Synthesis of oligodeoxynucleotides using fully protected Deoxynucleoside 3c-Phosphoramidite building blocks and base recognition of Oligodeoxynucleotides incorporating N3-Cyano-Ethylthymine

Article abstract of DOI:10.3390/molecules15117509

Oligodeoxynucleotide (ODN) synthesis, which avoids the formation of side products, is of great importance to biochemistry-based technology development. One side reaction of ODN synthesis is the cyanoethylation of the nucleobases. We suppressed this reaction by synthesizing ODNs using fully protected deoxynucleoside 3c-phosphoramidite building blocks, where the remaining reactive nucleobase residues were completely protected with acyl-, diacyl-, and acyl-oxyethylene-type groups. The detailed analysis of cyanoethylation at the nucleobase site showed that N³-protection of the thymine base efficiently suppressed the Michael addition of acrylonitrile. An ODN incorporating N³-cyanoethylthymine was synthesized using the phosphoramidite method, and primer extension reactions involving this ODN template were examined. As a result, the modified thymine produced has been proven to serve as a chain terminator.

Full text of DOI:10.3390/molecules15117509

Molecules 2010, 15, 7509-7531; doi:10.3390/molecules15117509
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of Oligodeoxynucleotides Using Fully Protected
Deoxynucleoside 3′-Phosphoramidite Building Blocks and Base
Recognition of Oligodeoxynucleotides Incorporating
N3-Cyano-Ethylthymine
Hirosuke Tsunoda, Tomomi Kudo, Akihiro Ohkubo, Kohji Seio and Mitsuo Sekine *
Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku,
Yokohama, Japan; E-Mails: htsunoda@bio.titech.ac.jp (H.T.); tkudo@bio.titech.ac.jp (T.K.);
aohkubo@bio.titech.ac.jp (A.O.); kseio@bio.titech.ac.jp (K.S.)
* Author to whom correspondence should be addressed; E-Mail: msekine@bio.titech.ac.jp;
Tel.: +81-45-924-5706; Fax: +81-45-924-5772.
Received: 27 August 2010; in revised form: 8 October 2010 / Accepted: 14 October 2010 /
Published: 27 October 2010
Abstract: Oligodeoxynucleotide (ODN) synthesis, which avoids the formation of side
products, is of great importance to biochemistry-based technology development. One side
reaction of ODN synthesis is the cyanoethylation of the nucleobases. We suppressed this
reaction by synthesizing ODNs using fully protected deoxynucleoside 3′-phosphoramidite
building blocks, where the remaining reactive nucleobase residues were completely
protected with acyl-, diacyl-, and acyl-oxyethylene-type groups. The detailed analysis of
cyanoethylation at the nucleobase site showed that N3-protection of the thymine base
efficiently suppressed the Michael addition of acrylonitrile. An ODN incorporating
N3-cyanoethylthymine was synthesized using the phosphoramidite method, and primer
extension reactions involving this ODN template were examined. As a result, the modified
thymine produced has been proven to serve as a chain terminator.
Keywords: protecting group; oligodeoxynucleotide synthesis; cyanoethylation

Molecules 2010, 15
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1. Introduction
Much attention has been focused recently on chemically synthesized oligodeoxyribonucleotides
(ODNs) for use in biochemical and therapeutic studies [1–3]. Many modified ODNs have, therefore,
been synthesized to improve their original sequence specificity, stability, hybridization ability, enzyme
resistance, and various functional properties. Demand for effective syntheses of longer ODNs has also
increased since the total synthesis of a bacterial gene of Mycoplasma genitalium was achieved by
Gibson et al. [4,5], who utilized extensive stepwise ligations of medium-size ODNs. At present, the
chemical synthesis of ODNs seems limited to a one hundred nucleotide-length level. This limitation is
due to the fact that side reactions of the synthesis increase as the ODN chain length increases. Among
the most serious side reactions observed is the cyanoethylation of the base residues during the
deprotection step. Several improved procedures to avoid this side reaction have been reported. From a
different viewpoint, however, this side reaction could be avoided altogether through the full protection
of all reactive functional groups on the base moieties. To date, no such example of a full protection
strategy with the phosphoramidite method has been reported, although as reported by our group, such
strategies were employed in the phosphotriester approach. In this paper, we report the synthesis of
ODNs using the full-protection mode and discuss the problems associated with this strategy.
The phosphoramidite approach for the chemical synthesis of ODNs is an established procedure
[6–9] that has achieved high reliability in coupling efficiency, practicality, and versatility. Through the
use of appropriately protected monomer building blocks, side reactions on the nucleobases have been
minimized. The 2-cyanoethyl group has been commonly utilized for the protection of internucleotidic
phosphate groups because of the ease of deprotection with the use of concentrated NH3 [10–12].
However, this protecting group generates the toxic and potentially carcinogenic compound
acrylonitrile through -elimination in the deprotection step [13,14]. The generated acrylonitrile has
also been proven to be reactive towards the amino or imido groups on the nucleobases [14–18] to give
addition products. It has also been reported that the imido NH group of the thymine residue is
predominantly cyanoethylated over the other nucleobases (A, G, and C) [19]. In fact, Ravikumar has
reported that ODN derivatives, cyanoethylated at the thymine residue, were isolated as side products
when concentrated NH3 (aq) was used for the deprotection step [20]. Some researchers have recently
studied the synthesis of ODNs without cyanoethylation at the nucleobase site because oligonucleotide
products contaminated with cyanoethylated species would affect the chemical and pharmaceutical
properties when used as DNA probes and nucleic acid drugs, respectively. Beaucage et al. has used the
4-[N-(2,2,2-trifluoroacetyl)amino]butyl group in place of the 2-cyanoethyl group for protection of the
phosphate group [21]. The 4-[N-(2,2,2-trifluoroacetyl)amino]butyl group has no addition ability after
the deprotection step because the protecting group is converted to a cycloaminoalkane derivative
through the deprotection process. Wada et al. reported that the formation of N3-cyanoethylthymidine
was efficiently suppressed by using nitromethane as a scavenger under the deprotection conditions [22].
Chang et al. indicated that the use of t-butylamine was efficient for avoidance of alkylation by
acrylonitrile at the deprotection step [23].
In this study, we synthesized ODNs with the focus of preventing the cyanoethylation at the
nucleobase site through the use of fully protected monomers. The three kinds of deoxynucleoside
3′-phosphoramidite building blocks (C, A, G) were completely protected by acyl type protecting

Molecules 2010, 15
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groups at the amino group of the nucleobases, as shown in Figure 1. In the thymine residue, the
benzoyl (Bz) group was used to block the addition reaction. In the cytosine residue, the phthaloyl
(phth) group was used to completely mask the two protons of the 4-amino substituent, because it was
known
that
4-N-benzoylcytidine is only slightly reactive toward acrylonitrile [21]. On the other hand, the adenine
and guanine residues were comparatively non-reactive towards cyanoethylation in comparison to the
thymine residue. The adenine and guanine moieties, however, were protected with phthaloyl and
isobutyryloxyethylene (iBu·dibe) groups, respectively, to entirely eliminate the possibility of the
cyanoethylation side reaction.
In this paper, we report the synthesis of ODNs using these fully protected monomers. In addition,
the stabilities of the fully protected base derivatives under various conditions were also examined with
the focus of whether these protecting groups could be effective in avoiding the acrylonitrile-mediated
Michael addition reaction. Lastly, we report the base recognition ability of N3-cyanoethylated T, when
introduced into DNA, based on Tm experiments and DNA polymerase-mediated insertion reactions.
Figure 1. Fully protected monomers.
O
O
O
O
O
N
O
N
N
N
N
O
O
O
N
OiBu
OiBu
N
iBu
N
N
N
N
N
N
O
N
iBu =
N
TBz
Cphth
Aphth
GiBu•dibe
2. Results and Discussion
2.1. Synthesis of Fully Protected Deoxynucleoside 3′-Phosphoramidite Building Blocks
To evaluate the deactivation of the fully protected nucleobases towards cyanoethylation with
acrylonitrile, we synthesized fully protected phosphoramidite units, as previously reported [24–34].
The benzoyl group was selected as a full protecting group for the thymine residue [24,25]. The
N3-protection of 2′-deoxythymidine by reaction with benzoyl chloride gave compound 2 quantitatively
via the transient O-trimethylsilylation. The tritylation of compound 2 with DMTrCl, as per standard
procedure, produced the 5′-protected product 3 in 98% yield. The phosphitylation of 3 with
bis(diisopropylamino)(2-cyanoethoxy)phosphine produced the fully protected phosphoramidite unit 4
in 73% yield (Scheme 1).
For the full protection of the cytosine and adenine residues, the phthaloyl group was utilized
[26–30]. This protecting group enabled us to protect the two protons of the amino group of the C or A
base simultaneously via formation of a five-membered ring. It was found that 2′-deoxyadenosine
derivatives protected with the phthaloyl group exhibited significantly greater suppression of
depurination under acid conditions than when protected with the simple benzoyl group. The phthaloyl
group also possesses a unique property that, even when ring opening occurred, re-cyclization could be
performed during the capping reaction with acetic anhydride. In this study, we developed a new route

