Synthesis of 5′-terminal capped oligonucleotides using O-N phosphoryl migration of phosphoramidite derivatives

Article abstract of DOI:10.1021/ol2026075

Trivalent phosphoramidite derivatives could be readily converted by reacting with 1-hydroxy-7-azabenzotriazole to phosphotriester intermediates; these intermediates reacted smoothly with phosphorylated compounds to give pyrophosphate derivatives. This new

Full text of DOI:10.1021/ol2026075

ORGANIC
LETTERS
Synthesis of 50-Terminal Capped
2012
Vol. 14, No. 1
10–13
Oligonucleotides Using OꢀN Phosphoryl
Migration of Phosphoramidite Derivatives
Akihiro Ohkubo, Nobuhiro Tago, Akira Yokouchi, Yudai Nishino, Ken Yamada,
Hirosuke Tsunoda, Kohji Seio, and Mitsuo Sekine*
Department of Life Science, Tokyo Institute of Technology, Nagatsuta, Midoriku,
Yokohama 226-8501, Japan
msekine@bio.titech.ac.jp
Received September 27, 2011
ABSTRACT
Trivalent phosphoramidite derivatives could be readily converted by reacting with 1-hydroxy-7-azabenzotriazole to phosphotriester
intermediates; these intermediates reacted smoothly with phosphorylated compounds to give pyrophosphate derivatives. This new
50
phosphorylation approach enabled a facile and rapid synthesis of 50-adenylated DNA oligomers (A ppDNA) on resins using a silyl-type
linker. Our new approach could be applied to the synthesis of a 20-OMe-RNA oligomer containing the 50-terminal 2,2,7-trimethylguanosine cap
structure.
Various nucleotides containing pyrophosphate or tri-
phosphate linkages play a very important role in biological
reactions.1 Among them, 50-terminal capped oligonucleo-
of mRNAs regulates their translation and degradation.5 In
addition, it has also been found that the 50-terminal 2,2,7-
0
trimethylguanosine cap structure (m32,2,7G5 pppNꢀ) of
U1snRNA, which serves as a component of RNP particles
formed in splicing, is produced in the cytoplasm by hy-
tides have unique biological properties. For example, 50-
0
adenylated DNAs (A5 ppDNAs) are naturally generated
as reaction intermediates catalyzed by DNA2 and RNA3
ligase and can serve as substrates in ligations catalyzed by
permethylation of the precursor of U1snRNA having the
0
m7G5 pppNꢀ structure and can bind to snurportin1
(a transport protein) that exclusively carries the hyper-
methylated molecule into the nucleus.6 The properties of
these 50-capped oligonucleotides would provide a new
insight into gene therapy and gene analysis if a new
approach for their efficient chemical synthesis could be
developed.
ribozymes and 20-deoxyribozymes.4 The well-known 50-
0
terminal 7-methylguanosine cap structure (m7G5 pppNꢀ)
(1) Hindsgaul, O.; Fukuda, M. Molecular Glycobiology; Oxford Uni-
versity Press: New York, 1994.
(2) (a) Weiss, B.; Jaquemin-Sablon, A.; Live, T. R.; Fareed, G. C.;
Richardson, C. C. J. Biol. Chem. 1968, 243, 4543–4555. (b) Harvey,
C. L.; Gabriel, T. F.; Wilt, E. M.; Richardson, C. C. J. Biol. Chem. 1971,
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Acids Res. 1976, 3, 1613–1623. (b) Uhlebeck, O. C.; Gumport, R. I. In
The Enzymes; Boyer, P. D., Ed.; Academic Press: New York, 1982; pp
31ꢀ58.
Chiuman and Li previously reported an enzymatic pre-
paration of A5 ppDNA using T4 DNA ligase.7 Piccirilli
et al. recently developed a new method for the chemical
0
50
synthesis of A ppDNA by using 50-phosphorimidazolidate
€
(4) (a) Li, Y.; Liu, Y.; Braker, R. R. Biochemistry 2000, 39, 3106–
3114. (b) Sreedhara, A.; Li, Y.; Breaker, R. R. J. Am. Chem. Soc. 2004,
126, 3454–3460.
(6) (a) Fischer, U.; Sumpter, V.; Sekine, M.; Satoh, T.; Luhrmann, R.
EMBO J. 1993, 12, 573–583. (b) Sekine, M.; Kadokura, M.; Satoh, T.;
€
Seio, K.; Wada, T.; Fischer, U.; Sumpter, V.; Luhrmann, R. J. Org.
Chem. 1996, 61, 4412–4422. (c) Ohkubo, A.; Sasaki, K.; Noma, Y.;
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(7) Chiuman, W.; Li, Y. Bioorg. Chem. 2002, 39, 332–349.
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242–254. (b) Parker, R.; Sheth, U. Mol. Cell 2007, 25, 635–646. (c)
Wickens, M.; Goldstrohm, A. Science 2003, 300, 753–755.
r
10.1021/ol2026075
Published on Web 12/14/2011
2011 American Chemical Society

