Oligonucleotides with clickable sugar residues: Synthesis, duplex stability, and terminal versus central interstrand cross-linking of 2′-O-propargylated 2-aminoadenosine with a bifunctional azide

Article abstract of DOI:10.1021/jo500392j

Duplex DNA with terminal and internal sugar cross-links were synthesized by the CuAAC reaction from oligonucleotides containing 2′-O-propargyl-2- aminoadenosine as a clickable site and a bifunctional azide (4). Stepwise click chemistry was employed to introduce cross-links at internal and terminal positions. Copper turnings were used as catalyst, reducing the copper load of the reaction mixture and avoiding complexing agents. For oligonucleotide building block synthesis, a protecting group strategy was developed for 2′-O-propargyl-2-aminoadenosine owing to the rather different reactivities of the two amino groups. Phosphoramidites were synthesized bearing clickable 2′-O-propargyl residues (14 and 18) as well as a 2′- deoxyribofuranosyl residue (10). Hybridization experiments of non-cross-linked oligonucleotides with 2,6-diaminopurine as nucleobase showed no significant thermal stability changes over those containing adenine. Surprisingly, an isobutyryl group protecting the 2-amino function has no negative impact on the stability of DNA-DNA and DNA-RNA duplexes. Oligonucleotide duplexes with cross-linked 2′-O-propargylated 2-aminoadenosine (1) and 2′-O-propargylated adenosine (3) at terminal positions are significantly stabilized (ΔT_m = +29 °C). The stability results from a molecularity change from duplex to hairpin melting and is influenced by the ligation position. Terminal ligation led to higher melting duplexes than corresponding hairpins, while duplexes with central ligation sites were less stable.

Full text of DOI:10.1021/jo500392j

Article
pubs.acs.org/joc
Oligonucleotides with “Clickable” Sugar Residues: Synthesis, Duplex
Stability, and Terminal versus Central Interstrand Cross-Linking of
2′-O-Propargylated 2‑Aminoadenosine with a Bifunctional Azide
Suresh S. Pujari,†,‡ Peter Leonard,† and Frank Seela*,†,‡
†Laboratory of Bioorganic Chemistry and Chemical Biology, Center for Nanotechnology, Heisenbergstraße 11, 48149 Munster,
̈
Germany
‡Laboratorium fur Organische und Bioorganische Chemie, Institut fur Chemie neuer Materialien, Universitat Osnabruck,
̈
̈
̈
̈
Barbarastraße 7, 49069 Osnabruck, Germany
̈
S
* Supporting Information
ABSTRACT: Duplex DNA with terminal and internal sugar cross-links were synthesized by the CuAAC reaction from
oligonucleotides containing 2′-O-propargyl-2-aminoadenosine as a clickable site and a bifunctional azide (4). Stepwise click
chemistry was employed to introduce cross-links at internal and terminal positions. Copper turnings were used as catalyst,
reducing the copper load of the reaction mixture and avoiding complexing agents. For oligonucleotide building block synthesis, a
protecting group strategy was developed for 2′-O-propargyl-2-aminoadenosine owing to the rather different reactivities of the
two amino groups. Phosphoramidites were synthesized bearing clickable 2′-O-propargyl residues (14 and 18) as well as a
2′-deoxyribofuranosyl residue (10). Hybridization experiments of non-cross-linked oligonucleotides with 2,6-diaminopurine as
nucleobase showed no significant thermal stability changes over those containing adenine. Surprisingly, an isobutyryl group
protecting the 2-amino function has no negative impact on the stability of DNA−DNA and DNA−RNA duplexes.
Oligonucleotide duplexes with cross-linked 2′-O-propargylated 2-aminoadenosine (1) and 2′-O-propargylated adenosine (3) at
terminal positions are significantly stabilized (ΔTm = +29 °C). The stability results from a molecularity change from duplex to
hairpin melting and is influenced by the ligation position. Terminal ligation led to higher melting duplexes than corresponding
hairpins, while duplexes with central ligation sites were less stable.
chemotherapy goes back to the toxic action of nitrogen mustard
acting as a bifunctional DNA cross-linker. Platinum(II)
compounds are used in cancer treatment for cross-linking
DNA.5 Metals such as copper, mercury, or silver ions can
be considered as cross-linking reagents as they replace the
canonical Watson−Crick base pairs with more stable metal base
pairs.6 Now, the Cu(I)-catalyzed azide−alkyne Huisgen−
Meldal−Sharpless cycloaddition (CuAAC) “click chemistry”
has emerged as a promising candidate for cross-linking because
of its bioorthogonal nature and robustness of the formed
triazole.7 Our laboratory has cross-linked alkynylated DNA or
RNA with bifunctional azides in a one-step process (bis-click)
or in a stepwise procedure (stepwise click).8
INTRODUCTION
■
The stabilization of nucleic acids assemblies (duplexes,
triplexes, and other supramolecular structures) plays a crucial
role in chemical and biochemical applications of DNA or RNA
and for the construction of nanoscopic devices in material
science.1 Stable supramolecular assemblies of DNA find use in
the arrangement of nanostructures by which DNA machines
such as DNA tweezers, walkers, and steppers are formed.2
Antisense therapeutics were stabilized by backbone modifica-
tion or replacement of canonical bases by modified nucleo-
sides.3 Such second-generation synthetic oligonucleotides
having 2′-O-modifications are promising candidates in
antisense technology (e.g., for the selective silencing of certain
genes).4
Another way to strengthen the duplex structure is to
introduce cross-links in DNA or RNA. The origin of cancer
Received: February 18, 2014
Published: April 2, 2014
© 2014 American Chemical Society
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Figure 1. (Top) Sugar propargylated nucleosides and bis-azide. (Bottom) Comparison of cross-linked and hairpin DNA.
Recently, our laboratory reported on the synthesis of 2′-O-
propargylated isoguanosine and on a significantly improved
synthesis of 2′-O-propargylated cytidine. It was found that
propargyl residues with clickable triple bonds introduced in the
sugar residue of nucleosides cause only a minor change on the
stability of duplexes since these units presumably lie outside the
helix. Parallel stranded cross-linked oligonucleotides were also
constructed using bifunctional azides.9
Among the various DNA and RNA constituents, the 2-amino-
adenine nucleosides 2a and 2b, which can be used as an analogue
of adenine, play a special role as stabilizing nucleosides as the
base eventually forms a third hydrogen bond with dT (Figure 1).10
Related base modified nucleosides with a pyrrolo[2,3-d]pyrimidine
or a pyrazolo[3,4-d]pyrimidine skeleton have also been studied,
and halogenated derivatives were used to adjust the dA−dT base
pair stability to that of a dG−dC pair.11
iodide as a phase transfer catalyst.13b Side-product formation
(e.g., cleavage of the silyl clamp) was suppressed by introducing
TBAF/THF into the mixture when the starting material was
completely consumed (∼20 min, TLC-monitoring). By this,
the propargylated isomer 1 was obtained as a main product in
66% yield together with a small amount of the 3′-O-isomer 6
(8%) (Scheme 1; for details, see Experimental Section). The
a
Scheme 1. Propargylation of 2-Aminoadenosine 2b
This study reports on the synthesis of 2′-O-propargylated
2-aminoadenosine (1), the selection of suitable protecting
groups for the two amino functionalities showing rather different
reactivity, and the conversion into phosphoramidite building
blocks. The installation of a clickable propargyl functionality in
the sugar residue in combination with an additional 2-amino
group into the adenine moiety is expected to stabilize duplexes
over those containing only 2′-O-propargylated adenosine (3).
Propargylated oligonucleotides were cross-linked with the
bifunctional azide 412 by a stepwise protocol. By this means,
terminal and internal cross-links were introduced in duplexes at
various positions in a site-specific way. Then, the thermal stability
of ligated and nonligated duplexes is compared. As the stepwise
click approach is flexible with respect to incorporation sites and
cross-links, propargylated oligonucleotides were cross-linked at
the center and the terminus of a duplex. Hairpins of similar
structure were synthesized to compare their thermal stability to
the melting of cross-linked duplexes (Figure 1).
a
Reagents and conditions: (i) (i-Pr)2ClSiOSi(i-Pr)2Cl, pyridine, rt, 12
h; (ii) (a) propargyl bromide, NaH, TBAI, DMF, 5−55 °C, 20 min;
(b) 1 M TBAF in THF, rt, 1 h, 73%.
position of propargylation was determined by NMR spectroscopy−
1
disappearance of the OH group in the H NMR spectrum and a
strong downfield shift of C-2′ in the 13C NMR spectrum. The yield
for the propargylated nucleoside 1 was significantly improved, from
32% to 59%, based on 2-aminoadenosine (2b) as a starting material
(2 steps).9
RESULTS AND DISCUSSION
■
1. Synthesis and Characterization of Phosphoramidite
Building Blocks. Earlier, the synthesis of 2′-O-propargyliso-
guanosine and 3′-O-propargylisoguanosine from the corre-
sponding 2′-O- and 3′-O-propargylated 2-aminoadenosine
precursors 1 and 6 was reported.9 However, the yield for
the propargylation of 2-aminoadenosine (2b) was low, and the
reaction time was rather long. Therefore, an alternative route
for the propargylation was explored. Compound 5, which was
synthesized according to the literature,13a was propargylated
using NaH and propargyl bromide with tetrabutylammonium
As many contradictory reports on the protection of the two
amino groups exist,14 a protecting group strategy has to be
developed for the propargylated 2-aminoadenosine phosphor-
amidite building block. Initially, the simultaneous protection of
both amino groups with dibutylaminomethylidene residues as
described for diamino pyrazolo[3,4-d]pyrimidine nucleosides
was investigated.14 In the case of 2′-O-propargylated compound 1,
we obtained the monoprotected (6-position) compound 7 as the
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the nucleobase. Thus, we went back to a stepwise protection
protocol using two different amino protecting groups. At first,
nucleoside 1 was selectively protected at the 2-amino group
with an isobutyryl residue (→ 11, 65%) followed by protection
of the 6-amino group by a butylamidine moiety (→ 12, 80%;
Scheme 4). 4,4′-Dimethoxytritylation (→ 13, 57%) and
phosphitylation gave phosphoramidite 14 (76%).
