Recent developments in the synthesis of novel xanthone-1,2,3-triazole dyads

The development of multi-target drugs for treating complex multifactorial diseases constitutes an active research ield. This kind of drugs has gained much importance as alternative strategy to combination therapy (“cocktail drugs”).1 A common way to design them brings together two different pharmacophores in one single molecule (so-called dyads). Following this idea and being aware that xanthones2 and 1,2,3-triazoles3 possess important pharmacological properties, we combined these two heterocycles in one molecule to create new dyads with improved therapeutic potential. In this work, new xanthone-1,2,3-triazole dyads were prepared from novel (E)-2-(4-arylbut-1-en-3-yn-1-yl)chromones by two different approaches to evaluate their eficiency and sustainability. Both methodologies involved Diels-Alder reactions to build the xanthone core, which were optimized using microwave irradiation as alternative heating method, and 1,3-dipolar cycloadditions to insert the 1,2,3-triazole moiety (Figure 1).4 All final and intermediate compounds were fully characterized by 1D and 2D NMR techniques.

(Table 1) Concentrations of phthalates in chloroform extracts from waters of the Northwest Indian Ocean and the Red Sea

Chloroform extracts of water-soluble organic matter collected in the water column from the surface to the bottom were studied by C-13 and H-1 NMR chromatographic mass spectrometry, and phthalate concentrations were determined by capillary gas-liquid chromatography. More than 14 compounds were found including diethyl phthalate, ethyl butyl phthalate, dibutyl phthalate, and di-2-ethylhexyl phthalate, phthalates with normal C4-C12 chains, phthalates partially esterified with methanol, and others, at total concentrations up to 0.4 mg/l. Possible reasons for presence of phthalates in oceans, sometimes in high concentrations, are discussed.

Structure of Petunia hybrida Defensin 1, a Novel Plant Defensin with Five Disulfide Bonds

The structure of a novel plant defensin isolated from the flowers of Petunia hybrida has been determined by H-1 NMR spectroscopy. P. hybrida defensin 1 (PhD1) is a basic, cysteine-rich, antifungal protein of 47 residues and is the first example of a new subclass of plant defensins with five disulfide bonds whose structure has been determined. PhD1 has the fold of the cysteine-stabilized alphabeta motif, consisting of an alpha-helix and a triple-stranded antiparallel beta-sheet, except that it contains a fifth disulfide bond from the first loop to the alpha-helix. The additional disulfide bond is accommodated in PhD1 without any alteration of its tertiary structure with respect to other plant defensins. Comparison of its structure with those of classic, four-disulfide defensins has allowed us to identify a previously unrecognized hydrogen bond network that is integral to structure stabilization in the family.

Embracing ligands. Synthesis, characterisation and the correlation between 59Co NMR and ligand field parameters of Co(III) complexes with a new class of nitrogen-thioether multidentate ligand

The syntheses of the hexadentate ligands 2,2,10,10-tetra(methyleneamine)-4,8-dithiaundecane (PrN(4)S(2)amp), 2,2,11,11-tetra(methyleneamine)-4,9-dithiadodecane (BuN(4)S(2)amp), and 1,2-bis(4,4-methyleneamine)-2-thiapentyl)benzene (XyN(4)S(2)amp) are reported and the complexes [Co(RN(4)S(2)amp)](3+) (R = Pr, Bu, Xy) characterised by single crystal X-ray study. The low-temperature (11 K) absorption spectra have been measured in Nafion films. From the observed positions of both spin-allowed (1)A(1g) --> T-1(1g) and (1)A(1g) --> T-1(2g) and spin forbidden (1)A(1g) --> T-3(1g) and (1)A(1g) --> T-3(2g) bands, octahedral ligand-field parameters (10D(q), B and C) have been determined. DFT calculations suggest that significant interaction between the d-d and CT excitations occurs for the complexes. The calculations offer an explanation for the observed deviations from linearity of the relationship between Co-59 magnetogyric ratio and beta(DeltaE)(-1) (beta = the nephelauxetic ratio; DeltaE the energy of the (1)A(1g) --> T-1(1g) transition) for a series of amine and mixed amine/thioether donor complexes.

NMR imaging of the diffusion of water at 37 degrees C into poly(2-hydroxyethyl methacrylate) containing aspirin or vitamin B-12

The ingress of water into poly(2-hydroxyethyl methacrylate), PHEMA, loaded with either one of two model drugs, vitamin B-12 or aspirin, was studied at 37 degreesC using three-dimensional NMR imaging. PHEMA was loaded with 5 and 10 wt % of the drugs. From the imaging profiles, it was observed that incorporation of vitamin B-12 into PHEMA resulted in enhanced crack formation on sorption of water and the crack healing behind the diffusion front was slower than for PHEMA without added drug. This was accounted for by the anti-plasticization of PHEMA by vitamin B-12. Crack formation was inhibited in the P-HEMA-aspirin systems because of the plasticizing effect of the aspirin on the PHEMA matrix. All of the polymers were found to absorb water according to an underlying Fickian diffusion mechanism. For PHEMA loaded with 5 wt % of aspirin or vitamin B-12, the best values of the water diffusion coefficients were both found to be 1.3 +/- 0.1 x 10(-11) m(2) s(-1) at 37 degreesC, while the values for the polymer loaded with 10 wt % of the drugs were slightly higher, 1.5 +/- 0.1 x 10(-11) m(2) s(-1).

