A family of click nucleosides for metal-mediated base pairing: Unravelling the principles of highly stabilizing metal-mediated base pairs

Article abstract of DOI:10.1002/chem.201402221

A family of artificial nucleosides has been developed by applying the Cu^I-catalyzed Huisgen 1,3-dipolar cycloaddition. Starting from 2-deoxy-β-D-glycosyl azide as a common precursor, three bidentate nucleosides have been synthesized. The 1,2,3-triazole involved in all three nucleobases is complemented by 1,2,4-triazole (TriTri), pyrazole (TriPyr), or pyridine (TriPy). Molecular structures of two metal complexes indicate that metal-mediated base pairs of TriPyr may not be fully planar. An investigation of DNA oligonucleotide duplexes comprising the new click nucleosides showed that they can bind Ag^I to form metal-mediated base pairs. In particular the mispair formed from TriPy and the previously established imidazole nucleoside is significantly stabilized in the presence of Ag ^I. A comparison of different oligonucleotide sequences allowed the determination of general factors involved in the stabilization of nucleic acids duplexes with metal-mediated base pairs. Artificial base pairs: The application of three closely related triazole-derived artificial nucleosides in metal-mediated base pairing is described (see figure). Depending on the complementary nucleoside, [2+2] and [2+1] coordination modes are possible. The investigation of different oligonucleotide sequences has enabled the derivation of general principles underlying the formation of highly stabilizing metal-mediated base pairs.

Full text of DOI:10.1002/chem.201402221

DOI: 10.1002/chem.201402221
Full Paper
&
Bioinorganic Chemistry
A Family of “Click” Nucleosides for Metal-Mediated Base Pairing:
Unravelling the Principles of Highly Stabilizing Metal-Mediated
Base Pairs
Tim Richters,[a] Olga Krug,[b] Jutta Kçsters,[a] Alexander Hepp,[a] and Jens Mꢀller*[a]
Abstract: A family of artificial nucleosides has been devel-
oped by applying the CuI-catalyzed Huisgen 1,3-dipolar cy-
cloaddition. Starting from 2-deoxy-b-d-glycosyl azide as
a common precursor, three bidentate nucleosides have been
synthesized. The 1,2,3-triazole involved in all three nucleo-
bases is complemented by 1,2,4-triazole (TriTri), pyrazole
(TriPyr), or pyridine (TriPy). Molecular structures of two
metal complexes indicate that metal-mediated base pairs of
TriPyr may not be fully planar. An investigation of DNA oli-
gonucleotide duplexes comprising the new “click” nucleo-
sides showed that they can bind AgI to form metal-mediated
base pairs. In particular the mispair formed from TriPy and
the previously established imidazole nucleoside is signifi-
cantly stabilized in the presence of AgI. A comparison of dif-
ferent oligonucleotide sequences allowed the determination
of general factors involved in the stabilization of nucleic
acids duplexes with metal-mediated base pairs.
Introduction
metal-mediated base pairs is also relevant from a fundamental
point of view: Several experiments and calculations have
shown that metal ions in neighboring metal-mediated base
pairs can interact with one another, for example, by ferromag-
netic or antiferromagnetic coupling.[12]
Nucleic acids are important building blocks in nanotechnolo-
gy.[1] They are regularly used for the directed assembly of func-
tional moieties.[2] One prominent possibility to introduce
metal-based functionality is the incorporation of a metal-medi-
ated base pair into a nucleic acid.[3] Metal-mediated base pairs
are formed when transition-metal ions coordinate to donor
atoms of complementary nucleobases. The corresponding nu-
cleosides can be either natural,[4] or they can comprise specifi-
cally designed ligands.[5] Depending on the type of ligand,
a metal-mediated base pair may contain up to two transition-
metal ions.[6] Moreover, different metal-mediated base pairs
can be incorporated into one duplex.[7] The structures of DNA
duplexes with metal-mediated base pairs show that the regular
B-type geometry is not necessarily disrupted by the introduc-
tion of the transition-metal ions.[8] This may be a reason why
metal-mediated base pairs can even be recognized and pro-
cessed by polymerases.[9] Several interesting applications have
been proposed for metal-mediated base pairs,[10] including the
introduction of improved charge-transfer properties into
DNA[11] as well as various sensor applications.[10] DNA with
To increase the scope of metal-mediated base pairing, we
devised a new family of bidentate triazole-based nucleosides.
