Synthesis and properties of oligodeoxyribonucleotides bearing a polyamino group at the 2' position via 2'-O-carbamoylmethyl and 2'-S-carbamoylmethyl groups

Article abstract of DOI:10.1080/15257770903307185

An oligodeoxyribonucleotide containing 2'-O-methoxycarbonylmethyluridine was synthesized and converted into several 2'-modified oligodeoxyribonucleotides by a postsynthetic modification method. Using this method, oligodeoxyribonucleotides bearing a polyam

Full text of DOI:10.1080/15257770903307185

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Synthesis and Properties of
Oligodeoxyribonucleotides Bearing a
Polyamino Group at the 2′ Position
via 2′-O-Carbamoylmethyl and 2′-S-
Carbamoylmethyl groups
Hiroaki Ozaki a , Shingo Nomura a , Shingo Momiyama a , Kazuyuki
Yokotsuka a , Masayasu Kuwahara a & Hiroaki Sawai a
a Department of Chemistry and Chemical Biology , Graduate School
of Engineering, Gunma University , Kiryu, Gunma, Japan
Published online: 20 Oct 2009.
To cite this article: Hiroaki Ozaki , Shingo Nomura , Shingo Momiyama , Kazuyuki Yokotsuka ,
Masayasu Kuwahara & Hiroaki Sawai (2009) Synthesis and Properties of Oligodeoxyribonucleotides
Bearing a Polyamino Group at the 2′ Position via 2′-O-Carbamoylmethyl and 2′-S-Carbamoylmethyl
groups, Nucleosides, Nucleotides and Nucleic Acids, 28:10, 943-952, DOI: 10.1080/15257770903307185
To link to this article: http://dx.doi.org/10.1080/15257770903307185
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Nucleosides, Nucleotides and Nucleic Acids, 28:943–952, 2009
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770903307185
SYNTHESIS AND PROPERTIES OF OLIGODEOXYRIBONUCLEOTIDES
BEARING A POLYAMINO GROUP AT THE 2ꢀ POSITION VIA
2ꢀ-O-CARBAMOYLMETHYL AND 2ꢀ-S-CARBAMOYLMETHYL GROUPS
Hiroaki Ozaki, Shingo Nomura, Shingo Momiyama, Kazuyuki Yokotsuka,
Masayasu Kuwahara, and Hiroaki Sawai
Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma
University, Kiryu, Gunma, Japan
An oligodeoxyribonucleotide containing 2 ꢁ-O-methoxycarbonylmethyluridine was synthesized
2
and converted into several 2 ꢁ-modified oligodeoxyribonucleotides by a postsynthetic modification
method. Using this method, oligodeoxyribonucleotides bearing a polyamine at the 2 ꢁ position were
easily prepared, which could form duplexes with either complementary DNA or RNA.
Keywords 2ꢁ-Modified oligonucleotides; postsynthetic modification; duplex stability
INTRODUCTION
Many modified DNAs have been prepared and their properties in-
vestigated. Modification of the major groove has been studied by many
researchers. We studied the modifications at the C5 position of pyrimidine
in the major groove. Furthermore, the 2ꢁ position of the nucleoside in
DNA is located in the minor groove, which is the binding site of DNA-
binding proteins such as nucleases, and is an interesting position for the
introduction of functional groups. The catalytic domain of RNase works in
the minor groove of RNA to cleave the strand.[1] It has been reported the
preparation of an oligodeoxyribonucleotide (ODN) bearing a functional
group, which acts as an acid-base catalysis, at the 2ꢁ position to obtain an
artificial nuclease.[2] A 2ꢁ-modified ODN has also been reported as a second-
generation antisense compound. 2ꢁ-O-[2-(Methoxy)ethyl]-modification[3]
and 2ꢁ-O-(3-aminopropyl)-modification[4] of ODN improved both the
Received 9 August 2009; accepted 2 September 2009.
Address correspondence to Hiroaki Ozaki, Department of Chemistry and Chemical Biology, Grad-
uate School of Engineering, Gunma University, Kiryu, Gunma 376–8515, Japan. E-mail: h-ozaki@chem-
bio.gunma-u.ac.jp
943

