Marylène Dias

Trafficking of Siderophore Transporters in Saccharomyces cerevisiae and Intracellular Fate of Ferrioxamine B Conjugates

We have studied the intracellular trafficking of Sit1 [ferrioxamine B (FOB) transporter] and Enb1 (enterobactin transporter) in Saccharomyces cerevisiae using green fluorescent protein (GFP) fusion proteins. Enb1 was constitutively targeted to the plasma membrane. Sit1 was essentially targeted to the vacuolar degradation pathway when synthesized in the absence of substrate. Massive plasma membrane sorting of Sit1 was induced by various siderophore substrates of Sit1, and by coprogen, which is not a substrate of Sit1. Thus, different siderophore transporters use different regulated trafficking processes. We also studied the fate of Sit1‐mediated internalized siderophores. Ferrioxamine B was recovered in isolated vacuolar fractions, where it could be detected spectrophotometrically. Ferrioxamine B coupled to an inhibitor of mitochondrial protoporphyrinogen oxidase (acifluorfen) could not reach its target unless the cells were disrupted, confirming the tight compartmentalization of siderophores within cells. Ferrioxamine B coupled to a fluorescent moiety, FOB‐nitrobenz‐2‐oxa‐1,3‐diazole, used as a Sit1‐dependent iron source, accumulated in the vacuolar lumen even in mutants displaying a steady‐state accumulation of Sit1 at the plasma membrane or in endosomal compartments. Thus, the fates of siderophore transporters and siderophores diverge early in the trafficking process.

Phase segregation on electroactive self-assembled monolayers: a numerical approach for describing lateral interactions between redox centers.

A numerical method is proposed in order to differentiate a random distribution from a phase segregation of redox centers on (mixed) SAMs. This approach is compared to Lavirons interactions model and voltammetric data of nitroxylalkanethiolate SAMs.

Nitroxyl Radical Self-Assembled Monolayers on Gold: Versatile Electroactive Centers in Aqueous and Organic Media

Since the pioneering work by Nuzzo and Allara in 1983, selfassembled monolayers (SAMs) of alkanethiols have gained much attention in the interfacial electrochemistry and other research fields. 3] During the last decade, 5] increasing attention has been dedicated to the design and elaboration of redox-responsive SAMs. Employing redox SAM-modified electrodes for catalysis, recognition and sensing have resulted in some elegant examples that incorporate sophisticated receptors on the electrode surfaces. However, to the best of our knowledge, no redox-responsive SAM was designed to be active and stable in both aqueous (on wide range of pH) and organic media. TEMPO (2,2,6,6-Tetramethylpiperidine-1-oxyl) and its derivatives (under their oxoammonium form) have been extensively studied in the search for organic synthesis as a redox mediator, mostly, for the oxidation of primary alcohols. The electrochemical oxidation of TEMPO is known to be a stable and reversible one-electron process in both aqueous and non-aqueous electrolytes. 13] Despite the wide electrochemical applications of nitroxyl radical, rare works have been devoted to design and elaborate of redox-responsive TEMPO SAMs. In 1997, Fuchigami et al. reported the first preparation of stable self-assembled TEMPO-modified electrodes in acetonitrile : the electrochemical stability was only reached with highly diluted mixed SAMs of nitroxyl radical derivative and hexadecanethiol. In 1999, Kashiwagi et al. reported electrocatalysis attempts of amines with a mixed SAM of chiral nitroxyl radical derivative and hexadecanethiol : as mentioned by authors, results could be applied to the determination of optical purity of chiral amines. After a ten-years pause, Finklea et al. worked on Au-SACHTUNGTRENNUNG(CH2)10C(O)N(H)-TEMPO SAM to the first estimation of the standard rate constant and the reorganization energies of TEMPO/TEMPO in 1 m H2SO4. Herein, we raised the challenge of a design of redox-responsive TEMPO SAMs to be active, stable and providing electrocatalytic activities in both aqueous and non-aqueous solvents. To reach this goal, we synthesized the three nitroxyl radical derivates 1 a, 1 b and 1 c shown in Scheme 1. The synthesis of 1 a, 1 b and 1 c were carried out as outlined in Scheme 1. w-thioACHTUNGTRENNUNGacetyl carboxylic acids with different carbon chains length 2 a–c were synthesized from the corresponding w-bromocarboxylic acids by nucleophilic displacement with potassium thioACHTUNGTRENNUNGacetate in DMF. 4-aminoTEMPO was coupled to w-thioacetyl carboxylic acids 2 a-c via the active ester method, using diACHTUNGTRENNUNGcyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBT) in methylene chloride (CH2Cl2), to give the TEMPO derivatives 3 a–c in good yields (77 to 84 %). The thioacetates 3 a–c were subsequently deprotected to thiols 1 a–c, under basic conditions, using CsOH.H2O in a mixture of THF and MeOH (yields: 50 to 91 %). Cyclic voltammograms (CVs) of 3 a, 3 b or 3 c exhibit a reversible one-electron process in CH2Cl2 and CH3CN, close to 0.56 V and 0.41 V (vs Ag/AgNO3 in 0.1 m Bu4NPF6) respectively. The kinetics for the formation of SAMs, prepared on Au substrate from 1 mm solution of 1 a, 1 b or 1 c in CH2Cl2 or CH3CN, were followed by using quartz crystal microbalance (QCM) measurements. By employing, the Langmuir adsorption isotherm model, it is possible to establish that deposition kiScheme 1. Synthesis of nitroxyl radical derivatives 1 a, 1 b and 1 c.