They are capable of engaging in either a [2+2] or a [2+1] coor-
dination mode, depending on the complementary nucleoside
(Scheme 1).
[a] T. Richters, J. Kçsters, Dr. A. Hepp, Prof. Dr. J. Mꢀller
Institut fꢀr Anorganische und Analytische Chemie
Westfꢁlische Wilhelms-Universitꢁt Mꢀnster
Corrensstrasse 28/30, 48149 Mꢀnster (Germany)
Fax: (+49)251-8336007
Scheme 1. Metal-mediated base pairs with bidentate triazole-based nucleo-
sides: [2+2] and [2+1] coordination mode.
E-mail: mueller.j@uni-muenster.de
[b] Dr. O. Krug
Results and Discussion
Fakultꢁt Chemie und Chemische Biologie
Technische Universitꢁt Dortmund
Otto-Hahn-Strasse 6, 44227 Dortmund (Germany)
Synthesis of the nucleosides
Three 1,2,3-triazole-based “click” nucleosides were synthesized
by using CuI-catalyzed Huisgen 1,3-dipolar cycloaddition
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201402221.
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(Scheme 2a). Depending on the alkyne, the 1,2,3-triazole
moiety is complemented by 1,2,4-triazole (TriTri), pyrazole
(TriPyr), or pyridine (TriPy) (Scheme 2b). The use of 2-deoxy-b-
d-glycosyl azide as a common precursor allows the modular
synthesis of a variety of 1,2,3-triazole nucleosides.
The synthesis of TriTri (and its phosphoramidite required for
automated DNA synthesis) has been reported recently.[13] The
other two nucleosides TriPyr and TriPy have been synthesized
accordingly (Scheme 3). The use of the TriPy nucleoside has re-
cently also been reported as a ligand for the generation of a lu-
minescent probe for the study of nucleoside transporters.[14] Its
incorporation into nucleic acids and its capability to form
metal-mediated base pairs has not yet been investigated
though.
Molecular structures of the model complexes
Scheme 2. a) Retrosynthesis of the bidentate triazole-based nucleosides;
b) Nucleosides reported in this work.
It is important to note that the methylene spacer in TriTri and
TriPyr might lead to the formation of a non-planar metal com-
plex. Such behavior has been reported for related ligands bear-
ing an alkyl or aryl substituent instead of the deoxyribose.[15]
These systems can be considered model nucleobases, which
are regularly applied to study the molecular structures of
metal complexes of nucleosides.[16] Such a model nucleobase
representing TriPy is known to support both fully planar and
tetrahedral complexes.[15,17] Accordingly, we synthesized
a model nucleobase for TriPyr (by formally replacing the sugar
moiety with a benzyl substituent) and investigated its metal-
binding properties. Two metal complexes of the re-
sulting ligand BnTriPyr are displayed in Figure 1 and
S1 (the Supporting Information). In both molecular
structures, the MꢀN distances are in the normal
range (Table 1). The CuII ion is weakly coordinated by
two additional solvent molecules in the axial posi-
tions, thereby completing a distorted octahedral co-
ordination environment. These methanol ligands are
easily cleaved off, as confirmed by mass spectrometry
and elemental analysis of the complex (see the Sup-
porting Information). Hence, they would not prohibit
the formal incorporation of such a metal complex as
a metal-mediated base pair into a nucleic acid
duplex. However, the transoid orientation of the
benzyl substituents observed in the molecular struc-
ture is not compatible with the regular B-DNA con-
formation, which requires a cisoid orientation of the
glycosidic bonds. The transoid orientation is probably
being adopted to avoid a steric clash of two closely
Table 1. Selected bond lengths [ꢂ] in the molecular structures of [Pd-
(BnTriPyr)2]Cl2·2H2O and [Cu(BnTriPyr)2(CH3OH)2](NO3)2.
