Silver(I)-mediated cytosine self-pairing is preferred over hoogsteen-type base pairs with the artificial nucleobase 1,3-dideaza-6-nitropurine

Article abstract of DOI:10.1080/15257770903451579

A 2′-deoxyribonucleoside containing 1,3-dideaza-6-nitropurine was synthesized and incorporated into oligonucleotides. The acid-base properties of this nucleoside and the corresponding N9-methylated derivative were investigated by pD-dependent 1H NMR spectroscopy. A possible formation of Hoogsteen-type base pairs with cytosine was studied by ultraviolet (UV) and circular dichroism (CD) spectroscopy in the presence and absence of Ag(I) and under neutral and acidic conditions, respectively. In each case, no indication for the formation of Hoogsteen-type base pairs was obtained, which is attributed to the higher affinity of cytosine to form self-complementary hemi-protonated base pairs under acidic conditions and metal-mediated homo base pairs in presence of Ag(I), respectively.

Full text of DOI:10.1080/15257770903451579

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Silver(I)-Mediated Cytosine Self-Pairing
is Preferred Over Hoogsteen-Type Base
Pairs with the Artificial Nucleobase 1,3-
Dideaza-6-Nitropurine
Dominik A. Megger a & Jens Müller a
a Westfälische Wilhelms-Universität Münster, Institut für
Anorganische und Analytische Chemie, Münster, Germany
Published online: 21 Dec 2009.
To cite this article: Dominik A. Megger & Jens Müller (2009): Silver(I)-Mediated Cytosine Self-Pairing
is Preferred Over Hoogsteen-Type Base Pairs with the Artificial Nucleobase 1,3-Dideaza-6-Nitropurine,
Nucleosides, Nucleotides and Nucleic Acids, 29:1, 27-38
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Nucleosides, Nucleotides and Nucleic Acids, 29:27–38, 2010
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770903451579
SILVER(I)-MEDIATED CYTOSINE SELF-PAIRING IS PREFERRED OVER
HOOGSTEEN-TYPE BASE PAIRS WITH THE ARTIFICIAL
NUCLEOBASE 1,3-DIDEAZA-6-NITROPURINE
Dominik A. Megger and Jens Mu¨ ller
Westfa¨lische Wilhelms-Universita¨t Mu¨nster, Institut fu¨r Anorganische und Analytische
Chemie, Mu¨nster, Germany
A 2ꢁ-deoxyribonucleoside containing 1,3-dideaza-6-nitropurine was synthesized and incorpo-
2
rated into oligonucleotides. The acid-base properties of this nucleoside and the corresponding
N9-methylated derivative were investigated by pD-dependent 1H NMR spectroscopy. A possible
formation of Hoogsteen-type base pairs with cytosine was studied by ultraviolet (UV) and circular
dichroism (CD) spectroscopy in the presence and absence of Ag(I) and under neutral and acidic
conditions, respectively. In each case, no indication for the formation of Hoogsteen-type base pairs
was obtained, which is attributed to the higher affinity of cytosine to form self-complementary
hemi-protonated base pairs under acidic conditions and metal-mediated homo base pairs in presence
of Ag(I), respectively.
Keywords Bioinorganic chemistry; metal-mediated base pairs; cytosine; Hoogsteen;
silver(I)
INTRODUCTION
During the last years many novel base pairs mediated by metal-binding
have been reported.[1–4] Most of these display a high thermal stability,
resulting in a significant increase of the melting temperature, Tm, even if
just one artificial base pair is present within the oligonucleotide double
helix. Recently, we have reported a Hoogsteen-type base pair between 1-
deazaadenine and a deprotonated thymine, mediated by hydrogen bonding
and metal-ion binding.[5] In this case, the binding of Ag(I) leads to an
Received 12 August 2009; accepted 23 October 2009.
Generous financial support by the Deutsche Forschungsgemeinschaft (MU1750/2-1, IRTG 1444)
is gratefully acknowledged.
Address correspondence to Jens Mu¨ller, Westfa¨lische Wilhelms-Universita¨t Mu¨nster, Institut
fu¨r Anorganische und Analytische Chemie, Corrensstr. 28/30, 48149 Mu¨nster, Germany. E-mail:
mueller.j@uni-muenster.de
27

