Determination of iron-porphyrin-like complexes at nanomolar levels in seawater

A new method for the non-specific determination of iron-porphyrin-like complexes in natural waters has been developed. It is based on the chemiluminescent oxidation of the luminol in the presence of dioxygen (O₂) at pH 13. The method has been implemented in a FIA manifold that allowed the direct injection of seawater. The limit of detection is 0.11 nM of equivalent hemin (Fe-protoporphyrin IX). Fe²⁺, Fe³⁺, H₂O₂, siderophore (deferoxamin mesylate), humic acid and phytic acid did not interfere when they were present at the concentrations expected in seawater. Metal free porphyrin and Mg, Cu, Co porphyrin complexes did not induce a significant chemiluminescent signal. Poisoned unfiltered samples could be stored for several weeks before analyses. The new method was successfully applied to the determination of the Fe-porphyrin complexes contained in cultured phytoplankton and in natural samples.     

A further contribution to the study of sagittamide a: synthesis of a pivotal intermediate belonging to a rare L-series

A key saggitamide intermediate corresponding to a rare sugar framework has been obtained. This approach should help to establish the overall configuration of more complex structures of the sagittamide family.     

Solvent effect on the excited-state dynamics of analogues of the photoactive yellow protein chromophore

We previously reported that two analogues of the Photoactive Yellow Protein chromophore, trans-p-hydroxycinnamic acid (pCA(2-)) and its amide derivative (pCM-) in their deprotonated forms, undergo a trans-cis photoisomerization whereas the thioester derivative, trans-p-hydroxythiophenyl cinnamate (pCT-), does not. pCT- is also the only one to exhibit a short-lived intermediate on its excited-state deactivation pathway. We here further stress the existence of two different relaxation mechanisms for these molecules and examine the reaction coordinates involved. We looked at the effect of the solvent properties (viscosity, polarity, solvation dynamics) on their excited-state relaxation dynamics, probed by ultrafast transient absorption spectroscopy. Sensitivity to the solvent properties is found to be larger for pCT- than for pCA(2-) and pCM-. This difference is considered to reveal that either the relaxation pathway or the reaction coordinate is different for these two classes of analogues. It is also found to be correlated to the electron donor-acceptor character of the molecule. We attribute the excited-state deactivation of analogues bearing a weaker acceptor group, pCA(2-) and pCM-, to a stilbene-like photoisomerization mechanism with the concerted rotation of the ethylenic bond and one adjacent single bond. For pCT-, which contains a stronger acceptor group, we consider a photoisomerization mechanism mainly involving the single torsion of the ethylenic bond. The excited-state deactivation of pCT- would lead to the formation of a ground-state intermediate at the "perp" geometry, which would return to the initial trans conformation without net isomerization.     

Potentiometric determination of the 'formal' hydrolysis ratio of aluminium species in aqueous solutions

The ‘formal’ hydrolysis ratio (h = C(OH⁻)_added/C(Al)_total) of hydrolysed aluminium-ions is an important parameter required for the exhaustive and quantitative speciation-fractionation of aluminium in aqueous solutions. This paper describes a potentiometric method for determination of the formal hydrolysis ratio based on an automated alkaline titration procedure. The method uses the point of precipitation of aluminium hydroxide as a reference (h = 3.0) in order to calculate the initial formal hydrolysis ratio of hydrolysed aluminium-ion solutions. Several solutions of pure hydrolytic species including aluminium monomers (AlCl₃), Al₁₃ polynuclear cluster ([Al₁₃O₄(OH)₂₄(H₂O)₁₂]⁷⁺), Al₃₀ polynuclear cluster ([Al₃₀O₈(OH)₅₆(H₂O)₂₆]¹⁸⁺) and a suspension of nanoparticulate aluminium hydroxide have been used as ‘reference standards’ to validate the proposed potentiometric method. Other important variables in the potentiometric determination of the hydrolysis ratio have also been optimised including the concentration of aluminium and the type and strength of alkali (Trizma-base, NH₃, NaHCO₃, Na₂CO₃ and KOH). The results of the potentiometric analysis have been cross-verified by quantitative ²⁷Al solution nuclear magnetic resonance (²⁷Al NMR) measurements. The ‘formal’ hydrolysis ratio of a commercial basic aluminium chloride has been measured as an example of a practical application of the developed technique.     

Photoswitching Kinetics and Phase‐Sensitive Detection Add Discriminative Dimensions for Selective Fluorescence Imaging

Non‐invasive separation‐free protocols are attractive for analyzing complex mixtures. To increase selectivity, an analysis under kinetic control, through exploitation of the photochemical reactivity of labeling contrast agents, is described. The simple protocol is applied in optical fluorescence microscopy, where autofluorescence, light scattering, as well as spectral crowding presents limitations. Introduced herein is OPIOM (out‐of‐phase imaging after optical modulation), which exploits the rich kinetic signature of a photoswitching fluorescent probe to increase selectively and quantitatively its contrast. Filtering the specific contribution of the probe only requires phase‐sensitive detection upon matching the photoswitching dynamics of the probe and the intensity and frequency of a modulated monochromatic light excitation. After in vitro validation, we applied OPIOM for selective imaging in mammalian cells and zebrafish, thus opening attractive perspectives for multiplexed observations in biological samples.     

Polarized transient absorption to resolve electron transfer between tryptophans in DNA photolyase

Transient absorption spectroscopy is a powerful tool for studying biological electron-transfer chains, provided that their members give rise to distinct changes of their absorption spectra. There are, however, chains that contain identical molecules, so that electron transfer between them does not change net absorption. An example is the chain flavin adenine dinucleotide (FAD)-W382-W359-W306 in DNA photolyase from E. coli. Upon absorption of a photon, the excited state of FADH* (neutral FAD radical) abstracts an electron from the tryptophan residue W382 in approximately 30 ps (monitored by transient absorption). The cation radical W382*+ is presumably reduced by W359 and W359*+ by W306. The latter two reactions could not be monitored directly so far because the absorption changes of the partners compensate in each step. To overcome this difficulty, we used linearly polarized flashes for excitation of FADH*, thus inducing a preferential axis in the a priori unoriented sample (photoselection). Because W359 and W306 are very differently oriented within the protein, detection with polarized light should allow us to distinguish them. To demonstrate this, W306 was mutated to redox-inert phenylalanine. We show that the resulting anisotropy spectrum of the initial absorption changes (measured at 10 ns time resolution) is in line with W359 being oxidized. The corresponding spectrum in wildtype photolyase is clearly different and identifies W306 as the oxidized species. These findings set an upper limit of 10 ns for electron transfer from W306 to W359*+ in wildtype DNA photolyase, consistent with previous, more indirect evidence [Aubert, C.; Vos, M. H.; Mathis, P.; Eker, A. P. M.; Brettel, K. Nature 2000, 405, 586-590].