[Pd(BnTriPyr)2]Cl2·2H2O
[Cu(BnTriPyr)2(CH3OH)2](NO3)2
M1ꢀN3t
M1ꢀN2p
M1ꢀO2m
1.9988(10)
2.0102(10)
–
2.0109(15)
2.0232(16)
2.4346(15)
Scheme 3. Synthesis of the artificial nucleosides and their phosphoramidites. a) THF/2-
propanol/H2O, CuSO4, sodium ascorbate, 12 h, RT; b) MeOH, NH3, 18 h, RT; c) Dimethoxy-
trityl chloride (DMT-Cl), 4-dimethylaminopyridine (DMAP), pyridine, 3 h, RT; d) 2-Cya-
noethyl N,N-diisopropylchlorophosphoramidite (CEDIP-Cl), diisopropylethylamine (DIPEA),
CH2Cl2, 30 min, RT.
located benzyl groups. As such a steric clash does
not exist in B-DNA, the complex still appears to be
a valid structural model for the corresponding poten-
tial metal-mediated base pair. Neither in the cation of
[Pd(BnTriPyr)2]Cl2·2H2O (Figure S1, the Supporting In-
formation) nor in that of [Cu(BnTriPyr)2(CH3OH)2]-
(NO3)2 (Figure 1) is the BnTriPyr ligand found to be planar. In
the PdII complex, the triazole and pyrazole moieties of this
ligand are tilted towards one another with an angle of
54.25(6)8. The corresponding angle in the CuII complex is a bit
smaller, amounting to 47.41(9)8. As we will see, the non-pla-
narity does not prohibit the use of TriPyr in metal-mediated
base pairs within an oligonucleotide duplex, even though fully
planar metal complexes can be additionally stabilized by p-
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Scheme 4. Oligonucleotide duplexes under investigation (X, Y=TriTri,
TriPyr, TriPy, or Imi).
been introduced in this study to enable an investigation of
nearest-neighbor effects. In addition to the bidentate triazole-
based nucleosides, the well-established monodentate imida-
zole nucleoside Imi[8a,b,18] has also been included in this study.
To probe the overall metal-binding preferences of the artifi-
cial nucleosides, the thermal stability of duplex I with X=
TriPyr and Y=TriPy was determined by temperature-depen-
dent UV spectroscopy in the presence of various transition-
metal ions.[25] Table 2 shows the resulting melting temperatures
Table 2. Melting temperature Tm [8C] of duplex I with X=TriPyr and Y=
TriPy (Scheme 5) in the absence and presence of various transition-metal
ions.[a]
Metal ion
Tm (0 equiv)
Tm (1 equiv)
DTm
AgI
HgII
CuII
ZnII
NiII
32.6
32.2
32.1
32.2
32.1
40.0
32.2
32.2
32.2
32.1
7.4
0.0
0.1
0.0
0.0
[a] Conditions: oligonucleotide duplex (1 mm), NaClO4 (150 mm), MOPS
(5 mm; pH 6.8). The estimated standard deviation of Tm amounts to 18C.
Figure 1. Molecular structure of the cation of [Cu(BnTriPyr)2(CH3OH)2](NO3)2
viewed from two different orientations.
stacking interactions with neighboring base pairs. In contrast,
the introduction of such non-planar nucleoside into an oligo-
nucleotide helps to unravel the principles of the stabilization
of metal-mediated base pairs.
Scheme 5. Proposed structure of the AgI-mediated base pair from TriPyr
and TriPy.
