化学修饰对反义寡核苷酸稳定性及抗流感病毒活性的影响

为了探讨 Ａ Ｓ Ｏ Ｄ Ｎ 化学修饰形式与 Ａ Ｓ Ｏ Ｄ Ｎ 稳定性,体外细胞毒性以及抗流感病毒活性之间的关系,合成了 ７ 种不同化学修饰形式的 Ａ Ｓ Ｏ Ｄ Ｎ：硫代 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端分别磷酸化和胆固醇修饰;３′与 ５′端硫代,中间为天然结构的混合骨架 Ａ Ｓ Ｏ Ｄ Ｎ;天然结构 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端分别磷酸化和胆固醇修饰等．测定了 ７ 种修饰体在小鼠血清, Ｍ Ｄ Ｃ Ｋ 细胞裂解液,含 ２％ 胎牛血清的 Ｄ Ｍ Ｅ Ｍ培养液以及水中的稳定性,体外细胞毒性和在细胞水平抗流感病毒活性．结果表明,混合骨架 Ａ Ｓ Ｏ Ｄ Ｎ,硫代 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端接磷酸和胆固醇的修饰形式在小鼠血清, Ｍ Ｄ Ｃ Ｋ 细胞裂解液与含２％ 胎牛血清的 Ｄ Ｍ Ｅ Ｍ 培养液中稳定性相对较高,作用 ２４～４８ ｈ 仅混合骨架 Ａ Ｓ Ｏ Ｄ Ｎ 与硫代 Ａ Ｓ Ｏ Ｄ Ｎ 发生部分降解;天然结构 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端接磷酸和胆固醇修饰体在 ２４ ｈ 内大部分降解．所有 Ａ Ｓ Ｏ Ｄ Ｎ 修饰体在水中具有很高稳定性,４８ ｈ 内未见降解作用．７ 种 Ａ Ｓ Ｏ Ｄ Ｎ 修饰形式在 Ｍ Ｄ Ｃ Ｋ 细胞中未表现明显的细胞毒性．硫代 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端接磷酸和胆     

Synthesis and properties of novel acyclic nucleotides.

Acyclic adenosine and thymidine analogs derived from L- and D-threonie were synthesised and incorporated into oligonucleotides by automated protocols using a standard phosphoramidite method. UV melting experiments with thus obtained oligonucleotides showed that incorporation of those acyclic nucleosides did not destabilize the hybrid duplexes and that the stabilities of them are influenced by the stereochemical structures of acyclic analogs. Modification of 3'-end of oligonucleotide with acyclic analogs protected the oligonucleotide against 3'-exonuclease.     

Synthesis and evaluation of novel dolastatin 10 derivatives for versatile conjugations

Dolastatin 10 (1) is a highly potent cytotoxic microtubule inhibitor (cytotoxicity IC₅₀?<?5.0?nM) and several of its analogs have recently been used as payloads in antibody drug conjugates. Herein, we describe the design and synthesis of a series of novel dolastatin 10 analogs useful as payloads for conjugated drugs. We explored analogs containing functional groups at the thiazole moiety at the C-terminal of dolastatin 10. The functional groups included amines, alcohols, and thiols, which are representative structures used in known conjugated drugs. These novel analogs showed excellent potency in a tumor cell proliferation assay, and thus this series of dolastatin 10 analogs is suitable as versatile payloads in conjugated drugs. Insights into the structure–activity relationships of the analogs are also discussed.     

Synthesis of chimeric oligonucleotides containing internucleosidic phosphodiester and S-pivaloylthioethyl phosphotriester residues

Novel oligonucleotide analogs that bear phosphodiester and bioreversible S-pivaloyl 2-mercaptoethyl (SPME) phosphate triester internucleosidic linkages are described. Their synthesis employs a novel methodology of oligonucleotide deprotection under mild, non-aqueous conditions.     

SYNTHESIS OF CHIMERIC OLIGONUCLEOTIDES CONTAINING INTERNUCLEOSIDIC PHOSPHODIESTER AND S–PIVALOYLTHIOETHYL PHOSPHOTRIESTER RESIDUES

Novel oligonucleotide analogs that bear phosphodiester and bioreversible Õ–pivaloyl 2–mercaptoethyl (SPME) phosphate triester internucleosidic linkages are described. Their synthesis employs a novel methodology of oligonucleotide deprotection under mild, non-aqueous conditions.     

Sequencing of oligonucleotide phosphorothioates based on solid-supported desulfurization.

We described a solid-supported desulfurization procedure allowing easy access to the sequence analysis of oligonucleotide phosphorothioates. The described method is based upon selective removal of the 2-cyanoethyl phosphate protecting groups, followed by iodine-promoted desulfurization of the resulting phosphorothioate diesters. Automatic oxidation of oligonucleotide phosphorothioates, anchored via an ester linkage to a standard solid support (LCAA/CPG), is combined with Maxam-Gilbert solid-support sequencing. The overall procedure allows rapid simultaneous sequence analysis of several oligonucleotide analogs.     

