Homo-N-oligonucleotides (N1/N9-C1′ methylene bridge oligonucleotides): Nucleic acids with left-handed helicity

Article abstract of DOI:10.1021/ja049865t

Oligonucleotides containing a methylene bridge between N1 or N9 of the heterocyclic base and C1′ of the pentofuranosyl ring (homo-N- oligonucleotides) were synthesized. Melting curves revealed that such homo-type oligomers could cross-pair with complement

Full text of DOI:10.1021/ja049865t

Published on Web 05/28/2004
Homo-N-oligonucleotides (N1/N9-C1′ Methylene Bridge
Oligonucleotides): Nucleic Acids with Left-Handed Helicity
Kouichi Ishiyama,* Gerald E. Smyth, Toshihiro Ueda, Yutaka Masutomi,
Tadaaki Ohgi, and Junichi Yano
Contribution from the DiscoVery Research Laboratories, Nippon Shinyaku Co. Ltd,
3-14-1 Sakura, Tsukuba City, Ibaraki 305-0003, Japan
Received January 9, 2004; E-mail: k.ishiyama@po.nippon-shinyaku.co.jp
Abstract: Oligonucleotides containing a methylene bridge between N1 or N9 of the heterocyclic base and
C1′ of the pentofuranosyl ring (homo-N-oligonucleotides) were synthesized. Melting curves revealed that
such homo-type oligomers could cross-pair with complementary homo-type or natural oligomers. Circular-
dichroic studies provide evidence that the homo-type dimers have a left-handed stacked conformation and
further suggest that single-stranded and double-stranded homo-type oligomers adopt a left-handed
conformation, while duplexes with natural oligomers or nucleic acids form RNA-like right-handed helices.
NMR spectroscopy (NOESY) provides supporting evidence for a left-handed stacked conformation of the
homo-type dimer, while atomic force microscopy indicates a left-handed helical conformation of homo-
type dsDNA. Homo-type dimers and oligomers showed high resistance to digestion by snake-venom and
calf-spleen phosphodiesterases and nuclease S1.
Introduction
when the glycosyl torsion angles are restricted to a high anti or
a low anti conformation, the helicity of the oligonucleotide can
There has been substantial interest in the synthesis and
applications of altered nucleic acids. We have investigated the
properties of various modified nucleic acids for potential
application to the development of therapeutic agents and
research reagents. In such applications, it is important for the
modified oligonucleotide to be resistant to nucleases yet to retain
the ability to form duplexes with complementary natural nucleic
acids. Many nucleic acid analogues have been synthesized with
modifications to the base, sugar, and phosphate regions.1-4 To
our knowledge, however, no oligonucleotide analogues have
been synthesized with an increase in the distance between the
sugar and the base or in the flexibility of their linkage. Work
in our laboratory on the development of altered nucleic acids
has led to an interest in the effects of such modifications on the
biochemical and physicochemical properties of nucleic acids
and especially on their helical structure and handedness. In this
latter regard, there has been discussion as to precisely why
natural nucleic acids adopt a right-handed helical structure.
Among the factors that are considered to determine the helical
sense of nucleic acids is the chirality of the sugar, and it is
known that nucleic acids with L-deoxyribose instead of the
natural D-deoxyribose show left-handed helicity.5,6 Another
factor is the conformation about the glycosyl bond. For example,
be reversed.7-9
We wondered what would be the effect on nucleic acid
helicity of increasing the conformational flexibility in the region
of the glycosyl bond. In one type of structure, a methylene group
is inserted between N1 or N9 of the heterocyclic base and C1′
of the pentofuranosyl ring, a modification that is expected to
introduce an extra degree of conformational flexibility compared
to natural nucleic acids. The corresponding homo-N-nucleosides
have previously been synthesized,10-14 but the biochemical and
physicochemical properties of the dimers or oligomers have not
yet been reported.
In this paper we describe the manual synthesis of homo-N-
nucleotide dimers and the automated synthesis of oligomers.
We present the physicochemical properties of homo-type dimers
and oligomers and propose a conformational model to explain
their properties. CD and NMR evidence is presented that the
dimers or oligomers have an inherent left-handed twist and that
they form left-handed duplexes with complementary homo-type
(7) Urata, H.; Miyagoshi, H.; Kumashiro, T.; Mori, K.; Shoji, K.; Akagi, M.
J. Am. Chem. Soc. 2001, 123, 4845-4846.
(8) Uesugi, S.; Tezuka, T.; Ikehara, M. J. Am. Chem. Soc. 1976, 98, 969-
973.
(9) Uesugi, S.; Yano, J.; Yano, E.; Ikehara, M. J. Am. Chem. Soc. 1977, 99,
2313-2323.
(1) Eschenmoser, A.; Loewenthal, E. Chem. Soc. ReV. 1992, 21, 1-16.
(2) De Mesmaeker, A.; Altmann, K.-H.; Waldner, A.; Wendeborn, S. Curr.
Opin. Struct. Biol. 1995, 5, 343-355.
(10) Montgomery, J. A.; Hewson, K. J. Heterocycl. Chem. 1970, 7, 443-445.
(11) Hossain, N.; Blaton, N.; Peeters, O.; Rozenski, J.; Herdewijn, P. Tetrahedron
1996, 52, 5563-5578.
(3) Matteucci, M. Perspect. Drug DiscoVery Des. 1996, 4, 1-16.
(4) Scho¨ning, K.-U.; Scholz, P.; Guntha, S.; Wu, X.; Krishnamurthy, R.;
Eschenmoser, A. Science 2000, 290, 1347-1351.
(12) Hossain, N.; Hendrix, C.; Lescrinier, E.; Van Aerschot, A.; Busson, R.;
De Clercq, E.; Herdewijn, P. Bioorg. Med. Chem. Lett. 1996, 6, 1465-
1468.
(5) Urata, H.; Shinohara, K.; Ogura, E.; Ueda, Y.; Akagi, M. J. Am. Chem.
Soc. 1991, 113, 8174-8175.
(13) Scremin, C. L.; Boal, J. H.; Wilk, A.; Phillips, L. R.; Beaucage, S. L. Bioorg.
Med. Chem. Lett. 1996, 6, 207-212.
(14) Boal, J. H.; Wilk, A.; Scremin, C. L.; Gray, G. N.; Phillips, L. R.; Beaucage,
S. L. J. Org. Chem. 1996, 61, 8617-8626.
(6) Hashimoto, Y.; Iwanami, N.; Fujimori, S.; Shudo, K. J. Am. Chem. Soc.
1993, 115, 9883-9887.
9
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J. AM. CHEM. SOC. 2004, 126, 7476-7485
10.1021/ja049865t CCC: $27.50 © 2004 American Chemical Society

Homo-N-oligonucleotides with a Left-Handed Twist
A R T I C L E S
Table 1. UV Spectral Characteristics of Monomers and Dimersa
Table 2. Melting Temperaturesa
compound
λmax
λmin
λmax/λmin
Tm (°C)
A
A
258.8
260.6
259.7
261.1
257.8
259.5
257.7
259.7
225.6
227.1
224.9
228.2
227.2
228.0
226.3
228.0
13.23
9.08
12.46
4.96
7.84
9.17
9.06
8.69
duplex
0.15 M NaCl
1 M NaCl
b
A2 + poly(U)
A2 + poly(U)
-
13.0
NTc
12.5
NT
19.5
6.3
-
dA
dA
A2
A2
dA2
dA2
-
dA2 + poly(U)
dA2 + poly(U)
dA2 + poly(dT)
dA2 + poly(dT)
dA16 + poly(dT)
dA15dA + poly(dT)
(dAdA)8 + poly(dT)
dT16 + poly(dA)
dT15dT + poly(dA)
(dTdT)8 + poly(dA)
dA16 + dT16
-
-
-
-
49.1
25.1
9.9
-
a The spectra were taken in 10 mM sodium phosphate, pH 7.0, containing
0.15 M NaCl. Italicized letters represent homo-type nucleotides.
-
46.4
NT
NT
43.9
39.7
65.8
62.3
56.5
44.0
50.0
46.7
37.8
27.7
-
-
-
oligomers but right-handed duplexes with natural nucleic acids.
