Fluorescence of the single tryptophan of cutinase: temperature and pH effect on protein conformation and dynamics

The cutinase from Fusarium solani pisi is an enzyme with a single L-tryptophan (Trp) involved in a hydrogen bond with an alanine (Ala) residue and located close to a cystine formed by a disulfide bridge between two cysteine (Cys) residues. The Cys strongly quenches the fluorescence of Trp by both static and dynamic quenching mechanisms. The Trp fluorescence intensity increases by about fourfold on protein melting because of the disruption of the Ala-Trp hydrogen bond that releases the Trp from the vicinity of the cystine residue. The Trp forms charge-transfer complexes with the disulfide bridge, which is disrupted by UV light irradiation of the protein. This results in a 10-fold increase of the Trp fluorescence quantum yield because of the suppression of the static quenching by the cystine residue. The Trp fluorescence anisotropy decays are similar to those in other proteins and were interpreted in terms of the wobbling-in-cone model. The long relaxation time is attributed to the Brownian rotational correlation time of the protein as a whole below the protein-melting temperature and to protein-backbone dynamics above it. The short relaxation time is related to the local motion of the Trp, whose mobility increases on protein denaturation.     

Diastereoselective synthesis of cularine alkaloids via enium ions and an easy entry to isoquinolines by aza-Wittig electrocyclic ring closure

In preliminary communications, we reported the diastereoselective synthesis of cularine and sarcocapnine via the intramolecular ring closure of nitrenium and oxenium ions, a new highly diastereoselective reductive methylation with (+)-8-phenylmenthyl chloroacetate followed by reduction with sodium borohydride, and a facile entry to the isoquinoline precursors by aza-Wittig electrocyclic ring closure. We now report the full details of the syntheses of (+)-O-demethylcularine, (+)-cularine, (+)-sarcocapnidine, (+)-sarcocapnine, and (+)-crassifoline and describe different methods of synthesis of their precursors.     

Carbonyl complexes of molybdenum and tungsten with sulfur donors: V. Reactions of carbonyl complexes of molybdenum(0) with uninegative (X,Y)-donor ligands (X,Y = S,N, O)

The reactions of the zerovalent carbonyl complexes Mo(CO)₆ and Mo(CO)₄(bipy) with a series of uninegative bidentate (X,Y)-donor ligands (X,Y = xanthates, dithiocarbamates, o-aminophenoxide, o-aminothiophenoxide, 2-picolinate and thioacetate) lead to new anionic tetracarbonyl complex anions [Mo⁰(X,Y)(CO)₄]^?. These anions, which can be isolated as their tetraphenylphosphonium salts, contain the (X,Y)-ligand as a bidentate group. In the case of (X,Y) = monothioacetate the decarbonylated species [PPh₄][Mo^II(TA)₃] is formed. The reacions of the new complexes with allyl bromide and methyl iodide are described.     

A Layer‐by‐Layer Film of Chitosan in a Taste Sensor Application

Chitosan is alternated with sulfonated polystyrene (PSS) to build layer‐by‐layer (LBL) films that are used as sensing units in an electronic tongue. Using impedance spectroscopy as the principle method of detection, an array using chitosan/PSS LBL film and a bare gold electrode as the sensing units was capable of distinguishing the basic tastes – salty, sweet, bitter, and sour – to a concentration below the human threshold. The suitability of chitosan as a sensing material was confirmed by using this sensor to distinguish red wines according to their vintage, vineyard, and brands.

PCA Plot for red wine samples obtained from impedance measurements at 1 kHz for the sensor array comprising a 3‐bilayer chitosan/PSS film and a bare gold electrode.     

Determination of manganese and zinc in powdered chocolate samples by slurry sampling using sequential multi-element flame atomic absorption spectrometry

In the present work, a slurry sampling flame atomic absorption spectrometric method to determine directly manganese and zinc in powdered chocolate samples is proposed. The optimization step was performed using univariate methodology involving the following factors: nature and concentration of the acid solution, sonication time, and particle size. The established conditions led to the use of a sample mass of 150 mg, 2.0 mol L^− 1 nitric acid solution, sonication time of 15 min, and a slurry volume of 50 mL. This method allows the determination of manganese and zinc with detection limit of 52 and 61 ng g^− 1, respectively, and a precision expressed as relative standard deviation (RSD) of 2.6% and 3.2% (both, n = 10) for contents of manganese and zinc of 52.4 and 100.0 μg g^− 1, respectively. The proposed method was applied for determination of manganese and zinc in five powdered chocolate samples. In these, the manganese content varied from 42.8 to 52.7 and from 88.6 to 102.4 μg g^− 1 of zinc. The analytical results were compared with the results obtained by analysis of these samples after digestion using open vessel and acid bomb digestion procedures and determination using FS-FAAS. The statistical comparison by t-test (95% confidence level) showed no significant difference between these results.     

