Oligonucleotide synthesis involving deprotection of amidine-type protecting groups for nucleobases under acidic conditions

Article abstract of DOI:10.1021/ol100676j

Amidine-type protecting groups, i.e., N,N-dimethylformamidine (dmf) and N,N-dibutylformamidine (dbf) groups, introduced into nucleobases were rapidly removed under mild acidic conditions using imidazolium triflate (IMT) or 1-hydroxybenztriazole (HOBt). This new deprotection strategy allowed a 2′-O-methyl-RNA derivative bearing a base-labile group to be efficiently synthesized using a silyl-type linker. It was also found that our new method could be applied to the synthesis of an unmodified RNA oligomer.

Full text of DOI:10.1021/ol100676j

ORGANIC
LETTERS
2010
Vol. 12, No. 11
2496-2499
Oligonucleotide Synthesis Involving
Deprotection of Amidine-Type Protecting
Groups for Nucleobases under Acidic
Conditions
Akihiro Ohkubo, Yasukazu Kuwayama, Yudai Nishino, Hirosuke Tsunoda,
Kohji Seio, and Mitsuo Sekine*
Department of Life Science, Tokyo Institute of Technology, J2-12, 4259 Nagatsuta,
Midoriku, Yokohama 226-8501, Japan
msekine@bio.titech.ac.jp
Received March 23, 2010
ABSTRACT
Amidine-type protecting groups, i.e., N,N-dimethylformamidine (dmf) and N,N-dibutylformamidine (dbf) groups, introduced into nucleobases
were rapidly removed under mild acidic conditions using imidazolium triflate (IMT) or 1-hydroxybenztriazole (HOBt). This new deprotection
strategy allowed a 2′-O-methyl-RNA derivative bearing a base-labile group to be efficiently synthesized using a silyl-type linker. It was also
found that our new method could be applied to the synthesis of an unmodified RNA oligomer.
Chemical modification plays an important role in improving
the properties of natural DNA and RNA molecules, including
hybridization, base recognition, and nuclease resistance.1 In
particular, several 2′-modified RNAs, such as 2′-O-alkylated
and 2′-fluoro-RNAs, have been applied to gene regulation
in antisense, antigene, and RNAi strategies.2 Further studies
for the introduction of additional functional groups into these
2′-modified RNAs are necessary to increase their therapeutic
effect in gene therapy. For example, the introduction of acyl-
type functional groups into nucleobases has proved to
increase hybridization and base-recognition abilities.3 In
contrast, oligonucleotides with acyloxymethyl and acylth-
ioethyl groups as hydroxyl and phosphate group modifiers,
(2) (a) Inoue, H.; Hayase, Y.; Imura, A.; Iwai, S.; Miura, K.; Ohtsuka,
E. Nucleic Acids Res. 1987, 15, 6131–6148. (b) Lesnik, E. A.; Sasmor,
H. M.; Bennett, C. F. J. Biol. Chem. 1997, 272, 11994–12000. (c) Prakash,
T. P.; Manoharan, M.; Fraser, A. S.; Kawasaki, A. M.; Lesnik, E. A.; Owens,
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A. M.; Lesnik, E. A.; Owens, S. R.; Manoharan, M. Org. Lett. 2003, 5,
403–406. (e) Saneyoshi, H.; Seio, K.; Sekiene, M. J. Org. Chem. 2005, 70,
10453–10460.
(3) Ohkubo, A.; Kasuya, R.; Sakamoto, K.; Miyata, K.; Nagasawa, H.;
Tsukahara, T.; Maki, Y.; Watanobe, T.; Seio, K.; Sekine, M. Nucleic Acids
Res. 2008, 36, 1952–1964.
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10.1021/ol100676j  2010 American Chemical Society
Published on Web 05/10/2010

Table 1. Deprotection of dbf Groups Incorporated in rA under
Acidic Conditions
Figure 1. Deprotection of N,N-dibutylformamidine (dbf) from
deoxyadenine (dA) via N-formyl-dA formation.
respectively, are expected to be useful as prodrugs.4 How-
ever, the synthesis of 2′-substituted RNAs containing these
functional groups through standard RNA synthesis proce-
dures is problematic because of their lack of stability under
basic conditions.