28
D. A. Megger and J. Mu¨ller
FIGURE 1 Proposed Hoogsteen-type base pairs between cytosine and 1,3-dideaza-6-nitropurine.
increase of Tm of around 2◦C per base pair, thus resembling the stability
of a natural base pair. As a result, a large number of metal ions can be
incorporated inside the DNA double helix without loss of the reversibility
of the hybridization process. We, therefore, set out to devise an analogous
base pair incorporating cytosine instead of thymine to create a pair of
orthogonal metal-mediated base pairs. This should expand our possibilities
to fine-tune duplex stability and metal-ion binding properties of artificial
DNA constructs.
Seela and coworkers had previously investigated the use of 1,3-dideaza-
6-nitropurine as a universal nucleobase.[6] In their study, the natural
nucleobases in Watson-Crick base pairs were successively exchanged by
1,3-dideaza-6-nitropurine, and the melting temperatures of the resulting
Watson-Crick duplexes were determined. Contrary to that work, we report
here an approach to use 1,3-dideaza-6-nitropurine as an artificial nucleobase
complementary to cytosine in a Hoogsteen-type metal-mediated base pair.
In this prospective base pair, one oxygen atom of the nitro group acts as a
hydrogen bond acceptor, and the N7 position acts as the metal-ion binding
site (Figure 1A). In principle, the metal ion in this base pair could also be
replaced by a proton (Figure 1B).
RESULTS AND DISCUSSION
Syntheses
Starting from 2,6-dinitroaniline, the DMT-protected phosphoramidite 7
was isolated after 6 steps with an overall yield of 25% (Scheme 1) according
to modified literature procedures.[7–10] The modifications included the
glycosylation of 1,3-dideaza-6-nitropurine 3 (reaction c) and the deprotec-
tion of the p-toluoyl-protected nucleoside (reaction d): NaH was used to
deprotonate 3 to afford the desired N9 glycosylated β-anomer 4a in 64%
yield, whereas the use of KOH/TDA-1 as reported in the literature[10]
resulted in a poor stereo- and regioselectivity. The deprotection of 4a was

Contiguous Silver(I)-Mediated Cytosine Self-Pairs
29
SCHEME 1 Synthesis of 1,3-dideaza-6-nitropurine 2ꢁ-deoxyribonucleoside 5a and conversion into a
DMT-protected phosphoramidite 7. (a) Na2S and NaHCO3 in H2O, 1.5 hours at 70◦C, 86%;[7] (b)
formic acid, 20 hours reflux, 87%;[8] (c) NaH, Hoffer’s chloro sugar, in MeCN, 30 minutes at 0◦C,
3 hours at 20◦C, 65%;[9] (d) NH3 (aq.) in MeOH, 24 hours at ambient temperature, 91%; (e) DMT-
Cl, DMAP, in pyridine, 2 hours at ambient temperature, 90%; (f) 2-cyanoethyl diisopropyl chloro
phosphoramidite, DIPEA, in CH2Cl2, 30 minutes at ambient temperature, 80%; (g) formaldehyde in
EtOH/HCl (3:1), 30 minutes, reflux, 40%.[11]
carried out with a solution of aqueous NH3 in methanol instead of sodium
methoxide. The N9-methylated derivative 8 was synthesized following litera-
ture procedures.[11]
Different oligonucleotides containing cytosine (C) and 1,3-dideaza-6-
nitropurine (X) were synthesized (Table 1). Oligonucleotides I—IV are
self-complementary and should be able to form antiparallel-stranded double
helices with CH+–X or C–Ag+–X base pairs. Oligonucleotides II and IV
were designed to investigate the alternative formation of parallel-stranded
duplexes in combination with I and III, respectively.
Oligonucleotide V represents one half of the strands III and IV and was
used as a reference.
TABLE 1 Oligonucleotides investigated in this study
(X = 1,3-dideaza-6-nitropurine)
Oligonucleotide
Sequence
I
5ꢁ-d(TACXCXCXTA)-3ꢁ
II
III
IV
V
5ꢁ-d(ATXCXCXCAT)-3ꢁ
5ꢁ-d(XXXXXXXXCCCCCCCC)-3ꢁ
5ꢁ-d(CCCCCCCCXXXXXXXX)-3ꢁ
5ꢁ-d(CCCCCCCC)-3ꢁ