Molecules 2010, 15
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to the amidite units using selective deacylation of the commercially available 4-benzoyldeoxycytidine
3′-phosphoramidite derivative, followed by acylation of the resulting N-free species 5 with phthaloyl
chloride [31]. Thus, the desired phosphoramidite building block 6 was obtained in 83% yield
(Scheme 2). In a similar manner, the 6-N-phthaloyldeoxyadenosine 3′-phosphoramidite derivative 8
was synthesized in 61% yield by treatment of the N-free derivative 7 with phthaloyl chloride in the
presence of diisopropylethylamine in THF.
Scheme 1. Synthesis of fully protected 2′-deoxythymidine derivative 4.
1)
iPr2NEt 5.0 eq
TMSCl 2.5 eq
O
O
O
O
O
NH
pyridine, r.t., 30 min
N
N
2)
3)
benzoyl chloride 1.5 eq
r.t., 1 h
N
O
N
O
N
O
DMTrCl 1.3 eq
HO
HO
DMTrO
O
O
O
pyridine, r.t., 5 h
98%
TFA 0.3 eq
MeOH-H2O (1:1, v/v),
r.t., 20 min
OH
OH
OH
2
3
1
quant
O
O
N
N
CEOP[N(iPr)2]2 1.1 eq
1H-tetrazole 0.6 eq
iPr2NH 0.6 eq
O
DMTrO
NC
O
CH2Cl2, r.t., 3 h
73%
O
P
O
N(iPr)2
4
Scheme 2. Synthesis of fully protected 2′-deoxycytidine derivative 6 and
2′-deoxyadenosine derivative 8.
O
O
O
N
N
NH2
N
N
phthaloyl chloride 1.1 eq
iPr2NEt 2.2 eq
N
O
DMTrO
NC
DMTrO
NC
O
P
O
THF, r.t., 2 h
83%
O
O
O
O
P
N(iPr)2
N(iPr)2
5
6
O
O
NH2
N
N
N
N
N
N
N
N
phthaloyl chloride 1.1 eq
iPr2NEt 5.0 eq
N
DMTrO
NC
DMTrO
NC
O
P
O
P
THF, r.t., 2 h
61%
O
O
O
O
N(iPr)2
N(iPr)2
7
8
For full protection of the guanine base, we used a 1,2-bis(isobutyryloxy)ethylene (iBu·dibe) group
as an additional protecting group with the widely used isobutyryl group on the 2-amino substituent.
This double protecting mode was used for the synthesis of G-rich oligodeoxynucleotides in the
phosphotriester approach, and proved to effectively avoid the side reactions associated with the
guanine base [32–34]. Moreover, the iBu·dibe group could be simultaneously removed under the

Molecules 2010, 15
7513
deprotection conditions of the other N-acyl protecting groups at T, C, and A. In this study, we
synthesized the phosphoramidite unit of deoxyguanosine, protected with the iBu·dibe group
(Scheme 3). 3′,5′-Bis(t-butyldimethylsilyl)-2-isobutyryl-2′-deoxyguanine derivative 9, having an
isobutyryl group at the 2-N-amino group, was allowed to react with excess glyoxal and isobutyric
anhydride in pyridine, which formed the fully protected derivative 10 in 98% yield. When the
N-unprotected 2′-deoxyguanosine derivative was used as the starting material in this reaction, the
desired compound 10 could not be obtained, but a complex mixture containing various glyoxal adducts
was formed. Treatment of compound 10 with Et3N·3HF in THF gave the desilylated product 11.
Tritylation of 11 with DMTrCl, as per usual conditions, produced the 5′-protected product 12 in 60%
yield. The phosphitylation of 12 with bis(diisopropylamino)(2-cyanoethoxy)phosphine produced the
fully protected phosphoramidite unit 13 in 93% yield.
Scheme 3. Synthesis of fully protected 2′-deoxyguanosine derivative 13.
O
N
O
O
N
OiBu
OiBu
OiBu
N
iBu
N
N
N
N
N
N
NH
NHiBu
N
N
OiBu
N
iBu
37% glyoxal aq. 20 eq
isobutyric anhydride 20 eq
Et3N•3HF 3.0 eq
Et3N 3.0 eq
N
TBDMSO
TBDMSO
HO
O
O
O
pyridine, r.t., 1 h
98%
THF, r.t., 12 h
quant
OTBDMS
OTBDMS
OH
9
10
11
O
O
OiBu
OiBu
N
iBu
OiBu
OiBu
N
iBu
N
N
N
N
N
CEOP[N(iPr)2]2 1.1 eq
1H-tetrazole 0.6 eq
iPr2NH 0.6 eq
N
N
N
DMTrO
DMTrO
DMTrCl 1.2 eq
O
P
O
CH2Cl2, r.t., 14 h
93%
pyridine, r.t., 16 h
60%
O
O
OH
NC
N(iPr)2
12
13
2.2. Evaluation of the Stability of the Fully Protected Nucleobases toward Cyanoethylation
We introduced the fully protected phosphoramidite units of TBz, dCphth, dAphth, and dGiBu·dibe into a
resin linked to UnyLinker™ in order to evaluate the resistance of the fully protected base moieties to
the addition reaction of acrylonitrile under the conditions required for removal of the cyanoethyl group
in the solid phase. Subsequently, each monomer loaded resin was treated with 100 equiv of
acrylonitrile in DBU/CH3CN, which corresponded to the conditions estimated when a 100mer ODN
was synthesized. To compare the stability of the fully protected monomers, stability of the common
protected monomers [T, 4-N-acetyl-dC (dCac), 6-N-phenoxyacetyl-dA (dApac), and
2-N-(4-isopropyl)phenoxyacetyl-dG (dGiPrpac)] was also examined under the same conditions.
Cyanoethylation for common protected T, which is the most reactive compared to the other bases,
was observed after 1 min by HPLC analysis. The N3-cyanoethylated derivative (TCE) of thymidine
increased as time passed and was formed in 52% yield after 24 h. The time course of the remaining T
monomer in the reaction of T or TBz with acrylonitrile is shown in Figure 2. Results indicated that T
decreased linearly in a time-dependent manner. In contrast, when TBz was used in this evaluation, TCE
was not detected by HPLC analysis within the 24 h period. Therefore, protection of T at the N3-amino
group proved to be very effective for suppression of cyanoethylation of T.

Molecules 2010, 15
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Figure 2. The remaining amount (%) of T or TBz in its reaction with acrylonitrile. Open
and filled circles refer to the remaining amounts (%) of T and TBz in use of T and TBz,
respectively. Conditions: 100 equiv of acrylonitrile in DBU/CH3CN.
100
80
60
40
20
0
0
5
10
15
20
25
time (h)
Cyanoethylation of the other fully protected monomers (Cphth, Aphth, and GiBu·dibe) did not occur
within 24 h under the same conditions as those used for the ODN synthesis using the common
protected monomers (Figure 3i). In the case of Cphth and Aphth, the minor product (peak a) was
phthalamide resulted from the ammonolysis of the phthaloyl group. In the case of GiBu·dibe, the side
product (peak b) was observed ahead of the peak of dG. This product is not an alkylated product but an
incompletely deprotected G residue because the product (peak b) was decreased by prolonged
treatment with concentrated NH3. To examine the stability of GiBu·dibe in DBU/CH3CN in detail, we
carried out the reaction of the GiBu·dibe derivative 10 with DBU/CH3CN. After the reaction for 5 min,
the decomposition of the GiBu·dibe derivative 10 was observed by TLC analysis. The decomposed
compounds did not involve 3´,5´-O-bis(tert-butyldimethylsilyl)-2´-deoxyguanosine. The side product
(peak b) might be an incompletely deprotected dG derivative or a compound having a moiety derived
from the iBu·dibe group.
On the other hand, cyanoethylation for the common protected monomers (Cac, Apac, and GiPrpac) was
not observed (Figure 3ii). The results indicated that the reactivity of the N-monoacylated C, A, and G
residues toward acrylonitrile is lower than that of the T residue. However, we found that
N-monoacylated C and A residues were alkylated by acrylonitrile in DBU/CH3CN in solution phase, as
shown in Table 1. The reaction of 4-N-acetyl-2′-deoxycytidine derivative 14 with acrylonitrile gave the
4-N-cyanoethylated derivative 18 in 15% yield. 6-N-Phenoxyacetyl-2′-deoxyadenosine derivative 15
was slightly alkylated under the same conditions to give the 6-N-cyanoethylated derivative 19 in 2%
yield. Therefore, there is a possibility that the common protected monomers Cac and Apac are alkylated
by acrylonitrile on the ODN synthesis even if the C and A residues are protected by the monoacylated
protecting group when DBU was used. It seems that the fully protected approach is important for the C
and A residues to avoid the cyanoethylation. Cyanoethylation of a 2-N-phenoxyacetyl-2′-
deoxyguanosine derivative was not detected by TLC and mass analysis after 24 h. It was found that the
N-monoacylated G residue could suppress the cyanoethylation by acrylonitrile.

