Anomeric 5-Aza-7-deaza-2′-deoxyguanosines in Silver-Ion-Mediated Homo and Hybrid Base Pairs: Impact of Mismatch Structure, Helical Environment, and Nucleobase Substituents on DNA Stability

Article abstract of DOI:10.1002/chem.201901276

Nucleoside configuration (α-d vs. β-d), nucleobase substituents, and the helical DNA environment of silver-mediated 5-aza-7-deazaguanine-cytosine base pairs have a strong impact on DNA stability. This has been demonstrated by investigations on oligonucleotide duplexes with silver-mediated base pairs of α-d and β-d anomeric 5-aza-7-deaza-2′-deoxyguanosines and anomeric 2′-deoxycytidines incorporated in 12-mer duplexes. To this end, a new synthetic protocol has been developed to access the pure anomers of 5-aza-7-deaza-2′-deoxyguanosine by glycosylation of either the protected nucleobase or its salt followed by separation of the glycosylation products by crystallization and chromatography. Thermal stability measurements were performed on duplexes with α-d/α-d and β-d/β-d homo base pairs or α-d/β-d and β-d/α-d hybrid pairs within two sequence environments, positions 6 or 7, of oligonucleotide duplexes. The respective T_m stability increases observed after silver ion addition differ significantly. Homo base pairs with β-d/β-d or α-d/α-d nucleoside combinations are more stable than α-d/β-d hybrid base pairs. The positional switch of silver-ion-mediated base pairs has a significant impact on stability. Nucleobase substituents introduced at the 5-position of the dC site of silver-mediated base pairs affect base pair stability to a minor extent. Our investigation might lead to applications in the construction of bioinspired nanodevices, in DNA diagnostics, or metal-DNA hybrid materials.

Full text of DOI:10.1002/chem.201901276

A Journal of
Accepted Article
Title: Anomeric 5-Aza-7-deaza-2`-deoxyguanosines in Silver Ion
Mediated Homo and Hybrid Base Pairs: Impact of Mismatch
Structure, Helical Environment and Nucleobase Substituents on
DNA Stability
Authors: Xinglong Zhou, Dasharath Kondhare, Peter Leonard, and
Frank Seela
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To be cited as: Chem. Eur. J. 10.1002/chem.201901276
Link to VoR: http://dx.doi.org/10.1002/chem.201901276
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10.1002/chem.201901276
Chemistry - A European Journal
Anomeric 5-Aza-7-deaza-2`-deoxyguanosines in Silver Ion Mediated Homo and Hybrid
Base Pairs: Impact of Mismatch Structure, Helical Environment and Nucleobase
Substituents on DNA Stability
Xinglong Zhou,[a] Dasharath Kondhare,[a] Peter Leonard,[a] and Frank Seela*[a,b]
[a] Laboratory of Bioorganic Chemistry and Chemical Biology, Center for Nanotechnology,
Heisenbergstrasse 11, 48149 Münster, Germany, [b]Laboratorium für Organische und
Bioorganische Chemie, Institut für Chemie neuer Materialien, Universität Osnabrück,
Barbarastrasse 7, 49069 Osnabrück, Germany
Corresponding author:
Prof. Dr. Frank Seela
Phone: +49 (0)251 53 406 500; Fax: +49 (0)251 53 406 857
E-mail: Frank.Seela@uni-osnabrueck.de
Homepage: www.seela.net
Supporting information for this article is available on the www under
http://dx.doi.org/10.1002/chem.2016xxxx.
1
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Abstract
Nucleoside configuration (α-D vs β-D), nucleobase substituents and the helical DNA
environment of silver-mediated 5-aza-7-deazaguanine-cytosine base pairs have a strong impact
on DNA stability. This is the result of investigations performed on oligonucleotide duplexes with
silver-mediated base pairs of α-D and β-D anomeric 5-aza-7-deaza-2’-deoxyguanosines and
anomeric 2'-deoxycytidines incorporated in 12-mer duplexes. To this end, a new synthesis
protocol was developed to access the pure anomers of 5-aza-7-deaza-2’-deoxyguanosine by
glycosylation of either the protected nucleobase or its salt followed by separation of
glycosylation products by crystallization and chromatography. Thermal stability measurements
were performed on duplexes with α-D/α-D and β-D/β-D homo base pairs or α-D/β-D and β-D/α-
D hybrid pairs within two sequence environments - positions 6 or 7 - of oligonucleotide
duplexes. The individual Tm stability increase observed after silver ion addition differs
significantly. Homo base pairs with β-D/β-D or α-D/α-D nucleoside combination were more
stable than α-D/β-D hybrid base pairs. The positional switch of silver ion mediated base pairs has
a significant impact on the stability. Nucleobase substituents introduced at the 5-position of the
dC site of silver-mediated base pairs effect base pair stability to a minor extent. Our investigation
might find application in the construction of bioinspired nanodevices, in DNA diagnostics or
metal DNA hybrid materials.
2
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Introduction
The DNA double helix as carrier of genetic information uses a four letter code system.
Nucleoside dA forms a Watson-Crick base pair with dT and dG with the complementary dC.[1]
The base pairs are held together by hydrogen bonds leading to a less stable bidentate dA-dT pair
and more stable tridentate dG-dC pair.
More than 50 years ago,[2] it was recognized that silver ions interact with DNA and several
binding motifs were proposed including those with silver ions replacing protons within the
Watson-Crick pairs. More recently, detailed information was collected regarding structure and
stability of silver-mediated base pairs by various laboratories including ours. The formation of
silver-mediated dC-dC base pair was established by single-crystal X-ray analyses.[3]A series of
publications appeared and reviews are available.[4,5]
Multiple incorporations of silver ions in DNA can form “metal” DNA in which all canonical
base pairs are replaced by silver-mediated pairs. [4b,e,f] By this means nanoscopic silver wires can
be constructed which have the potential to conduct electrons through the DNA molecule. Other
applications for silver-mediated base pairs is their use in DNA diagnostics for the detection of
mismatches in nucleic acids.[5d]
In principle, the programmable replacement of a H-bonded Watson-Crick base pair by a silver-
mediated pair is encountered with problems resulting from the presence of canonical base pairs.
Consequently, DNA mismatches were introduced to destabilize DNA at a particular site. Then,
silver ion binding is directed to the destabilized region. However, the amount of added silver ion
equivalents should be in the range of the mismatches. The dC-dC mismatch and the formation of
a silver-mediated dC-Ag-dC pair is a typical example for this phenomenon. If the amount of
silver ions is considerably increased not only mismatches but also canonical H-bonded Watson-
Crick base pairs form silver-mediated pairs. This occurs in a non-programmable way resulting in
the formation of DNA silver wires.[4b,f]
3
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Recently, we reported on a silver-mediated base pair formed by 5-aza-7-deaza-2’-
deoxyguanosine (1, β-dZ) with dC[6] (purine numbering is used throughout the manuscript). The
modified purine nucleoside 1[7] combines structural elements of dG and 7-deaza-dG (c7Gd) and
represents a shape mimic of the canonical nucleoside. Similar to 7-deaza-dG, 5-aza-7-deaza-dG
cannot form Hoogsteen base pairs due to the lack of nitrogen-7. Different from dG and c7Gd,
nitrogen-1 of 1 is now an acceptor site for protons and silver ions. Consequently, the dZ-dC pair
forms a mismatch and a metal ion mediated base pair in the presence of silver ions. This dZ-dC
pair represents a silver-mediated purine-pyrimidine pair that closely mimics the canonical dG-dC
pair.
In previous publications, we have shown that the silver-mediated α-dC/β-dC hybrid pair is more
stable than the β-dC-Ag+-β-dC homo pair[8]. The terms “homo” and “hybrid” correspond solely
to the stereochemistry at the anomeric center. Such a phenomenon let us anticipate that a similar
behavior might exist in the silver-mediated dZ-dC pair. To study this matter on oligonucleotide
duplexes, considerable amounts of stereochemically pure α-D and β-D-anomers of dZ (1 and 2)
have to be available. Unfortunately, the chemical synthesis of the pure 5-aza-7-deaza-2'-
deoxyguanosine α- and β-anomers 1 and 2 (Figure 1) is cumbersome as pure anomers are not
formed by the widely used stereoselective nucleobase anion glycosylation.[7b] Also, the almost
identical mobilities of anomers makes the separation of the anomeric mixture difficult.
