A Simple Synthesis of N4 -(6-Aminohexyl)-2′-Deoxy-5′-O-(4,4′-Dimethoxytrityl)Cytidine

Abstract

The title compound was synthesized by a transamination reaction between N⁴ -benzoyl-2′-deoxy-5′-O-(4,4′-dimethoxytrityl)cytidine and hexane-1,6-diamine in the presence of 1,5,7-triazabicyclo(4.4.0)dec-5-ene (TBD).     

Preparation of 5′-O-(1-Thiotriphosphate)-Modified Oligonucleotides Using Polymerase-Endonuclease Amplification Reaction (PEAR)

Antisense oligonucleotides (ASODNs) have been widely used as an important tool for regulating gene expression, and developed into therapeutics. Natural ODNs are susceptible to nuclease degradation, nucleic acid analogues, however, have less side effects, stronger stability and more potent activities. Large-scale de novo synthesis of a certain oligonucleotide has been very difficult and costly. In a previous preliminary study, we developed the polymerase-endonuclease amplification reaction (PEAR) for amplification and large-scale preparation of natural antisense ODNs. Here we extended the method in preparation of a widely used modified oligonucleotide with 5′-O-(1-Thiotriphosphate) modifications. Using electrospray ionization liquid chromatography mass spectrometry (ESI/LC/MS) detection, the purity of the PEAR product was measured as high as 100.0%. Using PEAR a large amount of a specific oligonucleotide can be produced starting from a small amount of synthetic seeds. It is suggested that PEAR can be a useful tool for large-scale production of modified oligonucleotides.     

Diastereomeric Process Control in the Synthesis of 2′-O-(2-Methoxyethyl) Oligoribonucleotide Phosphorothioates as Antisense Drugs

Abstract

Coupling of 2′-O-methoxyethylsubstituted nucleoside phosphoramidites to 5′-hydroxyl group of a nucleoside or nucleotide on solid support is under stereochemical process control and is independent of scale, concentration, synthesizer, ratio of amidite diastereomers, solid support etc. However, activators and phosphate protecting groups do play a role in influencing the ratio of phosphorothioate diesters obtained by sulfurization of phosphite triesters.     

Selective Biocatalytic Acylation Studies on 5′-O-(4,4′-Dimethoxytrityl)-2′,3′-Secouridine: An Efficient Synthesis of UNA Monomer

Lipozyme® TL IM (Theremomyces lanuginosus lipase immobilized on silica) in toluene catalyzes the acylation of the 2 ′-OH over the 3 ′-OH group in 5 ′-O-(4,4 ′-dimethoxytrityl)-2 ′,3 ′-secouridine (5 ′-O-DMT-2 ′,3 ′-secouridine) in a highly selective fashion in moderate to almost quantitative yields. The turn over during benzoyl transfer reactions mediated by vinyl benzoate or benzoic anhydride was faster than in acyl transfer reactions with vinyl acetate or C₁ to C₅ acid anhydrides; except in the case of butanoic anhydride. The 2 ′-O-benzoyl-5 ′-O-DMT-2 ′,3 ′-secouridine obtained by Lipozyme® TL IM catalyzed benzoylation of 5 ′-O-DMT-2 ′,3 ′-secouridine was successfully converted into its 3 ′-O-phosphoramidite derivative in satisfactory yield, which is a building block for the preparation of oligonucleotides containing the uracil monomer of UNA (unlocked nucleic acid).     

Improved Methods for the Preparation of 2′-Deoxyribonucleoside and Ribonucleoside 3′-Phosphoramidites with Allylic Protectors

Abstract

Improved methods of preparing 2′-deoxyribonucleoside and ribonucleoside 3′-phosphoramidites with allylic protecting groups, which serve as useful building blocks, particularly for the synthesis of base-labile derivatives, are developed.     

