A new approach to the synthesis of branched and branched cyclic oligoribonucleotides.

The six-step synthesis of the di-triethylammonium salt of 5[prime]-O -trityl-6-N-pivaloyladenosine-2[prime]-(H -phosphonate)-3'-[(2-chlorophenyl) phosphate]9 from 3', 5'- O -(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-6-N-pivaloyla denosine1in 68% overall yield is described. Compound9is converted into a branched pentaribonucleoside tetraphosphate 24 and a branched cyclic pentaribonucleotide ('lariat') 25 by solution phase triester chemistry involving both H-phosphonate and conventional phosphotriester coupling reactions. The monomeric building block 9 is proposed as a universal synthon for the preparation of branched and branched cyclic oligoribonucleotides derived from adenosine.     

Developing synthetic methods for bioactive phosphorus compounds using H-phosphonate chemistry: a progress report

In this paper a short account of our recent basic studies aiming toward development of new synthetic methods for the preparation of nucleotide analogues using H-phosphonate chemistry is presented.     

DEVELOPING SYNTHETIC METHODS FOR BIOACTIVE PHOSPHORUS COMPOUNDS USING H-PHOSPHONATE CHEMISTRY: A PROGRESS REPORT

In this paper a short account of our recent basic studies aiming toward development of new synthetic methods for the preparation of nucleotide analogues using H-phosphonate chemistry is presented.     

Synthesis and immunostimulatory properties of the phosphorothioate analogues of cdiGMP

The synthesis of mono- and bisphosphorothioate analogues of 3',5'-cyclic diguanylic acid (cdiGMP) via the modified H-phosphonate chemistry is reported. The immunostimulatory properties of these analogues were compared with those of cdiGMP.     

The Application of H-Phosphonate Chemistry in the HELP Synthesis of Oligonucleotides

Abstract

The use of the H-phosphonate chemistry for its possible utilization in the liquid phase synthesis of oligonucleotides has been investigated.     

Utilization of intrachain 4'-C-azidomethylthymidine for preparation of oligodeoxyribonucleotide conjugates by click chemistry in solution and on a solid support

4'-C-Azidomethylthymidine 3'-(H-phosphonate) monomer (10) was synthesized in high yield and three such monomers were incorporated by the H-phosphonate coupling into a 15-mer oligodeoxyribonucleotide. The unmodified 2'-deoxynucleosides could be coupled by either the H-phosphonate or phosphoramidite chemistry, indicating that the Staudinger reaction between the azido group and the phosphoramidite reagent severely hampered the coupling only when it took place intramolecularly. After chain assembly, three alkynyl group bearing ligands, viz., propargyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranoside (2), N-{4-[N-(trifluoroacetyl)aminomethyl]benzyl}-4-pentynamide (3) and N (1), N (3), N (2')-tris(trifluoroacetyl)-N (6')-(4-pentynoyl)neamine (4), were conjugated to the azido groups of the oligonucleotide by click chemistry both on a solid support and in solution. The products were deprotected by conventional ammonolysis and purified by HPLC chromatography. Melting temperature studies revealed that the mannose conjugated oligonucleotides formed more stable duplexes with 2'-O-methyl RNA than with DNA strand. With 2'-O-methyl RNA, a slight destabilization compared to an unmodified sequence was observed at low ionic strength, while at high salt content, the manno-conjugation was stabilizing.     

Dipentafluorophenyl carbonate--a reagent for the synthesis of oligonucleotides and their conjugates.

Dipentafluorophenyl carbonate has been successfully used as condensing agent for the internucleotide bond formation in the synthesis of oligonucleotides via H-phosphonate approach. The mechanism of a nucleotide component activation with this reagent has been investigated with the help of 31P NMR spectroscopy. It was shown that preactivation of deoxynucleoside H-phosphonate with dipentafluorophenyl carbonate has no influence on the efficiency of the synthesis. This reagent is highly reactive, nonhygroscopic and stable on storage at room temperature. The effectiveness of dipentafluorophenyl carbonate in the oligonucleotide chemistry has been demonstrated in the solid-phase synthesis of 10-50-mers on 0.2, 1 and 10 mumol scales. The use of this reagent for the derivatisation of polymer supports as well as for the synthesis of oligonucleotide conjugates with polyethylene glycol and a lipid is described.     

Influence of the incorporation of (S)-9-(3,4-dihydroxybutyl)adenine on the enzymatic stability and base-pairing properties of oligodeoxynucleotides.

