Synthesis of 1,8-naphthyridine C-nucleosides and their base-pairing properties in oligodeoxynucleotides: Thermally stable naphthyridine: imidazopyridopyrimidine base-pairing motifs

Article abstract of DOI:10.1002/anie.200461857

Out with the old, in with the new: Novel 1,8-naphthyridine C-nucleosides, Na-N^O and Na-O^N (see formulae), were synthesized through Heck coupling reactions. Oligodeoxynucleotides containing the Na-N^O and Na-O^N nu

Full text of DOI:10.1002/anie.200461857

Communications
Nonnatural Nucleotides
Synthesis of 1,8-Naphthyridine C-Nucleosides
and Their Base-Pairing Properties in
Oligodeoxynucleotides: Thermally Stable
Naphthyridine:Imidazopyridopyrimidine
Base-Pairing Motifs**
Sadao Hikishima, Noriaki Minakawa,*
Kazuyuki Kuramoto, Yuki Fujisawa, Maki Ogawa, and
Akira Matsuda*
A number of nucleoside analogues that contain non-natural
nucleobases have been synthesized and incorporated into
oligodeoxynucleotides (ODNs) with the aim of biological,
bioengineering, and therapeutic applications.[1,2] The develop-
ment of new base-pairing motifs beyond the Watson–Crick
hydrogen bonding (H bonding) model for thermal stability
and specificity is therefore still an area of active research.[3,4]
We recently reported the synthesis of imidazo[5’,4’:4,5]pyr-
ido[2,3-d]pyrimidine nucleosides with the ability to form four
H bonds and discussed their hybridization properties in
ODNs (Scheme 1A).[5,6] Accordingly, the Im-NO:Im-ON
base pair markedly stabilized a duplex when three of the
pairs were consecutively incorporated into ODNs. However,
incorporation of one pair into ODNs resulted in destabiliza-
tion of the duplex relative to those containing A:T and G:C
base pairs. These results were explained by the conflicting
effects of the Im-NO:Im-ON pair in ODNs, that is, the pair
stabilizes the duplex with four H bonds, but it widens of the
helix because the C1’–C1’ distance is longer than that in the
Watson–Crick base pair—a destabilizing factor for the duplex
that contains the pair. Since the goal of our continuing study is
to develop base-pairing motifs that stabilize and regulate
DNA structures, including a double-helix-independent mode
of incorporation of the new base pair(s) (i.e., one pair, three
nonconsecutive pairs, and three consecutive pairs in this
study), the novel 1,8-naphthyridine C-nucleosides 7 (which
bears an Na-NO base) and 9 (which bears an Na-ON base)
were designed.[6] These C-nucleosides are expected to form
two sets of naphthyridine:imidazopyridopyrimidine base-
pairing motifs (Na-ON:Im-NO and Na-NO:Im-ON) with four
hydrogen bonds when these are incorporated into ODNs
Scheme 1. A) Im-NO:Im-ON base-pairing motif. B) Newly designed
naphthyridine:imidazopyridopyrimidine base-pairing motifs.
(Scheme 1B). Furthermore, the new motifs can be regarded
as an expanded pyrimidine:purine-type base pair (with C1’–
C1’ distances similar to the Watson–Crick base pair), which,
unlike the Im-NO:Im-ON pair, would not distort the helical
structure.[5] Herein we describe the synthesis of the 1,8-
naphthyridine C-nucleosides 7 and 9, and the effects on the
thermal stabilities of the ODNs containing the naphthyri-
dine:imidazopyridopyrimidine base-pairing motifs.[7]
The synthetic route to the target compounds is illustrated
in Scheme 2. The synthesis started from 2-amino-7-hydroxy-
1,8-naphthyridine (1).[8] Iodination of 1 with N-iodosuccini-
mide (NIS) was followed by protection of the exocyclic amino
group to give the 6-iodo-1,8-naphthyridine derivative 2, a
substrate for the synthesis of 7. On the other hand, the
synthesis of 9 requires the 3-iodo-1,8-naphthyridine deriva-
tive. Treatment of 1 with excess NIS, followed by protection of
the exocyclic amino group gave the 3,6-diiodo derivative 3,
which was converted into the 3-iodo derivative 4 by treatment
with a stoichiometric amount of tributyltin hydride in the
presence of [Pd(PPh3)4]. This regioselective reduction of the
6-iodo group in 3 can be explained by the electron densities at
C3 and C6, which were estimated from the 13C NMR
spectrum (C3: d = 84.7 ppm and C6: d = 91.0 ppm).[9] Heck
coupling of the 6-iodo derivative 2 with the glycal 5[10] was
followed by deprotection and reduction[11] to afford the
desired 6 in 78% overall yield (from 2). In the same manner,
the reaction of 4 with 5 afforded 8 in 76% yield. Treatment of
6 and 8 with methanolic ammonia gave the free nucleosides 7
and 9, respectively. To incorporate both C-nucleosides 7 and 9
into ODNs, they were converted into the corresponding
phosphoramidites 10 and 11, respectively. For the conversion
of 9, the N-benzoyl group was the best choice as a protecting
group for the exocyclic amino function,[12] and methyl N,N-
diisopropylchlorophosphoramidite was used to give 11
because of purification problems that arose when 2-cyano-
ethyl N,N-diisopropylchlorophosphoramidite was used.
