O-selective condensation using P-N bond cleavage in RNA synthesis without base protection

Article abstract of DOI:10.1021/ol800911b

(Chemical Equation Presented) In RNA synthesis without base protection, a new method for O-selective condensation with more than 99% selectivity was developed by 6-nitro-HOBt-mediated cleavage of undesired P(III)-N bonds on nucleobase moieties. Moreover, we for the first time succeeded in synthesizing oligoRNAs without base protection.

Full text of DOI:10.1021/ol800911b

ORGANIC
LETTERS
2008
Vol. 10, No. 13
2793-2796
O-Selective Condensation Using P-N
Bond Cleavage in RNA Synthesis
without Base Protection
Akihiro Ohkubo,†,‡ Yasukazu Kuwayama,† Tomomi Kudo,† Hirosuke Tsunoda,†,‡
Kohji Seio,†,‡ and Mitsuo Sekine*,†,‡
Department of Life Science, Tokyo Institute of Technology, Nagatsuta, Midoriku,
Yokohama 226-8501, Japan, and CREST, JST (Japan Science and Technology
Agency), 4-1-8 Honcho, Kawaguchi 332-0012, Japan
msekine@bio.titech.ac.jp
Received April 20, 2008
ABSTRACT
In RNA synthesis without base protection, a new method for O-selective condensation with more than 99% selectivity was developed by
6-nitro-HOBt-mediated cleavage of undesired P(III)-N bonds on nucleobase moieties. Moreover, we for the first time succeeded in synthesizing
oligoRNAs without base protection.
Development of effective methods for the synthesis of
oligodeoxynucleotides having base-labile functional groups,
such as oxidatively damaged,1 N-acylated,2 and backbone-
modified3 DNA oligomers or aminoacylated RNA mol-
ecules,4 is highly desirable today because the diversification
of studies on nucleic acids has progressed considerably.
Previously, we reported a so-called “activated phosphite
method” for O-selective condensation using phosphite triester
intermediates in DNA synthesis.5 Its O-selectivity reached
more than 99% when HOBt or 6-nitroHOBt (HOnBt) was
used as an activator of N-unprotected deoxynucleoside 3′-
phosphoramidite building blocks. This high selectivity results
from the low reactivity of the phosphite triester intermediates
toward the exocyclic amino groups of the adenine and
cytosine base moieties compared with hydroxyl groups.
Moreover, we could also synthesize DNA oligomers contain-
ing base-labile deoxynucleoside derivatives such as N4-
acetyl-2′-deoxycytidine5a or N6-acetyl-8-aza-7-deaza-2′-
deoxyadenosine6 in our activated phosphite method using a
silyl-type linker5b,7 that can be cleaved under neutral
conditions.
† Tokyo Institute of Technology.
‡ CREST.
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10.1021/ol800911b CCC: $40.75
Published on Web 06/06/2008
2008 American Chemical Society