Molecules 2010, 15
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In the C and A monomers, the phth group for complete protection of the amino groups was effective
for avoidance of the cyanoethylation because N-monoacylated monomers proved to react with
acrylonitrile. The iBu·dibe group on the GiBu·dibe base was rapidly decomposed within 5 min during the
DBU/CH3CN treatment required for removal of the 2-cyanoethyl group at the internucleotidic
phosphate group as mentioned before but cyanoethylation for the GiBu·dibe residue was not observed
under the conditions of acrylonitrile-DBU/CH3CN (Figure 3i). However, the fully protected species of
TBz, Cphth, Aphth, and GiBu·dibe were completely removed by treatment with concentrated NH3 at 55 °C
for 6 h (Figure 3iii). Therefore, the full protection mode can be used when it is necessary to protect the
base moieties to avoid any side reactions which are expected when the usual monoacylated base
moieties are used for further modification of sugar or phosphate residues.
Figure 3. Analysis of cyanoethylation of (i) the fully protected monomers (Cphth, Aphth, and
GiBu·dibe) and (ii) the common protected monomers (Cac, Apac, and GiPrpac) by reverse-phase
HPLC; (iii) Analysis of deprotection of the fully protected monomers by concentrated NH3
at 55 °C for 6 h.

Molecules 2010, 15
Table 1. Cyanoethylation of the common protected monomers in solution phase reaction.
7516
Base
BaseCE
TBDMSO
TBDMSO
acrylonitrile 100 eq
O
O
DBU-CH3CN (1:9, v/v)
OTBDMS
OTBDMS
yield
baseCE
base
time
3 h
O
O
N
NC
HN
N
CH3
CH3
O
15%
N
N
O
N
14
18
O
O
N
OPh
NC
OPh
HN
N
N
5 h
2%
N
N
N
N
N
N
15
19
O
N
N
N
NH
O
24 h
not detected
OPh
N
H
16
O
O
CN
N
NH
1 h
83%
N
O
N
O
20
17
2.3. Synthesis of Oligonucleotides Using the Fully Protected Phosphoramidites
To evaluate if the full protection of the base residues was effective for the oligonucleotide synthesis,
we synthesized an ODN with the fully protected monomers using crosslinked polystyrene beads
(UnyLinker™ NittoPhase®). Selective removal of the 2-cyanoethyl groups at the internucleotidic
phoshate groups was performed by treatment with DBU/CH3CN (1:9, v/v) for 1 min [35,36]. The
successive release of the base-protected oligomer from the resin and complete deprotection of the other
base-labile protecting groups were simultaneously conducted by treatment with concentrated ammonia
at 55 °C for 14 h. The mixture thus obtained was analyzed by anion-exchange HPLC (Figure 4i). The
result showed that the desired ODN appeared as the main product. In contrast, several minor products
were also observed in this HPLC analysis. It was expected that treatment of dGiBu·dibe with
DBU/CH3CN produced these byproducts, as indicated the previous section. To confirm this
expectation, we prepared base-protected T10 and ATCATCATCG derivatives, which were synthesized
on the same resins by use of TBz, Cphth, Aphth, and GiPrpac phosphoramidite building blocks. In the latter
oligomer, the GiPrpac monomer unit was used in place of GiBu·dibe. These protected ODNs were treated
with DBU-CH3CN under the same conditions followed by treatment with concentrated NH3. The
analysis by HPLC suggested that the corresponding ODNs were obtained as single products and the
minor products were not produced as observed in the reaction using ODN containing the GiBu·dibe
monomer (Figures 4ii, iii). When a base-protected ATCATCATCG oligomer on the resin having fully

Molecules 2010, 15
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protected monomers (TBz, Cphth, Aphth, and GiBu·dibe) was deprotected by treatment with concentrated
NH3 at 55 °C for 6 h, the desired ODN was obtained without side products (Figure 4iv).
Figure 4. Anion-exchange HPLC profiles of ODNs obtained by using (i) the fully
protected monomers (TBz, Cphth, Aphth, and GiBu·dibe); (ii) only TBz monomer and (iii) TBz,
Cphth, Aphth, and GiPrpac monomers after removal of the cyaonoethyl groups of
the internucleotidic phosphate groups by treatment with DBU-CH3CN (1:9, v/v);
(iv) Anion-exchange HPLC profile of deprotection of ODN having the fully protected
monomers (TBz, Cphth, Aphth, and GiBu·dibe) by concentrated NH3.
When a longer ODN is synthesized in the solid phase, there are considerable concerns in addition to
cyanoethylation at the nucleobases. One expected problem is the depurination of the A and G residues
under acidic conditions, which is required for the repeated removal of the DMTr group during chain
elongation. In the synthesis of a longer ODN, the total time of the repeated acidic treatments increases
with chain length. Therefore, we examined the stability of the glycosyl bonds of the fully protected
Aphth and GiBu·dibe bases in 3% TCA/CH2Cl2, which is used for removal of the DMTr group in the
current DNA synthesis. To evaluate the stability of these nucleobases, we synthesized ODNs
containing the fully protected monomer Aphth or GiBu·dibe at the central position. The synthesized ODNs
on the resin were treated with 3% TCA in CH2Cl2 for 24 h. After the treatment with concentrated
ammonia, the mixtures thus obtained were analyzed by reverse-phase HPLC (Figure 5). In the reaction
mixture, the side products originating from the depurination of Aphth or GiBu·dibe were not detected. The
results showed that the Aphth and GiBu·dibe residues were stable under acidic conditions for extended
durations. These fully protecting groups on the purine bases will undoubtedly become useful for the
synthesis of long chain DNAs.

Molecules 2010, 15
Figure 5. Analysis of depurination using ODNs containing (A) Aphth or (B) GiBu·dibe by
7518
reverse-phase HPLC.
2.4. Evaluation of Base Recognition Ability of Cyanoethylated T by Tm Experiments
As mentioned above, cyanoethylation of the nucleobases, especially the T residue, is well known as
a side reaction in ODN synthesis. However, little attention has been devoted to study the properties of
ODNs containing TCE. We investigated the base recognition ability of TCE using Tm experiments and
DNA polymerase-mediated insertion reactions.
For the synthesis of ODN containing TCE, we synthesized the TCE phosphoramidite unit 23 from a T
derivative 21, as shown in Scheme 4. N3-Cyanoethylation of 21 with acrylonitrile gave the
cyanoethylated derivative 22 after acetylation of the 3′-hydroxy group. The phosphitylation of 22 with
bis(diisopropylamino)(2-cyanoethoxy)phosphine gave the cyanoethylated phosphoramidite unit 23 in
58% yield. Subsequently, we synthesized an ODN containing TCE using the phosphoramidite unit 23,
as shown in Table 2. At the deprotection step of the ODN synthesis, the N3-cyaoethyl moiety of T was
found to be very stable in concentrated NH3.
Scheme 4. Synthesis of TCE phosphoramidite unit 23.
O
O
N
O
N
CN
1)
Ac2O 1.1 eq
DMAP 10 mol%
pyridine, r.t., 2 h
CEOP(N(iPr)2)2 1.1 eq
1H-tetrazole 0.6 eq
iPr2NH 0.6 eq
N
CN
NH
N
N
O
O
O
DMTrO
NC
DMTrO
DMTrO
O
P
CH2Cl2, r.t., 12 h
58%
2)
3)
acrylonitrile 20 eq
DBU-CH3CN (1:9, v/v),
r.t., 2 h
2.0 M Ammonia in MeOH
r.t., 2 h
O
O
O
O
OH
OH
N(iPr)2
22
21
23
60%
Table 2. Sequences and isolated yields of ODNs containing TCE.
MALDI-TOF mass [M+H]+
ODN
Sequence
Yield (%)
calcd.
3326.6
7699.3
found
3329.4
7691.4
1
2
5′-d(CGTCTTCETCTGC)-3′
5′-d(TTAGACTCEGTAACCGGTCTTCGCGCG)-3′
48
33
To clarify the base pairing properties of TCE in DNA duplexes, Tm experiments were performed
using duplexes formed between modified ODNs and their complementary ODNs, with matched or
single-mismatched sequences at the central position, as shown in Table 3. A duplex with a matched