Synthesis of amberlite XAD-2-PC resin for preconcentration and determination of trace elements in food samples by flame atomic absorption spectrometry

A new chelating sorbent has been developed using Amberlite XAD-2 resin anchored with pyrocatechol through –N=C– group. This sorbent, characterised by elemental analysis and infrared (IR) spectra, was used as packing for the minicolumn in an on-line system preconcentration system for cadmium, cobalt, copper and nickel determination. Metal ions were sorbed in the minicolumn, from which it could be eluted directly to the nebulizer–burner system of the flame atomic absorption spectrometer (FAAS). Elution of all metals from minicolumn can be made with 0.50 mol L^− 1 HCl or HNO₃. The enrichment factors obtained were 16 (Cd), 24 (Co), 15 (Cu) and 19 (Ni), for 60 s preconcentration time, and 39 (Cd), 69 (Co), 36 (Cu) and 41 (Ni), if used 180 s preconcentration time. Under the optimum conditions, the proposed procedure allowed the determination of cadmium, cobalt, copper and nickel with detection limits of 0.31, 0.32, 0.39 and 1.64 μg L^− 1, respectively, when used preconcentration periods of 180 s. The accuracy of the developed procedure was sufficient and evaluated by the analysis of the certified reference materials NIST 1515 apple leaves and NIST 1570a spinach leaves. The method was applied to the analysis of food samples (spinach, black tea and rice flour).     

Singlet oxygen generation ability of squarylium cyanine dyes

The quantum yields for singlet oxygen generation of several squarylium cyanine dyes derived from benzothiazole, benzoselenazole and quinoline, displaying absorption within the so-called “phototherapeutic window” (600–1000 nm), were determined, envisioning their potential usefulness for photodynamic therapy (PDT). The determination was performed by a direct method measuring the luminescence decay of the dyes in the near infrared. Considering the absorption and the quantum yields displayed by some of the dyes, these seemed to be potential candidates as sensitizers for PDT.     

Novel approach for asymmetric synthesis of fluorinated beta-amino sulfones and allylic amines

[reaction: see text] Enantiomerically pure gamma-fluoroalkyl beta-amino sulfones are readily synthesized in three steps starting from fluorinated imidoyl chlorides and arylmethyl sulfones. A complementary two-step sequence starting from chiral fluorinated beta-amino sulfoxides has also been developed. To illustrate the application of this procedure, a new method for the synthesis of alpha-fluoroalkyl allylic amines in optically pure form involving a Julia methylenation-desulfonylation reaction is presented.     

Optically pure trans-2,3-disubstituted N-sulfinyl aziridines. Regio- and stereoselective opening mediated by the sulfinyl group

A new entry to optically pure trans-2,3-disubstituted N-sulfinyl aziridines starting from 1,2-aminosulfides, involving formation of a sulfonium salt intermediate followed by intramolecular nucleophilic attack by the sulfinamide nitrogen atom, is reported. The regio- and stereoselective opening of the aziridine ring can be achieved by anchimeric assistance of the sulfinyl group.     

Dynamic behavior of DNA base pairs containing 8-oxoguanine

The process by which DNA repair enzymes recognize and selectively excise damaged bases in duplex DNA is fundamental to our mechanistic understanding of these critical biological reactions. 8-Oxoguanine (8-oxoG) is the most common form of oxidative DNA damage; unrepaired, this lesion generates a G:C-->T:A mutation. Central to the recognition and repair of DNA damage is base extrusion, a process in which the damaged base lesion or, in some cases, its partner disengages from the helix and is bound to the enzyme's active site where base excision takes place. The conformation adopted by 8-oxoG in duplex DNA is affected by the base positioned opposite this lesion; conformational changes may also take place when the damaged base binds to its cognate repair enzyme. We performed unrestrained molecular dynamics simulations for several 13-mer DNA duplexes. Oligomers containing G:C and 8oxoG:C pairs adopted Watson-Crick geometries in stable B-form duplexes; 8oxoG showed increased local and global flexibility and a reduced barrier to base extrusion. Duplexes containing the G:A mismatch showed much larger structural fluctuations and failed to adopt a well-defined structure. For the 8oxoG:A mismatch that is recognized by the DNA glycosylase MutY, the damaged nucleoside underwent spontaneous and reproducible anti-->syn transitions. The syn conformation is thermodynamically preferred. Steric hindrance and unfavorable electrostatics associated with the 8oxoG O8 atom in the anti conformation were the major driving forces for this transition. Transition events follow two qualitatively different pathways. The overall anti-->syn transition rate and relative probability of the two transition paths were dependent on local sequence context. These simulations indicate that both the dynamic and equilibrium behavior of the duplex change as a result of oxidation; these differences may provide valuable new insight into the selective action of enzymes on damaged DNA.