Scheme 2. Amidine Protection of 2′-O-Propargyl-2-
a
aminoadenosine
Scheme 4. Synthesis of 2′-O-Propargyl-2-aminoadenosine
a
Phosphoramidite 14
a
Reagents and conditions: (i) N,N-dibutylformamide dimethylacetal,
MeOH, 40 °C.
main product (Scheme 2) accompanied by several side products,
although complete consumption of the staring material was
indicated by TLC.
To shed more light on this matter and to obtain a suitable
building block for oligonucleotide synthesis (see section 3), the
protection of 2-amino-2′-deoxyadenosine (2a) was investi-
gated. Although, the formation of new reaction products was
detected by TLC during butylamidine protection, they were
too labile to be isolated as clean material. Similar results have
been reported for formamidine protected 2-amino-2′-deoxy-
adenosine.14a Therefore, isobutyrylation using “transient protec-
tion” conditions was studied next. In this case, diisobutyrylated
compound 8 was obtained in 62% yield.14f 4,4′-Dimethoxytrity-
lation of the 5′-OH group (→ 9, 58%) and phosphitylation of the
3′-OH group gave 10 in 73% yield (Scheme 3).
a
Reagents and conditions: (i) i-BuCl, TMSCl, pyridine (−20 °C); (ii)
Scheme 3. Synthesis of 2-Amino-2′-deoxyadenosine
a
N,N-dibutylformamide dimethylacetal, MeOH, rt; (iii) 4,4′-dimethox-
ytrityl chloride, pyridine, rt; (iv) NC(CH2)2OP(Cl)N(i-Pr)2, DIPEA,
DCM, rt.
Phosphoramidite 10
Later, during oligonucleotide synthesis, we recognized that
cleavage of the isobutyryl residue from the 2-amino group
caused difficulties. Thus, the more labile methoxyacetyl group
was introduced, which has been successfully employed for other
2-aminoadenosine derivatives.14c For this, nucleoside 1 was
protected on the 2-amino group with the methoxyacetyl residue
(→ 15, 72%) followed by protection (isobutyryl) of the
6-amino group (→ 16, 52%) (Scheme 5). 4,4′-Dimethoxy-
tritylation (→ 17, 54%) and, finally, phosphitylation afforded
18 in 74% yield (Scheme 5).
From these experiments, it is obvious that the reactivity of
the two amino groups of propargylated 2-aminoadenosine (1)
is rather different. So, a special combination of protecting
groups is required composed of labile protection for the
2-amino group and a protecting group for the 6-amino group,
which is more stable than that for adenosine. Taken together,
the combination of the methoxyacetyl residue (2-amino) and
an isobutyryl group (6-amino) represents a suitable combina-
tion to design a 2′-O-propargylated 2-aminoadenosine building
block for oligonucleotide synthesis. However, base modified
2-aminoadenosine derivatives (pyrazolo[3,4-d]pyrimidine or
pyrrolo[2,3-d]pyrimidine analogues) require other protecting
groups as the other heterocylic skeleton changes the reactivity
of the amino groups.14
a
Reagents and conditions: (i) i-BuCl, trimethylsilyl chloride, pyridine;
(ii) 4,4′-dimethoxytrityl chloride, pyridine, rt; (iii) NC(CH2)2OP-
(Cl)N(i-Pr)2, DIPEA, DCM, rt.
Accordingly, isobutyryl protection was performed on the
2′-O-propargylated nucleoside 1. Surprisingly, an inseparable
mixture of products was obtained. As the behavior of the
propargylated nucleoside 1 was different from 2a during
isobutyryl protection, their pKa values were determined. Both
compounds showed similar pKa values, 4.2 for 1 and 4.3 for 2a,
and they are close to that of 2-aminoadenosine 2b (pKa = 4.4).
Therefore, we anticipated that side reactions occurring during
isobutyrylation are evoked by the propargyl residue and not by
2. Cross-Linking Performed on the 2′-O-Propargy-
lated 2-Aminoadenosine Nucleoside 1. Earlier, it was
reported that both the stepwise click and the bis-click protocol
can be performed when identical nucleosides are clicked
together, while a stepwise click reaction is required when the
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used to assign the 13C NMR signals (for details, see Experimental
Section, Table 5; Supporting Information, Table S1).
Scheme 5. Synthesis of 2′-O-Propargyl-2-aminoadenosine
a
Phosphoramidite 18
3. Synthesis and Thermal Stability of DNA Duplexes
and DNA−RNA Hybrids Containing 2′-O-Propargylated
2-Aminoadenosine (1). The syntheses of the propargylated
phosphoramidites of canonical purine and pyrimdine nucleosides
(Figure 2) have been already reported.16,17 The 2′-O-propargylated
a
Reagents and conditions: (i) trimethylsilyl chloride, methoxyacetyl
chloride, pyridine, 4 °C to rt; (ii) trimethylsilyl chloride, i-BuCl,
pyridine, 4 °C to rt; (iii) 4,4′-dimethoxytrityl chloride, pyridine, rt; (iv)
NC(CH2)2OP(Cl)N(i-Pr)2, DIPEA, DCM, rt.
nucleosides are different.15 Once we had 2′-O-propargylated
nucleoside 1 in hand, we studied the “click reaction” with
nucleoside 1 and bis-azide 4. First, the stepwise click
functionalization of nucleoside 1 was performed with copper
turnings and a 5-fold excess of bis-azide 4 furnishing the
monofunctionalized nucleoside 19 in 45% yield. In the next
step, 19 was converted to the cross-linked adduct 20 (42%).
Alternatively, nucleoside 1 was reacted with bis-azide 4 using a
nucleoside to bis-azide ratio of 2:1, affording the cross-linked
adduct 20 in 60% yield (Scheme 6) (for details, see
Figure 2. Structures of nucleosides used for hybridization studies on
DNA duplex formation.
2-aminoadenosine phosphoramidites 14 and 18, as well as the
corresponding 2′-deoxy building block 10, are described in this
work. These building blocks, together with those of canonical
nucleosides and the phosphoramidite of 1,3-propanediol18,
were used in the solid-phase synthesis of oligonucleotides. The
coupling yields were always higher than 95%. Modified
nucleosides were incorporated in both strands of the 12-mer
DNA oligonucleotides 5′-d(TAG GTC AAT ACT) (25) and
3′-d(ATC CAG TTA TGA) (26), which served as reference
duplex. The duplex stability of oligonucleotides incorporating
2′-O-propargylated 2-aminoadenosine 1 is compared with that
of oligonucleotides containing 2′-O-propargylated adenosine
(3) or the nonpropargylated nucleoside 2-amino-2′-deoxyade-
nosine (2a). The removal of the isobutyryl residue from the
2-amino group of compounds 2a and 1 was encountered with
difficulties during oligonucleotide synthesis, as this group
withstands standard deprotection conditions. However, it could
be cleaved under harsh conditions (MeNH2/aq NH3, 1:1,
60 °C, 1 h). In contrast, the methoxyacetyl group in the 2-position
was easily removed. Thus, a skillful choice of building blocks in
combination with the deprotection conditions made oligo-
nucleotides with or without isobutyryl residue easily accessible.
All oligonucleotides were characterized by MALDI-TOF mass
spectrometry (Table 1). Both the protected and nonprotected
oligonucleotides were used for hydridization studies (Table 2;
Experimental Section).
Scheme 6. Cu(I)-Catalyzed Stepwise Click and Bis-Click
a
Reaction Performed with Nucleoside 1 and Bis-azide 4
Earlier hybridization studies with canonical ribonucleosides
bearing 2′-O-propargyl residues were performed by Sproat and
co-workers. In their studies, thermal and enzymatic duplex
stabilization of the propargyl modification was compared to
2′-O-methyl or 2′-O-methoxyethyl derivatives, and a possible
application in antisense oligonucleotides was investigated.19
From hybridization studies on 2′-O-propargylated oligonucleo-
tides, they concluded that this modification increases the duplex
stability similar to other 2′-modifications.19
a
Reagents and conditions: (i) copper turnings, H2O/CH3CN, 10:1,
ratio 1/4 1:5; (ii) copper turnings, H2O/CH3CN, 10:1, ratio 1/4 2:1;
(iii) copper turnings, H2O/CH3CN, 10:1, ratio 1/19 1:1.
Experimental Section). All synthesized compounds were
characterized by 1H NMR, 13C NMR, and mass spectra.
DEPT-135 and H−13C gated-decoupled NMR spectra were
1
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Table 1. Molecular Masses of Oligonucleotides Measured by MALDI-TOF Mass Spectrometry
a
b
Calculated on the basis of molecular weight as [M + H]+. Determined by MALDI-TOF mass spectrometry as [M + H]+ in the linear positive
mode.
We selected 2′-O-propargylated 2-aminoadenosine (1) for
this study due to the stabilizing properties of 2-aminoadenine
replacing adenine in DNA−DNA duplexes or DNA−RNA
hybrids. In earlier studies on homopurine−homopyrimidine
polyribonucleotide duplexes with 2-aminoadenosine replacing
adenosine, a Tm increase of +33 °C was observed.20 For the
corresponding oligodeoxyribonucleotide duplexes, the incre-
ment is less but still high (about +12 °C). Later, optical melting
experiments on oligodeoxyribonucleotides showed that the
situation is more complex with regard to the stabilizing effect of the
2-aminoadenosine−dT base pair in different sequence motifs.21
The participation of the 2-amino group of 2-aminoadenosine
(2b) in a tridentate base pair with dT (bp1) leads to a higher
stability than the bidentate dA−dT (bp4), and the stabilizing
effect is similar to that of the tridentate dG−dC pair (bp3)
(Figure 3). However, due to ups and downs of thermal stability
data, this matter is not well understood. Only when we used
2-aminoadenine nucleoside analogues, which have a 7-deazapurine
or 8-aza-7-deazapurine skeleton, and a halogen substituent, which
were first studied in our laboratory and later used by others, was a
pronounced stability increase observed.11
Figure 3. Base pair motifs related to 2-amino-dA−dT, protected
2-amino-dA−dT, dA−dT, and dG−dC.
