Synthesis and hybridization affinity of oligodeoxyribonucleotides incorporating 4-N-(N-arylcarbamoyl)deoxycytidine derivatives

Article abstract of DOI:10.1016/j.tetlet.2004.10.116

Modified oligodeoxynucleotides incorporating 4-N-(N-arylcarbamoyl)-dC derivatives 1a-c were synthesized. The ¹H NMR spectra of 1a-c suggest that the carbamoyl group forms an intramolecular hydrogen bond with the cytosine ring nitrogen atom so t

Full text of DOI:10.1016/j.tetlet.2004.10.116

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 9365–9368
Synthesis and hybridization affinity of oligodeoxyribonucleotides
incorporating 4-N-(N-arylcarbamoyl)deoxycytidine derivatives
Kenichi Miyata,a,c Ryuji Tamamushi,a Akihiro Ohkubo,a,c Haruhiko Taguchi,a,c
Kohji Seiob,c and Mitsuo Sekinea,c,
*
aDepartment of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta, Midoriku, Yokohama 226-8501, Japan
bFrontier Collaborative Research Center, Tokyo Institute of Technology, 4259 Nagatsuta, Midoriku, Yokohama 226-8503, Japan
cCREST, JST (Japan Science and Technology Agency), 4259 Nagatsuta, Midoriku, Yokohama 226-8501, Japan
Received 24 September 2004; revised 20 October 2004; accepted 21 October 2004
Available online 5 November 2004
Abstract—Modified oligodeoxynucleotides incorporating 4-N-(N-arylcarbamoyl)-dC derivatives 1a–c were synthesized. The 1H
NMR spectra of 1a–c suggest that the carbamoyl groupforms an intramolecular hydrogen bond with the cytosine ring nitrogen
atom so that formation of a Watson–Crick base pair with the complementary guanine base is inhibited. The hybridization properties
of oligodeoxynucleotides containing 1a–c were investigated by use of Tm analysis. The hybridization properties of 4-N-(N-phenyl-
carbamoyl)-dC (1a) were similar to those of 4-N-(N-alkylcarbamoyl)-dC derivatives reported previously. In sharp contrast to 1a, it
turned out that 4-N-(N-napht-1-yl) and (N-quionol-5-yl)-dC (1b,c) have a unique property as a universal base.
Ó 2004 Published by Elsevier Ltd.
A variety of base-modified nucleosides have been exten-
sively synthesized to enhance their original functional
roles and to understand their effect on the neighboring
circumstance when introduced into the oligonucleotides.
Recently, we have reported the synthesis and hybridiza-
tion properties of oligodeoxynucleotides containing 4-
N-acyl-dC derivatives,1,2 4-N-alkoxycarbonyl-dC deriv-
atives,3 and 4-N-alkylcarbamoyl derivatives.4 Our
results showed that such modification of the cytosine
4-N-amino groupwith acyl or alkoxycarbonyl not only
allowed formation of the Watson–Crick base pair but
also increased their hybridization affinity for the com-
plementary guanine base owing to an intramolecular
hydrogen bond between the carbonyl oxygen atom
and the 5-H proton of the cytosine ring. On the other
hand, it was found that the carbamoyl groupof 4- N-
(carbamoyl)deoxycytidine has a Ôdistal Õ orientation due
to an intramolecular hydrogen bond between the carb-
amoyl groupand the cytosine ring nitrogen atom so that
formation of a Watson–Crick base pair with the comple-
mentary guanine base is inhibited.4
In this letter, we report the synthesis and properties of
deoxycytidine derivatives (1a–c) having various 4-N-
(N-arylcarbamoyl) groups, and also describe their
unique duplex stability and base recognition ability
when they were incorporated into oligodeoxynucleo-
tides. Tamura et al. reported the reaction of DNA with
2-naphtyl isocyanate gave 4-N-(napht-2-yl)carbamoyl-
dC as a degradation product.5 Recently, Sugimoto and
his co-workers have reported that 4-N-arylcarbonyl-
dA derivatives serve as base pair mimics in DNA
duplexes.6
The synthesis of nucleosides 1a-c is outlined in Scheme
1. Compounds 1a,b were obtained by selective reaction
of dC with aryl isocyanates in high yield. On the other
hand, 4-N-[N-(quinol-5-yl)carbamoyl]-dC (1c: dCqcm
)
was synthesized from 30,50-O-disilylated deoxycytidine
(3). The 1H NMR spectra of 1a–c thus obtained are simi-
lar to those of 4-N-(N-alkylcarbamoyl)-dC derivatives.