944
H. Ozaki et al.
FIGURE 1 Structures of reported 2ꢁ-modified nucleoside analogues.
stability of the ODN/RNA duplex and nuclease resistance. Series of 2ꢁ-O-(2-
aminoethyl)ribonucleosides and 2ꢁ-O-(2-meraptoethyl)ribonucleosides has
been synthesized for site-specific introduction of functional groups into
RNA.[5] Recently, 2ꢁ-O-(2-aminoethoxymethyl)ribonucleosides and 2ꢁ-O-(3-
aminopropyoxymethyl)ribonucleosides has been synthesized and incorpo-
rated into DNA for postsynthetic functionalization.[6] The 2ꢁ-position has
been used for introducing functional groups such as reactive groups,[7]
fluorescence groups,[8] lipophilic pendants,[9] or intercalating groups.[10]
Figure 1 shows the structures of reported 2ꢁ-modified nucleosides. Ha¨ner
et al. introduced several reporter groups via a carbamoylmethyl group
at the 2ꢁ position of the nucleoside (I, II, and III) and studies the ef-
fect on the ODNs affinity for RNA.[11,12] Sproat et al. showed that 2ꢁ-O-
carbamoylmethyl modification (IV) increased the stability of the duplex
with RNA.[13] Recently, Stetsenko et al. reported the synthesis of an ODN
bearing the carboxymethoxy group at the 2ꢁ-position and the conjuga-
tion of peptides (V and VI) to the ODN.[14–16] Manoharan and Egli et
al. demonstrated the crystal structure of the ODN containing 2ꢁ-O-(N -
methylcarbamoyl)methyl-modified thymidine (VII); this substitution did
not result in steric hindrance.[17] Praksh et al. synthesized ODNs bearing
2ꢁ-O-carbamoylmethyl (IV), 2ꢁ-O-N -methylcarbamoylmethyl (VII), and 2ꢁ-O-
(N -dimethylaminoethyl)carbamoylmethyl (VIII) modifications by postsyn-
thetic modification method. These reports suggest that modification at the
2ꢁ-position is a suitable for the modification of ODNs without distortion.
In the previous study, we synthesized an ODN containing 2ꢁ-
methoxycarbonylmethylthio-2ꢁ-deoxyuridine for introducing a variety of
functional groups at the 2ꢁ position.[18] The ODN was easily functionalized
by postsynthetic modification method to produce an ODN containing 2ꢁ-S-
modified 2ꢁ-deoxyuridine. In the present article, we describe the synthesis
of an ODN containing 2ꢁ-O-methoxycarbonylmethyluridine and its postsyn-
thetic modification to obtain a 2ꢁ-O-modified ODN containing an amine.
The stabilities of these ODN/DNA or ODN/RNA duplexes were measured
by a UV melting experiment.

DNA Bearing an Amino Group at the 2ꢁ Position
RESULTS AND DISCUSSION
945
The 2ꢁ-hydroxy group of uridine 1 was alkylated by methyl bromoacetate
in the presence of sodium hydride in DMF after blocking the 3-imino
and the 3ꢁ- and 5ꢁ-hydoxy groups as represented in Scheme 1. The 3-
imino group of uracil was protected with a benzoyl group to prevent N -
alkylation. The protected 2ꢁ-O-methoxycarbonylmethyluridine (5) was con-
verted to 5ꢁ-dimethoxytrityl-protected 2ꢁ-O-methoxycarbonylmethyluridine
3ꢁ-phosphoramidite (7) for incorporation into ODN.
SCHEME 1
First, we investigated the reactivity of the methoxycarbonylmethyl
group; 2ꢁ-O-Methoxycarbonylmethyluridine 8 derived from N 3-benzoyl-
2ꢁ-O-methoxycarbonylmethyluridine 5 was treated with ammonia or di-
aminoethane at 37◦C for 12 hours as shown in Scheme 2. The reaction
products were purified by HPLC and analyzed using ESI-MS. The structures
of the designed compounds 9 and 10 were confirmed by the ESI-MS data.
This result indicates that these amines are capable of reacting with the 2ꢁ-
methoxycarbonyl group in the ODN by an ester–amide exchange reaction.
ODNs containing the 2ꢁ-O-modified uridine (Figure 2) were synthesized
by an ester–amide exchange reaction on a solid support after incorporation
of N 3-benzoyl-2ꢁ-O-methoxycarbonylmethyluridine 5 into the ODN. An
ODN containing 5 (5ꢁ-CGC TTC TXC CTG CCA-3ꢁ, X = 5) was synthesized
using the protected nucleoside phosphoramidite 7 by a DNA synthesizer.