To this end, the nucleobase glycosylation of the 5-aza-7-deazaguanine base with the Hoffer
halogenose 7[9] was reinvestigated with the intention to improve the formation of one particular
anomer during the glycosylation reaction. Thereafter, 12-mer oligonucleotides were synthesized
from corresponding phosphoramidites incorporating combinations of the anomeric nucleosides
1-4 (Figure 1). Oligonucleotides were hybridized in the absence and the presence of silver ions
and the base pair stability was verified by Tm measurements. The anomeric silver-mediated base
pairs were introduced at two different positions of the DNA double helix to probe nearest
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neighbor influences and the impact of DNA helical environment on the helix stability. This
includes studies regarding the influence of space demanding substituents introduced in the dC
part of the anomeric dZ-dC base pairs.
Figure 1. Upper part: Anomeric 5-aza-7-deaza-2’-deoxyribonucleosides β-dZ (1) and α-dZ (2)
and anomeric dC nucleosides 3 and 4a. Lower part left: Mismatch and silver-mediated base pair.
Lower part right: Schematic view of silver-mediated homo and hybrid base pairs studied in this
work.
Results and Discussion
Synthesis and characterization of monomers
Earlier, both anomeric nucleosides 1 and 2 were synthesized by nucleobase anion glycosylation
of the isobutyryl protected 5-aza-7-deazapurine 5[7b,f] utilizing the halogenose 7 (Hoffer sugar).[9]
However, the α-D anomer 9 was the favored species and not the β-D anomer 8 as observed for
purines and 7-deazapurines. Therefore, attempts were made to shift the anomeric ratio of the
5
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glycosylation reaction. Serendipitously, we found that glycosylation forms predominantly the β-
D-anomer when the isobutyric acid salt 6a is used instead of the free base 6b. In detail, after
isobutyrylation the salt 6a was precipitated from the acidic reaction mixture and was used
without further recrystallization. The salt structure of 6a was confirmed by 1H NMR spectra
showing signals for two isobutyryl groups (Figure S9, Supporting Information).
Next, glycosylation was performed in MeCN, with TDA-1 and anhydrous potassium carbonate
utilizing a two-fold excess of the halogenose 7. Purification of the reaction mixture from by-
products by flash chromatography without separation of the anomers resulted in 92% yield of an
anomeric mixture of 8/9. The ratio of anomers determined by 1H NMR spectroscopy was 3:2 (β-
D to α-D). The column pre-purified anomeric reaction mixture was crystallized from MeOH
yielding the pure β-D nucleoside 8 in 46% yield. The content of the mother liquor was
chromatographed yielding the pure α-nucleoside 9 in 21% together with 8% of the deprotected
α-D anomer 10. This method represents so far the most efficient way to obtain both anomers in
sufficient yields using a combination of crystallization (β-anomer) and chromatography (α-
anomer) (Scheme) (for details, see Experimental Section). The phosphoramidites 13 and 14[7f] as
well as the phosphoramidites 15a-c and 16a-c[8b] were prepared as described (for structures see
Figure 3, Supporting Information).
6
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Scheme. Upper part: Synthesis of protected nucleobase residues and nucleobase-anion
glycosylation protocol for the synthesis of β-dZ (1) and α-dZ (2). i) (i-Bu)2O, H3PO4, reflux; ii)
precipitation from the reaction mixture; iii) recryst. aq. MeOH; iv) TDA-1, MeCN, K2CO3, r.t.,
1h, v) flash column (FC); vi) recryst. from aq. MeOH; vii) FC of the mother liquor; viii) sat.
NH3/MeOH. Lower part: Protected 5-aza-7-deaza-2’-deoxyguanosine nucleoside derivatives
used in this study.
All compounds were characterized by mass spectra and high resolution 1H, 13C NMR and 2D
NMR spectra (Figures S9-S84, Supporting Information). The UV spectra of the salt 6a and the
pure base 6b show identical UV maxima at 224 and 278 nm and the molar extinction coefficients
of both compounds are almost equal (14100 for 6a and 13600 for 6b, both measured at 278 nm).
The isobutyrylated nucleobase 6b shows two pKa values: 3.1 for protonation and 7.7 for
7
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deprotonation, whereas the anomeric nucleosides show identical pKa values only for protonation
at pKa = 3.8. (Table S1, Figures S1, S2, Supporting Information).
Synthesis and characterization of oligonucleotides
Next, oligonucleotides were prepared employing solid-phase synthesis using the
phosphoramidites of α- and β-dZ[7f] (13, 14; Scheme) and of α- and β-dC[8b] (Figure S3,
Supporting Information) together with standard building blocks. The 5-aza-7-deazaguanine α-
and β-anomers 1 (β-dZ) and 2 (α-dZ) were incorporated at two different positions of the 12-mer
duplex 5’-d(TAGGTCAATACT) (ODN-1) • 3’-d(ATC CAG TTA TGA) (ODN-2) replacing a
dA-dT (Table 2) or dG-dC (Table 3) base pair. ODN-1 • ODN-2 represents the well-
characterized standard duplex of our laboratory and has been used to study a series of nucleoside
analogues. After solid-phase synthesis, the oligonucleotides were cleaved from the solid support
and deprotected in concentrated 28% aqueous ammonia at 55 °C for 2 h and then at rt overnight
followed by purification by HPLC. Oligonucleotides incorporating anomeric dC residues with
and without functionalization of the base were prepared according to the literature.[8b] All
oligonucleotides used for this study and their masses are shown in Table 1.
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Table 1. Synthesized oligonucleotides and their molecular masses determined by MALDI-TOF
mass spectrometry.[a]
Mr calcd.[a]
Mr found[b]
Mr calcd.[a]
Mr found[b]
Entry
Oligonucleotides
Entry
Oligonucleotides
3661.4
3661.0
3630.4
3631.2
3756.3
3754.8
3734.5
3734.5
3747.3
3746.9
3670.4
3670.4
3725.5
3725.9
3661.4
3661.0
3670.4
3669.8
3630.4
3631.1
3756.3
3756.7
3734.5
3734.1
3747.3
3747.2
3725.5
3724.2
3685.4
3685.4
3603.6
3603.3
3730.3
3730.2
3709.5
3709.3
3770.3
3771.1
3645.4
3646.0
3749.6
3749.3
3605.4
3604.4
3730.3
3730.5
3709.5
3710.1
3770.3
3770.5
3749.6
3748.3
3685.4
3684.2
3645.2
3642.7
ODN-3
ODN-4
ODN-5
ODN-6
ODN-7
ODN-8
ODN-9
5’-d(TAGGTC2ATACT)
ODN-19 5’-d(TAGGT2AATACT)
ODN-20 5’-d(AGTATT4aACCTA) [8b]
ODN-21 5’-d(AGTATT4bACCTA)[8b]
ODN-22 5’-d(AGTATT4cACCTA)[8b]
ODN-23 5’-d(TAGGT4bAATACT) [8b]
ODN-24 5’-d(AGTATT2ACCTA)
ODN-25 5’-d(TAGGT4cAATACT)[8b]
ODN-26 5’-d(AGTATT3aACCTA)[8b]
ODN-27 5’-d(AGTATT3bACCTA) [8b]
ODN-28 5’-d(AGTATT3cACCTA) [8b]
ODN-29 5’-d(TAGGT3bAATACT) [8b]
ODN-30 5’-d(TAGGT3cAATACT) [8b]
ODN-31 5’-d(TAGGT1AATACT) [8a]
ODN-32 5’-d(AGTATT1ACCTA) [8a]
5’-d(AGTAT4aGACCTA) [8b]
5’-d(AGTAT4bGACCTA) [8b]
5‘-d(AGTAT4cGACCTA) [8b]
5’-d(TAGGTC4bATACT) [8b]
5’-d(AGTAT2GACCTA)
5’-d(TAGGTC4cATACT) [8b]
ODN-10 5’-d(TAGGTC1ATACT)
ODN-11 5’-d(AGTAT1GACCTA)
ODN-12 5’-d(AGTAT3aGACCTA) [8b]
ODN-13 5‘-d(AGTAT3bGACCTA) [8b]
ODN-14 5‘-d(AGTAT3cGACCTA) [8b]
ODN-15 5‘-d(TAGGTC3bATACT) [8b]
ODN-16 5‘-d(TAGGTC3cATACT) [8b]
[a] Calculated on the basis of the molecular mass of [M + H]+. [b] Determined by MALDI-TOF mass-spectrometry as [M + H]+ in
the linear positive mode.
