Stepwise "click" chemistry for the template independent construction of a broad variety of cross-linked oligonucleotides: Influence of linker length, position, and linking number on DNA duplex stability

Article abstract of DOI:10.1021/jo2004988

Cross-linked DNA was constructed by a "stepwise click" reaction using a bis-azide. The reaction is performed in the absence of a template, and a monofunctionalized oligonucleotide bearing an azido-function is formed as intermediate. For this, an excess of

Full text of DOI:10.1021/jo2004988

ARTICLE
pubs.acs.org/joc
Stepwise “Click” Chemistry for the Template Independent
Construction of a Broad Variety of Cross-Linked Oligonucleotides:
Influence of Linker Length, Position, and Linking Number
on DNA Duplex Stability
Hai Xiong†,‡ and Frank Seela*,†,‡
†Laboratory of Bioorganic Chemistry and Chemical Biology, Center for Nanotechnology, Heisenbergstrasse 11,
48149 M€unster, Germany
‡Laboratorium f€ur Organische und Bioorganische Chemie, Institut f€ur Chemie, Universit€at Osnabr€uck, Barbarastrasse 7,
49069 Osnabr€uck, Germany
S Supporting Information
b
ABSTRACT: Cross-linked DNA was constructed by a “step-
wise click” reaction using a bis-azide. The reaction is per-
formed in the absence of a template, and a monofunctionalized
oligonucleotide bearing an azido-function is formed as inter-
mediate. For this, an excess of the bis-azide has to be used
compared to the alkynylated oligonucleotide. The cross-linking
can be carried out with any alkynylated DNA having a terminal
triple bond at any position of the oligonucleotide, independent
of chain length or sequence with identical or nonidentical
chains. Short and long linkers with terminal triple bonds were introduced in the 7-position of 8-aza-7-deaza-20-deoxyguanosine
(1 or 2), and the outcome of the “stepwise” click and the “bis-click” reaction was compared. The cross-linked DNAs form cross-
linked duplexes when hybridized with single-stranded complementary oligonucleotides. The stability of these cross-linked duplexes
is as high as respective individual duplexes when they were ligated at terminal positions with linkers of sufficient length. The stability
decreases when the linkers are incorporated at central positions. The highest duplex stability was reached when two complementary
cross-linked oligonucleotides were hybridized.
’ INTRODUCTION
As bis-azides are easily accessible,28ꢀ30 we have used them to
cross-link DNA in one step in a template independent way with
alkynyl linkers.23 By the so-called “bis-click” protocol, only
identical strands can be cross-linked in a selective way; non-
identical strands lead to more than one cross-linked species.23
Four-stranded DNA consisting of two cross-linked oligonucleo-
tide duplexes was obtained after hydridization.
Continuing our research on cross-linking of DNA led to the
development of the “stepwise click” strategy. This approach
offers the possibility to cross-link nonidentical oligonucleotides
with short or long linker arms selectively at identical or different
linking positions. For this, a large excess of a bis-azide is used
leading to a monofunctionalized azide (“first click”). This azide
intermediate is isolated, and then a “second click” is performed
with an alkynylated oligonucleotide leading to the cross-linked
product. This “stepwise click” reaction was now carried out on
nucleosides and oligonucleotides. 8-Aza-7-deaza-20-deoxyguano-
sine with short (1) and long linker (2) arms was employed in the
“stepwise click” protocol together with the cross-linking reagent
DNA interstrand cross-links (ICLs) are mutagenic if not
repaired.1 Cross-linking is specific to particular DNA nucleo-
bases.2,3 Cross-links shut down gene replication,1 and cross-
linking drugs are used in cancer chemotherapy.4
Bifunctional reagents such as nitrogen mustards, nitrosoureas, or
platinum compounds can covalently connect nucleobases, which
ultimately leads to the interstrand cross-linking.1,4 Alternatively, inter-
strand cross-links can be induced by light5 or γ irradiations6 as well as
with naturally occurring molecules such as mitomycin,7 psoralen,8 etc.
The copper catalyzed azideꢀalkyne HuisgenꢀMeldalꢀSharpless
cycloaddition “click” reaction (CuAAC)9ꢀ11 has been applied
to nucleoside and oligonucleotide functionalization12ꢀ18 and to
cross-linking.19ꢀ23 The bio-orthogonal nature has increased the
credentials of this reaction which is suitable to be performed in
vitro and even in vivo.24ꢀ26 The created 1,2,3-triazole system is
inert to functional groups present in nucleic acids, proteins, and
other cellular components and is stable at physiological pH. Our
laboratory has reported on the “click” functionalization of the
four DNA building blocks modified with diynyl side chains at the
5-position of pyrimidine bases and the 7-position of 7-deazapur-
ine or 8-aza-7-deazapurine residues.27
Received: March 7, 2011
Published: May 18, 2011
r
2011 American Chemical Society
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Figure 1. Structures of nucleosides and mono- and bis-functionalized adducts used in this study.
azido-p-xylene 3. Thefirst click reaction afforded the monofunction-
alized azido intermediates 4 and 5, while the cross-linked nucleo-
sides 6 and 7 were formed in the second click reaction (Figure 1).
The ethynylated and octadiynylated 8-aza-7-deaza-20-deoxy-
guanosines 1 and 2 were incorporated into a series of 12-mer
oligonucleotides. These oligonucleotides bearing diynynl side
chains with terminal triple bonds at various positions were ligated
using azido-p-xylene (3) as cross-linking reagent. Thus, by
employing the “stepwise click” protocol, any DNA oligonucleo-
tide can be cross-linked selectively.
Novel cross-linked four-stranded duplexes were constructed
from cross-linked single strands. For this, hybridization experi-
ments were performed. Complementary free single-stranded
oligonucleotides as well as cross-linked strands were hybridized
with the cross-linked oligonucleotides. The novel DNA con-
structs were evaluated with respect to the influence of linker
length, linking position and number of the modification sites.
under standard conditions furnished the 50-O-DMT derivative
11 (83% yield). Phosphitylation with 2-cyanoethyl N,N-diiso-
propylphosphoramidochloridite furnished the phosphoramidite
12 (74%) (Scheme 1). The synthesis of the phosphoramidite 13
has already been reported.27c
Next, the potential of the bis-azide 3 in the cross-link reaction
was studied on nucleosides. For this, the click reaction was
performed at room temperature on nucleosides 1 or 2 with the
bis-azide 3 in a mixture of THF/H2O/t-BuOH with CuSO4 5
3
H2O as catalyst and sodium ascorbate as reducing agent. The
starting material was consumed within 12 h. Under these
conditions, monofunctionalization occurred when the molar
excess of the bis-azide 3 over the nucleosides 1 or 2 was 5: 1,
while bis-adducts were formed when the bis-azide/nucleoside
ratio was 0.5:1 (Scheme 2). In the bis-click reaction with 1, the
cross-linked product 6 precipitated. Because of its low solubility
it could not be chromatographed but was pure (52% yield) after
the filter cake was washed with methanol and water. From the
washing solution, the monofunctionalized intermediate 4 was
isolated in 7% yield (Experimental Section). The corresponding
reaction with compound 2, which is shown in the second part of
the Scheme 2, has already been reported.23
’ RESULTS AND DISCUSSION
1. Synthesis and Characterization of Monomers. 7-Iodo-8-
aza-7-deaza-20-deoxyguanosine (8)31 was used as starting ma-
terial for the synthesis of the phosphoramidite 12. Nucleoside 1
was synthesized by Sonogashira cross-coupling. The reaction was
performed in dry DMF in the presence of Et3N, [Pd0(PPh3)4],
and CuI, with a 7-fold excess of trimethylsilyl acetylene affording
9 (68%). The trimethylsilyl derivative was deprotected using
K2CO3 in methanol affording compound 1 (83%). Nucleoside 1
was protected with the N,N-dimethylaminomethylidene residue
to give the derivative 10 (80% yield). 4,40-Dimethoxytritylation
All compounds were characterized by elemental analyses as
1
well as by their H and 13C NMR spectra. The 13C NMR
chemical shifts are listed in the Experimental Section (see
Table 6). NMR chemical shift signals were assigned by
13
1Hꢀ C gated-decoupled spectra as well as by DEPT-135
NMR spectra (Table S1, Supporting Information). The mono-
functionalized adduct 4 and the bis-click adduct 6 can be easily
identified by 1H NMR spectroscopy. For compound 4, the two
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Scheme 1. Synthesis of Phosphoramidite 12
methylene bridges of the 1,4-phenylene moiety show two
separate singlets (5.75 ppm and 4.45 ppm; Figure S46, Support-
ing Information), while only one singlet is presented in com-
pound 6 (5.72 ppm; Figure S54, Supporting Information).
