Cyclorheniated Azabutadienes and Their Reactions with Unsaturated Molecules

This thesis reports the studies on the reaction of 1-azabutadienes with PhCH2Re(CO)5 to prepare the cyclometallated azabutadiene tetracarbonyl compounds. Three 1-azabutadienes with different reactivity on the N and C-1 carbons were prepared and their reactions with PhCH2Re(CO)5 were investigated. All the reactions gave a mixture of the cyclorheniated azabutadienes and the isomers of the substituted derivatives formed by substitution by a second azabutadiene. The substituted derivatives as the mixture were always the main products of the reactions and were obtained in good-to-excellent yield even under modified conditions intended to optimise the yield of the cyclorheniated azabutadienes. The yield could not be improved beyond 30 %. Isomerisation of the azabutadiene about the N=C bond provided very rare cis-azabutadiene metal complexes. NMR studies on the mixture of the two isomers and suggested that the product ratio depended on the molar ratio of PhCH2Re(CO)5 in the reaction mixtures. The reactions gave the Re analogues of the products obtained in the corresponding Mn reactions. All the compounds were characterised spectroscopically as well as by microanalysis and examples of all three types were structurally determined by X-ray crystallography. The reactions of the substituted derivative as the mixture of the two isomers with unsaturated molecules including phenyl acetylene, p-methoxyphenyl isocyanide, and phenyl isocyanate and methyl acrylate were investigated

Stabilisation effects of ferrocenylalkyl groups on hydrides of heavier main group elements

New hydrides of heavier p-block main group elements with a ferrocenylalkyl moiety, Fc(CH₂)nEHm (Fc = (CpFeC₅H₄-); E = P, As, Si, Ge or Se; n = 1, 2, 4, 6 or 11; m = 1, 2 or 3) and FcCH₂P(R)H (R = CH₃, C₆H₁₁ or p-CH₂C₆H₄NO₂), have been synthesised and characterised. Although some precursors of the desired hydrides, i.e. Fc(Cl)C=C(SnCl₃)H and (FcCH₂Te)₂, which are also new compounds, could be prepared, the syntheses of the corresponding desired hydrides, FcCH₂EHn, were unsuccessful probably due to their extreme instabilities. Some related primary phosphanes, [CpFeC₅H₃(CH₂OH)(CH₂PH₂)], RcCH₂PH₂ and Fc(CH₂)₆PH₂∙BH₃, phosphane oxide, FcCH₂P(O)H₂, and phosphinic acid, FcCH₂P(O)(OH)H, were also synthesised and reported. The X-ray crystal structures of Fc(CH₂)₆PH₂∙BH3 and FcCH₂SeCN are also presented in the present thesis. The stability of the hydrides of heavier p-block main group elements with a ferrocenyl or ruthenocenylalkyl moiety under ambient conditions has been investigated using NMR and/or IR spectroscopy. Ferrocenylalkyl and ruthenocenylmethyl primary phosphanes, Fc(CH₂)nPH₂ (n = 4, 6 or 11) and RcCH₂PH₂, respectively, exhibited a remarkable stability towards air oxidation in solution, i.e. ~1 year. In contrast, the secondary phosphanes were not as stable as expected, rapidly oxidising over several weeks or months. General trend for the oxidative stability of the secondary phosphanes could not be elucidated on the basis of the electronegativity, size or degree of conjugation of the substituent on the phosphorus. Ferrocenylmethyl primary arsane, FcCH₂AsH₂, was also unexpectedly air-sensitive, having been readily oxidised as a neat liquid or in solution upon the exposure to air. Ferrocenylethyl primary silane, Fc(CH₂)₂SiH₃, was stable both as a neat liquid and also in solution. It could be purified on a TLC plate in air and also stored in solution for up to 7 months. On the other hand, ferrocenylmethyl primary germane, FcCH₂GeH₃, was unstable, almost completely decomposing left overnight in solution, which was indicated by the disappearance of the germane proton NMR signal by ¹H NMR spectroscopy. Ferrocenylalkyl selenols, Fc(CH₂)nSeH (n = 1 or 4), were both found to be unstable as neat liquids or in solution. Handling the compounds in air caused significant oxidation, resulting in the formation of the corresponding diselenides which are the common oxidation products of selenols. Ferrocenylmethyl selenol, FcCH₂SeH, was completely oxidised in solution in air in 5 days while ferrocenylbutyl selenol, Fc(CH₂)₄SeH, in 3 days. The rapid oxidation of the latter was also observed by IR spectroscopy over a period of 10 minutes when exposed to air as a neat liquid. The oxidative stability of the air-sensitive primary phosphanes, PhPH₂ and camphylPH₂, in the presence of ferrocene, FcH, or its derivative, FcCH₂PH₂, in solution, was studied by ³¹P NMR spectroscopy. The study showed that the primary phosphanes could be stabilised by simple addition of FcH or FcCH₂PH₂. The corresponding ³¹P NMR study using known antioxidants, diphenyl picryl hydrazyl (DPPH) or nitrosobutane, in place of the ferrocene species also exhibited that PhPH₂ could be stabilised by addition of an antioxidant. These results suggest that FcH and FcCH₂PH₂ can be used to stabilise air-sensitive primary phosphanes in solution by simply adding them, probably acting as radical scavengers