Investigation of the oligonucleotide duplex I
Two different oligonucleotide duplexes were prepared by
using automated solid-phase synthesis according to the phos-
phoramidite method. Both duplexes comprise one artificial
metal-mediated base pair, six G:C base pairs, and six A:T base
pairs (G=guanine, C=cytosine, A=adenine, T=thymine). The
natural nucleosides are arranged in a way that the duplex con-
sists of one purine strand and one pyrimidine strand. Hence,
the two duplexes have an identical overall composition and
differ (amongst others) by the identity of the two canonical
base pairs located adjacent to the artificial base pair
(Scheme 4). One of these duplexes has previously been used in
other studies,[13,16b] thereby enabling a direct comparison of
the melting temperatures with those of nucleic acids compris-
ing other metal-mediated base pairs. The other duplex has
Tm. As can be seen, only AgI has a stabilizing effect on the oli-
gonucleotide duplex (Scheme 5). The same observation was
made for other combinations of the triazole-based bidentate
nucleosides. Hence, only the AgI-mediated base pairs were
studied subsequently in more detail. It is interesting to note
that CuII does not stabilize any of the base pairs, despite the
fact that a corresponding model structure could be structurally
characterized (Figure 1) and that several examples exist in the
literature of CuII-mediated base pairs.[7,9c,11b,12,21] Various explan-
ations are possible: 1) A CuII-mediated base pair is formed but
does not bring about a net stabilization of the duplex because
its formation is accompanied by other destabilizing conforma-
tional changes; 2) Most previously reported CuII-mediated base
pairs involve negatively charged ligands, that is, lead to the
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formation of a neutral complex. Such neutral entities might be
more easily accommodated within the base pair stack com-
pared with the potential CuII-mediated base pairs investigated
here carrying two positive charges.
Table 3 lists the increase in melting temperature (DTm) upon
the addition of one equivalent of AgI to duplex I with all possi-
ble combinations of TriTri, TriPyr, TriPy, and Imi in the posi-
tions X and Y.
Table 3. Increase of Tm [8C] of duplex I upon the addition of one equiva-
lent of AgI.[a]
X=Imi
X=TriTri
X=TriPyr
X=TriPy
Y=Imi
9.5
6.4
1.5
12.6
2.2
2.1
1.9
1.3
1.1
2.9
2.8
7.4
12.6
1.5
4.1
Y=TriTri
Y=TriPyr
Y=TriPy
1.4
Figure 2. Melting curves of duplex I with X=Imi and Y=TriPy in the pres-
ence of various amounts of AgI. Inset: Dependence of melting temperature
Tm on number of added equivalents of AgI. Conditions: oligonucleotide
duplex (1 mm), NaClO4 (150 mm), 4-morpholinepropanesulfonate (MOPS;
5 mm; pH 6.8).
[a] Conditions: oligonucleotide duplex (1 mm), NaClO4 (150 mm), MOPS
(5 mm; pH 6.8), AgNO3 (1 mm). The estimated standard deviation of Tm
amounts to 18C. The absolute melting temperatures in the absence and
presence of one equivalent of AgI are listed in Tables S1 and S2 (the Sup-
porting Information).