30
D. A. Megger and J. Mu¨ller
Acid-Base Properties
Prior to performing experiments with the oligonucleotides, the pKa
value of the protonated artificial nucleoside 5a was determined by pD-
1
dependent H NMR spectroscopy. The pKa value of 3.86 0.03 indicates
that under physiological conditions and even in the slightly acidic pH
range, a protonation at the N7 position can be excluded. The pKa value
of the N7 glycosylated side product 5b could not be determined because an
acidification of the sample to approximately pD 4 led to a depurination
of the nucleoside. The protonated N9-methyl derivative 8, acting as a
model nucleobase, has a pKa value of 4.03
0.02, obeying the empirical
rule that nucleosides are less basic than the corresponding methylated
nucleobases.[12,13]
pH-Dependent Hybridization Experiments in the Absence
of Silver(I)
To investigate the possible formation of a Hoogsteen-type base pair
between CH+ and X, ultraviolet (UV) melting curves and circular dichroism
(CD) spectra were recorded at pH 6.8 and pH 5.5, respectively. As cytosine
is not protonated at pH 6.8,[12] the resulting potential C–X base pair would
be held together by one hydrogen bond only and therefore be extremely
unstable. In fact, no cooperative melting was observed for any of the
oligonucleotides under investigation (I and III) at pH 6.8, and the CD
spectra showed no features characteristic of double helices. After decreasing
the pH to 5.5, neither cooperative melting nor changes in the CD spectrum
were observed in the case of I (data not shown). However, in the case of
III, a positive Cotton effect at 289 nm and a concomitant negative one at
267 nm were observed under acidic conditions (Figure 2A). The spectral
FIGURE 2 (A) CD spectra of III and V at different pH values. (B) Melting curves of the i-motif structures
formed by III and V. Conditions: 1 µM oligonucleotide (5 µM for the CD spectra of III at pH 5.5), 150
mM NaClO4, 5 mM MOPS (pH 6.8), or 5 mM MES (pH 5.5).

Contiguous Silver(I)-Mediated Cytosine Self-Pairs
31
properties observed are characteristic for an i-motif structure[14, 15] with
CH+–C instead of the desired CH+–X base pairs, as shown by control
experiments with oligonucleotide V. In the latter case, the minimum and
maximum are more distinct though (Figure 2A). The different spectral
intensities as well as the differences below 240 nm are probably a result of
the fact that the tetrameric i-motif structure of III contains four overhanging
d(X8) sequences that additionally influence the CD spectra.
The Tm values of the i-motif structures formed by III and V at pH
5.5, determined by temperature-dependent UV spectroscopy at 260 nm,
amount to 40◦C and 25◦C, respectively. Additionally, hyperchromicity values
of around 7% for both i-motif structures were observed (Figure 2B). The
fact that the Tm of III is higher by 15◦C compared to that of V might
be explained by further stabilization of the cytosine-based i-motif structure
via additional weak interactions (π stacking and/or other hydrophobic
interactions) involving the overhanging d(X8) regions.
Titrations with AgNO3
The oligonucleotides were titrated with AgNO3 to investigate the poten-
tial formation of metal-mediated base pairs in the presence of a linearly
coordinating metal ion. The CD spectrum of oligonucleotide I remains
unaffected by the addition of Ag(I) (data not shown), suggesting that the
desired C–Ag+–X base pairs are not formed. In contrast, the CD spectrum
of oligonucleotide III shows strong changes upon the addition of Ag(I):
two intense minima at 217 and 270 nm and a weak one at 357 nm appear
until eight Ag(I) ions per duplex are present (Figures 3A and 3B). However,
16 metal ions per duplex should be expected if only C–Ag+–X base pairs
were formed. The observed stoichiometry could be explained in two ways:
Either only every other binding site is occupied by a Ag(I) ion (similar to
the neighbor-exclusion principle[16] found for nucleic acid intercalators),
or the resulting duplex contains one sort of metalated homo base pairs only,
that is, either C–Ag+–C or X–Ag+–X base pairs. Inspection of a series of CD
spectra clearly points to the second possibility: The CD spectra of oligonu-
cleotide V (comprising only cytosine) with Ag(I) display the same spectral
features as those of III with Ag(I) (Figure 3C), except for the weak minimum
at 357 nm, which can most probably be attributed to an interaction of the
overhanging d(X8) regions (see below), strongly suggesting that C–Ag+–C
base pairs are formed in both cases. This conclusion is further corroborated
by the fact that oligonucleotide V can bind eight metal ions per duplex,
indicative of the formation of eight C–Ag+–C base pairs (Figure 3D).
However, no cooperative melting was observed by means of UV spec-
troscopy (at 260, 271 and 314 nm, with the latter two wavelengths represent-
ing the maxima of the UV spectra) for the duplexes of III and V. Possibly,
there are only weak π-stacking effects in a metalated poly-d(C) sequence