(S)-9-(3,4-dihydroxybutyl)adenine was used at several positions as nucleoside substitute in the synthesis of dimers and 13-mers. Therefore we used the phosporamidite and the H-phosphonate chemistry. The nuclease susceptibilities and the base-pairing properties of these oligomers have been evaluated.     

The Prooligonucleotide Approach: Synthesis of Mixed SATE-Phosphotriester Phosphodiester Oligonucleotides

Abstract

Synthesis of mixed SATE phosphotriester-phosphodiester prooligos was done following two ways. Phosphodiesters were generated by selective β-elimination of cyanoethyl using DBU or were introduced with H-phosphonate chemistry.     

DNA/RNA synthesis and labelling.

Reliable methods of machine-aided RNA synthesis have been established to complement those for DNA assembly. Oligonucleotides containing thio-modified backbones and 2'-O-alkyl sugars head the list of many newly available analogues. Biotin, fluorescent agents and many reporter groups can be conveniently introduced into oligonucleotides in multiples by phosphoramidite or H-phosphonate chemistry.     

The preparation and application of functionalised synthetic oligonucleotides: III. Use of H-phosphonate derivatives of protected amino-hexanol and mercapto-propanol or -hexanol.

Syntheses of H-phosphonate salts (4a-e) of N/S-protected alcohols such as 6-aminohexan-1-ol, 3-mercaptopropan-1-ol and 6-mercaptohexan-1-ol are described using 2-chloro-5,6-benzo-1,3,2-phosphorin-4-one (2) as the phosphonylating agent. The H-phosphonate salts (4a-e), in the presence of pivaloyl chloride or adamantoyl chloride as an activator, were coupled to the 5'-end of synthetic oligonucleotides on solid supports to produce amino or thio-linked oligonucleotides. Following deprotection and purification, fluorescent dyes, biotin derivatives and poly-L-lysine-maleimide were separately attached to the functionalised oligonucleotides. Identical derivatized oligomers were obtained with cyanoethyl-N,N-diisopropylamidite chemistry and amidites (5a-e) of the respective alcohols.     

Synthesis of a 2'-O-(carbomoylmethyl)ribonucleoside H-phosphonate building block and a model dinucleotide

In order to obtain higher quality 2'-O-carbamoylmethyl oligoribonucleotides we are conducting studies of this modification. Here we present synthesis of 2'-O-carbamoylmethyl containing H-phosphonate building blocks as well as synthesis of model dinucleotides needed for these studies.     

Synthesis of A 2′- O -(Carbomoylmethyl)Ribonucleoside H-Phosphonate Building Block and A Model Dinucleotide

In order to obtain higher quality 2 ′-O-carbamoylmethyl oligoribonucleotides we are conducting studies of this modification. Here we present synthesis of 2 ′-O-carbamoylmethyl containing H-phosphonate building blocks as well as synthesis of model dinucleotides needed for these studies.     

Synthesis of Oligoribonucleotides by the H-Phosphonate Approach Using Base Labile 2′-O-Protecting Groups. V. Recent Progress in Development of the Method

Abstract

A simple and inexpensive method for the preparation of oligoribonucleotides using N-isopropoxyacety(phenoxyacetyl)-2′-O-(2-chlorobenzoyl)-5′-O-dimethoxytritylnucleoside H-phosphonate building blocks has been developed.     

Phosphoramitoids—A submonomer approach to sequence defined N-substituted phosphoramidate polymers

There is a growing interest in new methods to generate bio-inspired, chemically diverse, sequence-defined synthetic polymers. Solid-phase submonomer approaches offer facile access to these types of materials, since they take advantage of readily available synthons. Submonomer approaches to date have been applied to peptidomimetics with oligo-amide backbones. Here we extend the approach to a phosphorous-containing backbone, where N-substituted phosphoramidate oligomers are constructed from a set of amine submonomers, diphenyl H-phosphonate, and cyclohexane diol. The key chemical steps in chain elongation are a chain extension reaction based on H-phosphonate (P III) chemistry, and a side chain attachment step based on the Atherton-Todd reaction. Cheap, stable chemical reagents are used without heating, all reaction times are 30 minutes or less and open to air, and no main-chain protecting groups are required. Phosphoramitoid tetramers and pentamers displaying a variety of side chain functionalities were synthesized by a three-step solid-phase submonomer method, typically with >85% crude purities.     