substoichiometric amounts of AgI are present (Figure 2). The
biphasic melting is a result of the kinetically inert Imi-Ag-TriPy
complex. As long as less than one equivalent of AgI is present
per base pair, two types of double helices exist, namely metal-
free ones (with an Imi:TriPy mispair) and metal-containing
ones (with an Imi-Ag-TriPy base pair). As the exchange of AgI
is slow, these duplexes do not interconvert during the record-
ing of the melting curves. A similar behavior had previously
been reported for metal-mediated salen base pairs.[19] As can
be seen from the inset of Figure 2, a significant increase in Tm
is observed up to one equivalent of AgI only. Excess AgI leads
to very little additional stabilization. This nicely confirms the
structure of the base pair proposed in Scheme 6. The addition-
al stabilization conferred by excess AgI may be explained by
non-specific binding events between the positively charged
metal ion and the polyanion DNA.[16b]
Several interesting conclusions can be drawn from the data
shown in Table 3. Firstly, the most stabilizing combination is
that of Imi and TriPy. Secondly, the well-known Imi-Ag-Imi
base pair is also significantly stabilized. Thirdly, not all combi-
nations of nucleosides lead to stabilizing metal-mediated base
pairs. Finally, not all DTm values are arranged symmetrically in
the Table. Estimating a standard deviation of Tm of 18C, the
stabilization of two base pairs depends on the positioning of
the nucleosides in the purine or pyrimidine strand: the duplex
comprising TriPyr-Ag-TriPy (DTm =7.48C) is stabilized to
a larger extent than that with TriPy-Ag-TriPyr (DTm =4.18C).
Similarly, the stabilization observed for Imi-Ag-TriTri (DTm =
6.48C) is much larger than that for TriTri-Ag-Imi (DTm =2.28C).
These findings will be discussed individually in the following.
Observation 1
Observation 2
A more detailed investigation of the AgI-mediated base pair
formed from Imi and TriPy (Scheme 6) indicates that it com-
prises a kinetically inert metal complex. This kinetic effect may
be a significant contributor to the observed large thermal sta-
bilization. As can be seen from the melting curves of duplex I
with an Imi-Ag-TriPy base pair, biphasic melting occurs when
As Imi-Ag-Imi base pairs have been found to be stabilizing in
a series of different oligonucleotide sequences,[8a,18b] it is not
surprising to see the formation of stable base pairs of this type
also for the sequence investigated here. Nonetheless, the stabi-
lization observed here (DTm =9.58C) is the largest one found
as yet for Imi-Ag-Imi base pairs.
Observation 3
Imidazole and pyridine are more basic ligands than 1,2,4-tria-
zole and pyrazole.[20] Accordingly, it is not surprising that Imi
and TriPy are involved more often in the formation of strongly
stabilizing metal-mediated base pairs than TriTri and TriPyr.
Moreover, it appears that non-planar metal-mediated base
pairs obtained by combining two “click” nucleosides compris-
Scheme 6. Proposed structure of the AgI-mediated base pair from Imi and
TriPy.
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ing a methylene linker do not have a significant stabilizing
effect, as they cannot interact efficiently with neighboring base
pairs through p-stacking. Similarly, the combination of two
TriPy nucleosides is expected to lead to a tetrahedral AgI com-
plex, in analogy to a previously reported structure of a related
model nucleobase.[17] Hence, no significant increase in Tm is ob-
served upon the addition of AgI to duplex I with X=Y=TriPy.
Comparison of duplexes I and II
As mentioned before, duplexes I and II have the same overall
composition and differ by the identity of the canonical base
pairs flanking the metal-mediated base pair. More precisely,
two guanine residues are located next to nucleoside X in
duplex I whereas X is surrounded by adenine residues in
duplex II. This difference has important effects on the thermal
stability of the duplexes. Table 4 lists the melting temperatures
Observation 4
Duplex I with Imi-Ag-TriTri is stabilized to a larger extent than
duplex I with TriTri-Ag-Imi (Table 3). Figure 3 confirms this by
showing the dependence of Tm on the number of added
Table 4. Melting temperature Tm [8C] and increase of Tm (DTm, 8C) of du-
plexes I and II (Y=Imi) upon the addition of one equivalent of AgI.[a]
Tm (0 equiv AgI)
Tm (1 equiv AgI)
DTm
X
duplex I duplex II duplex I duplex II duplex I duplex II
Imi
TriTri
TriPyr 25.6
TriPy 33.6
29.8
25.4
25.4
21.7
23.4
30.4
39.3
27.6
26.7
46.2
39.1
27.7
27.4
43.7
9.5
2.2
1.1
12.6
13.7
6.0
4.0
13.4
[a] Conditions: oligonucleotide duplex (1 mm), NaClO4 (150 mm), MOPS
(5 mm; pH 6.8). The estimated standard deviation of Tm amounts to 18C.
