Synthesis of oligodeoxyribonucleotides containing hydroxymethylphosphonate bonds in the phosphoramidite method and their hybridization properties

Article abstract of DOI:10.1016/j.tetlet.2005.10.082

A set of diastereomeric TpT phosphoramidite building blocks, 1-f and 1-s, containing an alkaline-labile hydroxymethylphosphonate (HMP) linkage were synthesized. Dodecadeoxynucleotides incorporating the HMP bond were also synthesized by use of each stereoc

Full text of DOI:10.1016/j.tetlet.2005.10.082

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 8953–8957
Synthesis of oligodeoxyribonucleotides containing
hydroxymethylphosphonate bonds in the phosphoramidite
method and their hybridization properties
Akihiro Ohkubo,a,c Katsufumi Aoki,a Yusuke Ezawa,a Yuichi Sato,a Haruhiko Taguchi,a,c
Kohji Seiob,c and Mitsuo Sekinea,c,
*
aDepartment of Life Science, Tokyo Institute of Technology, Nagatsuta, Midoriku, Yokohama 226-8501, Japan
bDivision of Collaborative Research for Bioscience and Biotechnology, Frontier Collaborative Research Center, Nagatsuta,
Midoriku, Yokohama 226-8501, Japan
cCREST, JST (Japan Science and Technology Agency), Nagatsuta, Midoriku, Yokohama 226-8501, Japan
Received 8 August 2005; revised 11 October 2005; accepted 14 October 2005
Available online 7 November 2005
Abstract—A set of diastereomeric TpT phosphoramidite building blocks, 1-f and 1-s, containing an alkaline-labile hydroxy-
methylphosphonate (HMP) linkage were synthesized. Dodecadeoxynucleotides incorporating the HMP bond were also synthesized
by use of each stereochemically pure dimer block under mild conditions in the N-unprotected phosphoramidite method. Moreover,
it turned out that HMP-oligonucleotides derived from a fast-eluted dimer block exhibit higher affinity for DNA single and double
strands than those containing a slow-eluted one.
Ó 2005 Elsevier Ltd. All rights reserved.
A variety of modified oligonucleotides have been synthe-
sized as inhibitors of gene expression in antisense,1 de-
coy,2 and RNAi3 strategies. Among them, backbone
modified DNA oligomers such as phosphorothioate,4
phosphoramidate,5 and methylphosphonate6 DNA have
proved to have high nuclease resistance to endonucleases
and exonucleases. Therefore, much attention has been
paid to their potential usefulness as antisense drugs. In
our previous study,7 we reported the chemical synthesis
of oligothymidylates having hydroxymethylphosphonate
(HMP)8 linkages instead of phosphate diester linkages,
as shown in Figure 1. However, three unavoidable
problems were reported as described below because
HMP oligonucleotides were synthesized by the H-phos-
phonate method.9 (1) It is very hard to isolate a single
isomer of an HMP-modified oligonucleotide because a
set of diastereomers having the HMP bond was gener-
ated at the modified phosphate linkage. (2) The detailed
analysis of HMP-modified duplexes is very difficult
because of the presence of this diastereomeric mixture.
(3) The isolated yields of modified oligonucleotides are
very low, especially, in the synthesis of longer than
10-mer oligonucleotides because of cleavage of the
H-phosphonate diester bonds due to intermolecular
cyclization in the H-phosphonate method.10
In this letter, we report the synthesis of phosphoram-
idite dimer building blocks, 1-f and 1-s (where f and s
refer to a fast-eluted product and a slow-eluted one,
respectively, in silica gel chromatography), containing
a HMP bond (Scheme 1) and modified DNA 12mers
having HMP bonds to overcome the problems described
above by use of 1-f and 1-s. Moreover, for the first time,
we revealed detailed properties of HMP-DNA such as
the ability of duplex formation and base recognition.
In order to obtain the dimer phosphoramidite units
1a,b, the thymidine dimer 3 having an H-phosphonate
internucleotidic linkage was synthesized by condensa-
tion of 50-O-(4,40-dimethoxytrityl)thymidine H-phos-
phonate (2) with 30-O-(tert-butyldimethylsilyl)thymidine
in the presence of N,N-bis(2-oxo-3-oxazolidinyl)phos-
phoric chloride (BOP-Cl) as a condensing agent for
30 min. After the extraction without further purifica-
tion, the resulting material was converted into the corre-
sponding silyl phosphite intermediate by treatment with
N,O-bis(trimethylsilyl)acetamide (BSA). The reaction of
*
Corresponding author. Tel.: +81 45 924 5706; fax: +81 45 924
5772; e-mail: msekine@bio.titech.ac.jp
0040-4039/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.10.082

