Phosphoramidite Approach Research Articles

The synthesis of modified oligonucleotides by the phosphoramidite approach and their applications

The synthesis of modified oligonucleotides by the phosphoramidite approach and their applications

An efficient synthesis of sphingosine-1-phosphate

The total synthesis of D- erythro-sphingosine-1-phosphate ( 2) via the phosphoramidite approach, starting from 3-O-TBDMS-protected D- erythro-azidosphingosine 3 is described.

Oligodeoxynucleotide analogues containing 3′-deoxy-3′- C- threo-hydroxymethylthymidine: Synthesis, hybridization properties and enzymatic stability

Novel Oligodeoxynucleotide analogues containing 3′- C- threo-methylene phosphodiester internucleoside linkages were synthesized on automated DNA-synthesizers using the phosphoramidite approach. The sugar modified phosphoramidite building block 5 was obtained by phosphitylation of 1-(2,3-dideoxy-5- O-(4,4′-dimethoxytrityl)-3- C-hydroxymethyl-β-D- threo-pentofuranosyl)thymine ( 4) which was synthesized in only three steps from 5′- O-(4,4′-dimethoxytrityl)thymidine ( 1). The hybridization properties and enzymatic stability of the oligonucleotide analogues were studied by UV experiments. 17-Mers having one or three modifications in the middle or two modifications in each end hybridized to DNA with moderate lowered affinity compared to unmodified 17-mers (ΔT m 1–3°C per modification). Furthermore, the end-modified and all-modified oligonucleotides were stable towards snake venom phosphodiesterase.

Large scale, liquid phase synthesis of oligonucleotides by the phosphoramidite approach.

A new method for the liquid phase synthesis of oligonucleotides is described which makes use of polyethylene glycol (PEG) as soluble support and phosphoramidite derivatives as synthons. The new synthetic protocol was applied to a quite large scale production (about 100 mumoles) of such compounds up to the 20mer level. This solution method, called HELP High Efficiency Liquid Phase) Plus, appears effective in terms of speed and coupling yield and can be evaluated for the production of large amount of oligonucleotides.

Chemical synthesis and biological characterization of phosphorothioate analogs of 2', 5'-3'-deoxyadenylate trimer.

In continued studies to elucidate the requirements for binding to and activation of the 2',5'-oligoadenylate (2-5A) dependent endoribonuclease (RNase L), four 2-5A trimer analogs were examined to evaluate the effect of chirality of phosphorothioate substitution on biological activity. The chemical syntheses and purification of the four isomers of P-thio-3'-deoxyadenylyl-(2'-5')-P-thio-3'- deoxyadenylyl-(2'-5')-3'-deoxyadenosine, by the phosphoramidite approach, is described. The isolated intermediates were characterized by elemental and spectral analyses. The fully deblocked compounds were characterized by 1H and 31P NMR and HPLC analyses. The 2',5'-(3'dA)3 cores with either Rp or Sp chirality in the 2',5'-internucleotide linkages will bind to but will not activate RNase L. This is in contrast to 2',5'-A3 core analogs with either RpRp or SpRp phosphorothioate substitution in the 2',5'-internucleotide linkages which can bind to and activate RNase L. There are also marked differences in the ability of the 2',5'-A3 analogs to activate RNase L following introduction of the 5'-monophosphate. For example, the 5'monophosphates of 2',5'-(3'dA)3-RpRp and 2',5'-(3'dA)3-SpRp can bind to and activate RNase L, whereas the 5'-monophosphates of 2',5'-(3'dA)3-RpSp and 2',5'-(3'dA)3-SpSp can bind to but can not activate RNase L.

Branched poly-labelled oligonucleotides: enhanced specificity of fork-shaped biotinylated oligoribonucleotides for antisense affinity selection.

Poly-labelled oligonucleotides can be synthesised using a modified nucleoside phosphoramidite approach (1, 2). However, the use of this approach for the integration of multiple labels into DNA and RNA is expensive and labour intensive because of the high cost of starting materials and the large number of synthetic steps involved. Misiura et al. (3) have developed a non-nucleoside phosphoramidite based on a 3-carbon glyceryl backbone for the introduction of multiple biotin and phosphotyrosine groups. In both these methods the labels are introduced by repeated successive addition during automated synthesis. Bazin et al. (4) described the use of a phosphoramidite based on 1, 4, 7-heptanetriol which was designed to introduce two primary hydroxyl groups onto the oligonucleotide during solid phase synthesis and was used for the preparation of poly-biotinylated probes using a biotin phosphoramidite based on a modified nucleoside. Here we describe the synthesis and applications of a new simple phosphoramidite based on commercially available 1,2,6-trihydroxyhexane which can be used to add multiple hydroxyl groups to the 5'-end of synthetic oligonucleotides during solid phase synthesis. The two primary hydroxyl functions of 1,2,6-trihydroxyhexane were selectively protected as their 4,4'-dimethoxytrityl ethers, by reaction with 4,4'-dimethoxytrityl chloride (2.2 eq) in pyridine to give 1,6-bis(4,4'-dimethoxytrityloxy)hexan2-ol (86%), which was subsequently phosphitylated on the secondary hydroxyl using 2-cyanoethyl N,N-diisopropyl-phosphoramidochloridite (1.1 eq) in the presence of N,N-diisopropylethylamine (1 eq), to give 2-cyanoethyl[ 1,6-bis(4,4'dimethoxytrityloxy)hex-2-yl]N,N-diisopropylamino phosphoramidite (83%, 'MuOH', Figure 1). The multiple hydroxyl monomer (MuOH) was used as a solution in acetonitrile (0.15 M) during automated solid phase synthesis on an ABI 380B DNA/RNA synthesiser and coupled at 93% (first addition) and 95 % (second addition). The wait step for the coupling reaction of the MuOHand the biotin phosphoramidite was 600 sec. The addition of one MuOH-phosphoramidite to the 5'-end of DNA/RNA during automated synthesis allows for the simultaneous addition of two biotin monomers (e.g. ref. 5) or for the addition of another two MuOH-phosphoramidites. The latter permits the attachment of four biotin moieties during one synthesis cycle. We have used the monomer for the automated synthesis of the following polybiotinylated antisense oligoribonucleotides, in conjunction with a commercially available single addition biotin phosphoramidite (5) (0.2M in acetonitrile) (Figure 2):