Tm as well as DTm for both duplexes. In all cases, Imi acts as ar-
tificial nucleoside Y in the pyrimidine strand.
Judging by the increase in Tm upon the addition of AgI
(shown in the two columns on the right), it appears that the
degree of thermal stabilization depends on the oligonucleotide
sequence. In all cases, duplex II is stabilized to a larger extent
than duplex I. However, a close inspection of the underlying
melting temperatures shows that both duplexes have identical
melting temperatures in the presence of AgI for X=Imi, TriTri,
and TriPyr (within standard deviation). Hence, the differences
in DTm must arise from different stabilities of the metal-free
X:Y mispairs. In the absence of transition-metal ions, duplex I
is consistently more stable than duplex II. This is likely to be
the result of better p-stacking capabilities of the flanking gua-
nine residues in duplex I compared with the flanking adenine
residues in duplex II.[22] As long as no metal ion is coordinated,
the nucleobases TriTri and TriPyr can adopt a planar confor-
mation and can therefore participate in p-stacking. Comprising
five-membered azole moieties only, their general p-stacking ca-
pability is low, as is that of Imi. The fact that the Tm value is
identical for both duplexes if X=Imi, TriTri, or TriPyr in the
presence of AgI indicates that non-planar metal-mediated base
pairs are formed, so that no additional p-stacking interactions
are possible. Hence, for duplex I the formation of a metal-
mediated base pair is accompanied by a decrease in stability
due to the loss of p-stacking interactions with guanine and
a concomitant increase in stability due to the formation of co-
ordinative bonds. In contrast, only the stabilization resulting
from the coordinative bonds is relevant for duplex II, because
comparatively weak p-stacking interactions with adenine resi-
dues are lost.
*
I
Figure 3. Melting temperature Tm of duplex I with Imi-Ag-TriTri ( ) or TriTri-
&
Ag-Imi ( ), depending on the number of added equivalents of Ag . Inset:
Change of the UV absorbance at 260 nm upon the addition of AgI to duplex
I with X=TriTri and Y=Imi.
equivalents of AgI for these two duplexes. The plot suggests
that in these base pairs the AgI ion is bonded in a labile fash-
ion, as Tm increases rather smoothly. Nonetheless, the 1:1 bind-
ing stoichiometry can clearly be discerned from the inset of
Figure 3, showing the change of the UV absorbance upon the
addition of AgI. A possible explanation for the differential stabi-
lization could be an additional coordination of the metal ion
by a donor atom above or below the base pair. In asymmetri-
cal [2+1] base pairs, the metal ion is not necessarily located di-
rectly in the center of the duplex, hence different additional
donor atoms might be present depending on the orientation
of the base pair. A similar behavior had been reported for
other metal-mediated base pairs before.[12c,21] Unfortunately, no
structural information exists on the systems studied here, so
no final conclusion can be drawn at this stage.
In this context it is interesting to discuss also the base pairs
involving TriPy. This artificial nucleobase is fully planar, as are
its [2+1] metal complexes. Moreover, being a six-membered
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aromatic heterocycle, the pyridine moiety is a good p-stacking
entity. Hence, no differential stabilization as discussed above
for Imi, TriTri, and TriPyr should be expected as both the
metal-free and the metal-containing base pairs are planar.
Indeed, the duplexes comprising TriPy have the largest melt-
ing temperature of all duplexes listed in Table 4. Furthermore,
both duplexes are stabilized by ꢁ138C upon the addition of
AgI. Hence, for a planar base pair such as TriPy-Ag-Imi the sta-
bilizing effect of the formation of coordinative bonds is rele-
vant only.
rect conclusion that no metal-mediated base pair is formed
at all.