Further confirmation for mono- vs bifunctionalized compounds
was obtained from the intensity ratio of the H-triazole (8.95
ppm) vs 1,4-phenylene-H signals (7.33ꢀ7.41 ppm) (Figure S46,
Supporting Information). The ratio is 1:4 for the monofunctio-
nalized compound 4 containing one triazole ring (1H,
H-triazole) and one 1,4-phenylene residue (4H, H-arom). The
bis-click compound 6 containing two triazole rings (2H, 2 ꢁ
H-triazole) and one 1,4-phenylene ring (4H, H-arom) shows an
intensity ratio (H-triazole vs 1,4-phenylene-H) of 2:4 (Figure
S54, Supporting Information). In the same way, it can be
distinguished between the monofunctionalized compound 5
and the bis-click adduct 7.
2. Evaluation of the Effect of Linker Lengths and Position
of Nucleosides 1 or 2 on Oligonucleotide Duplexes. In order
to evaluate the influence of side chains (long and short linker
arms and different linking positions) on the DNA duplex
stability, a series of oligonucleotides were prepared by solid-
phase synthesis using the phosphoramidite 12 or 13 as well as
standard phosphoramidites. The syntheses were performed at
1 μmol scale. The coupling yields were always higher than 95%.
Deprotection of the oligomers was performed in aqueous NH3
(25%) at 60 °C for 14 h. The oligonucleotides were purified
before and after detritylation by reversed-phase HPLC. The
homogeneity of the oligonucleotides was confirmed by HPLC
analysis, and their molecular weights were determined by LC-
ESI-TOF or MALDI-TOF mass spectrometry (Table S2, Sup-
porting Information).
Single replacements of dG by compound 1 at various positions
of the reference duplex 50-d(TAG GTC AAT ACT) (ODN-14)
and 30-d(ATC CAG TTA TGA) (ODN-15) were performed,
and the duplex stability was investigated by Tm measurements.
The Tm values listed in Table 1 were determined at high salt (1 M
NaCl) conditions at pH 7.0 with a 5 μM concentration of the
single strands. Tm values at low salt (0.1 M NaCl) are generally 3°
lower.27c From Table 1 the following conclusions can be drawn:
(i) replacement of one dG residue by nucleoside 1 (duplexes
14 16, 14 17, and 18 15) increases the Tm value (ΔTm
=
3
3
3
þ2.5ꢀ3 °C) compared to the reference duplex 14 15. (ii) The
3
Tm changes found for oligonucleotide duplexes containing 8-aza-
7-deaza-7-ethynyl-20-deoxyguanosine (1) are similar to those
incorporating the octadiynyl compound 2. (iii) The less space
demanding ethynyl side chain has almost the same positive
influence on DNA duplex stability as the long octadiynyl linker.
The positioning of the modified nucleoside is of less importance.
The melting hypochromicity (% h) of the oligonucleotide
duplexes is in the range of 15ꢀ17%.
3. Comments to the “Click” Cross-Linking of Alkynyl
Oligonucleotides with Bis-azides. Azido groups as well as
terminal triple bonds display functionalities which are highly
exergonic during adduct formation. Both functionalities were
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Scheme 2. Synthesis of the Monofunctionalized Conjugates 4 and 5 and the Bis-adducts 6 and 7
already introduced previously in individual oligonucleotides
strands, and “click” reactions thereof were performed by copper-
(I) salt assistance.15ꢀ17,20,21,23
The presence of the azido group and the triple bonds in two
separate strands leads to only one “clicked” cross-linked product.
This is a benefit of the strategy already reported by other
laboratories.15ꢀ17,20ꢀ22 However, azides are not compatible with
the protocol of phosphoramidite chemistry, so postmodific-
ation was necessary to introduce the azido group into the
oligonucleotide chain. This reaction uses conventional conjuga-
tion chemistry resulting in overall moderate yields.20a,32ꢀ34
Furthermore, subsequent click reactions are usually limited to
terminal positions of an oligonucleotide chain.
As we want to be free in the position of a cross-link and make use
of the highly efficient click reaction to link oligonucleotide chains
together, our strategy employs bis-azides and oligonucleotides with
diynyl chains. If click reactions take place simultaneously at a bis-
azide—“bis-click reaction”—only identical oligonucleotide strands
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Table 1. Tm Values of Oligonucleotide Duplexes Containing
the Modified 8-Aza-7-deaza-20-deoxyguanosines 1 and 2
Table 2. Tm Values of Duplexes Containing the Monofunc-
tional Derivatives 4 or 5
a Measured at 260 nm in a 1 M NaCl solution containing 100 mM MgCl2
and 60 mM Na-cacodylate (pH 7.0) with 5 μM þ 5 μM single-strand
concentration. b % h refers to the percentage of hypochromicity.
c ΔG°310 values are given with 15% error.
a Measured at 260 nm in a 1 M NaCl solution containing 100 mM MgCl2
and 60 mM Na-cacodylate (pH 7.0) with 5 μM þ 5 μM single-strand
c
concentration. b % h refers to percentage of hypochromicity. ΔG°310
values are given with 15% error.
4. Stepwise “Click” Cross-Linking of Oligonucleotides
Containing Nucleosides 1 and 2: Cross-Linking between
Identical or Nonidentical Strands. The synthetic route of the
stepwise click reaction to construct the cross-linked oligonucleo-
tides is shown in Scheme 3, starting with the ethynyl compound
ODN-16 or the octadiynyl derivative ODN-20. The click reac-
tion was performed at room temperature in aqueous solution
(H2O/DMSO/t-BuOH) employing a premixed 1:1 complex of
CuSO4ꢀTBTA (tris(benzyltriazoylmethyl)amine), TCEP (tris-
(carboxyethyl)phosphine), and NaHCO3. NaHCO3 was essential
to complete the reaction within 12 h. The ratio of bis-azide 3 over
the respective oligonucleotides was 15:1. With this excess of bis-
azide 3 only one azido group reacts (“first click”), thereby yielding
the monofunctionalized click intermediates ODN-22 and ODN-
26 (Scheme 3 and Table 3). Then, in the second step (“second
click”), the intermediates ODN-22 and ODN-26 were reacted
with another alkynylated oligonucleotide (ODN-16 or ODN-20)
(Scheme 3). The reaction conditions for the second step were the
same as those described for the “bis-click” reaction,23 except that
the alkynylated oligonucleotides (ODN-16 or ODN-20) were in
slight excess (4:3) over the intermediates ODN-22 and ODN-26.
Bothroutes, usingeitherODN-16 or ODN-20 asstarting material,
afforded the identical product ICL-33step.