Silver-mediated base pair formation
Recent studies on silver-mediated α-dC/β-dC hybrid base pairs[8] showed that very stable silver-
mediated duplexes are formed when one nucleoside in the base pair shows α-D configuration and
the other is the β-D anomer. As it was anticipated that the phenomenon is of general
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applicability, this work studies now silver-mediated anomeric dZ-dC hybrid base pairs and base
pair stability was compared with silver-mediated β-D/β-D and α-D/α-D homo pairs.
It became obvious that significant differences exist among silver-mediated base pairs of dZ with
dC and those of dC with dC. In the latter case, both nucleobases are identical and the two
pyrimidine moieties form a silver-mediated base pair that is slimmer than the corresponding
purine-pyrimidine Watson-Crick pair. On the contrary, the size of the silver-mediated dZ-dC pair
is close to that of the canonical dG-dC pair. Furthermore, the X-ray structure of the dC-Ag-dC
pair[3a] teaches us that in canonical DNA the nucleobases are in a cisoid alignment. However, in
the methylated base derivatives cisoid and transoid alignments have been reported.[4e,10] The
orientation of the dZ-Ag-dC base pair can be Watson-Crick like or reverse Watson-Crick like.
For the formation of a silver-mediated base pair, the stability of the silver-free mismatch plays an
important role. Promiscuous base pairing and dependence on rigidity or flexibility of nearest
neighbors segments have to be considered as well. Thus, the final increase of the helix stability
generated by a silver-mediated base pair depends on a number of factors caused by the mismatch
and the silver-ion mediated base pair within duplex DNA.
Figure 2 shows typical melting profiles of three selected duplexes in the absence and presence of
silver ions. In all cases, thermodynamically stable silver-mediated 5-aza-7-deazaguanine -
cytosine base pairs were formed indicated by a significant increase of Tm values. In order to
demonstrate the specificity of the process sodium bromide was added to the buffer solution.
Bromide ions can capture silver ions from the silver-mediated base pair and turn the metal pair
back to a silver ion free mismatch.[5i] The recovery of the initial melting profiles after halide
addition are shown in Figures 2a,c,d confirming the reactivation of the silver ion free duplex and
the stabilization by silver ions. Stoichiometric titration experiments are used to confirm the
consumption of silver-ion equivalents per base pair (Figure 2b).
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Figure 2. Thermal denaturation curves were measured at 2.5 µM + 2.5 µm single-strand
concentration in 100 mM NaOAc, 10 mM Mg(OAc)2, pH 7.4 at 260 nm in the absence (black
curves) and presence of two equivalents of Ag+-ions (red curves) followed by addition of 20 eq.
of NaBr (blue curve). a) 5’-d(TAGGTC2AT ACT) (ODN-3) • 3’-d(ATCCAG4aTATGA)
(ODN-4); c) 5’-d(TAGGTC2ATACT) (ODN-3) • 3’-d(ATCCAG4bTATGA) (ODN-5); d) 5’-
d(TAG GTC2ATACT) (ODN-3) • 3’-d(ATCCAG4cTATGA) (ODN-6); b) titration graph
displaying consumption of silver ions/duplex versus changes in UV absorbance (measured at 260
nm) for duplex ODN-3 • ODN-4. All duplexes contain α-D/α-D homo base pairs.
11
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In the following silver-mediated base pair stability was studied using temperature dependent
melting of mismatch DNA in the absence and presence of silver ions (Tables 2 and 3). Homo
and hybrid base pairs were investigated. The homo base pairs were either β-D/β-D or α-D/α-D
pairs whereas in the series of hybrid base pairs either the α-anomer of dZ paired with β-dC or the
β-anomer of dZ base pairs with α-dC. Furthermore, the positional impact of silver-mediated base
pair formation was examined by shifting the metal ion base pair from position-7 to position-6.
Also, the impact of space demanding residues (iodo, octadiynyl) located at the 5-position of dC
was evaluated with regard to a possible functionalization without disturbing the helical DNA
structure.
The Tm increase induced by silver ions was then used to establish silver-mediated base pair
formation. Table 2 and 3 summarize Tm data for mismatches incorporated in the standard duplex
ODN-1•ODN-2. In Table 2, a dA-dT base pair at position-7 was replaced by silver-mediated
base pairs, whereas in Table 3 a dG-dC base pair at position-6 was substituted by the silver ion
pair. All experiments were performed under identical conditions in a 100 mM NaOAc, 10 mM
Mg(OAc)2 buffer at pH 7.4 and were measured at 260 nm in the absence and the presence of
silver ions. Single-strand concentration was 2.5 µM. All curves show sigmoidal melting profiles
and no or very little hysteresis.
Table 2 shows Tm data for duplexes with dZ-dC mismatches at position-7 of the duplex in the
presence and the absence of silver ions. The upper part of the Table 2 displays homo pairs with
α-D/α-D mismatches (left) and β-D/β-D mismatches (right). The lower part of the Table shows
Tm data for α-D/β-D hybrid pairs under participation of the α-dZ anomer 2 (left) and the β-dZ
anomer 1 (right). A significant Tm increase was observed for α-dZ (2) in base pairs with α-dC
(homo pairs) or β-dC (hybrid pairs), whereas duplexes with β-dZ did not show any significant Tm
increase after silver ion addition, neither in homo pairs nor in hybrid pairs (right side of Table 2).
For the β-D/β-D homo pairs the small stability increase might be explained by the high stability
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10.1002/chem.201901276
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of the silver-free mismatches. These mismatches are too stable to complex silver ions; a situation
which is similar to the standard duplex that does not form silver-mediated base pairs under the
same conditions. However, this explanation does not hold for the hybrid pairs.
Furthermore, stability changes were observed when nucleoside residues in mismatches were
reversed (duplex 3•5 vs duplex 7•8). Thus, in the homo base pairs a positional impact of the
various dZ-dC silver pairs exists when the 5-aza-7-deazapurine nucleoside is moved from one
strand to the other. It should also be noted that the side chain modifications at the dC site have an
influence on the mismatch stability. This is shown for base pairs containing 5-iodo-2'-
deoxycytidines (3b, 4b) or 5-octadiynyl-2'-deoxycytidines (3c, 4c) with anomeric sugar
configurations. 5-Iodo and 5-octadiynyl substituents introduced at the dC side of the homo base
pairs have only a small impact on stability in the presence or absence of silver ions. On the
contrary, for the hybrid base pairs the situation is more complex. In case of the β-D/α-D hybrid
pair even a destabilizing effect is observed which is more pronounced for the 5-iodo substituent
than for the 5-octadiynyl side chain.