In previous studies of oligonucleotide synthesis, various
protecting groups have been reported for nucleobase amino
groups.5 Among them, acyl groups, such as acetyl, benzoyl,
and isobutyryl, have been commonly used in the phosphora-
midite approach to avoid side reactions associated with the
nucleobase amino groups. These acyl groups can be removed
by treatment with aqueous ammonia but are incompatible
with the synthesis of oligonucleotides bearing base-labile
functional groups.
Recently, we developed a new method called “activated
phosphite method without base protection” for the synthesis
of base-labile modified oligomers.6 In this method, the
O-selectivity of the internucleotidic bond formation in DNA
synthesis reached more than 99% using 6-nitroHOBt as an
activator of N-unprotected deoxynucleoside 3′-phosphora-
midite units. This high selectivity results from the low
reactivity of phosphite triester intermediates toward the
exocyclic amino groups of the adenine and cytosine base
moieties compared to the 5′-hydroxyl group. However, the
RNA synthesis required an additional P(III)-N bond cleav-
age step after each condensation reaction because of the low
O-selectivity (93-95%) of coupling steps involving RNA
monomer building blocks.7 In addition, the purification of
desired oligomers from shorter oligomers missing one and
two nucleotide residues was difficult because the capping
step using acetic anhydride was omitted to avoid undesired
N-acetylations.
a Estimated by TLC analysis.
(unpublished data), a modified DNA oligomer 2 having a
6-N-formyl-dA was rapidly converted to the unmodified
oligomer 3 by simple heating under neutral conditions, as
shown in Figure 1. In contrast, the formyl group was reported
to form from the N,N-dimethylformamidine group under
acidic conditions.8 N,N-Dialkylformamidine groups have
been usually used as base protecting groups that are removed
by aqueous ammonia treatment.
In this paper, we propose a new method for RNA synthesis
involving the deprotection of amidine-type groups under
acidic conditions.
In this study, we synthesized an 2′-OMe-RNA oligomer
containing an N-benzoyladenosine derivative as a representa-
tive base-labile model compound. To achieve this synthesis,
our interest was focused on the unique property of the formyl
group attached to the base amino groups. In a previous study
First, the deprotection of the N,N-dibutylformamidine (dbf)
group was examined using 6-N-(N,N-dibutylformamidine)ad-
enosine 4a (Table 1) and its deoxy counterpart 4b (Table
S1, Supporting Information) under various acidic conditions.
This dbf group is well-known as an adenine and cytosine
protecting group in DNA and RNA synthesis.9 When 4a was
treated with 80% AcOH and heated in a 10 mM sodium
citrate buffer at 50 °C for 9-10 h, it was completely
converted to 5a without decomposition (entries 1 and 2 in
Table 1). Subsequently, deprotection of the dbf group by
(4) (a) Parey, N.; Baraguey, C.; Vasseur, J.-J.; Debart, F. Org. Lett. 2006,
8, 3869–3872. (b) Tesquellas, G.; Alvarez, K.; Dell’Aquila, C.; Morvan,
F.; Vasseur, J.-J.; Imbach, J.-L.; Rayner, B. Nucleic Acids Res. 1998, 26,
2069–2074.
(5) Beaucage, S. L., Bergstrom, D. E., Glick, G. D., Jones, R. A., Eds.
Current Protocols in Nucleic Acid Chemistry; John Wiley & Sons, Inc.:
New York, 2000. and references cited therein.
(6) (a) Ohkubo, A.; Seio, K.; Sekine, M. Tetrahedron. Lett. 2004, 45,
363–366. (b) Ohkubo, A.; Seio, K.; Sekine, M. J. Am. Chem. Soc. 2004,
126, 10884–10896.
(8) Seio, K.; Sasami, T.; Twarada, R.; Sekine, M. Nucleic Acids Res.
2006, 34, 4324–4334.
(7) Ohkubo, A.; Kuwayama, Y.; Kudo, T.; Tsunoda, H.; Seio, K.; Sekine,
M. Org. Lett. 2008, 10, 2793–2796.
(9) McBride, L. J.; Kierzek, R.; Beaucage, S. L.; Caruthers, M. H. J. Am.
Chem. Soc. 1986, 108, 2040–2048.
