Calcium rubies: a family of red-emitting functionalizable indicators suitable for two-photon ca(2+) imaging

We designed Calcium Rubies, a family of functionalizable BAPTA-based red-fluorescent calcium (Ca(2+)) indicators as new tools for biological Ca(2+) imaging. The specificity of this Ca(2+)-indicator family is its side arm, attached on the ethylene glycol bridge that allows coupling the indicator to various groups while leaving open the possibility of aromatic substitutions on the BAPTA core for tuning the Ca(2+)-binding affinity. Using this possibility we now synthesize and characterize three different CaRubies with affinities between 3 and 22 μM. Their long excitation and emission wavelengths (peaks at 586/604 nm) allow their use in otherwise challenging multicolor experiments, e.g., when combining Ca(2+) uncaging or optogenetic stimulation with Ca(2+) imaging in cells expressing fluorescent proteins. We illustrate this capacity by the detection of Ca(2+) transients evoked by blue light in cultured astrocytes expressing CatCh, a light-sensitive Ca(2+)-translocating channelrhodopsin linked to yellow fluorescent protein. Using time-correlated single-photon counting, we measured fluorescence lifetimes for all CaRubies and demonstrate a 10-fold increase in the average lifetime upon Ca(2+) chelation. Since only the fluorescence quantum yield but not the absorbance of the CaRubies is Ca(2+)-dependent, calibrated two-photon fluorescence excitation measurements of absolute Ca(2+) concentrations are feasible.     

Phytotoxicity assessment of diclofenac and its phototransformation products

The occurrence of pharmaceuticals in the environment is an emerging issue. Several studies observed that the non-steroidal anti-inflammatory drug diclofenac is ubiquitously present in most of the surveyed surface waters, worldwide. Phototransformation of diclofenac was reported from laboratory assays as well as in natural water systems, raising the question of possible adverse effects of the phototransformation products of diclofenac to aquatic organisms. In this study the phytotoxicity of diclofenac exposed to natural sunlight was evaluated using synchronized cultures of the unicellular chlorophyte Scenedesmus vacuolatus. Diclofenac dissolved in ultra-pure water at 50 mg L⁻¹ was exposed to natural midsummer sunlight for a maximum of 145 h. Twice a day subsamples were taken for chromatography and parallel phytotoxicity assessment. Inhibition of algal reproduction of the initial diclofenac solution was in the mg L⁻¹ range indicating no specific toxicity of diclofenac towards S. vacuolatus. Fast degradation of diclofenac was observed with half lives between 3.3 and 6.4 h during the first and the third day of exposure, respectively. Phytotoxicity increased after 3.5 h of exposure of diclofenac to sunlight and showed a maximum of sixfold enhanced toxicity after 53 h of exposure to sunlight. Several phototransformation products were found during the experiment. The time courses of the relative concentration of three transformation products significantly correlated with enhanced phytotoxicity during the experiment. This indicates a high toxicity potential of phototransformation products of diclofenac at concentration levels that may come close to environmental concentrations of residual diclofenac after degradation. We conclude that toxicity assessment of phototransformation products should be included in the risk assessment of pharmaceuticals in the environment.     

Transesterification and amide cis-trans isomerization in Zn and Cd complexes of the chelating amino acid ligand Boc-Asp(Dpa)-OBzl

The amino acid derivative Boc-Asp-OBzl (Boc=N-butyloxycarbonyl; Asp=aspartic acid; Bzl=benzyl) was functionalized by coupling its carboxylate side chain to dipicolylamine. This yielded the tridentate nitrogen donor ligand Boc-Asp(Dpa)-OBzl (-OBzl). The compound -OBzl contains three different carbonyl groups: a tertiary amide linkage between Asp and Dpa, a C-terminal benzyl ester function, and an N-terminal urethane protecting group. NMR spectra were used to compare the reactivity of these moieties. The Boc protecting group gives rise to two isomers, (E, 9%) and (Z, 91%). Coordination of Cd(NO3)2 and Zn(NO3)2 yielded the complexes and. These compounds have significantly reduced barriers to rotation about the tertiary amide C-N bond compared with the free ligand (-OBzl:18.5 kcal mol-1 in CDBr3;: 12.9 kcal mol-1 in (CD3)2CO;: 13.8 kcal mol-1 in (CD3)2CO). Both complexes readily undergo transesterification in methanol or CD3OD. Experimental pseudo-first order rate constants were determined in CD3OD and (CD3)2CO:CD3OD (3:1;). It was found that the zinc complex (k=(2.28+/-0.02)x10(-4) s-1) is significantly more reactive than the cadmium complex (k=(1.41+/-0.03)x10(-6) s-1). In order to study their tertiary amide cis-trans isomerization, the cadmium complex [(-OCH3)Cd(NO3)2] was synthesized, and the zinc complex [(-OCD3)Zn(NO3)2] was generated in situ in (CD3)2CO:CD3OD (3:1). The barriers to rotation were determined (:14.1 kcal mol-1 in CD3OD;: 13.4 kcal mol-1 in (CD3)2CO:CD3OD (3:1)). Our results show that the stronger Lewis-acid zinc(II) is significantly more active than cadmium(II) in the acceleration of the transesterification. This is in marked contrast to the tertiary amide bond rotation which is comparably fast with both metal ions.     