946
H. Ozaki et al.
O
N
O
N
NH
NH
O
HO
O
HO
O
O
O
RNH2/MeOH orEtOH
O
OH
O
OH
O
O
NHR
8
R = H (9)
CH2CH2NH2 (10)
SCHEME 2
The coupling yield of 7 in the DNA synthesizer was 95% based on a
dimethoxytrityl cation assay with a longer coupling time (360 seconds)
and double coupling. The ODN was treated with amines followed by
methanolic ammonia on CPG support. The products (ODN1 and ODN2)
were purified by reversed-phase HPLC. The ODNs obtained were charac-
terized by nuclease digestion. The compositions of the nucleosides were
in agreement with those of the desired sequences as shown in Figure 3.
The 2ꢁ-S-modified ODNs (ODN3 and ODN4) were prepared as described
previously.[18]
The binding affinity of these ODNs to complementary DNA or RNA was
determined for the UV melting curves of the corresponding duplexes. The
melting temperatures are summarized in Table 1 together with the melting
temperatures of the unmodified ODN (ODNN) and the 2ꢁ-S-modified
ODNs (ODN3 and ODN4). The melting temperature of each modified
ODN with complementary DNA was by 4.6–6.0◦C lower than that of the
unmodified ODNN. The lowering of melting temperature was independent
of the substituent atom (oxygen or sulfur) at the 2ꢁ position and was slightly
dependent on the number of amino groups present on the substituent
group. On the other hand, the melting temperature of the 2ꢁ-O-modified
ODNs/RNA (ꢀT, approximately −4.2◦C to −4.3◦C) were higher than
those of the 2ꢁ-S-modified ODNs (ꢀT, apploximately −5.7◦C to −6.4◦C).
FIGURE 2 Structure of the ODNs containing 2ꢁ-modified 2ꢁ-deoxyuridine.

DNA Bearing an Amino Group at the 2ꢁ Position
947
TABLE 1 Melting temperatures of ODN/DNA and ODN/RNA
DNA (◦C)
RNA (◦C)
ODN
Tm
ꢀTm
Tm
ꢀTm
ODNN
ODN1
ODN2
ODN3
ODN4
61.5
55.5
56.9
55.7
56.4
—
68.8
64.6
64.5
63.1
62.4
—
−6.0
−4.6
−5.8
−5.1
−4.2
−4.3
−5.7
−6.4
ꢀTm is difference between Tm of modified and unmodified duplex.
We considered that the 2ꢁ-O-modified nucleoside preferred the C3ꢁ-endo
conformation, which fitted in the A-type DNA/RNA duplex, rather than
2ꢁ-S-modified nucleoside.[19,20] The stability of the duplex also depended
on the substituent group. It was reported that lipophilic modifications such
as an n-octyl group, decreased duplex stability but intercalating substituents,
such as 2-aminoanthraquinone and positively charged N,N -dimethylamino
residues, increased duplex stability.[12] In the present work, the number
of the amino groups present on the substituent group did not affect the
stability of the duplexes. These results suggest that the substituent groups in
ODN1 and ODN2 could not interact directly with the DNA backbone.
The 2ꢁ-O-carbamoylmethyl group caused some perturbations in the
duplexes but the effect was smaller than that of 2ꢁ-S-carbamoylmethyl
modification. Also, the methyl ester group of the methoxycarbonylmethyl
substituent is useful in postsynthetic functionalization. The modification of
ODN via the 2ꢁ-O-carbamoylmethyl group is suitable for the preparation of
a variety of 2ꢁ-modified oligodeoxyribonucleotides.
FIGURE 3 HPLC analysis of the digested ODN1 (a) and ODN2 (b) by snake venom phosphodiesterase
and alkaline phosphatase. HPLC condition: column, Wakosil 5C18 (4 mm i.d. × 250 mm); eluent, A, 50
mM TEAA (pH 7.2), B, 50 mM TEAA in 70% CH3CN; gradient, 3–53%B in 35 minutes; flow rate, 1.0
mL/min.

