Spider silk and amyloid fibrils: a structural comparison

Although spider silks have been studied for decades, the assembly properties of the underlying silk proteins have still not been unravelled. Previously, the detection of amyloid-like nanofibrils in the spider's silk gland suggested their involvement in the assembly process.Recombinantly produced spider silk also self-assembles into nanofibrils. In order to investigate the structural properties of such silk nanofibrils in more detail, they have been compared to amyloid-like fibrils to highlight structural similarities.     

Taking control of P1, P1' and double bond stereochemistry in the synthesis of Phe-Phe (E)-alkene amide isostere dipeptidomimetics

A protocol for the stereocontrolled independent preparation of both C-2 epimers of Phe-Phe trans-vinyl amide isostere dipeptidomimetics has been devised based on a Wittig-type reaction, in which two chiral building blocks were joined with excellent E-selectivity to give compounds of the type PhePsi[(E)-CH[double bond, length as m-dash]CH]-PheOH.     

Redox characteristics of a de novo quinone protein

The electrochemistry of 2,6-dimethylbenzoquinone (DMBQ) has been characterized for three different systems: DMBQ freely solvated in aqueous buffer; DMBQ bound to a neutral, blocked cysteine (N-acetyl-L-cysteine methyl ester) and the resulting DMBQ-bCys compound solvated in aqueous buffer; and DMBQ bound to a small model protein denoted alpha(3)C. The goal of this study is to detect and characterize differences in the redox properties of the protein-ligated DMBQ relative to the solvated quinones. The alpha(3)C protein used here is a tryptophan-32 to cysteine-32 variant of the structurally defined alpha(3)W de novo protein (Dai et al. J. Am. Chem. Soc. 2002, 124, 10952-10953). The properties of alpha(3)C were recently described (Hay et al. Biochemistry 2005, 44, 11891-11902). DMBQ was covalently bound to bCys and alpha(3)C through a sulfur substitution reaction with the cysteine thiol. In contrast to the solvated DMBQ and DMBQ-bCys compounds, diffusion controlled electrochemistry of DMBQ-alpha(3)C showed well-behaved and fully reversible n = 2 oxidation/reduction with a peak separation of approximately 30 mV between pH 5 and 9. DMBQ-alpha(3)C could also be immobilized on a gold electrode modified with a self-assembled monolayer of 3-mercaptopropionoic acid, allowing the measurement, by cyclic voltammetry, of an apparent rate of electron transfer of 22 s(-1). The (cysteine) sulfur substitution significantly lowers one of the hydroquinone pKA's from 10.4 in DMBQ to 6.8 in DMBQ-bCys. This pKA is slightly elevated in DMBQ-alpha(3)C to 7.0 and the E1/2 at pH 7.0 is raised by 110 mV from +190 mV in DMBQ-bCys to +297 mV in DMBQ-alpha(3)C.     

Calculation of measurement uncertainty in quantitative analysis of genetically modified organisms using intermediate precision--a practical approach

Quantitative characterization of nucleic acids is becoming a frequently used method in routine analysis of biological samples, one use being the detection of genetically modified organisms (GMOs). Measurement uncertainty is an important factor to be considered in these analyses, especially where precise thresholds are set in regulations. Intermediate precision, defined as a measure between repeatability and reproducibility, is a parameter describing the real situation in laboratories dealing with quantitative aspects of molecular biology methods. In this paper, we describe the top-down approach to calculating measurement uncertainty, using intermediate precision, in routine GMO testing of food and feed samples. We illustrate its practicability in defining compliance of results with regulations. The method described is also applicable to other molecular methods for a variety of laboratory diagnostics where quantitative characterization of nucleic acids is needed.     