Fluorescent pteridine nucleoside analogs

Pteridine nucleoside analog probes are highly fluorescent and offer different approaches to monitor subtle DNA interactions with other molecules. Similarities in structure and size to native nucleosides make it possible to incorporate these probes into oligonucleotides through the standard deoxyribose linkage. These probes are formulated as phosphoramidites and incorporated into oligonucleotides using automated DNA synthesis. Their position within the oligonucleotide renders them exquisitely sensitive to changes in structure as the oligonucleotide meets and reacts with other molecules. Changes are measured through fluorescence intensity, anisotropy, lifetimes, spectral shifts, and energy transfer. The fluorescence properties of pteridine nucleoside analogs as monomers and incorporated into single and double stranded oligonucleotides are reviewed. The two guanosine analogs, 3MI and 6MI, and two adenosine analogs, 6MAP and DMAP, are reviewed in detail along with applications utilizing them.     

Conjugation of oligonucleotides via an electrophilic tether: N-chloroacetamidohexyl phosphoramidite reagent

A novel method for the preparation of oligonucleotides conjugated with nucleophilic ligands is described. A new phosphoramidite building block derived from N-chloroacetyl-6-aminohexanol is attached at the 5′-terminus on the last step of oligonucleotide synthesis. Postsynthetic treatment of support-bound modified oligonucleotide with a variety of amines and mercaptans affords conjugates in high yield.     

Synthesis of Chimerical Oligonucleotides Containing Internucleosidic Phosphodiester and S-Pivaloyl Mercaptoethyl Phosphotriester Linkages

Abstract

Novel oligonucleotide analogs that bear phosphodiester and bioreversible S-pivaloyl 2-mercaptoethyl (SPME) phosphate triester internucleosidic linkages and their thioate analogs are described. Their synthesis involves new methodology for the deprotection of base-labile oligonucleotides.     

Stabilization of DNA double and triple helices by conjugation of minor groove binders to oligonucleotides

New conjugates containing two parallel or antiparallel carboxamide minor groove binders (MGB) attached to the same terminal phosphate of one oligonucleotide strand were synthesized. The conjugates interact with their target DNA stronger than the individual components. Effect of conjugated MGB on DNA duplex and triplex stability and their sequence specificity was demonstrated on the short oligonucleotide duplexes and on the triplex formed by model 16-mer oligonucleotide with HIV polypurine tract.     

Mutagenesis, directed by artificial oligonucleotide analogs. I. Participation of ribonucleotide fragments in DNA replication and repair

The mutation system has been developed to study the mutagenic properties of modified oligonucleotide analogs. The mutagenic properties of oligonucleotides containing one ribonucleotide have been examined. The presence of a ribonucleotide is shown not to induce any mutations. But when the oligonucleotide induces two marker deletions detached by 6 nucleotides they may be repaired separately, in this case the deletion bordering with the ribonucleotide is predominantly repaired.     

DNA or RNA oligonucleotide 2'-hydrazides for chemoselective click-type ligation with carbonyl compounds

An efficient method for the synthesis of DNA or RNA oligonucleotide 2'-hydrazides is described. Fully deprotected oligonucleotides containing a hydrazide group at the 2'-position of a uridine residue were obtained by a novel two-step procedure: periodate cleavage of an oligonucleotide with 1,2-diol group followed by conversion of the aldehyde to hydrazide with an extended linker arm using a homobifunctional reagent succinic dihydrazide and NaBH(3)CN. The resulting oligonucleotide 2'-hydrazides were efficiently conjugated by a click-type reaction at acidic pH to aliphatic or aromatic aldehydes with or without NaBH(3)CN reduction to afford novel 2'-conjugates.     

Incorporation of two modified nucleosides allows selective platination of an oligonucleotide making it suitable for duplex cross-linking

Platinated oligonucleotides are promising tools for the control of gene expression, since they may target and cross-link nucleic acid chains. Here we describe a method for the preparation of platinated oligonucleotides that has proved able to selectively cross-link complementary sequences, making use of 5-methylcytidine analogs with thioether or imidazole groups attached to the 4-position. These nucleoside analogs were derivatized as phosphoramidites and introduced in oligonucleotide chains using standard phosphite triester chemistry. Different oligonucleotide sequences containing either one or two analogs appending from the 5′-end were synthesized and used in preliminary platination studies. The reaction of transplatin with oligonucleotides containing the thioether-modified nucleobase was fast, but generally afforded unstable adducts and complex reaction mixtures. The imidazole-containing oligonucleotides reacted with transplatin much more slowly, in particular at slightly basic pH, and it was found that the imidazole-modified cytosine was less reactive than the natural nucleobases. In contrast, transplatin selectively reacted with the thioether and imidazole groups of oligonucleotides containing the two cytosine analogs in neighboring positions, even in the presence of the four nucleobases and particularly three guanines, affording platinated oligonucleotides suitable for cross-linking. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Incorporation of an aldehyde function in oligonucleotides

A nucleotide-like phosphoramidite building block that has the nucleic base replaced by the tert-butyldimethylsilyl-protected styrene glycol was synthesized. After the automatic synthesis of an oligonucleotide incorporating this synthon, the benzaldehyde function was generated by fluoride deprotection and oxidation by sodium periodate. In a similar manner, an oligonucleotide where a nucleic base was replaced by the (CH2)8CH=O chain was synthesized and conjugated with biotin derivatives.     

