Solid-phase parallel synthesis of 4-beta-D-ribofuranosylpyrazolo[4,3-d]pyrimidine nucleosides

The synthesis of pyrazolo[4,3-d]pyrimidine nucleoside library using solid-phase parallel synthesis methodology is described. Glycosylation of the trimethylsilyl (TMS) derivative of 1- and 2-(methyl)-1H and 2H-pyrazolo[4,3-d]pyrimidine-5,7-(4H, 6H)-dione (5) with 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose in the presence of TMS triflate provided two novel protected nucleosides 6 and 7. The structures of 6 and 7 were assigned by 1H and 2D NMR experiments. Nucleosides 6 and 7 were then transformed to the key intermediates 12 and 15 respectively. Reaction of 12 and 15 with MMTCl resin in the presence of 2,6-lutidine afforded the necessary scaffolds B and C. Different amines (96) were introduced selectively by nucleophilic substitution on scaffolds B and C using solid-phase parallel semi-automated synthesizer. Cleavage of the products from the solid support with 30% HFIP in a parallel fashion yielded nucleoside libraries simultaneously, and they were analyzed and characterized by high-throughput LC-MS.     

Synthesis, magnetic properties and MCD spectra of a four coordinate copper(II) complex with two chelated phenolate-substituted imino nitroxides

A reaction of Cu(II) nitrate and 4,4,5,5-tetramethyl-2-(2-hydroxophenyl)imidazolin-1-oxyl (IM2PhOH) with potassium methoxide in methanol gave a homoleptic bis(imino nitroxide) complex of [Cu(IM2PhO)₂] (1). The single-crystal X-ray analysis of 1 showed that the imino nitroxide anion, IM2PhO⁻, chelated to a Cu^II ion via an imino-N and a phenoxide-O atoms to form a six-membered chelate ring. The coordination geometry around the Cu(II) ion was a distorted square-planar polygon; the dihedral angle between the two coordination planes, each of which was defined by Cu and two ligating atoms of IM2PhO⁻, was 40.81°. The temperature dependences of the magnetic susceptibility and the EPR spectra of 1 indicate that the magnetic interaction between Cu(II) and the imino nitroxide is ferromagnetic, while there is a moderate antiferromagnetic interaction between two coordinated imino nitroxides. A balance between these opposite interactions attains the lowest molecular doublet spin-state in 1. The variable temperature magnetic circular dichroism (MCD) spectrum of the complex 1 also showed two negative components with a large C term, which may be due to the charge-transfer (CT) transition originated from the d orbital to the SOMO π^* orbital in the spin-coupled IM2PhO⁻ radicals; resulting in the largely split doublet excited states with the spin singlet and triplet d⁸ configurations.     

Isolation and characterization of a novel small cardioactive peptide-related peptide from the brain of Octopus vulgaris

A novel small cardioactive peptide (SCP)-related peptide (oct-SCPRP: Ser-Asn-Gly-Tyr-Leu-Ala-Leu-Pro-Arg-Gln-NH2) was isolated from the brain of the octopus (Octopus vulgaris). cDNA encoding this precursor protein was cloned. Oct-SCPRP was shown to evoke contraction in the radula protractor muscle, and the precursor protein was highly homologous to the SCP family in the Mollusk but did not encode a related peptide, SCPB. The expression of oct-SCPRP mRNA was present not only in the peripheral nervous system (PNS) which is a motor center for the control of feeding, but also in the central nervous system (CNS) which is capable of complex analysis, learning, and controls behaviors.     