Rapid multi-residue method for the quantitative determination and confirmation of glucocorticosteroids in bovine milk using liquid chromatography-electrospray ionization-tandem mass spectrometry

Dexamethasone, betamethasone and prednisolone are synthetic glucocorticosteroids authorised for therapeutic use in bovine animals within the European Union. Dexamethasone and betamethasone are used mainly for the treatment of metabolic and inflammatory diseases. Prednisolone is used to treat bovine mastitis. Maximum residue limits (MRLs) of 0.3 μg kg⁻¹ for both dexamethasone and betamethasone and 6.0 μg kg⁻¹ for prednisolone in bovine milk have been established. 6α-Methylprednisolone and flumethasone are not authorised for use in bovine animals and are completely banned in bovine milk. The proposed method is based on deprotenisation of milk using 20% (w/v) trichloroacetic acid. Samples are filtered using glass microfibre filters and subject to clean-up using OASIS HLB solid phase extraction. Separation was achieved on a Hypercarb 100 mm × 2.1 mm × 5 μm column. Mobile phase was: 90/10 acetonitrile/0.1% formic acid in water; flow rate was 600 μL min⁻¹. The method allowed the rapid identification and confirmation of the five glucocorticosteroids according to the criteria laid down in Commission Decision 2002/657/EC. Matrix calibration curves for all compounds were linear in the interval 0.0 MRL to 2.0 MRL with a correlation coefficient (r²) higher than 0.96. Relative recoveries ranged from 97% for betamethasone to 111% for prednisolone. Precision at the MRL ranged from 3.8% for prednisolone to 13.8% for betamethasone. Decision limits, CCα, and detection capability, CCβ have been calculated for all compounds.     

Synthesis of functionalized, unsymmetrical 1,3,4,6-tetrasubstituted 2,5-diketopiperazines

A general and efficient method for the synthesis of unsymmetrical 1,3,4,6-tetrasubstituted 2,5-diketo- piperazines (DKPs) is described. Cyclization of N-amide alkylated dipeptide methyl esters, followed by alkylation, furnished the corresponding tetrasubstituted DKPs in good overall yields. The influence of steric hindrance in the alkylation reactions appeared to be of lesser importance as long as reactive alkylating agents were used. Furthermore, we have demonstrated the use of tetrasubstituted DKPs as a scaffold for further chemical manipulations to produce novel DKPs with desired properties.     

Trapping of peroxidic intermediates with sulfur and phosphorus centered electrophiles

The trapping ability of a new peroxidic trapping agent relative to several well-established trapping agents was measured. Two different methods for this measurement were utilized. It was shown that adamantylidene adamantane reacts under the trapping conditions to give adamantylidene adamantane epoxide via a more complicated process than previously recognized and is consequently an inappropriate system to make this measurement. In contrast, the use of diethyl sulfide is straightforward and gives reliable values. © 1998 John Wiley & Sons, Inc. Heteroatom Chem 9:51–56, 1998     

Synthesis of Tetracyclic Analogues of Calcitriol (1α,25‐Dihydroxyvitamin D3) with Side‐Chain‐Locked Spatial Orientations at C(20)

We present our first results on the synthesis of a new class of conformationally restricted vitamin D analogues bearing an extra five‐membered ring formed by linking C(18) and C(21). Two analogues of calcitriol ( 1 ) with unsaturations at the extra ring and the lateral chain were prepared. The triene system was introduced by the convergent Wittig Horner approach developed by Lythgoe [8] and F. Hoffmann‐La Roche [9]. The key steps in the preparation of the requisite fragments were: i) the long‐distance functionalization of ketal 11 at C(18), ii) the ring closure on 15 through an intramolecular aldol condensation to give the α,β‐unsaturated ketone 10 , and iii) the Pd‐catalyzed installation of the side chains.     

Defect Formation,T-Atom Substitution and Adsorption of Guest Molecules in MSE-Type Zeolite Framework—DFT Modeling

We used computational modeling, based on Density Functional Theory, to help understand the preference for the formation of silanol nests and the substitution of Si by Ti or Al in different crystallographic positions of the MSE-type framework. All these processes were found to be energetically favorable by more than 100 kJ/mol. We suggested an approach for experimental identification of the T atom position in Ti-MCM-68 zeolite via simulation of infrared spectra of pyridine and acetonitrile adsorption at Ti. The modeling of adsorption of hydrogen peroxide at Ti center in the framework has shown that the molecular adsorption was preferred over the dissociative adsorption by 20 to 40 kJ/mol in the presence or absence of neighboring T-atom vacancy, respectively.