Stabilization of DNA Double and Triple Helices by Conjugation of Minor Groove Binders to Oligonucleotides

Abstract

New conjugates containing two parallel or antiparallel carboxamide minor groove binders (MGB) attached to the same terminal phosphate of one oligonucleotide strand were synthesized. The conjugates interact with their target DNA stronger than the individual components. Effect of conjugated MGB on DNA duplex and triplex stability and their sequence specificity was demonstrated on the short oligonucleotide duplexes and on the triplex formed by model 16-mer oligonucleotide with HIV polypurine tract.     

Base-pairing systems related to TNA containing phosphoramidate linkages: synthesis of building blocks and pairing properties

As part of a project that aims at screening TNA-related oligonucleotide systems in which threose backbone units may have some or all of their oxygen functions replaced by nitrogen, two TNA analogs containing (2'NH)- and (3'NH)-phosphoramidate groups, respectively, in place of phosphodiester groups were synthesized. They show base-pairing properties that are very similar to those of TNA itself. We also synthesized 2',3'-diamino analogs of alpha-L-threofuranosyl mononucleosides, yet attempts to convert them to TNA analogs containing phosphodiamidate linker groups were not successful. Such 2',3'-diamino derivatives of threofuranosyl nucleosides may be of interest, however, as building blocks of TNA analogs that contain non-phosphorous linker groups.     

RNase H cleavage of RNA hybridized to oligonucleotides containing methylphosphonate, phosphorothioate and phosphodiester bonds.

Three types of 14-mer oligonucleotides were hybridized to human beta-globin pre-mRNA and the resultant duplexes were tested for susceptibility to cleavage by RNase H from E. coli or from HeLa cell nuclear extract. The oligonucleotides contained normal deoxynucleotides, phosphorothioate analogs alternating with normal deoxynucleotides, or one to six methylphosphonate deoxynucleosides. Duplexes formed with deoxyoligonucleotides or phosphorothioate analogs were susceptible to cleavage by RNase H from both sources, whereas a duplex formed with an oligonucleotide containing six methylphosphonate deoxynucleosides alternating with normal deoxynucleotides was resistant. Susceptibility to cleavage by RNase H increased parallel to a reduction in the number of methylphosphonate residues in the oligonucleotide. Stability of the oligonucleotides in the nuclear extract from HeLa cells was also tested. Whereas deoxyoligonucleotides were rapidly degraded, oligonucleotides containing alternating methylphosphonate residues remained unchanged after 70 minutes of incubation. Other oligonucleotides exhibited intermediate stability.     

Synthesis and hybridization data of oligonucleotide analogs with triazole internucleotide linkages, potential antiviral and antitumor agents

Triazolyl-functionalized oligonucleotide (ON) analogs have received much attention as potential antitumor and antiviral agents. The most promising of such analogs are those exhibiting high binding affinity toward native DNA/RNA, since they may prove to be efficient antisense or siRNA agents. To date, relatively few ON analogs with triazole internucleotide linkages have been described. In this paper, we report an improved synthesis of a modified dinucleoside phosphoramidite and hybridization data of ON analogs with four-bond triazole internucleotide linkages. We believe these data are essential for comprehensive analysis of the relation between the length of triazole internucleotide linkages and duplex stability.     

Oligonucleotide therapy

Rapid progress in oligonucleotide therapeutics has continued over the past year as major programs established in the past four years have grown and begun to be productive. Important advances were reported in the medicinal chemistry of oligonucleotides and in understanding their pharmacodynamic properties. Significant progress was made in understanding the pharmacokinetic and toxicologic properties of first generation analogs, particularly phosphorothioates and one oligonucleotide, ISIS 2105, entered clinical trials. Additionally, combinatorial approaches designed to identify oligonucleotides that may bind to a variety of targets were reported.     

Label‐free DNA sensor based on fluorescent cationic polythiophene for the sensitive detection of hepatitis B virus oligonucleotides

Water‐soluble fluorescent conjugated polymers can be used as an optical platform in highly sensitive DNA sensors. Here we report a simple label‐free DNA sensor using poly(3‐alkoxy‐4‐methylthiophene) to recognize and detect different oligonucleotide targets related to the YMDD gene mutation of hepatitis B virus. The concentration of surfactant Triton X‐100, NaCl, the oligonucleotide capture probe and the oligonucleotide hybridization conditions have a great impact on fluorescence intensity. Under the optimum conditions, two types of oligonucleotide targets involving YMDD gene mutation of hepatitis B virus were successfully recognized. Moreover, there was a linear relationship between fluorescence intensity and the concentration of oligonucleotide target. The detection limit of the wild‐type hepatitis B virus target is 88 pmol L^?1. Copyright © 2009 John Wiley & Sons, Ltd.