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Table 2. Tm values of oligonucleotide duplexes containing α/α- and β/β-homo base pairs and α/β-hybrid base pairs of anomeric dZ nucleosides (1
and 2) with anomeric dC residues (3a-c and 4a-c) in the presence and absence of silver ions replacing a dA-dT base pair (position-7).[a]
Tm value [°C]
+1Ag+/
duplex
(∆Tm [°C])
Tm value [°C]
+2Ag+/
duplex
(∆Tm [°C])
Tm value [°C]
+1Ag+/
duplex
(∆Tm [°C])
48
Tm value [°C]
+2Ag+/
duplex
(∆Tm [°C])
47
Tm
value
[°C]
Duplexes
Duplexes
Tm value
[°C]
α-D/α-D Homo Pairs
β-D/β-D Homo Pairs
5’-d(TAG GTC AAT ACT) (ODN-1)
3'-d(ATC CAG TTA TGA) (ODN-2)
5’-d(TAG GTC AT ACT) (ODN-10)
3’-d(ATC CAG TA TGA) (ODN-12)
5’-d(TAG GTC AT ACT) (ODN-10)
3’-d(ATC CAG TA TGA) (ODN-13)
5’-d(TAG GTC AT ACT) (ODN-10)
3’-d(ATC CAG TA TGA) (ODN-14)
5’-d(TAG GTC AT ACT) (ODN-15)
3’-d(ATC CAG TA TGA) (ODN-11)
5’-d(TAG GTC AT ACT) (ODN-16)
3’-d(ATC CAG TA TGA) (ODN-11)
47
38
40
40
40
42
(+1)
40
(+2)
41
(+1)
40
(±0)
42
(±0)
41
(+3)
42
(+2)
41
(+1)
42
5’-d(TAG GTC AT ACT) (ODN-3)
3’-d(ATC CAG TA TGA) (ODN-4)
5’-d(TAG GTC AT ACT) (ODN-3)
3’-d(ATC CAG TA TGA) (ODN-5)
5’-d(TAG GTC AT ACT) (ODN-3)
3’-d(ATC CAG TA TGA) (ODN-6)
5’-d(TAG GTC AT ACT) (ODN-7)
3’-d(ATC CAG TA TGA) (ODN-8)
5’-d(TAG GTC AT ACT) (ODN-9)
3’-d(ATC CAG TA TGA) (ODN-8)
41
(+5)
41
(+5)
40
(+3)
39
(+4)
40
(+4)
43
(+7)
44
(+8)
43
(+6)
39
(+4)
40
(+4)
36
36
37
35
36
(+2)
43
(+1)
(+2)
44
(+2)
α-D/β-D Hybrid Pairs
β-D/α-D Hybrid Pairs
5’-d(TAG GTC AT ACT) (ODN-3)
3’-d(ATC CAG TA TGA) (ODN-12)
38
(+4)
39
(+5)
5’-d(TAG GTC AT ACT) (ODN-10)
3’-d(ATC CAG TA TGA) (ODN-4)
35
(±0)
34
(-1)
34
35
5’-d(TAG GTC AT ACT) (ODN-3)
3’-d(ATC CAG TA TGA) (ODN-13)
5’-d(TAG GTC AT ACT) (ODN-3)
3’-d(ATC CAG TA TGA) (ODN-14)
5’-d(TAG GTC AT ACT) (ODN-15)
3’-d(ATC CAG TA TGA) (ODN-8)
5’-d(TAG GTC AT ACT) (ODN-16)
3’-d(ATC CAG TA TGA) (ODN-8)
34
(+4)
34
(+2)
31
(+2)
34
(+3)
36
(+6)
36
(+4)
31
(+2)
33
(+2)
5’-d(TAG GTC AT ACT) (ODN-10)
3’-d(ATC CAG TA TGA) (ODN-5)
5’-d(TAG GTC AT ACT) (ODN-10)
3’-d(ATC CAG TA TGA) (ODN-6)
5’-d(TAG GTC AT ACT) (ODN-7)
33
(±0)
33
(-1)
32
(±0)
36
(-1)
32
(-1)
32
(-2)
32
(±0)
36
(-1)
30
32
29
31
33
34
32
37
3’-d(ATC CAG
TA TGA) (ODN-11)
5’-d(TAG GTC AT ACT) (ODN-9)
3’-d(ATC CAG TA TGA) (ODN-11)
[a] Measured at 260 nm with 2.5 µM + 2.5 µM single-strand concentration at a heating rate of 1.0 °C/min in 100 mM NaOAc, 10 mM Mg(OAc)2, pH 7.4 in the presence of various
concentrations of AgNO3 (0-2 eq. of silver ions/duplex). Tm values were calculated from the heating curves using the program Meltwin 3.0.[11] ∆Tm = Tm after the addition of AgNO3
– Tm before the addition of AgNO3.
14
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Next, mismatches were shifted from the 7-position to position-6 replacing a dG-dC pair of the
double helix (Table 3). Now, the mismatches are surrounded by two dA-dT Watson-Crick pairs.
In this position, the silver-free mismatches are less stable than those in position-7 (Table 2). This
is the result from the loss of a dG-dC pair in this series compared to a dA-dT pair shown in the
series of Table 2. In position-7, the most stable duplexes are formed by silver-mediated β-D/β-D
homo pairs, whereas α-D/α-D pairs show significantly lower Tm increases. Also, hybrid pairs
containing the α-dZ 2 show a Tm increase after silver ion addition. The higher Tm values of the β-
d/β-D homo base pairs compared to the α-D/α-D homo and α-D/β-D hybrid pairs result mainly
from the higher stability of the silver-free mismatches. To visualize the rather complex situation
bar diagrams were composed (Figure 3) summarizing the Tm values from Tables 2 and 3.
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Table 3. Tm values of oligonucleotide duplexes containing α/α- and β/β-homo base pairs and α/β-hybrid base pairs of anomeric dZ nucleosides (1
and 2) with anomeric dC residues (3a-c and 4a-c) in the presence and absence of silver ions replacing a dG-dC base pair (position-6).[a]
Tm value [°C]
Tm value [°C]
Tm value [°C] Tm value [°C]
+1Ag+/
+2Ag+/
Tm value
[°C]
+1Ag+/
duplex
(∆Tm [°C])
42
+2Ag+/
duplex
(∆Tm [°C])
42
Duplexes
α-D/α-D Homo Pairs
Duplexes
β-D/β-D Homo Pairs
Tm value
[°C]
duplex
(∆Tm [°C])
duplex
(∆Tm [°C])
5’-d(TAG GTAAAT ACT) (ODN-17)
3'-d(ATC CAT TTATGA) (ODN-18)
5’-d(TAG GT AAT ACT) (ODN-31)
3’-d(ATC CA TTA TGA) (ODN-26)
5’-d(TAG GT AAT ACT) (ODN-31)
3’-d(ATC CA TTA TGA) (ODN-27)
5’-d(TAG GT AAT ACT) (ODN-31)
3’-d(ATC CA TTA TGA) (ODN-28)
5’-d(TAG GT AAT ACT) (ODN-29)
3’-d(ATC CA TTA TGA) (ODN-32)
5’-d(TAG GT AAT ACT) (ODN-30)
3’-d(ATC CA TTA TGA) (ODN-32)
42
30
30
29
35
31
(±0)
(±0)
5’-d(TAG GT AAT ACT) (ODN-19)
3’-d(ATC CA TTA TGA) (ODN-20)
5’-d(TAG GT AAT ACT) (ODN-19)
3’-d(ATC CA TTA TGA) (ODN-21)
5’-d(TAG GT AAT ACT) (ODN-19)
3’-d(ATC CA TTA TGA) (ODN-22)
5’-d(TAG GT AAT ACT) (ODN-23)
3’-d(ATC CA TTA TGA) (ODN-24)
5’-d(TAG GT AAT ACT) (ODN-25)
3’-d(ATC CA TTA TGA) (ODN-24)
28
(+3)
27
(+3)
28
(+2)
27
(+2)
29
(+4)
29
(+4)
27
(+3)
29
(+3)
28
(+3)
31
(+6)
37
38
25
24
26
25
25
(+7)
33
(+8)
34
(+3)
(+4)
32
34
(+3)
37
(+5)
38
(+2)
(+3)
33
35
(+2)
(+4)
α-D/β-D Hybrid Pairs
β-D/α-D Hybrid Pairs
5’-d(TAG GT AAT ACT) (ODN-19)
3’-d(ATC CA TTA TGA) (ODN-26)
27
(+2)
29
(+4)
5’-d(TAG GT AAT ACT) (ODN-31)
3’-d(ATC CA TTA TGA) (ODN-20)
24
(±0)
24
(±0)
25
24
5’-d(TAG GT AAT ACT) (ODN-19)
3’-d(ATC CA TA TGA) (ODN-27)
5’-d(TAG GT AAT ACT) (ODN-19)
3’-d(ATC CA TTA TGA) (ODN-28)
5’-d(TAG GT AAT ACT) (ODN-29)
3’-d(ATC CA TTA TGA) (ODN-24)
5’-d(TAG GT AAT ACT) (ODN-30)
3’-d(ATC CA TTA TGA) (ODN-24)
22
(+2)
26
(+2)
27
(+3)
24
(+4)
23
(+3)
28
(+4)
27
(+3)
25
(+5)
5’-d(TAG GT AAT ACT) (ODN-31)
3’-d(ATC CA TTA TGA) (ODN-21)
5’-d(TAG GT AAT ACT) (ODN-31)
3’-d(ATC CA TTA TGA) (ODN-22)
5’-d(TAG GT AAT ACT) (ODN-23)
3’-d(ATC CA TTA TGA) (ODN-32)
5’-d(TAG GT AAT ACT) (ODN-25)
3’-d(ATC CA TTA TGA) (ODN-32)
24
(-1)
24
(-1)
24
(±0)
24
(±0)
22
(-3)
24
(-1)
24
(±0)
24
(±0)
20
24
24
20
25
25
24
24
[a] Measured at 260 nm with 2.5 µM + 2.5 µM single-strand concentration at a heating rate of 1.0 °C/min in 100 mM NaOAc, 10 mM Mg(OAc)2, pH 7.4 in the presence of
various concentrations of AgNO3 (0-2 eq. of silver ions/duplex). Tm values were calculated from the heating curves using the program Meltwin 3.0.[11] ∆Tm = Tm after the addition
of AgNO3 – Tm before the addition of AgNO3.