Water characteristics in cooked beef as influenced by ageing and high-pressure treatment -an NMR micro imaging study

The water characteristics in cooked pressure-heat treated (45 degreesC for 45 min prior to pressurisation at 150 MPa for 30 min) and non-pressurised, cooked (control) samples of beef Longissimus aged for 1, 3, 8 or 16 days were studied by nuclear magnetic resonance microscopy. A multi-echo sequence was used to obtain T2 images, and independent of ageing period, the T2 values were found to be lower in pressure-heat treated meat revealing alterations in water characteristics of pressure-treated, cooked meat compared with cooked meat. With increasing ageing duration, the T2 values in both pressure-treated, cooked and cooked meat decreased indicating that the water became more tightly trapped in the protein network. In addition, independent of length of ageing period the relationship between cooking loss in the cooked meat and transverse relaxation differed between non-pressurised and pressure-treated meat. which reveals that the mechanisms changing the water properties in beef during ageing are different from those occuring during pressure-heat treatment of meat. (C) 2003 Elsevier Ltd. All rights reserved.

The use of simultaneous H-1 & P-31 magic angle spinning nuclear magnetic resonance measurements to characterize energy metabolism during the conversion of muscle to meat

In order to investigate the potential of magic angle spinning nuclear magnetic resonance (MAS NMR) in the elucidation of post-mortem metabolism in muscle biopsies, simultaneous H-1 and (31)p MAS NMR measurements were made continuously on postmortem (20 min to 24 h) muscle longissimus samples from rabbits. The animals had either been or not been given adrenaline (0.5 mg kg(-1) 4 h pre-slaughter) to deplete stores of muscle glycogen. The intracellular pH was calculated from H-1 spectra, and the post-mortem rate of formation of lactate was followed and quantified. Comparison of measurements made on muscle samples from rabbits treated with adrenaline with measurements made on muscle samples from untreated' rabbits revealed significant effects of adrenaline treatment on both pH (pH24 h = 6.42 vs. pH24 It = 5.60) and formation of lactate (16 mmol g(-1) vs. 65 mmol g(-1)). The P-31 NMR spectra were used to follow the rate of degradation of ATP and phosphocreatine. The present study clearly shows that MAS NMR has potential for the study of post-mortem energy metabolism.

Discovery, structural determination, and putative processing of the precursor protein that produces the cyclic trypsin inhibitor sunflower trypsin inhibitor 1

Backbone-cyclized proteins are becoming increasingly well known, although the mechanism by which they are processed from linear precursors is poorly understood. In this report the sequence and structure of the linear precursor of a cyclic trypsin inhibitor, sunflower trypsin inhibitor 1 (SFTI-1) from sunflower seeds, is described. The structure indicates that the major elements of the reactive site loop of SFTI-1 are present before processing. This may have importance for a protease-mediated cyclizing reaction as the rigidity of SFTI-1 may drive the equilibrium of the reaction catalyzed by proteolytic enzymes toward the formation of a peptide bond rather than the normal cleavage reaction. The occurrence of residues in the SFTI-1 precursor susceptible to cleavage by asparaginyl proteases strengthens theories that involve this enzyme in the processing of SFTI-1 and further implicates it in the processing of another family of plant cyclic proteins, the cyclotides. The precursor reported here also indicates that despite strong active site sequence homology, SFTI-1 has no other similarities with the Bowman-Birk trypsin inhibitors, presenting interesting evolutionary questions.

Isolation and characterization of novel cyclotides from Viola hederaceae - Solution structure and anti-HIV activity of vhl-1, a leaf-specific expressed cyclotide

Based on a newly established sequencing strategy featured by its efficiency, simplicity, and easy manipulation, the sequences of four novel cyclotides (macrocyclic knotted proteins) isolated from an Australian plant Viola hederaceae were determined. The three-dimensional solution structure of V. hederaceae leaf cyclotide-1 ( vhl-1), a leaf-specific expressed 31-residue cyclotide, has been determined using two-dimensional H-1 NMR spectroscopy. vhl-1 adopts a compact and well defined structure including a distorted triple-stranded β- sheet, a short 310 helical segment and several turns. It is stabilized by three disulfide bonds, which, together with backbone segments, form a cyclic cystine knot motif. The three-disulfide bonds are almost completely buried into the protein core, and the six cysteines contribute only 3.8% to the molecular surface. A pH titration experiment revealed that the folding of vhl-1 shows little pH dependence and allowed the pK(a) of 3.0 for Glu(3) and ∼ 5.0 for Glu(14) to be determined. Met(7) was found to be oxidized in the native form, consistent with the fact that its side chain protrudes into the solvent, occupying 7.5% of the molecular surface. vhl-1 shows anti-HIV activity with an EC50 value of 0.87 μ m.