Typical examples for the reversed-phase HPLC profiles of the
crude reaction mixtures for selected monofunctionalized inter-
mediates obtained after the “first click” reaction are shown in the
Supporting Information (Figure S4a,b). Figure S4e,f (Support-
ing Information) depicts interstrand cross-linked oligonucleo-
tides obtained after the “second click” reaction. The monofunc-
tionalized oligonucleotides were separated and isolated by
reversed-phase HPLC. Moreover, HPLC profiles of mixtures
of the ethynyl or octadiynyl compounds and their adducts show
that the monofunctionalized intermediates migrate slower than
the starting materials but faster than the cross-linked oligonu-
cleotides (Figure S3, Supporting Information).
Figure 2. Scheme of the “stepwise click” (a) and the “bis-click” (b) reaction.
can be ligated selectively. Nonidentical oligonucleotides, being
uncomplementary, employed under those conditions give rise to
a mixture of various (three) cross-linked products complicating this
route greatly. Thus, the “stepwise” protocol was developed which
overcomes these limitations. The bis-click and stepwise click
strategies are illustrated in Figure 2.
We were able to start the stepwise click reaction with
various oligonucleotides (ODN-16ꢀODN-21) with short or
long linkers and various linking positions affording monoazide
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Scheme 3. Interstrand Cross-linking of Oligonucleotides by the Cu(I)-Catalyzed “Stepwise Click” Reaction Using Oligonu-
cleotide ODN-16 (Short Linker) or ODN-20 (Long Linker) in the “First Click” and Oligonucleotide ODN-20 (Long Linker) or
ODN-16 (Short Linker) in the “Second Click”
Scheme 4. Interstrand Cross-Linking of Oligonucleotides ODN-18 and ODN-21 by Cu(I)-Catalyzed “Bis-click” Reaction
intermediates ODN-22ꢀODN-27 (Table 3 and Figures S5ꢀS7,
Supporting Information). The monofunctionalized oligonucleo-
tides were always completely consumed while the remaining ex-
cess of the alkynylated oligonucleotides was still seen in the HPLC
profiles. The cross-linked oligonucleotides were separated and
purified by reversed-phase HPLC (RP-18 column) (Figures S5ꢀ
S7, Supporting Information). Accordingly, a series of cross-linked
oligonucleotides (ICL-28stepꢀICL-33step) as shown in Table 3
were prepared. All oligonucleotide conjugates were characterized by
LC-ESI-TOF or MALDI-TOF mass spectrometry (Table S3,
Supporting Information); even oligonucleotides bearing azido
groups did not decompose during mass spectrometry and gave
the expected molecular weights in all cases.
Table 3 summarizes the various cross-linked oligonucleo-
tides ICL-28stepꢀICL-33step as well as the oligonucleotide
intermediates still carrying one azido group (ODN-22ꢀODN-
27) obtained from the “stepwise click” reaction. The cross-
linked oligonucleotides are composed of (i) identical strands or
(ii) nonidentical strands employing a combination of (iii) two
short or (iv) two long linkers as well as (v) a combination
of a long and a short linker. The linking positions are placed
at (vi) a central or (vii) a terminal position. The diversity of
possible combinations demonstrates the powerful potential of
the “stepwise click” reaction as the three parameters oligonucleo-
tide sequence, linker length, and linking position can be varied
freely.
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Table 3. Oligonucleotide Intermediates with Azido Groups and Cross-linked Oligonucleotides with Identical and Nonidentical
Strands Prepared by the “Stepwise Click” Reaction with Different Linking Positions and Linkers of Different Lengtha
a 1-1, 2-2, 1-2, and 2-1 represent the cross-linked nucleosides formed by the cycloaddition of the azido group within one side chain and the terminal triple
bond of the other side chain. For detailed structures see Figure S2, Supporting Information. The various connectivities are as follows: CꢀC central position
connected with central position; TꢀT terminal position connected with terminal position; CꢀT central position connected with terminal position.
Table 4. Cross-Linked Oligonucleotides with Identical Strands Prepared by the “Bis-click” Reaction with Identical Linker Lengtha
a 1-1 or 2-2 represent the cross-linked nucleosides formed by the cycloaddition of the azido group within one side chain and the terminal triple bond of
the other side chain. For detailed structures see Figure S2, Supporting Information. The various connectivities are: CꢀC central position connected with
central position; T-T terminal position connected with terminal position.
The hybridization of oligonucleotides incorporating the azido
intermediates 4 or 5 with complementary oligonucleotide
strands results into slightly reduced Tm values compared to the
parent alkynylated oligonucleotide duplexes. Modifications at
central positions have a stronger destabilizing effect (about
ꢀ3 °C) than at terminal positions (around þ1 °C). These
findings are in line with previous observations on side-chain
modifications reporting on duplex destabilization when the side
chain gets too long.27aꢀc For further details, see Table 2.
The “stepwise click reaction” can also be carried out on
oligonucleotides with identical strands leading to the same
reaction products as obtained from the “bis-click reaction”. Both
reactions were performed in order to compare the cross-linked
reaction products of both protocols. Indeed, the stepwise
click and the bis-click reaction (Scheme 4) yielded identical
cross-linked oligonucleotides as demonstrated on ICL-28step,
ICL-29step, ICL-30step (Table 3) and ICL-28bis, ICL-29bis,
ICL-30bis (Table 4). For hybridization studies, the cross-linked
oligonucleotides ICL-34bisꢀICL-36bis were also prepared by
the bis-click reaction (Table 4). Although the reaction is tem-
plate-free, it proceeds smoothly forming the bis-click products as
major compounds (major peaks; ICL-34bis and ICL-35bis;
Figure S8, Supporting Information). Only a small amount
(minor peaks) of the respective monofunctionalized oligonu-
cleotides still carrying one azido group (ODN-24 and ODN-27)
were detected as shown in the reversed-phase HPLC profiles
(RP-18 column) of the crude reaction mixtures. The starting
material which migrates faster than the cross-linked or mono-
functionalized oligonucleotides was completely consumed. The
contents of the major peaks containing ICL-34bis and ICL-35bis
were separated by HPLC and isolated. The formation of cross-
linked oligonucleotides was further confirmed by the molecular
masses of the final reaction products determined by LC-ESI-
TOF and MALDI-TOF mass spectrometry (Supporting Infor-
mation, Table S4).
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Comparing the protocols of the bis-click and stepwise click
reaction, it can be concluded that for the synthesis of cross-linked
oligonucleotides with identical strands, the protocol of the bis-
click reaction is advantageous over the stepwise click reaction
affording cross-linked oligonucleotides with yields in the range of
50ꢀ70%. The stepwise click reaction requires the isolation of the
monofunctionalized intermediate. Thus, the overall yield over
two steps is generally lower (step 1, about 60ꢀ80%; step 2, about
40ꢀ50%). However, for the synthesis of cross-linked oligonu-
cleotides with nonidentical strands the stepwise click protocol
represents a versatile method.
Finally, ion-exchange chromatography was performed with
the starting materials and products of the stepwise-click and the
bis-click reaction (Figure 3). ODN-16 and ODN-22 show almost
identical retention times (11.5 and 12.2 min) because of 11
negative phosphate charges (Figure 3a), while the retention time
of the cross-linked ICL-28step is increased by 6 min (22 negative
phosphate charges). The retention time of cross-linked ICL-
28step is almost identical to the retention time of the duplex
14 1535 as the number of charges of the cross-linked oligonucleo-
Figure 3. Ion-exchange HPLC elution profiles of (a) oligonucleotide
ODN-16, azidomethylbenzyl-labeled ODN-22, and the interstrand
cross-linked ICL-28step. (b) Oligonucleotide ODN-20 and the inter-
strand cross-link ICL-36bis on a 4 ꢁ 250 mm DNA PA-100 column
using the following buffer system: (C) 25 mM Tris-HCl, 10% MeCN,
pH 7.0; (D) 25 mM Tris-HCl, 1.0 M NaCl, and 10% MeCN, pH 7.0.