The left-handed nature of the homo-type duplex was confirmed
by atomic force microscopy (AFM). As far as we know, this is
the first report of a D-sugar nucleic acid without restricted
glycosyl torsion angles that forms a left-handed helix under
physiological conditions.
57.1
-
dA15dA + dT15dT
dA50 + dT50
dA50 + dT50
80.4
67.1
-
(AU)8
(dAdT)8
-
(dAdT)7dAdT
-
dA25 + poly(U)
dA7dA11dA7 + poly(U)
dA25 + poly(U)
-
Results and Discussion
-
-
UV Spectra, Hyperchromicity, and Mixing Curves. The
spectra of the homo-type monomers and dimers were almost
identical to those of the corresponding natural compounds (Table
1). The homo-type compounds, however, displayed a small
increase in λmax (1.5-2.0 nm) and λmin (0.8-3.5 nm) relative
to the corresponding natural compounds.
a Tm values were derived from melting curves for nucleotide mixtures
recorded at 260 nm. Italicized letters represent homo-type nucleotides. The
spectra were taken in 10 mM sodium phosphate, pH 7.0, containing 0.15
or 1.0 M NaCl. b Not determined. c No transition observed.
meltinglike transition), whereas A2 and poly(U) displayed a
clear, cooperative hyperchromic effect characterized by a Tm
of 13.0 °C. The melting curve for dA2 and poly(U) under the
same conditions also showed no transition. In contrast, the curve
for dA2 and poly(dT) (data not shown) displayed a clear, coop-
erative hyperchromic effect with a Tm of 6.3 °C. (Poly(dT) alone
showed no meltinglike transition between 0 °C and 26 °C.) Tm
for dA2 and poly(dT) under these conditions was 19.5 °C.
Whereas at 0 °C and in 1 M NaCl the natural dimer dA2
formed a 1:2 complex with poly(dT), the corresponding homo-
type adenosine dimer dA2 formed a 1:1 complex. Therefore the
Tm values characterizing the interaction of the two adenosine
dimers with poly(dT) in the presence of 1 M NaCl cannot be
directly compared. In contrast to the melting curve of A2 and
poly(U), that of A2 and poly(U) provided no evidence for
complex formation. The dimer dA2 also showed no evidence
of interaction with poly(U) but did interact weakly with poly-
(dT). An attempt was made to compare the effect of NaCl on
Tm for both dA2‚poly(dT) and dA2‚poly(dT). The melting of
dA2‚poly(dT) was not measured at NaCl concentrations above
0.3 M to keep well below the concentration of 1 M where this
nucleotide mixture forms a triple-helical complex, and dA2‚poly-
(dT) melting could not be measured at NaCl concentrations
below 0.5 M because of the difficulty of measuring Tm values
close to 0 °C. There was, therefore, no common range of NaCl
concentrations over which Tm could be compared.
Each of the four dimers A2, A2, dA2, and dA2, upon
acidification, displayed an increase in absorbance at most
wavelengths below ∼300 nm (spectra not shown). (In this paper,
an italicized “A” or “T” represents the homo-type nucleoside.)
Each dimer also displayed a small decrease in λmax (∼0.8 nm)
accompanying the increase in absorbance. The hyperchromicity
values obtained were 8.15% for A2, 7.42% for A2, 10.26% for
dA2, and 6.05% for dA2. The hyperchromicity value for A2
(8.15%) obtained by the pH-shift method is less than the
published value of 12%15 obtained upon complete hydrolysis
of the phosphodiester linkage, suggesting that there are residual
stacking interactions between the bases even at low pH.
However, dA2 being resistant to enzymic degradation as
described below, the pH-shift method of estimating hyperchro-
micity was used as a basis for comparing the four adenosine
dimers. For both homo-type dimers, the hyperchromicity was
less than that of the corresponding natural dimer: for A2, it was
∼9% less, and for dA2, it was ∼41% less.
The mixing curve for dA2 and poly(dT) at 260 nm (not
shown) in the presence of 1 M NaCl revealed a break at dA2/
poly(dT) ≈ 1:2, indicating the formation of a dA/2dT complex
(the poly(dA)/2poly(dT) triple-helical complex forms in 1.0 M
NaCl15). In contrast, the corresponding mixing curve for dA2
and poly(dT) showed a break at dA2/poly(dT) ≈ 1:1, indicating
the formation of a 1:1 complex.
Melting Curves. Melting curves were taken for various
nucleotide mixtures or self-complementary oligomers to deter-
mine the melting (transition) temperature, Tm, which can be
taken as a measure of the strength of base-pairing (Table 2).
The melting curve for A2 and poly(U) in the presence of 1.0 M
NaCl and 10 mM sodium phosphate, pH 7.0, was indistinguish-
able from that of poly(U) alone (which showed a weak
Each of the three 16-mers dA16, (dAdA)8, and dA15dA, when
mixed with poly(dT) in the presence of 0.15 M NaCl, displayed
sharp melting transitions (T90 - T10, ∼6 °C) and hyperchro-
micity values greater than 35% (see Table 2 for Tm values).
The melting curve for the self-complementary (dAdT)7dAdT
recorded at 0.15 M NaCl yielded a Tm of 50 °C and a broad
melting range (T90 - T10, 22 °C), while the corresponding
natural 16-mer yielded a lower Tm of 44 °C and an even broader
melting range (T90 - T10, 44 °C; see Table 2). Tm for
(15) Nucleic Acids; Ikehara, M., Ed.; Asakura Press: Tokyo, 1979; pp 243-
307.
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A R T I C L E S
Ishiyama et al.
Figure 1. CD spectra of dA2 (A) and dA2 (B) at 0 °C (solid line), 18 °C (dashed line), 40 °C (dotted line), and 60 °C (dashed and dotted line) and of A2
(C) and A2 (D) at 0 °C (solid line), 20 °C (dashed line), 40 °C (dotted line), and 60 °C (dashed and dotted line). The base concentration was 0.1 mM.
(dAdT)7dAdT increased from ∼43 °C in the absence of NaCl
to ∼52 °C in 2.0 M NaCl (data not shown). The melting curves
for complexes formed between poly(U) and a series of a 25-
mers containing strings of natural residues of decreasing length
sandwiched by homo-type residues yielded a monotonically
decreasing series of Tm values from 47.8 °C for dAdA23dA‚
poly(U) to 27.7 °C for the all-homo dA25‚poly(U).
When compared with the melting curve for (dAdT)8, the
corresponding curve for (dAdT)7dAdT displayed a Tm value 6
°C higher (Table 2), as well as a narrower melting range and a
higher hyperchromicity. The melting curves for the nonself-
complementary complexes dA15dA‚dT15dT and dA16‚dT16 (Table
2) displayed the opposite trend: Tm for the homo-type complex
was about 4 °C lower than that for the natural complex. These
nonself-complementary complexes melted over a narrower range
(about 13.5 °C) than did either the homo-type or natural self-
complementary complexes (22.0 °C and 43.5 °C, respectively).
The broader melting ranges of the self-complementary com-
plexes may reflect the formation of intramolecular hairpin-type
complexes (or a mixture of intramolecular and intermolecular
complexes) and are consistent with a lower degree of cooper-
ativity.
point at ∼230 nm, and a positive maximum at ∼218.7 nm (θobs
,
43.6 mdeg), as well as dichroic points at 260, 238, and 213
nm. In complete contrast, the spectrum of dA2 (Figure 1B) is
close to a mirror image about the θ ) 0 axis of that of the
natural dimer, with a negative maximum at 271.5 nm (θobs
,
-19.4 mdeg), a positive maximum at 252.4 nm (θobs, 16.7
mdeg), a negative inflection point at ∼230 nm, and a negative
maximum at 219.9 nm (θobs, -20.9 mdeg), though the relative
intensities of the bands differ. The spectra taken at different
temperatures also display good isodichroic points at 261 and
214.5 nm, as well as an “isodichroic region” at 234-239 nm.