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Figure 3. Bar diagrams of Tm values for DNA duplexes containing homo and hybrid
base pairs of anomeric dZ ( ) with anomeric 5-substituted dC (3a 4a ) in the
absence (first bar) and presence (second bar) of silver ions according to Tables 2 and 3.
a) 5’-d(TAGGTC ATACT) • 3’-d(ATCCAGYTATGA) (7-position); b) 5’-
d(TAGGT AATACT) • 3’-d(ATCCA TTATGA) (6-position).
1
,
2
-c, -c
X
X
Y
From the figure the following conclusions can be drawn. Position-6: (i) the highest Tm
increases and most stable silver-mediated base pairs are observed when both nucleosides
display β-D configuration. (ii) The α-D/α-D homo pairs are less stable and show reduced Tm
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increases after silver ion addition. (iii) Strong Tm increases but less stable base pairs are
observed for the hybrid pairs with dZ in α-configuration, whereas the β/α pairs becomes
destabilized after silver ion addition.
Position-7: (i) the α/α combinations form the most stable silver-mediated base pairs and show
significant Tm increases. (ii) The β/β pairs are significantly less stable and the Tm increase
after silver addition is low. (iii) The α/β hybrid pairs are stabilized by silver ion addition,
when dZ is in α-configuration, whereas destabilization is observed when β-dZ participates in
the hybrid base pair. This is similar to position-6. Stability changes are also observed when
the nucleobase is functionalized. The 5-iodo- and 5-octadiynyl substituents have a moderate
but noticeable influence on mismatches and stability of silver-mediated DNA. However, these
residues are in general well accommodated in the metal DNA. For the homo base pairs the
effect of functionalization is almost negligible, whereas the situation is more complex in case
of the hybrid base pairs. In presence of silver ions a destabilizing effect is observed which is
more pronounced for the 5-iodo compared to the 5-octadiynyl substituent.
The global structure of DNA is extremely flexible and is able to accommodate distortions
introduced by mismatches without altering the overall structure. On the local level the
influence of mismatches can be moderate or strong. Taking into account that the stability of
Watson-Crick base pairs and silver-free mismatches depends already on nearest neighbors[12]
or even on larger areas of the double helix, the stability of silver-mediated dZ-dC base pairs
shows a similar behavior. The position of incorporation, the configuration of the nucleoside
residue and modifications by substituents are of importance. The studies on homo and hybrid
base pairs demonstrate that the helical DNA environment on both sides of a silver-mediated
base pair has a significant impact on its stability. In principle, each combination of α- and β-
anomers 1 and 2 with dC generates a particular mismatch and a corresponding silver-
mediated base pair of unique stability.
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A general stability increase induced by silver-mediated base pair formation is observed for
almost each silver-mediated base pair at both positions (6- and 7-substitution). However, the
individual Tm increases differ significantly. When already rather stable mismatches are
formed in the absence of silver ions, the tendency to form thermodynamically stable silver-
mediated base pairs is lower than in the case of low stability mismatches. Thus, the impact of
the local environment of the silver-mediated base pair can vary from moderate to very
extensive. Nevertheless, stability changes caused by substituents, configuration or nearest
neighbors is different in silver-mediated base pair mismatches compared to Watson-Crick
pairs.
It is also obvious, that the silver-mediated dZ-dC base pairs behave differently as the recently
investigated anomeric dC-dC pairs.[8] The distortion of the double helix by dC/dC mismatches
is almost negligible, whereas local changes by purine-pyrimidine mismatches are more
pronounced. In the latter cases, changes of the groove dimensions or breathing of the helix are
more likely. Thus, it is difficult to predict structures for the various mismatches and silver-
mediated base pairs. However, we anticipate that Watson-Crick like base pairs are formed in
homo base pair interactions (α/α and β/β) according to Figure 4. The higher stability of these
structures might be caused by the formation of one additional hydrogen bond, a phenomenon
that was also reported for other silver-mediated base pairs.[13] The formation of two hydrogen
bonds is unlikely because to the size of the silver ion. In the less stable hybrid base pairs
hydrogen bond formation is unlikely due to the possible reverse Watson-Crick like alignment
of the nucleobases in the base pair (Figure 4). To this end DFT calculations[14-17] were
performed for the dZ-dC base pair containing one silver ion. The position of the silver ion
was found to connect nitrogen-1 of the 5-aza-7-deazaguanine base with nitrogen-3 of the
cytosine residue (Figure 4e). The silver-mediated base pair forms a Watson-Crick like motif.
The dihedral angle N1(9)-Ag-N3(4) = 159° is not far from a linear connectivity. The
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hydrogen of the amino group of the 5-aza-7-deazaguanine moiety and the oxo group of
cytosine form an additional hydrogen bond (N-H…O = 176°, H…O = 2.11 Å).
When the calculation started with the reverse Watson-Crick like motif of the silver-mediated
dZ-dC pair the reverse structure collapsed during calculation to a Watson-Crick like structure.
This confirms the low stability of the reverse Watson-Crick motif. Nevertheless, final answers
on the individual structures of the silver-mediated base pairs cannot be given. The positional
dependence of the base pair stability reported in this work indicates that the DNA
environment surrounding the silver-mediated base pair has a strong impact on its stability, a
phenomenon which is already known from silver-free DNA mismatches.[12] This work also
shows that the high stability of silver-free mismatches can impede silver-mediated base pair
formation. Furthermore, it should be recalled that the DNA double helix backbone and
therefore the position of incorporation might even alter the molecular structure of an
individual silver-mediated base pair and base stacking has a significant impact on base pair
stability.
Figure 4. Silver-mediated base pairs with Watson-Crick like alignment and one hydrogen
bond (a,c) or reverse Watson-Crick like alignment (b) and the canonical Watson-Crick dG-dC
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base pair (d). Structure obtained from DFT calculations of the silver-mediated dZ-Ag+-dC
pair (e). S corresponds to 2’-deoxyribofuranosyl sugar residues.
Conclusion
The stability of silver mediated base pairs of α-D and β-D anomeric 5-aza-7-deaza-2’-
deoxyguanosines 1 and 2 with anomeric 2'-deoxycytidines 3 to 4 depends on nucleoside
configuration, nucleobase substituents and the helical environment that surrounds the metal
mediated DNA base pair. For this, the pure nucleoside anomers 1 and 2 were synthesized.