Elution gradient IV: 0ꢀ30 min 20ꢀ80% D in C with a flow rate of
3
tide is doubled compared to the non-cross-linked oligonucleotides
ODN-16 and ODN-22. Mobility shift analysis was also carried
out for the bis-click reaction of ODN-20 affording ICL-36bis
(Figure 3b).
5. Influence of DNA Interstrand Cross-Links on Oligonu-
cleotide Duplex Stability. The cross-linked oligonucleotides of
Tables 3 and 4 gave us the opportunity to construct DNA
assemblies by a combination of cross-linking and hybridization
which generated DNA structures with novel structural features.
For this, cross-linked oligonucleotides were hybridized with
individual complementary strands to form two double-stranded
helices covalently linked by the cycloadduct of the alkynyl side
chains and the azido groups. Herein, a pyrazolo[3,4-d]pyrimidine
base related to guanine was used as nucleobase at the modifica-
tion sites. The linkers which were always connected to the
7-position of the 8-aza-7-deazaguanine moiety were changed in
length and position. Cross-linked oligonucleotides of identical
and nonidentical strands were studied. Also, cross-linked strands
were hybridized with the corresponding complementary cross-
linked species. However, 7-substituted 7-deazapurines or 5-sub-
stituted pyrimidines related to the canonical DNA bases or even
universal bases and other residues can be used for this purpose.
A related strategy using the phosphodiester backbone as
connecting point was reported by Endo et al.36 However, this
method removed the negative charge from the internucleotide
phosphodiester linkage and generated Rp and Sp diastereoi-
somers which had to be separated. This resulted in a dramatic
Tm decrease. In the work of Endo et al., only identical strands
were ligated, and disulfide formation was used for cross-linking.36
We started our hydridization studies with cross-linked oligo-
nucleotides with two identical strands. They were hybridized
with individual complementary chains to give cross-linked dou-
ble-stranded helices. Tm measurements of 2.5 μM of cross-
linked DNA and 5 μM of the single-stranded complementary
oligonucleotides (ODN-14 or ODN-15) were performed. Then,
the influence of the linker length and position was evaluated.
As shown in Table 5, the duplex stability varies according to
the position of the cross-linker. When positioning a short
linker (combination of two ethynyl side chains) at a central
0.75 mL minꢀ1
.
(14 ICL-29step 14: ΔTm = ꢀ1.5 °C) for a short cross-link at a
3
3
terminal position.
A similar observation is made when the linker is longer
(combination of two octadiynyl chains). However, in this case,
the Tm decrease is significantly reduced compared to duplexes
with short linkers (34.0 °C for 14 ICL-28step 14 vs 45.0 °C for
3
3
14 ICL-30step 14 or 41.0 °C for 15 ICL-34bis 15 vs 47.0 °C
3
3
3
3
for 15 ICL-35bis 15; central positions). For the terminal cross-
linking position, even a small stabilization over the reference
duplex 14 15 was observed (14 ICL-36bis 14: ΔTm
3
3
=
3
3
3
þ1.0 °C).
The Tm value of duplex 14 ICL-31step 14 with a combina-
tion of a short (ethynyl) and a long (octadiynyl) linker intro-
3
3
duced at a terminal position is even more stable (ΔTm
þ2.0 °C).
=
In addition, we cross-linked oligonucleotides at a central
position of one strand and at a terminal position of the other
strand. For this, we used a combination of two ethynyl linkers
(14 ICL-32step 14) or of an ethynyl and octadiynyl linker
3
3
(14 ICL-33step 14). As shown in Table 5, duplex 14 ICL-
3
3
3
33step 14 (Tm = 48.5 °C) is significantly more stable than
14 ICL-32step 14 with two short linkers (Tm = 45.0 °C).
Next, two linker units were introduced in the cross-linked
duplexes. This was realized by hybridizing a cross-linked species
with identical strands with a second cross-linked species also with
identical strands but with complementary base sequence
(duplexes ICL-34 ICL-28 and ICL-35 ICL-28; Table 5 and
3
3
3
3
3
Figure 4c, d). Now, the Tm values increased significantly over
those of the cross-linked duplexes with only one cross-link
(56.0 °C for ICL-34 ICL-28 with two cross-links vs 41.0 °C
3
for 15 ICL-34bis 15 and 34.0 °C for 14 ICL-28step 14, each
3
3
3
3
position, the Tm value decreases by 17 °C (14 ICL-28step 14)
with one cross-link). As the cross-links were introduced near the
central position, the effect of double cross-linking over the single
cross-linking is rather strong with a Tm increase of 15 or 22 °C.
3
3
or 10 °C (15 ICL-34bis 15) compared to the reference duplex
3
3
14 15. On the contrary, the destabilization is almost negligible
3
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Table 5. Tm Values of Cross-Linked Oligonucleotides ICL-28ꢀICL-35 Prepared by the “Stepwise Click” and the “Bis-click”
Reaction
a Measured at 260 nm in a 1 M NaCl solution containing 100 mM MgCl2 and 60 mM Na-cacodylate (pH 7.0) with 2.5 μM of cross-linked DNA and
5 μM of the single-stranded complementary oligonucleotides. 1-1, 2-2, 1-2, or 2-1 represent the cross-linked nucleosides formed by the cycloaddition of
the azido group within one side chain and the terminal triple bond of the other side chain. For structural details of the cross-linked oligonucleotides, see
Figure S2, Supporting Information. Data in parentheses refer to measurements in 0.1 M NaCl2, 10 mM MgCl2, 10 mM Na-cacodylate, pH 7.0. b % h
refers to percentage of hypochromicity.
The stability of these double cross-linked duplexes is also spacer
for this phenomenon: (i) distortion of the helix, (ii) changes in
helix hydration by water molecules, (iii) hydrophobic effects
caused by the lipophilic linker, etc. Apparently, all these unfavorable
properties can be superimposed by the entropic gain induced by
the cross-linking of oligonucleotide chains in particular, when
each individual complementary species is cross-linked before
hybridization.
depended. A combination of two short linkers (ethynylꢀ
ethynyl; ICL-34 ICL-28: Tm = 56.0 °C) is more favorable than
3
a combination of a short and long linker (ethynylꢀethynyl and
octadiynylꢀoctadiynyl; ICL-35 ICL-28: Tm = 53.5 °C). The
3
stability of the double cross-linked duplexes is even higher than
that of the parent noncross-linked duplex 14 15. This results from
3
anentropicallymorefavorable situationforduplexes formedby two
complementary cross-linked strands compared to duplexes formed
by only one cross-linked strand and two individual strands. The
details of the various combinations and their influence on the
duplex stability are listed in Table 5.
’ CONCLUSIONS AND OUTLOOK
Various DNA cross-linking methods have already been report-
ed, including the so-called “bis-click” protocol.1,37ꢀ40 While the
application of the template-independent “bis-click” reaction is
limited to identical oligonucleotide strands, we developed the
“stepwise click” methodology which allows us to cross-link any
oligonucleotide bearing an alkynyl linker as a side chain in a
selective way with a bis-azide. For this, alkynylated 8-aza-7-deaza-
20-deoxyguanine residues with short and long linker arms (1 and
2) were incorporated into a series of 12-mer oligodeoxyribonu-
cleotides (ODNs) using solid-phase synthesis and employing
phosphoramidites 12 and 13. The stepwise click methodol-
ogy requires an excess of the bis-azide over the modified
The various types of cross-linked duplexes were modeled with
Hyperchem 8.0 (Hypercube Inc.) without energy minimization.