In the following, the properties of the propargylated
nucleoside 1 along with the related compounds 3, 11, 21 and
the 2-amino 2′-deoxyribonucleosides 2a and 22 were investigated
with regard to base pair stability (Table 2). Hybridization was
studied on DNA−DNA duplexes, as well as on DNA−RNA
hybrids having the modification always in the DNA strand, and the
Tm data are summarized in Table 2. Some propargylated and
azido-modified oligonucleotides served later as starting materials
for cross-linking reactions using the “stepwise click” reaction
(Tables 3 and 4).
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a
Table 2. Tm Values of Modified DNA Duplexes and DNA−RNA Hybrids Containing 2′-O-Propargylated Nucleosides
a
Measured at 260 nm in 1 M NaCl, 100 mM MgCl2, 60 mM Na-cacodylate (pH 7.0) with 2.5 + 2.5 μM single-strand concentration. Tm values were
determined from the melting curves by using the software MELTWIN 3.0.
The Tm of the reference RNA−RNA duplex 31·32 is 10 °C
higher than that of the corresponding DNA−DNA duplex 25·
26. The stability of DNA−RNA hybrids (25·32, 31·26)
corresponds to that of the DNA−DNA duplex and not to
that of the RNA−RNA duplex. A single modification of
oligonucleotide duplexes with 2′-O-propargyl residues has only
a minor impact on the thermal stability of DNA−DNA and
DNA−RNA duplexes (Table 2). Generally, duplexes incorpo-
rating 1 instead of propargylated dA opposite to dT show
slightly increased Tm values (+1 °C). Apparently, the 2-amino
group of 1 is too basic to act as an efficient proton donor to
form a third hydrogen bond with dT. Accordingly, formation of
a tridentate base pair of 1 with dT as shown in bp1 is unlikely
(Figure 3). This is supported by the finding that duplex 27·28
containing a dG−dC base pair (bp3) instead of a dA−dT pair
(bp4) shows a significantly higher Tm value compared to duplex
38·26 with a 1−dT base pair. Apparently, bp1 is only formed in
a particular environment (e.g., in homopolymers).22
Surprisingly, when the 2-amino group was still protected with
an isobutyryl residue (11 or 22), as in duplexes 44·26 and 25·
45 as well as in 46·26 and 25·48, there were few differences in
the Tm values compared to the 2-amino unprotected
compounds, even under the spatial demand of the isobutyryl
residue. Similar observations were already made on other
modified oligonucleotides with acetyl groups.23
4. Formation of Cross-Linked Duplexes by Stepwise
Click Ligation Using the Bifunctional Azide 4. In the next
series of experiments, oligonucleotides incorporating 2′-O-pro-
pargylated 2-aminoadenosine (1) were cross-linked to oligonu-
cleotides containing other propargylated nucleosides to increase
duplex stability. Cross-linking was performed between two
complementary strands. The cross-links were introduced within
base pairs or with proximal propargylated nucleosides of the
complementary strand. The click reaction was performed in a
stepwise manner with single-stranded azido derivatives prepared
by a first-click reaction. This methodology was applied for the
following reason: Although it is accepted knowledge that template-
assisted reactions occur much faster than those not assisted by the
template effect,24 the situation for terminal and internal cross-
linking is different. Cross-linking of terminal azido groups with
terminal alkyne residues of preformed DNA duplex is much faster
than on individual single strands.15,24 The situation changes when
a cross-link between two complementary strands has to be
installed in the center of a duplex and short linkers are employed.
This is valid for the formation of cross-links by the bis-click
reaction with bifunctional azides in the center of a preformed
duplex consisting of long oligonucleotides. In this case, the duplex
can have a negative impact on cross-linking. Inspection of the
DNA double helix structure clearly indicates that internal cross-
links are difficult to form due to steric hindrance. Thus, a positive
template effect can be expected only when all components of the
bis-click reaction are well-suited in the required space of the DNA
duplex. Consequently, when using bis-click conditions (stoichio-
metric amounts of bis-azide and alkynylated DNA), these effects
can cause side reactions. Possibly, the azido-modified DNA strand
can react with incompletely functionalized material of the same
strand instead of the complementary strand, thus forming homo-
DNA duplexes. This unfavorable situation can be circumvented if
the stepwise procedure is used either employing chelating bis-
azides or using an excess of bis-azide to form a monofunctionalized
azido−DNA intermediate.8,25
Accordingly, in the first step of the stepwise click reaction,
the propargylated oligonucleotides were converted into
monofunctionalized azido derivatives using a large excess of
azide 4 (ratio ODN/bis-azide 1:15). Usually, 3 to 4 OD of
propargylated ODN was dissolved in a mixture of 100 μL of
H2O/MeCN (10:1, v/v), followed by the addition of copper
turnings. By this means, the reaction was completed within 3 h.
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Table 3. Molecular Masses of Monofunctionalized Oligonucleotides Prepared by the Stepwise Click Reaction
a
b
Calculated on the basis of molecular weight as [M + H]+. Determined by MALDI-TOF mass spectrometry as [M + H]+ in the linear positive
mode.
a
Scheme 7. Stepwise Click Reaction of Sugar-Modified DNA
a
General scheme for the monofunctionalization and cross-linking of oligonucleotides demonstrated for 49 and ICL-1.
The sequences of the azido-modified oligonucleotides and their
molecular masses are depicted in Table 3.
linked ODNs are listed in Table 4 and were characterized by
MALDI-TOF and ion-exchange HPLC (Figure 4; Figure S3,
Supporting Information; for detailed structures, see Figure S2,
Supporting Information).
In the second step, the azido-functionalized ODNs were
cross-linked with alkyne-modified complementary duplexes.
The alkyne-modified ODN (2−3 OD) was dissolved in 100 μL
H2O/MeCN, then 50 μL of 0.1 M NaCl and monofunction-
alized oligonucleotide were added. The monofunctionalized
ODN was taken in slight excess to drive the reaction to
completion, leading to easy purification. Finally, copper
turnings were introduced, and the mixture was stirred in the
dark at normal atmosphere for 3 h (Scheme 7). The reaction
yield was in the range of 50−60% (for details, see Experimental
Section). The structures of monofunctionalized ODNs are
given in the Supporting Information (Figure S1). The cross-
It is worth mentioning that the conditions described above
using copper turnings and H2O/MeCN mixtures as reaction
medium, a method initially introduced by Jawalekar, is
particularly useful as the click reactions (monofunctionalization
and cross-linking) were performed quickly and efficiently.16b
The copper turnings have to be removed after 1 h. Then, the
reaction was continued for completion (2 h at rt) without
copper turnings. When the copper turnings were kept in the
solution for more than 1 h, the final product turned a greenish
color, and the yield of the click reactions was greatly reduced.
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Table 4. Tm Values of Sugar-Modified Duplexes, Cross-Linked Duplexes, and Hairpins
a
Measured at 260 nm in 1 M NaCl, 10 mM MgCl2, 10 mM Na-cacodylate (pH 7.0) with 2.5 + 2.5 μM single-strand concentration for non-cross-
b
linked oligonucleotides and 5 μM for cross-linked oligonucleotides. For hairpins, 5 μM of oligonucleotide was used. In 50 mM NaCl, 10 mM
Na3PO4, 0.1 mM EDTA with 2.5 + 2.5 μM single-strand concentration for non-cross-linked oligonucleotides and 5 μM for cross-linked
c
d
oligonucleotides. For hairpins, 5 μM of oligonucleotide was used. Data taken from visual inspection of the melting curve. Calculated on the basis
e
of molecular weight as [M + H]+. Determined by MALDI-TOF mass spectrometry as [M + H]+ in the linear positive mode. pd corresponds to
1,3-propanediol. All cross-linked oligonucleotides are shown in the Supporting Information (Figure S2). Tm values were determined from the
melting curves by using the software MELTWIN version 3.0.
Figure 4a−c displays ion-exchange HPLC profiles demonstrat-
ing the various mobilities of the alkynylated, azido-modified, and
cross-linked oligonucleotides, which were purified by reversed-
phase HPLC (for details, see Experimental Section). Figure 4d
shows the ion-exchange profile of an artificial mixture of all
components. Azido-modified oligonucleotides with identical base
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Figure 4. Ion-exchange HPLC elution profiles of (a) monofunctionalized ODN-49, ODN-38, and interstrand cross-linked ODN ICL-1; (b)
monofunctionalized ODN-55, ODN-43, and interstrand cross-linked ODN ICL-2; (c) monofunctionalized ODN-50, ODN-61, and interstrand
cross-linked ODN ICL-7; (d) artificial mixture of monofunctionalized ODN-54, ODN-39, and interstrand cross-linked ODN ICL-6. Ion-exchange
chromatography was performed on a 4 × 250 mm DNA Pac PA-100 column, using the following buffer system: (A) 25 mM Tris-HCl, 10% MeCN,
pH 7.0; (B) 25 mM Tris-HCl, 1.0 M NaCl, and 10% MeCN, pH 7.0. Elution gradient: 0−30 min 20−80% B in A with a flow rate of 0.75 mL min−1.
composition can be cleanly separated from the alkynylated
precursor molecules. Cross-linked duplexes migrate significantly
slower. The reaction shown in Scheme 7 is not limited to
propargylated 2-aminoadenosine. The stepwise click reaction was
also performed on propargylated derivatives of the canonical
nucleosides. A complete set of ion-exchange profiles of
propargylated, monofunctionalized, and cross-linked ODNs is
given in the Supporting Information (Figure S3; for reaction
profiles, see Figures S4 and S5).
