New imidazopyridopyrimidine:naphthyridine base-pairing motif, ImN N:NaOO, consisting of a DAAD:ADDA hydrogen bonding pattern, markedly stabilize DNA duplexes

Article abstract of DOI:10.1039/c1cc13805g

The new imidazopyridopyrimidine:naphthyridine base-pairing motifs, ImO ^O:NaN^N and ImN^N:NaO^O, were designed. Among the base pairs examined, DNA duplexes containing ImN^N:NaO ^O pair(s) consist

Full text of DOI:10.1039/c1cc13805g

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COMMUNICATION
New imidazopyridopyrimidine:naphthyridine base-pairing motif,
ImNN:NaOO, consisting of a DAAD:ADDA hydrogen bonding pattern,
markedly stabilize DNA duplexesw
Kazuyuki Kuramoto,a Noriko Tarashima,b Yasuyuki Hirama,a Yusaku Kikuchi,b
Noriaki Minakawa*b and Akira Matsuda*a
Received 26th June 2011, Accepted 11th August 2011
DOI: 10.1039/c1cc13805g
The new imidazopyridopyrimidine:naphthyridine base-pairing
motifs, ImOO:NaNN and ImNN:NaOO, were designed. Among
the base pairs examined, DNA duplexes containing ImNN:NaOO
pair(s) consisting of a DAAD:ADDA hydrogen bonding pattern
(D = donor, A = acceptor) were markedly stabilized thermally
and thermodynamically.
selective recognition of ImON:NaNO and ImNO:NaON pairs
by DNA polymerases.9,10 These successful results prompted us
to develop new base-pairing motifs consisting of a series of
Im:Na pairs. As can be seen in Fig. 1A and B, the previous
ImON:NaNO and ImNO:NaON pairs have alternate H-bonding
patterns of ADAD:DADA and DADA:ADAD, respectively
(A = acceptor, D = donor). Since it has been suggested that
the stability of hydrogen bonded complexes is affected by the
arrangement of H-bonds arising from a secondary interaction,11
we designed new base-pairing motifs, the ImOO:NaNN and
ImNN:NaOO pairs (Fig. 1C and D), that possess ADDA:DAAD
and DAAD:ADDA H-bonding patterns, which are expected to
be more stable than the alternate H-bonding patterns.
In this communication, we report the synthesis of the new
naphthyridine derivatives, NaNN and NaOO, and their
base-pairing property with ImOO and ImNN,4 respectively.
The base-pairing property of ImOO:NaNN was almost equal
to those of the ImON:NaNO and ImNO:NaON pairs. In contrast,
the ImNN:NaOO pair thermally and thermodynamically stabilized
its duplex more than the ImON:NaNO and ImNO:NaON pairs.
These results are also discussed.
Hydrogen bonding (H-bonding) is one of the most important
non-bonded molecular interactions. Therefore, it is important
to understand the molecular recognition process in biology
and in the design of functional organic supramolecules
through H-bonding. In the field of nucleic acid chemistry,
for example, specific H-bonding between adenine and thymine
(A:T) and guanine and cytosine (G:C), the Watson–Crick base
pairs, plays a critical role not only in conserving and transmitting
genetic information but also in duplex stability. Furthermore,
the development of an artificial base-pairing motif beyond the
Watson–Crick base pairs is an area of active research with the
aim of expanding biological, bioengineering, and therapeutic
applications.1–3
We have been working on a project to develop new base-
pairing motifs consisting of four hydrogen bonds (H-bonds)
designed to stabilize and regulate DNA structures.4–7 We have
already prepared two sets of base-pairing motifs consisting of
the imidazopyridopyrimidine:naphthyridine (Im:Na) pairs,
that is, the ImON:NaNO and ImNO:NaON pairs shown in
Fig. 1A and B.4,5 Since the resulting base-pairing motifs were
specific and markedly stabilized DNA duplexes (ca. +8 1C per
pair relative to an A:T pair) independent of the sequence
context, we attempted to develop thermally stabilized decoy
molecules.8 Furthermore, we have recently demonstrated the
a Fuculty of Pharmaceutical Sciences, Hokkaido University, Kita-12,
Nishi-6, Kita-ku, Sapporo 060-0812, Japan.
