Phosphoramidite Group Research Articles

Retro-1-Oligonucleotide Conjugates. Synthesis and Biological Evaluation.

Addition of small molecule Retro-1 has been described to enhance antisense and splice switching oligonucleotides. With the aim of assessing the effect of covalently linking Retro-1 to the biologically active oligonucleotide, three different derivatives of Retro-1 were prepared that incorporated a phosphoramidite group, a thiol or a 1,3-diene, respectively. Retro-1–oligonucleotide conjugates were assembled both on-resin (coupling of the phosphoramidite) and from reactions in solution (Michael-type thiol-maleimide reaction and Diels-Alder cycloaddition). Splice switching assays with the resulting conjugates showed that they were active but that they provided little advantage over the unconjugated oligonucleotide in the well-known HeLa Luc705 reporter system.

Synthesis of a protected ribonucleoside phosphoramidite-linked spin label via an alkynyl chain at the 5′ position of uridine

New, spin-labeled nucleosides, and an efficient synthetic route for a modified uridine amidite, were developed. The spin-labeled part was the 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) group, which was linked via an alkynyl chain at the 5 position of uridine. Three typical protecting groups, the t-butyldimethylsilyl (TBDMS) group at 2′, the dimethoxytrityl (DMTr) group at 5′, and the phosphoramidite group at 3′, were introduced to produce an automated nucleic acid synthesizer. The TEMPO group at the 5 position in the uridine structure affected introduction of bulky protecting groups, such as the DMTr group at the 5′ position and the TBDMS group at the 2′ position. The electron paramagnetic resonance (EPR) data revealed a nitroxyl radical in the structure of synthetic nucleoside compounds; however, RNA produced by automated synthesis using a TEMPO-linked uridine phosphoramidite building block was EPR silent.

Thio‐ and Seleno‐Dioxaphosphorinane‐Constrained Dinucleotides (D‐CNA): Synthesis and Conformational Study

Thio‐ and seleno‐α,β‐ and α,β,γ‐D‐CNA (constrained nucleic acid) dinucleotides in which two or three torsion angles of the sugar/phosphate backbone are controlled within a dioxaphosphorinane structure have been prepared. Their structural determination was carried out by means of NMR spectroscopy to show only slight variation on the torsion‐angle control in comparison with their oxo analogues. Unexpected selenium migration has been pointed out from selenophosphotriester moiety to phosphoramidite group, but this drawback can be overcome using H‐phosphonate chemistry.

Chimeric RNA Oligonucleotides Incorporating Triazole-Linked Trinucleotides: Synthesis and Function as mRNA in Cell-Free Translation Reactions.

A method for the synthesis of chimeric oligonucleotides was developed to incorporate purine nucleobases and multiple triazole linkers in natural, phosphate-linked structures of RNA. A solution-phase synthesis method for triazole-linked RNA oligomers via copper-catalyzed azide-alkyne cycloaddition reaction was optimized and tolerated purine nucleobases and protecting groups for further transformations. Three TLRNA trinucleotides with 5'-protected hydroxy and 3'-phosphoramidite groups were prepared, and one congener with a representative sequence was subjected to automated, solid-phase phosphoramidite synthesis. The synthesis allowed the efficient preparation of 13-mer chimeric RNA oligonucleotides with two triazole linkers, ten phosphate linkers and purine/pyrimidine nucleobases. The chimeric oligonucleotide was found applicable to a cell-free translation system as mRNA and provided the genetic code for dipeptide production.

Biodegradable Main-Chain Phosphate-Caged Fluorescein Polymers for the Evaluation of Enzymatic Activity

We describe here the biodegradable polymers consisted of the tandemly linked phosphate-caged fluorescein in the main chains. The caged-fluorescein monomer with the phosphoramidite groups at both hydroxyl groups was synthesized, and the polycondensations were executed in the presence of the diol derivatives which were promised to be the reactivity-tunable linker. After oxidation and the alkaline treatment for the deprotection, less-emissive polymeric products were obtained. The resulting polymers can release fluorescein by the digestion with alkaline phosphatase. We demonstrated that the enzymatic activity of the cell lysate can be quantified from the increase of the magnitude of fluorescence emission with the synthetic polymers.