5. Thermal Stability of Cross-Linked Duplexes with
Cross-Links between 2′-O-Propargylated 2-Aminoade-
nosine and Other Modified Nucleoside Residues. Differ-
ent methods have been employed for cross-linking DNA.26 Our
laboratory has reported on site-specific cross-linking of
oligonucleotides in which terminal triple bonds on the side
chain of a nucleobase were clicked together and the length of
the linker, the ligation position, or both were altered. In some
cases, Tm values of cross-linked duplexes were about 25 °C
higher than those for the non-cross-linked hybrids; in other
cases, the effect was lower.15 Later, cross-linking oligonucleo-
tides with 2′-O-propargylated sugar residues in DNA with
parallel and antiparallel chain orientation were reported.9 In this
work, 2′-O-propargylated nucleosides were introduced in both
strands of oligonucleotide duplexes (Table 4, left column).
Cross-linked duplexes show significantly increased Tm values
(Table 4, right column). This is valid not only for cross-links
with 2-aminoadenosine but also for other nucleoside
combinations.
From Table 4 (upper part), it is apparent that Tm values of
cross-linked duplexes ligated at the terminus of the duplex
(ICLs 1−4) are around 80 °C. Moving the ligation site toward
the center of the duplex leads to decreased duplex stability
(ICLs 5−12; Table 4, lower part). Thus, the connectivity of
cross-linked residues plays a key role in duplex stability.
From the physical point of view, the thermal stability of a
cross-linked duplex increases simply by the change of the
molecularity during melting or formation. The non-cross-linked
duplex dissociates in a bimolecular reaction to yield single-
stranded species, while in the cross-linked duplexes, base pair
melting occurs without strand separation.
The melting of a cross-linked duplex with a terminal ligation
site can be described as the melting of a hairpin. We
synthesized two hairpins (H1 and H2) to analyze this matter.
The hairpins consist of the sequence motif of the duplex 25 and
26 (Table 1) in the stem region and a loop of either five dT
(H1) or five propanediol (H2) residues. These two hairpins
exhibit similar Tm values (77 and 78 °C). All four
oligonucleotides with cross-links at the terminus (ICLs 1−4)
show slightly higher Tm values than the two hairpins H1 and
H2, in particular at low salt conditions. This reveals that not
only the change of the molecularity during the melting process
of cross-linked duplexes vs non-cross-linked duplexes but also
structural changes induced by the cross-link can have an effect
on the stability of the molecule. In the case of cross-linked
duplexes with terminal ligation sites, this effect is positive; for
cross-linked duplexes with internal ligation sites, a decreased
thermal stability is often observed. For cross-linked duplexes
with lower stability, see Table 4 (right column, lower part), and
for those with higher thermal stability, see Table 4 (right
column, upper part).
To support these findings, molecular dynamic simulation
studies (AMBER force field, HyperChem 8.0; Hypercube, Inc.,
Gainesville, FL, 2001) were conducted on ICL-1 and ICL-6
and are displayed in Figure 5. From the figure, it is evident that
a long run of base pairs is still intact in ICL-1 (terminal cross-
link). For ICL-6 (internal cross-link), two shorter regions of
matched base pairs exist. A closer inspection shows that in
ICL-1 as well as in ICL-6 the base pair of the cross-link and at
least one next to it are opened. Nevertheless, the disturbed base
pair is always replaced by a covalent linkage of the strands. A
higher stability is observed for duplexes with terminal cross-
links compared to those with central ligation sites, which might
result from a stronger distortion of the helix for central cross-
links, changes in stacking interactions, or a combination thereof.
Furthermore, helix hydration and ion-binding might be affected
in a different manner. In turn, this effect depends on the
participating nucleosides, their distance in the duplex, and cross-
linking within a base pair or between nucleosides residues in
proximal or more distant positions. Also, the length of the linker
is of utmost importance. Unpredictable Tm changes might occur
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25·26, the corresponding hairpin H1, and the cross-linked
duplex with terminal (ICL-1) or central (ICL-6) ligation sites
at high salt and low salt measuring conditions (for additional
melting curves measured under high and low salt conditions,
see Supporting Information, Figures S5 and S6).
From Figure 6, it is obvious that the duplex 25·26 and the
hairpin H1, as well as the cross-linked duplex with the near
terminal ligation site (ICL-1) connecting propargylated 2-
aminoadenosine with propargylated dT, show similar steep
melting curves with high cooperativity. In contrast, the duplex
with the cross-link near the center (ICL-6) shows a less
cooperative melting, which is particularly visible under high salt
conditions. Furthermore, the duplex with the central ligation
site began melting with a continuous increase of the UV-
absorbance followed by cooperative melting. The hypochro-
micity of cooperative melting was reduced over that observed
for the corresponding duplexes or hairpins under the same high
salt conditions. The cross-linked duplex with a terminal ligation
site (ICL-1) shows a steep melting profile similar to that of
hairpin H1 but has a different salt dependence.
Figure 5. Molecular models of (a) ICL-1 with a terminal cross-link;
(b) ICL-6 with a cross-link at the center of the duplex. The
modification sites are presented as colored, space-filling, overlapping
spheres. The models were constructed using HyperChem 8.0 and
energy minimized by using AMBER calculations.
CONCLUSION AND OUTLOOK
■
A protecting group strategy for 2′-O-propargylated 2-amino-
adenosine (1) containing two amino groups of rather different
reactivities was developed successfully, and various new
phosphoramidites were prepared as building blocks for solid
phase oligonucleotide synthesis. Hybridization experiments with
a series of oligonucleotide duplexes containing 2,6-diaminopurine
as nucleobase showed no significant duplex stabilization over
those containing adenine. Consequently, the formation of a
tridentate base pair was ruled out. An isobutyryl group protecting
when the cross-linked duplex has the choice to form another set
of base pairs.
We recognized that the salt dependence of the Tm values is
rather different between duplexes, hairpins, and cross-linked
species. Figure 6 compares melting profiles of the parent duplex
Figure 6. Melting profiles of the duplex 25·26, the hairpin H1, and the cross-linked duplexes ICL-1 and ICL-6. Tm curves in red represent heating
profiles measured in 1 M NaCl, 100 mM MgCl2, 60 mM sodium-cacodylate. Tm curves in blue represent heating profiles measured in 50 mM NaCl,
10 mM sodium-phosphate, 0.1 mM EDTA buffer. Δ corresponds to thermal melting.
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a b
,
Table 5. 13C NMR Chemical Shifts of 2-Aminoadenosine Derivatives
c
c
C(2)
C(4)
151.4
C(5)
113.4
C(6)
C(8)
135.8
CC
CH2
56.9
C(1′)
C(2′)
C(3′)
C(4′)
C(5′)
CO
CHMe2
CH
e
1
156.2
160.1
79.7
77.6
86.2
79.8
68.9
84.8
61.6
f
2a
7
156.1
160.0
151.2
153.3
113.5
119.3
160.0
160.1
135.7
137.3
83.1
84.5
39.4
79.8
71.0
68.9
87.6
86.1
61.9
61.4
77.7
79.8
56.8
c
8
9
152.2
152.2
152.9
152.2
152.8
152.1
151.8
151.6
156.2
156.2
152.6
152.4
150.3
152.7
152.1
149.9
149.9
149.7
151.4
151.4
121.0
121.0
116.1
122.1
122.2
115.9
120.3
119.9
113.4
113.4
149.9
149.9
156.1
159.8
159.8
155.9
152.6
152.2
160.1
160.1
141.7
141.7
138.5
140.1
139.9
138.4
141.6
141.3
135.9
135.9
83.1
d
d
70.7
70.5
68.8
68.7
69.1
68.5
68.7
68.9
69.0
87.9
86.2
86.1
86.1
83.8
84.8
86.1
85.3
86.0
86.1
61.7
64.3
61.3
61.2
63.9
61.0
61.1
63.6
61.6
175.6
175.2
175.5
175.0
175.0
34.2
34.3
29.6
34.2
34.1
c
83.2
11
12
13
15
16
17
19
20
79.9
77.6
79.8
77.5
79.9
77.6
79.6
77.3
80.2
77.6
79.7
77.4
57.0
57.1
57.3
84.8
84.8
85.4
85.8
85.2
85.6
85.1
85.1
80.1
80.2
79.8
79.8
79.8
79.5
80.2
80.1
19.4
19.3
19.3
19.2
19.3
19.2
174.9
174.8
168.3
34.3
34.2
56.8
58.3
58.5
57.2
58.2
57.2
49.8
49.2
48.6
175.5
169.2
175.2
168.8
19.2
19.0
34.3
34.1
69.0
61.6
49.0
a
b
c
d
e
f
Measured in DMSO-d6 at 298 K. Purine numbering. Tentative. Superimposed by DMSO. Reference no. 9. Reference no. 27.
1
was set to 2.50 ppm for H NMR and 39.50 ppm for 13C NMR. The
13C NMR signals were assigned on the basis of DEPT-135 and
1H−13C gated-decoupled NMR spectra (Table 5; for coupling
the 2-amino function does not destabilize DNA−DNA and
DNA−RNA duplexes, showing that this group is well
accommodated in complementary duplexes. Cross-linking
oligonucleotides containing 2′-O-propargylated 2-aminoadeno-
sine (1) led to significant duplex stabilization (Tm = 79 °C for
ICL-1 and 73 °C for ICL-6 compared to the reference duplex
25·26, Tm = 50 °C). These cross-linked duplexes were prepared
in a stepwise manner using copper turnings as a catalyst. It was
shown that duplex stabilization by cross-links does not result
simply from a molecularity change from duplex to hairpin
melting but also was influenced by the ligation position.