Photodegradation of poly(3-hydroxybutyrate)

The effect of ultraviolet radiation on the properties of poly(3-hydroxybutyrate) (PHB) was studied. The PHB investigated is produced from microbial fermentation using saccharose from sugarcane as the carbon source to the bacteria. The material was exposed to artificial UV-A radiation for 3, 6, 9 and 12 weeks. The photodegradation effect was followed by changes of molecular weight, of chemical and crystalline structures, of thermal, morphological, optical and mechanical properties, as well as of biodegradability. The experimental results showed that PHB undergoes both chain scission and crosslinking reactions, but the continuous decrease in its mechanical properties and the low amount of gel content upon UV exposure indicated that the scission reactions were predominant. Molar mass, melting temperature and crystallinity measurements for two layers of PHB samples with different depth suggested that the material has a strong degradation profile, which was attributed to its dark colour that restricted the transmission of light. Previous photodegradation initially delayed PHB biodegradability, due to the superficial increase in crystallinity seen with UV exposure. The possible reactions taking place during PHB photodegradation were presented and discussed in terms of the infrared and nuclear magnetic resonance spectra. A reference peak (internal standard) in the infrared spectra was proposed for PHB photodegradation.     

Miniaturised enzymatic conductometric biosensor with Nafion membrane for the direct determination of formaldehyde in water samples

A new conductometric enzyme-based biosensor was developed for the determination of formaldehyde (FA) in aqueous solutions. The biosensor was prepared by cross-linking formaldehyde dehydrogenase from Pseudomonas putida with bovine serum albumin in saturated glutaraldehyde vapours (GA) at the surface of interdigitated gold microelectrodes. Nicotinamide adenine dinucleotide cofactor (NAD⁺) was added in solution at each measurement to maintain enzyme activity. Addition of a Nafion layer over the enzyme modified electrode resulted in a significant increase of biosensor signal due to enhanced accumulation of protons generated by enzymatic reaction at the electrode surface. Different parameters affecting enzyme activity or playing a role in ionic transfer through the Nafion membrane were optimised. In optimal conditions (0.045 mg enzyme, 30 min exposure to GA, 0.3 μL of a 1 % (v/v) Nafion solution deposit, measurement in 5 mM phosphate buffer pH 7 containing 20 μM NAD⁺), the biosensor signal was linear up to 10 mM FA, and the detection limit was 18 μM. Relative standard deviations calculated from five consecutive replicates of FA solutions were lower than 5 % in the 1–10 mM range. The biosensor was successfully applied to the determination of FA in spiked water samples (tap water and Rhone river water), with recoveries in the 95–110 % range.

New Homoleptic Rare‐Earth Metal Complexes Comprising the Unsymmetrically Substituted Amidinate Ligand [MeC(NEt)(NtBu)]–

New homoleptic complexes of selected rare‐earth elements containing the unsymmetrically substituted amidinate ligand [MeC(NEt)(NtBu)]^– [= (L)^–] were synthesized and fully characterized. Treatment of in situ‐prepared Li(L) ( 1 ) with anhydrous lanthanide(III) chlorides, LnCl₃ (Ln = Sc, La, Ce, Ho), afforded three different types of amidinate complexes depending on the ionic radius of the central metal atom. The large La³⁺ formed the octa‐coordinate DME solvate La(L)₃(DME) ( 2 ). Using Ce³⁺, the octa‐coordinate “ate” complex Li(THF)[Ce(L)₄] ( 3 ) was formed. Depending on the crystallization conditions, compound 3 could be crystallized in two modifications differing in the coordination environment around Li. In the case of the smaller Sc³⁺ and Ho³⁺ ions, six‐coordinate homoleptic Sc(L)₃ ( 4 ) and Ho(L)₃ ( 5 ) were isolated. The title compounds were fully characterized by spectroscopic and analytical methods as well as single‐crystal X‐ray diffraction. With Ln = La and Ce, several by‐products incorporating lithium, chlorine and/or oxygen were also isolated and structurally characterized.     