0
Scheme 1. Synthesis of Adenosine 50-Phosphoramidite Unit 1
Scheme 2. Synthesis of A5 ppd[CAGT]3 9 on Polymer Supports
Containing a Succinyl Linker
Figure 1. OꢀN phosphoryl rearrangement of phosphoramidite
compound 1 mediated by HOAt.
in a solid phase.8 However, the 50-terminal adenylation of
polythymidylate having more than 10 nucleobases required
a considerably longer time, that is, 16ꢀ48 h.
We found that trivalent phosphoramidite derivatives
could be converted to phosphotriester intermediates by
reacting with 1-hydroxybenzotriazole (HOBt) via an OꢀN
phosphoryl rearrangement.9 These intermediates reacted
smoothly with not only phosphomonoesters but also
phosphodiesters to give the corresponding pyrophosphate
derivatives. Therefore, we were able to demonstrate the
Figure 2. Anion-exchange HPLC profiles of crude mixtures
obtained in the synthesis of A5 ppd[CAGT]3 9 by treatment
with (a) NH4OH and (b) Et3N-3HF.
0
0
0
efficient synthesis of A5 ppDNA and m32,2,7G5 pppRNA
by this new phosphorylation using phosphoramidite
derivatives.
We first synthesized adenosine 50-phosphoramidite
unit 1 by phosphitylation of compound 2, as shown in
Scheme 1. 31P NMR analysis showed that, within 10 min,
phosphoramidite unit 1 (150 ppm) immediately changed to
the corresponding phosphotriester compound 5 (0 ppm)
via the OꢀN phosphoryl rearrangement of phosphite
intermediate 3 when the starting material 1 was treated
with 5 equiv of HOAt (Figure 1).
followed by hydrolysis, afforded H-phosphonate com-
pound 7.10 Compound 7 was subsequently oxidized using
(2R, 8S)-(þ)-(camphorylsulfonyl)oxaziridine in the pre-
sence of N,O-bis(trimethylsilyl)acetamide (BSA). In these
steps for 50-phosphorylation, 2-cyanoethyl groups of inter-
nucleotidic phosphate residues were preserved to avoid
side reactions in adenylation because, as mentioned earlier,
similar phosphorylating reagents reacted easily with not
only phosphomonoesters but also phosphodiesters. The
condensation of the in situ generated reactive phospho-
triester 5 with resin 8 was carried out at room temperature
for 1 h. After the removal of the 2-cyanoethyl and
trimethylsilyl groups, the target oligomer 9 was released
from the resin by treatment with 28% NH4OH for
8 h. Figure 2a shows the HPLC profiles 0 of the crude
mixture obtained in the synthesis of A5 ppd[CAGT]3
To demonstrate the efficiency of compound 5 as a
phosphorylating reagent for pyrophosphate bond forma-
0
tion, the synthesis of A5 ppd[CAGT]3 9 was carried out on
highly cross-linked polystyrene (HCP) resins containing a
succinyl linker, as shown in Scheme 2. After chain elonga-
tion by using a DNA synthesizer, the treatment of resin 6
with 100 equiv of diphenyl phosphonate in pyridine,
(8) Dai, Q.; Saikia, M.; Li, N.; Pan, T.; Piccirilli, J. A. Org. Lett. 2009,
11, 1067–1070.
(9) Ohkubo, A.; Aoki, K.; Seio, K.; Sekine, M. Tetrahedron Lett.
2004, 45, 979–982.
(10) (a) Ohkubo, A.; Seio, K.; Sekine, M. J. Am. Chem. Soc. 2004,
126, 10884–10896. (b) Ohkubo, A.; Kasuya, R.; Aoki, K.; Kobori, A.;
Taguchi, H.; Seio, K.; Sekine, M. Bioorg. Med. Chem. 2008, 16, 5345–
5351.
Org. Lett., Vol. 14, No. 1, 2012
11