Their access was significantly improved by the use of the isobutyrylated nucleobase 6b or its
salt 6a for a particular anomer formed by nucleobase anion glycosylation. Efficient separation
of anomeric glycosylation products was accomplished by combination of crystallization and
chromatography. A series of phosphoramidites were prepared and used to synthesize 12-mer
oligonucleotides with all four possible combinations of anomeric 5-aza-7-deaza-2'-
deoxyguanosine and anomeric 2'-deoxycytidines. They were hybridized to form α-D/α-D and
β-D/β-D homo base pairs as well as α-D/β-D and β-D/α-D hybrid pairs at position-6 or
position-7 of duplex DNA. The individual stability of mismatches increases after silver ion
addition and differs significantly between the various series. α-D/β-D hybrid base pairs were
less stable in this series, whereas homo base pairs with β/β or even α/α nucleoside
combination form the most stable complexes. 5-Iodo and 5-octadiynyl substituents introduced
at the dC side of the homo base pairs have only a small impact on stability while the situation
is more complex for hybrid base pairs. In case of the β-D/α-D hybrid pair even a destabilizing
effect is observed, which is more pronounced for the 5-iodo derivatives than those with 5-
octadiynyl side chain. The studies performed on silver-mediated homo and hybrid base pairs
have the potential to be used for the development of new metal/DNA hybrid materials or as
analytical tools for DNA diagnostics and detection.
21
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Experimental Section
General methods and materials
All chemicals and solvents were of laboratory grade as obtained from commercial suppliers
and were used without further purification. Reversed-phase HPLC was carried out using a
HITACHI 655A-12 liquid chromatograph connected to L6200A intelligent pump and 655A
variable wavelength UV monitor on a 4 x 250 mm RP-18 (10 μm) LiChrospher 100 column
with a HPLC pump connected with a variable wavelength monitor, a controller and an
integrator. The molecular masses of the oligonucleotides were determined by MALDI-TOF
mass spectrometry on a Bruker Autoflex Speed in the linear positive mode with 3-
hydroxypicolinic acid (3-HPA) as a matrix. The thermal melting curves were measured with
an Agilent Technologies Cary 100 Bio UV-vis spectrophotometer equipped with a
thermoelectrical controller. The temperature was measured continuously in the reference cell
with a Pt-100 resistor with a heating rate of 1 °C min-1. Tm values were determined from the
melting curves using the software MELTWIN, version 3.0.[11]
Computational details
DFT calculations were used to optimize the molecular structure of the silver-mediated homo
base pair. The calculation was performed using Gausssian 09[14] program package with
B3LYP-D3[15] functional. The 6-311 G(d,p)[16] and LANL2DZ[17] basis sets were employed
for light atoms (C, H, O, N) and Ag+-ion, respectively.
Oligonucleotide syntheses and characterization
Solid-phase oligonucleotide syntheses were performed on an ABI 392-08 synthesizer at 1
µmol scale (trityl-on mode) employing the phosphoramidites of α- and β-dZ,[7f] (13, 14;
Scheme) and of α- and β-dC[8b] (15a-c, 16a-c; for structures see Figure S3 in the Supporting
22
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Information) as well as the standard building blocks with an average coupling yield over 95%.
After cleavage from the solid support, the oligonucleotides were deprotected in 28% aqueous
ammonia at 55 °C for 2 h. The DMT-containing oligonucleotides were purified by reversed-
phase HPLC (RP-18) with the gradient system at 260 nm: (A) MeCN, (B) 0.1 M (Et3NH)OAc
(pH 7.0)/MeCN, 95:5; gradient I: 0-3 min 10-15% A in B, 3-15 min 15-50% A in B; flow rate
0.7 mL/min. The purified “trityl-on” oligonucleotides were treated with 2.5%
CHCl2COOH/CH2Cl2 for 2 min at 0 °C to remove the 4,4′-dimethoxytrityl residues. The
detritylated oligomers were purified again by reversed-phase HPLC with gradient II: 0-20 min
0-20% A in B; 20-25 min, 20% A in B; flow rate 0.7 mL/min. The oligonucleotides were
desalted on a reversed-phase column (RP-18) using water for elution of salt, while the
oligonucleotides were eluted with H2O/MeOH (2:3). The oligonucleotides were lyophilized
on a Speed-Vac evaporator to yield colorless solids which were frozen at -24 °C. The purity
of all oligonucleotides was confirmed by RP-18 HPLC (Figure S4, Supporting Information)
and MALDI-TOF mass spectrometry (Table 1). The extinction coefficients ε260 (H2O) of the
nucleosides are: dA 15400, dG 11700, dT 8800, dC 7300, β-dZ (1) 14100, α-dZ (2) 13700,
(3b) 3100 (MeOH)[8b], (3c) 5600 (MeOH)[8b], (4b) 3400 (MeOH)[8b], (4c) 5900 (MeOH)[8b]
The extinction coefficients of the oligonucleotides were calculated from the sum of the
extinction coefficients of nucleoside constituents.
.
2-[(2-Methylpropionyl)amino]-8H-imidazo[1,2-a]-s-triazin-4-one • ibuOH (6a).
2-Aminoimidazo[1,2-a]-s-triazin-4(8H)-one[7a] (5) (1.5 g, 9.93 mmol) was suspended in
isobutyric anhydride (35 mL). Then, two drops of 85% phosphoric acid were added to this
solution and the reaction mixture was refluxed until the solution became clear (1.5 h). The
reaction mixture was filtered and the remaining clear solution was stored at r.t. overnight.
During this period the product precipitated. The precipitate was filtrated and washed by ice-
23
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cold water (3 x 10 mL) furnishing compound 6a as colorless solid (2.10 g, 68%). Rf = 0.5
(CH2Cl2/CH3OH 9:1);1H NMR (600 MHz, [D6]DMSO, 26°C): δ = 12.51 (bs, 1H; NH), 10.68
(bs, 1H; NH), 7.53 (d, J = 1.3 Hz, 1H; H-7), 7.38 (s, 1H; H-6), 2.82-2.86 (m, 1H; CH), 2.38-
2.43 (m, 1H; CH), 1.04-1.07 ppm (m, 12H; CH3); 13C NMR (101 MHz, [D6]DMSO, 26°C): δ
= 18.91 (CH(CH3)2), 19.05 (CH(CH3)2), 33.13 ((CH(CH3)2), 37.80 ((CH(CH3)2), 109.02 (C-
7), 149.43 (C-8a), 177.84 (C=O); UV/Vis (CH3OH): λmax (ε) = 224 (18000), 278 nm (14100
mol-1dm3cm-1); HRMS (ESI-TOF): m/z calcd for C9H11N5O2Na: 244.0805 [M + Na]+; found
244.08025.
2-[(2-Methylpropionyl)amino]-8H-imidazo[1,2-a]-s-triazin-4-one (6b).[7b] Compound 6a
(1.0 g, 3.23 mmol) was dissolved in 10% aq. MeOH (10 mL) by gentle warming. When the
base was completely dissolved, the solution was immediately cooled to 0°C (ice-bath). After
10 min., white crystals appeared which were filtered and dried furnishing 6b (410 mg, 57%).
Evaporation of the mother liquor gave another crop of 275 mg (38%) of 6b. The materials
obtained from crystallization and evaporation of mother liquor were identical. Rf = 0.5
(CH2Cl2/CH3OH 9:1);1H NMR (600 MHz, [D6]DMSO, 26°C): δ = 12.85 (bs, 1H; NH), 10.65
(bs, 1H; NH), 7.53 (d, J =2.0 Hz, 1H; H-7), 7.38 (s, 1H; H-6), 2.82-2.86 (m, 1H; CH), 1.06
ppm (d, J = 6.9 Hz, 6H; CH3); 13C NMR (101 MHz, [D6]DMSO, 26°C): δ = 22.13
(CH(CH3)2), 37.80 ((CH(CH3)2), 112.1 (C-7), 152.95 (C-8a), 180.21 (C=O); UV/Vis
(CH3OH): λmax (ε) = 224 (15900), 278 nm (13600 mol-1dm3cm-1); HRMS (ESI-TOF): m/z
calcd for C9H11N5O2Na: 244.0805 [M + Na]+; found 244.08025.
Glycosylation of 2-aminoimidazo[1,2-a]-s-triazin-4(8H)-one • ibuOH (6b) with 2-deoxy-
3,5-di-O-(p-toluoyl)-β-D-erythro-pentofuranosyl chloride (7). The nucleobase 6b (0.50 mg,
1.62 mmol) was dissolved in dry CH3CN (60 mL) under gentle warming. After cooling to r.t.