Isotactic Glycero Oligothymidylate. a Convenient Preparation of (R) and (S) 1′, 2′-Seco 2′-Nor Thymidine

Abstract (R) and (S) dimethoxytrityl derivatives of 1′, 2′-seco 2′-nor thymidine were synthesized in an efficient way. Isotactic dodecaoligoglycerothymidylate was obtained by a solid support phosphoramidite approach. The lack of hybridization with poly rA makes this acyclooligonucleotide useless as antisense or sense agent.

Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach

Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach

The use of oligonucleotide probes containing 2′-deoxy-2′-fluoronucleosides for regiospecific cleavage of RNA by RNase H from Escherichia coli

Protected 2'-deoxy-2'-fluorouridine and 2'-deoxy-2'-fluorocytidine suitable for incorporation into oligonucleotides via the phosphoramidite approach have been prepared. Five modified and two unmodified oligonucleotides have been synthesized to investigate the regiospecific cleavage of a 5S RNA from Escherichia coli by RNase H. In order to show whether the modified oligonucleotides are able to hybridize with the RNA the physico-chemical properties (melting curves, CD spectra) of analogous DNA/oligodeoxyribonucleotide duplexes have been examined. The modified oligonucleotides are shown to form stable duplexes with a DNA-matrix which exist in an A-like form. Two of the modified probes containing four 2'-deoxy-2'-fluorocytidines or two 2'-deoxy-2'-fluorouridines direct the splitting by RNase H of only one phosphodiester bond of the RNA.

Solid phase synthesis of oligoribonucleotides by the phosphoramidite approach using 2′-O-1-(2-chloroethoxy)ethyl protection

The new type protecting group, 1-(2-chloroethoxy)ethyl (Cee) group has been employed for the protection of the 2′-OH groups of ribonucleoside residues in the synthesis of oligoribonucleotides by the phosphoramidite approach en a solid support, using the acid-labile 5′-O-dimethaxytrityl (DMTr) group. This group is completely stable under the acidic conditions required to remove the 5t́-terminal protecting groups in oligonucleotide synthesis on a solid support, and yet is easily removable under mild condition of acidic hydrolysis (pH 2.0) for the final unblocking step. The Cee-protected ribonucleoside 3′-phosphoramidite units were evaluated in the synthesis of a series of oligoribonucleotides consisting of the homopolymers of cytidine, the box 9R and 9R' sequences of Tetrahymena rRNA, and a leader sequence of phage Qβ-A protein mRNA. A full data for the deprotection and purification of synthetic oligoribonucleotides are also described.

Solid-Phase Synthesis of Oligoribonucleotides Using 5′-9-Fluorenylmethoxycarbonyl and 2′-1-(Isopropoxyl)ethyl Protection

Abstract The 1-(isopropoxy)ethyl group (Ipe) has been employed for the protection of the 2′-hydroxy groups of ribonucleoside residues in the synthesis of oligoribonucleotides by the phosphoramidite approach on a solid support, using an base-labile 5′-9-fluorenylmethoxycarbonyl (Fmoc) group.

A Comparative Study of the Phosphotriester Versus the Phosphoramidite Approach to Nitroxide Labeled Oligo dT

Abstract Deoxyuridine labeled in C-5 with a one-atom-tehered 6-memberedring mitroxide (DUTA) was incorporated into the oligomer d(T)7(DUTA)d(T)7 by either the phosphotriester or phosphoramidite method. While the EPR specific activity (AEPR) of the phosphotriester product concurs with EPR data of nucleic acids spin labeled enzymatically, a lower AEPR is observed with the phosphoramidite product. The effects of the chemically harsher phosphoramidite conditions the DUTA label are held accountable for these results.