For both dimers, the intensity of the bands decreased as the
temperature was raised from 0 °C to 60 °C. For dA2, the
decrease was approximately linear and amounted to 42-45%,
depending on the band; but for dA2, the decrease is better
described by an exponential function and amounted to as much
as 75-90%.
The spectra of A2 (Figure 1C) are similar to previously
reported spectra,16 with a positive maximum at 271.3 nm (θobs
,
43.2 mdeg), a negative maximum at ∼250 nm, a positive
inflection point at ∼230 nm, and a negative maximum at 252.3
nm (θobs, -42.8 mdeg), as well as isodichroic regions at 260-
261.5, 228.5-237, and 215-216 nm. In contrast, the spectrum
Circular Dichroism. The circular dichroic (CD) spectrum
of dA2 (Figure 1A) is similar to previously reported spectra,16
with a positive maximum at 269.8 nm (θobs, 20.7 mdeg; this
and subsequent values are reported for 0 °C), a negative
maximum at 250.3 nm (θobs, -23.6 mdeg), a positive inflection
of A2 (Figure 1D) has an inverted CD band at 220.2 nm (θobs
,
-26.9 mdeg), a low-intensity inverted band at 251.3 nm (θobs
,
3.1 mdeg), and an isodichroic point at 213.5 nm. The remainder
of the spectrum consists of one or two low-intensity bands that
are difficult to relate to the 270-nm band of A2. For both
(16) Kang, H.; Chou, P.-J.; Johnson, W. C., Jr.; Weller, D.; Huang, S.-B.;
Summerton, J. E. Biopolymers 1992, 32, 1351-1363.
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Homo-N-oligonucleotides with a Left-Handed Twist
A R T I C L E S
Figure 2. (A) CD spectra for dA15dA (solid line), dA16 (dashed line), and poly(A) (dotted line) at 10 °C and a base concentration of 0.05 mM. (B) Spectra
for dA15dA‚poly(dT) (solid line), dA16‚poly(dT) (dashed line), and (dAdA)8‚poly(dT) (dotted line), at 10 °C (natural oligomer) or 0 °C and a base concentration
of 0.05 mM. (C) Spectra for (dAdT)7dAdT (solid line), (dAdT)8 (dashed line), and (AU)12 (dotted line) at 10 °C and a base concentration of 0.1 mM. (D)
Spectra for dA15dA‚dT15dT (solid line), poly(A)‚dT16 (dashed line), and dA16‚dT16 (dotted line) at 10 °C and a base concentration of 0.05 mM in each
species.
compounds, the intensity of the major bands decreased nonlin-
early as the temperature was raised from 0 °C to 60 °C.
and (dAdT)8 are very different, and in fact the spectrum of
(dAdT)7dAdT resembles a mirror image about the θ ) 0 axis
of that of the RNA-type self-complementary oligomer (AU)12
(Figure 2C). The spectra of dA15dA‚dT15dT resemble a mirror
image about the θ ) 0 axis of that of poly(A)‚dT16 (Figure
2D).
The CD spectrum for dA2, being an approximate reflection
about the θ ) 0 axis of the spectrum for dA2, can be interpreted
as evidence for a left-handed stacked conformation, in contrast
to the right-handed stacked conformation of dA2. The good
isodichroic points observed in the dA2 spectra taken at various
temperatures are consistent with a two-state model of the
stacking-unstacking transition.16 The spectrum for A2, being
clearly inverted in only one CD band, does not provide strong
evidence for a left-handed stacked conformation. A left-handed
stacked conformation of nucleotide dimers has previously been
reported for dGdA at low pH17 and for the conformationally
restricted A°pA°, a dinucleoside monophosphate of O-cycload-
enosine.18 The apparently exponential decrease in the intensity
of the CD bands of dA2 as the temperature was increased
suggests that its stacking conformation is less stable than that
of dA2.
The similarity of the CD spectra for the natural, alternating,
and homo-type 16-mer complexes with poly(dT) (Figure 2B)
provides evidence that (dAdA)8‚poly(dT) and dA15dA‚poly(dT)
adopt a right-handed helical conformation similar to that of
dA16‚poly(dT). Deviation spectra (not shown) suggest that the
spectrum for d(AA)8‚poly(dT) is close to the average of the
spectra for dA16‚poly(dT) and dA15dA‚poly(dT). The CD
spectrum of (dAdT)7dAdT is drastically different from that of
(dAdT)8 (Figure 2C). Though the two spectra are not precisely
reflected about the θ ) 0 axis, the opposite sign of the major
bands may reflect opposite helical twist (right-handed for the
natural oligomer and left-handed for the homo-type). Our results
suggest a closer structural resemblance, though with opposite
helical twist, between the homo-type self-complementary oli-
godeoxyribonucleotide and RNA-type oligomers (Figure 2C).
The spectra for nonself-complementary oligomer duplexes show
a corresponding similarity between homo-type oligodeoxyri-
bonucleotides and RNA/DNA hybrids, so that homo-type
duplexes may resemble a left-handed version of A-type dsRNA
or RNA/DNA hybrids.
The spectrum of dA15dA is similar to that of dA2 and strikingly
resembles a mirror image about the θ ) 0 axis of that of natural
poly(A) (Figure 2A). The spectra for d(AA)8‚poly(dT) and dA15-
dA‚poly(dT) display patterns similar to that of dA16‚poly(dT)
(Figure 2B). The spectra of the self-complementary (dAdT)7dAdT
(17) Dolinnaya, N. G.; Fresco, J. R. Proc. Natl. Acad. Sci. U.S.A. 1992, 89,
9242-9246.
(18) Ikehara, M.; Uesugi, S.; Yano, J. Nature (London), New Biol. 1972, 240,
16-17.
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A R T I C L E S
Ishiyama et al.
Figure 3. NMR (NOESY) analysis of dA2. (A) Contour plot of part of the NOESY spectrum. (B) Schematic of the dimer showing the observed interresidue
and intraresidue proton proximity relationships.
Figure 5. Atomic force microscopy of natural and homo-type 50-mers.
(A) AFM image of dA50‚dT50, (B) AFM image of dA50‚dT50, (C) sectional
image of dA50‚dT50, and (D) sectional image of dA50‚dT50.
Figure 4. Tentative model of the conformation of dA2 derived from NOESY
measurements. Side view showing the interresidue proximity relationships
of the H-8, H-1′, and H-4′ protons.
dsDNA. The AFM images obtained revealed structures of
opposite helicity (Figure 5A, B). A sectional image for natural
dsDNA (Figure 5C) shows the expected shift of the points of
maximal height to the right in the upper part, indicating a right-
handed helix. In contrast, the sectional image for homo-type
dsDNA shows a shift of the points of maximal height to the
left in the upper part (Figure 5D), indicating a left-handed helix.
These images provide further evidence for a left-handed helical
conformation of homo-type dsDNA.
Nuclease Digestion. Snake venom phosphodiesterase is an
exonuclease that cleaves DNA or RNA nonprocessively in the
3′f 5′ direction to produce 5′-mononucleotides.24 Venom
phosphodiesterase readily digested dA2 to yield 5′-dAMP and
dA. By contrast, the enzyme did not detectably digest dA2
(chromatograms not shown). Similarly, dA16 was largely
digested by an hour’s treatment with 0.36 U of the enzyme,
whereas dA15dA remained essentially intact under the same
conditions, except for the cleavage of the single natural residue.