Terminal ligation led to higher melting duplexes than
hairpins, while duplexes with central ligation sites were less
stable than hairpins due to helix distortion. With the help of
the stepwise click procedure, any DNA strand with an
alkynyl side chain (independent of the position) can be
converted into an azido DNA. This applies to the formation
of homodimers and heterodimers. Azido DNA can be con-
nected to any other alkynylated biomolecule and can be used
for the construction of supramolecular assemblies and nano-
scopic devices.
constants, see Table S1, Supporting Information). Reversed-phase
HPLC was carried out on a 250 × 4 mm RP-18 LiChrospher 100
column with an HPLC pump connected with a variable wavelength
monitor, a controller, and an integrator. Gradients used for HPLC
chromatography: A = MeCN; B = 0.1 M (Et3NH)OAc (pH 7.0)/
MeCN, 95:5. Conditions: (I) 3 min 15% A in B, 12 min 15−50% A in
B, and 5 min 50−10% A in B, flow rate 0.7 mL min−1; (II) 0−25 min
0−20% A in B, flow rate 0.7 mL min−1. Melting curves were measured
with a UV−vis spectrophotometer equipped with a thermoelectrical
controller with a heating rate of 1 °C/min. The Tm values were
determined from the melting curves using the software MELTWIN,
version 3.0 (J. A. McDowell, 1996). ESI-TOF mass spectra of the
nucleosides were measured with a Micro-TOF spectrometer.
Molecular masses of oligonucleotides were determined by MALDI-
TOF mass spectrometry in the linear positive mode with 3-hydroxy-
picolinic acid (3-HPA) as a matrix (Tables 1, 3, and 4).
Synthesis, Purification, and Characterization of Oligonu-
cleotides. The syntheses of oligonucleotides 25−48 and 59−65 were
performed on a DNA synthesizer at a 1 μmol scale (trityl-on mode)
using the phosphoramidites 10, 14, and 18 and the phosphoramidites
of 2′-O-propargyladenosine, 2′-O-propargylguanosine, 2′-O-propar-
gylthymidine, 2′-O-propargylcytidine, and 1,3-propanediol, as well as
the standard phosphoramidite building blocks following the synthesis
protocol for 3′-O-(2-cyanoethyl)phosphoramidites (user’s manual for
the 392 and 394 DNA synthesizer, Applied Biosystems, Weiterstadt,
Germany). After cleavage from the solid support, the oligonucleotides
were deprotected in 25% aqueous ammonia solution for 18 h at 60 °C.
The isobutyryl residue of the 2-amino group of 2-amino-2′-
deoxyadenosine (2a) and the 2′-O-propargylated derivative 1
(phosphoramidites 10 and 14) withstands the standard deprotection
conditions. Therefore, this group was cleaved under harsh conditions
using MeNH2/aq. NH3 (1:1, v/v, at 60 °C for 1 h). Then, the
oligonucleotides were dried and purified. The purification of the
“trityl-on” oligonucleotides was carried out on reversed-phase HPLC
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. Thin-layer chromatography
(TLC) was performed on TLC aluminum sheets covered with silica
gel 60 F254 (0.2 mm). Flash column chromatography (FC): silica gel
60 (40−60 μM) at 0.4 bar. UV spectra were recorded on a
spectrophotometer: λmax in nm, ε in dm3 mol−1 cm−1. NMR spectra were
1
measured at 300.15 MHz for H, 75.48 MHz for 13C, and 121.52 MHz
for 31P. The J values are given in hertz (Hz); δ values in parts per
million (ppm) relative to Me4Si as an internal standard. For NMR
spectra recorded in DMSO-d6, the chemical shift of the solvent peak
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1
(RP-18 column; gradient system I). The purified “trityl-on”
oligonucleotides were treated with 2.5% of Cl2CHCOOH/CH2Cl2
for 5 min at 0 °C to remove the dimethoxytrityl residues. The
detritylated oligomers were purified by reversed-phase HPLC
(gradient II). The oligomers were desalted on a short column using
distilled water for elution of salt, while the oligonucleotides were
eluted with H2O/MeOH (2:3). The solvent was evaporated using a
Speed Vac evaporator to yield colorless solids, which were then frozen
at −24 °C. The molecular masses of the oligonucleotides were
determined by MALDI-TOF mass spectrometry in the linear positive
mode. Extinction coefficients ε260 (H2O) of nucleosides are dA, 15
400; dG, 11 700; dT, 8800; dC, 7300; 3, 14 800 (MeOH); 21, 8600
(MeOH); 23, 8200 (MeOH); 24, 13 000 (MeOH). For the extinction
coefficients of 2-aminoadenosine derivatives, see Supporting Informa-
tion (Table S2).
cm−1 16 200), 276 (16 700). H NMR (DMSO-d6, 300 MHz): δ 1.05−
1.14 (m, 6H, CH3), 2.32−2.41 (m, 1H, C2′−H), 2.83−2.92 (m, 2H, C2′−
H, CH), 3.00−3.15 (m, 2H, C5′−H, CH), 3.26−3.32 (m, 1H, C5′−H),
3.69, 3.71 (2s, 6H, 2 × OCH3), 3.94−3.97 (m, 1H, C4′−H), 4.52−4.55
(m, 1H, C3′−H), 5.34 (d, J = 4.5 Hz, 1H, C3′−OH), 6.39 (‘t’, J = 6.3 Hz,
1H, C1′−H), 6.37−6.41 (m, 1H, 4H, Ar−H), 7.16−7.19 (m, 7H, Ar−H),
7.29−7.32 (m, 2H, Ar−H) 8.40 (s, 1H, C8−H), 10.30 (br s, 1H, NH),
10.52 (br s, 1H, NH). ESI-TOF m/z calcd for C39H44N6O7 [M + Na]+,
731.3164; found, 731.3164.
9-[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-β-D-erythro-pentofur-
anosyl]-N2,N6-diisobutyryl-purin-2,6-diamine 3′-(2-Cyanoethyl)-
N,N-diisopropylphosphoramidite (10). To a solution of compound
9 (0.30 g, 0.42 mmol) in dry CH2Cl2 (10 mL) were added (i-Pr)2NEt
(0.097 g, 125 μL, 0.72 mmol) and 2-cyanoethyl diisopropylphosphor-
amidochloridite (0.13 g, 125 μL, 0.56 mmol), and the reaction mixture
was stirred for 15 min at rt. Then, the reaction mixture was diluted
with CH2Cl2 (30 mL), poured into 5% NaHCO3 solution (20 mL),
and extracted with CH2Cl2 (3 × 20 mL). The combined organic layers
were dried over Na2SO4, and the solvent was evaporated. The residual
foam was applied to FC (silica gel, CH2Cl2/acetone, 80:20).
Evaporation of the main zone afforded 10 (0.277 g, 73%) as a
N6-[(Di-n-butylamino)methylidene]-9-[2-O-(propargyl)-β-D-
ribofuranosyl]purin-2,6-diamine (7). Compound 1 (0.25 g,
0.78 mmol) was dissolved in MeOH; then N,N-dibutylaminomethy-
lidene dimethylacetal (500 μL, 2.14 mmol) was added and the solution
was stirred at 40 °C for 1 h. The solvent was removed, and the remaining
residue was adsorbed on silica gel and subjected to FC (silica gel,
CH2Cl2/MeOH, 9:1), affording compound 7 (0.147 g, 41%) as a
colorless solid. TLC (silica gel, CH2Cl2/MeOH, 90:10) Rf 0.4. λmax
(MeOH)/nm 253 (ε/dm3 mol−1 cm−1 13 900), 279 (14 500), 325
colorless foam. TLC (silica gel, CH2Cl2/acetone, 80:20) Rf 0.5, 0.6. 31
P
NMR (CDCl3, 121.5 MHz): 148.77. ESI-TOF m/z calcd for
C48H62N8O8P [M + H]+, 909.4423; found, 909.4423.
1
(20 600). H NMR (DMSO-d6, 300 MHz): δ 0.87−0.94 (m, 6H, 2 ×
9-[(2-O-propargyl)-β-D-ribofuranosyl]purin-2,6-diamine (1)
and 9-[(3-O-propargyl)-β-D-ribofuranosyl]purin-2,6-diamine
(6) from 3′,5′-(1,1,3,3-Tetraisopropyl-1,3-disiloxan-1,3-yl)-9-
(β-D-ribofuranosyl)purin-2,6-diamine (5). Compound 513a (0.5
g, 0.95 mmol) was dissolved in anhydrous DMF (10 mL) under argon
atmosphere. The solution was cooled to 5 °C, and NaH (0.038 g,
1.28 mmol, 60% dispersion in mineral oil) was added, followed by the
addition of TBAI (0.16 g, 0.42 mmol) and propargyl bromide (0.14 g,
105 μL, 1.18 mmol). The reaction mixture was allowed to stir for
20 min at 55 °C. After completion of the reaction (TLC monitoring),
the solvent was evaporated under reduced pressure. The residual
suspension was dissolved in THF (10 mL) and treated with 0.1 M
TBAF in THF (8 mL). The resulting mixture was stirred at rt for 1 h.
The solvent was evaporated under reduced pressure, and the
remaining residue was subjected to FC (CH2Cl2/MeOH, 95:5).
From the faster migrating zone, 9-[(2-O-propargyl)-β-D-ribofuranosyl]-
purin-2,6-diamine (1) was obtained as a colorless foam (0.2 g, 66%).
TLC (silica gel, CH2Cl2/MeOH, 90:10) Rf 0.6. λmax (MeOH)/nm
256 (ε/dm3 mol−1 cm−1 9 400), 260 (8 800), 281 (10 900). Anal. Calcd
for C13H16N6O4 (320.3): C, 48.75; H, 5.03; N, 26.24. Found: C, 48.62;
H, 5.10; N, 26.09. From the slower migrating zone, 9-[(3-O-propargyl)-
β-D-ribofuranosyl]purin-2,6-diamine (6) was isolated as colorless solid
(0.023 g, 8%). TLC (silica gel, CH2Cl2/MeOH, 90:10) Rf 0.5. λmax
(MeOH)/nm 260 (ε/dm3 mol−1 cm−1 9 800), 281 (11 400). Anal.