Redox Isomerization of Allylic Alcohols Catalyzed by Osmium and Ruthenium Complexes Containing a Cyclopentadienyl Ligand with a Pendant Amine or Phosphoramidite Group: X-ray Structure of an η3-1-Hydroxyallyl-Metal-Hydride Intermediate

Complexes [MCl2(η6-p-cymene)]2 (M = Os (1a), Ru (1b)) react with Li(C5H4CH2CH2NHMe) (LiCpN) and KPF6 to give the sandwich derivatives [M(η5-CpN)(η6-p-cymene)]PF6 (M = Os (2a), Ru (2b)). Treatment of 2a and 2b with (2,2′-biphenol)PCl leads to [M(η5-CpP)(η6-p-cymene)]PF6 (M = Os (3a), Ru (3b); CpP = C5H4CH2CH2N(Me)P(2,2′-biphenol)). The photolysis of 2a, 2b, 3a, and 3b in acetonitrile produces the release of the p-cymene group and the coordination of the cyclopentadienyl pendant substituent to the metal center to afford [M(η5-C5,κ-N-CpN)(CH3CN)2]PF6 (M = Os (4a), Ru (4b)) and [M(η5-C5,κ-P-CpP)(CH3CN)2]PF6 (M = Os (5a), Ru (5b)). Complex 4a, which has been characterized by X-ray diffraction analysis, is a more efficient catalyst precursor than 4b for the redox isomerization of primary allylic alcohols, while the latter is more efficient than the former for the redox isomerization of secondary allylic alcohols. From the catalytic solutions containing 4a and 2-methyl-2-propen-1-ol, the η3-1-hydroxyallyl comple...

Synthesis of Triphosphorous Bidentate Phosphine–Phosphoramidite Ligands: Application in the Highly Enantioselective Hydrogenation of ortho‐Substituted Aryl Enamides

New couple: A novel combination of phosphine and phosphoramidite groups is applied to the preparation of pseudo-C2-symmetric triphosphorous bidentate phosphine–phosphoramidite ligands 1. Unprecedented enantioselectivities toward ortho-substituted aryl enamides and a 1-naphthylenamide in Rh-catalyzed hydrogenation reactions are obtained.

Synthesis of an elongated linear oligo(phenylene ethynylene)-based building block for application in DNA-programmed assembly

The synthesis of an elongated linear oligonucleotide-functionalised module (ELOM) is described. The ELOM structure is based on an oligo(phenylene ethynylene) backbone substituted with two decyloxy groups. The two termini constitute two salicylaldehyde moieties acting as chemical cross-linkers. Before incorporation into an oligonucleotide sequence the organic part of the module, the elongated linear module (ELM), is functionalised with a dimethoxytrityl group and a phosphoramidite group. This enables incorporation into the middle of 30-mer oligonucleotide sequences by automated DNA synthesis. The obtained ELOMs were characterised by polyacrylamide gel electrophoresis and MALDI-TOF mass spectrometry. In analogy with previously reported LOM and TOM structures the coupling reactions of the ELOM modules were tested.

Nucleoside Phosphoramidites Containing Cleavable Linkers

Phosphoramidite reagents (linker phosphoramidites) containing a cleavable 3'-ester linkage between the nucleoside and the phosphoramidite group can be used to attach the first nucleoside to a solid-phase support. Inexpensive underivatized supports such as LCAA-CPG can then be used as universal supports for oligonucleotide synthesis. No modifications to synthesis coupling conditions and no 3'-dephosphorylation are required. Only oligonucleotides with terminal 3'-OH ends are produced. Phosphoramidites containing both a succinate and a sulfonyldiethanol linkage are particularly useful and create oligonucleotides with both a 3'-OH and 5'-phosphate. In addition, by using these reagents, one oligonucleotide sequence can be added onto the 5'-end of another (tandem synthesis) to produce a string of multiple oligonucleotides linked end-to-end. Deprotection releases the oligonucleotides from each other to yield a mixture of oligonucleotides. This approach is particularly useful for making pairs of PCR primers or both strands of a double-stranded sequence in a single operation.