[*] S. Hikishima, Prof. N. Minakawa, K. Kuramoto, Y. Fujisawa,
M. Ogawa, Prof. A. Matsuda
Graduate School of Pharmaceutical Sciences
Hokkaido University
Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812 (Japan)
Fax: (+81)11-706-4980
E-mail: noriaki@pharm.hokudai.ac.jp
matuda@pharm.hokudai.ac.jp
[**] This work was supported in part by a Grant-in-Aid for Scientific
Research on Priority Areas and Encouragement of Young Scientists
from the Ministry of Education, Science, Sports, and Culture of
Japan. This paper constitutes Part 229 of Nucleosides and
Nucleotides. Part 228 is reference [15].
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
596
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/anie.200461857
Angew. Chem. Int. Ed. 2005, 44, 596 –598

Angewandte
Chemie
Table 1: Sequences of ODNs and hybridization data.
Duplex
X
Y
Tm
DTm
[8C][a]
[8C][b]
ODNI:ODNII
Im-ON
Im-NO
Im-ON
Na-NO
G
Na-NO
Na-ON
Im-NO
Na-ON
C
57.2
56.4
44.0
50.1
49.1
47.8
+9.4
+8.6
À3.8
+2.3
+1.3
5’-GCACCGAAXAAACCACG-3’
3’-CGTGGCTTYTTTGGTGC-5’
A
T
ODNIII:ODNIV
Im-ON
Im-NO
Im-ON
Na-NO
G
Na-NO
Na-ON
Im-NO
Na-ON
C
82.2
80.9
53.3
48.9
56.7
+34.4
+33.1
+5.5
+1.1
+8.9
5’-GCXCCGAAXAAACCXCG-3’
3’-CGYGGCTTYTTTGGYGC-5’
ODNV:ODNVI
Im-ON
Im-NO
Im-ON
Na-NO
G
Na-NO
Na-ON
Im-NO
Na-ON
C
80.2
81.0
70.4
68.1
55.2
+32.4
+33.2
+22.6
+20.3
+7.4
5’-GCACCGAXXXAACCACG-3’
3’-CGTGGCTYYYTTGGTGC-5’
[a] Experimental conditions are described in the Supporting Information.
The data presented are averages of triplicates. [b] The DTm values were
obtained by subtracting data for the Tm possessing X:Y=A:T from that
for each duplex.
results.[5] Although the Na-NO:Na-ON pair stabilized the
duplex by
Scheme 2. Reagents and conditions: a) NIS (1.1 equiv), DMF; b) dime-
thylformamide dimethylacetal, DMF, 808C; c) NIS (2.9 equiv), DMF,
808C; d) dibutylformamide dimethylacetal, DMF; e) Bu3SnH,
[Pd2dba3]·CHCl3, PPh3, DMF, 608C; f) 5, Pd(OAc)2, AsPh3, Bu3N, DMF,
608C; g) TBAF, THF; h) NaBH(OAc)3, AcOH, CH3CN; i) NH3/MeOH,
808C; j) DMTrCl, pyridine; k) 2-cyanoethyl N,N-diisopropylchlorophos-
phoroamidite, iPr2NEt, CH2Cl2; l) 1) TMSCl, pyridine then BzCl,
2) NH4OH; m) methyl N,N-diisopropylchlorophosphoroamidite,
iPr2NEt, DMAP, CH2Cl2. NIS=N-iodosuccinimide; DMF=N,N-dime-
thylformamide; dba=dibenzylideneacetone; TBAF=tetrabutylammo-
nium fluoride; DMTr=4,4’-dimethoxytrityl; TMS=trimethylsilyl;
DMAP=4-(dimethylamino)pyridine.