Synthesis and Properties of Non-Hammerhead RNA Using 1-(2-Chloroethoxy)-ethyl Group for the Protection of 2′-Hydroxyl Function

Abstract The new type protecting group, 1- (2-chloroethoxy)ethyl (Cee) group has been employed for the protection of the 2′-hydroxyl functions of ribonucleoside residues in the synthesis of oligoribonucleotides by the phosphoramidite approach on a solid support, using an acid-labile 5′-dimethoxytrityl group. This group is completely stable under the acidic conditions required to remove the 5′-terminal protecting groups in oligonucleotide synthesis on a solid support, and yet is easily removable under the similar conditions to that of the tetrahydropyranyl (Thp) in the region of pH 2-3 for the final unblocking step. The synthesis of the Cee-protected ribonucleoside 3′-phosphoramidite units proceed smoothly. The Cee-protected ribnucleoside 3′-phosphoramidite units were evaluated in the synthesis of oligoribonucleotides of up to 20 mer residues. The autocleavage of a precursor RNA from bacteriophage T4 (p2Sp1 RNA; precursor of species 1) does not contain the “hammerhead” sequence required for the authocleava...

ChemInform Abstract: Organometallic Methodologies for Nucleic Acid Synthesis

A new, general preparation of 2'-5'-linked oligoadenylates has been developed, which relies on: (1) chemoselective 0- phosphorylation of N-unblocked nucleosides via hydroxyl activation by Grignard reagents, (2) one-pot construction of internucleotide linkage using bifunctional phosphorylating agents, and (3) selective production of a 3',5'-di-O-protected adenosine. This solution-phase synthesis allows large-scale preparation of a wide range of related oligomers. Palladium chemistry coupled with allylic protection of functional groups leads to the development of an efficient solid-phase synthesis of DNA oligomers via a phosphoramidite approach. Ally1 groups on internucleotide linkages and allyloxycarbonyl groups on amino moieties of nucleobases are removable all at once on the solid supports by exposure to a palladium(0) complex and nucleophiles. This procedure has been utilized for synthesis of a DNA 60mer of unprecedented high purity. In addition, this marks the realization of the first efficient preparation of solid-anchored DNA oligomers. Oligonucleotides are one of the most significant classes of compounds from both the academic and industrial point of view. The chemical synthesis, however, poses an array of problems in the transformation and isolation of products, and high overall efficiency is secured only by using a combination of truly appropriate methods. Organometallic chemistry, though exerting general intense influence on contemporary organic synthesis, has seldom been used in nucleic acids synthesis, perhaps because of the multifunctional, polar nature of the substrates. We demonstrate here the potent utility of metallo-organic methodologies in this area by describing a solution-phase synthesis of some short-chain oligomers having unusual structures and a solid-phase preparation of a long-sequence DNA.

Large scale synthesis of oligoribonucleotides on a solid support: synthesis of a catalytic RNA duplex

Three oligoribonucleotides, octaadenosine heptaphosphate [(Ap) 7A]and two fragments of self-cleaving RNA, have been synthesized on a 10 μ mol scale by the phosphoramidite approach using the tetrahydrofuranyl and levulinyl groups for protection of the 2'- and 5'-hydroxyl functions respectively.

Synthesis of new adenosine-2'- and 3'-O-phosphodiesters via the phosphoramidite approach

Synthesis of new adenosine-2'- and 3'-O-phosphodiesters via the phosphoramidite approach

Organometallic methodologies for nucleic acid synthesis

Abstract

Synthesis and characterization of a ribavirin-3′, 5′-phosphate pentadecamer homoribopolymer bearing a 5′-amino tether group and a 3′-thymidine

The synthesis of a pentadecamer of the 5'-phosphate of the antiviral nucleoside ribavirin (1'-beta-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide) has been achieved. This homoribopolymer is terminated at the 5'-position with an (6-aminohexyl)phosphate group to permit conjugation to a carrier and at the 3'-position by a thymidine-5'-phosphate. The synthesis was accomplished using the methyl phosphoramidite approach to oligoribonucleotides. The homoribopolymer was insensitive to ribonuclease A but was sensitive to ribonuclease T2 digestion.

ChemInform Abstract: Nucleotides. Part 29. Solution Synthesis of Protected Di‐2′‐deoxynucleoside Phosphotriesters and Thiophosphotriesters via the Phosphoramidite Approach.

ChemInform Abstract: Nucleotides. Part 29. Solution Synthesis of Protected Di‐2′‐deoxynucleoside Phosphotriesters and Thiophosphotriesters via the Phosphoramidite Approach.

Nucleotides XXIX.1Solution Synthesis of Protected Di-2′-deoxynucleoside Phosphotriesters and Thiophosphotriesters via the Phosphoramidite Approach

The synthesis of the fully protected 16 possible natural di-2′-deoxynucleoside phosphotriesters 6aa-dd and 16 thiophosphotriesters 7aa-dd was achieved by the phosphoramidite approach in solution in preparative scale. The 5′-O-dimethoxytrityl-2′-deoxyribonucleoside 3′-[2-(p-nitrophenyl)ethyl] phosphoromorpholidites 3a-d and the 3′-O-benzyl-2′-deoxyribonucleosides 4a-d were used as starting materials.