948
H. Ozaki et al.
MATERIAL AND METHODS
General
Thin-layer chromatography (TLC) was performed on Kieselgel 60F254
(Art. 5554, E. Merck). Silica-gel column chromatography was performed on
Silica gel 60 (63–200 µm or 40–63 µm, Merck). High-performance liquid
chromatography (HPLC) was carried out on Wakosil 5C18 columns (4 mm
× 250 mm length or 10 mm × 250 mm length, Wako, Osaka, Japan).
The eluent was acetonitrile gradient in 50 mM triethylammonium acetate
1
(TEAA, pH 7.0). H NMR spectra and 31P NMR spectra were obtained
with a JEOL AL-300 or a JEOL λ-500 spectrometer (JEOL Co., Akishima,
Japan). Mass spectra were measured by a Perkin Elmer Sciex API-100 in-
strument (MDS/Sciex, Concord, ON, Canada) in ESI mode. Oligodeoxyri-
bonucleotides were synthesized by a phosphoramidite chemistry on an
Applied Biosystems 392 DNA synthesizer (Applied Biosystems, Foster City,
CA, USA). Normal nucleoside phosphoramidites were purchased from
Glen Research (Sterling, VA, USA). Dichloro(2-cyanoethoxy)phosphine
and chloro(diisopropylamino)-2-cyanoethoxyphosphine were prepared by
the described method.[9] Snake venom phosphodiesterase(SVPD) was pur-
chased from Worthinton (Lakewood, NJ, USA). Nuclease P1 was purchased
from Yamasa Co (Chiba, Japan). Alkaline phosphatase(AP) was purchased
from Boehringer Mannheim BmbH (Mannheim, Germany). All other
reagents were purchased from Wako (Osaka, Japan) or Kanto Chemical
Co., Inc. (Tokyo, Japan). All organic solvents for reactions were dried and
distilled in the usual manner.
N3-Benzoyl-2ꢁ-O-methoxycarbonylmethyl-1ꢁ,3ꢁ-O-(1,1,3,3-
tetraisopropyldisiloxane)uridine (4)
Sodium hydride (0.03 g, 1.08 mmol) and methyl bromoacetate (0.65
mL, 6.86 mmol) were added to the solution of N 3-Benzoyl-1ꢁ,3ꢁ-O- (1,1,3,3-
tetraisopropyldisiloxane)uridine[21] (3, 0.58 g, 7.20 mmol) in DMF under
Ar at 0◦C. After stirring at 0◦C for 6.5 hours, a small amount of methanol
was added to the reaction solution and the solution was evaporated to
dryness. The residue was dissolved in ethyl acetate (50 mL) and washed with
saturated sodium bicarbonate solution and saturated sodium chloride solu-
tion. The organic layer was dried over magnesium sulfate and evaporated
to dryness. The product was purified by silica gel column chromatography
using 2% methanol in dichloromethane as an eluent. Yield of 3 was 88%
(0.57 g).
1H NMR (CDCl3) δ 8.01 (1H, d, H6, J = 8.0 Hz), 7.93–7.42 (5H, m,
N-3-benzoyl), 5.86 (1H, d, H5, J = 8.4 Hz), 5.67 (1H, d, H1ꢁ, J = 5.1 Hz), 4.53
(1H, dd, H2ꢁ, J = 5.1 and 9.0 Hz), 4.28 (1H, d, H3ꢁ, J = 13.5 Hz), 4.17 (1H,