Molecules 2010, 15
7519
natural T-A base pair showed a Tm value of 58.1 °C. In contrast, the Tm value of a duplex with a
N3-cyanoethylated TCE-A base pair decreased to 36.0 °C, which is 22.1 °C lower than that of the
natural T-A matched base pair. In addition, the Tm values of DNA duplexes containing the mismatched
pairs (for T, C, and G) also decreased, showing a Tm of −7.2 °C, −6.8 °C, and −8.4 °C, respectively.
These results indicated that the base pairs having TCE were unstable and the Tm values (for all bases) of
DNA duplexes containing TCE became similar to each other. Therefore, it was found that the sequence
dependence of ODN having TCE was completely lacking. A significant drop of the thermal stability of
the duplex especially was observed when X was the matched A base. This is apparent because the
hydrogen bonding ability of TCE was lost by addition of acrylonitrile to the N3-NH group, which is an
accepter site of a hydrogen bond for the A-T base pair.
Table 3. Tm values for DNA–DNA 11-mer duplexes containing TCE.
d(GCA GAX AGA CG)-5′
DNA–DNA duplex
5′-d(CGT CTY TCT GC)
Y = T
Tm (°C)a
58.1
Y = TCE
Tm (°C)a
36.0
X
A
T
Tm (°C)b
−22.1
−7.2
43.6
36.4
C
G
42.0
44.0
35.2
35.6
−6.8
−8.4
aThe Tm values are accurate within ±0.5 °C. The Tm measurements were performed in a buffer
containing 10 mM sodium phosphate (pH 7.0), 1 M NaCl, 0.1 mM EDTA, and 2 µM duplex. bTm
is the difference in the Tm values between the unmodified and modified ODNs.
2.5. Evaluation of the Base Recognition Ability of Cyanoethylated T by DNA Polymerase-Mediated
Insertion Reactions
To study the template specific incorporation of dNTPs using DNA polymerase, we examined the
single insertion reaction of dNTPs toward the opposite TCE in templates using Taq polymerase [37], a
well-known thermostable enzyme used in PCR reactions [38,39]. The template incorporating T or TCE
at position X was annealed with a 5′-FAM-labeled 18-nt primer in the presence of DNA polymerase.
Following the enzymatic reaction, the products were analyzed by PAGE, as shown in Figure 6B.
Consequently, the incorporation of dATP was observed using the template containing a natural T base.
In contrast, dNTPs were not incorporated into the opposite site of TCE in the template by Taq DNA
polymerase. It was found that N3-cyanoethylation of the T residue caused the inhibition of the base
recognition by DNA polymerase and TCE in the template terminated the primer extension to give no
incorporation of dNTPs at the opposite site. Therefore, if synthesized ODNs including cyanoethylated
species would be used as PCR templates, PCR would be stopped at the modified base to produce
shorter duplicated products. Since PCR is a system that enables amplification of a trace amount of
DNA, a minor contamination of N3-cyanoethylated T does not affect the whole PCR process. TCE,
therefore, could be used as a new type of chain terminator in DNA duplications.

Molecules 2010, 15
Figure 6. Single dNTP insertion reactions using Taq DNA polymerases. (A) Sequences of
7520
5′-FAM labeled 18-nt primer and 25-nt templates; (B) PAGE analysis of
single-insertion reactions.
3. Experimental
General
1H-, 13C-, and 31P-NMR spectra were recorded at 500, 126 and 203 MHz, respectively. The
1
13
chemical shifts were measured from tetramethylsilane for H NMR spectra, CDCl3 (77 ppm) for C-
NMR spectra and 85% phosphoric acid (0 ppm) for 31P NMR spectra. UV spectra were recorded on a
U-2000 spectrometer. Column chromatography was performed with silica gel C-200 and a minipump
for a goldfish bowl was conveniently used to attain sufficient pressure for rapid chromatographic
separation. HPLC was performed using the following systems. Reversed-phase HPLC was done on a
system with a 3D UV detector and a C18 column (4.6 x 150 mm). A linear gradient (0-30%) of solvent
I [0.03 M ammonium acetate buffer (pH 7.0)] in solvent II (CH3CN) was used at 30 °C at a rate of
1.0 mL/min for 30 min. Anion-exchange HPLC was done on an apparatus with a 3D UV detector and
a FAX column (Waters, 4.6 × 100 mm). A linear gradient (0-60%) of Solvent III [1 M NaCl and 25
mM phosphate buffer, 10% CH3CN (v/v)] in solvent IV [25 mM phosphate buffer, 10% CH3CN (v/v)]
was used at 50 °C at a flow rate of 1.0 mL/min for 45 min. ESI mass was performed by use of
MarinerTM (PerSeptive Biosystems Inc.). MALDI-TOF mass was performed by use of Bruker
Daltonics [Matrix: 3-hydroxypicolinic acid (100 mg/ml) in H2O-diammonium hydrogen citrate (100
mg/ml) in H2O (10:1, v/v)]. Compounds 1 and 21 were purchased from GeneACT, Inc. Compounds 5,
7 [31], 9 [40], 14 [26], and 16 [41] were prepared according to the published procedure. Common
protected phosphoramidites were purchased from Glen Research Corporation.
N3-Benzoyl-5´-O-(4, 4'-dimethoxytrityl)thymidine (3). Compound 2 (2.42 g, 7.0 mmol) was rendered
anhydrous by repeated coevaporation with dry pyridine (3 mL × 1) and dissolved in dry pyridine
(70 mL). To the solution was added DMTrCl (3.08 g, 9.1 mmol) and the mixture was stirred at room
temperature for 5 h. The reaction was quenched by addition of saturated aqueous NaHCO3. The

Molecules 2010, 15
7521
mixture was partitioned between CHCl3 and H2O. The organic phase was collected, dried over
Na2SO4, filtered, and evaporated under reduced pressure. The residue was chromatographed on a
column of silica gel with CHCl3-MeOH (100:0–98:2, v/v) containing 1% Et3N to give the fractions
containing 3. The fractions were collected and evaporated under reduced pressure. The residue was
finally evaporated by repeated coevaporation three times each with toluene and CHCl3 to remove the
last traces of Et3N to give 3 (4.45 g, 98%). 1H-NMR (CDCl3)  1.50 (s, 3H), 2.36–2.42 (m, 2H), 3.40
(dd, 1H, J = 2.2 Hz, J = 9.5 Hz), 3.52 (dd, 1H, J = 2.7 Hz, J = 9.2 Hz), 3.81 (s, 6H), 4.05–4.06 (m,
1H), 4.60 (m, 1H), 6.39 (t, 1H, J = 6.6 Hz), 6.85–6.87 (m, 4H), 7.27–7.32 (m, 7H), 7.41 (d, 2H, J = 7.6
Hz), 7.49 (t, 2H, J = 7.6 Hz), 7.64 (t, 1H, J = 7.6 Hz), 7.70 (s, 1H), 7.94 (d, 1H, J = 7.6 Hz); 13C-NMR
(CDCl3)  12.1, 41.4, 55.5, 63.7, 72.7, 85.2, 86.4, 87.3, 111.5, 113,57, 113.59, 127.5, 128.30, 128.37,
129.3, 130.3, 130.7, 131.9, 135.2, 135.50, 135.57, 135.63, 144.5, 149.5, 159.0, 163.1, 169.3. HRMS
+
(ESI) m/z (M+Na)+: calcd for C38H36N2NaO8 671.2364; found, 671.2364.
N3-Benzoyl-5´-O-(4,4'-dimethoxytrityl)thymidine 3'-(2-cyanoethyl N,N-diisopropylphosphor-amidite)
(4). Compound 3 (380 mg, 0.59 mmol) was rendered anhydrous by repeated coevaporation with dry
pyridine (1 mL × 1), dry toluene (1 mL × 1), dry CH3CN (1 mL × 1) and dissolved in dry CH2Cl2 (6
mL). To the solution was added bis(diisopropylamino)(2-cyanoethoxy)phosphine (205 g, 0.65 mmol),
1H-tetrazole (25 mg, 0.35 mmol), diisopropylamine (50 L, 0.35 mmol) and the mixture was stirred at
room temperature for 3 h. The reaction was quenched by addition of saturated aqueous NaHCO3. The
mixture was partitioned between CHCl3 and aqueous NaHCO3. The organic phase was collected, dried
over Na2SO4, filtered, and evaporated under reduced pressure. The residue was chromatographed on a
column of silica gel with CHCl3-MeOH (100:0–98:2, v/v) containing 1% Et3N to give the fractions
containing 4. The fractions were collected and evaporated under reduced pressure. The residue was
finally evaporated by repeated coevaporation three times each with toluene and CHCl3 to remove the
last traces of Et3N to give 4 (338 mg, 73%). 1H-NMR (CDCl3)  1.07 (d, 3H,
J = 6.5 Hz), 1.16 (d, 9H, J = 5.6 Hz), 1.48 (s, 3H), 2.39 (t, 1H, J = 6.0 Hz), 2.48 (m, 1H), 2.55 (t, 1H,
J = 6.0 Hz), 2.61 (m, 1H), 3.40 (t, 1H, J = 10.1 Hz), 3.54–3.71 (m, 5H), 3.76 (s, 6H), 4.18, 4.24 (2s,
1H), 4.75 (m, 1H), 6.42–6.45 (m, 1H), 6.86–6.89 (m, 4H), 7.24 (m, 1H), 7.32–7.37 (m, 6H), 7.43–7.48
13
(m, 4H), 7.59 (t, 1H, J = 7.3 Hz), 7.79, 7.84 (2s, 1H), 7.94 (d, 1H, J = 7.3 Hz); C-NMR (CDCl3) 
12.1, 12.1, 20.4, 20.5, 20.6, 20.6, 24.8, 40.5, 43.4, 43.5, 43.5, 43.6, 55.5, 55.5, 55.5, 58.3, 58.4, 58.6,
63.3, 63.4, 73.7, 74.0, 85.2, 85.8, 86.1, 87.3, 111.4, 111.4, 113.5, 113.6, 117.9, 118.1, 127.5, 128.3,
128.5, 128.5, 129.4, 130.4, 130.7, 131.9, 135.3, 135.5, 135.5, 135.6, 135.6, 135.9, 144.6, 144.6, 149.6,
31
149.7, 159.1, 163.1, 169.5; P-NMR (CDCl3)  149.5, 149.9 (2s). HRMS (ESI) m/z (M+Na)+: calcd
for C47H53N4NaO9P+ 871.3442; found, 871.3441.
4-N-Phthaloyl-5´-O-(4,4'-dimethoxytrityl)-2´-deoxycytidine 3'-(2-cyanoethyl N,N-diisopropylphos-
phoramidite) (6). Compound 5 (869 mg, 1.19 mmol) was rendered anhydrous by repeated
coevaporation with dry pyridine (1 mL × 1), dry toluene (1 mL × 1), dry CH3CN (1 mL × 1), and
dissolved in dry THF (12 mL). To the solution was added phthaloyl chloride (189 L, 1.31 mmol),
diisopropylethylamine (456 L, 2.62 mmol) at 0 C and the mixture was stirred at room temperature
for 2 h. The reaction was quenched by addition of H2O. The mixture was partitioned between ethyl
acetate and H2O. The organic phase was collected, dried over Na2SO4, filtered, and evaporated under