Disulfide bond mutagenesis and the structure and function of the head-to-tail macrocyclic trypsin inhibitor SFTI-1

SFTI-1 is a novel 14 amino acid peptide comprised of a circular backbone constrained by three proline residues, a hydrogen-bond network, and a single disulfide bond. It is the smallest and most potent known Bowman-Birk trypsin inhibitor and the only one with a cyclic peptidic backbone. The solution structure of [ABA(3,11)]SFTI-1, a disulfide-deficient analogue of SFTI-1, has been determined by H-1 NMR spectroscopy. The lowest energy structures of native SFTI-1 and [ABA(3,11)]SFTI-1 are similar and superimpose with a root-mean-square deviation over the backbone and heavy atoms of 0.26 +/- 0.09 and 1.10 +/- 0.22 Angstrom, respectively. The disulfide bridge in SFTI-1 was found to be a minor determinant for the overall structure, but its removal resulted in a slightly weakened hydrogen-bonding network. To further investigate the role of the disulfide bridge, NMR chemical shifts for the backbone H-alpha protons of two disulfide-deficient linear analogues of SFTI-1, [ABA(3,11)]SFTI-1[6,5] and [ABA(3,11)]SFTI-1[1,14] were measured. These correspond to analogues of the cleavage product of SFTI-1 and a putative biosynthetic precursor, respectively. In contrast with the cyclic peptide, it was found that the disulfide bridge is essential for maintaining the structure of these open-chain analogues. Overall, the hydrogen-bond network appears to be a crucial determinant of the structure of SFTI-1 analogues.

Equilibrium swelling measurements of network and semicrystalline polymers in supercritical carbon dioxide using high-pressure NMR

The extent of swelling of cross-linked poly(dimethylsiloxane) and linear low-density poly(ethylene) in supercritical CO2 has been investigated using high-pressure NMR spectroscopy and microscopy. Poly(dimethylsiloxane) was cross-linked to four different cross-link densities and swollen in supercritical CO2. The Flory-Huggins interaction parameter, x, was found to be 0.62 at 300 bar and 45 degrees C, indicating that supercritical CO2 is a relatively poor solvent compared to toluene or benzene. Linear low-density poly(ethylene) was shown to exhibit negligible swelling upon exposure to supercritical CO2 up to 300 bar. The effect Of CO2 pressure on the amorphous region of the poly(ethylene) was investigated by observing changes in the H-1 T-2 relaxation times of the polymer. These relaxation times decreased with increasing pressure, which was attributed to a decrease in mobility of the polymer chains as a result of compressive pressure.

Analysis of lipoproteins using 2D diffusion-edited NMR spectroscopy and multi-way chemometrics

This study represents the first application of multi-way calibration by N-PLS and multi-way curve resolution by PARAFAC to 2D diffusion-edited H-1 NMR spectra. The aim of the analysis was to evaluate the potential for quantification of lipoprotein main- and subtractions in human plasma samples. Multi-way N-PLS calibrations relating the methyl and methylene peaks of lipoprotein lipids to concentrations of the four main lipoprotein fractions as well as 11 subfractions were developed with high correlations (R = 0.75-0.98). Furthermore, a PARAFAC model with four chemically meaningful components was calculated from the 2D diffusion-edited spectra of the methylene peak of lipids. Although the four extracted PARAFAC components represent molecules of sizes that correspond to the four main fractions of lipoproteins, the corresponding concentrations of the four PARAFAC components proved not to be correlated to the reference concentrations of these four fractions in the plasma samples as determined by ultracentrifugation. These results indicate that NMR provides complementary information on the classification of lipoprotein fractions compared to ultracentrifugation. (C) 2004 Elsevier B.V. All rights reserved.

Chemical synthesis and structure elucidation of bovine kappa-casein (1-44)

The caseins (alpha(s1), alpha(s2), beta, and kappa) are phosphoproteins present in bovine milk that have been studied for over a century and whose structures remain obscure. Here we describe the chemical synthesis and structure elucidation of the N-terminal segment (1-44) of bovine K-casein, the protein which maintains the micellar structure of the caseins. K-Casein (1-44) was synthesised by highly optimised Boc solid-phase peptide chemistry and characterised by mass spectrometry. Structure elucidation was carried out by circular dichroism and nuclear magnetic resonance spectroscopy. CD analysis demonstrated that the segment was ill defined in aqueous medium but in 30% trifluoroethanol it exhibited considerable helical structure. Further, NMR analysis showed the presence of a helical segment containing 26 residues which extends from Pro(8) to Arg(34). This is the first report which demonstrates extensive secondary structure within the casein class of proteins. (c) 2006 Elsevier Inc. All rights reserved.