An Improved Synthesis of 2′-Deoxy-9-deazaadenosine and an N-7 Blocked Derivative Useful for the Synthesis of Modified Oligonucleotides Containing 2′-Deoxy-9-deazaadenosine

Abstract

As an epimerization resistant synthon in the synthesis of oligo-nucleotides consisting of C-nucleoside analogues, hitherto unknown 5-benzyloxy-methyl-3-(2-deoxy-β-D-erythro-pentofuranosyl)pyrrolo[3,2-d]pyrimpyrimidine (7-benzyloxymethyl-2′-deoxy-9-deazaadenosine) was prepared in seven steps from the known 3-amino-2-cyano-4-(2,3-O-isopropylidene-5-O-trityl-β-D-ribofuranosyl)-pyrrolpyrrole (1). Treatment of 1 with benzyl chloromethyl ether in the presence of potassium t-butoxide and 18-crown-6 afforded the N-protected pyrrole 2, which was converted into the 9-deazapurine derivative 3 in high yield by heating in EtOH. 7-Benzyloxymethyl-9-deazaadenosine 4 was obtained from 3 by acid hydrolysis in 2.5% methanolic hydrogen chloride. After protection of the hydroxyl groups of 4 with Markievicz's reagent, the product 5 was converted into the 2′-O-phenoxythiocarbonyl derivative 6. Reduction of 6 with butyltin hydride in the presence of 2,2′-azobis(2-methylpropionitrile), followed by desilylation with triethylammonium fluoride, afforded the desired 7-benzyloxymethyl-2′-deoxy-9-deazaadenosine (8) in high overall yield. The benzyloxymethyl group of 8 was removed by hydrogenolysis over palladium hydroxide (Degussa type) to give 2′-deoxy-9-deazaadenosine (9) in quantitative yield. The structure of 9 is discussed.     

Characterization of a Small Auxin-Up RNA (SAUR)-Like Gene Involved in Arabidopsis thaliana Development

The root of Arabidopsis thaliana is used as a model system to unravel the molecular nature of cell elongation and its arrest. From a micro-array performed on roots that were treated with aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, a Small auxin-up RNA (SAUR)-like gene was found to be up regulated. As it appeared as the 76th gene in the family, it was named SAUR76. Root and leaf growth of overexpression lines ectopically expressing SAUR76 indicated the possible involvement of the gene in the division process. Using promoter::GUS and GFP lines strong expression was seen in endodermal and pericycle cells at the end of the elongation zone and during several stages of lateral root primordia development. ACC and IAA/NAA were able to induce a strong up regulation of the gene and changed the expression towards cortical and even epidermal cells at the beginning of the elongation zone. Confirmation of this up regulation of expression was delivered using qPCR, which also indicated that the expression quickly returned to normal levels when the inducing IAA-stimulus was removed, a behaviour also seen in other SAUR genes. Furthermore, confocal analysis of protein-GFP fusions localized the protein in the nucleus, cytoplasm and plasma membrane. SAUR76 expression was quantified in several mutants in ethylene and auxin-related pathways, which led to the conclusion that the expression of SAUR76 is mainly regulated by the increase in auxin that results from the addition of ACC, rather than by ACC itself.     

Ancient Evolutionary Trade-Offs between Yeast Ploidy States

The number of chromosome sets contained within the nucleus of eukaryotic organisms is a fundamental yet evolutionarily poorly characterized genetic variable of life. Here, we mapped the impact of ploidy on the mitotic fitness of baker''s yeast and its never domesticated relative Saccharomyces paradoxus across wide swaths of their natural genotypic and phenotypic space. Surprisingly, environment-specific influences of ploidy on reproduction were found to be the rule rather than the exception. These ploidy–environment interactions were well conserved across the 2 billion generations separating the two species, suggesting that they are the products of strong selection. Previous hypotheses of generalizable advantages of haploidy or diploidy in ecological contexts imposing nutrient restriction, toxin exposure, and elevated mutational loads were rejected in favor of more fine-grained models of the interplay between ecology and ploidy. On a molecular level, cell size and mating type locus composition had equal, but limited, explanatory power, each explaining 12.5%–17% of ploidy–environment interactions. The mechanism of the cell size–based superior reproductive efficiency of haploids during Li⁺ exposure was traced to the Li⁺ exporter ENA. Removal of the Ena transporters, forcing dependence on the Nha1 extrusion system, completely altered the effects of ploidy on Li⁺ tolerance and evoked a strong diploid superiority, demonstrating how genetic variation at a single locus can completely reverse the relative merits of haploidy and diploidy. Taken together, our findings unmasked a dynamic interplay between ploidy and ecology that was of unpredicted evolutionary importance and had multiple molecular roots.     