The Synthesis of the Sixteen Possible 2′-O-Methyl MMI Dimer Phosphoramidites: Building Blocks for the Synthesis of Novel Antisense Oligonucleotides

Abstract

The synthesis of Methylene(methylimino) or MMI linked nucleoside dimers in all sixteen possible configurations has been accomplished via a reductive coupling of a nucleosidic aldehyde with an hydroxylamine. This has allowed us to prepare all of the necessary 2′-O-methyl MMI dimer building blocks necessary for use in an antisense motif.     

Semisynthesis of 6-Chloropurine-2′-deoxyriboside 5′-Dimethoxytrityl 3′-(2-Cyanoethyl-N,N-diisopropylamino)Phosphoramidite and its Use in the Synthesis of Fluorescently Labeled Oligonucleotides

An efficient enzymatic synthesis of 6-chloropurine-2′-deoxyriboside from the reaction of 6-chloropurine with 2′-deoxycytidine catalyzed by nucleoside-2′-deoxyribosyltransferase (E.C. 2.4.2.6) followed by chemical conversion into the 5′-dimethoxytrityl 3′-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite derivative is described. The phosphoramidite derivative was incorporated site-specifically into an oligonucleotide and used for the introduction of a tethered tetramethylrhodamine-cadaverine conjugate. The availability of an efficient route to 6-chloropurine-2′-deoxyriboside 5′-dimethoxytrityl 3′-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite enables the facile synthesis of oligonucleotides containing a range of functional groups tethered to deoxyadenosine residues.     

The Synthesis of 5′-O-Triphosphate-4N-(ω-aminoalkyl)deoxycytidine A Useful Precursor to the Generation of Differently Labeled Triphosphates

Abstract

A chemical synthesis is described for 5′-O-triphosphate-4N-[6-(-γ-aminopropylamidosuccinylamido)-hexyl]deoxycytidine (13), the substrate for synthesis of 5′-O-triphosphate deoxycytidine derivatives (17-19) labeled with biotin, fluorescein and photoreactive azide.     

The Preparation of a 3′-(2-Cyanoethyl)phosphoramidite of 5′-O-(3-Thiopropyl)methylphosphorylthymidine

Abstract

A high-yielding three-step reaction sequence to a useful novel phosphoramidite, using 3′-O-acetylthymidine as starting material, is reported.     

Synthesis of Protected 3′,5′-Di-2′-Deoxythymidine-(α-hydroxy-2-nitrobenzyl)-phosphonate Diesters as Dimer Building Blocks for Oligonucleotides

Abstract

The synthesis of 3′-succinyl-CPG bound 3′,5′-di-2′-deoxythymidyl-(α-hydroxy-2-nitrobenzyl)-phosphonate diester 1 and the 3′-phosphoamidite derivative 2 is descibed. The hydroxyl-groups of the backbone modification were protected with trialkylsilyl groups: TES and TBS. Compounds 1, 2 are suitable blocks for oligonucleotide synthesis.     

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.     

Synthesis of 2′,3′-Dideoxy-2′-methylene Pyrimidine Nucleosides as Potential Anti-Human Immunodeficiency Virus (HIV) Agents

Abstract

Several 2′,3′-dideoxy-2′-methylene pyrimidine nucleosides, 2′,3′-dideoxy-2′-methylenecytidine hydrochloride (20), 2′,3′-dideoxy-2′-methyleneuridine (21), and 2′,3′-dideoxy-2′-methylene-5-methyluridine (22), have been synthesized via a multi-step synthesis from uridine and 5-methyluridine, respectively. These compounds were tested for their cytotoxicity against L1210, S-180, CCRF-CEM, and P388 cells in culture and their antiviral activity is under investigation.     

3′/5′-Regioselectivity of Introduction of the 9-Fluorenyl-Methoxycarbonyl Group to 2′-o-Tetrahydropyran-2-YL-and 2′-O-(4-Methoxytetrahydropyran-4-YL-)-Nucleosides: Useful Intermediates for Solid-Phase-Rna-Synthesis

Abstract

X-ray structure analysis of the more laevorotatory isomers of 2′-O-tetrahydropyranyl-4N-benzoylcytidine (4b) and of 2′-O-tetrahydropyranyluridine (5b) confirmed their chirality at the satellite anomeric centre C2″ to be S. The other diastereomers (4a resp. 5a) exhibited an unexpected reversal of 3′/5′-regio-selectivity when treated with 9-fluorenylmethoxycarbonyl chloride in pyridine. The X-ray crystallographic results form the basis for a mechanistic proposal.     