If the O-selectivity of the condensation is very high in
this strategy using N-unprotected ribonucleoside 3′-phos-
phoramidite units, we can similarly synthesize RNA oligo-
mers containing base-labile functional groups. For example,
it becomes possible to synthesize non-natural aminoacylated
tRNAs4 by solid-phase synthesis or 2′-O-acetoxymethyl
RNAs8 as prodrugs of siRNAs.To examine the selectivity
of condensation using N-unprotected RNA phosphoramidite
units in our activated phosphite method, we performed the
synthesis of 2′-OMe-ApT and 2′-OMe-CpT dimers, as shown
in Scheme 1. The N-unprotected 2′-OMe RNA monomer
be efficiently cleaved by treatment with pyridinium hydro-
chloride as an acidic promoter in the presence of aniline
before the oxidation step. However, this reaction was slow
and in equilibrium with the reverse reaction, as shown in
Figure 1.
Scheme 1. Synthesis of 2′-OMe-ApT (3a) and 2′-OMe-CpT
(3b) Dimers in the Activated Phosphite Method Using HOnBt
Figure 1. Trivalent P-N bond cleavage of a nucleobase using (a)
pyridinium chloride and aniline or (b) HOBt derivatives.
We considered that if HOBt derivatives could be used for
the P-N bond cleavage in place of pyridinium hydrochloride
and aniline, the reverse reaction might be considerably
suppressed. Therefore, we investigated the efficiency of P-N
bond cleavage using HOBt derivatives.
First, we examined this efficiency in the DNA dimer
synthesis of d[ApT] (5a) and d[CpT] (5b). After the coupling
of N-unprotected deoxynucleoside 3′-phosphoramidite de-
rivatives 4a,b5 with dT-loaded HCP 2 in the presence of
benzimidazolium triflate (BIT),12 P-N bond cleavage was
performed by treatment with pyridinum hydrochloride or
HOBt derivatives for 1 min. Subsequently, oxidation of the
resulting phosphite intermediate and release of the desired
dimers from resin were performed by treatment with concd
ammonia. The O-selectivity of each two-step coupling
reaction was evaluated by anion-exchange HPLC analysis
of the reaction mixture obtained, as shown in Table 1.
When the post-treatment was eliminated, the O-selectivities
of condensation in the synthesis of d[ApT] and d[CpT] were
56% and 90%, respectively. The selectivity of the synthesis
of d[ApT] with P-N bond cleavage using pyridinium
hydrogen chloride significantly increased to 96%, but it was
still not enough to synthesize oligonucleotides. The selectivi-
ties in the synthesis of d[ApT] and d[CpT] using HOBt were
similar to that using pyridinium hydrochloride.
building units 1a,b were synthesized by treatment of com-
mercially available N-acylated 2′-OMe RNA phosphoramid-
ite units with methylamine in THF.9 These units reacted with
the hydroxyl group of thymidine on highly cross-linked
polystyrene (HCP) resins10 2 in the presence of 6-nitro-HOBt
(HOnBt) for 10 min at room temperature. The resulting
dimers were released from the resins by treatment with concd
ammonia. The reaction selectivity was estimated by HPLC
analysis of the mixture thus obtained. Unfortunately, these
condensations resulted in very low selectivities of 93% and
95% for 2′-OMe-ApT (3a) and 2′-OMe-CpT (3b), respec-
tively. These results suggested that a new strategy superior
to the activated phosphite method was required for RNA
synthesis without base protection.Therefore, to increase
O-selectivity, we focused on the pioneering study reported
by Gryaznov and Letsinger on the N-unprotected synthesis
of DNA oligomers using a two-step reaction of condensation
and successive P-N bond cleavage.11 In their strategy using
tetrazole or pyridinum chloride as an activator, N-branched
oligonucleotides containing trivalent P-N bonds were gener-
ated as byproducts. They found that these P-N bonds could
To increase the efficiency of P-N bond activation of the
undesired byproducts, we employed two methods using
HOBt derivatives. One involves addition of BIT to the
reaction mixture, based on our previous report that the
coupling efficiency of phosphoramidite units using HOBt
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Org. Lett., Vol. 10, No. 13, 2008

Table 1. O-Selectivitya of Phosphorylation with P-N Bond
Cleavage in d[ApT] (5a) and d[CpT] (5b) Synthesis
Table 2. O-Selectivitya of Phosphorylation with P-N Bond
Cleavage in d[TpApT] (8a) and d[TpCpT] (8b) Synthesis
a Estimated by anion-exchange HPLC analysis.
a Estimated by anion-exchange HPLC analysis.
derivatives and BIT was higher than that using only HOBt
derivatives.5b However, O-selectivity decreased to 89% in
the d[ApT] synthesis, as shown in Table 1. The other method
was devised to increase the acidity of HOBt derivatives by
addition of electron-withdrawing groups, such as trifluoro
or nitro groups. As a result, the efficiency of P-N bond
cleavage increased when using 6-trifluoro-HOBt (HOtfBt) or
HOnBt in place of HOBt. In particular, the selectivity using
HOnBt was more than 99% in each synthesis. These results
demonstrated that the efficiency of P-N cleavage using
HOnBt was much higher than that reported in the previous
method using pyridinum hydrochloride and aniline.
synthesis in the previous activated phosphite method using
HOnBt and BIT as activators.
Table 3. O-Selectivitya of Phosphorylation with P-N Bond
Cleavage in 2′-OR-AT and 2′-OR-CT Synthesis
Next, d[TpApT] (8a) and d[TpCpT] (8b) trimers were
synthesized to examine the O-selectivity of condensation using
a thymidine phosphoramidite unit 3c toward the nucleobase (A
or C) containing an amino group on polymer supports, as shown
in Table 2. d[ApT]- and d[CpT]-loaded HCP resins 6a,b were
synthesized in advance by the method depicted in Table 1. The
resins 6a,b were treated with an excess thymidine phosphora-
midite unit 7 in the presence of BIT with or without HOBt
derivatives. When the condensation did not involve a post-
treatment of P-N bond cleavage, the selectivities in the
synthesis of d[TpApT] (8a) and d[TpCpT] (8b) were 91% and
90%, respectively. Among the HOBt derivatives tested as P-N
bond cleavage reagents, HOtfBt and HOnBt proved to give
O-selectivities of more than 99%. Interestingly, the efficiency
of P-N bond cleavage using HOBt and BIT increased to 96%
in the d[TpApT] (8a) synthesis unlike the decrease in the
efficiency in the d[ApT] (5a) synthesis.
a Estimated by anion-exchange HPLC analysis.
Figure S1 (Supporting Information) shows the HPLC
profile of the crude mixture obtained in the synthesis of an
oligodeoxynucleotide containing 20 bases by condensation
using 0.2 M BIT and P-N bond cleavage using 0.1 M
HOnBt. The target 20-mer oligonucleotide was obtained as
the main product and satisfactorily isolated in 22% yield.
This result was similar to the results of the usual DNA
Subsequently, we also examined the efficiency of P-N
bond cleavage with HOnBt in RNA synthesis using N-
unprotected phosphoramidite units 1a,b, 9a,b, and 10a,b
having methyl (Me), tert-butyldimethylsilyl (TBDMS), and
triisopropylsilyloxymethyl (TOM) groups, respectively, as
2′-protecting groups, as summarized in Table 3. Compounds
Org. Lett., Vol. 10, No. 13, 2008
2795