Sphere models are shown in Figure 4. In all cases, the side linkers
protrude into the major groove of B-DNA. It is expected that the
double helix geometry should not be affected significantly when
linker arms are positioned in such uncritical positions, e.g., the
7-position of the 8-aza-7-deazaguanine bases, even when rather
short linkers or two linkers units are introduced. Nevertheless, as
indicated by the decrease of Tm values (Table 5), cross-linking in
central positions affects duplex stability. Many factors can account
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Figure 4. Schematic representations of the molecular models of two double helices connected by (a) the short linker and (b) by a long linker, both in a
central position of the helix and (c) with a short linker in a central and a long linker in a near terminal position and (d) with two long linkers in terminal
and central positions. The models were constructed using Hyperchem 8.0.
oligonucleotide and yields a monofunctionalized intermediate
which still carries one reactive azido group. These oligonucleo-
tides were then cross-linked in a second click reaction with the
terminal triple bond of a second oligonucleotide in a selective
way. Homocoupling of identical strands is excluded. By this
route, cross-linked oligonucleotides are accessible having the
cross-link at any position of the chain with linker arms of various
lengths. Also, the linking positions can be varied freely.
We have hybridized the various cross-linked oligonucleotides
with complementary oligonucleotides including those which
were already cross-linked before. As in the functionalized mol-
ecules the linker protrudes into the major groove of the DNA,
only small distortions of the duplex structures were expected.
Indeed, stable cross-linked duplexes were formed with short and
long linker arms as displayed in Figure 4a,b. Nevertheless, cross-
linking near terminal positions using long linkers results in more
stable helices than those modified near or at the center of duplex
DNA with a short linker. We anticipate that our methodology
might find broad application in chemical biology and the con-
struction of nanostructures. Also, the stepwise click method can
be applied for other molecules including natural biopolymers
such as carbohydrates or polypeptides or in material science.41ꢀ47
Currently, the stepwise click reaction is studied on solid-phase in
order to simplify the two-step protocol. Also, we were able to
show that a template-controlled bis-click reaction can be per-
formed in a selective way when complementary oligonucleotide
strands are ligated with bis-azides within a duplex structure.
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 (40ꢀ60 μM) at
0.4 bar. UV spectra: λmax in nm, ε in dm3 molꢀ1 cmꢀ1. NMR spectra:
measured at 300.15 MHz for 1H, 75.48 MHz for 13C and 121.52 MHz
for 31P. The J values are given in Hz and δ in ppm. For NMR spectra
recorded in DMSO, the chemical shift of the solvent peak was set to
1
2.50 ppm for H NMR and 39.50 ppm for 13C NMR (see Table 6).
Reversed-phase HPLC was carried out on a 250 ꢁ 4 mm RP-18
LiChrospher 100 column with a 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 50ꢀ10% A in B, flow rate 0.7 mL minꢀ1; (II) 0ꢀ25 min
0ꢀ20% A in B, flow rate 0.7 mL minꢀ1; (III) 0ꢀ15 min 0ꢀ20% A in B,
15ꢀ18 min 20ꢀ40% A in B, flow rate 0.7 mL minꢀ1. ESI-TOF mass
spectra of the nucleosides and oligonucleotides were measured with a
Micro-TOF spectrometer. Molecular mass of oligonucleotides were
determined by LC-ESI-TOF mass spectrometry or by MALDI-TOF
mass spectrometry in the linear negative mode with 3-hydroxypicolinic
acid (3-HPA) as a matrix. The detected masses were in line with the
calculated values (Tables S2ꢀS4, Supporting Information).
Synthesis, Purification, and Characterization of Oligonu-
cleotides. The syntheses of oligonucleotides were performed on a
DNA synthesizer at a 1 μmol scale (trityl-on mode) using the phos-
phoramidite 12 or 13 and the standard phosphoramidite building blocks
following the synthesis protocol for 30-O-(2-cyanoethyl)phosphora-
midites.48 After cleavage from the solid support, the oligonucleotides
were deprotected in 25% aqueous ammonia solution for 12ꢀ16 h at 60 °C.
The purification of the “trityl-on” oligonucleotides was carried out on
reversed-phase HPLC (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 4,40-dimethoxytrityl residues.
’ EXPERIMENTAL SECTION
General Methods. All chemicals and solvents were of laboratory
grade as obtained from commercial suppliers and were used without
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Table 6. C-NMR Chemical Shifts of 8-Aza-7-deaza-20-deoxyguanosine Derivativesa
13
C(2)b
C(6)c
C(4)b
C(7a)c
C(5)b
C(3a)c
C(6)b
C(4)c
C(7)b
C(3)c
CtC
triazole
C(10)
C(20)
C(30)
C(40)
C(50)
1
155.6d
155.5d
158.8d
158.7d
156.3d
155.5d
156.3d
155.5d
155.4d
155.3d
154.7d
154.6d
155.0d
155.2d
155.0d
155.2d
83.8
88.9
84.0
83.8
96.6
93.4
96.6
93.4
157.0d
156.8d
159.7d
159.5d
158.0d
157.1d
158.0d
157.1d
129.2
129.5
129.3
129.6
138.4
130.4
138.4
130.4
100.4, 75.9
100.3, 97.0
102.9, 76.0
102.9, 76.1
83.3
83.2
83.5
83.1
83.2
83.1
83.2
83.1
38.7
38.7
38.7
38.7
38.7
38.7
38.7
38.6
70.9
70.9
71.1
70.5
71.1
71.0
71.1
70.9
87.6
87.5
87.7
85.3
87.6
87.5
87.6
87.5
62.4
62.3
62.5
64.1
62.5
62.4
62.5
62.4
9
10
11
4
125.8, 139.9
122.1, 147.1
125.8, 139.9
122.1, 147.0
5
100.0, 73.1
100.0, 73.1
6
723
a Measured in DMSO-d6 at 298 K. b Purine numbering. c Systematic numbering. d Tentative.
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). Then, the solvent was evaporated using a
SpeedVac evaporator to yield colorless solids which were frozen at
ꢀ24 °C. The molecular masses of the oligonucleotides were determined
by LC-ESI-TOF mass spectrometry or by MALDI-TOF mass spectro-
metry in the linear negative mode (for spectra, see Figures S12ꢀS27,
Supporting Information).
Ion-Exchange HPLC Analysis of DNA Interstrand Cross-
Linked Oligonucleotides. Mobility Shift Analyses. The ion-
exchange chromatography was performed on a 4 ꢁ 250 mm DNA PA-
100 column with a precolumn using a HPLC apparatus. Elution profiles
were recorded at 260 nm. The alkynylated oligonucleotides ODN-16,
ODN-20, azidomethylbenzyl-labeled ODN-22 and interstrand cross-
linked oligonucleotides ICL-28step and ICL-36bis (0.1 A260 units each)
were dissolved in 100 μL of water and then directly injected into the
apparatus. The compounds were eluted using gradient IV: 0ꢀ30 min
with 20ꢀ80% D in C with a flow rate of 0.75 mL minꢀ1 ((C) 25 mM
Tris-HCl, 10% MeCN, pH 7.0; (D) 25 mM Tris-HCl, 1.0 M NaCl, 10%
MeCN, pH 7.0).
