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Solid-phase synthesis and hybrization behavior of partially 2'/3'-O-acetylated RNA oligonucleotides.

Xu J, Duffy CD, Chan CK, Sutherland JD - J. Org. Chem. (2014)

Bottom Line: Strong-base stability of the photolabile linker allowed on-column nucleobase and phosphate deprotection, followed by a mild cleavage of the acetylated oligonucleotides from the solid support with UV light.In order to further investigate the effect of 2'/3'-O-acetyl groups on the stability of RNA duplex structure, two complementary bis-acetylated RNA oligonucleotides were also expediently obtained with the newly developed protocols.UV melting curves of 2'-O-acetylated RNA duplexes showed a consistent ~3.1 °C decrease in Tm per 2'-O-acetyl group.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory of Molecular Biology , Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, U.K.

ABSTRACT
Synthesis of partially 2'/3'-O-acetylated oligoribonucleotides has been accomplished by using a 2'/3'-O-acetyl orthogonal protecting group strategy in which non-nucleophilic strong-base (DBU) labile nucleobase protecting groups and a UV-light cleavable linker were used. Strong-base stability of the photolabile linker allowed on-column nucleobase and phosphate deprotection, followed by a mild cleavage of the acetylated oligonucleotides from the solid support with UV light. Two 17nt oligonucleotides, which were synthesized possessing one specific internal 2'- or 3'-acetyl group, were used as synthetic standards in a recent report from this laboratory detailing the prebiotically plausible ligation of RNA oligonucleotides. In order to further investigate the effect of 2'/3'-O-acetyl groups on the stability of RNA duplex structure, two complementary bis-acetylated RNA oligonucleotides were also expediently obtained with the newly developed protocols. UV melting curves of 2'-O-acetylated RNA duplexes showed a consistent ~3.1 °C decrease in Tm per 2'-O-acetyl group.

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Sugar pucker at a 2′,5′-linkage site (inotherwise3′,5′-linked RNA) can be C2′-endo or C3′-endo,although the former is preferred in A-form duplex. It is thought thatthe increase in Tm that is observed onacetylation of the 3′-hydroxyl of a 2′,5′-linkagesite is caused by an increase in C2′-endo sugar pucker preferencedue to an increase in the magnitude of the σC–H2′ → σ*C–O3′ stereoelectroniceffect as the antibonding orbital becomes a better acceptor due tothe increased electron withdrawal.
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fig3: Sugar pucker at a 2′,5′-linkage site (inotherwise3′,5′-linked RNA) can be C2′-endo or C3′-endo,although the former is preferred in A-form duplex. It is thought thatthe increase in Tm that is observed onacetylation of the 3′-hydroxyl of a 2′,5′-linkagesite is caused by an increase in C2′-endo sugar pucker preferencedue to an increase in the magnitude of the σC–H2′ → σ*C–O3′ stereoelectroniceffect as the antibonding orbital becomes a better acceptor due tothe increased electron withdrawal.

Mentions: As expected, inclusion of an internal 2′,5′-linkagewithin a duplex led to a reduction in the Tm (ΔTm = −6.9 °C) anddecreased the duplex stability (ΔΔG°37 = +19.4 kJmol–1, Table 3, entries 1 and 2).58 On acetylationof the 3′-hydroxyl at the internal 2′,5′-linkagethe Tm increased (ΔTm = +4.2 °C) and a favorable decrease in ΔG°37 (ΔΔG°37 = −14.5 kJmol–1, Table 3, entries 2 and 4) was calculated that indicateda more stable duplex. The reason for the stabilization effect of 3′-acetylationon the RNA duplexes is not clear thus far. Szostak and co-workershave shown that partially 2′,5′-linked RNA can forman A type duplex with C2′-endo sugar puckering predominatingat the 2′,5′-linkage site.59 The C2′-endo (south) sugar pucker can potentially be stabilizedby acetylation of the 3′-hydroxyl group by enhancing the σC–H2′ → σ*C–O3′ stereoelectronic effect. The increased electronegativity of 3′-OAcrelative to 3′-OH is thought to further stabilize the alreadypreferred C2-endo sugar pucker (Figure 3).This could explain the relatively higher stability of the duplex with3′-O-acetylated RNA compared to its 3′-nonacetylatedcounterpart.