Calcd for C13H16N6O4 (320.3): C, 48.75; H, 5.03; N, 26.24. Found: C,
48.93; H, 5.15; N, 26.10. The analytical data of compounds 1 and 6 are
identical to reported values.9
CH3), 1.26−1.36 (m, 4H, CH2), 1.54−1.61 (m, 4H, 2 × CH2), 3.34 (m,
superimposed by DMSO, NCH2), 3.38 (t, J = 2.1 Hz, 1H, CCH),
3.53−3.63 (m, 4H, 2 x C5′−H, NCH2), 3.92−3.95 (m, 1H, C4′−H),
4.15−4.32 (m, 3H, OCH2, C3′−H), 4.53 (dd, J = 4.8 Hz, J = 5.1 Hz, 1H,
C2′−H), 5.30 (m, 2H, C5′−OH, C3′−OH), 5.90 (d, J = 6.3 Hz, 1H,
C1′−H), 6.00 (br s, 2H, 2-NH2), 8.04 (s, 1H, C8−H), 8.79 (s, 1H, N
CH). ESI-TOF m/z calcd for C22H33N7O4 [M + H]+, 460.2667; found,
460.2657.
9-(2-Deoxy-β-D-erythro-pentofuranosyl)-N2,N6-diisobutyryl-
purin-2,6-diamine (8). Compound 2a (0.5 g, 1.88 mmol) was dried
by repeated coevaporation with pyridine (3 × 5 mL) and dissolved in
pyridine (20 mL). Then, trimethylsilyl chloride (1.9 g, 2.26 mL, 17.42 mmol)
was added, and the solution was stirred at rt for 15 min. Then,
isobutyryic chloride (1.62 g, 1.60 mL, 15.2 mmol) was added dropwise
over a period of 5 min, and the reaction mixture was stirred at rt for 3 h.
After completion of the reaction (TLC monitoring), the reaction
mixture was cooled in an ice-bath, H2O (5 mL) was added, and,
subsequently, 5 min later, 28% aq ammonia (5 mL) was introduced.
Stirring was continued for 30 min at rt. The solvent from the reaction
mixture was evaporated, and the remaining oily residue was
coevaporated with toluene (3 × 10 mL) and subjected to FC (silica
gel, CH2Cl2/MeOH, 90:10) to give compound 8 (0.474 g, 62%) as a
colorless solid. TLC (silica gel, CH2Cl2/MeOH, 90:10) Rf 0.6. λmax
(MeOH)/nm 265 (ε/dm3 mol−1 cm−1 11 500), 287 (11 200). 1H NMR
(DMSO-d6, 300 MHz): δ 1.07−1.13 (m, 6H, 2 x CH3), 2.26−2.34 (m,
1H, C2′−H), 2.69−2.78 (m, 1H, C2′−H), 2.85−2.92 (m, 1H, CH),
2.99−3.08 (m, 1H, CH), 3.49−3.64 (m, 2H, 2 x C5′−H), 3.84−3.88
(m, 1H, C4′−H), 4.43−4.44 (m, 1H, C3′−H), 4.91 (t, J = 5.4 Hz, 1H,
C5′−OH), 5.33 (d, J = 4.2 Hz, 1H, C3′−OH), 6.36 (‘t’, J = 6.6 Hz, 1H,
C1′−H), 8.51 (s, 1H, C8−H), 10.35 (br s, 1H, NH), 10.51 (br s, 1H,
NH). ESI-TOF m/z calcd for C18H26N6O5 [M + Na]+, 429.1857;
found, 429.1857.
9-[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-β-D-erythro-pentofur-
anosyl]-N2,N6-diisobutyryl-purin-2,6-diamine (9). Compound 8
(0.30 g, 0.74 mmol) was dried by repeated coevaporation with dry
pyridine (3 × 10 mL). The residue was dissolved in dry pyridine
(5 mL), and the mixture was stirred with 4,4′-dimethoxytrityl chloride
(0.400 g, 1.18 mmol) until the starting material was completely
consumed (TLC monitoring). Then, the reaction mixture was diluted
with CH2Cl2 (20 mL) and washed with 5% aq NaHCO3 solution
(20 mL) and water (20 mL). The organic layer was dried over Na2SO4, the
solvent was evaporated under reduced pressure, and the residue was
subjected to FC (silica gel, CH2Cl2/acetone, 1:1). Evaporation of the main
zone afforded 9 (0.30 g, 58%) as a pale yellow foam. TLC (silica gel,
CH2Cl2/acetone, 1:1) Rf 0.4. λmax (MeOH)/nm 268sh (ε/dm3 mol−1
N2-Isobutyryl-9-[2-O-(propargyl)-β-D-ribofuranosyl]purin-
2,6-diamine (11). Compound 1 (0.2 g, 0.62 mmol) was dried by
repeated coevaporation with pyridine, (3 × 5 mL) and dissolved in
pyridine (8 mL). Trimethylsilyl chloride (0.34 g, 0.4 mL, 3.12 mmol)
was added and the mixture was stirred at rt for 15 min. Then, the
solution was cooled to −20 °C, isobutyryl chloride (0.073 g, 0.072 mL,
0.68 mmol) was added dropwise over 5 min, and the reaction mixture
was stirred at −20 °C for 2.5 h followed by 1 h at rt. After completion
of the reaction (TLC monitoring), the reaction mixture was cooled in
an ice-bath, ethanol (2 mL) was added, and subsequently, 5 min later,
28% aq ammonia was added. Stirring was continued for 30 min at rt.
The solvent from the reaction mixture was evaporated, and the
resulting oily residue was coevaporated with toluene and subjected to
FC (silica gel, CH2Cl2/MeOH, 95:5) to give compound 11 (0.158 g,
65%) as a colorless solid. TLC (silica gel, CH2Cl2/MeOH, 90:10) Rf
1
0.4. λmax (MeOH)/nm 271 (ε/dm3 mol−1 cm−1 15 800). H NMR
(DMSO-d6, 300 MHz): δ 1.04−1.07 (2 s, 6H, 2 × CH3), 2.79−2.88
(m, 1H, CH), 3.37 (t, J = 2.1 Hz, 1H, CCH), 3.53−3.58 (m, 1H,
C5′−H), 3.61−3.66 (m, 1H, C5′−H), 3.92−3.96 (m, 1H, C4′−H),
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4.24 (d, J = 2.4 Hz, 1H, OCH2), 4.27 (d, J = 2.4 Hz, 1H, OCH2),
4.32−4.37 (m, 1H, C3′−H), 4.56 (t, J = 5.4 Hz, 1H, C2′−H), 5.10
(t, J = 5.4 Hz, 1H, C5′−OH), 5.31 (d, J = 5.1 Hz, 1H, C3′−OH), 5.95
(d, J = 6.3 Hz, 1H, C1′−H), 7.24 (br s, 2H, 6-NH2), 8.26 (s, 1H, C8−H),
9.85 (s, 1H, NH). ESI-TOF m/z calcd for C17H22N6O5 [M + Na]+,
413.1544; found, 413.1539.
chloride (430 μL, 3.4 mmol), and the solution was stirred for 1 h at
4 °C. The reaction mixture was diluted with MeCN (6 mL), kept at
4 °C, treated with methoxyacetyl chloride (110 μL, 1.2 mmol), and
finally stirred for 40 min at 4 °C. After CH2Cl2 (15 mL) was added,
the mixture was poured into H2O (15 mL). The organic phase was
dried over Na2SO4 and evaporated, and the residue was coevaporated
with toluene (3 × 10 mL). The resulting foam was dissolved in
MeOH/THF/28% aq NH3 solution 1:1:1 (8 mL). After the solution
stirred for 1 h at rt, the solvent was evaporated, and the remaining
residue was coevaporated with toluene (3 × 10 mL) and finally
subjected to FC (silica gel, CH2Cl2/MeOH, 93:7) to afford compound
15 (0.093 mg, 72%) as a colorless solid. TLC (silica gel, CH2Cl2/
MeOH, 90:10) Rf 0.6. λmax (MeOH)/nm 271 (ε/dm3 mol−1 cm−1
N6-Dibutylaminomethylidene-N2-isobutyryl-9-[2-O-(prop-
argyl)-β-D-ribofuranosyl]purine (12). Compound 11 (0.35 g,
0.90 mmol) was dissolved in methanol (10 mL) and treated with
N,N-dibutylformamid dimethylacetal (1.5 mL) under argon atmos-
phere at rt for 2 h. The solution was evaporated, and the remaining
oily residue was applied to FC (petroleum ether/EtOAc, 4:1, 500 mL;
then CH2Cl2/MeOH, 95:5) to give compound 12 (0.38 g, 80%) as a
colorless foam. TLC (silica gel, CH2Cl2/MeOH, 90:10) Rf 0.5.
λmax (MeOH)/nm 260 (ε/dm3 mol−1 cm−1 20 700), 320 (29 500). 1H
NMR (DMSO-d6, 300 MHz): δ 0.68−0.93 (m, 6H, 2 × CH3), 1.06−
1.08 (d, J = 6.9 Hz, 6H, 2 × CH3), 1.26−1.43 (m, 4H, 2 × CH2),
1.57−1.58 (m, 4H, 2 × CH2), 2.84 (t, J = 6.6 Hz, 1H, CH), 3.17 (m,
1H, C5′−H), 3.39−3.41 (m, 1H, CCH, superimposed by H2O),
3.47−3.57 (m, 4H, NCH2), 3.61−3.68 (m, 1H, C5′−H), 3.92−3.94
(m, 1H, C4′−H), 4.18−4.28 (m, 2H, OCH2), 4.33−4.37 (m, 1H,
C3′−H), 4.57 (t, J = 5.4 Hz, 1H, C2′−H), 5.07 (t, J = 5.1 Hz, 1H,
C5′−OH), 5.30 (d, J = 5.4 Hz, 1H, C3′−OH), 6.00 (d, J = 6.0 Hz, 1H,
C1′−H), 8.36 (s, 1H, C8−H), 8.92 (s, 1H, NCH), 10.10 (s, 1H, NH).
ESI-TOF m/z calcd for C26H39N7O5 [M + H]+, 530.3085; found,
530.3080.
