also compatible with the mild deprotection conditions.
0
Protection of 5 -OH of 5 with DMTr (91%) followed by
0
3
-phosphitylation afforded the phosphoramidite III
88%). The synthesis entails six steps in 50% overall
(
yield starting from 1.
Phosphoramidite III was then incorporatedintoa model
0
sequence 5 -TCXGA (X = 5-fC) using ultramild reagents
with the modified phosphoramidite III using double cou-
pling to give resin-attached ODN1. We divided the resin
into two parts and deprotected them with the following
two procedures, respectively. The first part was treated
with 0.1 M K CO in MeOH/H O (1:1 v/v) at rt for 2 h
2
3
2
followed by addition of AcOH to neutralize the final
solution to neutral pH; the second part was treated with
concentrated NH OH at rt for 2 h. The two samples were
4
then subjected to reverses-phase HPLC analysis. In both
cases, the desired 5mer was produced as the only major
product (peak a, Figure 3A,B). The small peak b in
Figure 3B is probably the imine intermediate; this peak
disappeared completely after overnight standing, suggest-
ing that the imine intermediate was cleanly hydrolyzed into
aldehyde (Figure 3C).
2 3
Figure 3. (A) ODN1 was deprotected by treatment of 0.1 M K CO
Recently, we reported the synthesis of 5-hmC phosphor-
3
in MeOH/H O (1:1 v/v) at rt for 2 h. Peak a is the fully deprotected
2
5mer, [MH] = 1491. (B) ODN1 was deprotected by treatment of
1
þ
amidite with TBDMS as a 5-CH OH protecting group.
2
NH
ODN1 obtained in the same way as (B) was dissoved in H
allowedtostandovernightatrt,after which peak b disappeared. (D)
ODN2 was deprotected by treatment of 0.1 M K CO in MeOH/
4
OH at rt for 2 h; peak b is the proposed imine intermediate. (C)
Although the synthesis is highly efficient and entailed eight
steps in 32% overall yield, the generation of 5-CH OH was
2
O and
2
accomplished through the reduction of the corresponding
-CHO analogue, and the resulting 5-CH OH was subse-
2
2
3
5
H O (1:1 v/v) at rt for 2 h; peak c is the fully deprotected 5mer,
2
[MH] = 1493. (E) ODN2 was deprotected by NH OH at rt for
þ
quently protected as a TBDMS ether. Since 5-fC phos-
phoramidite III could be synthesized more efficiently in six
steps in 50% overall yield and it is completely compatible
with DNA synthesis and the subsequent deprotection, we
reasoned that 5-fC-containing DNA might be converted to
4
2
h. (F) ODN3 made from phoshoramidite IV was deprotected
by treatment of 0.1 M K CO in MeOH/H O (1:1 v/v) at rt
2
3
2
þ
overnight. Peak d is the fully deprotected 5mer, [MH] = 1507;
peak e is the 5mer with 5-trifluoroethoxycarbonyl-dC modifi-
cation, [MH] = 1589. (G) ODN3 made from phoshoramidite
þ
5
-hmC-containing DNA by postsynthetic reduction so
2 3 2
V was deprotected by treatment of 0.1 M K CO in MeOH/H O
(
amidite V was treated with NH OH at rt for 2 h. Peak f is the
1:1 v/v) at 40 °C overnight. (H) ODN3 made from phoshor-
that two steps (introduction and removal of TBDMS)
could be spared.
4
þ
5
g is the 5mer with a 5-methoxycarbonyl-dC modification,
mer with a 5-formamide modification, [MH] = 1506; peak
To facilitate isolation of the DNA, we chose to reduce
the 5-fC-containing DNA when it was still attached with
resin. Thus, resin-attached ODN1 was treated with 0.1 M
CeCl 7H O in MeOH (1 mL) followed by addition of 1
þ
[
MH] = 1521.
3
3
2
mg of NaBH . After 15 min, the resin was treated with the
4
group that can be hydrolyzed to 5-carboxylic acid in
postsynthetic deprotection. Nomura et al. reported the
synthesis of the phosphoramidite IV (Figure 2) with a
same two mild conditions as those used for deprotection of
ODN1 to give ODN2. The HPLC analysis showed that in
both cases the peak of 5-fC-containing oligo ODN1 was
shifted to peak c (Figure 3D,E), which is the corresponding
1
4
5-trifluoroethyl ester functionality. After incorporation
into DNA, it was used to react with amines to generate
amide analogues withtrifluoroethoxy asthe leaving group.
We prepared phosphoramidite IV by following the litera-
ture procedure and found that, for unknown reasons, the
palladium-catalyzed carbonylation reaction always led to
formation of a significant amount of reduced byproduct,
5
-hmC-containing oligo ODN2. This new method pro-
vides a more efficient and convenient approach for synth-
esis of 5-hmC-containing DNA.
Toprepare 5-caC inDNA, we searchedfor the oxidation
conditions that can postsynthetically oxidize the 5-formyl
group of 5-fC in DNA to the corresponding 5-carboxyl
functionality. Although we have screened various oxidiz-
ing reagents, we have yet to identify the proper oxidizing
conditions. As an alternative approach, we sought to
synthesize the phosphoramidite that contains a 5-ester
4
0
N -Ac-2 -deoxycytidine. In addition, we found that the
trifluoroethyl ester could not be hydrolyzed efficiently
under mild basic conditions. After phosphoramidite IV
was incorporated into a short oligo, 5-TCXGA (ODN3),
and deprotected with 0.1 M K CO in 1:1 MeOH/H O
2
3
2
overnight at rt, HPLC analysis showed that only 65% of
the fully deprotected DNA (peak d, Figure 3F) was
obtained in addition to another 35% oligo (peak e,
Figure 3F) still containing trifluoroethyl ester.
(
13) Dai, Q.; Song, C.-X.; Pan, T.; He, C. J. Org. Chem. 2011, 76,
182.
14) Nomura, Y.; Haginoya, N.; Ueno, Y.; Matsuda, A. Bioorg.
Med. Chem. Lett. 1996, 6, 2811.
4
(
3
448
Org. Lett., Vol. 13, No. 13, 2011