Org. Lett., Vol. 12, No. 11, 2010
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Table 2. Deprotection of dbf and dmf Groups in
O-Toluoylnucleosides Using HOBt or IMT
Table 3. Deprotection of dbf and dmf Groups Incorporated into
Nucleotides Using HOBt or IMT on HCP Resins
entry starting compd
B
reagent time (h) yield (%)
1
2
3
4
5
6
7
8
9
6a
6a
6b
6b
6c
6c
6d
6d
6e
6e
6f
Ad
Ad
Ad
Ad
Cy
Cy
Cy
Cy
Gu
Gu
Gu
Gu
HOBt
IMT
HOBt
IMT
HOBt
IMT
HOBt
IMT
HOBt
IMT
HOBt
IMT
0.5
1
98
87
92
quant
quant
81
90
quant
87
0.2
1.5
0.5
1.5
0.2
1.5
1
22
1.3
24
10
11
12
68
68
70
6f
treatment with acid-azole or acid-pyridine complexes was
examined at 50 °C (entries 3-6 and 9). Interestingly, the
deprotection rate using benzimidazolium triflate (BIT, entry
6) was higher than with 4,5-dichloroimidazolium triflate
(dcIMT, entry 3), 1,2,4-trizazolium triflate (TRT, entry 4),
and pyridinium chloride (PyHCl, entry 5), despite a higher
pH value for BIT compared to dcIMT, TRT, and PyHCl.
These results suggest that the important factors were not only
the acidity of reagents and solvents used to hydrolyze the
amidine group by conversion of the dbf group into a formyl
group but also the nucleophilicity of the reagents used to
deprotect the formyl group. Surprisingly, the deprotection
rate under mild acidic conditions using imidazolium triflate
(IMT, pH 5.3, entry 9) was also higher than that using
PyHCl. This two-step deprotection strategy might be useful
for nucleoside and nucleotide chemistry because the depu-
rination and decomposition of various functional groups
might be suppressed under these mild acidic conditions. On
the other hand, deprotections using 1H-tetrazole and 1-hy-
droxybenztriazole (HOBt) were found to be faster than with
the acidic-azole complexes (entries 6 and 9). In particular,
the dbf group was removed in only 1 h by treatment with
HOBt.
entry
B
R′
reagent
timea (h)
1
2
3
4
5
6
Ad
Ad
Cy
Cy
Gu
Gu
Bu
Bu
Bu
Bu
Me
Me
HOBt
IMT
HOBt
IMT
HOBt
IMT
12
4.5
3.5
1
3.5
24
a Estimated by RP-HPLC analysis.
Next, the deprotection of dbf and N,N-dimethylformami-
dine (dmf) groups from O-toluoyl (Tol) nucleoside deriva-
tives 6a-f using HOBt and IMT under acidic conditions was
investigated. These results are summarized in Table 2. The
formamidine group was rapidly deprotected without decom-
position of the ester groups and depurinaton by treatment
with HOBt for all nucleobases (entries 1, 3, 5, 7, 9, and 11).
The dbf group introduced into adenine and cytosine bases
was removed within 1.5 h by treatment with IMT, although
reactions using IMT were slower than those using HOBt
(entries 2, 4, 6, and 8). However, the removal of the dmf
group from guanine was very slow. Reaction times were 22
and 24 h for compounds 6e and 6f, respectively (entries 10
and 12). The deprotection time might be extended because
2-N-formylguanine is stable under conditions using IMT.
Subsequently, the stability of silyl-type protecting groups
under acidic conditions using IMT was examined (Scheme
S1, Supporting Information). The dbf group was removed
Deprotection results obtained for 4b (entries 1 and 2 in
Table S1, Supporting Information) using BIT, HOBt, and
IMT were similar to those of 4a. However, depurination was
observed under acidic conditions using PyHCl (entry 1 in
Table S1, Supporting Information). Moreover, an increase
in reaction temperature from 50 to 80 °C was found to
significantly accelerate the deprotection in all cases.
2498
Org. Lett., Vol. 12, No. 11, 2010

from 2′,3′,5′-O-tris(tert-butyldimethylsilyl)adenosine (tri-
TBDMS-A) derivative 8 by treatment with IMT without
decomposition of the silyl-type linker. The isolated yield of
the desired product 9 was 81%. These results indicate that
the acyl- and silyl-type protecting groups are stable under
conditions for removal of formamidine-type protecting groups
from nucleobases.
