Improved Methods for 3′-O-Succinylation of 2′-Deoxyribo- and Ribonucleosides and Their Covalent Anchoring on Polymer Supports for Oligonucleotide Synthesis

Abstract

A more convenient and efficient method is described for the preparation of 3′-O-succinates of 2′-deoxyribo- and ribonucleosides, in a variety of solvents. Also, a new one-pot procedure has been developed for anchoring these succinates to polymer supports, suitable for solid phase synthesis of oligonucleotides.     

An Inverse Approach in Oligodeoxyribonucleotide Synthesis

Abstract

We synthesized 3′-O-dimethoxytrityl-5′O-phosphoramidites and 5′-O-succinates which can be used as monomeric building blocks for the built up of oligodeoxyribonucleotides in the alternative 5′-3′ direction. With this inverse strategy oligonucleotide 3′-conjugates as well as 3′-3′ and 5′-5′ internucleotidic linkages can be easily formed.     

A New Solid Phase Method for the Synthesis of Oligonucleotides with Terminal -3′-Phosphate

Abstract

A novel method for the synthesis of oligonucleotides with terminal-3′-phosphate using universal CPG polymer support is described.     

Solid Phase Synthesis of 2′, 5′-Oligoadenylates Containing 3′-Fluorinated Ribose

Abstract

2′, 5′-phosphodiester bond-linked oligoadenylate trimers with 3′-fluoro-3′-deoxyadenosine residues incorporated at specific positions of the nucleotide sequence were synthesized by the solid phase phosphite triester (phosphoramidite) method. The syntheses were in the 2′ to 5′ direction and were performed manually using commercially available microcolumns. The oligonucleotides were 5′-end phosphorylated on the support before deprotection.     

The Chemical Synthesis of Oligoribonucleotides with Selectively Placed 2′-O-Phosphates

Abstract

A phosphoramidite, solid support method for the chemical synthesis of oligoribonucleotides containing 2′-O-phosphate at a selected position is presented. Synthesis of these oligoribonucleotides is based on uridine- and adenosine-(2′-O-phosphate)-3′-phosphoramidites, and a new condition for removal of 2′-O-phosphate protecting groups, which does not cleave internucleotide bonds. The structure of oligoribonucleotides with 2′-O-phosphate has been proven by enzymatic digestions and dephosphorylation by yeast 2′-phosphotransferase.     

3′-Terminal Modification of Oligonucleotides Using a Universal Solid Support

Abstract

A practical method to prepare 3′-terminally labeled oligonucleotides using a universal solid support (1) was developed. This novel method was demonstrated by incorporating thirteen different label moieties on the 3′-terminus of a common (dT)₇ sequence. Label incorporations were verified by ion exchange HPLC analysis and MALDI TOF mass spectrometry.     

Study on Disulfur-Backboned Nucleic Acids: Part 3. Efficient Synthesis of 3′,5′-Dithio-2′-Deoxyuridine and Deoxycytidine

A general method is described for synthesizing 3′,5′-dithio-2′-deoxypyrimidine nucleosides 6 and 13 from normal 2′-deoxynucleosides. 2,3′-Anhydronucleosides 2 and 9 are applied as intermediates in the process to reverse the conformation of 3′-position on sugar rings. The intramolecular rings of 2,3′-anhydrothymidine and uridine are opened by thioacetic acid directly to produce 3′-S-acetyl-3′-thio-2′-deoxynucleosides 3 or 5. To cytidine, OH^? ion exchange resin was used to open the ring and 2′-deoxycytidine 10 was abtained in which 3′-OH group is in threo-conformation. The 3′-OH is activated by MsCl, and then substituted by potassium thioacetate to form the S,S′-diacetyl-3′,5′-dithio-2′-deoxycytidine 12. The acetyl groups in 3′,5′ position are removed rapidly by EtSNa in EtSH solution to afford the target molecules 6 and 13. The differences of synthetic routes between uridine and cytidine are also discusssed.     

Synthesis of High Quality Phosphorothioate Oligonucleotides as Antisense Drugs. Use of I-Linker in the Elimination of 3′-Terminal Phosphorothioate Monoesters

Abstract

Detritylation of a 5′-O-DMT-2′-deoxyadenosine moiety attached to solid support under acidic condition leads to depurination during oligonucleotide synthesis. Deprotection followed by reversed phase HPLC purification leads to desired oligonucleotide contaminated with significant levels of 3′-terminal phosphorothiaote (3′-TPT) monoester (n?1)-mer. However, it is demonstrated that attachment of dA nucleoside through its exocyclic amino group to solid support leads to substantial reduction of 3′-TPT formation thereby improving the quality of oligonucleotide synthesized.     

A Universal Glass Support for Oligonucleotide Synthesis

Abstract

A single type of controlled pore glass derivatized with 3-anisoyl-2′(3′)-O-benzoyluridine 5′-O-succinyl residues can be used as the support in solid phase syntheses of either oligoribo- or oligodeoxyribonucleotides.     