2-Amino-alpha-2'-deoxyadenosine increased duplex stability of methoxyethylphosphoramidate alpha-oligodeoxynucleotides with RNA target

A new efficient synthesis of 2-amino-alpha-2'-deoxyadenosine and its incorporation into methoxyethylphosphoramidate alpha-oligodeoxynucleotides (ODNs) via H-phosphonate chemistry were reported. Thermal denaturation experiments demonstrated a significant stabilization of the complexes formed between these analogues and their RNA target (+2 degrees C/NH2A) relative to adenosine-containing phosphoramidate alpha-oligonucleotides. Concerning the binding specificity of these modified ODNs, unlike natural ODNs, discrimination against G pairing is higher and against C pairing is lower.     

Evaluation of 2'-hydroxyl protection in RNA-synthesis using the H-phosphonate approach.

A number of different protecting groups were compared with respect to their usefulness for protection of 2'-hydroxyl functions during synthesis of oligoribonucleotides using the H-phosphonate approach. The comparison was between the t-butyldimethylsilyl (t-BDMSi), the o-chlorobenzoyl (o-CIBz), the tetrahydropyranyl (THP), the 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp), the 1-(2-chloro-4-methylphenyl)-4-methoxypiperidin-4-yl (Ctmp), and the 1-(2-chloroethoxy)ethyl (Cee) protecting groups. All these groups were tested in synthesis of dodecamers, (Up)11U and (Up)11A, using 5'-O-(4-monomethoxytrityl) or (4,4'-dimethoxytrityl) uridine H-phosphonate building blocks carrying the respective 2'-protection. The performance of the t-BDMSi and o-CIBz derivatives were also compared in synthesis of (Up)19U. The most successful syntheses were clearly those where the t-butyldimethylsilyl group was used. The o-chlorobenzoyl group also gave satisfactory results but seems somewhat limited with respect to synthesis of longer oligomers. The results with all tested acetal derivatives (Fpmp, Ctmp, Cee, THP) were much less successful due to some accompanying cleavage of internucleotidic H-phosphonate functions during removal of 5'-O-protection (DMT).     

Global phosphorylation at Ser16 of the 32-residue cytoplasmic domain of phospholamban: Comparison of di-t-butyl- and dibenzyl-N,N-diisopropylphosphoramidites

Summary Phospholamban (PLB), a 52-residue integral membrane protein of the cardiac sarcoplasmic reticulum, is known as the regulator of the Ca²⁺-ATPase pump via its phosphorylation-dephosphorylation at Ser¹⁶. In order to investigate the structural effects of the phosphorylation on the cytoplasmic domain of PLB, we synthesized PLB 2–33 in its nonphosphorylated and phosphorylated forms using Fmoc chemistry. PLB 2–33 was phosphorylated using the global phosphorylation method. Phosphorylation with di-t-butyl-N,N-diisopropylphosphoramidite led to an incomplete reaction (nonphosphorylated peptide was recovered) and to the formation of an H-phosphonate. In contrast, the use of dibenzyl-N,N-diisopropylphosphoramidite yielded the desired phosphorylated peptide quantitatively and did not give rise to the H-phosphonate. Our results show the effectiveness of the dibenzyl-protected phosphoramidite when the site to be phosphorylated is particularly hindered.     

Synthesis and Properties of Oligoribonucleotide Analogues Having Amide (3″-CH2-CO-NH-5′) Internucleoside Linkages

Abstract

Amide linked ribonucleoside dimer 5 was prepared and converted into selectively protected H-phosphonate building block 9. Oligoribonucleotide duplexes having amide linkages at selected sites were synthesized and their stability was studied by UV melting experiments.     

Use of new phosphonylating and coupling agents in the synthesis of oligodeoxyribonucleotides via the H-phosphonate approach.

New phosphonylating and coupling agents for the synthesis of oligodeoxyribonucleotides via H-phosphonate approach have been developed. Tris(1,1,1,3,3,3-hexafluoro-2-propyl) phosphite, prepared by the reaction of lithium salt of 1,1,1,3,3,3-hexafluoro-2-propoxide with PCl3, reacts with deoxyribonucleosides in the presence of a catalytic amount of triethylamine to produce in the high yield the corresponding deoxyribonucleoside 3'-H-phosphonate units. The use of a new coupling reagent, 1,3-dimethyl-2-chloro-imidazolinium chloride (DMCI) for the internucleotidic H-phosphonate bond formation via the H-phosphonate approach is also discussed in detail.