This result suggests that the orientation of the carb-
amoyl groups is Ôdistal Õ.
The synthesis of target phosphoramidites 7a–c is out-
lined in Scheme 2. Treatment of 1a–c with DMTrCl
gave the 50-O-dimethoxytritylated products 6a–c. Com-
pounds 6a–c were converted to the phosphoramidite
units 7a–c by phosphitylation in the usual way.7
Keywords: 4-N-carbamoyldeoxycytidine; Oligonucleotide; Tm; Univer-
sal base.
*
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 2004 Published by Elsevier Ltd.
doi:10.1016/j.tetlet.2004.10.116

9366
K. Miyata et al. / Tetrahedron Letters 45 (2004) 9365–9368
O
1a (dCpcm): R =
H
R
NH2
N
N
H
N
N
N
1b (dCncm): R =
i
N
O
O
HO
HO
O
O
OH
OH
2
O
N
O
N
O
N
H
H
H
O
H
H
N
N
N
N
N
N
H
N
N
N
N
H
N
OPh
N
N
O
O
O
O
ii
iii
iv
TBSO
TBSO
TBSO
HO
O
O
O
OTBS
OTBS
OTBS
OH
1c (dCqcm
)
3
4
5
Scheme 1. Reagents and conditions: (i) aryl isocyanate (1.0equiv), triethylamine (1.0equiv), DMF, rt, 30min (1a: 92%, 1b: 84%); (ii) phenyl
chloroformate (1.5equiv), CH2Cl2–pyridine (1.1, v/v), rt, 1h (86%); (iii) 5-aminoqunoline (1.5equiv), pyridine, 70°C, 3h (60%); (iv) triethylamine–
3HF (5equiv), rt, 7h (84%).
O
R
H
O
O
N
O
N
N
H
N
N
H
R
H
R
N
H
N
H
N
N
N
N
N
O
DMTrO
i
ii
O
O
DMTrO
HO
O
O
O
P
CN
OH
OH
phenyl (1a)
N
O
napht-1-yl (1b)
quinol-5-yl (1c)
R =
6a-c
7a-c
Scheme 2. Reagents and conditions: (i) DMTrCl (1.2equiv), pyridine, rt, 4h (6a: 87%, 6b: 89%, 6c: 85%); (ii) chloro(2-cyanoethoxy)(N,N-
diisopropylamino)phosphine (1.1equiv), diisopropylethyamine (1.3equiv), CH2Cl2, rt, 30min (7a: 84%, 7b: 78%, 7c: 75%).
The solid-phase synthesis of oligodeoxynucleotides
containing 4-N-(N-arylcarbamoyl)-dC derivatives was
carried out with a DNA/RNA synthesizer by use of
the standard phosphoramidite method.8 The oligomer
was released and deprotected from the polymer support
by treatment with concd aq NH3 for 1h. The product
was purified on C18-cartridge by the DMTr-ON purifi-
cation method, analyzed by anion-exchange HPLC
and reversed-phase HPLC, and characterized by MAL-
DI-TOF mass. These results are summarized in Table 1.
sides 1a–c are summarized in Table 2. As references,
the Tm values of duplexes containing 4-N-(N-methyl-
carbamoyl)-dC (dCmcm
;
entry 2), 4-N-(N-ethyl-
entry 3), and 4-N-(N-
carbamoyl)-dC (dCecm
;
butylcarbamoyl)-dC (dCbcm; entry 4) are also shown in
Table 2.
It turned out that the Tm values of the duplex containing
dCpcm-G (entry 5, 49.3°C), dCncm-G (entry 6, 47.5°C),
and dCqcm-G (entry 7, 48.7°C) base pair were quite low-
er than that of the unmodified control duplex (entry 1,
53.2°C). The stability of these modified duplexes was
almost the same as that of the duplex substituted with
a T–A base pair (entry 7, 49.2°C). It is inferred that
the carbamoyl groups of modified deoxycytidines chan-
ged their orientation from Ôdistal Õ to Ôproximal Õ that does
not interrupt the formation of a Watson–Crick base pair
with deoxyguanosine, as shown in Figure 1. Compared
with the Tm values of the duplexes containing a 4-N-alk-
ylcarbamoyl-dC derivative (entries 2–4), these results
are in good agreement with those of oligodeoxynucleo-
tides having a 4-N-(N-alkylcarbamoyl)-dC derivative.
The stability of the modified duplexes highly depends
on the hydrophilicity of a substituent of the carbamoyl
group.
From the previous studies of 4-N-(N-alkylcarbamoyl)-
dC derivatives,4 it was expected that 4-N-(N-aryl-
carbamoyl) modified nucleosides would form
a
Watson–Crick base pair with deoxyguanosine, with
their conformational change from Ôdistal Õ to Ôproximal Õ.
Therefore, the thermal stability of DNA duplexes con-
taining a modified nucleoside was investigated in sodium
phosphate buffer (pH7.0) containing 1.0M NaCl. The
Tm values of the duplexes containing modified nucleo-
Table 1. Sequence and isolated yield of oligodeoxynucleotides contain-
ing 4-N-(N-arylcarbamoyl)-dC derivatives
Sequence
Yield (%)
Founda
Calcd
d(TTTCTCCpcm TTCTCT)
d(TTTCTCCncm TTCTCT)
d(TTTCTCCqcm TTCTCT)
36
36
66
3934.0
3986.5
3987.1
3935.7
3985.7
3986.7
The Tm values of the DNA duplexes having a mis-
matched base pair (Y = T, C, A) are also summarized
in Table 2. The Tm values of the duplexes containing a
a MALDI-TOF mass.

K. Miyata et al. / Tetrahedron Letters 45 (2004) 9365–9368
9367
Table 2. Tm valuesa for DNA 13 mer duplexes containing 4-N-carbamoyldeoxycytidine derivatives
50-d(TTTCTCXTTCTCT)-30 10mM sodium phosphate (pH7.0), 1.0M NaCl,
30-d(AAAGAGYAAGAGA)-50 0.1mM EDTA, 2lM duplex
Entry
X
Y = G
DTm
Y = T
DTm
Y = C
DTm
Y = A
DTm
Selectivityc
Ranged
b
b
b
b
Tm
Tm
Tm
Tm
1
C
53.2
51.7
49.7
48.2
49.3
47.5
48.7
37.8
39.6
38.8
38.0
43.3
47.7
48.7
30.7
32.5
32.4
36.1
44.1
49.4
49.3
32.8
38.1
38.8
40.3
42.2
46.4
47.1
49.2
+14.4
+12.1
+10.9
+7.9
24.5
18.2
17.3
12.1
6.1
2e
3e
4e
5
Cmcm
Cecm
ꢀ1.5
ꢀ3.5
ꢀ5.0
ꢀ3.9
ꢀ5.7
ꢀ4.5
+1.8
+1.0
+0.2
+5.5
+9.9
+1.8
+1.7
+5.4
+5.3
+6.0
+7.5
+9.4
Cbcm
Cpcm (1a)
Cncm (1b)
Cqcm (1c)
T
+13.4
+18.7
+18.6
+5.2
6
+13.6
+14.3
ꢀ1.9
ꢀ0.5
3.0
2.2
7
+10.9
8
(°C)
a The Tm values are accurate within 0.5°C.
b DTm is the difference in the Tm value between the duplex having a modified base and that having a natural base.
c ÔSelectivityÕ is the difference between the Tm value of the duplex containing a C*–G natch base pair and the highest Tm value of duplexes containing
a mismatch base pair.
d ÔRangeÕ is the difference between the maximum and minimum Tm value.
e Ref. 4; dCmcm, deecm, and dCbcm represent 4-N-(N-methylcarbamoyl)-dC, 4-N-(N-ethylcarbamoyl)1-dC, and 4-N-(N-butylcarbamoyl)-dC,
respectively.
Table 3. Tm valuesa for DNA 13mer duplexes containing 4-N-
carbamoyldeoxycytidine derivatives and abasic site
conformational
distal
Proximal
change
O
R
H
N
Entry
X
Y = abasic
DTmb(°C)
H
R
N
N
N
N
N
H
O
H
dR
H
N
Tm
O
H
N
hybiridize
N
N
H
N
1
2
3
4
5
6
C
31.4
33.9
37.6
45.5
48.8
51.1
O
N
HO
Cmcm
Cbcm
Cpcm
Cncm
Cqcm
+2.5
+6.2
O
H
O`
N
dR
+14.1
+17.4
+19.7
OH
1a-c
dC*-dG base pair
1a; R = phenyl,4-N-(N-phenylcarbamoy)l-dC (dCpcm
1b; R = napht-1-yl, 4-N-[N-(napht-1-yl)carbamoyl]-dC (dCncm
1c; R = quinol-5-yl,4-N-[N-(quinol-5-yl)carbamoyl]-dC (dCqcm
)
a The Tm values are accurate within 0.5°C. These sequences of
duplexes and used buffer are indicated in Table 2.
b DTm is the differences in the Tm value between the duplex having a
modified base and that having a natural base.
)
)
Figure 1. Structures of dCpcm (1a), dCncm (1b), and dCqcm (1c).
4-N-(N-arylcarbamoyl)-dC derivative are higher than
those of the duplexes containing a 4-N-alkylcarba-
moyl-dC derivative. The base recognition abilities of
modified dC derivatives are summarized in the column
ÔselectivityÕ. The selectivity of the 4-N-(N-aryl-
carbamoyl)-dC derivatives was markedly low when
compared with those of 4-N-(N-alkylcarbamoyl)-dC
and unmodified dC derivatives. Particularly, the Tm val-
ues of the DNA duplexes having a C*–C mismatch base
pair (entry 6, dCncm; 49.4°C; entry 7, dCqcm ; 49.3°C)
were higher than those of the DNA duplexes having a
C*–G match base pair (entry 6, dCncm, 47.5°C; entry
7, dCqcm, 48.7°C). The values in the column ÔrangeÕ rep-
resent the difference between the highest and the lowest
Tm values in each lane. To our surprise, the range of 4-
N-[N-(quinol-5-yl)carbamoyl]-dC (entry 7, 2.2°C) was
small enough to be called a universal base. It seemed
to us that the orientation of the carbamoyl groupwas
ÔdistalÕ in the mismatched base pair, and the stabilization
of the duplex having a mismatch base pair was caused
by increase of the stacking effect of the substituent on
the carbamoyl groups.
These results are summarized in Table 3. It is clear that
the Tm value increases with an increase of the surface
area of the substituent of the carbamoyl group. The
Tm values of the duplexes having a C*–abasic pair were
almost close to those of the duplexes having a mis-
matched base pair. These results suggested that the sta-
bilization of the duplexes containing a mismatch base
pair is due to the stacking effect of the substituent of
the carbamoyl group. However, the geometry of the
mismatch base pair containing a 4-N-carbamoyl-dC
derivative is still unclear. In addition, the Tm value of
the duplex containing dCqcm (entry 6, 51.1°C) was quite
higher than that of the duplex containing dCncm (entry
5, 48.8°C). This is due to the nitrogen atom in the quin-
ol-5-yl group.
The fluorescence of 2-aminopurine is known to be sensi-
tive to a delicate change in its surrounding DNA struc-
ture. Particularly, it is strongly quenched when
hybridized with a base. This inherent property of ap
has been used to detect the local structure of bases
flipped out by enzymes such as uracil DNA glycosylase9
and adenine-specific DNA methyltransferase.10 There-
fore, we used this base as a site-specific fluorescent probe
in DNA duplexes to see if the mismatched base is flipped
out. As the result, the fluorescence of 2-aminopurine in
To confirm this assumption, the Tm values of DNA du-
plexes containing a C*–abasic pair were measured. We
used a tetrahydrofuran derivative as an abasic site.

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K. Miyata et al. / Tetrahedron Letters 45 (2004) 9365–9368
Table 4. Fluorescence intensitya of 2-aminopurine (ap) in DNA 13mer
duplexes containing dCncm
Acknowledgements
I: 50-d(AGAGAAapGAGAAA)-30,
This work was supported by a Grant-in-Aid for Scien-
tific Research from the Ministry of Education, Culture,
Sports, Science and Technology, Japan. This work was
also supported by CREST of JST (Japan Science and
Technology) and partially supported by the COE21
project.
II: 50-d(TTTCTCCTTCTCT)-30,
III: 50-d(TTTCTCCncmTTCTCT)-30
Entry
Oligo
Fluorescence intensity
1
2
3
Only I
I–II
I–III
62
15
13
a Recorded with an exicitation wavelength of 310nm and an emission
wavelength at 370nm.
References and notes
1. Wada, T.; Kobori, A.; Kawahara, S.; Sekine, M. Tetra-
hedron Lett. 1998, 39, 6907–6910.
DNA duplexes containing a Cncm–apmismatch base
pair was actually quenched as shown in Table 4 (entries
1–3). These results suggest that the mismatched base
moiety opposite to 4-N-[N-(napht-1-yl)carbamoyl]-dC
is not flipped out but is involved in stacking of the
DNA duplex. However, owing to the steric hindrance
of the N-(napht-1-yl)carbamoyl group, the modified
and complementary base moieties are expected to stack
with each other in the DNA duplex.
2. Wada, T.; Kobori, A.; Kawahara, S.; Sekine, M. Eur. J.
Org. Chem. 2001, 4583–4593.
3. Kobori, A.; Miyata, K.; Ushioda, M.; Seio, K.; Sekine, M.
J. Org. Chem. 2002, 67, 476–485.
4. Miyata, K.; Kobori, A.; Seio, K.; Sekine, M., J. Org.
Chem., unpublished work.
5. Tamura, N.; Aoki, K.; Lee, M. S. Carcinogenesis 1990, 11,
2009–2014.
6. Nakano, S.; Uotani, Y.; Nakashıma, S.; Anno, Y.; Fujii,
M.; Sugimoto, N. J. Am. Chem. Soc. 2003, 125, 8086–
8087.
7. Current Protocols in Nucleic Acid Chemistry; Beaucage, S.
L., Bergstrom, D. E., Glick, G. D., Jones, R. A., Eds.;
John Wiley & Sons, Inc.: New York, 2000.
8. Sinha, N. D.; Biernat, J.; Koster, H. Tetrahedron Lett.
1983, 24, 5843–5846.
9. Stivers, J. T.; Pankiewicz, K. W.; Watanabe, K. A.
Biochemistry 1999, 38, 952–963.
10. Beuck, C.; Singh, I.; Bhattacharya, A.; Hecker, W.;
Parmar, V. S.; Seitz, O.; Weinhold, E. Angew. Chem.,
Int. Ed. 2003, 42, 3958–3960.
11. Nichols, R.; Andrews, P. C.; Zhang, P.; Bergstrom, D. E.
Nature 1994, 369, 492–493.
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4039–4043.
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6191–6192.
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Harrit, N. H.; Wengel, J. Org. Biomol. Chem. 2004, 2, 80–
89.
The conventional universal bases would be classified
into two groups. Most artificial universal bases such as
3-nitropyrrole11 and 5-nitroindole12 do not stack with
the opposite base moiety. On the other hand, intercala-
tor-like nucleobases such as pyrenyl C-nucleoside13,14
act as not only universal bases but also intercalators.
Our results mentioned above suggest that 4-N-[N-(quin-
ol-5-yl)carbamoyl]-dC acts as an intercalator-type
universal. On the other hand, it was suggested that,
when the guanine base is located on the opposite site
of the modified cytosine, the orientation of the N-phen-
ylcarbamoyl groupchanges to ÔproximalÕ so that a
Watson–Crick base pair is formed in the DNA duplex.
These results would provide new insight into the design
of artificial universal bases that induce change in their
geometry. Future work should be done to elucidate
the geometry of the Cqcm–G base pair in more detail.