In addition, we investigated the deprotection of dbf and
dmf groups incorporated into polymer-supported oligonucle-
otides. Phosphoramidite units 12a-c were easily synthesized
by reacting N,N-dialkylformamide dimethyl acetal (3 equiv)
with N-unprotected phosphoramidite units 11a-c,10 which
were derived from N-acylated phosphoramidite units 10a-c
(Scheme S2, Supporting Information). After chain elongation
using phosphoramidite derivatives 12a-c and 14, the remov-
al of 2-cyanoethyl groups on T-loaded highly cross-linked
polystyrene (HCP) resins having a silyl-type linker and the
deprotection of dbf and dmf groups using HOBt and IMT
were successively carried out (Table 3). The dbf or dmf
group was removed from the adenine base over prolonged
periods of time at 50 °C (12 h, entry 1) but was deprotected
in only 3.5 h from cytosine and guanine bases using HOBt
(entries 3 and 5). The slow deprotection of the dbf group
from the adenine base results from the slow hydrolysis of
the amidine group into a formyl group. The dbf group on
the adenine and cytosine bases was deprotected within 4.5 h
using IMT (entries 2 and 4). On the other hand, the
deprotection of the dmf group on the guanine base required
a prolonged time (24 h, entry 6) similar to the deprotection
of guanosine monomers 6e and 6f.
Furthermore, we synthesized a 2′-OMe-RNA-DNA chi-
meric oligomer (17), which is a 21-base siRNA analogue of
Maitogen-protein kinase 1411 and a base-labile 2′-OMe-
RNA oligomer (18) having a 6-N-benzoyladenine base (Abz)
by deprotection of amidine-type groups under acidic condi-
tions. In these syntheses, IMT was chosen because of its
milder acidity compared to HOBt. Oligomer chain elongation
was performed on HCP resins having a silyl-type linker12
according to the general procedure involving the capping
step using Ac2O. When the resin-bound 21-mer oligomer
protected with dbf and dmf groups was treated with IMT
for 24 h after removal of the 2-cyanoethyl groups, ca. 70%
of product 17 was unexpectedly found to be released from
the resin because of Si-O bond cleavage. A solution
containing the released oligomer was therefore mixed with
a solution obtained by treating the residual resin with 0.2 M
Et3N-3HF in THF for 4 h. HPLC analysis of the resulting
mixture showed that compound 17 was obtained as the main
product and was isolated in 18% yield (Figure 2a). Figure
2b shows the anion exchange HPLC profile of the crude
mixture obtained for the synthesis of 2′-OMe-RNA oligomer
18. The desired oligonucleotide was obtained as the main
product, although several peaks of byproducts containing the
Figure 2. Anion-exchange HPLC profiles of crude mixtures
obtained by RNA-DNA chimeric and RNA oligomer syntheses:
(a) 2′-OMe-[CCUACAGAGAACUGCGGUU]-TT (17), (b) 2′-
OMe-[GAUACAbzUUGACCU] (18), and (c) r[CUCUCUCUCU]-
T (19).
oligomer containing an acyl group were observed in HPLC
and MALDI-TOF mass spectroscopy (Supporting Informa-
tion). Oligomer 18 was isolated in 19% yield and was
characterized by MALDI-TOF mass spectroscopy.
Finally, this method was applied to the synthesis of an
unmodified RNA oligomer (19). The triisopropylsilyloxym-
ethyl (TOM) group was chosen to protect the 2′-hydroxyl
group, since the TBDMS group was unstable even under
weak acidic conditions because of the neighboring 3′-
phosphate group.13 Figure 2c shows the anion-exchange
HPLC profile of the crude mixture obtained from the
synthesis of RNA oligomer 19 using 2′-TOM-Cdbf phos-
poramidite 20. The desired oligonucleotide was obtained as
the main product and was isolated in 19% yield. No 3′f2′
isomerization of the internucleotidic phosphodiester linkages
of oligomer 19 was confirmed by its complete digestion with
nuclease P1.14 These results demonstrate that our new
method can be applied to the synthesis of unmodified RNA
oligomers.
In summary, we developed a new method for the depro-
tection of dbf and dmf groups on nucleosides and nucleotides
under acidic conditions using HOBt and IMT. Moreover,
our new strategy allowed the first synthesis of an RNA
oligomer carrying a base-labile acyl group on adenine. These
results have prompted us to study the introduction of various
base-labile functional groups into 2′-substituted and unmodi-
fied RNA. These additional studies are now underway.
Acknowledgment. This study was supported by a Grant-
in-Aid for Scientific Research from the Ministry of Educa-
tion, Culture, Sports, Science and Technology, Japan. This
study was also supported in part by a grant of the global
COE project from the Ministry of Education, Culture, Sports,
Science and Technology, Japan.
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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OL100676J
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