EST assembly supported by a draft genome sequence: an analysis of the Chlamydomonas reinhardtii transcriptome

Clustering and assembly of expressed sequence tags (ESTs) constitute the basis for most genomewide descriptions of a transcriptome. This approach is limited by the decline in sequence quality toward the end of each EST, impacting both sequence clustering and assembly. Here, we exploit the available draft genome sequence of the unicellular green alga Chlamydomonas reinhardtii to guide clustering and to correct errors in the ESTs. We have grouped all available EST and cDNA sequences into 12 063 ACEGs (assembly of contiguous ESTs based on genome) and generated 15 857 contigs of average length 934 nt. We predict that roughly 3000 of our contigs represent full-length transcripts. Compared to previous assemblies, ACEGs show extended contig length, increased accuracy and a reduction in redundancy. Because our assembly protocol also uses ESTs with no corresponding genomic sequences, it provides sequence information for genes interrupted by sequence gaps. Detailed analysis of randomly sampled ACEGs reveals several hundred putative cases of alternative splicing, many overlapping transcription units and new genes not identified by gene prediction algorithms. Our protocol, although developed for and tailored to the C. reinhardtii dataset, can be exploited by any eukaryotic genome project for which both a draft genome sequence and ESTs are available

A High-Density SSR Linkage Map of Red Clover and Its Transferability to Other Legumes

A high-density linkage map of red clover was constructed based on SSR and RFLP markers. In order to construct a linkage map with user (breeder) friendly markers; i.e. informative and easy detection, two policies were adopted for marker development. One was that the markers should be derived from cDNA or gene-rich regions, and the other was that the SSR markers should be detected polymorphisms on agarose gels. We also discuss the transferability of the markers on the map to other red clover germplasm and legumes. Such highly transferable markers could be used to screen anchor markers for both on a consensus map of red clover and other legume species

Suppression of γ-Aminobutyric Acid (GABA) Transaminases Induces Prominent GABA Accumulation, Dwarfism and Infertility in the Tomato (Solanum lycopersicum L.)