Automated synthesis of new ferrocenyl-modified oligonucleotides: study of their properties in solution.

We have developed new ferrocenyl-modified oligonucleotide (ODN) probes for electrochemical DNA sensors. A monofunctional ferrocene containing phosphoramidite group has been prepared, and a new bisfunctional ferrocene containing phosphoramidite and dimethoxytrityl (DMT) groups has been developed. These ferrocenyl-phosphoramidites have been directly employed in an automated solid-phase DNA synthesizer using phosphoramidite chemistry. The advantages of this method are that it allows a non-specialist in nucleotide chemistry to access labeled ODNs and that it has demonstrated good results. ODNs modified at the 3' and/or 5' extremities have been prepared, with the incorporation of the ferrocenyl group into the chain. The 5' position appears to be more important due to its particular behavior. The thermal stability and electrochemical properties of these new ODN ferrocenes were analyzed before and after hybridization with different ODNs. The feasibility of using these new ferrocenyl-labeled ODNs in DNA sensors has been demonstrated.

Linker phosphoramidite reagents for the attachment of the first nucleoside to underivatized solid-phase supports.

New linker phosphoramidite reagents containing a cleavable 3'-ester linkage are used for attaching the first nucleoside to the surface of a solid- phase support. Inexpensive, underivatized amino supports, such as long chain alkylamine controlled-pore glass, can serve as universal supports. No modifications to phosphoramidite coupling conditions are required and, after synthesis, treatment with NH(4)OH releases the products with 3'-OH ends. No 3'-dephosphorylation is required. Phosphoramidite reagents containing a succinate and sulfonyl diethanol linkage between the nucleoside and phosphoramidite group are particularly advantageous and can be used to create both 3'-OH and 5'-phosphate ends on oligonucleotides. Reproducibility and quality of oligonucleotide synthesis is demonstrated for either column and 96-well plate formats on low-, medium- or high-loading CPG supports.

Solid-phase development of chiral phosphoramidite ligands for enantioselective conjugate addition reactions.

The development of a method for the optimization of chiral ligands for the steric steering of enantioselective Cu-catalyzed conjugate additions of Znalkyls to enones is described. The method is based on combinatorial principles and solid-phase techniques. It includes the combinatorial synthesis of chiral bispidine-derived ligands embodying a phosphoramidite group on the solid phase and their investigation in immobilized form in the conjugate addition of ZnEt2 to cyclohexenone as test reaction. The best identified ligands were also synthesized separately and investigated in its soluble form. The results obtained for the polymer-bound ligands correctly mirrored the performance of the soluble ligands. The library embodied members giving ee values varying between 3 and 67%. The "positional scanning" approach proved to be invalid for the study of the ligand system, indicating that this approach in general should be applied with care. Taken together, the method allowed for rapid and efficient optimization of the ligands and led to the development of the first enantioselective, Cu-catalyzed conjugate addition reaction with a polymer-bound ligand.

Electrospray mass spectrometry of phosphoramidites, a group of acid-labile compounds

In contemporary solid-phase synthesis of oligonucleotides, phosphoramidites containing O-beta-cyanoethyl and N,N -diisopropyl groups are the most widespread monomer units. The N,N -diisopropyl phosphoramidite group can be activated by mild acidic treatment and then it easily reacts with nucleophiles (alcohols, water, etc.) to furnish the required phosphodiester linkage efficiently and cleanly. Owing to these properties, these compounds cannot be investigated using classical electrospray ionization. Their mass spectometric analysis is further hampered by the fact that they are often transiently protected with acid-sensitive groups (4, 4'-dimethoxytrityl, 4-monomethoxytrityl or trityl), which give intense signals in the spectra. Nanoelectrospray measurements from non-aqueous solvents (e.g. acetonitrile, methanol, tetrahydrofuran) were carried out in order to eliminate the nucleophilic water. Different types of alkali metal salts were used to form adduct ions. Among these salts, lithium chloride was found to be the most suitable for the analysis of amidites. Fairly abundant [M+Li](+) and [M+Cl](-) ions are formed in the positive and negative ion mode, respectively. These ions represent the base peaks in most cases whereas the intensities of the peaks corresponding to the protecting group are reduced by approximately 20%. This method is a powerful tool for the mass spectrometric identification of phosphoramidites. Copyright 1999 John Wiley & Sons, Ltd.

