Synthesis and properties of morpholino chimeric oligonucleotides

Chimeric oligonucleotides with the novel morpholino modification and the phosphoramidate linkers have been synthesized and characterized. These oligonucleotides showed moderate thermal stability with complementary RNA and DNA, and enhanced resistance toward the nuclease (t_1/2 > 10 h). The phosphoramidate linker made the synthesis of such oligonucleotides applicable on a DNA synthesizer. Under the acidic condition (pH 3.0), the phosphoramidate linkers were readily cleaved, and such property might be useful for the DNA-sequence determination.     

A versatile reagent for the synthesis of 5′-phosphorylated, 5′-thiophosphorylated or 5′-phosphoramidate-conjugated oligonucleotides

We report the synthesis of a new phosphorylating reagent that is easily accessible and allows not only the chemical synthesis of 5′-phosphorylated and 5′-thiophosphorylated oligonucleotides but also the 5′-conjugation through a phosphoramidate linkage. 5′-Amino-linker and 5′-alkyne oligonucleotides were obtained and the latter was conjugated by a 1,3-dipolar cycloaddition (click chemistry) with a galactosylated azide derivative to afford 5′-galactosyl oligonucleotide with high efficiency.     

Phosphoramidite coupling to oligonucleotides bearing unprotected internucleosidic phosphate moieties

The coupling of 2-cyanoethyl thymidine phosphoramidite to solid-support-bound, phosphate-unprotected oligothymidylates and their phosphorothioate analogues was studied. The yield of the coupling reaction depended on the pK(BH)()+ values of protonated nitrogen bases that served as counterions to the phosphodiester functions of oligonucleotides. To maximize the coupling efficiency, the oligonucleotides were detritylated and washed with a mixture of 0.1 M DMAP and 0.1 M 1H-tetrazole, which resulted in a 98+% coupling efficiency. The utility of the results was demonstrated in the preparation of oligonucleotides with a mixed backbone that required the successive use of H-phosphonate and phosphoramidite methods of synthesis. Using this approach, 20-mer antisense oligonucleotides containing 2'-O-(2-methoxyethyl) ribonucleoside residues and phosphorothioate and phosphoramidate internucleosidic linkages were synthesized in high yield.     

Synthesis of chimeric oligonucleotides containing phosphodiester, phosphorothioate, and phosphoramidate linkages

[reaction: see text] 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 Et(3)N in CCl(4)/Pyr/H(2)O (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.     

A phosphoramidate substrate analog is a competitive inhibitor of the Tetrahymena group I ribozyme

BACKGROUND: Phosphoramidate oligonucleotide analogs containing N3'-P5' linkages share many structural properties with natural nucleic acids and can be recognized by some RNA-binding proteins. Therefore, if the N-P bond is resistant to nucleolytic cleavage, these analogs may be effective substrate analog inhibitors of certain enzymes that hydrolyze RNA. We have explored the ability of the Tetrahymena group I intron ribozyme to bind and cleave DNA and RNA phosphoramidate analogs. RESULTS: The Tetrahymena group I ribozyme efficiently binds to phosphoramidate oligonucleotides but is unable to cleave the N3'-P5' bond. Although it adopts an A-form helical structure, the deoxyribo-phosphoramidate analog, like DNA, does not dock efficiently into the ribozyme catalytic core. In contrast, the ribo-phosphoramidate analog docks similarly to the native RNA substrate, and behaves as a competitive inhibitor of the group I intron 5' splicing reaction. CONCLUSIONS: Ribo-N3'-P5' phosphoramidate oligonucleotides are useful tools for structural and functional studies of ribozymes as well as protein-RNA interactions.     

Structural arrangement of DNA constrained by a cross-linker

Self-complementary cross-linked oligonucleotides with a disulfide linkage were designed and synthesized. Double helix and hairpin structures were controlled using the different diastereochemistry of phosphoramidate where the cross-linker was introduced. The structures of the strained cross-linked DNAs were estimated by gel mobility, circular dichroism spectra, and melting profiles.     

Cleavage of oligonucleotides containing a P3'→N5' phosphoramidate linkage mediated by single-stranded oligonucleotide templates

Double-stranded DNA (dsDNA) templates can hybridize to and accelerate cleavage of oligonucleotides containing a P3'→N5' phosphoramidate (P-N) linkage. This dsDNA-templated cleavage of P-N linkages could be due to conformational strain placed on the linkage upon triplex formation. To determine whether duplex formation also induced conformational strain, we examined the reactivity of the oligonucleotides with a P-N linkage in the presence of single-stranded templates, and compared these reactions to those with dsDNA templates. P-N oligonucleotides that are cleaved upon duplex formation could be used as probes to detect single-stranded nucleic acids.     