Solid-phase synthesis and hybrization behavior of partially 2'/3'-O-acetylated RNA oligonucleotides.

Xu J, Duffy CD, Chan CK, Sutherland JD - J. Org. Chem. (2014)

Sugar pucker at a 2′,5′-linkage site (inotherwise3′,5′-linked RNA) can be C2′-endo or C3′-endo,although the former is preferred in A-form duplex. It is thought thatthe increase in Tm that is observed onacetylation of the 3′-hydroxyl of a 2′,5′-linkagesite is caused by an increase in C2′-endo sugar pucker preferencedue to an increase in the magnitude of the σC–H2′ → σ*C–O3′ stereoelectroniceffect as the antibonding orbital becomes a better acceptor due tothe increased electron withdrawal.
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Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4323357&req=5

fig3: Sugar pucker at a 2′,5′-linkage site (inotherwise3′,5′-linked RNA) can be C2′-endo or C3′-endo,although the former is preferred in A-form duplex. It is thought thatthe increase in Tm that is observed onacetylation of the 3′-hydroxyl of a 2′,5′-linkagesite is caused by an increase in C2′-endo sugar pucker preferencedue to an increase in the magnitude of the σC–H2′ → σ*C–O3′ stereoelectroniceffect as the antibonding orbital becomes a better acceptor due tothe increased electron withdrawal.
Mentions: As expected, inclusion of an internal 2′,5′-linkagewithin a duplex led to a reduction in the Tm (ΔTm = −6.9 °C) anddecreased the duplex stability (ΔΔG°37 = +19.4 kJmol–1, Table 3, entries 1 and 2).58 On acetylationof the 3′-hydroxyl at the internal 2′,5′-linkagethe Tm increased (ΔTm = +4.2 °C) and a favorable decrease in ΔG°37 (ΔΔG°37 = −14.5 kJmol–1, Table 3, entries 2 and 4) was calculated that indicateda more stable duplex. The reason for the stabilization effect of 3′-acetylationon the RNA duplexes is not clear thus far. Szostak and co-workershave shown that partially 2′,5′-linked RNA can forman A type duplex with C2′-endo sugar puckering predominatingat the 2′,5′-linkage site.59 The C2′-endo (south) sugar pucker can potentially be stabilizedby acetylation of the 3′-hydroxyl group by enhancing the σC–H2′ → σ*C–O3′ stereoelectronic effect. The increased electronegativity of 3′-OAcrelative to 3′-OH is thought to further stabilize the alreadypreferred C2-endo sugar pucker (Figure 3).This could explain the relatively higher stability of the duplex with3′-O-acetylated RNA compared to its 3′-nonacetylatedcounterpart.

Bottom Line: Strong-base stability of the photolabile linker allowed on-column nucleobase and phosphate deprotection, followed by a mild cleavage of the acetylated oligonucleotides from the solid support with UV light.In order to further investigate the effect of 2'/3'-O-acetyl groups on the stability of RNA duplex structure, two complementary bis-acetylated RNA oligonucleotides were also expediently obtained with the newly developed protocols.UV melting curves of 2'-O-acetylated RNA duplexes showed a consistent ~3.1 °C decrease in Tm per 2'-O-acetyl group.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory of Molecular Biology , Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, U.K.

ABSTRACT
Synthesis of partially 2'/3'-O-acetylated oligoribonucleotides has been accomplished by using a 2'/3'-O-acetyl orthogonal protecting group strategy in which non-nucleophilic strong-base (DBU) labile nucleobase protecting groups and a UV-light cleavable linker were used. Strong-base stability of the photolabile linker allowed on-column nucleobase and phosphate deprotection, followed by a mild cleavage of the acetylated oligonucleotides from the solid support with UV light. Two 17nt oligonucleotides, which were synthesized possessing one specific internal 2'- or 3'-acetyl group, were used as synthetic standards in a recent report from this laboratory detailing the prebiotically plausible ligation of RNA oligonucleotides. In order to further investigate the effect of 2'/3'-O-acetyl groups on the stability of RNA duplex structure, two complementary bis-acetylated RNA oligonucleotides were also expediently obtained with the newly developed protocols. UV melting curves of 2'-O-acetylated RNA duplexes showed a consistent ~3.1 °C decrease in Tm per 2'-O-acetyl group.

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