Calf-spleen phosphodiesterase is an exonuclease that hy-
drolyses single-stranded DNA or RNA in the 5′f3′ direction
NOESY Spectra. To further investigate the base-stacking
conformation of dA2, we carried out a NOESY NMR study
under the conditions used in the CD experiments. An NMR
spectrum of dA2 was acquired at 500 MHz. NOESY intraresidue
cross-peaks included two between the H-8 proton of Ap (the
3′-nucleotidyl unit) and the H-6′ and H-6′′ protons of Ap and
two between the H-8 proton of pA (the 5′-nucleotidyl unit) and
the H-6′ and H-6′′ protons of pA. There were two strong
interresidue cross-peaks, one between the H-8 proton of pA and
the H-4′ proton of Ap and one between the H-8 proton of pA
and the H-1′ proton of Ap (Figure 3). Weak interresidue cross-
peaks were also observed between adenine base protons. Most
other cross-peaks observed were intraresidue cross-peaks of
pentofuranose. A hand-built model of the dimer constructed to
fit the observed proximity relationships displayed a left-handed
twist (Figure 4). The observed interresidue distances, which
would be expected to be very much greater in a right-handed
model of the dimer, corroborate the CD evidence for left-
handedness.
(19) Mou, J.; Czajkowsky, D. M.; Zhang, Y.; Shao, Z. FEBS Lett. 1995, 371,
279-282.
(20) Tanigawa, M.; Okada, T. Anal. Chim. Acta 1998, 365, 19-25.
(21) Mu¨ller, D. J.; Fotiadis, D.; Engel, A. FEBS Lett. 1998, 430, 105-111.
(22) Oesterhelt, F.; Oesterhelt, D.; Pfeiffer, M.; Engel, A.; Gaub, H. E.; Mu¨ller,
D. J. Science 2000, 288, 143-146.
Atomic-Force Microscopy (AFM). AFM has recently been
applied to the investigation of biological macromolecules at the
subnanometer level.19-23 Because the technique is now suf-
ficiently powerful to distinguish between right-handed and left-
handed double-stranded DNA molecules,19 we applied it to a
comparison of natural (dA50‚dT50) and homo-type (dA50‚dT50)
(23) Tahirov, T. H.; Sato, K.; Ichikawa, E.; Sasaki, M.; Inoue, T.; Shiina, M.;
Kimura, K.; Takata, S.; Fujikawa, A.; Morii, H.; Kumasaka, T.; Yamamoto,
M.; Ishii, S.; Ogata, K. Cell 2002, 108, 57-70.
(24) Laskowski, M. In The Enzymes. Vol. 4: Venom Exonuclease, 3rd ed.; Boyer,
P. S., Ed.; Academic Press: New York, 1971; pp 313-328.
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Homo-N-oligonucleotides with a Left-Handed Twist
A R T I C L E S
to produce 3′-mononucleotides.25 Its specificity is thus comple-
mentary to that of venom phosphodiesterase. The dimer dA2
was completely digested after incubation for 1 h with 0.02 U
of the enzyme. In contrast, dA2 was only digested to the extent
of ∼3% under these conditions and even after an overnight
incubation with 0.02 U almost 90% of the original dimer
remained. The homo-type dimer was therefore digested at least
630 times more slowly than the natural dimer. Similarly, dA16
was 96% digested after overnight incubation with 0.02 U,
whereas dA15dA under these conditions was not digested by
more than 20%. In other words, oligomers containing homo-
type nucleotide residues were degraded several 100-fold more
slowly by spleen phosphodiesterase than were the corresponding
natural oligomers.
Nuclease S1 is a Zn2+-dependent glycoprotein that acts both
endonucleolytically and exonucleolytically on single-stranded
nucleic acids, releasing 5′-terminal monophosphate products.26
The 25-mers dAdA23dA, dA3dA19dA3, dA5dA15dA5, dA7dA11dA7,
dA9dA7dA9, and dA11dA3dA11 were all substantially digested by
1 U of the enzyme. The all-homo-type 25-mer dA25, in contrast,
showed not a trace of digestion by up to 10 U and, even at 100
U, showed only a trace of monomer. The 16-mer dA15dA was
virtually intact after overnight incubation in 66% fetal bovine
serum, conditions that resulted in the complete digestion of dA16.
While all-homo-type deoxyadenosine oligomers were digested
extremely slowly, if at all, by nuclease S1, oligomers containing
at least three natural residues in an otherwise all-homo-type
chain were susceptible to degradation.
salt conditions with no requirement for alternating pyrimidine-
purine sequences and that have an unrestricted glycosyl torsion
angle.
Experimental Section
General Methods. UV spectra, mixing curves, and melting curves
were recorded with a Hitachi U-3200 double-beam spectrophotometer.
Circular dichroic spectra were taken with a Jasco J-720 spectropola-
rimeter interfaced with an NEC PC-9801DX personal computer and
1
fitted with a Jasco temperature controller. H and 13C NMR spectra
were recorded on a Varian Gemini 2000, Varian Unity Plus 300, or
Bruker DRX 500 spectrometer, with tetramethylsilane or sodium 2,2-
dimethyl-2-silapentane sulfonate as the internal standard. FAB mass
spectra were recorded on a JEOL JMS-SX 102 mass spectrometer.
High-pressure liquid chromatography (HPLC) was done on a Shimadzu
system with an LC-6A liquid chromatograph coupled to an SPD-6A
UV spectrophotometric detector operated from an SCL-6A system
controller linked to a C-R3A data processor.
Thin-layer chromatography (TLC) was carried out on Merck silica
gel 60 F254 precoated plates (Merck, Whitehouse Station, New Jersey).
Analytical plates were 20 cm × 20 cm × 0.2 mm, and preparative
plates were 20 cm × 20 cm × 2 mm. Column chromatography was
performed on Wako silica gel C-300. We used published extinction
coefficients15 for adenosine and deoxyadenosine dimers and oligomers.
Materials. Phosphodiesterase from Crotalus adamanteus (oligo-
nucleate 5′- nucleotidohydrolase; EC 3.1.4.1) was purchased from Koch-
Light Ltd (now NBS Biologicals, Hatfield, Hertfordshire, U.K.), and
phosphodiesterase from calf spleen (oligonucleate 3′-nucleotidohydro-
lase; EC 3.1.16.1) and nuclease S1 (EC 3.1.30.1) from Aspergillus
oryzae were purchased from Boehringer (Mannheim, Germany). Sodium
polyuridylate (poly(U); A280/A260, 0.35; A250/A260, 0.78; S20,w, 6.0; 2.75
µmol/mg) was from Yamasa Corp. (Choshi, Chiba Prefecture, Japan).
Unmodified phosphoramidite monomers, DNA solid supports, and other
synthetic reagents were from Applied Biosystems (now PE Biosystems,
Foster City, California). A long chain alkylamine controlled pore glass
solid support (LCAA-CPG; 0.081 mmol amino groups/g) was from
CPG Inc. (Lincoln Park, New Jersey).
Synthesis of Homo-N-dinucleoside Monophosphate 4 (Scheme 1).