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A. Ohkubo et al. / Tetrahedron Letters 46 (2005) 8953–8957
OTMS
NTMS
H2CO
t-BuNH2
O
N
O
O
N
O
NH
CH3
NH
NH
O
N
O
O
DMTrO
O
O
HO
O
HO
O
O
N
O
N
O
H
O
O
NH
O
NH
P
P
P
-
O
+
O
O
O
H
O
O
HOCH2
HNEt3
O
9
9
O
OH
Figure 1. Previous method for the synthesis of HMP-DNA oligomers in the H-phosphonate method.
O
N
O
O
N
O
N
O
O
N
NH
NH
NH
NH
O
O
O
DMTrO
DMTrO
DMTrO
O
c, d
O
O
a
b
O
DMTrO
O
O
N
O
N
O
N
O
H
O
O
NH
NH
NH
P
P
P
O
O
O
O
O
O
O
O
O
TMSO
HOCH2
P
O
O
-
+
O
H
HNEt3
2
3
4
5
OTBS
OTBS
OTBS
O
N
O
O
N
O
O
NH
NH
NH
O
O
DMTrO
DMTrO
O
O
N
O
O
f
e
g
DMTrO
O
N
O
O
O
O
O
NH
NH
P
P
O
NH
O
N
O
O
O
P
AcOCH2
AcOCH2
O
O
N
O
O
AcOCH2
O
O
OTBS
7
6
1
P
OH
N
CEO
Scheme 1. Regents and conditions: (a) 30-O-(tert-butyldimethylsilyl)thymidine (1.05 equiv), BOP-Cl (1.1 equiv), pyridine, rt, 0.5 h; (b) BSA
(10 equiv), CH3CN, rt, 0.5 h; (c) H2CO (gas), rt, 2 min; (d) tert-butylamine–MeOH (1:9, v/v), rt, 15 min; (e) Ac2O (1.5 equiv), DMAP (0.04 equiv), rt,
2 h; (f) TBAF (2 equiv), AcOH (2 equiv), rt, 4 h; (g) ClP(OCE)NiPr2 (1.5 equiv), EtNiPr2 (1.5 equiv), THF, rt, 15 min.
the resulting silyl phosphite derivative with H2CO gas
followed by treatment with 10% tBuNH2/MeOH for
15 min gave the diastereoisomers 5-f and 5-s, which
could be separated by silica gel column chromatography
and isolated in 34% and 32% yields, respectively. Treat-
ment of each 5-f and 5-s with Ac2O gave 6-f and 6-s,
which in turn were converted to 7-f and 7-s by treatment
with tetrabutylammonium fluoride (TBAF)–AcOH in
overall yields of 61% and 62%, respectively. In these
TBAF-mediated reactions, decomposition of the
hydroxymethylphophonate linkage was observed. A
similar decomposition was also observed when Et3N-
3HF was used. Finally, the desired phosphoramidite di-
mer units 1a and 1b were synthesized in 90% and 79%
yields, respectively, by the usual phosphitylation of 7-f
and 7-s with ClP(OCE)NiPr2.
promoter for 1 min at room temperature, as shown in
Scheme 2. After the coupling, oxidation was carried
out by using I2 in pyridine–water. Subsequently, the
DMTr group of the terminal 50-hydroxyl group was
removed by the acid treatment using CCl3COOH. The
resulting oligomer 9 was deprotected and released from
the resin by treatment with concd NH3 for 30 min, and
analyzed by HPLC. However, complicated decomposi-
tion of the target material 10 was observed, as shown
in Figure 2a. To avoid the cleavage of the HMP bond,
milder conditions for deprotection, that is, 5% nPrNH2
for 30 min, were used instead of concd NH3 for
30 min. Consequently, the side reactions could be signi-
ficantly suppressed, as shown in Figure 2b. The oligo-
mer 10-f was isolated in satisfactory yield (63%) and
characterized by ESI mass spectrometry.
The synthesis of modified trithymidylate derivatives 10-f
having a HMP bond was carried out to examine
the chemical stability of the HMP bond under the
conditions prescribed for the DNA synthesis involving
detritylation, coupling, and oxidation (Scheme 2). Con-
densation was performed by use of 20 equiv of the phos-
phoramidite unit 1-f with a T-loaded highly cross-linked
polystyrene resin11 having an oxalyl linker12 in the pres-
ence of 40 equiv of benzimidazolium triflate (BIT)13 as a
Moreover, we synthesized three 12mer oligonucleotides
containing one or three HMP bonds under similar mild
conditions. However, the protecting groups of the nucleo-
bases such as benzoyl and acetyl could not be com-
pletely removed by treatment with 5% nPrNH2/MeOH
for 30 min. Therefore, we tried to apply the activated
phosphite method14 without base protection to the syn-
thesis of oligonucleotides using an ABI 392 synthesizer.
These oligomers were also isolated in satisfactory yields