Fax: +81 (0)11-706-3228; Tel: +81 (0)11-706-4980
b Graduate School of Pharmaceutical Sciences, The University of
Tokushima, Shomachi 1–78–1, Tokushima 770–8505, Japan.
E-mail: minakawa@ph.tokushima-u.ac.jp;
Fax: +81 (0)88-633-7288; Tel: +81 (0)88-633-7288
w Electronic supplementary information (ESI) available: Experimental
procedures and characterization data of all new compounds, and
properties of oligodeoxynucleotides containing a series of Im:Na base
pairs. See DOI: 10.1039/c1cc13805g
Fig. 1 Imidazopyridopyrimidine:naphthyridine (Im:Na) base-pairing
motifs consisting of four H-bonds. (A) ImON:NaNO, (B) ImNO:NaON,
(C) ImOO:NaNN, and (D) ImNN:NaOO.
c
10818 Chem. Commun., 2011, 47, 10818–10820
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Table 1 Sequences of ODNs and hybridization data
a
Experimental conditions are described in the ESI.w The data
b
presented are average of triplicates. The DTm values were obtained
by subtracting data for the Tm possessing X:Y = A:T from each duplex.
synthesized. As shown in Table 1, the first class consists of
duplexes (a series of ODN I:ODN II) containing one X:Y pair
at the center of the duplexes (with the noncanonical and
natural bases in their X or Y positions). The second class is
made up of duplexes (a series of ODN III:ODN IV) having
three non-consecutive X:Y pairs, and the third class (a series of
ODN V:ODN VI) is made up of three consecutive X:Y pairs.
These duplexes were used in a series of studies,4,5,7 where the
measurement of the thermal stability was carried out in a
buffer of 10 mM sodium cacodylate (pH 7.0) containing 1 mM
NaCl. The resulting melting temperature Tms and the DTms
values calculated based on the Tm of the duplex (X:Y = A:T,
common to all duplexes) were listed in Table 1.12 In the ODN
I:ODN II case, the ImON:NaNO and ImNO:NaON pairs
stabilized the duplexes by +7.8 1C and +7.5 1C, respectively,
relative to the one containing the A:T pair. These results are
consistent with our previous data.5 When X:Y was substituted to
the ImOO:NaNN pair, one of the newly designed base-pairing
motifs, the DTm value was +7.9 1C, which is almost equal to
those of the ImON:NaNO and ImNO:NaON pairs. On the other
hand, the ImNN:NaOO pair stabilized the duplex by +11.4 1C,
which was the highest among the four pairs. The preferable
base-pairing motif by the ImNN:NaOO pair was emphasized in
the series of ODN III:ODN IV and ODN V:ODN VI. Thus,
both duplexes containing three ImNN:NaOO pairs were
stabilized by ca. +40 1C, which is a dramatic improvement.
In Table 1, the results of the base pairs illustrated in Fig. 1
(i.e., 4 matched pairs) are listed. However, 16 combinations of
the Im:Na pairs, for example ImNO:NaNO and ImNN:NaNN
(i.e., an additional 12 mismatched pairs), are possible. To
determine the specificity of these base pairs, we measured the
Tms for all possible combinations (see the ESIw). As a result,
ImON:NaNO, ImNO:NaON, ImOO:NaNN, and ImNN:NaOO
illustrated in Fig. 1 were found to be well matched pairs,
respectively, and the ImNN:NaOO pair was the most thermally
stable base-pairing motif among all possible combinations.
All the Im:Na base-pairing motifs illustrated in Fig. 1 are
expected to form base pairs with four H-bonds. Therefore, the
Scheme 1 Reagents and conditions: (a) Ac2O, DMAP, Et3N, DMF;
(b) POCl3; (c) NH3/MeOH, 80 1C; (d) liq. NH3, 120 1C; (e) TIPSCl,
imidazole, DMF, 55 1C; (f) NaNO2, AcOH; (g) NH3/MeOH, 60 1C;
(h) TBAF, THF.