Preparation of compound 2: To a suspension of 1 (916 mg, 3.26 mmol)
in pyridine at 0 °C (15 mL) was added benzoyl chloride (2.75 g, 19.6
mmol). After 2 h, the reaction mixture was poured into chloroform
(100 mL) containing ice (115 g) and sodium bicarbonate (9.0 g). The
aqueous layer was extracted twice with chloroform, and the organic
layers were combined and concentrated. The residue was dissolved in
a mixture of ethanol (9.8 mL) and pyridine (6.5 mL), and the solution
was treated with a mixture of 2 N NaOH (13 mL) and ethanol (13
mL) at room temperature for 30 min. The reaction mixture was
neutralized with 2 N HCl (13 mL), concentrated, and applied to a
reverse-phase column (ODS, 50 × 80 mm2; Waters, Milford, Mas-
sachusetts), which was developed with a discontinuous methanol
gradient (solvent system, methanol-water; step size, 5%) over 1000
mL. Appropriate fractions were combined and evaporated under reduced
pressure to give 9-N-[(â-D-ribo-pentofuranosyl)methyl]-N6-benzoylad-
enine (1.2 g, 95% yield). To a solution of this compound (160 mg,
0.42 mmol) and imidazole (113 mg, 1.66 mmol) in N,N-dimethylfor-
mamide (DMF; 1.1 mL), tetraisopropyldisiloxane dichloride (153 mg,
0.49 mmol) was added. The reaction mixture was stirred at room
temperature for 3 h, poured into water, and extracted with dichlo-
romethane. The organic layer was washed with cool dilute HCl and
then with water and concentrated under reduced pressure. The residue
was purified by preparative TLC with dichloromethane-methanol (95:
5) as the solvent to give 9-N-[(3′,5′-O-(tetraisopropyldisiloxane-1,3-
diyl)-â-D-ribo-pentofuranosyl)methyl]-N6-benzoyladenine (182 mg, 70%
yield). This compound (150 mg, 0.24 mmol) was dissolved in dioxane,
and then 2,3-dihydrofuran (336 mg, 4.8 mmol) was added in the
presence of pyridinium p-toluene sulfonate (70 mg, 0.28 mmol) and
Conclusions
We have synthesized adenine (both ribo- and deoxyribo-types)
and thymine homo-N-nucleoside monomers and adenine homo-
N-nucleotide dimers and shown that homo-N-nucleotide oligo-
mers can be synthesized from the monomers by means of
standard phosphoramidite chemistry on an automated DNA
synthesizer. The glycosyl conformation of natural nucleic acids
is one of the important factors determining their helicity.7 In
homo-type nucleic acids, the greater distance between sugar and
base imposed by the inserted methylene group appears to confer
an extra degree of conformational flexibility compared to the
direct sugar-base link of natural nucleic acids. Such flexibility
may explain the ability of homo-type oligomers to adopt a left-
handed conformation in both the single-stranded and the double-
stranded state while allowing a right-handed conformation in
heteroduplexes with natural nucleic acids. A left-handed helical
conformation has been observed for nucleic acids or nucleic
acid analogues with alternating pyrimidine-purine sequences
(at high salt concentrations; Z-DNA), with L-deoxyribose instead
of D-deoxyribose as the sugar27 and with the glycosyl torsion
angle restricted to force a high anti conformation.8,9 The
modified nucleic acids described in the present paper, however,
are to our knowledge the first reported example of D-sugar
nucleic acids that form a left-handed double-helix under low
(25) Bernardi, A.; Bernardi, G. In The Enzymes. Vol. 4: Spleen Acid Exonuclease,
3rd ed.; Boyer, P. S., Ed.; Academic Press: New York, 1971; pp 329-
336.
(26) Fraser, M. J.; Low, R. L. In Nucleases: Fungal and Mitochondrial
Nucleases, 2nd ed.; Linn, S. M., Lloyd, R. S.; Roberts, R. J., Eds.; Cold
Spring Harbor Laboratory Press: New York, 1993; pp 171-207.
(27) Urata, H.; Ogura, E.; Shinohara, K.; Ueda, Y.; Akagi, M. Nucleic Acids
Res. 1992, 20, 3325-3332.
9
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A R T I C L E S
Ishiyama et al.
Scheme 1. Synthesis of Homo-N-diadenosine Monophosphatea
a Reagents and conditions: (a) (i) BzCl, pyridine, 0 °C, 2 h; (ii) 2 N NaOH, aq EtOH, THF, pyridine, rt, 30 min; (b) tetraisopropyldisiloxane dichloride,
imidazole, DMF, rt, 3 h; (c) 2,3-dihydrofuran, pyridinium p-toluenesulfonate, dioxane, rt, 20 h; (d) n-Bu4NF, THF, rt, 30 min; (e) DMTrCl, pyridine, rt, 2-5
h; (f) 2-chlorophenylphosphoditriazolidate, 1-methylimidazole, dioxane, rt, 2 h; (g) Ac2O, pyridine, rt, 1 h; (h) 80% AcOH, rt, 1 h; (i) MSNT, pyridine, rt,
1 h; (j) concentrated NH4OH, pyridine, rt, 2 days.
the solution was stirred at room temperature for 20 h. Pyridine (0.3
mL) and dichloromethane (10 mL) were added, and the mixture was
washed with saturated sodium bicarbonate and water, after which the
organic layer was dried with Na2SO4 and concentrated under reduced
pressure. The residue was then dried by coevaporation with pyridine
and toluene and treated with 0.5 M tetrabutylammonium fluoride
(TBAF) in tetrahydrofuran solution (1.5 mL) at room temperature for
30 min. The solvent was removed under reduced pressure, and the
residue was dissolved in 1.5 mL of pyridine-methanol (3:1, v/v). The
resulting solution was passed through a cation exchange column (Dowex
50w × 2; 3 mL), washed with 10 mL of pyridine-methanol (3:1, v/v),
and concentrated under reduced pressure. The residue was then purified
by TLC with dichloromethane-methanol (94:6) as the solvent to give
9-N-[(2′-O-(tetrahydrofuranyl)-â-D-ribo-pentofuranosyl)methyl]-N6-ben-
zoyladenine (101 mg, 92% yield). This compound (100 mg, 0.22 mmol)
was coevaporated with pyridine and dissolved in the same solvent (0.7
mL). To this solution, 4,4′-dimethoxytrityl chloride (DMTrCl; 89 mg,
0.26 mmol) was added. The reaction mixture was stirred at room
temperature for 5 h, treated with methanol (5 drops) for 5 min at room
temperature, and evaporated to dryness under reduced pressure. The
residue was then dissolved in dichloromethane (10 mL), washed with
water, and concentrated. The residue was purified by TLC with
dichloromethane-methanol (95:5) containing 0.1% pyridine as the
solvent to give 9-N-[(5′-O-(4,4′-dimethoxytrityl)-2′-O-(tetrahydrofura-
nyl)-â-D-ribo-pentofuranosyl)methyl]-N6-benzoyladenine (107 mg, 64%
yield). To 107 mg (0.14 mmol) of this compound, a solution of
2-chlorophenylphosphoditriazolidate in anhydrous dioxane (0.65 mL;
0.28 M stock-solution) and 1-methylimidazole (58 mg, 0.72 mmol)
was added. After 2 h of stirring, 50% pyridine-water (1.0 mL) was
added. The reaction mixture was then diluted with dichloromethane
(10 mL) and washed with 0.1 M triethylammonium bicarbonate (pH
7, 10 mL). The organic layer was concentrated to give 2 (140 mg),
which was used without further purification for the next step.
DMTrCl (172 mg, 0.51 mmol) was added. The reaction mixture was
stirred at room temperature for 2 h, treated with methanol (5 drops)
for 5 min at room temperature, and evaporated under reduced pressure.
The residue was then dissolved in dichloromethane (10 mL), washed
with water, and concentrated. The residue was purified by TLC with
dichloromethane-methanol (93:7) containing 0.1% pyridine as the
solvent, to give 9-N-[(5′-O-(4,4′-dimethoxytrityl)-â-D-ribo-pentofura-
nosyl)methyl]-N6-benzoyladenine (202 mg, 74% yield). This compound
(200 mg, 0.29 mmol) was dissolved in pyridine (1.0 mL), and after
the addition of acetic anhydride (0.5 mL), the reaction mixture was
stirred at room temperature for 1 h. The solvent was evaporated under
reduced pressure, the residue was treated with 80% acetic acid (4.5
mL) at room-temperature for 1 h, methanol (5 drops) was added, and
the solvent again was evaporated under reduced pressure. The residue
was purified by TLC with dichloromethane-methanol (93:7) to give
9-N-[(2′,3′-O-diacetyl-â-D-ribo-pentofuranosyl)methyl]-N6-benzoylad-
enine (3; 136 mg, 65% yield).
Preparation of compound 4: To a solution of 2 (135 mg, 0.14 mmol)
and 3 (47 mg, 0.1 mmol) in pyridine (1.0 mL) 1-(mesitylene-2-
sulfonyl)-3-nitro-1,2,4-triazole (MSNT; 83 mg, 0.28 mmol) was added,
and the reaction mixture was stirred at room temperature for 1 h.