+ 2.38C, the value was much less than those of Im-ON:Na-NO
and Im-NO:Na-ON, and similar to that of the G:C pair. The
preferable base-pairing motifs by Im-ON:Na-NO and Im-
NO:Na-ON were emphasized in a series of ODNIII:ODNIV.
Both pairs stabilized the duplexes by more than + 308C, and
the effects of Im-ON:Im-NO and Na-NO:Na-ON were insuffi-
cient, despite the expected base-pairing motifs with four
H bonds. In the series ODNV:ODNVI, not only the Im-
ON:Na-NO and Im-NO:Na-ON pairs but also the Im-ON:Im-NO
and Na-NO:Na-ON pairs stabilized the duplexes much more
than G:C and A:T pairs, although the first pairs are generally
considered more effective for thermal stability. From these
results, it can be concluded that the newly designed base
pairing motifs Im-ON:Na-NO and Im-NO:Na-ON thermally
stabilized the duplex by nearly 108C more per pair than the
A:T pair and 88C more than the G:C pair independent of the
mode of incorporation of the new base pair(s) into the ODNs.
This effect is presumably caused by the noncanonical base
pairs consisting of four H bonds and the stacking effect of the
expanded aromatic surfaces.[14] Furthermore, the fact that the
shape of the pairs resembles a pyrimidine:purine base pair
(i.e., shape complementarity) would also be critical for their
effect because of the sequence-dependent thermal stabilizing
effect of the Im-ON:Im-NO and Na-NO:Na-ON pairs. As we
expected, the Im-ON:Na-NO and Im-NO:Na-ON pairs did not
cause the disruption of the helical structure, unlike the Im-
ON:Im-NO pair. Although some shift in the base-pairing phase
from the usual pyrimidine:purine base pairing could occur to
complete the base pairing of Im-ON:Na-NO and Im-NO:
Na-ON (see Scheme 1B), the effect of this shift should be
negligible for the thermally stable duplex formation, since
To investigate the base-pairing properties of Na-NO and
Na-ON, three classes of complementary duplexes were
synthesized. As shown in Table 1, the first class consists of
duplexes (a series of ODNI:ODNII) that contain one X:Y
pair in the center of the duplexes (containing Na-NO, Na-ON,
Im-NO, Im-ON, or natural bases in their X or Y positions). The
second class is made up of duplexes (a series of ODNIII:OD-
NIV) that contain three nonconsecutive X:Y pairs, and the
last class (a series of ODNV:ODNVI) is made up of three
consecutive X:Y pairs. The thermal stability of all duplexes
was measured by thermal denaturation in a buffer of 10 mm
sodium cacodylate (pH 7.0) containing 1 mm NaCl.[13] The
resulting melting temperatures Tms and the DTms values
calculated based on the Tm of the duplex (X:Y= A:T,
common to ODNI:ODNII, ODNIII:ODNIV, and ODN-
V:ODNVI) are listed in Table 1. As we expected, the Im-
ON:Na-NO and Im-NO:Na-ON pairs stabilized the duplex by
+ 9.48C and + 8.68C, respectively, relative to that containing
the A:T pair. In contrast, the Im-ON:Im-NO pair destabilized
the duplex by À3.88C, which agreed with our previous
Angew. Chem. Int. Ed. 2005, 44, 596 –598
www.angewandte.org
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
597

Communications
ON. The aglycons of 7 and 9, which contain naphthyridine
nucleobases, are referred to as Na-NO and Na-ON, respectively.
[7] Recently, the synthesis and properties of peptide nucleic acids
that contain 1,8-naphthyridine bases were reported: A. B.
Eldrup, C. Christensen, G. Haaima, P. E. Nielsen, J. Am.
Chem. Soc. 2002, 124, 3254 – 3262.
both pairs stabilized the duplex, irrespective of the mode of
incorporation.
To clarify the specificity of the naphthyridine:imidazopyr-
idopyrimidine base pairs, the base-pairing properties of Na-
NO with natural bases, as an example, were examined in a
series of ODNI:ODNII and ODNV:ODNVI. As can be seen
in Table 2, the resulting Tms were all lower than that of A:T
[8] G. R. Newkome, S. J. Garbis, V. K. Majestic, F. R. Fronczek, G.