32
D. A. Megger and J. Mu¨ller
FIGURE 3 (A and C) CD spectra of III and V in the absence and presence of various amounts of
AgNO3. (B and D) Changes of molar ellipticity of III and V upon the addition of AgNO3. Conditions: 1
µM oligonucleotide, 150 mM NaClO4, 5 mM MOPS (pH 6.8).
that in turn lead to a small hyperchromicity. Therefore, temperature-
dependent CD spectra of the oligonucleotides III and V were recorded
in the presence of Ag(I) to obtain information on the thermal stability
of the metalated species. The spectra display their largest temperature
dependence at 270 and 280 nm. At the latter wavelength, cooperative
melting can be observed, revealing melting temperatures of Tm(III) =
63◦C and Tm(V) = 73◦C (both with eight Ag(I) per duplex; Figure 4A). A
possible explanation for the higher stability of V as compared with III will be
discussed in the following chapter. It should be noted that—contrary to the
behavior of III and V under acidic conditions (see above)—the formation
of a tetra-stranded structure like the i-motif is unlikely in the presence of
Ag(I), and therefore the different effect of the single-stranded overhang in
III on the thermal stability of the resulting helices is not unexpected. The
assumption that each nucleobase of the overhanging d(X8) region might act
as a competitive potential binding site for the added metal ions appears to
be an unlikely explanation for the higher stability of V as compared with III.

Contiguous Silver(I)-Mediated Cytosine Self-Pairs
33
FIGURE 4 (A) CD melting curves of III and V in the presence of eight Ag(I) per duplex, and (B) Tm
values upon addition of various amounts of AgNO3 (B). Conditions: 1 µM oligonucleotide, 150 mM
NaClO4, 5 mM MOPS (pH 6.8).
Under these conditions, different slopes for the plot of Tm versus number
of Ag(I) ions should be expected for III and V. However, identical slopes
are observed (Figure 4B).
Investigation of a Parallel-Stranded Duplex Formation
To examine the potential formation of parallel-stranded duplexes with
C–Ag+–X base pairs, the above-mentioned titrations and hybridization
experiments were repeated with equimolar mixtures of I+II and III+IV
instead of I only and III only. For both mixtures, results similar to those
reported above for oligonucleotides I and III alone were obtained, revealing
that no parallel-stranded double helices with C–Ag+–X or CH+–X base pairs
are formed either. Instead, formation of C–Ag+–C base pairs appears to
be predominant also in the cases of III and III+IV. Oligonucleotides I
and I+II, not being able to form C–Ag+–C base pairs as a result of their
sequences, do not adopt any double helical structure (data not shown).
Nonetheless, these investigations helped to shed light onto the question
of the strand-orientation of duplexes with consecutive C–Ag+–C base pairs:
The CD spectra of III and III+IV display analogous changes upon the
addition of Ag(I) (data not shown) with the exception of the region of
350–370 nm. In this wavelength region, the CD spectrum of III+IV does
not change when adding Ag(I) whereas the spectrum of III exhibits a small
yet significant negative Cotton effect (Figure 5A). This differential behavior
could be conveniently explained by the formation of a parallel-stranded
duplex: A parallel duplex of C–Ag+–C base pairs formed by III+IV would
locate the overhanging d(X8) region on opposite ends of the helix whereas
an analogous duplex formed by III would result in overhanging d(X8)
regions located in close vicinity to each other (Figure 5D), probably for