Molecules 2010, 15
7522
reduced pressure. The residue was chromatographed on a column of silica gel with hexane-ethyl
acetate (40:60–30:70, v/v) containing 1% pyridine to give the fractions containing 6. The fractions
were collected and evaporated under reduced pressure. The residue was finally evaporated by repeated
coevaporation three times each with toluene and CHCl3 to remove the last traces of pyridine to give 6
(846 mg, 83%). 1H-NMR (CDCl3)  1.04–1.17 (m, 12H), 2.40–2.45 (m, 2H), 2.62 (t, 1H, J = 6.3 Hz),
2.72–2.82 (m, 1H), 3.40–3.61 (m, 6H), 3.78 (s, 6H), 4.20–4.21 (m, 1H), 4.59–4.69 (m, 1H), 6.22–6.32
(m, 2H), 6.81–6.86 (m, 4H), 7.26–7.39 (m, 9H), 7.79–7.82 (m, 2H), 7.94–7.97 (m, 2H), 8.51, 8.60 (2d,
13
1H, J = 7.3 Hz); C-NMR (CDCl3) 20.1, 20.2, 20.4, 24.4, 24.5, 24.6, 40.7, 41.1, 43.1, 43.2, 43.3,
53.4, 55.2, 58.1, 58.4, 61.5, 61.9, 70.9, 71.2, 71.8, 72.1, 85.8, 86.9, 87.5, 87.6, 100.5, 113.2, 117.3,
117.5, 124.3, 127.1, 128.0, 128.1, 128.2, 130.07, 130.13, 131.4, 135.1, 135.3, 144.0, 145.2, 154.6,
31
158.6, 159.1, 164.9; P-NMR (CDCl3)  149.6, 150.2 (2s). HRMS (ESI) m/z (M+H)+: calcd for
C47H51N5O9P+ 860.3419; found, 860.3411.
6-N-Phthaloyl-5´-O-(4,4'-dimethoxytrityl)-2´-deoxyadenosine
3'-(2-cyanoethyl
N,N-diisopropyl-
phosphoramidite) (8). Compound 7 (1.06 g, 1.40 mmol) was rendered anhydrous by repeated
coevaporation with dry pyridine (1 mL × 1), dry toluene (1 mL × 1), dry CH3CN (1 mL × 1) and
dissolved in dry THF (14 mL). To the solution was added phthaloyl chloride (221.7 L, 1.54 mmol),
diisopropylethylamine (1.22 mL, 7.0 mmol) at 0 C and the mixture was stirred at room temperature
for 2 h. The reaction was quenched by addition of H2O. The mixture was partitioned between CHCl3
and H2O. The organic phase was collected, dried over Na2SO4, filtered, and evaporated under reduced
pressure. The residue was chromatographed on a column of silica gel with hexane-ethyl acetate
(40:60–30:70, v/v) containing 1% pyridine to give the fractions containing 8. The fractions were
collected and evaporated under reduced pressure. The residue was finally evaporated by repeated
coevaporation three times each with toluene and CHCl3 to remove the last traces of pyridine to give 8
(761 mg, 61%). 1H-NMR (CDCl3) 1.13–1.22 (m, 12H), 2.48 (t, 1H, J = 6.3 Hz), 2.62–2.68 (m, 3H),
2.72–2.75 (m, 1H), 3.35–3.37 (m, 1H), 3.59–3.77 (m, 9H), 4.33–4.35 (m, 1H), 4.77–4.78 (m, 1H), 6.55
(t, 1H, J = 5.7 Hz), 6.79–6.81 (m, 4H), 7.18–7.30 (m, 7H), 7.40 (d, 2H, J = 7.3 Hz), 7.82–7.83 (m,
2H), 8.01–8.02 (m, 2H), 8.34, 8.36 (2s, 1H), 8.96 (d, 1H, J = 3.7 Hz); 13C-NMR (CDCl3) 20.4, 20.6,
24.65, 24.70, 24.76, 24.8, 39.7, 39.8, 43.4, 43.5, 55.35, 55.36, 58.4, 58.5, 63.3, 63.5, 73.6, 73.7, 74.3,
74.4, 85.11, 85.13, 86.7, 113.3, 117.5, 117.6, 124.5, 127.08, 127.11, 128.0, 128.2, 128.3, 130.11,
130.15, 130.19, 130.23, 130.27, 132.1, 134.9, 135.6, 135.7, 135.8, 144.44, 144.48, 144.5, 152.5, 152.5,
153.5, 153.6, 158.7, 165.7; 31P-NMR (CDCl3)  149.9, 150.2 (2s). HRMS (ESI) m/z (M+H)+: calcd for
C48H51N7O8P+ 884.3531; found, 884.3533.
2-N-Isobutyryl-1,N2-[1,2-di(isobutyryloxy)ethylene]-3´,5´-O-bis(tert-butyldimethylsilyl)-2´-deoxy-
guanosine (10). 37% Glyoxal (5 mL, 40 mmol) was added to compound 9 (1.26 g, 2.0 mmol) at room
temperature. The mixture was rendered anhydrous by repeated coevaporation with dry pyridine (1 mL
x1) and dissolved in dry pyridine (20 mL). After the solution was stirred at room temperature for 3 h,
isobutyric anhydride (6.5 mL, 40 mmol) was added, and the mixture was stirred at room temperature
for 1 h. The reaction was quenched by addition of saturated aqueous NaHCO3. The mixture was
partitioned between CHCl3 and H2O. The organic phase was collected, dried over Na2SO4, filtered, and
evaporated under reduced pressure. The residue was chromatographed on a column of silica gel with