9a,b, and 10a,b were also synthesized by treatment of the
corresponding N-acylated phosphoramidite units with me-
thylamine, similar to the synthesis of 1a,b.
As shown in Table 3, the synthesis without P-N bond
cleavage resulted in poor O-selectivities of 74-93%, which
were similar to those in the DNA synthesis described above.
However, the selectivity increased to more than 99% for
P-N bond cleavage using HOnBt for prolonged periods of
2 min regardless of the kind of 2′-protecting group, while
the selectivity of synthesis with the P-N bond cleavage for
1 min was not adequate (96-99%).
Moreover, 2′-OR-[UpA]pT (19a, 20a, 21a) and 2′-OR-
[UpC]pT (19b, 20b, 21b) trimers, where R is methyl, TBDMS,
or TOM, were synthesized to examine the O-selectivity of the
uridine units 16, 17, and 18 toward the nucleobase A or C
containing an amino group on polymer supports, as shown in
Scheme 2. The selectivity also increased to more than 99% for
Figure 2. Anion-exchange HPLC profile of the crude mixture
obtained in the sysnthesis of RNA-DNA chimeric oligomers and
an RNA oligomer: (a) 2′-OMe-[UCAG]T, (b) r[U4CU5], (c)
r[U4AU5], and (d) 2′-OMe-[CCUACAGAGAACUGCGGUU]-TT.
Scheme 2. Synthesis of 2′-OR-[UpA]pT and 2′-OR-[UpC]pT
Trimers with P-N Bond Cleavage Using HOnBta
of byproducts containing P-N bonds (data not shown)
because the O-selectivity of the condensation gradually
decreased due to increase in the number of amino groups.
Therefore, we added HOnBt to a solution of the activator,
BIT, to suppress the reactivity of the reaction intermediate
toward the amino groups. Figure 2d shows the anion-
exchange HPLC profile of the crude mixture obtained in the
synthesis of the RNA-DNA chimeric oligonucleotide 21-
mer. The target oligonucleotide was obtained as a main
product although peaks of byproducts were also observed.
The oligomer was isolated in 11% yield, as shown in Figure
S2, and characterized by MALDI-TOF mass spectroscopy.
In summary, we determined that the O-selectivity of
condensation increased to more than 99% by the P-N bond
cleavage using HOnBt not only in DNA synthesis but also
in RNA synthesis without base protection. Moreover, we
could for the first time synthesize RNA oligomers using our
new strategy. These results encourage us to study the
synthesis of base-labile RNAs such as an aminoacylated
RNA. Further studies are now under way in this direction.
aEstimated by anion-exchange HPLC analysis.
P-N bond cleavage using HOnBt for 2 min.
Furthermore, we synthesized RNA-DNA chimeric oli-
gomers having 5 and 21 bases (an siRNA analogue of
Maitogen-protein kinase 1413) and two RNA 10-mers using
P-N bond cleavage with HOnBt. In the synthesis of 2′-OMe-
[UCAG]T, r[U4CU5], and r[U4AU5] using N-unprotected
phosphoramidite units having the 2′-O-methyl or TBDMS
group (including the N-unprotected 2′-OMeG unit 22),
elongation was performed by condensation using 0.2 M BIT
followed by P-N bond cleavage using 0.1 M HOnBt. Thus,
the target oligonucleotides could be obtained as the main
products and isolated in 64%, 14%, and 17% yields,
respectively (Figure 2a-c). Finally, we tried to synthesize
an RNA-DNA chimeric oligonucleotide 21-mer in a similar
manner. However, the crude mixture involved large amounts
Acknowledgment. This study was supported by a grant
from CREST of Japan Science and Technology (JST) and a
Grant-in-Aid for Scientific Research from the Ministry of
Education, Culture, Sports, Science and Technology, Japan.
This study was also supported in part by a grant of the
Genome Network Project from the Ministry of Education,
Culture, Sports, Science and Technology, Japan, and by the
global COE project.
Supporting Information Available: Experimental pro-
cedures and full spectroscopic data for all new compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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J.; Mao, M.; Li, B.; Cavent, G.; Linsley, P. S. Nat. Biotechnol. 2003, 21,
635–637.
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