Pyridine-water (50%; 1.0 mL) was added, the reaction mixture was
diluted with dichloromethane (5 mL) and washed with water (5 mL),
and the organic layer was dried with MgSO4 and concentrated under
reduced pressure. The residue was purified by TLC with dichlo-
romethane-methanol (95:5) to give a fully protected dimer (131 mg,
94% yield). The protected dimer (131 mg, 0.094 mmol) was dissolved
in pyridine (10 mL) and concentrated ammonium hydroxide (10 mL)
and deprotection carried out at room temperature for 2 days. The solvent
was then removed under reduced pressure, and the residue was
chromatographed on a reverse-phase column (ODS, 10 × 100 mm2;
Waters) with a linear gradient from 0 to 40% acetonitrile in 0.05 M
triethylammonium acetate, pH 7.0, over 400 mL. The main UV-
absorbing fractions were combined and concentrated to dryness under
reduced pressure. The purified DMTr-dimer was treated with 80% acetic
acid at room temperature for 2 h, and after removal of the acid under
Preparation of compound 3: 9-N-[(â-D-ribo-pentofuranosyl)methyl]-
N6-benzoyladenine (150 mg, 0.39 mmol) was coevaporated with
pyridine and dissolved in the same solvent (1.2 mL). To this solution
9
7482 J. AM. CHEM. SOC. VOL. 126, NO. 24, 2004

Homo-N-oligonucleotides with a Left-Handed Twist
A R T I C L E S
Scheme 2. Synthesis of Homo-N-deoxydiadenosine Monophosphatea
a Reagents and conditions: (a) (i) BzCl, pyridine, 0 °C, 1 h; (ii) 2 N NaOH, aq EtOH, THF, pyridine, rt, 30 min; (b) DMTrCl, pyridine, rt, 4 h; (c)
2-chlorophenylphosphoditriazolidate, dioxane, rt, 2 h; (d) tert-butyldimethylchlorosilane, imidazole, DMF, rt, 4 h; (e) 80% AcOH, rt, 2 h; (f) MSNT, pyridine,
rt, 2 h; (g) n-Bu4NF, THF, rt, 30 min; (h) concentrated NH4OH, pyridine, rt, 2 days.
reduced pressure, the residue was dissolved in water (10 mL) and
washed with ethyl acetate (2 × 10 mL). The aqueous solution was
concentrated to give the dimer product, which was further purified by
reverse-phase chromatography under the same conditions and then by
anion-exchange chromatography on a Dowex column (Dowex 1 × 2,
Cl- form; 10 × 50 mm2) with a linear gradient from 0 to 1 M NaCl
over 200 mL. The main UV-absorbing fractions were combined,
desalted on an ODS column, and freeze-dried to give 4 (36 mg, 59%
Preparation of compound 8: To a solution of 6 (125 mg, 0.13 mmol)
and 7 (48 mg, 0.1 mmol) in pyridine (1.0 mL) MSNT (77 mg, 0.26
mmol) was added, and the reaction mixture was stirred at room
temperature for 2 h. Pyridine-water (50%; 1.0 mL) was added, the
reaction mixture was diluted with dichloromethane (5 mL) and washed
with water (5 mL), and the organic layer was dried with MgSO4 and
concentrated under reduced pressure. The residue was purified by TLC
with dichloromethane-methanol (94:6) to give a fully protected dimer
(109 mg, 83% yield). The protected dimer (75 mg, 0.056 mmol) was
treated with 0.2 M TBAF in tetrahydrofuran solution (1 mL) at room
temperature for 30 min, methanol (0.5 mL) was added, and the solvent
was evaporated to dryness under reduced pressure. The residue was
dissolved in methanol (2.0 mL) and concentrated ammonium hydroxide
(20 mL), and deprotection was allowed to proceed at room temperature
for 2 days, after which the solvent was removed under reduced pressure.
This residue was chromatographed on a reverse-phase column (ODS,
10 × 100 mm2; Waters) with a linear gradient from 0 to 35% acetonitrile
in 0.1 M triethylammonium acetate, pH 7.0, over 400 mL. The main
UV-absorbing fractions were combined and concentrated to dryness
under reduced pressure. The purified DMTr-dimer was deprotected with
80% acetic acid at room temperature for 30 min, and after removal of
the acid under reduced pressure, the residue was dissolved in water
(10 mL) and washed with ethyl acetate (2 × 10 mL). The aqueous
solution was concentrated to give the dimer product. The product was
further purified by reverse-phase chromatography under the same
conditions and then by anion-exchange chromatography on a Dowex
column (Dowex 1 × 2, Cl- form; 10 × 50 mm2) with a linear gradient
from 0 to 0.5 M NaCl over 200 mL. The main UV-absorbing fractions
were combined, desalted on an ODS column, and freeze-dried to give
1
yield). H NMR (200 MHz, D2O): δ 3.60-3.80 (m, 4H), 3.80-4.80
(m, 12H), 8.06 (bs, 2H), 8.14 (bs, 2H). FAB-MS: m/z 623 (M-H)-.
Anal. Calcd for C22H28N10PNa2‚2.5H2O: C, 36.98; H, 4.66; N, 19.60.
Found: C, 37.19; H, 4.72; N, 19.21.
Synthesis of Homo-N-deoxydinucleoside Monophosphate 8 (Scheme
2). Preparation of compound 6: Compound 6 was prepared from 5
(133 mg, 0.50 mmol) essentially as described for 2 and used without
1
further purification for the next step. H NMR (300 MHz, CDCl3): δ
1.88 (m, 1H, H-2′), 2.41 (m, 1H, H-2′′), 3.15 (m, 2H, H-5′ and H-5′′),
3.78 (s, 6H, -OMe), 4.28 (m, 2H, H-4′ and H-6′), 4.56 (m, 2H, H-3′
and H-6′′), 5.00 (m, 1H, H-1′), 6.80 (m, 4H), 6.93 (m, 1H), 7.22 (m,
8H), 7.65 (m, 5H), 8.05 (m, 2H), 8.10 (s, 1H, H-2), 8.83 (s, 1H, H-8).
Preparation of compound 7: To a solution of 9-N-[5′-O-(4,4′-
dimethoxytrityl)-(2′-deoxy-â-D-ribo-pentofuranosyl)methyl]-N6-benzoy-
ladenine (80 mg, 0.11 mmol; prepared from 5) and imidazole (41 mg,
0.55 mmol) in DMF (1.6 mL) tert-butyldimethylchlorosilane (54 mg,
0.33 mmol) was added, and the reaction mixture was stirred at room
temperature for 4 h. Methanol (2 drops) was added, and the reaction
mixture was concentrated and coevaporated with toluene. This material
was deprotected with 80% acetic acid (5.5 mL) at room temperature
for 2 h, and then methanol (2 drops) was added and the solvent was
evaporated under reduced pressure. The residue was coevaporated with
toluene and purified by TLC with dichloromethane-methanol (94:6)
1
8 (28 mg, 81% yield). H NMR (500 MHz, D2O): δ 1.84 (m, 2H,
H-2′ (Ap) and H-2′ (pA)), 2.05 (m, 1H, H-2′′ (pA)), 2.21 (m, 1H, H-2′′
(Ap)), 3.58 (m, 2H, H-5′ and H-5′′ (Ap)), 3.76 (m, 2H, H-5′ and H-5′′
(pA)), 4.01 (m, 1H, H-4′ (pA)), 4.06 (dd, J ) 14.69, 7.28 Hz, 1H, H-6′
(pA)), 4.13 (m, 1H, H-6′ (Ap)), 4.17 (bs, 1H, H-4′ (Ap)), 4.25 (bs, 1H,
H-6′′ (pA)), 4.28 (bs, 1H, H-3′ (pA)), 4.33 (bs, 1H, H-6′′ (Ap)), 4.39
(m, 1H, H-1′ (Ap)), 4.47 (m, 1H, H-1′ (pA)), 4.63 (bs, 1H, H-3′ (Ap)),
1
to give 7 (48 mg, 83% yield from DMTr derivative). H NMR (200
MHz, D2O): δ 0.06 (s, 6H), 0.86 (s, 9H), 1.86 (m, 1H, H-2′), 2.04 (m,
1H, H-2′′), 3.50 (dd, J ) 12, 4 Hz, 1H, H-5′), 3.66 (dd, J ) 12, 3 Hz,
1H, H-5′′), 3.78 (m, 1H, H-4′), 4.20-4.60 (m, 4H), 7.46-7.70 (m,
3H), 8.00-8.10 (m, 2H), 8.12 (s, 1H, H-2), 8.80 (s, 1H, H-8).