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Chemistry - A European Journal
K2CO3 (996 mg, 3.6 mmol) and tris[2-(2-methoxyethoxy)ethyl]amine (100 μL, 0.3 mmol)
were added to the solution and stirring was continued for 20 min at rt. Then, the halogenose 7
(1.75 g, 4.52 mmol) was added in three portions over 10 min. Stirring at r.t. was continued
until the base was completely consumed (1 h, TLC-monitoring). Then, the reaction mixture
was filtrated and the solvent was evaporated. The remaining residue was dissolved in CH2Cl2
(5 mL) and applied to FC (silica gel, column 14 x 4 cm, CH2Cl2/MeOH 100:0 →99:1). From
the main zone a mixture of the anomers 8 and 9 (851 mg, 92%) in a ratio of 3/2 (β/α) was
obtained as colorless foam. The anomeric ratio was calculated on the basis of the 1H NMR
chemical shifts.
8-[2-Deoxy-3,5-di-O-(p-toluoyl)-β-D-erythro-pentofuranosyl]-2-[(2-methylpropionyl)
amino-8H-imidazo[1,2-a]-s-triazin-4-one (8).[7b] The anomeric mixture of 8 and 9 obtained
by glycosylation (358 mg) was dissolved in MeOH (25 mL) by reflux for 2 min. While
rapidly cooling the hot solution to r.t the β-D-anomer 8 crystallized as colorless needles.
Filtration and drying afforded β-D nucleoside 8 (428 mg, 46%). Rf = 0.50 (CH2Cl2/CH3OH
95:5);1H NMR (400 MHz, [D6]DMSO, 26°C): δ = 10.39 (s, 1H; NH), 7.93 (d, J = 8.2 Hz,
2H; Ar-H), 7.84 (d, J = 8.2 Hz, 2H; Ar-H), 7.72 (d, J = 2.8 Hz, 1H; H-6), 7.62 (d, J = 2.7 Hz,
1H; H-7), 7.37 (d, J = 8.0 Hz, 2H; Ar-H), 7.29 (d, J = 8.0 Hz, 2H; Ar-H), 6.41 (t, J = 7.0 Hz,
1H; H-1'), 5.78 (dt, J = 6.6, 2.6 Hz, 1H; H-3’), 4.65-4.51 (m, 3H; H-4’, H-5’), 3.25-3.17 (m,
1H; Hβ-2’), 2.94-2.85 (m, 1H; CH), 2.73 (d, J = 18.0 Hz, 1H; Hα-2’), 2.40, 2.36 (s, 6H; Ar-
CH3), 1.04 ppm (d, J = 6.8 Hz, 6H; CH(CH3)2); 1H NMR (600 MHz, CDCl3, 26°C): δ = 8.09
(s, 1H; NH), 7.95 (d, J = 8.2 Hz, 2H; Ar-H), 7.86 (d, J = 8.2 Hz, 2H, Ar-H), 7.37 (d, J = 2.7
Hz, 1H; H-6), 7.28 (d, J = 8.4 Hz, 2H; Ar-H), 7.23 (d, J = 8.0 Hz, 2H; Ar-H), 7.11 (d, J = 2.7
Hz, 1H; H-7), 6.49-6.46 (m, 1H; H-1'), 5.74 (dt, J = 5.1, 2.3 Hz, 1H; H-3’), 4.76 (dd, J = 11.5,
2.9 Hz, 1H; H-4’), 4.66-4.60 (m, 2H; H-5’), 3.15-3.13 (m, 1H; Hβ-2’), 2.91-2.81 (m, 2H; Hα-
25
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10.1002/chem.201901276
Chemistry - A European Journal
2’, CH), 2.44, 2.41 (s, 6H; Ar-CH3), 1.23 ppm (dd, J = 6.9, 2.3 Hz, 6H; CH(CH3)2); 13C NMR
(101 MHz, [D6]DMSO, 26°C): δ = 175.64 (C=O), 165.39 (arom. C), 165.16 (arom. C),
160.72 (C-4), 149.94 (C-2), 149.40 (C-8a), 144.08 (arom. C), 143.78 (arom. C), 129.43
(arom. C), 129.32 (arom. C), 129.26 (arom. C), 129.23 (arom. C), 126.58 (arom. C), 126.48
(arom. C), 116.82 (C-7), 108.98 (C-6), 84.44 (C-1’), 81.92 (C-4’), 74.85 (C-3’), 64.06 (C-5’),
35.47 (CH(CH3)2), 35.46 (C-2’), 21.19, 21.15 (CH3, (toluoyl)), 19.05, 19.03 (CH(CH3)2);
UV/Vis (CH3OH): λmax (ε) = 277 (13800), 242 nm (37600 mol-1dm3cm-1); HRMS (ESI-TOF):
m/z calcd for C30H31N5O7Na: 596.2116 [M + H]+; found 596.2115.
8-[2-Deoxy-3,5-di-O-(p-toluoyl)-α-D-erythro-pentofuranosyl]-2-[(2-methylpropionyl)
amino-8H-imidazo[1,2-a]-s-triazin-4-one (9). [7b] The mother liquor obtained after
crystallization of the β-D-anomer 8 was evaporated and applied to FC (silica gel, column 14 x
4 cm, CH2Cl2/MeOH 100:0 →98:2). From the faster migrating zone the α-D anomer 9 was
obtained (198 mg, 21%) as colorless foam. Rf = 0.55 (CH2Cl2/CH3OH 95:5); 1H NMR (400
MHz, [D6]DMSO, 26°C): δ = 10.31 (s, 1H; NH), 7.92 (d, J = 8.2 Hz, 2H; Ar-H), 7.72 (d, J =
8.2 Hz, 2H; Ar-H), 7.69 (d, J = 2.7 Hz, 1H; H-6), 7.60 (d, J = 2.7 Hz, 1H; H-7), 7.36 (d, J =
8.0 Hz, 2H; Ar-H), 7.28 (d, J = 8.0 Hz, 2H; Ar-H), 6.43 (t, 1H; H-1'), 5.60 (dt, J = 5.3, 3.3 Hz,
1H; H-3’), 5.06 (td, J = 4.6, 2.4 Hz, 1H; H-4’), 4.56-4.47 (m, 2H; H-5’), 2.99 (t, J = 5.3 Hz,
2H; H-2’), 2.91 (m, J = 6.8 Hz, 1H; CH), 2.37, 2.39 (s, 6H; Ar-CH3), 1.05 ppm (d, J = 6.8 Hz,
6H; CH(CH3)2); 1H NMR (600 MHz, CDCl3, 26°C) δ = 8.43 (s, 1H; NH), 7.91 (d, J = 8.3 Hz,
2H; Ar-H), 7.63 (d, J = 8.3 Hz, 2H; Ar-H), 7.46 (d, J = 2.7 Hz, 1H; H-6 ), 7.28 (d, J = 2.7 Hz,
1H; H-7), 7.25 (d, J = 7.4 Hz, 2H; Ar-H), 7.18 (d, J = 7.9 Hz, 2H; Ar-H), 6.50 (dd, J = 6.7,
1.8 Hz, 1H; H-1'), 5.63 (dt, J = 6.4, 1.5 Hz, 1H; H-3’), 4.86 (td, J = 4.0, 1.6 Hz, 1H; H-4’),
4.57 (d, J = 4.1 Hz, 2H; H-5’), 3.10-2.97 (m, 3H, H-2’; CH), 2.40, 2.37 (s, 6H; Ar-CH3), 1.20
ppm (dd, J = 6.9, 1.0 Hz, 6H; CH(CH3)2); 13C NMR (101 MHz, [D6]DMSO, 26°C): δ =
26
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10.1002/chem.201901276
Chemistry - A European Journal
175.66 (C=O), 165.47 (arom. C), 165.10 (arom. C), 160.59 (C-4), 150.01 (C-2), 148.56 (C-
8a), 143.97 (arom. C), 143.91 (arom. C), 129.41 (arom. C), 129.34 (arom. C), 129.26 (arom.