Experimental Section
DNA syntheses were performed in the DMT-off mode on a K&A La-
borgerꢃte H8 DNA/RNA synthesizer by following standard proto-
cols.[16b] The oligonucleotides were identified by MALDI-TOF mass
spectrometry (see the Supporting Information). MALDI-TOF mass
spectra were recorded on a Bruker Reflex IV instrument using a 3-
hydroxypicolinic acid/ammonium citrate matrix and applying
a commercially available oligonucleotide with a molecular mass of
4577 Da as internal reference. NMR spectra were recorded using
Bruker Avance(I) 400 and Bruker Avance(III) 400 spectrometers at
300 K. Chemical shifts were referenced to residual CD3OH (CD3OD,
d=4.78 ppm), TSP (D2O, d=0 ppm), TMS (CDCl3, d=0 ppm), or
H3PO4 (31P NMR, d=0 ppm). UV/Vis spectra were recorded on
a Varian CARY BIO 100 spectrophotometer. Temperature-depen-
dent UV spectra were recorded between 10 and 708C with a heat-
ing/cooling rate of 18Cminꢀ1 and a data interval of 0.58C. Absorb-
ance was normalized according to Anorm =(AꢀAmin)/(AmaxꢀAmin) at
260 nm. Melting temperatures have been determined as the maxi-
mum of the derivative of the annealing curves. 2-Deoxy-3,5-di-O-
(p-toluoyl)-b-d-erythro-pentofuranosyl azide, orthogonally protect-
ed imidazole nucleoside, 1-propargyl-1H-pyrazol, 1-propargyl-1H-
1,2,4-triazole, and BnTriPyr were prepared according to literature
procedures, respectively.[18a,23]
Conclusion
To increase the scope of metal-mediated base pairing, a new
family of 1,2,3-triazole-based “click” nucleosides was devel-
oped. The use of 2-deoxy-b-d-glycosyl azide as a common pre-
cursor allows the modular synthesis of a family of 1,2,3-triazole
nucleosides through CuI-catalyzed Huisgen 1,3-dipolar cycload-
dition. Three of these nucleosides (TriTri, TriPyr, TriPy) were
tested with respect to their applicability in metal-mediated
base pairs. Several conclusions can be drawn that are impor-
tant for the future development of new metal-mediated base
pairs and for the characterization of metal-mediated base pairs
in general.
1) The introduction of “click” chemistry into the generation of
metal-mediated base pairs enables a fast and easy synthe-
sis of a variety of closely related artificial nucleosides.
Hence, a series of ligands can be created with a gradually
changing metal-binding behavior. Such a series of nucleo-
sides may be useful for the development of tailored metal
ion sensors based on nucleic acids with metal-mediated
base pairs. So far, nucleobases containing nitrogen donor
atoms only have been synthesized with the “click” ap-
proach. The introduction of another donor atom such as
oxygen is a goal to be established next.
In the following, only the general syntheses are reported. The
spectral data of the resulting compounds are listed in the Support-
ing Information.
Syntheses
Toluoyl-protected nucleosides: (Scheme 3, reaction a): To a mixture
of alkyne (1.2 equiv) and 2-deoxy-3,5-di-O-(p-toluoyl)-b-d-erythro-
pentafuranosyl azide (1 equiv) in THF/isopropanol (40 mL, 4/1), a so-
lution of CuSO4·5H2O (0.2 equiv) in H2O (8 mL) and sodium ascor-
bate (0.4 equiv) were added. After stirring for 12 h at room temper-
ature the reaction was quenched by addition of EtOAc (150 mL).
The organic layer was washed with aqueous ethylenediaminete-
traacetate (EDTA) solution (0.5%) until the aqueous layer was color-
less. The organic phase was dried (MgSO4). After filtration the sol-
vent was removed. The crude product was purified by column
chromatography.
