Synthesis and metal complexation of chiral 3-mono-or 3, 3-bis-allyl-2-hydroxypyrrolopyrazine-1, 4-diones

A novel synthesis of chiral cyclic hydroxamic acids (4, 6, 8 and 10) related to cyclodipeptides is described. The crucial reduction of the nitro group of the N-nitroacetyl derivatives of (S)-α-amino acid esters is brought about by zinc-aq. ammonium chloride. The FeIII and CuII complexes of one such cyclic hydroxamic acid 10a have been prepared and their DNAse activity investigated

Metal complexation and DNA cleavage activities of N-substituted hydroxamic acids

712-715Hydroxamic acid, RCON(R')OH, is a bidentate oxygen ligand possessing affinity for a variety of metal ions. The Cu(II) complexes of three N-substituted hydroxamic acids [R=C6H5; R'=C6H5, 4-CH3 -C6H4 and R'= C6H5.CH2] have been synthesized and characterized. Their DNA cleavage activities have been investigated using plasmid DNA. An oxidative mechanism for DNA cleavage by hydroxy radicals via abstraction of a H atom from sugar unit has been proposed.</span

Polarity sensing by fluorescent oligonucleotides: first demonstration of sequence-dependent microenvironmental changes in the DNA major groove

DNA duplex recognition by macromolecules (proteins, enzymes) and small molecules (drugs, metal complexes) occurs in the major or minor grooves of DNA via hydrogen bonding and electrostatic or hydrophobic interactions, whose strengths depend on the medium. It is therefore important to understand the local environments of the major/minor grooves of DNA. It is known that small molecules bind in the minor groove that have nonpolar character (ε = 22) and are organic-like. By employing fluorescent oligonucleotides, we demonstrated recently that the major groove of DNA is more polar than the minor groove (ε = 55) and the DNA double helix has different polarity in its two grooves. Our experimentally measured values were recently validated independently by two theoretical groups, employing calculations by two different methodologies. In this paper, a hitherto unknown property of DNA double helix ¯ sequence dependent local polarity or microenvironment - is experimentally demonstrated. This is shown by fluorescence experiments using oligonucleotides containing the modified base 5-amino-dU in place of dT and the polarity varied between ε ≈ 40 and 60, in different sequences. This is the first experimental demonstration of sequence-dependent microenvironmental effects in DNA in solution, although structural effects are well-known by X-ray data. The technique used has a good potential to investigate differing microenvoronmental effects in various secondary structures, DNA polymorphs, and chemically modified DNA and their hybrids

5-amido-(carboxyfluorescein)-2'-dU-oligonucloetides: novel primers for fluorescent detection of PCR amplified DNA

The synthesis of oligonucleotides containing a novel nucleobase fluorescent derivative 5-NH<SUB>2</SUB>-(carboxyfluorescein)-2'-deoxyuridine, in which the chromophore fluorescein is directly linked to nucleobase of 2'dU at C-5 via an amido function is described. The fluorescent oligonucleotides have been used as primers for PCR amplification of Human &#946;-globin gene fragment. Oligonucleotides containing multiple insertions of such fluorophores have potential applications in molecular biotechnology

Silencing Myostatin Using Cholesterol-conjugated siRNAs Induces Muscle Growth

Short interfering RNAs (siRNAs) are a valuable tool for gene silencing with applications in both target validation and therapeutics. Many advances have recently been made to improve potency and specificity, and reduce toxicity and immunostimulation. However, siRNA delivery to a variety of tissues remains an obstacle for this technology. To date, siRNA delivery to muscle has only been achieved by local administration or by methods with limited potential use in the clinic. We report systemic delivery of a highly chemically modified cholesterol-conjugated siRNA targeting muscle-specific gene myostatin (Mstn) to a full range of muscles in mice. Following a single intravenous injection, we observe 85–95% knockdown of Mstn mRNA in skeletal muscle and >65% reduction in circulating Mstn protein sustained for >21 days. This level of Mstn knockdown is also accompanied by a functional effect on skeletal muscle, with animals showing an increase in muscle mass, size, and strength. The cholesterol-conjugated siRNA platform described here could have major implications for treatment of a variety of muscle disorders, including muscular atrophic diseases, muscular dystrophy, and type II diabetes

Post-Synthetic Modification of Oligonucleotides via Orthogonal Amidation and Copper Catalyzed Cycloaddition Reactions

An efficient multicomponent orthogonal protocol was developed for post-synthetic oligonucleotide modification using commercially available 2′-<i>O</i>-methyl ester and 2′-<i>O</i>-propargyl nucleoside scaffolds. Amidation of methyl esters with primary amines was achieved in the presence of 2′-propargyl groups which were utilized for subsequent copper catalyzed cycloaddition with GalNAc-azide. The methodology was applied to generate siRNA composed of multiple amide and triazole conjugates. Computational methods were used to illustrate the impact of substitution at the 2′-position. This a powerful post-oligomerization technique for rapidly introducing diversity to oligonucleotide design

Facile Synthesis, Geometry, and 2′-Substituent-Dependent in Vivo Activity of 5′‑(<i>E</i>)- and 5′‑(<i>Z</i>)‑Vinylphosphonate-Modified siRNA Conjugates

(<i>E</i>)-Vinylphosphonate ((<i>E</i>)-VP), a metabolically stable phosphate mimic at the 5′-end of the antisense strand, enhances the in vivo potency of siRNA. Here we describe a straightforward synthetic approach to incorporate a nucleotide carrying a vinylphosphonate (VP) moiety at the 5′-end of oligonucleotides under standard solid-phase synthesis and deprotection conditions by utilizing pivaloyloxymethyl (POM) protected VP-nucleoside phosphoramidites. The POM protection enhances scope and scalability of 5′-VP-modified oligonucleotides and, in a broader sense, the synthesis of oligonucleotides modified with phosphonate moieties. Trivalent <i>N</i>-acetylgalactosamine-conjugated small interfering RNA (GalNAc-siRNA) comprising (<i>E</i>)-geometrical isomer of VP showed improved RISC loading with robust RNAi-mediated gene silencing in mice compared to the corresponding (<i>Z</i>)-isomer despite similar tissue accumulation. We also obtained structural insights into why bulkier 2′-ribosugar substitutions such as 2′-<i>O</i>-[2-(methylamino)-2-oxoethyl] are well tolerated only when combined with 5′-(<i>E</i>)-VP

RNAi-mediated rheostat for dynamic control of AAV-delivered transgenes

Here the authors propose an RNA interference-based switch for dynamic control of AAV transgene expression. In this approach, transgene expression may be silenced by RNAi and subsequently recovered using REVERSIR oligonucleotides