Generation of a monoclonal antibody against the myelin protein CNP (2′,3′-cyclic nucleotide 3′-phosphodiesterase) suitable for biochemical and for immunohistochemical investigations of CNP

The functional role of CNP (2,3-cyclic nucleotide 3-phosphodiesterase), a minor component of central and peripheral myelin is still unclear. Here we describe preparation of a monoclonal antibody directed against CNP. The antibody, of the immunoglobulin IgG₁ type, raised with a basic 46 kDa membrane-associated protein solubilized from pig cerebellar membranes, can be used to detect immunoreactivity in solubilized brain homogenates from pig, mouse, rat, sheep, cow and man, in cerebrum and cerebellum, but not in other tissues such as liver, skeletal and heart muscle. The antibody recognizes the CNP doublet band and shows no cross-reactivity with any of the other brain proteins solubilized. In tissue sections from paraformaldehyde-fixed rat brain the antigen was localized in oligodendrocytes. In cultured glial cells from newborn mice the antibody stained cells which were identified as oligodendrocytes by co-localization of myelin basic protein. Even cells from a C6 rat glioma cell line, which contain very little of CNP, were labeled by the monoclonal antibody. Thus the monoclonal antibody recognizing CNP from several species is suitable for immunocytochemical investigations and also for biochemical studies of CNP, since the antibody has been employed for immunoprecipitation and immunopurification of CNP in crude brain homogenates.     

A New Approach to the Synthesis of Isoflavones, 2′-Hydroxyisoflavones and an Alternative Synthesis of (±)-Pterocarpin

A new approach to the synthesis of isoflavones and 2′-hydroxyisoflavones, useful intermediates for synthesis of pterocarpinoids, are described. The 2′-hydroxyisoflavone (VIII; R=CH₃, R′=H) synthesized by this method, was converted into (±)-pterocarpin (VII; R=CH₃, R′=H). Attempts to obtain the corresponding isoflavane or isoflavene from the coumarine (XVII) failed.     

A Convenient Method for the Synthesis of 2-(β-D-Glycopyranosylthio) Pyridines

Abstract

Treatment of piperidinium salts of dihydropyridinethiolates 3 with glycosyl bromides 4 in dry acetone provides a convenient and high yielding synthesis of 1,4-dihydro-3-cyanopyridine thioglycosides 5. The structures of 5 were confirmed by oxidation as well as by ¹H NMR and ¹³C NMR spectral analysis.     

Synthesis of An Acid-Stable 2,5′-Cyclo-2-oxo Analogue of Wyosine (Nucleosides and Nucleotides. Part 611)

Abstract

Methylation of a 4-desmethylwyosine derivative fixed in anti-conformation has afforded a higher yield of fluorescent N-4-methyl isomer, 2,5′-cyclo-2-oxo-2′,3′-O-isopropylidenewyosine (7), which has been shown to be relatively stable in acidic media.     

Conjugates of Fluorescein and Saenta (5′-S-(2-Aminoethyl)-N 6-(4-nitrobenzyl)-5′-thioadenosine): Flow Cytometry Probes for the ES Nucleoside Transporter Elements of the Plasma Membrane

Abstract

SAENTA was linked to the C-5 or C-6 positions of fluorescein through several structures to form conjugates that were bound tightly to plasma membrane sites associated with es nucleoside transport activity. The conjugates imparted fluorescence to cells that expressed es nucleoside transport activity and served as es-selective plasma membrane stains suitable for flow cytometry. Prior treatment of es-expressing cells with nitrobenzylthioinosine prevented fluorescent staining with the conjugates. Seven SAENTA-fluorescein conjugates served as flow cytometric stains with high affinities for es sites, despite substantial differences in the SAENTA-fluorescein linkage structures.