34
D. A. Megger and J. Mu¨ller
FIGURE 5 (A) CD spectra of III and III+IV in presence of eight Ag(I) per duplex. (B) UV spectra
of 5a (0.1 mM in water) in the absence and presence of AgNO3. (C) CD melting curves of III and
III+IV in presence of eight Ag(I) per duplex. (D) Schematic representation of the proposed parallel-
stranded double helices formed by III and III+IV, respectively. Conditions: 1 µM oligonucleotide, 150
mM NaClO4, 5 mM MOPS (pH 6.8).
steric reasons adopting some preferred non-random conformation and
thereby evoking the negative Cotton effect. The following observations are
supportive of such an interpretation:
1. A comparison with the CD spectra of V shows (Figure 3C) the negative
Cotton effect around 360 nm cannot be attributed to cytosine. It must,
therefore, originate from the artificial nucleobases.
2. It cannot be caused by a mere binding of excess Ag(I) to the d(X8)
region because in this case no differences would be expected for III
and for III+IV, respectively. Furthermore, such binding should also
influence the UV spectrum of the nucleoside 5a which does not show
any significant changes upon the addition of AgNO3 (Figure 5B).
3. The thermal stabilities of the duplexes with C–Ag+–C base pairs formed
by III and III+IV vary significantly: In the presence of comparable
amounts of Ag(I), both III+IV (with the d(X8) regions on opposite ends

Contiguous Silver(I)-Mediated Cytosine Self-Pairs
35
of the duplex) and V (with no d(X8) region) have melting temperatures
of 73◦C, whereas III displays a Tm of only 63◦C (Figures 4A and 5C).
Assuming a parallel strand orientation, this would suggest that the duplex
formed by III is destabilized by the above-mentioned interaction of the
neighboring overhanging d(X8) regions.
Taken together, this evidence is supportive of a parallel-stranded orien-
tation of duplexes with contiguous C–Ag+–C base pairs, contrary to what
is expected for single C–Ag+–C base pairs surrounded by regular Watson-
Crick base pairs.[17] Provided that the glycosidic bond angles are in the usual
anti range, the metal-mediated base pairs are comprised of nucleobases with
transoid glycosidic bonds (Figure 5D).
CONCLUSIONS
Oligonucleotides containing the artificial nucleobase 1,3-dideaza-6-
nitropurine were synthesized and characterized. By UV and CD spectro-
scopic experiments the potential formation of a Hoogsteen-type base pair
with cytosine was investigated. Under none of the conditions investigated
experimentally (decreased pH, presence of Ag(I) or complementary par-
allel strands) the formation of a stable duplex with CH+–X base pairs
was detected. For the alternating sequences d(TACXCXCXTA) (I) and
d(ATXCXCXCAT) (II), no indication was found for the formation of a
stable double-helical structure at all. On the contrary, the oligonucleotide
sequences d(X8C8) (III) and d(C8X8) (IV), respectively, revealed the for-
mation of C–Ag+–C base pairs in presence of Ag(I) as well as the formation
of an i-motif structure with CH+–C under slightly acidic conditions. It
can, therefore, be concluded that cytosine self-pairing is preferred over
the formation of Hoogsteen-type base pairs between cytosine and 1,3-
dideaza-6-nitropurine, both in the presence and in the absence of Ag(I).
The experiments are supportive of a parallel-stranded orientation of the
duplexes containing consecutive C–Ag+–C base pairs.
EXPERIMENTAL
Nucleoside and Phosphoramidite Synthesis
The nucleobase 1,3-dideaza-6-nitropurine 3,[7,8] the N9-methylated
derivative 8,[11] Hoffer’s chloro sugar,[9] the DMT-protected nucleoside
6,[10] and phosphoramidite 7[10] were synthesized following literature
procedures. For the glycosylation of 3 and the deprotection of 4 these
procedures were modified as follows.