2) The formation of asymmetric metal-mediated base pairs
may result in a significantly different stabilization of the
double helix, depending on whether X-M-Y or Y-M-X is
formed. Structural information will be necessary to eluci-
date whether this effect can be explained by the presence
of additional axial ligands.
Free nucleosides: (Scheme 3, reaction b): The toluoyl-protected nu-
cleoside was dissolved in MeOH (100 mL), and aqueous NH3 (25%,
50 mL) was added. After stirring overnight, the solvent was re-
moved and the crude product was purified by column chromatog-
raphy.
3) Metal-mediated base pairs do not necessarily have to be
fully planar to be incorporated into a double helix. If the
energy gained from forming the coordinative bonds is suffi-
ciently large to compensate the loss of p-stacking interac-
tions, slightly tilted metal-mediated base pairs are accepta-
ble, too.
DMT-protected nucleosides: (Scheme 3, reaction c): Free nucleo-
side (1 equiv) was co-evaporated three times with dry pyridine
(20 mL) and then dissolved in dry pyridine (50 mL). To this reaction
mixture, catalytic amounts of 4-dimethylaminopyridine (DMAP)
were added under an argon atmosphere, followed by the addition
of 4,4’-dimethoxytrityl chloride (DMT-Cl; 1.2 equiv). The mixture
was stirred for 3 h at room temperature. The solution was diluted
with CH2Cl2 (100 mL) and washed with water (3ꢄ20 mL). The or-
ganic phase was dried (Na2SO4) and evaporated to dryness. The
crude product was purified with column chromatography.
4) It is important to choose properly the oligonucleotide se-
quence when investigating metal-mediated base pairs. Par-
ticularly in those cases in which a metal-mediated base pair
is not necessarily planar, the stabilization of the DNA
duplex may depend significantly on the stability of the
metal-free mispair. Taking this idea to the extreme, a rela-
tively stable mispair in combination with an only slightly
stabilizing metal-mediated base pair may lead to the incor-
Orthogonally protected nucleosides: (Scheme 3, reaction d): The
DMT-protected nucleoside (1 equiv) was dissolved in freshly dis-
Chem. Eur. J. 2014, 20, 7811 – 7818
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ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
tilled CH2Cl2 (35 mL). N,N-Diisopropylethylamine (DIPEA; 3 equiv)
and N,N-diisopropylchlorophosphoramidite (CEDIP-Cl; 2 equiv)
were then added to the nucleoside/CH2Cl2 solution under an argon
atmosphere. After 30 min of stirring at room temperature the mix-
ture was diluted with EtOAc (75 mL). The organic phase was
washed with saturated aqueous NaHCO3 solution (30 mL) and
dried (MgSO4). The solvent was evaporated to dryness and the
crude product was purified by column chromatography.
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
Acknowledgements
This work was supported by the Deutsche Forschungsgemein-
schaft (MU 1750/2–1).
Metal complexes: A metal salt/metal precursor complex (1 equiv)
in MeOH (5 mL) was added to a solution of BnTriPyr (2 equiv) in
MeOH (5 mL). After stirring for 3 h with heating at reflux the solu-
tion was kept at 48C for several days, until crystals suitable for
single-crystal X-ray diffraction are obtained.
Keywords: bioinorganic chemistry
cycloaddition · DNA · silver
·
click chemistry
·
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[Pd(BnTriPyr)2]Cl2·2H2O
[Cu(BnTriPyr)2(CH3OH)2](NO3)2
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C26H30Cl2N10O2Pd
C28H32CuN12O8
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[I>2s(I)]
0.48ꢄ0.23ꢄ0.16
0.22ꢄ0.20ꢄ0.07
2.78, 30.07
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Chem. Eur. J. 2014, 20, 7811 – 7818
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Published online on May 18, 2014
Chem. Eur. J. 2014, 20, 7811 – 7818
www.chemeurj.org
7818
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim