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Permanent or reversible conjugation of 2'-O- or 5'-O-aminooxymethylated nucleosides with functional groups as a convenient and efficient approach to the modification of RNA and DNA sequences.

Cieslak J, Grajkowski A, Ausín C, Gapeev A, Beaucage SL - Nucleic Acids Res. (2011)

Bottom Line: The reaction of these novel ribonucleosides with 1-pyrenecarboxaldehyde results in the efficient formation of stable and yet reversible ribonucleoside 2'-conjugates in yields of 69-82%.Although the versatility and uniqueness of 2'-O-aminooxymethyl ribonucleosides in the preparation of modified RNA sequences is demonstrated by the single or double incorporation of a reversible pyrenylated uridine 2'-conjugate into an RNA sequence, the conjugation of 2'-O-aminooxymethyl ribonucleosides with aldehydes, including those generated from their acetals, provides reversible 2'-O-protected ribonucleosides for potential applications in the solid-phase synthesis of native RNA sequences.The synthesis of a chimeric polyuridylic acid is presented as an exemplary model.

View Article: PubMed Central - PubMed

Affiliation: Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Food and Drug Administration, 8800 Rockville Pike, Bethesda, MD 20892, USA.

ABSTRACT
2'-O-Aminooxymethyl ribonucleosides are prepared from their 3',5'-disilylated 2'-O-phthalimidooxymethyl derivatives by treatment with NH(4)F in MeOH. The reaction of these novel ribonucleosides with 1-pyrenecarboxaldehyde results in the efficient formation of stable and yet reversible ribonucleoside 2'-conjugates in yields of 69-82%. Indeed, exposure of these conjugates to 0.5 M tetra-n-butylammonium fluoride (TBAF) in THF results in the cleavage of their iminoether functions to give the native ribonucleosides along with the innocuous nitrile side product. Conversely, the reaction of 5-cholesten-3-one or dansyl chloride with 2'-O-aminooxymethyl uridine provides permanent uridine 2'-conjugates, which are left essentially intact upon treatment with TBAF. Alternatively, 5'-O-aminooxymethyl thymidine is prepared by hydrazinolysis of its 3'-O-levulinyl-5'-O-phthalimidooxymethyl precursor. Pyrenylation of 5'-O-aminooxymethyl thymidine and the sensitivity of the 5'-conjugate to TBAF further exemplify the usefulness of this nucleoside for modifying DNA sequences either permanently or reversibly. Although the versatility and uniqueness of 2'-O-aminooxymethyl ribonucleosides in the preparation of modified RNA sequences is demonstrated by the single or double incorporation of a reversible pyrenylated uridine 2'-conjugate into an RNA sequence, the conjugation of 2'-O-aminooxymethyl ribonucleosides with aldehydes, including those generated from their acetals, provides reversible 2'-O-protected ribonucleosides for potential applications in the solid-phase synthesis of native RNA sequences. The synthesis of a chimeric polyuridylic acid is presented as an exemplary model.

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Ribonucleoside 2′-conjugates produced from the reaction of 5a or 5b with cholesten-3-one (9) or with aldehydes derived from various acetals (11, 13, 15 and 17).
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gkr896-F1: Ribonucleoside 2′-conjugates produced from the reaction of 5a or 5b with cholesten-3-one (9) or with aldehydes derived from various acetals (11, 13, 15 and 17).

