction, rate of glycoside elimination

Abstract The synthesis of new 3′-deamino-3′-hydroxy-daunorubicin analogs is reported and the rate of glycoside elimination under electrochemical reduction in an aprotic medium is compared to known anthracyclines.

Kinetics of enzyme action on surface-attached substrates: a practical guide to progress curve analysis in any kinetic situation.

In the present work, exact kinetic equations describing the action of an enzyme in solution on a substrate attached to a surface have been derived in the framework of the Michaelis-Menten mechanism but without resorting to the often-used steady-state approximation. The here-derived kinetic equations are cast in a workable format, allowing us to introduce a simple and universal procedure for the quantitative analysis of enzyme surface kinetics that is valid for any kinetic situation. The results presented here should allow experimentalists studying the kinetics of enzyme action on immobilized substrates to analyze their data in a perfectly rigorous way.

Novel monolabeled redox biomaterials: Amino-ended poly(ethylene oxide) ferrocenes and their biotin-conjugates

Abstract Treatment of ferrocenoyl chloride with symmetrical poly(oxyalkylene) diamine 1 in excess affords the monofonctionalized derivative 2 in moderate to good yields. This one-step procedure provides a convenient entry to novel monolabeled redox precursors such as the biotin derivative 3 for more elaborate architectures.

Double fragmentation in cation radicals. An example in the NADH analogues series

The cation radical of 9-tert-butyl-N-methylacridan, generated electrochemically or photochemically, offers, in the presence of strong bases, a remarkable example of a double fragmentation. Whereas in acidic or weakly basic media the tert-butyl radical is cleaved with concomitant formation of the methylacridinium cation, the presence of a strong base triggers the cleavage of both the methyl group borne by the nitrogen atom and the tert-butyl group on C-9 leading to acridine, formaldehyde and the tert-butyl anion, even though methylacridinium cation is stable under these conditions. The origin of this unprecedented behavior resides in the prior deprotonation of the methyl group borne by the nitrogen atom which outruns the usual deprotonation at the 9-carbon because this is slowed by the steric hindrance due to the presence of the tert-butyl group.

Electrochemical reduction of the antitumour anthrapyrazole Cl-941: mechanism of formation and isolation of the leuco form

The title compound undergoes two successive one-electron reductions in the aprotic solvent DMF and an apparent one-step two-electron reduction in the presence of proton donors. Both the leuco form, resulting from the addition of two electrons and two protons, and the product of its hydrolysis in acidic media, have been isolated. All of the observed reactions proceed according to thermodynamic and kinetic parameters (redox potentials, pKas and rate constants) which are quantitatively analysed.

Electrochemical Imaging of Dense Molecular Nanoarrays

The aim of the present work is to explore the combination of atomic force electrochemical microscopy, operated in molecule touching mode (Mt/AFM-SECM), and of dense nanodot arrays, for designing an electrochemically addressable molecular nanoarray platform. A high density nanoarray of single grained gold nanodots (∼15 nm-diameter nanoparticles, 100 nm pitch) is decorated by a model molecular system, consisting of ferrocene (Fc) labeled polyethylene glycol (PEG) disulfide chains. We show that the high resolution of Mt/AFM-SECM enables the electrochemical interrogation of several hundreds of individual nanodots in a single image acquisition. As a result, the statistical dispersion of the nanodot molecular occupancy by Fc-PEG chains can be reliably quantified, evidencing that as little as a few tens of copies of redox-labeled macromolecules immobilized on individual nanodots can be detected. The electrochemical reactivity of individual nanodots can also be reliably sampled over a large population of nanodots. We evidence that the heterogeneous rate constant characterizing the electron transfer between the nanodots and the Fc heads displays some quantifiable variability but that the electron transfer remains in any case in the quasi-reversible regime. Overall, we demonstrate that Mt/AFM-SECM enables high throughput reading of dense nanoarrays, with a sensitivity and a read-out speed considerably higher than previously reported for scanning electrochemical microscopy (SECM) imaging of molecular microarrays.

1,4-Diamino-and 1,4-dibutylamino-anthraquinones: reduction and/or deprotonation-initiated elimination of the butyl groups in dipolar aprotic media

The standard redox potentials of the one- and two-electron reductions of the title compounds have been determined. The deprotonated form of the dibutylamino compound underwent a base-initiated elimination of the butyl groups and the basicity of the radical anion resulting from one-electron reduction was sufficient to provoke the same type of cleavage through an initial father–son reaction. A multi-step mechanism is proposed for the elimination on the basis of the identification of intermediates.