A. Ohkubo et al. / Tetrahedron Letters 46 (2005) 8953–8957
8955
Th
O
DMTrO
O
O
Th
N
P
BIT
O
O
AcOCH
H
N
2
+
-
OTf
Th
O
N
H
HO
P
O
CEO
1
0.1 M
I
3% CCl COOH
3
2
O
O
O
CH CN
Py - H O ( 9:1, v/ v )
2
CH Cl
2 2
3
NH
r.t., 1 min
r.t., 2 min
r.t., 2 min
8
Th
HO
O
Th
HO
O
O
O
P
Th
O
O
O
O
AcOCH
deprotection
2
P
Th
O
HOCH
O
2
O
P
O
O
Th
CEO
O
O
O
O
O
P
Th
O
-
O
O
O
9
10
OH
NH
Scheme 2.
1-f increased compared with that of the unmodified
duplex (DTm = +0.6 °C). This increase might result
from the reduction of an anion charge of the backbone.
To the contrary, that of the duplex containing a HMP
(slow) linkage greatly decreased (DTm = ꢀ3.9 °C). It is
well known that modified DNA duplexes having an
Rp isomer of methylphosphonate is thermodynamically
more stable than those of the duplexes having an Sp iso-
mer because of steric hindrance between the methyl
group of the Sp isomer and H-30.15 Therefore, these re-
sults strongly suggested that the HMP (fast) is an Rp
isomer and the HMP (slow) is an Sp isomer. In addition,
the duplex having three fast HMP linkages was thermo-
dynamically more stable than the natural duplex or the
duplex having a HMP (fast) linkage (DTm = +1.7 °C).
Analysis of the CD spectra of these modified DNA
duplexes suggested that they have a typical B-form (data
is not shown).
Figure 2. The anion-exchange HPLC of the crude mixture obtained in
the synthesis of 10 having a hydroxymethylphosphonate linkage. (a)
Deprotection by treatment with concd NH3 for 30 min; (b) 5%
nPrNH2/MeOH for 30 min.
(44%, 37% and 29%) and characterized by MALDI-
TOF mass spectrometry.
The measurements of Tm values were carried out to
evaluate the thermal stability of duplexes formed
HMP-DNAs and their complementary DNA, as shown
in Table 1. As a result, it turned out that thermal stabil-
ity of the duplex containing a dimer block derived from
The Tm values of the DNA duplexes having a mis-
matched base pair are also summarized in Table 2.
The base recognition ability of the DNA duplex having
Table 1. Tm valuesa,b for DNA 12mer duplexes containing HMP bonds
HMP-DNA 12mer
Target DNA 12mer
5'-d(TpTxTpCpTyTpCpCpTxTpCpT)-3'
3'-d(ApApApGpApApGpGpApApGpA)-3'
HMP modification
x
y
Tm (°C)
DTm (°C)
None
Phosphodiester
Phosphodiester
Phosphodiester
HMP (fast)
Phosphodiester
HMP (fast)
HMP (slow)
HMP (fast)
34.6
35.2
30.7
36.3
—
1
1
3
+0.6
ꢀ3.9
+1.7
a Tm values were measured under the following conditions; 10 mM sodium phosphate (pH 7), 100 mM NaCl, 0.1 mM EDTA, 2 lM duplex.
b DTm is the difference in Tm between the duplex having HMP linkages and that having natural phosphodiester.