Synthesis and Biological Properties of 2′-5′ Oligodeoxyribonucleotide,an Isomer of Biologic DNA

Abstract

Oligonucleotide having 2′-5′ phosphodiester linkage has been synthesised on solid support using indigenously prepared 3′-deoxy-2′-phosphoramidites. The 2′-5′ oligonucleotide showed higher half-life when subjected to 3′-exonuclease, SVPD, digestion. This oligonucleotide formed a stable duplex with complementary RNA but not with DNA. Similarly, it did not form triplex as well either with DNA or RNA duplex.     

Tetraethylene Glycol-Derived Spacer for Oligonucleotide Synthesis

Abstract

3,6,9-Trioxaundecane-1,11-diisocyanate (6) was synthesised from tetraethylene glycol in 5 steps and 48 % overall yield. Spacer 6 was monofunctionalised with the fully protected adenosyl-3′-O-succinate derivative 7 and linked to aminomethyl polystyrene affording a solid support suitable for oligoribonucleotide synthesis (loading: ～20 μmol/g). The HPLC analysis of a crude oligoribonucleotide synthesis and the yield of the full-length product show that this spacer compares well to hexamethylene diamine.     

A Simple Solid-Phase Based Purification Procedure for Oligodeoxynucleotides

Abstract

A two-cartridge method for routine purification of DNA oligomers has been investigated. The full-length target oligonucleotides are purified using a method that select for intact 3′- and 5′-termini. The procedure results in purified DNA without the use of PAGE gels or HPLC.     

Synthesis of Deoxycytidine (dC) Building Blocks for Amide-Linked Dimers or Oligomers: Example of a Deoxycytidine-amide-thymidine Dimer

Abstract

5′-Azido-3′-carbomethoxymethyl-4-N-benzoyl-2′,3′,5′-trideoxy-cytidine 17 and 5′-O-t-butyldimethylsilyl-3′-carboxymethyl-4-N-benzoyl-2′,3′-dideocytidine 22 were efficiently synthesized from 2′-deoxyuridine via a new method which transformed the uracil heterocycle to 4-N-benzoylcytosine (four steps, 60 % overall yield). The amide-linked deoxycytidine-thymidine dimer analog was synthesized.     

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.     

o-Chlorobenzoyl Protected Nucleoside Succinates for Functionalization of the Solid Support Used in Oligoribonucleotide Synthesis

Abstract

In the final stages of automated oligonucleotide synthesis the oligomer has to be cleaved from the solid support. This is usually carried out using ammonolysis since the 3′-end of the oligomer is most commonly attached to the support via a succinate ester linkage. The t-butyldimethylsilyl (TBDMS) group is currently the most widely used 2′-hydroxyl in RNA-synthesis and is used together with phosphoroamidites¹ as well as with H-phosphonates². The nucleoside directly attached to the support, often carries the same TBDMS-protection on the secondary hydroxyl next to the succinate linker. The use of more labile acyl groups for N-protection in RNA-synthesis was suggested in reports where partial loss of the TBDMS groups during ammonolysis was detected^3,4. This has since been introduced^5,6 and is now general practice. However, one can question if all oligomer will be released from the support under the milder ammonolytic conditions used to remove these more labile N-protecting groups.     

A Facile Synthetic Method for 3′-α-Fluoro- 2′,3′-dideoxyadenosine

Abstract

A facile method for the synthesis of 3′-α-fluoro-2′,3′-dideoxyadenosine (5) has been developed using a novel rearrangement of 3′-β-bromine to the 2′-β position during 3′-α fluorination.     

Purification of 5′-O-Trityl-on Oligoribonucleotides. Investigation of Phosphate Migration During Purification and Detritylation.

Abstract

Synthetic oligoribonucleotides (RNA) are efficiently prepared with 2′-O-tert-butyldimethylsilyl nucleoside 3′-O-phosphoramidites with labile base-protection; A_dmf or A_Pac, G_dmf, C_ibu, U. After cleavage from the polystyrene support, the exocyclic amine protecting groups are removed with conc. NH₄OH: ethanol/3:1 by heating at 55°C for 3–5 h. The 2′-O- silyl protecting groups are removed with tetra-n-butylammonium fluoride in THF or more conveniently with neat triethylamine trihydrofluoride. To gain the advantages of increased capacity on reverse phase HPLC and the convenience of cartridge based purification (OPC, Oligonucleotide Purification Cartridge), the 5′ trityl was left on the RNA as the final protecting group to be removed. The mild conditions which are effective for trityl removal are shown to preserve 3′-5′ phosphate linkage integrity in RNA. The absence of phosphate migration is demonstrated by model studies, utilizing N⁴ -isobutyryl-5′-O-DMT-3′-O-TBDMS-2′-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite) as a control monomer and digestion by 3′-5′ selective P1 nuclease and alkaline phosphatase and HPLC analysis. Oligoribonucleotides were analyzed by Microgel capillary electrophoresis, anion-exchange HPLC, and the enzymatic digest/HPLC method.