(15 mL) was treated with CuI (120 mg, 0.6 mmol), [Pd0[P(Ph3)4] (360
mg, 0.3 mmol), anhydrous Et3N (220 μL, 3.0 mmol), and 7.0 equiv of
(trimethylsilyl)acetylene (2.06 g, 21.0 mmol). The reaction mixture was
stirred under N2 for 2 days (TLC monitoring). The mixture was diluted
with MeOH/CH2Cl2, 1:1 (60 mL), and Dowex HCO3 (100 ( 200
h
mesh; 3.0 g) was added. After the mixture was stirred for 15 min, the
evolution of gas ceased. Stirring was continued for another 30 min, and
the resin was filtered off and washed with MeOH/CH2Cl2, 1:1
(200 mL). The combined filtrate was evaporated, and the oily residue
was adsorbed on silica gel and loaded on the top of a column. FC (silica
gel, column 15 ꢁ 3 cm, eluted with CH2Cl2/MeOH, 95:5 f 90:10)
afforded one main zone. Evaporation of the solvent furnished 9 as a
colorless foam (760 mg, 68%): TLC (CH2Cl2/MeOH, 90:10) Rf 0.75;
λmax (MeOH)/nm 246 (ε/dm3 molꢀ1 cmꢀ1 28400), 260 (15800), 280
1
(6000); H NMR (DMSO-d6, 300 MHz) (δ, ppm) 0.24 (s, 9H, 3 ꢁ
CH3), 2.10ꢀ2.18 (m, 1H, 20-HR), 2.61ꢀ2.69 (m, 1H, 20-Hβ),
3.42ꢀ3.50 (m, 2H, 50-H2), 3.73ꢀ3.78 (m, 1H, 40-H), 4.31ꢀ4.37 (m,
1H, 30-H), 4.74 (t, J = 5.7 Hz, 1H, 50-OH), 5.24ꢀ5.25 (d, J = 4.2 Hz, 1H,
30-OH), 6.27 (t, J = 6.6 Hz, 1H, 10-H), 6.77 (br s, 2H, NH2), 10.72 (s,
1H, NH). Anal. Calcd. for C15H21N5O4Si (363.44): C, 49.57; H, 5.82;
N, 19.27. Found: C, 49.61; H, 5.76; N, 19.10.
Tm Measurements. The oligonucleotides were dissolved in 1 M
NaCl, 100 mM MgCl2, and 60 mM Na-cacodylate buffer, pH 7.0 or
100 mM NaCl, 10 mM MgCl2 and 10 mM Na-cacodylate, pH 7.0. For
non-cross-linked oligonucleotide duplexes 5 μM þ 5 μM single-stranded
concentration was used. Hybridization experiments to obtain cross-linked
duplexes were carried out with 2.5 μM of the cross-linked oligonucleotide
and 5 μM of the complementary non-cross-linked oligonucleotide.
Hybridization experiments to obtain double cross-linked duplexes were
carried out with 2.5 μM of each cross-linked oligonucleotide.
The melting temperature curves were measured with a UVꢀvis
spectrophotometer equipped with a thermoelectrical controller. The tem-
perature was measured continuously in the reference cell with a Pt-100
resistor with a heating rate of 1 °C minꢀ1, and the absorbance at 260 nm was
recorded as a function of the temperature. The thermodynamic data of
duplex formation were calculated by the MeltWin (version 3.0) program
using the curve fitting of the melting profiles according to a two-state model.49
6-Amino-1-(2-deoxy-β-D-erythro-pentofuranosyl)-1,5-di-
hydro-3-ethynyl-4H-pyrazolo[3,4-d]pyrimidin-4-one (1). A
solution of compound 9 (650 mg, 1.79 mmol) and anhyd K2CO3 (160
mg, 1.16 mmol) in MeOH (40 mL) was stirred for 6 h at rt. After
evaporation, the residue was adsorbed on silica gel and loaded on the top of
a column. FC (silica gel, column 15 ꢁ 3 cm, eluted with CH2Cl2/MeOH,
95:5 f 90:10 f 85:15) afforded one main zone. The isolated product was
recrystallized from MeOH, and 1 (430 mg, 83%) was obtained as a
colorless solid: TLC (CH2Cl2/MeOH, 80:20) Rf 0.47; λmax (MeOH)/nm
1
240 (ε/dm3 molꢀ1 cmꢀ1 24500), 260 (10900), 280 (5800); H NMR
(DMSO-d6, 300 MHz) (δ, ppm) 2.08ꢀ2.19 (m, 1H, 20-HR), 2.60ꢀ2.69
(m, 1H, 20-Hβ), 3.43ꢀ3.51 (m, 2H, 50-H2), 3.73ꢀ3.78 (m, 1H, 40-H),
4.34ꢀ4.38 (m, 2H, 30-H, CtCH), 4.73 (t, J = 5.4 Hz, 1H, 50-OH),
5.23ꢀ5.25 (d, J = 4.2 Hz, 1H, 30-OH), 6.28 (t, J = 6.6 Hz, 1H, 10-H), 6.79
(br s, 2H, NH2), 10.79 (s, 1H, HN). Anal. Calcd for C12H13N5O4
(291.10): C, 49.48; H, 4.50; N, 24.04. Found: C, 49.35; H, 4.52; N, 23.91.
6-[[(Dimethylamino)methylidene]amino]-1-(2-deoxy-β-
D-erythro-pentofuranosyl)-1,5-dihydro-3-ethynyl-4H-pyra-
zolo[3,4-d]pyrimidin-4-one (10). To a solution of compound 1
(592 mg, 2.0 mmol) in MeOH (20 mL) was added N,N-dimethylformamide
dimethyl acetal (7 mL, 50.0 mmol). The mixture was stirred for 2 h at rt.
The solvent was evaporated, and the residue was applied to FC (silica
gel, column 15 ꢁ 3 cm, eluted with CH2Cl2/MeOH, 95:5 f 90:10).
Evaporation of the main zone afforded 10 as colorless foam (560 mg,
80%): TLC (CH2Cl2/MeOH, 90:10) Rf 0.40; λmax (MeOH)/nm 246
The hypochromicity (h (%) = [(Emonomer ꢀ Epolymer)(Emonomer
)
ꢀ1)]-
100%) of the oligonucleotides was determined from the melting curves.
The extinction coefficients of the oligonucleotides were calculated from
the sum of the extinction coefficients of the monomeric 20-deoxyribo-
nucleosides divided by the hyperchromicity.50 Extinction coefficients
ε260 (MeOH) of the nucleosides: dA 15400, dG 11700, dT 8800, dC
7300, 1 10900, 2 11500, 4 26000, 5 23100, 6 31800 (0.1 M aq NaOH),
7 23000.
6-Amino-1-(2-deoxy-β-D-erythro-pentofuranosyl)-1,5-di-
hydro-3-[2-(trimethylsilyl)ethynyl]-4H-pyrazolo[3,4-d]pyri-
midin-4-one (9). A solution of 831 (1.2 g, 3.0 mmol) in dry DMF
1
(ε/dm3 molꢀ1 cmꢀ1 17500), 255 (21700), 300 (21400); H NMR
(DMSO-d6, 300 MHz) (δ, ppm) 2.18ꢀ2.22 (m, 1H, 20-HR), 2.63ꢀ2.72
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(m, 1H, 20-Hβ), 3.05 (s, 3H, NCH3), 3.18 (s, 3H, NCH3), 3.42ꢀ3.50
(m, 2H, 50-H2), 3.76ꢀ3.81 (m, 1H, 40-H), 4.38ꢀ4.40 (m, 2H, 30-H,
CtCH), 4.76 (t, J = 5.7 Hz, 1H, 50-OH), 5.30ꢀ5.31 (d, J = 4.2 Hz, 1H,
30-OH), 6.45 (t, J = 6.3 Hz, 1H, 10-H), 8.67 (s, 1H, NdCH), 11.36 (s,
1H, HN). Anal. Calcd for C15H18N6O4 (346.14): C, 52.02; H, 5.24; N,
24.27. Found: C, 52.54; H, 5.16; N, 24.46.
6.78 (br s, 2H, NH2), 7.32ꢀ7.41 (m, 4H, H-phenylene), 8.95 (s, 1H,
H-triazole), 10.75 (s, 1H, HN); ESI-TOF calcd for C20H21N11O4Na (M
þ Naþ) 502.1670, m/z found 502.1671. Anal. Calcd for C20H21N11O4
(479.45): C, 50.10; H, 4.41; N, 32.14. Found: C, 50.10; H, 4.51; N, 31.95.