Chiari, J. Org. Chem. 1981, 46, 833 – 839.
[9] W.-S. Kim, H.-J. Kim, C.-G. Cho, J. Am. Chem. Soc. 2003, 125,
14288 – 14289.
[10] R. S. Coleman, M. L. Madaras, J. Org. Chem. 1998, 63, 5700 –
5703.
[11] H.-C. Zhang, G. Doyle Daves, Jr., J. Org. Chem. 1992, 57, 4690 –
4696.
[12] As the reaction of 8 with DMTrCl gave the desired product in
poor yield, a protecting group for the exocyclic amino function
was examined.
Table 2: Sequences of ODNs and hybridization data of Na-NO with
natural bases.
duplex
X
Y
T
m [8C][a]
DTm [8C][b]
ODNI:ODNII
Na-NO
Na-NO
Na-NO
Na-NO
G
A
G
C
T
C
T
61.0
58.4
54.3
59.0
64.8
63.6
À2.6
À5.2
À9.3
À4.6
+1.2
5’-GCACCGAAXAAACCACG-3’
3’-CGTGGCTTYTTTGGTGC-5’
[13] In a buffer containing 100 mm NaCl (conditions used in the
previous paper[5]), some duplexes showed Tms higher than 958C.
Therefore, 1 mm NaCl was used in this study.
A
[14] For example, the stacking ability of Na-NO was higher than those
of purine bases and similar to those of the imidazopyridopyr-
imidine bases (Im-NO and Im-ON).
ODNV:ODNVI
Na-NO
Na-NO
Na-NO
Na-NO
G
A
G
C
T
60.3
55.6
45.2
60.0
69.0
À3.3
À8.0
5’-GCACCGAXXXAACCACG-3’
3’-CGTGGCTYYYTTGGTGC-5’
À18.4
À3.6
[15] S. Hoshika, N. Minakawa, A. Matsuda, Nucleic Acids Res. 2004,
32, 3815 – 3825.
C
+5.4
[a] Experimental conditions are described in the Supporting Information.
The data presented are averages of triplicates. [b] The DTm values were
obtained by subtracting data for the Tm possessing X:Y=A:T from that
for each duplex.
pair. Although the adenine base is expected to form a base
pair with Na-NO like the A:T pair, Na-NO:A pair also
destabilized the duplex.
In conclusion, the novel 1,8-naphthyridine C-nucleosides
7 and 9 with the ability to form four H bonds were synthesized
through Heck coupling. The ODNs containing 7 and 9 formed
extremely stable duplexes by the base-pairing motifs Im-
ON:Na-NO and Im-NO:Na-ON. Furthermore, these motifs are
specific, so that these would be versatile in stabilizing and
regulating a variety of DNA structures.
Received: September 1, 2004
Published online: December 21, 2004
Keywords: DNA recognition · hydrogen bonds · nucleobases ·
.
nucleosides · oligonucleotides
[1] For recent reviews, see: a) E. T. Kool, Acc. Chem. Res. 2002, 35,
936 – 943; b) A. A. Henry, F. E. Romesberg, Curr. Opin. Chem.
Biol. 2003, 7, 727 – 733; and references therein.
[2] W. M. Flanagan, J. J. Wolf, P. Olson, D. Grant, K.-Y. Lin, R. W.
Wagner, M. D. Matteucci, Proc. Natl. Acad. Sci. USA 1999, 96,
3513 – 3518.
[3] For a recent review, see: C. R. Geyer, T. R. Battersby, S. A.
Benner, Structure 2003, 11, 1485 – 1498, and references therein
[4] H. Liu, J. Gao, S. R. Lynch, Y. D. Saito, L. Maynard, E. T. Kool,
Science 2003, 302, 868 – 871.
[5] N. Minakawa, N. Kojima, S. Hikishima, T. Sasaki, A. Kiyosue, N.
Atsumi, Y. Ueno, A. Matsuda, J. Am. Chem. Soc. 2003, 125,
9970 – 9982.
[6] For the sake of simplicity, the imidazopyridopyrimidine nucle-
obases shown in Scheme 1 A are referred to as Im-NO and Im-
598
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
Angew. Chem. Int. Ed. 2005, 44, 596 –598