Tomatoes accumulate γ-aminobutyric acid (GABA) at high levels in the immature fruits. GABA is rapidly converted to succinate during fruit ripening through the activities of GABA transaminase (GABA-T) and succinate semialdehyde dehydrogenase (SSADH). Although three genes encoding GABA-T and both pyruvate- and α-ketoglutarate-dependent GABA-T activities have been detected in tomato fruits, the mechanism underlying the GABA-T-mediated conversion of GABA has not been fully understood. In this work, we conducted loss-of-function analyses utilizing RNA interference (RNAi) transgenic plants with suppressed pyruvate- and glyoxylate-dependent GABA-T gene expression to clarify which GABA-T isoforms are essential for its function. The RNAi plants with suppressed SlGABA-T gene expression, particularly SlGABA-T1, showed severe dwarfism and infertility. SlGABA-T1 expression was inversely associated with GABA levels in the fruit at the red ripe stage. The GABA contents in 35S::SlGABA-T1RNAi lines were 1.3–2.0 times and 6.8–9.2 times higher in mature green and red ripe fruits, respectively, than the contents in wild-type fruits. In addition, SlGABA-T1 expression was strongly suppressed in the GABA-accumulating lines. These results indicate that pyruvate- and glyoxylate-dependent GABA-T is the essential isoform for GABA metabolism in tomato plants and that GABA-T1 primarily contributes to GABA reduction in the ripening fruits

Comprehensive Resources for Tomato Functional Genomics Based on the Miniature Model Tomato Micro-Tom

Tomato (Solanum lycopersicum L., Solanaceae) is an excellent model plant for genomic research of solanaceous plants, as well as for studying the development, ripening, and metabolism of fruit. In 2003, the International Solanaceae Project (SOL, www.sgn.cornell.edu ) was initiated by members from more than 30 countries, and the tomato genome-sequencing project is currently underway. Genome sequence of tomato obtained by this project will provide a firm foundation for forthcoming genomic studies such as the comparative analysis of genes conserved among the Solanaceae species and the elucidation of the functions of unknown tomato genes. To exploit the wealth of the genome sequence information, there is an urgent need for novel resources and analytical tools for tomato functional genomics. Here, we present an overview of the development of genetic and genomic resources of tomato in the last decade, with a special focus on the activities of Japan SOL and the National Bio-Resource Project in the development of functional genomic resources of a model cultivar, Micro-Tom

Mechanistic Insights into Validoxylamine A 7\u27-Phosphate Synthesis by VldE Using the Structure of the Entire Product Complex

The pseudo-glycosyltransferase VldE catalyzes non-glycosidic C-N coupling between an unsaturated cyclitol and a saturated aminocyclitol with the conservation of the stereochemical configuration of the substrates to form validoxylamine A 7′-phosphate, the biosynthetic precursor of the antibiotic validamycin A. To study the molecular basis of its mechanism, the three-dimensional structures of VldE from Streptomyces hygroscopicus subsp. limoneus was determined in apo form, in complex with GDP, in complex with GDP and validoxylamine A 7′-phosphate, and in complex with GDP and trehalose. The structure of VldE with the catalytic site in both an open and closed conformation is also described. With these structures, the preferred binding of the guanine moiety by VldE, rather than the uracil moiety as seen in OtsA could be explained. The elucidation of the VldE structure in complex with the entirety of its products provides insight into the internal return mechanism by which catalysis occurs with a net retention of the stereochemical configuration of the donated cyclitol. © 2012 Cavalier et al

Genome Structure of the Legume, Lotus japonicus

The legume Lotus japonicus has been widely used as a model system to investigate the genetic background of legume-specific phenomena such as symbiotic nitrogen fixation. Here, we report structural features of the L. japonicus genome. The 315.1-Mb sequences determined in this and previous studies correspond to 67% of the genome (472 Mb), and are likely to cover 91.3% of the gene space. Linkage mapping anchored 130-Mb sequences onto the six linkage groups. A total of 10 951 complete and 19 848 partial structures of protein-encoding genes were assigned to the genome. Comparative analysis of these genes revealed the expansion of several functional domains and gene families that are characteristic of L. japonicus. Synteny analysis detected traces of whole-genome duplication and the presence of synteny blocks with other plant genomes to various degrees. This study provides the first opportunity to look into the complex and unique genetic system of legumes