Molecules 2010, 15
7523
hexane-ethyl acetate (90:10–70:30, v/v) to give the fractions containing 10. The fractions were
collected and evaporated under reduced pressure to give 10 (1.51 g, 98%). 1H-NMR (CDCl3)  0.11 (s,
12H), 0.92 (s, 18H), 1.15–1.19 (m, 12H), 1.25–1.31 (m, 6H), 2.35–2.38 (m, 2H), 2.54–2.59 (m, 2H),
3.80 (m, 2H), 4.00–4.04 (m, 2H), 4.51 (m, 1H), 6.29 (t, 1H, J = 6.5 Hz), 6.78 (d, 1H, J = 10.0 Hz),
13
6.83 (d, 1H, J = 6.3 Hz), 8.016, 8.021 (2s, 1H); C-NMR (CDCl3) -5.4, -5.3, -4.65, -4.63, 18.0,
18.46, 18.50, 18.53, 18.55, 18.57, 18.6, 18.7, 18.80, 18.83, 18.9, 19.0, 25.8, 26.1, 33.85, 33.94, 33.97,
42.6, 63.1, 72.3, 72.4, 78.6, 81.6, 84.3, 88.3, 120.6, 137.1, 148.1, 148.3, 153.7, 174.2, 174.4, 175.6.
+
HRMS (ESI) m/z (M+H)+ : calcd for C3 6 H6 2 N5 O9 Si2 764.4081; found, 764.4021.
2-N-Isobutyryl-1,N2-[1,2-di(isobutyryloxy)ethylene]-2´-deoxyguanosine (11). Compound 10 (1.0 g, 1.3
mmol) was dissolved in dry THF (6 mL). To the solution was added triethylamine (544 L,
3.9 mmol) and Et3N·3HF (635 L, 3.9 mmol) and the mixture was stirred at room temperature for
12 h. The mixture was partitioned between CHCl3-iPrOH (3:1, v/v) and aqueous NaHCO3. The organic
phase was collected, dried over Na2SO4, filtered, and evaporated under reduced pressure. The residue
was chromatographed on a column of silica gel with CHCl3-MeOH (100:0–95:5, v/v) to give the
fractions containing 11. The fractions were collected and evaporated under reduced pressure to give 11
1
quantitatively. H-NMR (DMSO) 1.05–1.11 (m, 12H), 1.17–1.22 (m, 6H), 2.36–2.40 (m, 1H), 2.59–
2.64 (m, 3H), 3.52–3.57 (m, 2H), 3.87 (m, 1H), 3.96 (m, 1H), 4.36 (m, 1H), 4.95–4.96 (m, 1H), 5.36
(d, 1H, J = 4.2 Hz), 6.23–6.24 (m, 1H), 6.70 (s, 1H), 6.77 (s, 1H), 8.29, 8.30, 8.31 (3s, 1H); 13C-NMR
(DMSO)  18.7, 18.8, 18.8, 19.1, 19.1, 19.4, 19.5, 33.7, 33.8, 40.8, 61.9, 62.0, 70.9, 71.0, 78.6, 79.7,
82.2, 84.6, 84.6, 88.4, 120.4, 120.5, 139.2, 139.4, 148.2, 148.3, 148.7, 153.8, 174.8, 175.0, 175.0,
+
176.0. HRMS (ESI) m/z (M+H)+: calcd for C24H34N5O9 536.2351; found, 536.2348.
2-N-Isobutyryl-1,N2-[1,2-di(isobutyryloxy)ethylene]-3´,5´-O-(4,4'-dimethoxytrityl)-2´-deoxy-guanosine
(12). Compound 11 (696 mg, 1.3 mmol) was rendered anhydrous by repeated coevaporation with dry
pyridine (1 mL × 1), and dissolved in dry pyridine (13 mL). To the solution was added DMTrCl (533
mg, 1.6 mmol), and the mixture was stirred at room temperature for 16 h. The reaction was quenched
by addition of saturated aqueous NaHCO3. The mixture was partitioned between CHCl3 and H2O. The
organic phase was collected, dried over Na2SO4, filtered, and evaporated under reduced pressure. The
residue was chromatographed on a column of silica gel with CHCl3-MeOH (100:0–98:2, v/v)
containing 1% Et3N to give the fractions containing 12. The fractions were collected and evaporated
under reduced pressure. The residue was finally evaporated by repeated coevaporation three times each
with toluene and CHCl3 to remove the last traces of Et3N to give 12 (657 mg, 60%). 1H-NMR (CDCl3)
1.14–1.19 (m, 12H), 1.26–1.31 (m, 6H), 2.55–2.59 (m, 4H), 3.80 (s, 6H), 3.92–4.03 (m, 3H), 4.74–
4.75 (m, 1H), 6.28 (d, 1H, J = 5.4 Hz), 6.77, 6.78 (2s, 1H), 6.81–6.84 (m, 4H), 7.17 (d, 1H, J = 8.8
13
Hz), 7.22–7.32 (m, 9H), 8.60, 8.64 (2s, 1H); C -NMR (CDCl3) 18.6, 18.7, 18.9, 19.0, 19.1, 34.0,
34.1, 41.3, 41.6, 55.4, 64.2, 72.3, 78.4, 78.5, 81.8, 81.9, 84.5, 86.7, 86.8, 86.9, 113.4, 120.7, 120.8,
127.2, 128.1, 128.3, 130.2, 130.3, 130.3, 135.7, 135.8, 135.9, 137.7, 137.9, 144.7, 148.3, 148.4, 148.5,
153.9, 158.8, 174.4, 175.8, 175.9. HRMS (ESI) m/z (M+H)+: calcd for C45H52N5O11+ 838.3658; found,
838.3659.

Molecules 2010, 15
7524
2-N-Isobutyryl-1,N2-[1,2-di(isobutyryloxy)ethylene]-3´,5´-O-(4,4'-dimethoxytrityl)-2´-deoxy-guanosine
3'-(2-cyanoethyl N,N-diisopropylphosphramidite) (13). Compound 12 (210 mg, 0.25 mmol) was
rendered anhydrous by repeated coevaporation with dry pyridine (1 mL × 1), dry toluene (1 mL × 1),
dry CH3CN (1 mL × 1), and dissolved in dry CH2Cl2 (2.5 mL). To the solution was added
bis(diisopropylamino)(2-cyanoethoxy)phosphine (89 L, 0.28 mmol), 1H-tetrazole (11 mg, 0.15
mmol), diisopropylamine (21 L, 0.15 mmol), and the mixture was stirred at room temperature for 14
h. The reaction was quenched by addition of saturated aqueous NaHCO3. The mixture was partitioned
between CHCl3 and aqueous NaHCO3. The organic phase was collected, dried over Na2SO4, filtered,
and evaporated under reduced pressure. The residue was chromatographed on a column of silica gel
with CHCl3-MeOH (100:0–98:2, v/v) containing 1% Et3N to give the fractions containing 13. The
fractions were collected and evaporated under reduced pressure. The residue was finally evaporated by
repeated coevaporation three times each with toluene and CHCl3 to remove the last traces of Et3N to
1
give 13 (239 mg, 93%). H-NMR (CDCl3)  1.11–1.29 (m, 30H), 2.42 (m, 1H), 2.53–2.62 (m, 5H),
3.32–3.35 (m, 2H), 3.57–3.76 (m, 8H), 3.81–3.99 (m, 2H), 4.29–4.32 (m, 1H), 4.64 (m, 1H), 6.28 (m,
1H), 6.79–6.84 (m, 6H), 7.18–7.20 (m, 1H), 7.24–7.30 (m, 6H), 7.38–7.40 (m, 2H), 7.85, 7.90 (2s,
13
1H); C-NMR (CDCl3)  18.6, 18.7, 18.9, 19.1, 19.2, 20.4, 20.6, 24.8, 24.9, 34.0, 40.7, 40.9, 41.2,
43.5, 43.6, 55.5, 58.3, 58.4, 63.6, 63.7, 74.2, 74.3, 74.6, 78.5, 78.6, 81.7, 81.8, 84.3, 84.4, 86.0, 86.4,
86.9, 113.5, 117.6, 117.7, 120.9, 121.0, 127.2, 128.2, 128.4, 130.2, 130.3, 135.6, 135.7, 135.8, 137.2,
31
137.3, 144.6, 148.4, 148.5, 153.8, 158.9, 174.3, 174.4, 175.7, 175.8; P-NMR (CDCl3) 150.0, 150.4,
150.5 (3s). HRMS (ESI) m/z (M+H)+: calcd for C54H69N7O12P+ 1038.4736; found, 1038.4064.
Evaluation of the resistance for addition of acrylonitrile to protected monomers. Fully protected
monomers and common protected monomers were introduced into UnyLinker™ NittoPhase® by use of
the ABI 392 DNA synthesizer. The protected monomer (125 nmol) loading UnyLinker™ NittoPhase®
were treated with acrylonitrile (0.82 L, 12.5 mol) in 4 mM DBU-CH3CN (200 L). After the
reaction for appropriate time, the reaction solution was removed by filtration. The DMTr group was
removed by treatment with 3% trichloroacetic acid in CH2Cl2 (1 mL) for 1 min, and the resin was
washed with CH2Cl2 (1 mL × 3), and CH3CN (1 mL × 3). The monomer was deprotected and released
from UnyLinker™ NittoPhase® by treatment with concentrated NH3 aq (500 µL) at 55 °C for 6 h. The
polymer support was removed by filtration and washed with distilled water (1 mL × 3). The filtrate
was evaporated and purified by reversed-phase HPLC.
6-N-Phenoxyacetyl-3´,5´-O-bis(tert-butyldimethylsilyl)-2´-deoxyadenosine (15). 3´,5´-O-Bis(tert-
butyldimethylsilyl)-2´-deoxyadenosine (960 mg, 2.0 mmol) was rendered anhydrous by repeated
coevaporation with dry pyridine (1 mL × 1) and dissolved in dry pyridine (10 mL). To the solution was
added phenoxyacetic anhydride (1.37 mg, 4.8 mmol), and the mixture was stirred at room temperature
for 3 h. To the reaction was added concentrated NH3 aq (5 mL), and the mixture was stirred at room
temperature for 10 min. The mixture was partitioned between CHCl3 and aqueous NaHCO3. The
organic phase was collected, dried over Na2SO4, filtered, and evaporated under reduced pressure. The
residue was chromatographed on a column of silica gel with CHCl3-MeOH (100:0, v/v) to give the
fractions containing 15. The fractions were collected and evaporated under reduced pressure to give 15
1
1
(831 mg, 68%). H NMR (CDCl3)  H NMR (CDCl3)  0.10 (s, 12H), 0.91 (s, 18H), 2.45–2.49 (m,

