Post-synthetic functionalization of oligodeoxyribonucleotides at the 2′-position

Article abstract of DOI:10.1016/S0040-4039(00)01926-2

Oligodeoxyribonucleotides bearing 2′-methoxycarbonylmethylthio-2′-deoxyuridine were synthesized. This reactive substituent at the 2′-position could be used for post-synthetic functionalization.

Full text of DOI:10.1016/S0040-4039(00)01926-2

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 677–680
Post-synthetic functionalization of oligodeoxyribonucleotides at
the 2%-position
Hiroaki Ozaki, Shingo Momiyama, Kazuyuki Yokotsuka and Hiroaki Sawai*
Department of Chemistry, Faculty of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan
Received 20 September 2000; accepted 27 October 2000
Abstract—Oligodeoxyribonucleotides bearing 2%-methoxycarbonylmethylthio-2%-deoxyuridine were synthesized. This reactive sub-
stituent at the 2%-position could be used for post-synthetic functionalization. © 2001 Elsevier Science Ltd. All rights reserved.
There has been growing interest recently in 2%-modified
oligodeoxyribonucleotides (ODN) as the second-gener-
ation antisense molecules. The 2%-position in a duplex is
placed in a minor groove, which is a binding site of
some nucleic acid-binding proteins. For example,
RNase H, which cleaves the RNA strand of RNA/
DNA hybrids, is thought to bind to the minor groove.1
Therefore, the 2%-position is interesting in the study of
nucleic acid–protein interaction. An ODN bearing a
lanthanide complex at the 2%-position of a thymidine
residue, which was designed to attack the bulged nucle-
otide across the minor groove in the ODN/RNA
duplex, was shown to cleave the RNA more efficiently
than an ODN bearing the lanthanide complex at the
C5-position of the thymidine residue, which was
designed to act through the major groove.2 From this
point of view, the syntheses of ODNs bearing an acid–
base catalyst at the 2%-position of cytidine or 2%-amino-
2%-deoxyuridine were reported recently.3
2%-Methoxycarbonylmethylthio-2%-deoxyuridine (5) was
synthesized from uridine (1) as shown in Scheme 1.
2,2%-Anhydrouridine5 (2), 2%-(4-methoxybenzylthio)-2%-
deoxyuridine6 (3), and 2%-mercapto-2%-deoxyuridine7 (4)
were prepared by the method described previously.
Compound 4 was allowed to react with methyl bro-
moacetate in dichloromethane containing triethylamine,
giving
2%-methoxycrbonylmethylthio-2%-deoxyuridine
(5).8 In this reaction, slight alkylation of N3 in the
uracil moiety was observed, and the product was
purified by silica gel column chromatography. Com-
pound 5 was subjected to 5%-dimethoxytritylation giving
compound 6 and the subsequent 3%-phosphitylation (7)
for the chemical synthesis of ODNs.
To deduce the reactivity of the methoxycarbonylthio
group with an amine, a dimer (dU*pT) containing
2%-methoxycarbonylmethylthio-2%-deoxyuridine (dU*)
was prepared. The coupling yield of compound 7 on a
DNA synthesizer was ca. 60–70% even for 360 s as the
coupling time. This may be due to steric hindrance of
the substituent at the 2%-position. The dimer attached to
the CPG was treated with each amine (Scheme 2; 50%
solution of tris(2-aminoethyl)amine, ethylenediamine,
or heptylamine in dry ethanol, or methanolic ammonia
overnight at rt). The crude products were analyzed on
reversed-phase HPLC as shown in Fig. 1. Each chro-
matogram has two main peaks. One peak at TR:12
min was thymidine, which was a failure product that
could not react with the modified nucleoside phospho-
ramidite, and the other peak was the desired product.
The main products (8–10) of each reaction were
purified by HPLC and analyzed by ESI-MS spec-
troscopy,9 except for the sample treated with methano-
lic ammonia. The product (11) obtained by treatment
We have been developing a post-synthetic functional-
ization method as a convenient way to introduce sev-
eral functional groups into ODN. Previously, we
reported incorporation of reactive groups, such as
methoxycarbonylmethyl and cyanomethoxycarbonyl-
methyl groups, into the C5-position of 2%-deoxyuridine
and the introduction of a functional group by post-syn-
thetic functionalization.4 In this paper, the synthesis of
2%-methoxycarbonylmethylthio-2%-deoxyuridine,
its
incorporation into ODN, and functionalization of the
ODN by a post-synthetic method are reported.
Keywords: modified nucleoside; DNA; post-synthetic functionaliza-
tion.
* Corresponding author. Fax: 81-277-30-1224; e-mail: sawai@
chem.gunma-u.ac.jp
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01926-2