9
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A R T I C L E S
Ishiyama et al.
7.80 (s, 1H, H-2 (pA)), 7.82 (s, 1H, H-8 (pA)), 7.99 (s, 1H, H-8 (Ap)),
8.02 (s, 1H, H-2 (Ap)). 13C NMR (125 MHz, D2O): δ 35.80 (C-2′,
Ap), 36.13 (C-2′, pA), 46.93 (C-6′, pA), 47.07 (C-6′, Ap), 61.53 (C-5′,
Ap), 65.52 (C-5′, pA), 72.25 (C-3′, pA), 76.75 (C-1′, Ap and pA), 76.79
(C-3′, Ap), 85.10 (C-4′, pA), 85.80 (C-4′, Ap), 117.16 (C-5, pA), 117.24
(C-5, Ap), 142.07 (C-8, pA), 142.11 (C-8, Ap), 147.65 (C-4, pA), 147.71
(C-4, Ap), 151.22 (C-2, pA), 151.72 (C-2, Ap), 154.33 (C-6, pA), 154.64
(C-6, Ap). FAB-MS: m/z 591 (M-H)-. Anal. Calcd for C22H28N8-
PNa2‚4H2O: C, 37.27; H, 5.11; N, 19.74. Found: C, 36.94; H, 5.02;
N, 19.82.
Preparation of Homo-N-Nucleoside Phosphoramidite. 9-N-[5′-
O-(4,4′-Dimethoxytrityl)-2′-deoxy-3′-O-[(2-cyanoethoxy)-N,N-diisopro-
pylaminophosphino]-â-D-ribo-pentofuranosyl]methyl]-N6-benzoylade-
nine 9 and 1-N-[5′-O-(4,4′-dimethoxytrityl)-2′-deoxy-3′-O-[(2-cyano-
ethoxy)-N,N-diisopropylaminophosphino]-â-D-ribo-pento-
furanosyl]methyl]thymine 10 were prepared from the corresponding
homo-N-nucleosides as described by Boal et al.14
as described by Sinha et al.28 Fully protected oligonucleotides containing
a 5′-terminal DMTr group were deprotected in concentrated ammonium
hydroxide at 55 °C overnight. The crude oligomers were purified by
reverse-phase HPLC (ODS, 10 × 100 mm2; Waters) at a flow rate of
2 mL/min with a linear gradient from 0 to 35% acetonitrile in 0.1 M
triethylammonium acetate, pH 7.0, over 200 mL. The main UV-
absorbing fractions were combined and concentrated under reduced
pressure, and the terminal DMTr group was removed by treatment with
80% acetic acid at room temperature for 30 min. After removal of the
acid under reduced pressure, the residue was dissolved in water (10
mL) and washed with ethyl acetate (2 × 10 mL), and the aqueous
solution was concentrated to give the oligodeoxyribonucleotide product.
Hyperchromicity. The UV spectrum for each dimer (final concen-
tration, 0.05 mM) was recorded in the presence of 10 mM sodium
phosphate, pH 7.0, and 0.15 M NaCl in a total volume of 3 mL. Then
5 M HCl (60 µL) was added to the solution, and the spectrum was
rerecorded. The percentage increase in absorbance at 260 nm upon
acidification was calculated for each dimer, taking into account the
dilution factor of 1.02. We also took the spectrum of each of the
corresponding nucleosides A, A, dA, and dA at pH 7.0 and 1.0 to allow
for changes in the absorbance of the bases as a function of pH. Each
nucleoside displayed a decrease of ∼5% in A260 upon acidification (and
also a small decrease in λmax of ∼2.3 nm; data not shown). These
absorbance decreases were assumed to have canceled part of the
hyperchromic effect displayed by the dimers, so the absorbance increase
observed upon acidification was corrected by the appropriate factor to
obtain the hyperchromicity value.
Compound 9 1H NMR (300 MHz, CDCl3): δ 1.00-1.35 (m, 14H),
1.80 (m, 1H), 2.15 (m, 1H), 2.42 (t, J ) 6.3 Hz 1H), 2.59 (t, J ) 6.3
Hz, 1H), 3.05 (m, 2H), 3.60 (m, 2H), 3.78 (s, 6H), 4.10 (m, 1H), 4.30
(m, 1H), 4.40 (m, 1H), 4.53 (m, 1H), 4.60 (m, 1H), 6.80 (m, 4H), 7.25
(m, 7H), 7.60 (m, 3H), 8.03 (d, J ) 6.9 Hz, 2H), 8.11 (d, J ) 3.0 Hz,
1H), 8.82 (s, 1H), 9.04 (bs, 1H). FAB-MS: m/z 872 (M + H)+.
Compound 10 1H NMR (200 MHz, CDCl3): δ 1.00-1.30 (m, 14H),
1.74 (s, 3H), 1.80 (m, 1H), 2.10 (m, 2H), 2.42 (t, J ) 6 Hz, 1H), 2.58
(t, J ) 6 Hz, 1H), 3.12 (m, 2H), 3.60 (m, 2H), 3.78 (s, 6H), 4.10 (m,
2H), 4.15 (m, 1H), 4.40 (m, 2H), 6.80 (m, 4H), 7.08 (m, 1H), 7.25 (m,
7H), 7.40 (m, 2H), 7.90 (bs, 1H). FAB-MS: m/z 757 (M-H)-.
Preparation of Homo-N-Nucleoside-Derivatized CPG Support.
To a solution of 9-N-[(5′-O-(4,4′-dimethoxytrityl)-2′-deoxy-D-ribo-
pentofuranosyl)methyl]-N6-benzoyladenine (470 mg, 0.70 mmol) in
dichloromethane (3 mL) were added 4-(dimethylamino)pyridine (123
mg, 1.05 mmol) and succinic anhydride (105 mg, 1.05 mmol). This
mixture was stirred at room temperature for 3 h, dichloromethane was
added, and the reaction mixture was washed with 0.5 M KH2PO4 (2 ×
10 mL) and water (10 mL), after which the solvent was removed under
reduced pressure. The residue (509 mg) was dissolved in DMF (5 mL),
pentachlorophenol (190 mg, 0.71 mmol) and N,N′-dicyclohexylcarbo-
diimide (200 mg, 0.97 mmol) were added, and the reaction mixture
was stirred at room temperature for a further 20 h. The precipitate that
formed was removed by filtration and discarded, and the filtrate,
containing the product, was concentrated under reduced pressure. The
concentrated filtrate was dissolved in dichloromethane (10 mL) and
precipitated by adding the solution to n-hexane (150 mL). The
precipitate was collected by centrifugation for 5 min at 2000 rpm and
dried in a desiccator under reduced pressure to give the 3′-succinyl
Mixing Curves. Poly(dT) and dA2 or dA2 were mixed in various
proportions to maintain a constant base concentration. Each nucleotide
mixture contained, in a total volume of 3 mL, nucleotide dimer and/or
polymer (total base-concentration, 0.1 mM), 1 M sodium chloride, and
10 mM sodium phosphate, pH 7.0. The mixture was scanned at 0 °C
from 300 to 205 nm, and from each spectrum, A260 was determined
and plotted against the proportion of poly(dT).