Molecules 2010, 15
7525
1H), 2.65–2.70 (m, 1H), 3.78 (dd, 1H, J = 2.9 Hz, J = 9.8 Hz), 3.87 (dd, 1H, J = 4.2 Hz, J = 9.2 Hz),
4.037–4.043 (m, 1H), 4.62–4.63 (m, 1H), 4.87 (s, 2H), 6.50 (t, 1H, J = 6.3 Hz), 7.08 (d, 3H, J = 8.3
Hz), 7.35 (t, 2H, J = 7.9 Hz), 8.33 (s, 1H), 8.79 (s, 1H), 9.41 (s, 1H); 13C-NMR (CDCl3)  -5.3, -5.2, -
4.6, -4.5, 18.1, 18.5, 26.0, 26.1, 40.9, 62.8, 68.6, 72.1, 84.9, 88.1, 115.0, 117.6, 122.1, 123.0, 129.8,
+
142.5, 148.7, 151.5, 152.3, 157.5, 167.9. HRMS (ESI) m/z (M+H)+: calcd for C30H48N5O5Si2
614.3188; found, 614.3186.
General procedure for evaluation of cyanoethylation of compounds 14-17. An appropriate compound
(0.30 mmol) was dissolved in DBU-CH3CN [3 mL, (1:9, v/v)]. To the solution was added acrylonitrile
(1.96 mL, 30 mmol). After being stirred at room temperature for appropriate time, as shown in Table
1, the mixture was partitioned between CHCl3 and H2O. The organic phase was collected, dried over
Na2SO4, filtered, and evaporated under reduced pressure. The residue was chromatographed on a
column of silica gel with CHCl3-MeOH (100:0–99:1, v/v) to give the fractions containing the
cyanoethylated compound. The fractions were collected and evaporated under reduced pressure to give
the cyanoethylated compound. Thus, the following compounds 18–20 were obtained.
4-N-Acetyl-4-N-2-cyanoethyl-3´,5´-O-bis(tert-butyldimethylsilyl)-2´-deoxycytidine
(18).
The
compound 18 was obtained (25.3 mg, 15%). 1H NMR (CDCl3)  0.11 (s, 12H), 0.90 (s, 18H), 2.13–
2.18 (m, 1H), 2.47 (s, 3H), 2.52–2.57 (m, 1H), 2.84–2.93 (m, 2H), 3.78 (dd, 1H, J = 2.6 Hz, J = 10.8
Hz), 3.95–3.97 (m, 2H), 4.26 (t, 2H, J = 6.6 Hz), 4.38–4.41 (m, 1H), 6.61–6.23 (m, 1H), 6.88 (d, 1H, J
= 7.3 Hz), 8.39 (d, 1H, J = 7.6 Hz); 13C-NMR (CDCl3)  -5.4, -5.3, -4.8, -4.4, 17.5, 18.1, 18.5, 22.6,
25.8, 26.0, 42.3, 42.4, 61.8, 70.0, 87.1, 88.0, 99.3, 144.0, 154.9, 164.2, 172.1. HRMS (ESI) m/z
+
(M+H)+: calcd for C26H47N4O5Si2 551.3080; found, 551.3079.
6-N-2-Cyanoethyl-6-N-phenoxyacetyl-3´,5´-O-bis(tert-butyldimethylsilyl)-2´-deoxyadenosine (19). The
compound 19 was obtained (3.4 mg, 2%). 1H NMR (CDCl3)  0.12 (s, 12H), 0.92 (s, 18H), 2.46–2.51
(m, 1H), 2.57–2.62 (m, 1H), 2.93 (t, 2H, J = 7.3 Hz), 3.79 (dd, 1H, J = 2.7 Hz, J = 9.9 Hz), 3.89 (dd,
1H, J = 3.9 Hz, J = 9.3 Hz), 4.05–4.06 (m, 1H), 4.57–4.60 (m, 2H), 4.62–4.64 (m, 1H), 5.17 (d, 2H, J
= 3.9 Hz), 6.52 (t, 1H, J = 6.2 Hz), 6.67 (d, 2H, J = 8.1 Hz), 6.93 (t, 1H, J = 7.3 Hz), 7.20 (t, 2H, J =
13
7.9 Hz), 8.42 (s, 1H), 8.70 (s, 1H); C-NMR (CDCl3)  -5.3, -5.2, -4.6, -4.5, 17.3, 18.2, 18.6, 25.9,
26.1, 41.7, 43.6, 62.8, 68.9, 71.9, 84.9, 88.3, 114.6, 117.6, 121.7, 125.5, 129.6, 142.7, 151.4, 151.6,
+
152.7, 157.7, 170.8. HRMS (ESI) m/z (M+H)+: calcd for C33H51N6O5Si2 667.3454; found, 667.3454.
N3-(2-Cyanoethyl)-3´,5´-O-bis(tert-butyldimethylsilyl)thymidine (20). The compound 20 was obtained
1
(127.3 mg, 83%). H NMR (CDCl3)  0.11 (s, 12H), 0.90 (s, 18H), 1.93 (s, 3H), 1.95–2.03 (m, 1H),
2.25–2.28 (m, 1H), 2.73–2.76 (m, 2H), 3.76 (d, 1H, J = 11.0 Hz), 3.86 (d, 1H, J = 11.5 Hz), 3.95 (m,
1H), 4.24–4.29 (m, 2H), 4.39 (m, 1H), 6.34 (t, 1H, J = 6.8 Hz), 7.50 (s, 1H); 13C-NMR (CDCl3)  -5.3,
-5.2, -4.7, -4.5, 13.3, 16.2, 18.1, 18.5, 25.9, 26.1, 36.8, 41.6, 63.1, 72.4, 85.8, 88.1, 110.2, 117.3, 134.3,
+
150.6, 163.1. HRMS (ESI) m/z (M+H)+: calcd for C25H46N3O5Si2 524.2971; found, 524.2913.