DNA Bearing an Amino Group at the 2ꢁ Position
949
d, H4ꢁ, J = 8.1 Hz), 4.13–4.02 (2H, m, H5ꢁ), 3.74 (3H, s, OCH3), 2.56 (2H, d,
2ꢁ-CH2-, J = 5.4 Hz), 1.12–0.97 (28H, m, isopropyl). Mass m/z 685.4 (MNa+,
685.3 calcd. for C31H46N2O10NaSi2).
N3-Benzoyl-2ꢁ-O-methoxycarbonylmethyluridine (5)
A mixture of N 3-benzoyl-2ꢁ-O-methoxycarbonylmethyl-1ꢁ,3ꢁ-O- (1,1,3,3-
tetraisopropyldisiloxane)uridine (4, 0.46 g, 0.69 mmol) and triethylamine
trihydrofluoride (0.57 mL, 3.47 mmol) in THF (7 mL) was stirred overnight
at room temperature. A small amount of methanol was added to the
reaction mixture and the solution was evaporated to dryness. The residue
was dissolved in ethyl acetate (50 mL) and washed with saturated sodium
bicarbonate solution and saturated sodium chloride solution The organic
layer was dried over magnesium sulfate and evaporated to dryness. The
product was purified by silica gel column chromatography using 1%
methanol in dichloromethane as an eluent. Yield of 3 was 62% (0.18 g).
1H NMR (CDCl3) δ 7.48–7.97 (6H, m, N-3-benzoyl and H6), 5.84 (1H, d, H5,
J = 8.1 Hz), 5.75 (1H, d, H1ꢁ, J = 3.3 Hz), 4.46–4.15 (3H, m, H2ꢁ, H3ꢁ and
H4ꢁ), 4.03–3.80 (2H, m, H5ꢁ), 3.76 (3H, s, OCH3), 3.47 (2H, s, 2ꢁ-CH2-).
N3-Benzoyl-5ꢁ-O-dimethoxytrityl-2ꢁ-O-
methoxycarbonylmethyluridine (6)
5 (0.8 g, 1.90 mmol) was react with 4,4ꢁ-dimethoxytrityl chloride (0.77
g, 2.28 mmol) in pyridine (5 mL) under Ar at room temperature for 17
hours. A small amount of methanol was added to the reaction mixture and
the solution was evaporated to dryness. The residue was dissolved in ethyl
acetate (30 mL) and washed with saturated sodium bicarbonate solution
and saturated sodium chloride solution The organic layer was dried over
magnesium sulfate and evaporated to dryness. The product was purified by
silica gel column chromatography using 5% methanol in dichloromethane
as an eluent. Yield of 3 was 89% (1.12 g). 1H NMR (CDCl3) δ 7.95–7.39 (6H,
m, N-3-benzoyl and H6), 7.34–6.81 (13H, m, DMTr), 5.86 (1H, s, H1ꢁ), 5.37
(1H, d, H5, J = 8.1 Hz), 4.54–4.48 (2H, m, H3ꢁ and H4ꢁ), 4.48–4.33 (1H, m,
H2ꢁ), 4.15–4.06 (2H, m, H5ꢁ), 3.78 (6H, s, OCH3), 3.73 (3H, s, OCH3), 3.59
(2H, s, 2ꢁ-CH2-).
3ꢁ-O-[Cyanoethoxy-(N,N-diisopropylamino)phosphino]-N3-
benzoyl-5ꢁ-O-dimethoxytrityl-2ꢁ-O-methoxycarbonylmethyluridine
(7)
2-Cyanoethyl-N,N -iisopropylchlorophosphoramidite was added to 6
(0.22 g, 0.30 mmol) in dichloromethane (3.7 mL) under Ar at room
temperature. After stirring at room temperature for 2 hours, a small amount