Melting Curves. Melting curves (absorbance-temperature profiles)
were recorded in the presence of 10 mM sodium phosphate, pH 7.0,
containing various concentrations of NaCl. Each nucleotide species was
used at a base concentration of 0.05 mM to give a total base
concentration of 0.1 mM. The absorbance was monitored at 260 nm
as the temperature was increased from 0 °C at a rate of 0.5 °C/min.
Before each melting curve was recorded, the nucleotide mixture was
cooled through the melting transition to the starting temperature at a
rate of not more than 1 °C/min. Tm values were determined as the
temperature corresponding to half of the total absorbance increase, and
the sharpness of the melting transition was characterized by the
temperature range over which the absorbance went from 10 to 90% of
the total increase (T90 - T10).
1
ester (492 mg, 75% yield). H NMR (300 MHz, CDCl3): δ 1.98 (m,
1H, H-2′), 2.16 (m, 1H, H-2′′), 2.75 (t, J ) 6.3 Hz, 2H), 3.02 (t, J )
6.3 Hz, 2H), 3.15 (dd, J ) 10.2, 4.2 Hz, 1H, H-5′), 3.27 (dd, J ) 10.2,
3.6 Hz, 1H, H-5′′), 3.78 (s, 6H, -OMe), 4.08 (m, 1H, H-4′), 4.27 (dd,
J ) 14.1, 7.2 Hz, 1H, H-6′), 4.50 (m, 1H, H-1′), 4.63 (dd, J ) 14.1,
2.4 Hz, 1H, H-6′′), 5.35 (m, 1H, H-3′), 6.81 (m, 4H), 7.25 (m, 7H),
7.39 (m, 2H), 7.58 (m, 3H), 8.03 (m, 2H), 8.07 (s, 1H, H-2), 8.82 (s,
1H, H-8). FAB-MS: m/z 1018 (M + H)+.
CD Spectroscopy. The spectropolarimeter was calibrated with (+)-
10-camphorsulfonic acid. Spectra were recorded in a 1-cm path length
quartz cell at a speed of 50 nm/min, and the recorded spectra were
digitized at 0.1-nm resolution and stored on a floppy disk. The
bandwidth was 1 nm, and the response time, 0.25 s. For each
determination, at least five spectra were accumulated, and the averaged
spectrum was subjected to noise reduction.
LCAA-CPG solid support (2 g, 0.16 mmol), succinyl ester (412 mg,
0.40 mmol), and triethylamine (0.055 mL, 0.44 mmol) were added to
anhydrous DMF (10 mL) in a round-bottom flask and shaken at room
temperature for 3 days. Unreacted amino groups on the support were
acetylated with acetic anhydride/pyridine (1:3 v/v), after which the
support was filtered, washed with pyridine and dichloromethane, and
dried under reduced pressure. The nucleoside loading was determined
by trityl analysis to be 39.5 µmol/g support.
Synthesis of Homo-N-nucleoside-containing Oligodeoxyribo-
nucleotides. Oligodeoxyribonucleotides containing homo-N-nucleosides
were synthesized on a 1-µmol scale by the cyanoethyl phosphoramidite
method on an Applied Biosystems 380B solid-phase DNA synthesizer
NMR Spectroscopy (NOESY). Homo-N-deoxydiadenosine 8 was
dissolved in D2O containing 10 mM phosphate buffer and 0.15 M NaCl
(the NaH2PO4/Na2HPO4 ratio was calculated to give pH 7.0 in H2O
and was not corrected for D2O). NOESY spectra were acquired at 4
°C with suppression of the residual solvent peak by presaturation.
Spectra were taken at mixing times of 0.2, 0.5, and 1 s, and the spectral
width was 4006 Hz, with 2048 data points in t2, 128 increments in t1,
and 16 scans per increment. The interresidue cross-peaks at the different
mixing times were essentially the same. Data were zero-filled to 1024
(28) Sinha, N. D.; Biernat, J.; McManus, J.; Ko¨ster, H. Nucleic Acids Res. 1984,
12, 4539-4557.
9
7484 J. AM. CHEM. SOC. VOL. 126, NO. 24, 2004

Homo-N-oligonucleotides with a Left-Handed Twist
A R T I C L E S
data points in t1, apodized with Gaussian line-broadening and expo-
nential line-narrowing, and analyzed with XWIN NMR software
(Bruker).
or 0.36 U of enzyme.29 The final A260 for each oligomer was 4.0, and
the final reaction volume was 0.1 mL. After 1 h, the reaction was
quenched by the addition of ethylenediaminetetraacetic acid (EDTA;
final concentration, 50 mM). For a zero time point, EDTA was added
before the enzyme. A sample of each reaction mixture (20 µL) was
analyzed by reverse-phase HPLC.
Atomic Force Microscopy (AFM). Samples (250 µg/mL) were
diluted with phosphate-buffered saline (PBS) to 0.5-1 µg/mL, made
10 mM in MgCl2, and, after 5 min, anchored to a mica substrate, rinsed
with 5 × 1 mL PBS, and dried in a vacuum oven at 40 °C for 10 min.
AFM was carried out with a NanoScope IIIa multimode system (Digital
Instruments, Santa Barbara, California) operated in the tapping mode
in air at room temperature. The Super Sharp Silicon cantilevers
(Nanosensors, Wetzlar-Blankenfeld, Germany) were 119 µm long with
a spring constant of 36 Nm-1. Typical resonant frequencies of the tip
were 345 kHz. After an initial scan of a 1 µm × 1 µm area, the
parameters were optimized for a scan of an 80 nm × 80 nm area, and
256 × 256 pixel images were collected at a rate of one scan line per
second.
HPLC. Reverse-phase HPLC for enzyme-digestion experiments was
carried out with a C18 Merck 50734 LiChrospher 100 RP-18 endcapped
stainless steel cartridge (4 × 125 mm) maintained at 35 °C in a
Shimadzu CTO-6A column oven. The HPLC buffer stock solution was
1 M triethylammonium acetate (TEAA), pH 7.0 (1 M in triethylamine,
titrated to pH 7.0 with acetic acid), and the HPLC buffers were prepared
by mixing the appropriate amounts of 1 M TEAA, pH 7.0, and
acetonitrile with distilled deionized water. The column was operated
at a flow rate of 1 mL/min, and the effluent was monitored at 260 nm.
Nuclease Digestion. Snake venom phosphodiesterase: Each oligo-
mer was incubated at 37 °C in a reaction mixture containing 0.1 M
Tris‚HCl, pH 8.9, 0.1 M NaCl, and 14 mM MgCl2 and either 0.036 U
Calf spleen phosphodiesterase: Each oligomer was incubated at 37
°C in a reaction mixture containing 0.1 M ammonium succinate, pH
5.9, 1 mM EDTA, and either 0.001 U or 0.02 U of enzyme. The final
A260 for each oligomer was 4.0, and the final reaction volume was 0.1
mL. The reaction was quenched by immersing the reaction tube in
boiling water for at least 4 min, and a sample of each reaction mixture
(20 µL) was subjected to reverse-phase HPLC.
Nuclease S1: Nuclease S1 digestion was carried out in 33 mM
sodium acetate, pH 4.5, containing 50 mM NaCl and 0.03 mM ZnSO4.
The reaction mixture (50 µL), which contained 25-mer (final A260, 4.0)
and 0.1, 1.0, or 100 U of enzyme, or no enzyme, was incubated for 24
h at 37 °C. A sample of each reaction mixture (20 µL) was subjected
to reverse-phase HPLC.
Acknowledgment. AFM imaging and analysis were carried
out by Dr T. Okada, Mr R. Hirota, and Mr H. Sugasawa, of the
Research Institute of Biomolecule Metrology, Tsukuba, Ibaraki,
Japan.
JA049865T
(29) Higuchi, H.; Endo, T.; Kaji, A. Biochemistry 1990, 29, 8747-8753.
9
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