Structural Characterization of Arabidopsis Leaf Arabinogalactan Polysaccharides

Proteins decorated with arabinogalactan (AG) have important roles in cell wall structure and plant development, yet the structure and biosynthesis of this polysaccharide are poorly understood. To facilitate the analysis of biosynthetic mutants, water-extractable arabinogalactan proteins (AGPs) were isolated from the leaves of Arabidopsis (Arabidopsis thaliana) plants and the structure of the AG carbohydrate component was studied. Enzymes able to hydrolyze specifically AG were utilized to release AG oligosaccharides. The released oligosaccharides were characterized by high-energy matrix-assisted laser desorption ionization-collision-induced dissociation mass spectrometry and polysaccharide analysis by carbohydrate gel electrophoresis. The Arabidopsis AG is composed of a β-(1→3)-galactan backbone with β-(1→6)-d-galactan side chains. The β-(1→6)-galactan side chains vary in length from one to over 20 galactosyl residues, and they are partly substituted with single α-(1→3)-l-arabinofuranosyl residues. Additionally, a substantial proportion of the β-(1→6)-galactan side chain oligosaccharides are substituted at the nonreducing termini with single 4-O-methyl-glucuronosyl residues via β-(1→6)-linkages. The β-(1→6)-galactan side chains are occasionally substituted with α-l-fucosyl. In the fucose-deficient murus1 mutant, AGPs lack these fucose modifications. This work demonstrates that Arabidopsis mutants in AGP structure can be identified and characterized. The detailed structural elucidation of the AG polysaccharides from the leaves of Arabidopsis is essential for insights into the structure-function relationships of these molecules and will assist studies on their biosynthesis.Arabinogalactans (AGs) are structurally complex large-branched polysaccharides attached to Hyp residues of many plant cell wall polypeptides. Most proteins glycosylated with AGs (AGPs) have both AG glycosylated domains (glycomodules) and structural or enzymatic domains. However, typical AGPs commonly contain less than 10% protein, suggesting that the AG is the functional part of the molecule (Clarke et al., 1979; Fincher et al., 1983; Kieliszewski and Lamport, 1994; Borner et al., 2003; Xu et al., 2008). Hyp is the most characteristic amino acid present at the glycosylated domain of the AGP, but other amino acids such as Ser, Ala, and Thr are also very common. Type II AG polysaccharides share common structural features based on a β-(1→3)-galactan backbone with β-(1→6)-linked galactan side chains and can be found both on AGPs and rhamnogalacturonan-I (RG-I) pectin (Renard et al., 1991). The galactopyranosyl (Galp) residues can be further substituted with l-arabinofuranosyl (l-Araf) and occasionally also l-rhamnosyl (l-Rha), l-fucosyl (l-Fuc), and glucuronosyl (GlcA; with or without 4-O-methylation) residues (Tsumuraya et al., 1988; Tan et al., 2004; Tryfona et al., 2010). (Sugars mentioned in this work belong to the D-series unless otherwise stated.)The structure of AGs is poorly characterized, and this is mainly due to the great heterogeneity of glycan structures, not only between different AGPs but also even on the same peptide sequence in the same tissue (Estévez et al., 2006). The glycan structure can also be different depending on the developmental stage and tissue type (Tsumuraya et al., 1988), adding to the great heterogeneity of these molecules and therefore limiting their detailed characterization. Molecular and biochemical evidence has indicated that AGPs have specific functions during root formation, promotion of somatic embryogenesis (van Hengel et al., 2002), and attraction of pollen tubes to the style (Cheung et al., 1995). In addition, enhanced secretion efficiency or stability in the cell wall are properties that the AG may confer on the glycosylated protein (Borner et al., 2003). However, it has been difficult to differentiate one species of AGP from another in plant tissues and to assign specific roles to individual AGPs.l-Fuc is present in AGPs in Arabidopsis (Arabidopsis thaliana; van Hengel et al., 2002), radish (Raphanus sativus; Nakamura et al., 1984; Tsumuraya et al., 1984a, 1984b, 1988), and several other dicot plants such as thyme (Thymus vulgaris; Chun et al., 2001) and celery (Apium graveolens; Lin et al., 2011). Reduction in l-Fuc by 40% in roots of murus1 (mur1) plants resulted in a decrease of 50% in root cell elongation, and eel lectin binding assays suggested that the phenotype was the result of alterations in the composition of root AGPs (van Hengel and Roberts, 2002). An α-(1→2)-fucosyltransferase (FUT) activity for radish primary root AGPs has been described, where an α-l-Araf-(1→3)-β-Galp-(1→6)-Galp trisaccharide was used as exogenous substrate acceptor to mimic an AG polysaccharide in the enzymatic assay (Misawa et al., 1996). Linkage analysis, reactivity with eel lectin, and digestion with α-(1→2)-fucosidase indicated that the l-Fuc residues added are terminal and attached via an α-linkage to the C-2 position of an adjacent l-Araf residue (Nakamura et al., 1984; Tsumuraya et al., 1984a, 1984b, 1988). Recently, Wu et al. (2010) identified AtFUT4 and AtFUT6 genes encoding FUT proteins specific to AGPs, but the structures of the fucosylated AG generated have not been fully characterized.To gain insights into the synthesis and function of plant AGPs, it would be useful to have mutants altered in their carbohydrate moieties. However, no AG-specific biosynthetic mutants have been characterized, and this, among other reasons, is due to the very limited knowledge of the structure of Arabidopsis AGs (Qu et al., 2008). Moreover, characterization of AG in candidate mutants remains challenging. Even though the structures of some AGs have been proposed using NMR and sugar linkage analyses, the complete structural elucidation of a native AG still remains a formidable task, because NMR spectroscopy and methylation analysis have been largely used to provide information regarding the amount and type of linkages between adjacent glycosyl residues, and AG heterogeneity can confound attempts to build complete structural models. Recently, a modular structure was proposed for AGs on heterologously expressed proteins in tobacco (Nicotiana tabacum; Tan et al., 2010). Tan et al. (2010) proposed that approximately 15-residue repeating blocks of decorated β-(1→3)-trigalactosyl subunits connected by β-(1→6)-linkages were the building blocks of type II AG polysaccharides and concluded that these molecules are far less complex than commonly supposed. Most characterized β-(1→6)-galactan side chains in AGs are reported to be short, of one or two residues (Neukom and Markwalder, 1975; Gane et al., 1995; Gaspar et al., 2001). On the contrary, there are reports of long β-(1→6)-galactan side chains in radish root AGPs (Haque et al., 2005). Similarly, we recently found evidence that wheat (Triticum aestivum) flour endosperm AGP extracts contained long β-(1→6)-galactan side chains heavily substituted with l-Araf at C-3 (Tryfona et al., 2010). This partial structure of the carbohydrate component of wheat flour AGP isolated from water extracts of wheat endosperm was elucidated utilizing a combination of analytical approaches, such as the use of enzymes able to release oligosaccharides specifically from AGs, high-energy matrix-assisted laser desorption ionization (MALDI)-collision-induced dissociation (CID) mass spectrometry (MS), and polysaccharide analysis by carbohydrate gel electrophoresis (PACE; Tryfona et al., 2010). In this work, we applied these techniques to study the carbohydrate component of Arabidopsis leaf AGPs. AG-specific enzyme digestion products were analyzed by PACE and MS, allowing a partial structure to be proposed. We show that endogenous Arabidopsis leaf AG is composed of a β-(1→3)-galactan backbone with β-(1→6)-galactan side chains. These side chains are substituted with l-Araf residues via α-(1→3)-linkages and can vary in length from one up to at least 20 Galp residues. We also found that the β-(1→6)-galactan side chains are substituted mainly with 4-O-methyl-glucuronosyl (4-O-Me-GlcA) at their nonreducing termini, while occasional l-Fuc substitutions were also present via α-(1→2)-linkages on l-Araf residues. In addition, AG oligosaccharides from leaves of the mur1 mutant were identified, and their structures were compared with those isolated from wild-type plants.     