Synthesis and characterization of monodeoxynucleotide tethered platinum-(II) and -(IV) complexes †

Improved syntheses of platinum-(II) and -(IV) complexes with pendant hydroxy groups have been elaborated. When the hydroxy groups of the platinum(II) complexes were subjected to phosphoramidite coupling agents to synthesize the corresponding phosphoramidites, intramolecular displacement of the chloride on the platinum by the incoming phosphoramidite group occurred. This generated chloro{N-2-[(2-cyanoethyl)(diisopropylamino)phosphinoxy]ethylenediamine}platinum(II) chloride, which was characterized by a number of physical techniques including X-ray diffraction study. It crystallized from methanol–diethyl ether as white block crystals. Single crystal X-ray analysis revealed that the platinum adopts a four-co-ordinate square planar configuration, provided by the two amine nitrogens, one phosphorus, and one chloride ion. The co-ordination of the phosphoramidite group prevents further manipulation of this group to connect with other hydroxy groups. Intramolecular electron transfer reaction occurred when the platinum(IV) complexes were used in the same reaction. A new strategy has been developed to couple monodeoxynucleotides with the hydroxy groups of the tethered platinum-(II) and -(IV) complexes through the use of monodeoxynucleotide phosphoramidite agents. Every peak in the 1H, 13C-{1H} and 195Pt NMR spectra for all the compounds reported has been assigned through the combination of 1-D 1H, 13C-{1H}, 2-D COSY, XHCORR (heteronuclear chemical shift correlation), HMQC (heteronuclear multiple quantum correlation), and the comparison of chemical shifts among analogs.

Oligo-2'-fluoro-2'-deoxynucleotide N3'-->P5' phosphoramidates: synthesis and properties

Uniformly modified oligodeoxyribonucleotide N3'-->P5' phosphoramidates containing 2'-fluoro-2'-deoxy-pyrimidine nucleosides were synthesized using an efficient interphase amidite transfer reaction. The 3'-amino group of solid phase-supported 2'-fluoro-2'-deoxynucleoside was used as an acceptor and 5'-diisopropylamino phosphoramidite as a donor of a phosphoramidite group in the tetrazole-catalyzed exchange reaction. Subsequent oxidation with aqueous iodine resulted in formation of an internucleoside phosphoramidate diester. The prepared oligo-2'-fluoro-nucleotide N3'-->P5' phosphoramidates form extremely stable duplexes with complementary nucleic acids: relative to isosequential phosphodiester oligomers, the melting temperature Tm of their duplexes with DNA or RNA was increased approximately 4 or 5 degrees C per modification respectively. Moreover, these compounds are highly resistant to enzymatic hydrolysis by snake venom phosphodiesterase and they are 4-5 times more stable in acidic media (pH 2.2-5.3) than the parent oligo-2'-deoxynucleotide N3'-->P5' phosphoramidates. The described properties of the oligo-2'-fluoronucleotide N3'-->P5' phosphoramidates suggest that they may have good potential for diagnostic and antisense therapeutic applications.

5′-5′ tethered oligonucleotides via nucleic bases: A potential new set of compounds for alternate strand triple-helix formation

The solid-phase synthesis of 5′-5′-linked oligonucleotides tethered via nucleic bases and with opposite polarities has been performed using a modified dinucleoside bearing a phosphoramidite group at the 3′-position of one nucleoside and a dimethoxytrityl group at the 3′-position of the second nucleoside.