Mentions: Over the past decade, the 2′-hydroxy function of ribonucleosides has been extensively modified for the purpose of identifying the biophysical and biochemical parameters necessary for effective and lasting RNA interference-mediated gene silencing activities (1–4). Actually, 2′-hydroxy modifications are known to impart high binding affinity to RNA sequences, increased lipophilicity, enhanced chemical stability and resistance to nucleases (1,2,5). The 2′-hydroxyl group of ribonucleosides is also an attractive function for conjugation reactions; there are numerous examples of ribonucleoside 2′-conjugates that have been reported in various structural studies (6,7) as well as in therapeutic and diagnostic applications (8,9). Although 2′-O-alkylation of ribonucleosides with functional groups has often been employed in the synthesis of ribonucleoside 2′-conjugates (8,9), this method is generally lacking the regioselectivity needed for the production of conjugates free of isomeric impurities. An alternate strategy to the preparation of ribonucleosides 2′-conjugates is the use of the oxyamino-aldehyde coupling reaction (10–12), which incidentally has extensively been applied to the derivatization of oligonucleotides (13–20). Although the reversibility of the oxyamino-aldehyde coupling reaction has, to the best of our knowledge, never been demonstrated, we rationalized that the conjugation of 2′-O-aminooxymethyl ribonucleosides with various functional groups may provide a powerful tool for the preparation and incorporation of permanent or reversible ribonucleoside 2′-conjugates into RNA sequences. Furthermore, reversible ribonucleoside 2′-conjugates may especially be useful in identifying novel ribonucleoside 2′-hydroxyl protecting groups, which have historically been shown to be of critical importance in RNA synthesis (21) and may lead to an improved approach to the solid-phase synthesis of native or modified RNA sequences. Given that the preparation of 2′-O-aminooxymethyl ribonucleosides has not been described in the scientific literature, we are now reporting an efficient method for the synthesis of these ribonucleosides (5a–d, Scheme 1) and that of several permanent or reversible 2′-conjugates (Figure 1). With the objective of demonstrating the reversibility of 2′-O-aminooxymethyl ribonucleoside conjugates, the details of an unprecedented fluoride-mediated conversion of conjugates 6a–d, 12, 14, 16 and 18 to their native ribonucleosides (Scheme 2 and Figure 2) will be discussed. Furthermore, 5′-O-aminooxymethyl thymidine (25, Scheme 3) has also been prepared for the first time and the addition of its pyrenylated conjugate 26 to the 5′-terminus of a DNA sequence serves as a relevant example for the permanent or reversible functionalization of DNA sequences at their 5′-termini. A single or a double incorporation of the 2′-O-pyrenylated ribonucleoside conjugate 6a into a chemically synthesized oligoribonucleotide (21-mer) is performed to further substantiate the permanent/reversible properties of the modified RNA sequence. Moreover, the phosphoramidite derivative of the reversible uridine 2′-conjugate 29 (Scheme 4) is incorporated into a chimeric polyuridylic sequence (21-mer) in order to provide convincing evidence of the usefulness and versatility of 2′-O-aminooxymethyl ribonucleoside conjugates in the design and implementation of novel 2′-hydroxyl protecting groups for potential applications in the synthesis of modified or native RNA sequences.Figure 1.


Permanent or reversible conjugation of 2'-O- or 5'-O-aminooxymethylated nucleosides with functional groups as a convenient and efficient approach to the modification of RNA and DNA sequences.

Cieslak J, Grajkowski A, Ausín C, Gapeev A, Beaucage SL - Nucleic Acids Res. (2011)

Ribonucleoside 2′-conjugates produced from the reaction of 5a or 5b with cholesten-3-one (9) or with aldehydes derived from various acetals (11, 13, 15 and 17).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC3300013&req=5

gkr896-F1: Ribonucleoside 2′-conjugates produced from the reaction of 5a or 5b with cholesten-3-one (9) or with aldehydes derived from various acetals (11, 13, 15 and 17).
Mentions: Over the past decade, the 2′-hydroxy function of ribonucleosides has been extensively modified for the purpose of identifying the biophysical and biochemical parameters necessary for effective and lasting RNA interference-mediated gene silencing activities (1–4). Actually, 2′-hydroxy modifications are known to impart high binding affinity to RNA sequences, increased lipophilicity, enhanced chemical stability and resistance to nucleases (1,2,5). The 2′-hydroxyl group of ribonucleosides is also an attractive function for conjugation reactions; there are numerous examples of ribonucleoside 2′-conjugates that have been reported in various structural studies (6,7) as well as in therapeutic and diagnostic applications (8,9). Although 2′-O-alkylation of ribonucleosides with functional groups has often been employed in the synthesis of ribonucleoside 2′-conjugates (8,9), this method is generally lacking the regioselectivity needed for the production of conjugates free of isomeric impurities. An alternate strategy to the preparation of ribonucleosides 2′-conjugates is the use of the oxyamino-aldehyde coupling reaction (10–12), which incidentally has extensively been applied to the derivatization of oligonucleotides (13–20). Although the reversibility of the oxyamino-aldehyde coupling reaction has, to the best of our knowledge, never been demonstrated, we rationalized that the conjugation of 2′-O-aminooxymethyl ribonucleosides with various functional groups may provide a powerful tool for the preparation and incorporation of permanent or reversible ribonucleoside 2′-conjugates into RNA sequences. Furthermore, reversible ribonucleoside 2′-conjugates may especially be useful in identifying novel ribonucleoside 2′-hydroxyl protecting groups, which have historically been shown to be of critical importance in RNA synthesis (21) and may lead to an improved approach to the solid-phase synthesis of native or modified RNA sequences. Given that the preparation of 2′-O-aminooxymethyl ribonucleosides has not been described in the scientific literature, we are now reporting an efficient method for the synthesis of these ribonucleosides (5a–d, Scheme 1) and that of several permanent or reversible 2′-conjugates (Figure 1). With the objective of demonstrating the reversibility of 2′-O-aminooxymethyl ribonucleoside conjugates, the details of an unprecedented fluoride-mediated conversion of conjugates 6a–d, 12, 14, 16 and 18 to their native ribonucleosides (Scheme 2 and Figure 2) will be discussed. Furthermore, 5′-O-aminooxymethyl thymidine (25, Scheme 3) has also been prepared for the first time and the addition of its pyrenylated conjugate 26 to the 5′-terminus of a DNA sequence serves as a relevant example for the permanent or reversible functionalization of DNA sequences at their 5′-termini. A single or a double incorporation of the 2′-O-pyrenylated ribonucleoside conjugate 6a into a chemically synthesized oligoribonucleotide (21-mer) is performed to further substantiate the permanent/reversible properties of the modified RNA sequence. Moreover, the phosphoramidite derivative of the reversible uridine 2′-conjugate 29 (Scheme 4) is incorporated into a chimeric polyuridylic sequence (21-mer) in order to provide convincing evidence of the usefulness and versatility of 2′-O-aminooxymethyl ribonucleoside conjugates in the design and implementation of novel 2′-hydroxyl protecting groups for potential applications in the synthesis of modified or native RNA sequences.Figure 1.