6-Amino-1-(2-deoxy-β-D-erythro-pentofuranosyl)-1,5-di-
hydro-3-[[1-(4-azidomethylbenzyl)-1H-(1,2,3-trizol-4-yl)]hexyl-
idene]-4H-pyrazolo[3,4-d]pyrimidin-4-one (5). To a solution of
compound 2 (74.2 mg, 0.2 mmol) and 3 (188 mg, 1.0 mmol) in
THFꢀH2Oꢀt-BuOH, 3:1:1 (3 mL) was added a freshly prepared 1 M
solution of sodium ascorbate (159 μL, 0.16 mmol) in water, followed by the
addition of copper(II) sulfate pentahydrate 7.5% in water (128 μL, 0.038
mmol). The reaction mixture was stirred in the dark at room temperature for
12 h. After completion of the reaction (monitored by TLC), the solvent was
evaporated, and the residue was applied to FC (silica gel, column 8 ꢁ 3 cm,
eluted with CH2Cl2/MeOH, 95:5 f 90:10 f 80:20). The main zone
afforded 5 as a colorless solid (67 mg, 60%): TLC (CH2Cl2/MeOH, 90:10)
Rf 0.58; λmax (MeOH)/nm 243 (ε/dm3 molꢀ1 cmꢀ1 51200), 280 (sh)
(13100); 1H NMR (DMSO-d6, 300 MHz) (δ, ppm) 1.55ꢀ1.60 (m, 2H,
CH2), 1.73ꢀ1.77 (m, 2H, CH2), 2.12ꢀ2.15 (m, 1H, 20-HR), 2.45ꢀ2.50
(m, 3H, CH2, 20-Hβ), 2.64ꢀ2.68 (m, 4H, 2x CH2), 3.43ꢀ3.49 (m, 1H, 50-
H), 3.75ꢀ3.76 (m, 1H, 40-H), 4.35 (s, 1H, 30-H), 4.42(s, 2H, PhCH2), 4.73
(m, 1H, 50-OH), 5.23 (s, 1H, 30-OH), 5.55 (s, 2H, PhCH2), 6.27 (t, J = 6.3
Hz, 1H, 10-H), 6.73 (br s, 2H, NH2), 7.29ꢀ7.37 (m, 4H, H-phenylene),
7.94 (s, 1H, H-triazole), 10.68 (s, 1H, HN); ESI-TOF calcd for
C26H29N11O4Na (M þ Naþ) 582.2296, m/z found 582.2300. Anal.
Calcd for C26H29N11O4 (559.58): C, 55.81; H, 5.22; N, 27.53. Found: C,
56.17; H, 5.24.
6-[[(Dimethylamino)methylidene]amino]-1-[2-deoxy-5-O-
(4,40-dimethoxytriphenylmethyl)-β-D-erythro-pentofuranosyl]-
1,5-dihydro-3-ethynyl-4H-pyrazolo[3,4-d]pyrimidin-4-one
(11). Compound 10 (550 mg, 1.6 mmol) was dried by repeated
coevaporation with anhydrous pyridine and then dissolved in dry
pyridine (15 mL) and stirred with 4,40-dimethoxytrityl chloride (810
mg, 2.4 mmol) in the presence of N,N-diisopropylethylamine (430 μL,
2.4 mmol) at room temperature. After 6 h, the solution was poured into
5% aqueous NaHCO3 (100 mL) and extracted with CH2Cl2 (3 ꢁ
80 mL). The combined organic layers were dried (Na2SO4), the solvent
was evaporated, and the remaining oily residue was coevaporated with
toluene (3 ꢁ 10 mL) to afford a foamy residue which was applied to FC
(silica gel, column 10 ꢁ 4 cm, CH2Cl2/MeOH, 95:5 f 90:10).
Evaporation of the main zone afforded 11 as colorless foam (855 mg,
83%): TLC (CH2Cl2/MeOH, 90:10) Rf 0.53; λmax (MeOH)/nm 236
1
(ε/dm3 molꢀ1 cmꢀ1 33700), 246 (29400), 300 (25900); H NMR
(DMSO-d6, 300 MHz) (δ, ppm) 2.19ꢀ2.28 (m, 1H, 20-HR), 2.65ꢀ2.72
(m, 1H, 20-Hβ), 3.06 (s, 3H, NCH3), 3.19 (s, 3H, NCH3), 3.36 (m, 2H,
50-H2), 3.71 (s, 6H, 2 ꢁ OCH3), 3.87ꢀ3.89 (m, 1H, 40-H), 4.41 (s, 1H,
CtCH), 4.47ꢀ4.51 (m, 1H, 30-H), 5.32ꢀ5.34 (d, J = 4.8 Hz, 1H, 30-
OH), 6.48 (t, J = 4.2 Hz, 1H, 10-H), 6.77ꢀ6.82 (m, 4H, H-phenyl),
7.16ꢀ7.34 (m, 9H, H-phenyl), 8.71 (s, 1H, NdCH), 11.43 (s, 1H, HN).
Anal. Calcd for C36H36N6O6 (648.27): C, 66.65; H, 5.59; N, 12.96.
Found: C, 66.58; H, 5.66; N, 12.83.
Bis[6-Amino-1-(2-deoxy-β-D-erythro-pentofuranosyl)-1,5-di-
hydro-3-[[1-methylbenzyl-1H-(1,2,3-trizol-4-yl)]hexylidene]-4H-
pyrazolo[3,4-d]pyrimidin-4-one] (7). Evaporation of the minor zone
gave 7 as a white powder (14 mg, 15%). Analytical data are identical with
already published data.23
6-[[(Dimethylamino)methylidene]amino]-1-[2-deoxy-5-O-
(4,40-dimethoxytriphenylmethyl)-β-D-erythro-pentofuranosyl]-
1,5-dihydro-3-ethynyl-4H-pyrazolo[3,4-d]pyrimidin-4-one
30-[(2-Cyanoethyl)-N,N-(diisopropyl)]phosphoramidite (12).
A solution of 11 (560 mg, 0.86 mmol) in anhydrous CH2Cl2 (10 mL)
was treated with anhydrous (iPr)2NEt (300 μL, 1.34 mmol) at rt. Then
2-cyanoethyl diisopropylphosphoramidochloridite (320 μL, 1.77 mmol)
was added. After 3 h, the solution was washed with saturated NaHCO3
and extracted with CH2Cl2 (2 ꢁ 100 mL). The combined organic layer
was dried (Na2SO4), and the solvent was evaporated. FC (silica gel,
column 8 ꢁ 3 cm, CH2Cl2/MeOH, 95:5) afforded 12 as a colorless foam
(540 mg, 74%): TLC (CH2Cl2/MeOH, 95: 5) Rf 0.74; 31P NMR
(CDCl3, 121.5 MHz) (δ, ppm) 148.36. Anal. Calcd for C45H53N8O7P
(848.38): C, 63.67; H, 6.29; N, 13.20. Found: C, 63.89; H, 6.36; N, 13.20.