C), 129.22 (arom. C), 126.56 (arom. C), 126.39 (arom. C), 116.52 (C-7), 108.32 (C-6), 85.86
(C-1’), 83.54 (C-4’), 74.63 (C-3’), 64.01 (C-5’), 37.02 (C-2’), 34.58 (CH(CH3)2), 21.18 (CH3,
(toluoyl)), 19.13, 19.05 (CH(CH3)2); UV/Vis (CH3OH): λmax (ε) = 280 (13100), 240 nm
(39600 mol-1dm3cm-1); HRMS (ESI-TOF): m/z calcd for C30H31N5O7NNa: 596.2116 [M +
H]+; found 596.2114.
2-Amino-8-[2-deoxy-3,5-di-O-(p-toluoyl)-α-D-erythro-pentofuranosyl]-8H-imidazo[1,2-
a]-s-triazin-4-one (10). The mother liquor obtained after crystallization of the β-D-anomer 8
was evaporated and applied to FC (silica gel, column 14 x 4 cm, CH2Cl2/MeOH 100:0
→95:5). From the slower migrating zone compound 10 was obtained (78 mg, 8.4%) as
colorless foam. Rf = 0.25 (CH2Cl2/CH3OH 95:5);1H NMR (600 MHz, [D6]DMSO, 26°C): δ =
7.91 (d, J = 8.2 Hz, 2H; Ar-H ), 7.78 (d, J = 8.2 Hz, 2H; Ar-H), 7.44 (d, J = 2.8 Hz, 1H; H-6),
7.37 (d, J = 2.8 Hz, 1H; H-7), 7.35 (d, J = 8.0 Hz, 2H; Ar-H), 7.32 (d, J = 7.9 Hz, 2H; Ar-H),
6.94 (s, 2H, NH2), 6.33 (dd, J = 7.1, 2.3 Hz, 1H; H-1'), 5.60 (dt, J = 6.9, 2.0 Hz, 1H; H-3’),
4.97-4.93 (m, 1H; H-4’), 4.51-4.45 (m, 2H; H-5’), 2.99 (dd, J = 14.6, 7.5 Hz, 1H; Hβ-2’), 2.77
(dt, J = 15.1, 1.9 Hz, 1H; Hα-2’), 2.38 ppm (d, J = 6.2 Hz, 6H; CH(CH3)2 ); 13C NMR (101
MHz, [D6]DMSO, 26°C): δ = 175.66 (C=O), 165.47 (arom. C), 165.20 (C-4), 165.19 (arom.
C), 149.93 (C-2), 149.63 (C-8a), 144.03 (arom. C), 143.95 (arom. C), 129.41 (arom. C),
129.34 (arom. C), 126.55 (arom. C), 126.40 (arom. C), 114.35 (C-7), 107.82 (C-6), 84.75 (C-
1’), 83.35 (C-4’), 74.74 (C-3’), 64.08 (C-5’), 36.98 (C-2’), 21.19, 21.18 (CH3, (toluoyl));
UV/Vis (CH3OH): λmax (ε) = 243 nm (37900 mol-1dm3cm-1); HRMS (ESI-TOF): m/z calcd for
C26H25N5O6H: 504.1878 [M + H]+; found 504.1869.
27
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10.1002/chem.201901276
Chemistry - A European Journal
2-Amino-8-(2-deoxy-β-D-erythro-pentofuranosyl)-8H-imidazo[1,2a]-s-triazin-4-one (1).
[7b] Compound 8 (1.15 g, 2.0 mmol) was suspended in NH3/MeOH (150 mL) and the reaction
mixture was stirred overnight until a clear solution was obtained After evaporation of the
solvent, the remaining residue was adsorbed on silica gel and applied to FC (silica gel,
column 15 x 4 cm, CH2Cl2/MeOH 4:1). From the main zone compound 8 was obtained as
colorless solid (520 mg, 94%). Rf = 0.5 (CH2Cl2/CH3OH 4:1); 1H NMR (600 MHz,
[D6]DMSO, 26°C): δ =7.45 (d, J = 2.8 Hz, 1H; H-7), 7.36 (d, J = 2.8 Hz, 1H; H-6), 6.94 (s,
2H; NH2), 6.20-6.15 (m, 1H; H-1'), 5.29 (d, J = 4.0 Hz, 1H; 3’-OH), 4.97 (t, J = 5.4 Hz, 1H;
5’-OH), 4.32 (dq, J = 6.3, 3.2 Hz, 1H; H-3’), 3.83-3.79 (m, 1H; H-4’), 3.58-3.48 (m, 2H; H-
5’), 2.38 (ddd, J = 13.3, 7.6, 5.7 Hz, 1H; Hβ-2’), 2.18 ppm (ddd, J = 13.2, 6.1, 3.2 Hz, 1H; Hα-
2’); UV/Vis (CH3OH): λmax (ε) = 258 nm (14100 mol-1dm3cm-1). HRMS (ESI-TOF): m/z calcd
for C10H13N5O4Na:290.0860 [M + Na]+; found 290.0863.
2-Amino-8-(2-deoxy-α-D-erythro-pentofuranosyl)-8H-imidazo[1,2a]-s-triazin-4-one (2).
[7b] As described above with compound 9 (1.70 g, 3.0 mmol), NH3/MeOH (150 mL). FC
(silica gel, column 15 x 4 cm, CH2Cl2/MeOH 4:1) gave compound 2 as colorless solid (710
mg, 86 %). Rf = 0.5 (CH2Cl2/CH3OH 4:1); 1H NMR (600 MHz, [D6]DMSO, 26°C): δ = 7.53
(d, J = 2.8 Hz, 1H; H-7), 7.33 (d, J = 2.8 Hz, 1H; H-6), 6.97-6.83 (m, 2H; NH2), 6.18 (dd, J =
8.0, 2.5 Hz, 1H; H-1'), 5.52 (d, J = 3.4 Hz, 1H; 3’-OH), 4.86 (t, J = 5.6 Hz, 1H; 5’-OH), 4.29
(dq, J = 5.5, 2.3 Hz, 1H; H-3’), 4.12-4.08 (m, 1H; H-4’), 3.43-3.39 (m, 2H; H-5’), 2.68 (ddd,
J = 14.4, 7.9, 6.6 Hz, 1H; Hβ-2’), 2.11 ppm (dt, J = 14.4, 2.3 Hz, 1H; Hα-2’); UV/Vis
(CH3OH): λmax (ε) = 258 (13700 mol-1dm3cm-1). HRMS (ESI-TOF): m/z calcd for
C10H13N5O4Na:290.0860 [M + Na]+; found 290.0863.
28
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10.1002/chem.201901276
Chemistry - A European Journal
Acknowledgements
We thank Dr. Simone Budow-Busse for critical reading of the manuscript. We would like to
thank Dr. M. Letzel, Universität Münster, Germany, for the ESI-TOF spectra and Prof. Dr. B.
Wünsch, Institut für Pharmazeutische und Medizinische Chemie, Universität Münster,
Germany to provide us with 600 MHz NMR spectra. We thank Mrs. Oxana Korzhenko for
the synthesis of oligonucleotides. We thank Prof. Dr. Jianping Hu, Sichuan Industrial Institute
of Antibiotics, Chengdu University, China for the DFT calculation. Funding by ChemBiotech,
Münster, Germany is gratefully acknowledged.
Conflict of interest
The authors declare no conflict of interest.
Key Words: DNA, 5-aza-7-deaza-2’-deoxyguanosine, silver ions, base pairing, duplex
stability
References
[1] J. D. Watson, F. H. C. Crick, Nature 1953, 171, 737-738.
[2] a) T. Yamane, N. Davidson, Biochim. Biophys. Acta 1962, 55, 609-621; b) T. Yamane, N.
Davidson, Biochim. Biophys. Acta 1962, 55, 780-782; c) R. H. Jensen, N. Davidson,
Biopolymers 1966, 4, 17-32; d) M. Daune, C. A. Dekker, H. K. Schachman, Biopolymers
1966, 4, 51-76; e) G. L. Eichhorn, J. J. Butzow, P. Clark, E. Tarien, Biopolymers 1967, 5,
283-296; f) N. Dattagupta, D. M. Crothers, Nucleic Acids Res. 1981, 9, 2971–2985; g) R. M.
Izatt, J. J. Christensen, J. H. Rytting, Chem. Rev. 1971, 71, 439- 481.
29
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