The desired NaNN and NaOO (5 and 10) were prepared
from the NaON derivative 15 (Scheme 1). Thus, the 30 and
50-hydroxyl groups of 1 were protected by acetyl groups to
give 2. In order to convert the 7-oxo group of 2 into an amino
group, 2 was treated with POCl3 at room temperature to give
the 7-chloro derivative 3 in 85% yield. The resulting 3 was
treated with NH3 in MeOH at 80 1C to afford the free
nucleoside 4. Since the 7-chloro group was inactive under
the above conditions, 4 was treated with liq. NH3 at 120 1C to
give the desired NaNN (5) in 88% yield.
For the synthesis of NaOO (10), 1 was first converted into
the di-O-TIPS derivative 6. The protecting group of the
exocyclic amino group was then removed by treatment with
NH3 in MeOH at 80 1C. If the resulting amino group could
have been converted easily into an oxo group via diazotization,
the desired NaOO derivative 9 would have been available.
However, the reaction of 7 with sodium nitrite in aqueous
solution afforded a complex mixture, not the desired 9.
Consequently, 7 was treated with sodium nitrite in acetic acid
to give the 2-acetate 8, which was then deprotected by NH3 in
MeOH to give the NaOO derivative 9 in 88% yield for the two
steps. Deprotection of the TIPS groups of 9 was effected by
treatment with TBAF in THF to give the desired NaOO (10).
The resulting nucleosides 5 and 10 were finally converted into
the corresponding phosphoramidite units after appropriate
protection of the nucleobase moieties for introduction into
the oligodeoxynucleotides (ODNs) (see the ESIw).
In order to investigate the base-pairing properties of NaNN
and NaOO, three classes of complementary duplexes were
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should be the same as those of the ImNN:NaOO pair. However
the thermal stability of this pair (+7.9 1C) was rather low
compared with the ImNN:NaOO pair and almost the same as
those of the ImON:NaNO and ImNO:NaON pairs. In our
previous study,4 we suggested that the ImOO exists as a
tautomeric form, represented as ImOO(t), possessing an
ADAA H-bonding pattern. This being the case, then the base
pair between ImOO(t) and NaNN should have four repulsive
and two attractive secondary interactions together with three
primary H-bonds (Fig. 2C). Accordingly, the overall strength
of interaction of the ImOO(t):NaNN pair can be estimated as
three H-bonds and ꢀ2 of secondary interactions, which was
thermally similar to those of four H-bonds and ꢀ6 of secondary
interactions. The attached ꢀDG0 data also support these
considerations. Thus, the ImNN:NaOO pair by giving rise to
a favorable contribution to the enthalpy of formation was
most thermodynamically stable, and the remaining pairs
showed similar thermodynamic parameters (see the ESIw).
The synthesis of the new 1,8-naphthyridine nucleosides NaNN
(5) and NaOO (10) and their incorporation into ODNs have
been accomplished. Comparison of the base-pairing properties
of a series of Im:Na pairs revealed that the ImNN:NaOO pair
possessing a DAAD:ADDA H-bonding pattern was most
thermally and thermodynamically stable. Our results showed
the importance of the secondary interaction, depending on the
arrangement of H-bonds, together with the number of H-bonds
and the stacking effect of the expanded aromatic surfaces in the
duplex stability.17 Application of this markedly stable
base-pairing motif toward developing alternative stable base
pairs other than the Watson–Crick base pairs is underway.
Fig. 2 Consideration of thermal and thermodynamic stability of a series
of Im:Na base-pairing motifs. The dotted line represented repulsive
secondary interaction and the bold line represented attractive secondary
interaction. The index represented the sum of both secondary interactions.