thus obtained. HPLC purification gave dimer 12 in 40%
yield, and the isolated material was characterized by
0
Scheme 3. Synthesis of A5 ppd[CAGT]3 9 on Polymer Supports
Containing a Silyl-type Linker
MALDI-TOF mass spectroscopy. These results indicate
that A5 ppDNA can be efficiently synthesized without
0
0
Scheme 4. Synthesis of A5 ppdC-NH2 12
9 by treatment with NH4OH. The decomposition of the
target oligonucleotide was unexpectedly observed at 25ꢀ27
min. Similar decomposition was also observed in the synthesis
0
of A5 ppODN containing other sequences (data not shown).
To avoid the decomposition resulting from treatment
with NH4OH, we used HCP resins containing a silyl
linker,10 which can be cleaved under neutral conditions,
instead of a succinyl linker (Scheme 3). After the phos-
phorylation and adenylation of an N-acylated oligomer
immobilized on the resin were performed in a manner
similar to that described above, the resin was treated with
2 M methylamine/THF to cleave the protecting groups
used for the amino groups of the nucleobases.11 The target
oligomer 9 was subsequently released from the resin by
treatment with 0.2 M Et3N-3HF for 12 h, as shown in
Figure 2b. The decomposition of the 50-adenylated oligo-
mer was surprisingly decreased by using the neutral con-
ditions, and the target oligomer 9 could be isolated in 54%
yield. The oligomer was characterized by MALDI-TOF
mass spectroscopy. Note that the reaction time for adeny-
lation was considerably reduced by using our phosphor-
ylating reagent 5 because the previous methods for the
adenylation of oligomers required a considerably longer
time of more than 16 h.
Figure 3. OꢀN phosphoryl rearrangement of phosphoramidite
compound 14 mediated by HOAt.
2,2,7 50
Scheme 5. Synthesis of m3
G ppp[20-OMe(AUAU6)TFT] 18
Next, 0 we synthesized 50-adenylated 50-deoxycytidylic
acid (A5 ppdC-NH2 12) with an aminoalkyl group, which
can be efficiently linked to the 30 terminus of RNAs by
ligation with T4 RNA ligase.12 After the adenylation of 50-
phosphorylated compound 11 and the removal of the
protecting groups, the resulting resin was treated with
2 M methylamine/THF for 8 h, as shown in Scheme 4.
The solution of methylamine was removed by filtration,
and the resin was rinsed with anhydrous acetonitrile.
Finally, the desired product was eluted from the resin
using water according to Dellinger’s procedure.13 Figure
4a shows the reversed phase HPLC profiles of the mixture
(11) Ohkubo, A.; Sakamoto, K.; Miyata, K.; Taguchi, H.; Seio, K.;
Sekine, M. Org. Lett. 2005, 7, 5389–5392.
(12) Sekine, M.; Kusuoku, H. Nucleosides Nucleotides 1992, 11,
1713–1730.
ꢀ
(13) Dellinger, D. J.; Timar, Z.; Myerson, J.; Sierzchala, A. B.;
ꢀ
Turner, J.; Ferreira, F.; Kupihar, Z.; Dellinger, G.; Hill, K. W.; Powell,
J. A.; Sampson, J. R.; Caruthers, M. H. J. Am. Chem. Soc. 2011, 133,
11540–11556.
12
Org. Lett., Vol. 14, No. 1, 2012

treatment with 1 M tetrabutylammonium fluoride in the
presence of 0.5 M AcOH for 8 h, as shown in the HPLC
profiles in Figure 4b. The target oligomer 18 could be
isolated in 12% yield and was characterized by MALDI-
TOF mass spectroscopy. These results show that our ap-
proach is useful for not only the 50-adenylation of oligomers
but also the formation of the 50-terminal triphosphate bond.
In conclusion, we have established a new convenient
approach for the 50-terminal capping reaction of oligonu-
cleotides on polymer supports by using the phosphorami-
ditecompound. Thus, 50-adenylatedDNA oligomers9 and
12 and 50-(2,2,7-trimethyl)guanosine capped 20-OMe-
RNA 18 could be efficiently and rapidly synthesized.
One advantage of our approach is that the reaction time
can be considerably reduced compared with that of
previous methods. Another advantage is that phosphor-
amidite derivatives used in these reactions are more stable
than general reagents, such as phosphorimidazolidates,
during purification and preservation. These results indi-
cate that our approach might be very useful for 50-terminal
capping modification of gene therapeutic oligonucleotides.
Further studies in this direction are currently in progress.
Figure 4. Reversed phase HPLC or anion-exchange profiles of
the crude mixtures obtained in the synthesis of 50-terminal
0
capped oligonucleotides 12 and 18: (a) A5 ppdC-NH2 12 and
(b) m3
2,2,7 50
G ppp[20-OMe(AUAU6)TFT] 18.
serious decomposition by using methylamine, even on the
resin containing a succinyl linker.
Moreover, employing ournewapproach, wealsocarried
2,2,7
5
0
out the synthesis of m3 G pppRNA on polymer sup-
ports using OꢀN phosphoryl rearrangement (Scheme 5).
Trivalent reagent 14 for phosphorylation could be readily
converted to phosphotriester intermediate 15 by treatment
with 5 equiv of HOAt within 10 min in a manner similar to
that described for the OꢀN phosphoryl rearrangement of
compound 1 (Figure 3). The 50-terminal pyrophosphate
bond was formed by the reaction of the protected oligo-
nucleotide immobilized on the resin with the in situ
generated compound 15 for 15 min, as shown in Scheme 5.
After the deprotection of the 2-cyanoethyl, sulfonylethyl,
and N-acyl groups, the 50-terminal capping reaction was
performed by treating the residue with m32,2,7G phosphor-
imidazolide unit 17.6c The target oligomer 18 was subse-
quently deprotected and released from the resin by
Acknowledgment. This study was supported by a
Grant-in-Aid for Scientific Research from the Ministry
of Education, Culture, Sports, Science and Technology,
Japan, andtheIzumi Scienceand TechnologyFoundation.
This study was also partly supported by a grant from the
global COE project, Ministry of Education, Culture,
Sports, Science and Technology, Japan.
Supporting Information Available. Experimental pro-
cedures and full spectroscopic data for all new com-
pounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
Org. Lett., Vol. 14, No. 1, 2012
13