Base-pairing systems related to TNA: alpha-threofuranosyl oligonucleotides containing phosphoramidate linkages

(3'NH)- and (2'NH)-TNA, two isomeric phosphoramidate analogues of TNA (alpha-threofuranosyl-(3'-->2') oligonucleotides), are shown to be efficient Watson-Crick base-pairing systems and to undergo intersystem cross-pairing with TNA, RNA, and DNA. [reaction: see text]     

Synergistic stabilization of nucleic acid assembly by oligo-N3'-->P5' phosphoramidate modification and additions of comb-type cationic copolymers

Synergic stabilization of DNA triplexes by oligo-N3'-->P5' phosphoramidate (PN) modification and additions of comb-type cationic copolymers was demonstrated. The combination of the copolymer and the PN modification increased triplex K(a) about 4 orders of magnitude. Kinetic analysis revealed that observed stabilization resulted from kinetic complimentarity between increased association rates by the copolymer and decreased dissociation rates by the PN modification of triplex forming oligonucleotides. No countering interference between these stabilizing effects was observed. We propose that kinetic analyses of stabilizing effects permit selection of a rational combination of stabilizing methods for successful synergy in stabilizing complex formation.     

Insertion of Chemical Handles into the Backbone of DNA during Solid-Phase Synthesis by Oxidative Coupling of Amines to Phosphites

Conjugation of molecules or proteins to oligonucleotides can improve their functional and therapeutic capacity. However, such modifications are often limited to the 5′ and 3′ end of oligonucleotides. Herein, we report the development of an inexpensive and simple method that allows for the insertion of chemical handles into the backbone of oligonucleotides. This method is compatible with standardized automated solid-phase oligonucleotide synthesis, and relies on formation of phosphoramidates. A unique phosphoramidite is incorporated into a growing oligonucleotide, and oxidized to the desired phosphoramidate using iodine and an amine of choice. Azides, alkynes, amines, and alkanes have been linked to oligonucleotides via internally positioned phosphoramidates with oxidative coupling yields above 80 %. We show the design of phosphoramidates from secondary amines that specifically hydrolyze to the phosphate only at decreased pH. Finally, we show the synthesis of an antibody-DNA conjugate, where the oligonucleotide can be selectively released in a pH 5.5 buffer.     

Phosphorylation Within DNA Duplexes. Synthesis of Modified Oligodeoxyribonucleotide

Abstract

An effective method was suggested for the activation of phos-phomonoester groups in nicks of a double-strand DNA (1,2). This approach allows to incorporate various sugar phosphate backbone modifications at a particular site when DNA duplexes are being assembled. A modifying group is first introduced at the 5′- or 3′-termini of oligonucleotides, then a duplex is formed and oligomers are coupled on the complementary template using water-soluble l-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide or cyanogen bromide as the condensing agents. Various DNA duplexes containing not only natural phosphodiester but also phosphoramidate and pyrophospha-te internucleotide bonds, as well as phosphodiester bonds between nucleotide residues with modified sugar analogs (ribo-, arabino- and xylo-derivatives) were assembled by this method.     

Synthesis and structure of silicon-containing <Emphasis Type="Italic">N</Emphasis>-methyland <Emphasis Type="Italic">N</Emphasis>-benzoylamides of diisopropylphosphoric acid

Diisopropyl N-benzoyl-N-(trimethylsilyl)phosphoramidate reacts with ClCH₂SiMe₂Cl under mild conditions to form diisopropyl N-benzoyl-N-[(chlorodimethylsilyl)methyl]phosphoramidate (III). Diisopropyl N-methyl-N-(trimethylsilyl)phosphoramidate with ClCH₂SiMe₂Cl affords an N-transsilylation product which does not rearrange into diisopropyl N-[(chlorodimethylsilyl)methyl]-N-methylphosphoramidate (XV) even under severe conditions (4 h, 130°C). Compound XV was prepared by the reaction of diisopropyl phosphorochloridate with N-[(methoxydimethylsilyl)methyl]-N-methylamine followed by treatment of diisopropyl N-[(methoxydimethylsilyl)methyl]-N-methylphosphoramidate with boron trichloride. Analysis of experimental and calculated ²⁹Si chemical shifts points to a five-coordinate silicon atom in compound III and a fourcoordinate silicon atom in compound XV. According to B3LYP calculations with due regard to solvent effects, compound III is an isomer with a C=O→Si bond. By variation of substituents at silicon, phosphorus, and carbonyl carbon atoms, chelate structures with either C=O→Si or P=O→Si dative bonds can be obtained.     

H-Phosphonate oligonucleotides from phosphoramidite chemistry

A novel phosphoramidite methodology allowing the formation of H-phosphonate oligonucleotides was employed to introduce backbone modifications into oligonucleotides. Novel N,N-diisopropylamino-para-methoxybenzylphosphoramidites were prepared and used in a two-step synthesis cycle during the elongation process. The coupling step was directly followed by an acidic treatment performing concomitant deprotection and phosphite deprotection through an Arbuzov reaction to yield an H-phosphonate linkage.     

Intramolecular hydrogen abstraction promoted by N-radicals: synthesis of chiral 7-oxa-2-azabicyclo[2.2.1]heptane and 8-oxa-6-azabicyclo[3.2.1]octane ring systems

Homochiral 7-oxa-2-azabicyclo[2.2.1]heptane and 8-oxa-6-azabicyclo[3.2.1]octane ring systems can be synthesized by reaction of specifically protected phosphoramidate derivatives of carbohydrates with (diacetoxyiodo)benzene or iodosylbenzene and iodine. The reaction mechanism goes through homolytic fragmentation of a hypothetical iodoamide intermediate. The N-radicals so generated participate in an intramolecular hydrogen abstraction reaction (IHA) to give the aforementioned bicycles.     