950
H. Ozaki et al.
of methanol was added to the reaction mixture and the solution was
evaporated to dryness. The residue was dissolved in ethyl acetate (20 mL)
and washed twice with saturated sodium bicarbonate solution and saturated
sodium chloride solution. The organic layer was dried over magnesium
sulfate and evaporated to dryness. The product was purified by silica gel col-
umn chromatography using 50% ethyl acetate in dichloromethane contain-
ing 5% triethylamine as an eluent. The appropriate fraction was collected,
evaporated, and precipitated with a small amount of dichloromethane into
hexane to give white precipitates. Yield of 6 was 89% (0.25 g). 31P NMR
(CDCl3) δ 151.44 and 151.07.
Reaction of 2ꢁ-Methoxycarbonylmethyluridine (8) with Amines
Compound
8
was derived from of N 3-benzoyl-2ꢁ-O-methoxy-
carbonylmethyluridine (5). 8 was treated with methanolic ammonia
or 50% 1,2-diaminoetrhane in ethanol at 37◦C for 12 hours. The products
were analyzed and purified by reversed-phase HPLC. These yields which
estimated by HPLC analyses, were about 60%. 9: Mass m/z 324.1 (MNa+,
323.9 calcd. for C11H15N3O7Na). 10: Mass m/z 345.1 (MH+, 345.1 calcd.
for C13H21N4O7).
Syntheses of ODNs and Modified ODNs
Oligodeoxyribonucleotides analogs were prepared using normal phos-
phoramidite coupling procedure on a DNA synthesizer. The oligodeoxyri-
bonucleotides bearing the 2ꢁ-O-modified uridine in place of thymidine
were synthesized along with the normal oligodeoxyribonucleotides. The
modified nucleoside phosphoramidite (7) was incorporated into the
oligodeoxyribonucleotides at the appropriate position by using the normal
synthetic cycle except that the reaction time for the coupling step was 5
minutes and the coupling was performed twice. For the synthesis of an
oligodeoxyribonucleotide containing the modified base (ODN-1 or ODN-
2), the CPG support was treated with methanolic ammonia at 37◦C for
24 hours or 50% 1,2-diaminoethane in ethanol at 37◦C for 23 hours,
then concentrated aqueous ammonia at 55◦C for 4 hours, reapectively.
The deprotection and cleavage from CPG support for nomal ODNs was
carried out by treatment with concentrated aqueous ammonia solution at
50◦C for 18 hours. All modified oligodeoxyribonucleotides with 5ꢁ-(4,4ꢁ-
dimethoxytrityl) group were isolated by reversed-phase HPLC on a Wakosil
5C18 column(10 mm × 250 mm length) using 50 mM TEAA (pH 7.0)
with a gradient of 15.0% to 40.0% acetonitrile in 25 minutes. The isolated
compound was treated with 10% acetic acid by the usual procedure to re-
move a 4,4ꢁ-dimethoxytrityl group followed by desalting on a Sephadex G-25
column. The modified oligodeoxyribonucleotides were further purified by

DNA Bearing an Amino Group at the 2ꢁ Position
951
reversed-phase HPLC after deprotection of 5ꢁ-(4,4ꢁ-dimethoxytrityl) group.
Isolated yields: ODN-1, 7.5%; ODN-2, 25.1%.
Nuclease Digestion of Oligodeoxyribonucleotides Containing the
Modified Base
The modified oligomers (ca. 0.2 OD260 nm) were treated overnight with
nuclease P1 (ca. 4 units) in 0.18 M sodium acetate (pH 5.3) at 37◦C. To
the reaction mixtures, 500 mM Tris-HCl(pH 9.0) containing 10 mM MgCl2,
snake venom phosphodiesterase (0.25 units) and alkaline phosphatase (2
units) were added, then the reaction mixtures were incubated overnight at
37◦C. The reaction mixtures were analyzed by reversed-phase HPLC. The
nucleoside composition ratios were calculated from areas of the peaks in
the HPLC chart.
Tm Measurements
UV absorbance was measured with a Hitachi UV-3000 spectrophotome-
ter equipped with a Hitachi Temperature Controller SPR-10 and a temper-
ature data collector AM-7002 (Anritsu Meter Co., Ltd., Tokyo, Japan). The
rate of heating or cooling was 0.5◦C/min. Tm values were obtained in 10
mM sodium phosphate buffer (pH 7.0) containing 150 mM sodium chloride
and 10 µM EDTA at a duplex concentration of 2 × 10−6 M. Alternatively,
Tm was measured with Shimazu UV-2550 spectrophotometer equipped with
TMSPC-8 Tm analysis system.
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