Evaluation of a new matrix-free laser desorption/ionization method through statistic studies: comparison of the DIAMS (desorption/ionization on self-assembled monolayer surface) method with the MALDI and TGFA-LDI techniques

This work demonstrates that the desorption/ionization on self-assembled monolayer surface (DIAMS) mass spectrometry, a recent matrix-free laser desorption/ionization (LDI) method based on an organic target plate, is as statistically repeatable and reproducible as matrix assisted laser desorption ionization (MALDI) and thin gold film-assisted laser desorption/ionization (TGFA-LDI) mass spectrometries. On lipophilic DIAMS of target plates with a mixture of glycerides, repeatability/reproducibility has been estimated at 15 and 30% and the relative detection limit has been evaluated at 0.3 and 3 pmol, with and without NaI respectively. Salicylic acid and its d(6)-isomer analysis confirm the applicability of the DIAMS method in the detection of compounds of low molecular weight.

Selective detection of alkaloids in MALDI-TOF: the introduction of a novel matrix molecule

The current manuscript presents 3-[5′-(methylthio)-2,2′-bithiophen-5-ylthio]propanenitrile (MT3P), as a novel matrix molecule, which facilitates the selective ionization of alkaloids in matrix-assisted laser desorption/ionization mass spectrometry. Exhibiting strong ionizing properties at low levels of laser energy, MT3P was evaluated on 55 compounds belonging to various chemical families. The observed molecular ion yields induced by MT3P were compared with those obtained by commercially available matrices such as 1,8-dihydroxy-9,10-dihydroanthracen-9-one, α-cyano-4-hydroxycinnamic acid, 2,2′:5′,2″-terthiophene and 2,5-dihydroxybenzoic acid. In conclusion, MT3P displayed excellent ionization properties for 23 out of 25 investigated alkaloids, while showing little to no interaction with compounds from different chemical origin. Further, in comparison to other tested matrices, MT3P generally facilitated better ionization of alkaloids. Eventually, levels of laser energy were adjusted to obtain spectra with significantly reduced matrix noise.

Self-assembled monolayer-assisted mass spectrometry

This article deals with the use of self-assembled monolayers (SAMs) for the formation and characterization of gaseous ions in mass spectrometry (MS). The first part reviews the different results reported in the literature concerning the use of SAMs for surface induced dissociation (SID) of produced ions into a mass spectrometer. In SID/MS, the ion collision at a given kinetic energy allows the accumulation of internal energy for reaching the activation energy of fragmentation reactions. The different chemical structures of SAMs in SID/MS are described and their influence on the amount of the kinetic energy (Ekin) converted into internal energies (Eint) is reported. The second part is dedicated to the implication of SAMs in the laser desorption–ionization (LDI) methods allowing gas-phase ion formation and highlights the specifications required for the SAMs elaboration in the LDI/MS application field. The matrix-free LDI method is more particularly described. The results obtained with the so-called DIAMS technique (desorption–ionization on self-assembled monolayer surface) are reported and the organization and stability of SAMs are pointed out to obtain reliable results in LDI/MS.

A facile route to steady redox-modulated nitroxide spin-labeled surfaces based on diazonium chemistry

A TEMPO derivative was covalently grafted onto carbon and gold surfaces via the diazonium chemistry. The acid-dependent redox properties of the nitroxyl group were exploited to elaborate electro-switchable magnetic surfaces. ESR characterization demonstrated the reversible and permanent magnetic character of the material.

Triazolobithiophene Light Absorbing Self-Assembled Monolayers: Synthesis and Mass Spectrometry Applications

The synthesis of five light absorbing triazolobithiophenic thiols, which were utilized for producing self-assembled monolayers (SAMs) on gold surfaces, is presented. The monolayer formation was monitored by cyclic voltammetry, indicating excellent surface coverage. The new triazolobithiophenic compounds exhibited an absorption maximum around 340 nm, which is close to the emission wavelength of a standard nitrogen laser. Consequently these compounds could be used to aid ionization in laser desorption mass spectrometry (MS).

Electrochemical transduction on self-assembled monolayers: are covalent links essential?

Electrochemical transduction without covalent links between redox and complexant units in a complexing self-assembled monolayer has been established. The results demonstrate that transduction depends on the crown ether/ferrocene ratio and appears to be tunable.

Free and immobilized matrix molecules: impairing ionization by quenching secondary ion formation in laser desorption MS.

Within the last 25 years, matrix-assisted laser desorption ionization (MALDI) has become a powerful analytical tool in mass spectrometry (MS). While the method has been successfully applied to characterize large organic molecules such as proteins, sugars and polymers, its utilization for small molecules (≤ 600 Da) is significantly impaired by the coformation of matrix ions. Reducing or eliminating matrix-related signals has been subject of many studies. Some of which propose the enhancement of so-called matrix suppression effects, while others suggest the replacement of matrix molecules by materials such as microporous silicon. Alternatively, the immobilization of matrix molecules by utilizing them as self-assembled monolayers (SAMs) has been discussed. In continuation of this research, the current manuscript focuses on the elucidation of ion formation processes occurring on the surface of light absorbing SAMs. Ion yields obtained by free and immobilized matrix molecules as well as those generated by matrix-free gold film-assisted laser desorption ionization (GF-LDI) were compared. Experiments showed that the formation of strong analyte signals essentially required the presence of free matrix molecules, while the immobilization of the latter severely impaired ionization. The observed effect inversely correlated with the surface coverage of SAMs determined by cyclic voltammetry (CV). Based on these findings, the MS signal generated on light absorbing SAMs could be used supplementary to CV for determining the surface coverage of light absorbing SAMs.