Trunculins X and Y from an Okinawan sponge Sigmosceptrella sp.

Two new norsesterterpenoid cyclic peroxides, trunculins X and Y, were isolated from an Okinawan sponge Sigmosceptrella sp. Their structures were determined by spectroscopic analyses on intact molecules and derivatives and also by crystallographic study. The compounds showed cytotoxicity in a range of IC₅₀ 0.32–20 μM against three cell lines.     

Spectroscopic and structural investigations reveal the signaling mechanism of a luminescent molybdate sensor

A heteroditopic ligand H(2)-L consisting of a dihydroxybenzene (catechol)-unit linked via an amide bond to a pyridyl-unit and its methyl-protected precursor Me(2)-L were synthesized, characterized, and their photophysical properties investigated. The three accessible protonation states of the ligand, H(3)-L(+), H(2)-L, and H-L(-), showed distinct (1)H NMR, absorption and emission spectroscopic characteristics that allow pH-sensing. The spectroscopic signatures obtained act as a guide to understand the signaling mechanism of the luminescent pH and molybdate sensor [Re(bpy)(CO)(3)(H(2)-L)](+). It was found that upon deprotonation of the 2-hydroxy group of H(2)-L, a ligand-based absorption band emerges that overlaps with the Re(dπ)→bpy metal-to-ligand charge transfer (MLCT) band of the sensor, reducing the quantum yield for emission on excitation in the 370 nm region. In addition, deprotonation of the catechol-unit leads to quenching of the emission from the Re(dπ)→bpy (3)MLCT state, consistent with photoinduced electron transfer from the electron-rich, deprotonated catecholate to the Re-based luminophore. Finally, reaction of 2 equiv of [Re(bpy)(CO)(3)(H(2)-L)](+) with molybdate was shown to give the zwitterionic Mo(VI) complex [MoO(2){Re(CO)(3)(bpy)(L)}(2)], as confirmed by electrospray ionization (ESI) mass spectrometry and X-ray crystallography. The crystal structure determination revealed that two fully deprotonated sensor molecules are bound via their oxygen-donors to a cis-dioxo-MoO(2) center.     

Thermal and odd–even behaviour in a homologous series of lithium n-alkanoates

Thermotropic phase transition temperatures, enthalpies and entropies of phase changes and odd–even alternation, in a homologous series of anhydrous lithium n-alkanoates, LiC_nH_2n?1O₂ (LiC_8–19 inclusive), have been investigated by differential scanning calorimetry (d.s.c.), hot stage polarizing microscopy and solid state ¹³C NMR spectroscopy. The number of phases observed, between the room temperature microcrystalline solid and isotropic melt, shows a clear dependence on chain length. For LiC_8–13, only one intermediate lamellar II crystalline phase is observed. For LiC_14–19, a lamellar II and high temperature phase are evident. The high temperature phase is characterized by pre-melting and disordering of hydrocarbon chains as they change from nearly all-trans to one with increased gauche conformers. It is probably a solid rotator phase. Odd–even alternation in melting temperature, density and some thermodynamic data result from the relative distance between methyl groups, from opposite layers in a bi-layer. Molecular models indicate that the methyl groups in odd chains are more favourably orientated which lead to a more energetically favoured staggered conformer. As a consequence, the methyl groups, for odd chains, are in closer proximity than even chains. This subtle change in the molecular lattice could account for the presence of polymorphic structures on cooling from the melt.     

A mild synthesis of bis(indolyl)methanes using a deep eutectic solvent

Deep eutectic solvents (such as the combination of urea and choline chloride) are effective solvents/organocatalysts for the condensation of indole and aryl or alkenyl aldehydes to form bis(indolyl)methanes. The reaction conditions are quite mild and do not require additional Bronsted or Lewis acid catalyst, though they fail with ketones or aliphatic aldehydes. Given the inexpensive, non-toxic, and recyclable nature of the DES, these reaction conditions are simple and highly environmentally friendly.