     

Synthesis of 2′,3′-Dideoxy-2′-fluoro-3′-thioarabinothymidine and Its 3′-Phosphoramidite Derivative

Abstract

An efficient method for the synthesis of 5′-O-monomethoxytrityl-2′,3′-dideoxy-2′-fluoro-3′-thioarabinothymidine [^5′-MMTaraF-T_3′SH, (5)] and its 3′-phosphoramidite derivative (6) suitable for automated incorporation into oligonucleotides, is demonstrated. A key step in the synthesis involves reaction of 5′-O-MMT-2,3′-O-anhydrothymidine (4) (Eleuteri, A.; Reese, C.B.; Song, Q., J. Chem. Soc. Perkin Trans. 1 1996, 2237 pp.) with sodium thioacetate to give ^5′-MMTaraF-T_3′SAc (5) (Elzagheid, M.I.; Mattila, K.; Oivanen, M.; Jones, B.C.N.M.; Cosstick, Lönnberg, H. Eur. J. Org. Chem. 2000, 1987–1991). This nucleoside was then converted to its corresponding phosphoramidite derivative, 6, as described previously ((a) Sun, S.; Yoshida, A.; Piccirilli, J.A. RNA, 1997, 3, 1352–1363; (b) Matulic-Adamic, J.; Beigelman, L. Helvetica Chemica Acta 1999, 82, 2141–2150; (c) Fettes, K.J.; O’Neil, I.; Roberts, S.M.; Cosstick, R. Nucleosides, Nucleotides and Nucl. Acids 2001, 20, 1351–1354).     

Binding of Bis-linked Netropsin Derivatives in the Parallel-Stranded Hairpin Form to DNA

Abstract

Cis-diammine Pt(II)- bridged bis-netropsin and oligomethylene-bridged bis-netropsin in which two monomers are linked in a tail-to-tail manner bind to the DNA oligomer with the sequence 5′-CCTATATCC-3′ in a parallel-stranded hairpin form with a stoichiometry 1:1. The difference circular dichroism (CD) spectra characteristic of binding of these ligands in the hairpin form are similar. They differ from CD patterns obtained for binding to the same duplex of another bis-netropsin in which two netropsin moieties were linked in a head-to-tail manner. This reflects the fact that tail-to-tail and head-to-tail bis-netropsins use parallel and antiparallel side-by-side motifs, respectively, for binding to DNA in the hairpin forms. The binding affinity of cis -diammine Pt(II)- bridged bis-netropsin in the hairpin form to DNA oligomers with nucleotide sequences 5′-CCTATATCC-3′ (I), 5′-CCTTAATCC-3′ (II), 5′-CCTTATTCC-3′ (III), 5′-CCTTTTTCC-3′ (IV) and 5′-CCAATTTCC-3′ (V) decreases in the order I = II > III > IV> V. The binding of oligomethylene-bridged bis-netropsin in the hairpin form follows a similar hierarchy. An opposite order of sequence preferences is observed for partially bonded monodentate binding mode of the synthetic ligand.     

Nucleosides and Nucleotides. 104. Radical and Palladium-Catalyzed Deoxygenation of the Allylic Alcohol Systems in the Sugar Moiety of Pyrimidine Nucleosides§,1

Abstract

New methods for the synthesis of 2′,3′-didehydro-2′,3′-dideoxy-2′ (and 3′)-methyl-5-methyluridines and 2′,3′-dideoxy-2′ (and 3′)-methylidene pyrimidine nucleosides have been developed from the corresponding 2′ (and 3′)-deoxy-2′ (and 3′)-methylidene pyrimidine nucleosides. Treatment of a 3′-deoxy-3′-methylidene-5-methyluridine derivative 8 with 1,1′-thiocarbonyldiimidazole gave the allylic rearranged 2′,3′-didehydro-2′,3′-dideoxy-3′-[(imidazol-1-yl)carbonylthiomethyl] derivative 24. On the other hand, reaction of 8 with methyloxalyl chloride afforded 2′-O-methyloxalyl ester 25. Radical deoxygenation of both 24 and 25 gave 26 exclusively. Palladium-catalyzed reduction of 2′,5′-di-O-acetyl-3′-deoxy-3′-methylidene-5-methyluridine (32) with triethylammonium formate as a hydride donor regioselectively afforded the 2′,3′-dideoxy-3′-methylidene derivative 35 and 2′,3′-didehydro-2′,3′-dideoxy-3′-methyl derivative 34 in a ratio of 95:5 in 78% yield. These reactions were used on the corresponding 2′-deoxy-2′-methylidene derivatives. An alternative synthesis of 2′,3′-dideoxy-2′-methylidene pyrimidine nucleosides (43, 52, and 54) was achieved from the corresponding 1-(3-deoxy-β-D-thero-pentofuranosyl)pyrimidines (44 and 45). The cytotoxicity against L1210 and KB cells and inhibitory activity of the pathogenicity of HIV-1 are also described