Novel oligonucleotide analogues containing a morpholinoamidine unit

Morpholinoamidines were devised as new cationic units in oligonucleotides, by combining morpholino-nucleosides (to simplify the nomenclature, we will use the term morpholino-nucleosides to refer to nucleoside analogues in which the ribose ring was transformed into a morpholine) with internucleoside guanidines. Here, methodology was developed to synthesize oligonucleotides containing morpholinoamidines formed by morpholino-uridine and 5′-amino-5′-deoxythymidine. Morpholinoamidine was produced by solid-phase reaction of Alloc-morpholinocarbothioamide with 5′-aminonucleoside resin and Mukaiyama's reagent activation. Two 14-mer oligonucleotides containing a single morpholinoamidine were synthesized and their affinity properties were investigated by forming DNA double and triple helices. Duplexes were slightly stabilized by a 3′ unit, but were less stable if internally positioned. Notably, triplexes were significantly stabilized at pH 7.0.     

Free solution mobility of DNA molecules containing variable numbers of cationic phosphoramidate internucleoside linkages

The free solution electrophoretic mobility of an 118-base pair DNA fragment containing zero, three, six or nine cationic phosphoramidate internucleoside linkages has been measured by capillary electrophoresis. The electrophoretic mobility decreases with the increasing number of cationic phosphoramidate linkages, as expected because of the reduced negative charge on the DNA molecules. The decrease in mobility is approximately linear for DNA molecules containing three and six cationic phosphoramidate linkages, but begins to level off when nine cationic phosphoramidate linkages have been added. The mobility also varies somewhat depending on whether the modified phosphoramidate linkages are located at the 5'- or 3'-end of the DNA molecule.     

Synthesis of 2′-O-[2-[(N,N-dialkylamino)oxy]ethyl]-modified oligonucleotides: hybridization affinity, resistance to nuclease, and protein binding characteristics

Synthesis of a series of 2′-O-[2-[(N,N-dialkylamino)oxy]ethyl]-modified 5-methyluridine nucleoside phosphoramidites and solid supports are described. Using these monomers, modified oligonucleotides containing phosphodiester linkages were synthesized in high yields. These modified oligonucleotides showed enhanced binding affinity to the complementary RNA (and not to DNA) and excellent nuclease stability with t_1/2>24 h. The human serum albumin binding properties of modified oligonucleotides have been evaluated to assess their transport and toxicity properties.     

Acyclovir phosphoramidates as potential anti-HIV drugs

A number of phosphoramidate derivatives of acyclovir (ACV) were obtained. These compounds revealed themselves as latent forms of acyclovir monophosphate (ACV-MP). The optimized route to the title phosphoramidates involves the synthesis of intermediate phosphorodichloridates followed by their treatment with various amines. The compounds obtained showed moderate antiviral activity both in vitro in a controlled system of secure screening for anti-HIV agents and in a tissue system enabling ex vivo determination of the efficiency of the drug and its effect on HIV replication in two weeks.     

Facile synthesis of a cis-syn thymine dimer building block and its incorporation into oligodeoxynucleotides

The synthesis of a building block containing the photobiologically relevant cis-syn thymine cyclobutane photoproduct and its incorporation into oligonucleotides by the phosphoramidite-based solid-phase synthesis is reported. Compared to previous syntheses, this route is extremely short and allows such modified oligonucleotides to be easily available for biological studies.     

Derivatives of 3′-Azidothymidine with 6-Cyanopyridone as Base or as Phosphoramidate Ester and their Antiretroviral Activity

Strongly pairing ethynylpyridone C-nucleosides are attractive surrogates for thymidine in oligonucleotides. Exploratory work on the antiviral activity of 3′-azidothymidine (AZT) derivatives with ethynylpyridone as base had identified strong lipophilicity as a limiting factor. Two strategies are being pursued to overcome this issue. In order to make the base more polar, the ethynyl group has been replaced with a cyano group, leading to a cyanopyridone C-nucleoside, whose eleven-step synthesis is reported here, together with the synthesis of a 3′-azido-2′,3′-dideoxynucleoside derivative. The base pairing with adenine in a DNA duplex was studied by UV melting analysis of a self-complementary hexamer containing the 6-cyano-2′-deoxynucleoside instead of thymidine. A melting point increase of 2 °C compared to the unmodified control was found. The other strategy employs a phosphoramidate prodrug design with less lipophilic amino acid esters. Here, anti-HIV test of the alaninyl and prolinyl methyl esters of AZT gave promising results in cell culture experiments, increasing the selectivity index up to 5.8-fold for the III_B strain and up to 5-fold for the ROD strain of the virus, as compared to the parent nucleoside. These findings help to design the next generation of pyridone C-nucleosides with potential applications as antivirals.