The listed DTm and ꢀDG0 values were obtained from a series of ODN
I:ODN II.
complementarity of H-bonds is one of the important factors
for duplex stability. However, the thermal stability of these
Im:Na base pairs was different. As described in the introduction,
Jorgensen and Pranata suggested the importance of secondary
interaction for the stability of the hydrogen bonded complexes.11
The validity of their consideration is well demonstrated and
evaluated in many complexes possessing a variety of H-bonding
patterns.13–16 Our results can also be understood in view of their
hypothesis. Thus, the ImON:NaNO pair has a DADA:ADAD
H-bonding pattern (Fig. 2A). In this pair, six repulsive
secondary interactions (represented by dotted lines) arising
from D–D and A–A repulsion have to be considered together
with four primary H-bonds. Accordingly, the overall strength
of interaction of the ImON:NaNO pair can be estimated as
four primary H-bonds and six repulsive secondary interactions
(ꢀ6) represented as ‘‘index’’. The ImNO:NaON pair possessing
an ADAD:DADA H-bonding pattern is expected to have the
same overall strength of interaction (Fig. 2B). This estimation
agrees well with the calculated Tm values of the ImON:NaNO
and ImNO:NaON pairs (+7.8 1C vs. +7.5 1C). Since the
ImNN:NaOO pair has a DAAD:ADDA H-bonding pattern
(Fig. 2D), this pair is expected to have four repulsive and two
attractive secondary interactions (represented by bold lines)
together with four primary H-bonds. Accordingly, the overall
strength of interactions of the ImNN:NaOO pair can be
estimated as four primary H-bonds and –2 of secondary
interactions, which was, in fact, thermally more stable than
the ImON:NaNO and ImNO:NaON pairs (+11.4 1C vs. +7.8
and +7.5 1C). For the ImOO:NaNN pair, if this pair has an
ADDA:DAAD H-bonding pattern as illustrated in Fig. 1C,
the overall strength of interaction of this pair can be estimated
as four H-bonds and ꢀ2 of secondary interactions, which
Notes and references
1 S. A. Benner and A. M. Sismour, Nat. Rev. Genet., 2005, 6, 533.
2 I. Hirao, Curr. Opin. Chem. Biol., 2006, 10, 622.
3 A. T. Krueger and E. T. Kool, Chem. Biol., 2009, 16, 242.
4 N. Minakawa, N. Kojima, S. Hikishima, T. Sasaki, A. Kiyosue,
N. Atsumi, Y. Ueno and A. Matsuda, J. Am. Chem. Soc., 2003,
125, 9970.
5 S. Hikishima, N. Minakawa, K. Kuramoto, Y. Fujisawa,
M. Ogawa and A. Matsuda, Angew. Chem., Int. Ed., 2005, 44, 596.
6 Y. Hirama, H. Abe, N. Minakawa and A. Matsuda, Tetrahedron,
2010, 66, 8402.
7 Y. Hirama, N. Minakawa and A. Matsuda, Bioorg. Med. Chem.,
2011, 19, 352.
8 S. Hikishima, N. Minakawa, K. Kuramoto, S. Ogata and
A. Matsuda, ChemBioChem, 2006, 7, 1970.
9 N. Minakawa, S. Ogata, M. Takahashi and A. Matusda, J. Am.
Chem. Soc., 2009, 131, 1644.
10 S. Ogata, M. Takahashi, N. Minakawa and A. Matsuda, Nucleic
Acids Res., 2009, 37, 5602.
11 W. L. Jorgensen and J. Pranata, J. Am. Chem. Soc., 1990,
112, 2008.
12 The Tm values of duplexes containing ImON:NaNO and ImNO:NaON
pairs have been already reported in our previous paper. However, all
Tms were measured again for accurate comparison.
13 T. J. Murray and S. C. Zimmerman, J. Am. Chem. Soc., 1992,
114, 4010.
14 H. C. Ong and S. C. Zimmerman, Org. Lett., 2006, 8, 1589.
15 F. H. Beijer, R. P. Sijbesma, H. Kooijman, A. L. Spek and
E. W. Meijer, J. Am. Chem. Soc., 1998, 120, 6761.
16 S. Djurdjevic, D. A. Leigh, H. McNab, S. Parsons, G. Teobaldi
and F. Zerbetto, J. Am. Chem. Soc., 2007, 129, 476.
17 The stacking abilities of a series of naphthyridine bases were
higher than those of purine bases and similar to those of the
imidazopyridopyrimidine bases (see the ESIw).
c
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This journal is The Royal Society of Chemistry 2011