Molecules 2010, 15
7526
Synthesis of ODN using fully protected monomers: The synthesis of ODN d[ATCATCATCG] by use
of an ABI 392 DNA synthesizer was carried out. After chain elongation was finished, the fully
protected ODN (125 nmol) on UnyLinker™ NittoPhase® beads was treated with DBU-CH3CN [200
L, (1:9, v/v)] for 1 min to remove the 2-cyanoethyl groups from the phosphate moieties. The ODN
was deprotected and released from the resin by treatment with concentrated NH3 aq (500 µL) at 55 °C
for 14 h. The polymer support was removed by filtration and washed with distilled water (1 mL × 3).
The filtrate was evaporated and purified by anion-exchange HPLC. MALDI-TOF Mass (M+H)+: calcd
+
for C97H125N35O58P9 2986.55; found 2987.91.
Evaluation of depurination using ODNs containing Aphth or GiBu·dibe. ODN d[TTTTXTTTT] (X = Aphth
or GiBu·dibe) (0.5 µmol) on UnyLinker™ NittoPhase® beads was treated with 3% trichloroacetic acid in
CH2Cl2 (1 mL) for 24 h. After the filtration of the reaction solution, the resin was washed with CH2Cl2
(1 mL × 3) and CH3CN (1 mL × 3). The ODN was deprotected and released from UnyLinker™
NittoPhase® by treatment with concentrated NH3 aq (500 µL) at 55 °C for 6 h. The polymer support
was removed by filtration and washed with distilled water (1 mL × 3). The filtrate was evaporated and
analyzed by reversed-phase HPLC. d[TTTTAphthTTTT]: MALDI-TOF Mass (M+H)+ calcd for
+
C90H118N21O59P8 2684.48; found 2686.58. d[TTTTGiBu·dibeTTTT]: MALDI-TOF Mass (M+H)+ calcd
+
for C90H118N21O60P8 2700.47; found 2702.94.
N3-(2-Cyanoethyl)-5´-O-(4, 4'-dimethoxytrityl)thymidine (22). Compound 21 (437 mg, 0.80 mmol) was
rendered anhydrous by repeated coevaporation with dry pyridine (1 mL × 1), and dissolved in dry
pyridine (8 mL). To the solution was added acetic anhydride (83 L, 0.88 mmol), N,N-
dimethtylaminopyridine (10 mg, 0.08 mmol), and the mixture was stirred at room temperature for 2 h.
The reaction was quenched by addition of saturated aqueous NaHCO3. The mixture was partitioned
between CHCl3 and aqueous NaHCO3. The organic phase was collected, dried over Na2SO4, filtered,
and evaporated under reduced pressure. The residue was dissolved in DBU-CH3CN (8 mL, 1:9, v/v),
and to the solution was added acrylonitrile (1.05 mL, 16.0 mmol). After being stirred at room
temperature for 2 h, the mixture was partitioned between CHCl3 and H2O. The organic phase was
collected, dried over Na2SO4, filtered, and evaporated under reduced pressure. To the residue was
added 2.0 M ammonium in MeOH (10 mL), and the mixture was stirred at room temperature for 2 h.
The mixture was evaporated under reduced pressure, chromatographed on a column of silica gel with
CHCl3-MeOH (100:0–98:2, v/v) containing 1% Et3N to give the fractions containing 22. The fractions
were collected and evaporated under reduced pressure. The residue was finally evaporated by repeated
coevaporation three times each with toluene and CHCl3 to remove the last traces of Et3N to give 22
(289 mg, 60%). 1H NMR (CDCl3)  1.50 (s, 3H), 2.34–2.37 (m, 1H), 2.41–2.42 (m, 1H), 2.76 (t, 2H, J
= 7.1 Hz), 3.39 (dd, 1H, J = 2.9 Hz, J = 10.3 Hz), 3.50 (dd, 1H, J = 3.2 Hz, J = 8.9 Hz), 3.80 (s, 6H),
4.05–4.06 (m, 1H), 4.26–4.30 (m, 2H), 4.59 (m, 1H), 6.42 (t, 1H, J = 6.7 Hz), 6.85 (d, 4H, J = 8.5 Hz),
7.25–7.32 (m, 7H), 7.40 (d, 2H, J = 7.3 Hz), 7.61 (s, 1H); 13C-NMR (CDCl3)  12.6, 16.2, 36.8, 41.2,
55.4, 63.5, 72.5, 85.5, 86.2, 87.2, 110.6, 113.5, 117.4, 127.3, 128.2, 128.3, 129.2, 130.2, 134.4, 135.41,
+
135.47, 144.4, 150.7, 158.9, 163.1. HRMS (ESI) m/z (M+Na)+: calcd for C34H35N3NaO7 620.2367;
found, 620.2368.

Molecules 2010, 15
7527
N3-(2-Cyanoethyl)-5´-O-(4,
4'-dimethoxytrityl)thymidine
3'-(2-cyanoethyl
N,
N-
diisopropylphosphramidite) (23). Compound 22 (239 mg, 0.40 mmol) was rendered anhydrous by
repeated coevaporation with dry pyridine (1 mL × 1), dry toluene (1 mL × 1), dry CH3CN (1 mL × 1),
and dissolved in dry CH2Cl2 (4 mL). To the solution was added bis(diisopropylamino)
(2-cyanoethoxy)phosphine (123 L, 0.44 mmol), 1H-tetrazole (17 mg, 0.24 mmol), diisopropylamine
(34 L, 0.24 mmol), and the mixture was stirred at room temperature for 12 h. The reaction was
quenched by addition of saturated aqueous NaHCO3. The mixture was partitioned between CHCl3 and
aqueous NaHCO3. The organic phase was collected, dried over Na2SO4, filtered, and evaporated under
reduced pressure. The residue was chromatographed on a column of silica gel with hexane-ethyl
acetate (70:30–50:50, v/v) containing 1% Et3N to give the fractions containing 23. The fractions were
collected and evaporated under reduced pressure. The residue was finally evaporated by repeated
coevaporation three times each with toluene and CHCl3 to remove the last traces of Et3N to give 23
1
(179 mg, 58%). H NMR (CDCl3) 1.07 (d, 3H, J = 6.8 Hz), 1.17-1.19 (m, 9H), 1.44 (s, 3H),
2.35–2.37 (m, 1H), 2.43 (t, 1H, J = 6.2 Hz), 2.49–2.58 (m, 1H), 2.63 (t, 1H, J = 6.2 Hz), 2.76 (t, 2H, J
= 7.1 Hz), 3.32–3.35 (m, 1H), 3.48–3.68 (m, 5H), 3.80, 3.81 (2s, 6H), 4.15–4.19 (m, 1H), 4.26–4.28
(m, 2H), 4.66–4.68 (m, 1H), 6.40–6.44 (m, 1H), 6.83–6.86 (m, 4H), 7.25–7.31 (m, 7H), 7.40–7.42 (m,
2H), 7.65, 7.69 (2s, 1H); 13C-NMR (CDCl3)  12.5, 16.2, 20.3, 20.4, 20.55, 20.61, 24.60, 24.66, 24.69,
24.7, 24.8, 36.8, 43.35, 43.43, 43.5, 55.41, 55.43, 58.2, 58.4, 63.1, 63.3, 73.5, 73.6, 73.8, 74.0, 85.6,
85.7, 85.9, 87.1, 110.6, 113.4, 117.4, 117.5, 117.7, 127.32, 127.35, 128.1, 128.3, 128.4, 130.29,
130.31, 130.3, 134.47, 134.51, 135.46, 135.51, 144.4, 150.67, 150.70, 158.9, 163.1; 31P NMR (CDCl3)
149.7, 150.1 (2s). HRMS (ESI) m/z (M+Na)+: calcd for C42H50N5NaO8P+ 820.3446; found, 820.3450.
Enzymes and ODNs. AmpliTaq Gold DNA polymerase was purchased from applied biosystemsTM, and
dNTPs were purchased from Takara Bio, Inc. ODNs used in enzyme reactions and Tm experiments
were purchased from Sigma-Aldrich Japan. ODNs 1 and 2 in Table 2 were synthesized by use of an
ABI 392 DNA synthesizer.
Tm experiments. An appropriate ODN (2 M) and its complementary 2 M ssDNA 11-mer were
dissolved in a buffer consisting of 1 M NaCl, 10 mM sodium phosphate, and 0.1 mM EDTA adjusted
to pH 7.0. The solution was maintained at 80 °C for 10 min for complete dissociation of the duplex to
single strands and cooled at the rate of UV-1700TM (Shimadzu) by increasing the temperature at the
rate of 0.5 °C/min. During this process of annealing and melting, the absorption at 260 nm was
recorded and used to draw UV melting curves. The Tm value was calculated as the temperature at
which the first derivative of the UV melting curve had a maximum.
Single dNTP Insertion Reaction Using Taq DNA Polymerase. A mixture containing PCR Gold buffer,
1.8 mM MgCl2, and 0.25 unit AmpliTaq Gold DNA polymerase was incubated at 95 C for
3 min and slowly cooled to room temperature. To the mixture was added 100 nM (final concentration)
5′-FAM-labeled primer/template, and 10 M (final concentration) dNTP (N = A, C, G, or T). The
mixture (10 L) was incubated at 74 C for 10 min, and the reactions were terminated by adding 30 L
of stop solution (95% formamide, 20 mM EDTA). After being gently vortexed, the samples were

Molecules 2010, 15
7528
separated by electrophoresis using 20% denaturing polyacrylamide gel containing 7 M urea and
visualized by Fujifilm FLA-7000.
4. Conclusions
In this study, we have synthesized the fully protected monomers (TBz, dCphth, dAphth, and dGiBu·dibe),
and an ODN using these monomers. The TBz monomer can be obtained efficiently from T in three
steps. The dCphth and dAphth monomers were obtained from the corresponding N-unprotected
phosphoroamidite units by facile acylation. The full protection of dG was achieved and used in an
addition reaction with glyoxal at the N1- and 2-N-amino groups. To examine the stability of the fully
protected monomers toward addition of acrylonitirile, we carried out the reaction of these monomers
with excess acrylonitirile in DBU/CH3CN. As a result, TBz suppressed the addition of acrylonitrile.
Full deprotection of the protecting groups used in the oligonucleotide synthesis revealed the presence
of side reactions associated with dGiBu·dibe. Further improvement was necessary to avoid such side
reactions. In contrast, it was found that an ODN having TCE formed an unstable DNA duplex in
comparison with the unmodified DNA duplex. In addition, TCE on the template exhibited complete
inhibitory effect on chain elongation of a primer in DNA polymerase reactions.
Acknowledgements
This work was supported by Grant-in-Aids for Scientific Research from the Ministry of Education,
Culture, Sports, Science and Technology, Japan and in part by Health Sciences Research Grants for
Research on Psychiatric and Neurological Diseases and Mental Health from the Ministry of Health,
Labor and Welfare of Japan, and by the global COE project.
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Sample Availability: Samples of the compounds are available from the authors.
© 2010 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).