36
D. A. Megger and J. Mu¨ller
1,3-dideaza-6-nitropurine-N7/N9-β-[2ꢁ-deoxy-3ꢁ,5ꢁ-di-(p-toluoyl)-
ribonucleoside] 4
To a suspension of 1,3-dideaza-6-nitropurine 3 (1.522 g, 9.330 mmol)
in acetonitrile (40 mL), NaH (60% suspension in mineral oil, 511.0 mg,
12.78 mmol) was added at 0◦C. After stirring the reaction mixture for
1 hour at 0◦C, Hoffer’s chloro sugar (2.467 g, 6.345 mmol) was added
in 4 portions every 20 minutes. The suspension was stirred for further
4 hours (0◦C → room temperature), filtered, and the resulting solution
evaporated to dryness. The resulting brownish syrup was purified by column
chromatography (SiO2, cyclohexane (100) : ethylacetate (60) : CH2Cl2 (5))
yielding the desired N9-glycosylated β-anomer 4a as a colorless foam (2.108
g, 4.082 mmol, 64%) as well as the N7-glycosylated β-anomer 4b (148.8 mg,
1
288.1 µmol, 5%). 4a: H NMR (500 MHz, CDCl3): δ/ppm = 8.33 (s, 1H,
H8); 8.12 (d, 1H, H1); 7.98 (d, 2H, PG); 7.90 (d, 1H, H3); 7.83 (d, 2H,
PG); 7.30 (d, 2H, PG); 7.24 (m, 1H, H2); 7.23 (d, 2H, PG); 6.45 (pt, 1H,
H1ꢁ); 5.77 (m, 1H, H3ꢁ); 4.78 (m, 1H, H5ꢁ); 4.67 (m, 1H, H4ꢁ); 4.65 (m,
1H, H5ꢁꢁ); 2.96 (m, 1H, H2ꢁ); 2.85 (m, 1H, H2ꢁꢁ); 2.45 (s, 3H, CH3); 2.42 (s,
3H, CH3). 13C NMR (125 MHz, CDCl3): δ/ppm = 166.4 (PG); 166.3 (PG);
145.1 (PG); 144.8 (PG); 143.9 (C8); 137.9 (C6); 135.6 (C4); 130.1 (C5);
129.6 (PG); 126.6 (PG); 126.5 (PG); 122.8 (C2); 120.1 (C1); 117.7 (C3);
86.0 (C1ꢁ); 83.2 (C4ꢁ); 74.6 (C3ꢁ); 63.7 (C5ꢁ); 38.5 (C2ꢁ); 22.0 (CH3); 21.9
(CH3). Anal. Calcd for C28H25N3O7: C, 65.2; H, 4.9; N, 8.2; found: C, 65.3;
H, 4.9; N, 7.9%. HRMS (ESI): Calcd for C28H26N3O7 (M+H)+: 516.17653,
1
found 516.17592. 4b: H NMR (200 MHz, CDCl3): δ/ppm = 8.51 (s, 1H,
H8); 8.07 (d, 1H, H1); 8.01 (d, 1H, H3); 7.96 (d, 2H, PG); 7.80 (d, 2H, PG);
7.35 (t, 1H, H2); 7.27 (d, 2H, PG); 7.18 (d, 2H, PG); 6.79 (dd, 1H, H1ꢁ);
5.62 (m, 1H, H3ꢁ); 4.64 (m, 3H, H4ꢁ, H5ꢁ, H5ꢁꢁ); 3.00 (m, 1H, H2ꢁ); 2.68 (m,
1H, H2ꢁꢁ); 2.43 (s, 3H, CH3); 2.38 (s, 3H, CH3).
1,3-dideaza-6-nitropurine-N9-β-2ꢁ-deoxyribonucleoside 5a
To a solution of 4a (984.4 mg, 1.910 mmol) in methanol (100 mL),
aqueous NH3 (25%, 100 mL) was added, and the mixture stirred for
24 hours at room temperature. The solvent was evaporated, and the
oily residue treated with CH2Cl2 (50 mL). The resulting suspension was
sonicated for 10 minutes and filtered. The white solid was dried at 40◦C
to yield the deprotected deoxyribonucleoside 5a (483. 8mg, 1.733 mmol,
91%). 1H NMR (500 MHz, DMSO-d6): δ/ppm = 8.78 (s, 1H, H8); 8.25 (d,
1H, H1); 8.21 (d, 1H, H3); 7.47 (t, 1H, H2); 6.47 (pt, 1H, H1ꢁ); 5.39 (d, 1H,
3ꢁ-OH); 5.02 (t, 1H, 5ꢁ-OH); 4.42 (m, 1H, H3ꢁ); 3.90 (m, 1H, H4ꢁ); 3.57 (m,
2H, H5ꢁ, H5ꢁꢁ); 2.63 (m, 1H, H2ꢁ); 2.38 (m, 1H, H2ꢁꢁ). 13C NMR (125 MHz,
DMSO-d6): δ/ppm = 145.3 (C8); 138.6 (C6); 136.7 (C4); 135.5 (C5); 122.1
(C2); 118.6 (C1); 118.3 (C3); 87.8 (C4ꢁ); 85.0 (C1ꢁ); 70.2 (C3ꢁ); 61.2 (C5ꢁ);
39.7 (C2ꢁ). Anal. Calcd for C12H13N3O5: C, 51.6; H, 4.7; N, 15.1%; found:

Contiguous Silver(I)-Mediated Cytosine Self-Pairs
37
C, 51.7; H, 5.0; N, 15.1. HRMS (ESI): Calcd for C12H14N3O5 (M+H)+:
280.09280, found: 280.09288.
1,3-dideaza-6-nitropurine-N7-β-2ꢁ-deoxyribonucleoside 5b
Following the above-mentioned procedure, 4b (573.6 mg, 1.112 mmol)
was deprotected to yield 5b as a brown solid (236.9 mg, 848.3 µmol, 76%).
1H NMR (500 MHz, DMSO-d6): δ/ppm = 8.83 (s, 1H, H8); 8.10 (d, 1H, H1);
7.97 (d, 1H, H3); 7.42 (t, 1H, H2); 6.43 (pt, 1H, H1ꢁ); 5.33 (d, 1H, 3ꢁ-OH);
4.92 (t, 1H, 5ꢁ-OH); 4.32 (m, 1H, H3ꢁ); 3.82 (m, 1H, H4ꢁ); 3.43 (m, 2H, H5ꢁ,
H5ꢁꢁ); 2.58 (m, 1H, H2ꢁ); 2.45 (m, 1H, H2ꢁꢁ). 13C NMR (125 MHz, DMSO-
d6): δ/ppm = 144.5 (C8); 136.35 (C6); 126.2 (C5); 124.6 (C4); 121.5 (C2);
120.1 (C1); 117.7 (C3); 87.4 (C4ꢁ); 86.8 (C1ꢁ); 69.6 (C3ꢁ); 60.7 (C5ꢁ); 50.0
(C2ꢁ). Anal. Calcd for C12H13N3O5: C, 51.6; H, 4.7; N, 15.1; found: C, 52.1;
H, 4.3; N, 15.1%. LRMS (MALDI): Calcd for C12H13N3O5Na (M+Na)+: 302,
found: 302.
Oligonucleotide Synthesis, Purification, and Characterization
Oligonucleotides I-IV were synthesized on an Expedite 8909 DNA syn-
thesizer (PerSeptive Biosystems, Framingham, MA, USA) in DMT-off mode
and purified by HPLC with a Nucleogen 60–7 DEAE column (Macherey-
Nagel, Du¨ren, Germany). The following linear gradients were used during
HPLC purification: I and II: 0–10 minutes: 100–80% A, 10–40 minutes:
80–75% A, 40–45 minutes: 75–0% A; III and IV: 0–10 minutes: 100–80% A,
10–40 minutes: 80–50% A, 40–45 minutes: 50–0% A (1.5 mL/min, solvent A:
20 mM sodium acetate (pH 6) in H2O/acetonitrile (4:1), solvent B: solvent
A + 2 M LiCl). After desalting via NAP 10 columns, the oligonucleotides
were characterized by MALDI-TOF mass spectrometry on a Bruker Autoflex
II instrument (I: Calcd for (M-H)−: 3062, found: 3063; II: Calcd for (M-H)−:
3062, found: 3063; III: Calcd for (M-H)−: 4979, found: 4977; IV: Calcd for
(M+Na-2H)−: 5000, found: 4999). The oligonucleotide V was purchased
from Eurogentec (Seraing, Belgium).
Titrations and Hybridization Experiments
For the hybridization experiments, oligonucleotide solutions (1–5 µM)
containing 150 mM NaClO4 and 5 mM MOPS (adjusted to pH 6.8) or 5 mM
MES (adjusted to pH 5.5) were prepared. UV melting curves were recorded
on a Varian CARY BIO 100 spectrometer at 260 nm (heat rate: 1◦C/min,
data interval: 0.2◦C). CD spectra and melting curves were recorded on a
Jasco J-815 (CD spectra: 10◦C, scan rate: 100 nm/min, 5 accumulations; CD
melting curves: 10–90◦C, heat rate: 1◦C/min, data interval: 1◦C). Melting
temperatures were determined as the maxima of the first derivatives of the
melting curves.

38
D. A. Megger and J. Mu¨ller
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