Reconstruction of putative DNA virus from endogenous rice tungro bacilliform virus-like sequences in the rice genome: implications for integration and evolution

Background  

Plant genomes contain various kinds of repetitive sequences such as transposable elements, microsatellites, tandem repeats and virus-like sequences. Most of them, with the exception of virus-like sequences, do not allow us to trace their origins nor to follow the process of their integration into the host genome. Recent discoveries of virus-like sequences in plant genomes led us to set the objective of elucidating the origin of the repetitive sequences. Endogenous rice tungro bacilliform virus (RTBV)-like sequences (ERTBVs) have been found throughout the rice genome. Here, we reconstructed putative virus structures from RTBV-like sequences in the rice genome and characterized to understand evolutionary implication, integration manner and involvements of endogenous virus segments in the corresponding disease response.     

Rac is activated by tumor necrosis factor alpha and is involved in activation of Erk.

Tumor necrosis factor alpha (TNFalpha) activates various signal transduction pathways including those involving phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinases (Erk), c-Jun N-terminal protein kinases (JNK), and p38 kinases. Using the Rac binding domain of PAK (PAK-RBD) as an activation-specific probe, here we demonstrate that TNFalpha very rapidly and transiently activates the Rho family GTPase Rac in L929 cells. The PI3K inhibitor LY294002 significantly inhibited TNFalpha activation of Rac as well as Erk and abolished that of the PI3K target Akt, without showing any inhibitory effects on JNK and p38 activation. Furthermore, TNFalpha activation of Erk was abolished by a dominant negative Rac mutant, Rac17N, or by an activated Rac mutant, Rac12V. These findings suggest that Rac is activated by a mechanism that is at least partly dependent on PI3K in TNFalpha stimulated cells and plays a critical role in activation of the Erk signaling pathway.     

Total synthesis of human chymase inhibitor methyllinderone and structure--activity relationships of its derivatives.

Total synthesis of human chymase inhibitor methyllinderone has been achieved in only four steps with an overall yield of 21% from dimethyl squarate. We developed an efficient synthetic method for obtaining methyllinderone derivatives and found the active compound. In addition, we propose the inhibition mechanism of the active compound against human chymase using calculations.