1
14 800). H NMR (DMSO-d6, 300 MHz): δ 3.37 (t, J = 2.4 Hz, 1H,
CCH), 3.52−3.59 (m, 1H, C5′−H), 3.62−3.68 (m, 1H, C5′−H),
3.92−3.96 (m, 1H, C4′−H), 4.20 (br s, 2H, OCH2), 4.25 (d, J = 2.1
Hz, 1H, OCH2), 4.28 (d, J = 2.4 Hz, 1H, OCH2), 4.33−4.38 (m, 1H,
C3′−H), 4.58 (t, J = 5.4 Hz, 1H, C2′−H), 5.09 (t, J = 5.4 Hz, 1H,
C5′−OH), 5.30 (d, J = 5.1 Hz, 1H, C3′−OH), 5.95 (d, J = 5.7 Hz, 1H,
C1′−H), 7.34 (br s, 2H, 6-NH2), 8.27 (s, 1H, C8−H), 11.58 (s, 1H,
NH). ESI-TOF m/z calcd for C16H20N6O6 [M + H]+, 393.1517;
found, 393.1512.
N6-Isobutyryl-N2-(methoxyacetyl)-9-[2-O-(propargyl)-β-D-
ribofuranosyl]purin-2,6-diamine (16). Compound 15 (0.165 g,
0.42 mmol) dissolved in pyridine (5 mL) at 4 °C was treated with
Me3SiCl (0.446 g, 525 μL, 4.1 mmol), and the solution stirred for 2 h
at 4 °C. The reaction mixture was diluted with MeCN (8 mL), cooled
to 4 °C, treated with isobutyryl chloride (0.054 g, 53 μL, 0.5 mmol),
and finally stirred for 40 min at 4 °C. After CH2Cl2 (10 mL) was
added, the reaction solution was poured into H2O (10 mL). The
organic phase was evaporated, and the residue was coevaporated with
toluene (3 × 10 mL). The resulting foam was dissolved in MeOH/
AcOH, 3:1 (10 mL). After 1 h at rt, the solution was evaporated, and
the remaining residue was coevaporated with MeOH (3 × 10 mL) and
toluene (3 × 10 mL). The resulting foam was applied to FC (silica gel,
CH2Cl2/MeOH, 95:5) to afford compound 16 (0.100 g, 52%) as a
colorless solid. TLC (silica gel, CH2Cl2/MeOH, 90:10) Rf 0.7. λmax
N6-[(Di-n-butylamino)methylidene]-N2-isobutyryl-9-[2-O-
(propargyl)-5-O-(4,4′-dimethoxytrityl)-β-D-ribofuranosyl]-
purin-2,6-diamine (13). Compound 12 (0.22g, 0.42 mmol) was
dissolved in anhydrous pyridine (5 mL), 4,4′-dimethoxytrityl chloride
(0.23 g, 0.67 mmol) was added, and the reaction mixture was stirred at
rt for 12 h. After completion of the reaction (TLC monitoring), 10 mL
of CH2Cl2 was added and the solution was washed with 5% aq
NaHCO3 (10 mL). The organic layer was dried over Na2SO4,
evaporated to dryness, and coevaporated with toulene (3 × 10 mL).
The residue was applied to FC (silica gel, CH2Cl2/acetone, 90:10) to
obtain compound 13 (0.198 g, 57%) as a light yellow foam. TLC
(silica gel, CH2Cl2/acetone, 90:10) Rf 0.3. λmax (MeOH)/nm 260
1
(MeOH)/nm 265 (ε/dm3 mol−1 cm−1 10 000), 286 (12 000). H
1
(ε/dm3 mol−1 cm−1 27 900), 320 (30 700). H NMR (DMSO-d6,
NMR (DMSO-d6, 300 MHz): δ 1.10 (s, 3H, CH3), 1.13 (s, 3H, CH3),
2.99−3.08 (m, 1H, CH), 3.36 (s, 4H, CCH, OCH3), 3.54−3.61 (m,
1H, C5′−H), 3.65−3.72 (m, 1H, C5′−H), 3.91−3.98 (m, 1H, C4′−
H), 4.22−4.32 (m, 4H, CH2), 4.36−4.40 (m, 1H, C3′−H), 4.63 (t, J =
5.1 Hz, 1H, C2′−H), 5.08 (t, J = 5.4 Hz, 1H, C5′−OH), 5.35 (d, J =
5.4 Hz, 1H, C3′−OH), 6.05 (d, J = 5.7 Hz, 1H, C1′−H), 8.55 (s, 1H,
C8−H), 10.17 (s, 1H, NH), 11.64 (s, 1H, NH). ESI-TOF m/z calcd
for C20H26N6O7 [M + H]+, 463.1936; found, 463.1923.
300 MHz): δ 0.92 (t, 6H, J = 7.2 Hz, 2 × CH3), 1.05 (t, J = 5.4 Hz,
6H, 2 × CH3), 1.25−1.39 (m, 4H, 2 × CH2), 1.55−1.61 (m, 4H, 2 ×
CH2), 2.80−2.87 (m, 1H, CH), 3.16−3.19 (m, 1H, C5′−H), 3.38−
3.47 (m, 3H, CCH, NCH2), 3.45−3.59 (m, 2H, NCH2), 3.70 (s,
3H, OCH3), 3.71 (s, 3H, OCH3), 4.04−4.06 (m, 1H, C4′-H), 4.33 (d,
J = 2.4 Hz, 1H, OCH2), 4.36 (d, J = 2.4 Hz, 1H, OCH2), 4.52−4.58
(m, 1H, C3′−H), 4.67 (t, J = 5.1 Hz, 1H, C2′−H), 5.29 (d, J = 6.0 Hz,
1H, C3′−OH), 6.08 (d, J = 4.5 Hz, 1H, C1′−H), 6.77−6.82 (m, 4H,
Ar−H), 7.16−7.26 (m, 8H, Ar−H), 7.34 (d, J = 7.2 Hz, 2H, Ar−H),
8.22 (s, 1H, C8−H), 8.93 (s, 1H, NCH), 10.07 (s, 1H, NH). ESI-TOF
m/z calcd for C47H57N7O7 [M + H]+, 832.4392; found, 832.4260.
N6-[(Di-n-butylamino)methylidene]-N2-isobutyryl-9-[2-O-
(propargyl)-5-O-(4,4′-dimethoxytrityl)-β-D-ribofuranosyl]-
purin-2,6-diamine 3′-(2-Cyanoethyl)-N,N-diisopropylphos-
phoramidite (14). To a solution of compound 13 (0.114 g,
0.15 mmol) in dry CH2Cl2 (10 mL) were added (i-Pr)2NEt (0.035 g,
45 μL, 0.26 mmol) and 2-cyanoethyl diisopropylphosphoramido-
chloridite (0.048 g, 45 μL, 0.20 mmol), and the reaction mixture was
stirred for 3 h at rt. After completion of the reaction (TLC
monitoring), the reaction mixture was diluted with CH2Cl2 (30 mL),
poured into 5% NaHCO3 solution (20 mL), and extracted with
CH2Cl2 (3 × 20 mL). The combined organic layers were dried over
Na2SO4, and the solvent was evaporated. The residual foam was
applied to FC (silica gel, CH2Cl2/acetone, 90:10). Evaporation of the
main zone afforded 14 (0.108 g, 76%) as a colorless foam. TLC (silica
gel, CH2Cl2/acetone, 90:10) Rf 0.6. 31P NMR (CDCl3, 121.5 MHz):
150.33, 150.76. ESI-TOF m/z calcd for C56H74N9O8P [M + H]+,
1032.5471; found, 1032.5447.
N6-Isobutyryl-N2-(methoxyacetyl)-9-[5-O-(4,4′-dimethoxytri-
tyl)-2-O-(propargyl)-β-D-ribofuranosyl]purin-2,6-diamine (17).
To a solution of compound 16 (0.355 g, 0.77 mmol) in anhydrous
pyridine (5 mL) was added 4,4′-dimethoxytrityl chloride (0.39 g,
1.16 mmol). The mixture was stirred at rt for 4 h. After completion of
the reaction (TLC monitoring), 20 mL of CH2Cl2 was added, and the
reaction mixture was poured into 5% aq NaHCO3 (20 mL). The
organic layer was dried over Na2SO4, evaporated to dryness, and
coevaporated with toulene (3 × 10 mL), and the residue was applied
to FC (silica gel, CH2Cl2/acetone, 30:20) to obtain compound 17
(0.32 g, 54%) as a colorless foam. TLC (silica gel, CH2Cl2/acetone,
30:20) Rf 0.6. λmax (MeOH)/nm 267 (ε/dm3 mol−1 cm−1 14 000), 284
(15 900). 1H NMR (DMSO-d6, 300 MHz): δ 1.09 (s, 3H, CH3), 1.12
(s, 3H, CH3), 3.00−3.05 (m, 1H, CH), 3.15−3.18 (m, 1H, C5′−H),
3.34 (s, 3H, OCH3, superimposed by H2O), 3.42 (t, J = 2.4 Hz, 1H,
CCH), 3.69, 3.70 (2s, 6H, 2 × OCH3), 4.06−4.08 (m, 1H, C4′−
H), 4.28 (s, 2H, CH2), 4.33 (d, J = 2.1 Hz, 1H, OCH2), 4.36 (d, J =
2.1 Hz, 1H, OCH2), 4.53−4.56 (m, 1H, C3′−H), 4.72 (t, J = 5.1 Hz,
1H, C2′−H), 5.33 (d, J = 6.0 Hz, 1H, C1′−H), 6.10 (d, J = 4.5 Hz,
1H, C3′−OH), 6.79 (t, J = 8.7 Hz, 4H, Ar−H), 7.16−7.34 (m, 9H,
Ar−H), 8.40 (s, 1H, C8−H), 10.08 (s, 1H, NH), 10.65 (s, 1H, NH).
ESI-TOF m/z calcd for C41H44N6O9 [M + Na]+, 787.3062; found,
787.3041.
