Synthesis and properties of a new fluorescent bicyclic 4-N- carbamoyldeoxycytidine derivative

Article abstract of DOI:10.1021/ol053125n

A bicyclic 4-N-carbamoyldeoxycytidine derivative (1, dC^hpp) geometrically locked was synthesized as a new fluorescent nucleobase. The hybridization properties of oligodeoxynucleotides containing dC^hpp were investigated by use of T

Full text of DOI:10.1021/ol053125n

ORGANIC
LETTERS
2006
Vol. 8, No. 8
1545-1548
Synthesis and Properties of a New
Fluorescent Bicyclic
4-N-Carbamoyldeoxycytidine Derivative
Kenichi Miyata,†,‡ Ryuji Tamamushi,† Akihiro Ohkubo,†,‡ Haruhiko Taguchi,†,‡
|
Kohji Seio,‡,§ Tomofumi Santa, and Mitsuo Sekine*,†,‡
Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta, Midoriku,
Yokohama 226-8501, Japan, DiVision of CollaboratiVe Research for Bioscience and
Biotechnology, Frontier CollaboratiVe Research Center, Tokyo Institute of Technology,
4259 Nagatsuta, Midoriku, Yokohama 226-8503, Japan, CREST, JST (Japan Science
and Technology Agency), and Graduate School of Pharmaceutical Sciences, UniVersity
of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan
msekine@bio.titech.ac.jp
Received December 24, 2005
ABSTRACT
A bicyclic 4-N-carbamoyldeoxycytidine derivative (1, dChpp) geometrically locked was synthesized as a new fluorescent nucleobase. The
hybridization properties of oligodeoxynucleotides containing dChpp were investigated by use of Tm analysis. It was found that dChpp forms
stable base pairs not only with the complementary guanine base, but also with the adenine base. Interestingly, the fluorescence of dChpp was
suppressed only when a dChpp-dG base pair was formed.
A variety of base-modified nucleosides have been synthe-
sized to introduce new attractive functions into oligonucleo-
tides. Recently, much attention has been paid to modified
nucleosides having fluorescent nucleobases that are sensitive
to the proximal environment as easily detectable probes for
DNA hybridization and SNP detection. Most of these
fluorescent nucleobases have been designed on the basis of
the following requirements: (a) conjugation of fluorescent
molecules such as pyrene or fluorene to pyrimidine and
deazapurine bases1 and (b) expansion of the conjugation
† Department of Life Science, Tokyo Institute of Technology.
‡ CREST, JST.
§ Frontier Collaborative Research Center, Tokyo Institute of Technology.
| Graduate School of Pharmaceutical Sciences, University of Tokyo.
(1) (a) Huber, R.; Fiebig, T.; Wagenknecht, H. A. Chem. Commun. 2003,
1878-1879. (b) Hwang, G. T.; Seo, Y. J.; Kim, B. H. Tetrahedron Lett.
2005, 46, 1475-1477. (c) Hwang, G. T.; Seo, Y. J.; Kim, S. J.; Kim, B. H.
Tetrahedron Lett. 2004, 45, 3543-3546. (d) Okamoto, A.; Kanatani, K.;
Saito, I. J. Am. Chem. Soc. 2004, 126, 4820-4827. (e) Hwang, G. T.; Seo,
Y. J.; Kim, B. H. J. Am. Chem. Soc. 2004, 126, 6528-6529. (f) Hurley, D.
J.; Seaman, S. E.; Mazura, J. C.; Tor, Y. Org Lett. 2002, 4, 2305-2308.
(g) Jiao G.-S.; Kim, T. M.; Topp, M. R. Org Lett. 2004, 6, 1701-1704.
Figure 1. Chemical structures of dChpp (1) and dCcmy (2)
nucleosides.
10.1021/ol053125n CCC: $33.50
© 2006 American Chemical Society
Published on Web 03/18/2006

Scheme 1. Synthesis of Bicyclic 4-N-Carbamoyldeoxycytidine (1, dChpp
)
system of natural nucleobases.2 Here, we describe a new
fluorescent nucleoside dChpp (1) (Figure 1).
the 5-hydroxymethyldeoxyuridine (4). The reaction of 4 with
chlorotrimethylsilane in dioxane followed by treatment with
sodium azide in DMF gave 5-azidomethyldeoxyuridine (5).5
The usual silylation of 5 with TBSCl gave the 3′,5′-O-
protected compound 6. The reaction of 6 with 2,4,6-
triisopropylbenzenesulfonyl chloride followed by ammonoly-
sis gave 5-azidomethyldeoxycytidine derivative (7).6 This
strategy proved to be much superior to the well-known
alternative method by use of POCl3/1,2,4-triazoles7 that gave
a complex mixture. The Pd/C-catalyzed hydrogenation of 7
gave the 5-aminomethyldeoxycytidine 8 in 91% yield.
Intramolecular cyclization of 8 with 1,1′-carbonyldiimidazole
afforded the bicyclic derivative 9 in 84% yield. The silyl
groups of 9 were removed by treatment with tetrabutylam-
monium fluoride hydrate to give dChpp (1) in 89% yield.
The UV absorption spectra of dC, dCcmy, and dChpp
nucleosides in phosphate buffer are shown in Figure 2A.
Different from dC and dCcmy, the spectrum of dChpp exhibited
an interesting absorption band at 300 nm. Furthermore, dChpp
exhibited an emission spectrum (λmax ) 360 nm) when
excited at 300 nm, as shown in Figure 2B.
The quantum yield of dChpp in 10 mM sodium phosphate
(pH 7.0) was caluculated to be 0.12. The absorption spectra
of 4-N-(N-methylcarbamoyl)deoxycytidine derivatives which
have acyclic N-methylcarbamoyl groups at the cytosine
amino group are similar to those of dCcmy, but these acyclic
derivatives did not exhibit any fluorescent properties (data
not shown).
The synthesis of the phosphoramidite 14 is shown in
Scheme 2. For the convenient synthesis of 14, the DMTr
group was first introduced to the 5′-OH group of compound
5. To avoid the deprotection of the DMTr group during
Recently, we have reported the synthesis and properties
of oligodeoxynucleotides containing 4-N-carbamoyldeoxy-
cytidine derivatives.3 It was revealed that the geometry of
the carbamoyl group of 4-N-carbamoyldeoxycytidine (Ccmy
)
derivatives changes depending on the polarity of the solvent.
The NMR analysis of dCcmy in D2O suggested that the
carbamoyl group formed an intramolecular hydrogen bond
with the cytosine ring nitrogen atom so that formation of a
Watson-Crick base pair with the complementary guanine
base was inhibited. However, the Tm analysis showed that
carbamoylation of the 4-amino group of deoxycytidine
allowed the base pairing with a guanine base following the
conformational change of the carbamoyl group. The stability
of DNA duplexes containing a dCcmy-dG base pair was
somewhat low (∆Tm ) -0.6 °C) compared with that of the
unmodified DNA duplex. On the other hand, the acylation
or alkoxycarbonylation4 of the 4-amino group of deoxycy-
tidine did not affect significantly the base pairing with the
guanine. These results imply that destabilization of DNA
duplexes containing dCcmy is due to the energy loss resulting
from the necessity of conformational change of the 4-N-
carbamoyl group. In this paper, we report the synthesis and
properties of a new bicyclic deoxycytidine derivative that
has a conformationally locked 4-N-carbamoyl group. The
carbamoyl group of dChpp and the 5-position of the cytosine
ring are bridged via a methylene linker so that the modified
group does not inhibit the formation of a Watson-Crick base
pair.
The synthesis of dChpp (1) is outlined in Scheme 1.
Treatment of deoxyuridine (3) with paraformaldehyde gave
(2) (a) Okamoto, A.; Tainaka, K.; Saito, I. J. Am. Chem. Soc. 2003, 125,
4972-4973. (b) Okamoto, A.; Tanaka, K.; Fukuta, T.; Saito, I. J. Am. Chem.
Soc. 2003, 125, 9296-9297. (c) Inoue, H.; Imura, A.; Ohtsuka, E. Nucleic
Acids Res. 1985, 13, 7119-7128. (d) Berry, D. A.; Jung, K.-Y.; Wise, D.
S.; Sercel, A. D.; Pearson, W. H.; Mackie, H.; Randolph, J. B.; Somers, R.
L. Tetrahedron Lett. 2004, 45, 2457-2461.
(5) Seio, K.; Wada, T.; Sekine, M. HelV. Chim. Acta 2000, 83, 162-
180.
(6) (a) Sekine, M. J. Org. Chem. 1989, 54, 2321-2326. (b) Matsuda,
A.; Yasuoka, J.; Sasaki, T.; Ueda, T. J. Med. Chem. 1991, 34, 999-1002.
(c) Awano, H.; Shuto, S.; Miyashita, T.; Ashida, N.; Machida, H.; Kira,
T.; Shigeta, S.; Matsuda, A. Arch. Pharm. Pharm. Med. Chem. 1996, 329,
66-72.
(7) (a) Matsuda, A.; Obi, K.; Miyasaka, T. Chem. Pharm. Bull. 1985,
33, 2575-2578. (b) Hodge, R. P.; Brush, C. K.; Harris, C. M.; Harris, T.
M. J. Org. Chem. 1991, 56, 1553-1564.
(3) Miyata, K.; Kobori, A.; Tamamushi, R.; Ohkubo, A.; Taguchi, H.;
Seio, K.; Sekine, M. submitted to Eur. J. Org. Chem.
(4) Wada, T.; Kobori, A.; Kawahara, S.; Sekine, M. Eur. J. Org. Chem.
2001, 4583-4593.
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Org. Lett., Vol. 8, No. 8, 2006

Table 1. Tm Valuesa for DNA 13mer Duplexes Containing
dChpp
entry
X
Y
Tmb (°C)
∆Tmc (°C)
+1.1
1
2
C
Chpp
G
G
57.1
58.2
3
4
C
A
A
40.6
50.8
Chpp
+10.2
-0.2
5
6
C
Chpp
C
C
30.6
30.4
7
8
C
Chpp
T
T
41.4
44.3
+2.9
a 2 µM duplex, 100 mM NaCl, 0.1 mM EDTA, 50 mM sodium phosphate
buffer, pH 7.0. b The Tm values are accurate within (0.5 °C. c ∆Tm is the
difference in the Tm value between the duplex having a modified base and
that having a natural base.
) -1.5 °C).3 These results indicate that the conformational
fixation of the 4-N-carbamoyl group resulted in direct binding
to the guanine base without energy loss.
The Tm values of DNA duplexes having mismatched base
pairs (Y ) A, C, and T) are also summarized in Table 1.
Interestingly, modification of the cytosine amino group with
the bridged carbamoyl group stabilized markedly the C-A
mismatched base pair. The Tm value of the duplex containing
Chpp-A was significantly higher (∆Tm ) +10.2 °C) than
that of the duplex containing a C-A mismatch base pair
(entry 3, 40.6 °C).
Figure 2. (A) UV absorption spectra of dC, dCcmy, and dChpp: 80
µM nucleoside, 10 mM sodium phosphate, pH 7.0. (B) Fluorescent
spectra of dChpp: 5 µM nucleoside, 10 mM sodium phosphate, pH
7.0.
reduction of the azide group with Pd-C, ammonium formate
was used as a hydrogen source for the catalytic hydrogena-
tion. Oligonucleotides containing dChpp were synthesized by
use of a DNA/RNA synthesizer according to the standard
protocol.
The thermal stability of DNA duplexes containing dChpp
was investigated in sodium phosphate buffer (pH 7.0)
containing 1.0 M NaCl. As shown in Table 1, the Tm value
of the duplex containing dChpp (entry 2, 58.2 °C) was slightly
higher by 1.1 °C than that of the control unmodified duplex
(entry 1, 57.1 °C). We have recently reported that modifica-
tion of the cytosine amino group with an acyclic N-
methylcarbamoyl group destabilized the duplex stability (∆Tm
dChpp can form a base pair with an adenine base with the
geometry of a reverse-Watson-Crick base pair. However,
it is not clear whether dChpp changes the tautomerism of
cytosine from the amino form to the imino form in order to
form a Watson-Crick base pair with the adenine base.
Further experiments are needed to determine the tautomerism
of the Chpp-A base pair in DNA duplexes. To investigate
the local environmental effect of this fluorescence nucleo-
Scheme 2. Synthesis of Bicyclic 4-N-Carbamoyldeoxycytidine Phosphoramidite (14)
Org. Lett., Vol. 8, No. 8, 2006
1547

base, the fluorescent spectra of single- or double-strand
oligodeoxynucleotides containing dChpp were measured
(Figure 3). It was found that the fluorescent properties of
containing a Chpp-G base pair was significantly suppressed
(Figure 3, blue line). These phenomena are similar to those
of benzopyridopyrimidine (BPP) nucleoside derivatives
reported by Okamoto et al.8 However, it is interesting that
the conjugated system of the cytosine ring of dChpp is much
smaller than that of BPP derivatives, suggesting the mini-
mized structure for the base-discriminating fluorescent
properties of BPP derivatives.
In conclusion, we synthesized a new fluorescence nucleo-
base that can form a stable base pair with both guanine and
adenine bases. Although the conjugated system of the
cytosine ring of dChpp is similar to dCcmy reported previously,
dChpp was found to serve as a strong fluorescent analogue
of deoxycytidine, which proved to have a base-discriminating
fluorescent property. A tautomeric study of dChpp is now
underway.
Acknowledgment. This work was supported by a Grant
from CREST of JST (Japan Science and Technology
Agency) and in part by a grant of the Genome Network
Project from the Ministry of Education, Culture, Sports,
Science and Technology, Japan. This work was also sup-
ported by the COE21 project.
Figure 3. Fluorescent spectra of oligodeoxynucleotides containing
dChpp (5 µM nucleoside, 10 mM sodium phosphate, pH 7.0,
excitation 300 nm).
dChpp remained in an oligodeoxynucleotide 13mer 15 con-
taining dChpp at the center position. The λmax value of the
excitation and the emission spectra of the oligonucleotide
were identical to those of dChpp (Figure 2B and Figure 3,
green line).
When the Chpp base faced a mismatch base dA as
exemplified by a DNA duplex formed between the modifed
oligomer 15 and a mismatched oligomer 16, the fluorescence
intensity (Figure 3, red line) was very similar to that (Figure
3, green line) of the single-strand oligonucleotide containing
Chpp. In contrast, the fluorescent spectrum of a DNA duplex
Supporting Information Available: Experimental pro-
cedure and characterization data for all new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL053125N
(8) (a) Okamoto, A.; Tainaka, K.; Saito, I. J. Am. Chem. Soc. 2003, 125,
4972-4973. (b) Okamoto, A.; Tainaka, K.; Saito, I. Chem. Lett. 2003, 32,
684-685. (c) Okamoto, A.; Tainaka, K.; Saito, I. Photomed. Photobiol.
2003, 25, 39-40.
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Org. Lett., Vol. 8, No. 8, 2006