The reactivity of 5-amino-3H-1,2,4-dithiazole-3-thiones substituted at their amino group and 5-amino-3H-1,2-dithiole-3-thiones substituted at their amino group and C4 toward compounds containing ...P(III) atoms has been studied. N,N-Disubstituted-N′-(3-thioxo-3H-1,2,4-dithiazol-5-yl)methanimidamides were selected as novel efficient sulfur transfer reagents suitable for DNA and RNA synthesis.
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Huntington's disease is an autosomal dominant neurodegenerative disease caused by a toxic gain of function mutation in the huntingtin gene (Htt). Silencing of Htt with RNA interference using direct ...CNS delivery in rodent models of Huntington's disease has been shown to reduce pathology and promote neuronal recovery. A key translational step for this approach is extension to the larger non-human primate brain, achieving sufficient distribution of small interfering RNA targeting Htt (siHtt) and levels of Htt suppression that may have therapeutic benefit. We evaluated the potential for convection enhanced delivery (CED) of siHtt to provide widespread and robust suppression of Htt in nonhuman primates. siHtt was infused continuously for 7 or 28days into the nonhuman primate putamen to analyze effects of infusion rate and drug concentration on the volume of effective suppression. Distribution of radiolabeled siHtt and Htt suppression were quantified by autoradiography and PCR, respectively, in tissue punches. Histopathology was evaluated and Htt suppression was also visualized in animals treated for 28days. Seven days of CED led to widespread distribution of siHtt and significant Htt silencing throughout the nonhuman primate striatum in an infusion rate and dose dependent manner. Htt suppression at therapeutic dose levels was well tolerated by the brain. A model developed from these results predicts that continuous CED of siHtt can achieve significant coverage of the striatum of Huntington's disease patients. These findings suggest that this approach may provide an important therapeutic strategy for treating Huntington's disease.
► Chronic convection enhanced delivery of siRNA targeting Huntingtin in primate brain. ► Widespread drug distribution results in Huntingtin lowering throughout striatum. ► Empirical model of data enables scaling of siRNA delivery. ► Potential as key disease-modifying therapeutic approach for Huntington's disease.
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This unit attempts to provide a reasonably complete inventory of over 280 solid supports available to oligonucleotide chemists for preparation of natural and 3'-modified oligonucleotides. Emphasis is ...placed on non-nucleosidic solid supports. The relationship between the structural features of linkers and their behavior in oligonucleotide synthesis and deprotection is discussed wherever the relevant observations are available.
A novel, conformationally preorganized nonnucleosidic universal solid support for oligonucleotide synthesis was developed. The solid support featured two chemically equivalent hydroxy groups locked ...in syn-periplanar orientation and orthogonally protected with 4,4‘-dimethoxytrityl and acetyl groups. The solid support was extensively tested in the preparation of oligonucleotides and their phosphorothioate analogues containing 2‘-deoxy, 2‘-O-methyl, and 2‘-O-methoxyethylnucleoside residues at the 3‘-terminus. Upon completion of oligonucleotide chain assembly, the support-bound oligonucleotide material was treated with concentrated ammonium hydroxide, which removed the O-acetyl protection. The deprotected hydroxy group then effected the transesterification of a phosphate linkage between the solid support and the 3‘-terminal nucleoside residue to result in a facile release of the oligonucleotide to solution. The kinetics of the release process was studied in a continuous flow of concentrated aqueous ammonium hydroxide at a temperature of 300.15 K. Optimal conditions for the release of oligonucleotides depending on the chemistry of the backbone and 3‘-terminal nucleoside residue were formulated.
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Specific step-by-step instructions for conversion of 5'-O-(4,4'-dimethoxytrityl)- and base-protected nucleosides and other mono-O-(4,4'-dimethoxytrityl)-protected diols to their hemisuccinate esters ...and their coupling to CPG (controlled-pore glass) supports bearing aminopropyl or long chain aminoalkyl groups are presented. Additional guidelines are provided for selecting a coupling protocol and performing in-process control.
A number of 5‘-O-(4,4‘-dimethoxytrityl)thymidine N,N-diisopropylamino phosphoramidites protected at P(III) with derivatives of 2-benzamidoethanol were synthesized and incorporated into synthetic ...oligonucleotides. Depending on substitution patterns at the alkyl chain, amido group, and phenyl ring, the time required for removal of these protecting groups using concentrated ammonium hydroxide varied from 48 h at 55 °C to 25 min at 25 °C. Of the 11 groups studied, 2-N-isopropyl-N- (4-methoxybenzoyl)aminoethyl- (H) and ω-(thionobenzoylamino)alkyl protections (I and K) were most easily removed. Derivatives of the 2-N-methyl-N-benzoylaminoethyl group (E−G) demonstrated moderate stability, but those of the 2-(N-benzoylamino)ethyl group (A−C) were the most stable. For the most reactive group, H, a phosphitylating reagent, bisamidite 60, was synthesized and used in the preparation of four deoxynucleoside phosphoramidites 28 and 65−67, plus the 2‘-O-(2-methoxyethyl)-5-methyluridine phosphoramidite 68. All of these novel building blocks were successfully tested in the preparation of natural, 20-mer oligonucleotides and their phosphorothioate analogues. With the model phosphotriester 37, the mechanism of deprotection was studied and revealed, in the case of group H, a pH-independent formation of the 2-oxazolinium cation 47. Under aqueous conditions, 47 gave 54, which in turn was converted in the presence of ammonia to a number of identified products. It is important to note that none of the products formed was reactive toward the oligonucleotide backbone or nucleic bases. Thus, a general strategy for protection of internucleosidic phosphodiester groups is described, which may also find application in synthetic organic chemistry of phosphorus(III) and (V).
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H-Phosphonate monomers of 2‘-O-(2-methoxyethyl) ribonucleosides have been synthesized. Oxidation of oligonucleotide H-phosphonates has been optimized to allow the synthesis of oligonucleotides ...containing either 2‘-deoxy or 2‘-O-(2-methoxyethyl) ribonucleoside residues combined with three different phosphate modifications in the backbone, i.e., phosphodiester (PO), phosphorothioate (PS), and phosphoramidate (PN). Phosphodiester linkages were introduced by oxidation with a cocktail of 0.1 M Et3N in CCl4/Pyr/H2O (5:9:1) without affecting phosphorothioate or phosphoramidate linkages. For the synthesis of phosphoramidate-modified oligonucleotides, N 4-acetyl deoxycytidine-3‘-H-phosphonate monomers were used to avoid transamination during the oxidation step.
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A novel, selective labeling of oligonucleotides with two different reporter groups is described. The oligonucleotide is synthesized using a stable 2-(4-methoxybenzamido)ethyl protection for a ...selected internucleosidic thiophosphate (PS) and a labile 2-(N-isopropyl-4-methoxybenzamido)ethyl for the 3‘-terminal PS and internucleosidic phosphates. The latter group and the base protection are removed, and the 3‘-terminal PS is labeled. The former protection is then cleaved by a prolonged ammonolysis, and the second reporter is introduced at the internucleosidic PS.
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Two novel phosphoramidite building blocks and a solid support that allow an efficient solid-phase phosphorylation or thiophosphorylation of synthetic oligonucleotides were developed. The utility of ...these synthetic tools was demonstrated in the preparation of 5′- or 3′-thiophosphorylated oligonucleotides, which were subsequently labeled at the termini with fluorescent reporters.
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