N2-(Methoxyacetyl)-9-[2-O-(propargyl)-β-D-ribofuranosyl]-
purin-2,6-diamine (15). Compound 1 (0.105 g, 0.33 mmol) was
dissolved in pyridine (4 mL) at 4 °C and treated with trimethylsilyl
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N6-Isobutyryl-N2-(methoxyacetyl)-9-[5-O-(4,4′-dimethoxytri-
tyl)-2-O-(propargyl)-β-D-ribofuranosyl]purin-2,6-diamine 3′-(2-
Cyanoethyl)-N,N-diisopropylphosphoramidite (18). To a sol-
ution of compound 17 (0.26 g, 0.34 mmol) in dry CH2Cl2 (5 mL)
were added (i-Pr)2NEt (0.076 g, 100 μL, 0.59 mmol) and 2-cyanoethyl
diisopropylphosphoramidochloridite (0.106 g, 100 μL, 0.46 mmol),
and the reaction mixture was stirred for 4 h at rt. After completion of
the reaction (TLC monitoring), the reaction mixture was diluted with
CH2Cl2 (20 mL) and poured into 5% NaHCO3 solution (25 mL). The
organic layer was dried over Na2SO4, and the solvent was evaporated.
The residual foam was applied to FC (silica gel, CH2Cl2/acetone,
70:30). Evaporation of the main zone afforded 18 (0.242 g, 74%) as a
colorless foam. TLC (silica gel, CH2Cl2/acetone, 40:10) Rf 0.6. 31P
NMR (CDCl3, 121.5 MHz): 150.55, 150.67. ESI-TOF m/z calcd for
C50H61N8O10P [M + Na]+, 987.4140; found, 987.4115.
9-[2-O-(2-(2-Azidoethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-
methoxy)]-β-D-ribofuranosyl-purin-2,6-diamine (19). To a sol-
ution of compound 1 (0.1 g, 0.31 mmol) in H2O/MeCN (10:1, 2 mL)
was added bis-azide 4 (0.243 g, 1.56 mmol) and copper turnings, and
the reaction mixture was stirred in the dark at rt overnight. After
completion of the reaction (TLC monitoring), the copper turnings
were filtered off, the solvent was evaporated, and the remaining residue
was subjected to FC (silica gel, CH2Cl2/MeOH, 95:5) to obtain 19
(0.070 g, 45%) as a colorless foam. TLC (silica gel, CH2Cl2/MeOH,
90:10) Rf 0.9. λmax (MeOH)/nm 282 (ε/dm3 mol−1 cm−1 10 000). 1H
NMR (DMSO-d6, 300 MHz): δ 3.36 (m, CH2, superimposed with
D2O), 3.56 (t, J = 2.4 Hz, 3H, C5′−H, CH2), 3.60−3.65 (m, 1H, C5′−
H), 3.79 (t, J = 5.1 Hz, 2H, CH2), 3.94−3.96 (m, 1H, C4′−H), 4.29−
4.33 (m, 1H, C3′−H), 4.46−4.56 (m, 4H, 2 × CH2), 4.70 (d, J = 12.1
Hz, 1H, C2′−H), 5.25 (d, J = 5.1 Hz, 1H, C3′−OH), 5.41−5.45
(m, 1H, C5′−OH), 5.75 (br s, 2H, 2-NH2), 5.85 (d, J = 6.6 Hz, 1H,
C1′−H), 6.78 (br s, 2H, 6-NH2), 7.87 (s, 1H, triazole-H), 7.89 (s, 1H,
C8−H). ESI-TOF m/z calcd for C17H24N12O5 [M + H]+, 477.2065;
found, 477.2056.
Second Click Reaction of Nucleoside 1 with Monofunction-
alized Nucleoside 19. To compound 1 (0.033 g, 0.10 mmol) in
H2O/MeCN (10:1, 2 mL) was added monofunctionalized nucleoside
19 (0.050 g, 0.10 mmol) and copper turnings, and the reaction
mixture was stirred in the dark at rt overnight. After completion of the
reaction (TLC monitoring), the copper turnings were filtered off, the
solvent was evaporated, and the residue was coevaporated with
methanol (2 × 2 mL). The residue was further washed successively
with methanol (2 mL) and dichloromethane (2 mL) and dried under
reduced pressure to obtain 20 (0.075 g, 60%) as a colorless solid. The
analytical data are identical to compound 20 as given below.
Bis[({1,1′-(oxybis(ethane-2,1-diyl)methylene)}-1H-1,2,3-tria-
zole-4,1-diyl)]-9-(β-D-ribofuranosyl)purin-2,6-diamine (20). To
compound 1 (0.10 g, 0.30 mmol) in H2O/MeCN (10:1, 2 mL) was
added bis-azide 4 (0.024 g, 0.16 mmol) and copper turnings, and the
reaction mixture was stirred in the dark at rt overnight. After
completion of the reaction (TLC monitoring), the copper turnings
were filtered, the solvent was evaporated, and the residue was
coevaporated with methanol (2 × 2 mL). The residue was further
washed successively with methanol (2 mL) and dichloromethane
(2 mL) and dried under reduced pressure to obtain 20 (0.035 g, 42%)
as a colorless solid. TLC (silica gel, CH2Cl2/MeOH, 80:20) Rf 0.1. λmax
(MeOH)/nm 282 (ε/dm3 mol−1 cm−1 27 700). 1H NMR (DMSO-d6,
300 MHz): δ 3.17 (d, 1H, J = 4.8 Hz, CH2), 3.56−3.62 (m, 4H, 2 ×
C5′−H), 3.69−3.72 (m, 4H, 2 × CH2), 3.95 (br s, 2H, 2 × C4′−H),
4.33−4.36 (m, 2H, 2 × C3′−H), 4.41−4.44 (m, 4H, 2 × CH2), 4.50−
4.54 (m, 4H, 2 × CH2), 4.67 (m, 2H, 2 × C2′−H), 5.28 (d, J = 5.1 Hz,
2H, 2 × C3′−OH), 5.41−5.45 (m, 2H, 2 × C5′−OH), 5.78 (br s, 4H, 2 ×
2-NH2), 5.85 (d, J = 6.3 Hz, 2H, 2 × C1′−H), 6.81 (br s, 4H, 2 × 6-NH2),
7.77 (s, 2H, 2 × triazole-H), 7.87 (s, 2H, 2 × C8−H). ESI-TOF m/z calcd
for C30H40N18O9 [M + Na]+, 819.3118; found, 819.3116.
stock solution in THF/H2O, 1:1). After copper turnings were added,
the reaction mixture was stirred in the dark at room temperature for
1 h. Then, the copper turnings were removed, and the reaction was
continued at room temperature for 2 h. The reaction mixture was
concentrated in a SpeedVac and dissolved in 0.2 mL of bidistilled
water. The reaction mixture was further purified by reversed-phase
HPLC, using gradient I to give the corresponding monofunctionalized
ODN 49 (2.0 A260 units, 70%). In a similar way, monofunctionalized
oligonucleotides 50−58 were prepared in 60−70% yields. These
monofunctionalized oligonucleotides were also prepared by the click
reaction procedure as reported in the literature.9,15
General Procedure for the Cross-Linking of Oligonucleo-
tides by the Stepwise Click Functionalization (“Second Click”).
To the single-stranded oligonucleotide 43 (3.0 A260 units, 30 μmol) in
a H2O/CH3CN mixture (10:1, 100 μL) were added 0.1 M NaCl
(50 μL) and the monofunctionalized oligonucleotide 49 (3.1 A260 units).
After copper turnings were added, the reaction mixture was stirred in the
dark at room temperature for 1 h. Then, the copper turnings were
removed, and the reaction was continued at room temperature for 2 h.
After completion of the reaction, the reaction mixture was concentrated in
a SpeedVac and dissolved in 0.2 mL of bidistilled water. The reaction
mixture was further purified by reversed-phase HPLC, using gradient I to
give the corresponding cross-linked ICL-1 (1.8 A260 units, 55%). In a
similar way, the other cross-linked oligonucleotides ICLs 2−12 were
prepared in 50−60% yields. These cross-linked oligonucleotides were also
prepared by the click reaction procedure as reported in the literature.9,15
Ion-Exchange Chromatography. Ion-exchange chromatography
was performed on a 4 × 250 mm DNA PA-100 column with a
precolumn using an HPLC apparatus. Elution profiles were recorded
at 260 nm. The alkynylated, monofunctionalized, and cross-linked
oligonucleotides (0.1 A260 units each) were dissolved in 100 μL of
water and then directly injected into the apparatus. The compounds
were eluted using the following gradient: (A) 25 mM Tris-HCl, 10%
MeCN, pH 7.0; (B) 25 mM Tris-HCl, 1.0 M NaCl, and 10% MeCN,
pH 7.0. Elution gradient: 0−30 min 20−80% B in A with a flow rate of
0.75 mL min−1.
ASSOCIATED CONTENT
■
S
* Supporting Information
Coupling constants of nucleosides, structures of cross-linked
and monofunctionalized oligonucleotides, HPLC purity profiles
of selected oligonucleotides, melting curves of cross-linked
duplexes, extinction coefficients, 1H, 13C NMR, DEPT-135 and
1H−13C gated-decoupled NMR spectra of the nucleoside
derivatives and click conjugates. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone, +49 (0) 251 53406 500; Fax, +49 (0) 251 53406 857;
E-mail, frank.seela@uni-osnabrueck.de; Homepage, www.seela.net.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We would like to thank Dr. S. Budow-Busse for helpful
discussions and support while preparing the manuscript. We
also thank Mr. H. Mei for measuring the NMR spectra and Mr.
Nhat Quang Tran for the oligonucleotide syntheses. We would
like to thank Dr. H. Luftmann, Organisch-Chemisches Institut,
General Procedure for the Synthesis of Azido-Modified
Oligonucleotides by the Stepwise Click Functionalization
Using Bis-azide 4 (“First Click”). To the single-stranded
oligonucleotide 38 (3.0 A260 units, 30 μmol) in a H2O/CH3CN
mixture (10:1, 100 μL) was added bis-azide 4 (37.5 μL of a 20 mmol
Universitat Munster, Germany, for the measurement of the
̈
̈
MALDI spectra. Financial support by ChemBiotech, Munster,
̈
Germany, is highly appreciated.
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Article
13. (g) Okamoto, A.; Tanaka, K.; Saito, I. Bioorg. Med. Chem. Lett.
2002, 12, 97−99.
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