Bottom Line: The reaction of these novel ribonucleosides with 1-pyrenecarboxaldehyde results in the efficient formation of stable and yet reversible ribonucleoside 2'-conjugates in yields of 69-82%.Although the versatility and uniqueness of 2'-O-aminooxymethyl ribonucleosides in the preparation of modified RNA sequences is demonstrated by the single or double incorporation of a reversible pyrenylated uridine 2'-conjugate into an RNA sequence, the conjugation of 2'-O-aminooxymethyl ribonucleosides with aldehydes, including those generated from their acetals, provides reversible 2'-O-protected ribonucleosides for potential applications in the solid-phase synthesis of native RNA sequences.The synthesis of a chimeric polyuridylic acid is presented as an exemplary model.

View Article: PubMed Central - PubMed

Affiliation: Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Food and Drug Administration, 8800 Rockville Pike, Bethesda, MD 20892, USA.

ABSTRACT
2'-O-Aminooxymethyl ribonucleosides are prepared from their 3',5'-disilylated 2'-O-phthalimidooxymethyl derivatives by treatment with NH(4)F in MeOH. The reaction of these novel ribonucleosides with 1-pyrenecarboxaldehyde results in the efficient formation of stable and yet reversible ribonucleoside 2'-conjugates in yields of 69-82%. Indeed, exposure of these conjugates to 0.5 M tetra-n-butylammonium fluoride (TBAF) in THF results in the cleavage of their iminoether functions to give the native ribonucleosides along with the innocuous nitrile side product. Conversely, the reaction of 5-cholesten-3-one or dansyl chloride with 2'-O-aminooxymethyl uridine provides permanent uridine 2'-conjugates, which are left essentially intact upon treatment with TBAF. Alternatively, 5'-O-aminooxymethyl thymidine is prepared by hydrazinolysis of its 3'-O-levulinyl-5'-O-phthalimidooxymethyl precursor. Pyrenylation of 5'-O-aminooxymethyl thymidine and the sensitivity of the 5'-conjugate to TBAF further exemplify the usefulness of this nucleoside for modifying DNA sequences either permanently or reversibly. Although the versatility and uniqueness of 2'-O-aminooxymethyl ribonucleosides in the preparation of modified RNA sequences is demonstrated by the single or double incorporation of a reversible pyrenylated uridine 2'-conjugate into an RNA sequence, the conjugation of 2'-O-aminooxymethyl ribonucleosides with aldehydes, including those generated from their acetals, provides reversible 2'-O-protected ribonucleosides for potential applications in the solid-phase synthesis of native RNA sequences. The synthesis of a chimeric polyuridylic acid is presented as an exemplary model.

Show MeSH
Related in: MedlinePlus