Monofunctionalization of 1,4-Bis-azidomethylbenzene
(3) with Nucleosides 1 and 2. 6-Amino-1-(2-deoxy-β-D-ery-
thro-pentofuranosyl)-1,5-dihydro-3-[1-(4-azidomethylbe-
nzyl)-1H-[1,2,3-trizol-4-yl]]-4H-pyrazolo[3,4-d]pyrimidin-4-
one (4). To a solution of compound 1 (58.3 mg, 0.2 mmol) and 3 (188
mg, 1.0 mmol) in THFꢀH2Oꢀt-BuOH, 3:1:1 (3 mL) was added a
freshly prepared 1 M solution of sodium ascorbate (159 μL, 0.16 mmol)
in water, followed by the addition of copper(II) sulfate pentahydrate
7.5% in water (128 μL, 0.038 mmol). The reaction mixture was stirred in
the dark at room temperature for 12 h. After completion of the reaction
(monitored by TLC), the solvent was evaporated, and the residue was
applied to FC (silica gel, column 8 ꢁ 3 cm, eluted with CH2Cl2/MeOH,
95:5 f 90:10). Evaporation of the main zone gave 4 as a pale yellow
solid (71 mg, 74%): TLC (CH2Cl2/MeOH, 80:20) Rf 0.60; λmax
(MeOH)/nm 246 (ε/dm3 molꢀ1 cmꢀ1 48600), 260 (26000), 280
(12000). 1H NMR (DMSO-d6, 300 MHz) (δ, ppm) 2.15ꢀ2.23 (m, 1H,
20-HR), 2.74ꢀ2.83 (m, 1H, 20-Hβ), 3.40ꢀ3.44 (m, 1H, 50-H),
3.51ꢀ3.56 (m, 1H, 50-H), 3.77ꢀ3.80 (m, 1H, 40-H), 4.44ꢀ4.49 (m,
3H, 30-H, PhCH2), 4.79 (t, J = 5.7 Hz, 1H, 50-OH), 5.26ꢀ5.27 (d, J = 4.2
Hz, 1H, 30ꢀOH), 5.75 (s, 2H, PhCH2), 6.36 (t, J = 6.3 Hz, 1H, 10-H),
“Bis-click” Cross-LinkingofNucleoside 1 with1,4-Bis-azido-
methylbenzene (3). Bis[6-Amino-1-(2-deoxy-β-D-erythro-
pentofuranosyl)-1,5-dihydro-3-[1-methylbenzyl-1H-(1,2,3-
trizol-4-yl)]-4H-pyrazolo[3,4-d]pyrimidin-4-one] (6). To a so-
lution of compound 1 (116.6 mg, 0.4 mmol) and 3 (37.6 mg, 0.2 mmol)
in THFꢀH2Oꢀt-BuOH, 3: 1: 1 (6 mL) was added a freshly prepared 1
M solution of sodium ascorbate (318 μL, 0.32 mmol) in water, followed
by the addition of copper(II) sulfate pentahydrate 7.5% in water (256
μL, 0.076 mmol), and the reaction mixture was stirred at room
temperature for 12 h. After completion of the reaction (monitored by
TLC), the mixture was filtered and the solid residue was washed with
methanol (5 mL) and water (5 mL). Compound 6 was obtained as a
yellow solid (80 mg, 52%): λmax (0.1 M NaOH)/nm 242 (ε/dm3
1
molꢀ1 cmꢀ1 47 500), 260 (sh) (31800); H NMR (DMSO-d6, 300
MHz) (δ, ppm) 2.11ꢀ2.18 (m, 2H, 20-HR), 2.76ꢀ2.80 (m, 2H, 20-Hβ),
3.51ꢀ3.55 (m, 4H, 50-H2), 3.78ꢀ3.79 (m, 2H, 40-H), 4.43ꢀ4.47 (m,
2H, 30-H), 4.78 (t, J = 5.1 Hz, 2H, 50-OH), 5.25ꢀ5.26 (d, J = 3.3 Hz, 2H,
30-OH), 5.72 (s, 4H, 2 ꢁ PhCH2), 6.35 (t, J = 6.0 Hz, 2H, 10-H), 6.76 (br
s, 4H, 2 ꢁ H2N), 7.33ꢀ7.40 (m, 4H, H-phenylene), 8.94 (s, 2H, 2 ꢁ
H-triazole), 10.74 (s, 2H, 2 ꢁ HN). Anal. Calcd for C32H34N16O8
(770.71): C, 49.87; H, 4.45; N, 29.08. Found: C, 49.49; H, 4.22; N, 28.65.
6-Amino-1-(2-deoxy-β-D-erythro-pentofuranosyl)-1,5-di-
hydro-3-[1-(4-azidomethylbenzyl)-1H-[1,2,3-trizol-4-yl]]-4H-
pyrazolo[3,4-d]pyrimidin-4-one (4). The remaining solution was
evaporatedtodryness, andtheresiduewasappliedtoFC(silicagel, column
8 ꢁ 3 cm, eluted with CH2Cl2/MeOH, 95:5 f 90:10). Compound 4 was
isolated as a white powder (14 mg, 7%). For analytical data of 4, see above.
General Procedure for the “Stepwise Click” Huisgenꢀ
SharplessꢀMeldal [3 þ 2] Cycloaddition. Azidomethylben-
zyl-Labeled Oligonucleotides (ODN-22ꢀODN-27) Using
Oligonucleotides ODN-16ꢀODN-21 and the Bis-Azide 3
(“1.Click”). To the single-stranded oligonucleotide (5 A260 units) were
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The Journal of Organic Chemistry
ARTICLE
added a mixture of the CuSO4ꢀTBTA ligand complex (50 μL of a
20 mM stock solution in H2O/DMSO/t-BuOH, 4:3:1 for TBTA; 50 μL
of a 20 mM stock solution in H2O/DMSO/t-BuOH, 4:3:1 for CuSO4),
tris(carboxyethyl)phosphine (TCEP; 50 μL of a 20 mM stock solution
in water), 1,4-bis-azidomethylbenzene 3 (37.5 μL of a 20 mM stock
solution in dioxane/H2O, 1: 1), sodium bicarbonate (50 μL of a 20 mM
aq solution), and 30 μL of DMSO, and the reaction proceeded at room
temperature for 12 h. The reaction mixture was concentrated in a
SpeedVac and dissolved in 1 mL of bidistilled water and centrifuged for
20 min at 12000 rpm. The supernatant solution was collected and further
purified by reversed-phase HPLC (gradient III) to give about 60ꢀ80%
isolated yield of the azidomethylbenzyl oligonucleotides ODN-22ꢀODN-
27. The molecular masses of the azidomethylbenzyl oligonucleotides
ODN-22ꢀODN-27 were determined by MALDI-TOF or LC-ESI-TOF
mass spectrometry (Table S3, Supporting Information).
for the measurement of the MALDI spectra. Financial support by
ChemBiotech, Muenster, Germany, is gratefully acknowledged.
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Supporting Information. Mass spectra of non-cross-
b
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the stepwise and bis-click derivatives, HPLC purification profiles
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oligonucleotides, melting curves of ICLs, 1H NMR, 13C NMR,
DEPT-135 and Hꢀ C-gated decoupled NMR spectra of the
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1
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’ AUTHOR INFORMATION
Corresponding Author
*Tel: þ49 (0)251-53406-500. Fax: þ49 (0)251-53406-587.
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E-mail: frank.seela@uni-osnabrueck.de. Web: www.seela.net.
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