8956
A. Ohkubo et al. / Tetrahedron Letters 46 (2005) 8953–8957
Table 2. Tm valuesa,b for DNA 12mer duplexes containing HMP bonds to evaluate their base recognition ability
z
Tm, °C (DTm, °C)
Mismatch
Match
-ApA-
-GpA-
-CpA-
-TpA-
-ApG-
-ApC-
-ApT-
Phosphodiester
HMP (fast)
HMP (slow)
34.6
28.7
21.0
23.5
24.1
20.1
24.2
35.2 (+0.6)
30.7 (ꢀ3.9)
28.4 (ꢀ0.3)
20.7 (ꢀ0.3)
22.8 (ꢀ0.7)
24.1 (0)
21.3 (ꢀ2.8)
18.9 (ꢀ1.2)
22.3 (ꢀ1.9)
22.0 (ꢀ6.7)
14.7 (ꢀ6.3)
19.2 (ꢀ4.3)
14.1 (ꢀ6.0)
17.5 (ꢀ4.8)
a Tm values were measured under the following conditions; 10 mM sodium phosphate (pH 7), 100 mM NaCl, 0.1 mM EDTA, 2 lM duplex.
b DTm is the difference in Tm between the duplex having HMP linkages and that having natural phosphodiester.
Table 3. Tm valuesa,b for DNA 12mer duplexes containing HMP bonds
HMP modification
x
y
Tm, °C (DTm, °C)
pH 7.0
pH 6.2
pH 5.4
None
Phosphodiester
Phosphodiester
Phosphodiester
HMP (fast)
Phosphodiester
HMP (fast)
HMP (slow)
HMP (fast)
7.1
20.1
28.8
1
1
3
8.3 (+1.1)
5.7 (ꢀ1.4)
9.5 (+2.4)
20.9 (+0.8)
14.0 (ꢀ6.1)
23.9 (+3.8)
29.5 (+0.7)
22.2 (ꢀ6.6)
34.1 (+5.3)
a Tm values were measured under the following conditions; 10 mM sodium cacodylate (pH 7.0, 6.2 and 5.4), 100 mM NaCl, 0.1 mM EDTA, 2 lM
triplex.
b DTm is the difference in Tm between the triplex having HMP linkages and that having natural phosphodiester.
a fast HMP was higher than that of the natural DNA
duplex in all cases. The difference in the Tm value
between HMP-DNA duplexes having a matched base
pair and those having a mismatched base pair increased
in the 0.6–2.5 °C region. In measurement of base recog-
nition ability of the DNA duplex having a slow HMP, it
turned out that the base recognition ability was main-
tained similarly to the case of a fast HMP though these
Tm values greatly decreased.
containing HMP (fast) linkage have not only high abil-
ity of duplex or triplex formation but also high ability of
base recognition. These results suggest the usefulness of
an oligonucleotide containing HMP (fast) as a DNA
drug in antisense methodology or a DNA probe of
DNA microarray. Further studies are now underway
in this direction.
Acknowledgements
Furthermore, we examined the thermal stability of the
triplex containing HMP-DNA, as shown in Table 3.
The triplex-formation of HMP-DNA 12mer toward
the hairpin DNA 34mer was carried out in sodium caco-
dylate buffers (pH 7.0, 6.2 and 5.4) containing 100 mM
NaCl. Consequently, the thermal stability of the triplex
containing HMP-DNA was almost similar to those of
duplexes containing HMP-DNA. The thermal stability
of the triplex containing a HMP (fast) linkage was high-
er than that of the triplex containing a HMP (slow) link-
age and the triplex containing three HMP (fast) linkages
was the most stable among them.
This work was supported by a Grant from CREST of
JST (Japan Science and Technology Agency) and a
Grant-in-Aid for Scientific Research from the Ministry
of Education, Culture, Sports, Science and Technology,
Japan. This work was also supported by the COE21
project.
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