678
H. Ozaki et al. / Tetrahedron Letters 42 (2001) 677–680
O
N
O
N
O
N
HN
O
HN
O
HO
HO
N
HO
4-Methoxy-α-
toluenethiol
O
O
O
O
Diphenyl carbonate
DMF, NaHCO3
TMG, DMF
OH
OH
OH
OH
S
OCH3
2 (87%)
3 (72%)
1
O
O
N
HN
HN
O
O
O
O
HO
N
HO
BrCH2CO2CH3
Et3N, CH2Cl2
O
TFA, Phenol
OH
SH
OH
S
OCH3
5 (73%)
4 (88%)
O
O
HN
O
HN
O
DMTrO
N
DMTrO
N
ClP(N(iPr)2)OCH2CH2CN
DIPEA, CH2Cl2
DMTr-Cl
O
O
O
O
DMAP, pyridine
OH
S
O
P
S
OCH3
OCH3
OCH2CH2CN
N
6 (74%)
7 (74%)
Scheme 1.
with methanolic ammonia was analyzed by HPLC after
nuclease digestion. The ratio of thymidine and 2%-car-
bamoylmethylthio-2%-deoxyuridine10 was 1:1. This result
suggests that the reactions of the 2%-methoxycarbonyl
group with the amines used in this study proceeded
quantitatively.
nuclease P1 and alkaline phosphatase. The modified
deoxynucleosides were confirmed by comparing the re-
tention times with those of authentic samples synthe-
sized from 2%-methoxycarbonylmethylthio-2%-deoxyuri-
dine and the corresponding amines.11 The composition
of the normal and modified deoxynucleosides corre-
sponded to the desired sequence.
Finally, we applied this post-synthetic strategy to 15mer
ODN (d5%(CGC TTC TXC CTG CCA)3%, X=5) to
prepare ODNs bearing a carbamoylmethylthio or bis(2-
aminoethyl)aminoethylcarbamoylmethylthio group as a
functional group at the 2%-position of the 2%-de-
oxyuridine residue. HPLC profiles of the crude ODN
bearing dimethoxytrityl group at the 5%-end are shown
in Fig. 2(a) and (b). The prepared 15mer ODNs were
characterized by nuclease digestion. Fig. 2(c) and (d)
shows the HPLC profiles of the digested products by
In conclusion, we have prepared ODNs bearing a reac-
tive group at the 2%-position of a 2%-deoxyuridine
residue and successfully incorporated a variety of func-
tional groups into the ODNs by post-synthetic func-
tionalization. Since this functional group at the
2%-position will be placed in the minor groove, it is
expected that it will act as a catalyst such as RNase.
Further study on the properties of the modified ODNs
and its application to RNA cleavage is in progress.
HO
U
HO
U
O
O
O
O
O
S
O
S
NHR
-O P O
OCH3
NC(CH2)2O P O
O
O
T
T
NH3/MeOH
or
O
O
amine/EtOH
OH
O
8-11
CPG
R=CH2CH2N(CH2CH2NH2)2 (8), CH2CH2NH2 (9), (CH2)6CH3 (10), H (11)
Scheme 2.

H. Ozaki et al. / Tetrahedron Letters 42 (2001) 677–680
679
Figure 1. HPLC profiles of crude-modified dimers. The dimers treated with (a) ammonia, (b) tris(2-aminoethyl)amine, (c)
ethylenediamine, and (d) heptylamine. HPLC conditions: column, Wakosil 5C18 (4×25 cm); eluent, a linear gradient of acetonitrile
(2.1 to 37.1% in 35 min) in 50 mM triethylammonium acetate (pH 7.2); flow rate, 1 mL/min; detection, UV at 260 nm.
Figure 2. HPLC profiles of the crude 15mer ODNs (a and b) and the digested 15mer ODNs (c and d) by nuclease P1 and alkaline
phosphatase. The 15mer ODNs treated with (a and c) ammonia and (b and d) tris(2-aminoethyl)amine. dUNH2 and dUTAEA in
the chromatograms were 2%-carbamoylmethyl-2%-deoxyuridine and 2%-[bis(2-aminoethyl)-aminoethylcarbamoylmethylthio]-2%-
deoxyuridine, respectively. The HPLC conditions for (c) and (d) were the same as in Fig. 1. The HPLC conditions for (a) and (b)
were the same as in Fig. 1 except for the eluent. Eluent: A 100 mM triethylammonium acetate, B acetonitrile; gradient, (a) 15 to
40% B in 30 min; (b) 20 to 30% B in 10 min and then 30% B in 10 min.

680
H. Ozaki et al. / Tetrahedron Letters 42 (2001) 677–680
6. Divaker, K. J.; Reese, C. B. J. Chem. Soc., Perkin Trans.
Acknowledgements
1 1982, 1625–1628.
7. Divaker, K. J.; Mottoh, A.; Reese, C. B.; Sanghvi, Y. S.
J. Chem. Soc., Perkin Trans. 1 1990, 969–974.
8. 2%-Methoxycarbonylmethylthio-2%-deoxyuridine (5): 1H
NMR (CDCl3) l 3.38 (2H, d, CH2), 3.76 (3H, s, OCH3),
3.90 (3H, m, H2% and H5%), 4.27 (1H, m, H4%), 4.34 (1H,
dd, H3%), 5.62 (1H, d, H1%), 5.79 (1H, dd, H5), 7.53 (1H,
d, H6), 8.47 (1H, s, NH). MS 333.2 (MH+, 333.1 calcd
for C12H16N2O7S).
9. Compound 8 (tris(2-aminoethyl)amine); MS 751.4 ([M+
H]+, 751.25, calcd for C27H44N8O13PS): 9 (ethylenedi-
amine); MS 663.2 ([M−H]−, 663.15, calcd for
C23H32N6O13PS): 10 (heptylamine); MS 718.4 ([M−H]−,
718.22, calcd for C28H41N5O13PS).
This research was supported by Grant No. 11780414
from Japanese Society for the Promotion of Science.
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1
5 with methanolic ammonia. H NMR (D2O) l 3.15 (2H,
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.
.