Schisandra henryi C. B. Clarke in vitro cultures: a promising tool for the production of lignans and phenolic compounds

Plant Cell, Tissue and Organ Culture (PCTOC) - We initiated and optimized in vitro culture conditions of the endemic Chinese plant species—Schisandra henryi C. B. Clarke. Different types of...     

Involvement of Schizosaccharomyces pombe rrp1+ and rrp2+ in the Srs2- and Swi5/Sfr1-dependent pathway in response to DNA damage and replication inhibition

Previously we identified Rrp1 and Rrp2 as two proteins required for the Sfr1/Swi5-dependent branch of homologous recombination (HR) in Schizosaccharomyces pombe. Here we use a yeast two-hybrid approach to demonstrate that Rrp1 and Rrp2 can interact with each other and with Swi5, an HR mediator protein. Rrp1 and Rrp2 form co-localizing methyl methanesulphonate–induced foci in nuclei, further suggesting they function as a complex. To place the Rrp1/2 proteins more accurately within HR sub-pathways, we carried out extensive epistasis analysis between mutants defining Rrp1/2, Rad51 (recombinase), Swi5 and Rad57 (HR-mediators) plus the anti-recombinogenic helicases Srs2 and Rqh1. We confirm that Rrp1 and Rrp2 act together with Srs2 and Swi5 and independently of Rad57 and show that Rqh1 also acts independently of Rrp1/2. Mutants devoid of Srs2 are characterized by elevated recombination frequency with a concomitant increase in the percentage of conversion-type recombinants. Strains devoid of Rrp1 or Rrp2 did not show a change in HR frequency, but the number of conversion-type recombinants was increased, suggesting a possible function for Rrp1/2 with Srs2 in counteracting Rad51 activity. Our data allow us to propose a model placing Rrp1 and Rrp2 functioning together with Swi5 and Srs2 in a synthesis-dependent strand annealing HR repair pathway.     

47 Incorporation of alternative nucleic acid chemistries into G-quadruplex structure

Nucleic acids that form G-quadruplex (G4) structure have found applications in a host of research and technology regimes. Numerous G4 based aptamer drugs have been identified with pharmacological activity against cancer, HIV, prions, and blood coagulation (1). In the field of nanotechnology, G4 based sensors and nano-machines have also received much attention. The ability to synthesize nucleic acid ex-vivo allows for the site-specific incorporation of non-natural chemistries into nucleic acids that can be used to tune their physical and pharmacological properties. We summarize the results of a series of studies investigating the effective incorporation of alternative nucleic acid chemistries into G4 DNA. These modified chemistries include C8-modified guanine bases, as well as 2′-F, 2′-F-ANA, and Locked nucleic acid (LNA) modifications to the ribose sugar. We report primarily on the effect of these modifications on G-quadruplex folding topology, thermal stability, and structure. The substitution of LNA-guanosine into the core guanine tetrads disrupts structure in specific structural environments. On the other hand, 2′-F- and 2′-F-ANA guanosine can generally be incorporated without disrupting the structure when substituted into guanine bases in certain structural conformations. We find that 2′-F-ANA-guanosine and 2′-F-guanosine are powerful tools for controling the conformation of G4 structures (2). Functionalization at the C8 of the guanine base stabilizes in a manner dependent on the glycosidic conformation of the base, with different modification chemistries stabilizing to varying extents (3). The results of these studies provide useful insight on how to effectively incorporate some useful chemical tools from the growing toolbox of modified nucleic acid chemistries into G-quadruplex nucleic acid.     

Transgenerational effects and the cost of ant tending in aphids

Ant protection of extrafloral nectar (EFN)-secreting plants is a common form of mutualism found in most habitats around the world. However, very few studies have considered these mutualisms from the ant, rather than the plant, perspective. In particular, a whole-colony perspective that takes into account the spatial structure and nest arrangement of the ant colonies that visit these plants has been lacking, obscuring when and how colony-level foraging decisions might affect tending rates on individual plants. Here, we experimentally demonstrate that recruitment of Crematogaster opuntiae (Buren) ant workers to the EFN-secreting cactus Ferocactus wislizeni (Englem) is not independent between plants up to 5 m apart. Colony territories of C. opuntiae are large, covering areas of up to 5,000 m², and workers visit between five and 34 EFN-secreting barrel cacti within the territories. These ants are highly polydomous, with up to 20 nest entrances dispersed throughout the territory and interconnected by trail networks. Our study demonstrates that worker recruitment is not independent within large polydomous ant colonies, highlighting the importance of considering colonies rather than individual workers as the relevant study unit within ant/plant protection mutualisms.