Unprecedented C-selective interstrand cross-linking through in situ oxidation of furan-modified oligodeoxynucleotides

Article abstract of DOI:10.1021/ja1048169

Chemical reagents that form interstrand cross-links have been used for a long time in cancer therapy. They covalently link two strands of DNA, thereby blocking transcription. Cross-link repair enzymes, however, can restore the transcription processes, causing resistance to certain anti-cancer drugs. The mechanism of these cross-link repair processes has not yet been fully revealed. One of the obstacles in this study is the lack of sufficient amounts of well-defined, stable, cross-linked duplexes to study the pathways of cross-link repair enzymes. Our group has developed a cross-link strategy where a furan moiety is incorporated into oligodeoxynucleotides (ODNs). These furan-modified nucleic acids can form interstrand crosslinks upon selective furan oxidation with N-bromosuccinimide.We here report on the incorporation of the furan moiety at the 20-position of a uridine through an amido or ureido linker. The resulting modified ODNs display an unprecedented selectivity for cross-linking toward a cytidine opposite the modified residue, forming one specific cross-linked duplex, which could be isolated in good yield. Furthermore, the structure of the formed cross-linked duplexes could be unambiguously characterized.

Full text of DOI:10.1021/ja1048169

ARTICLE
pubs.acs.org/JACS
Unprecedented C-Selective Interstrand Cross-Linking through in Situ
Oxidation of Furan-Modified Oligodeoxynucleotides
Marieke Op de Beeck and Annemieke Madder*
Laboratory for Organic and Biomimetic Chemistry, University of Ghent, Krijgslaan 281 S4, B-9000 Ghent, Belgium
S Supporting Information
b
ABSTRACT: Chemical reagents that form
interstrand cross-links have been used for a
long time in cancer therapy. They cova-
lently link two strands of DNA, thereby
blocking transcription. Cross-link repair
enzymes, however, can restore the tran-
scription processes, causing resistance to
certain anti-cancer drugs. The mechanism
of these cross-link repair processes has
not yet been fully revealed. One of the
obstacles in this study is the lack of sufficient
amounts of well-defined, stable, cross-linked
duplexes to study the pathways of cross-link repair enzymes. Our group has developed a cross-link strategy where a furan
moiety is incorporated into oligodeoxynucleotides (ODNs). These furan-modified nucleic acids can form interstrand cross-
links upon selective furan oxidation with N-bromosuccinimide. We here report on the incorporation of the furan moiety at the
20-position of a uridine through an amido or ureido linker. The resulting modified ODNs display an unprecedented selectivity
for cross-linking toward a cytidine opposite the modified residue, forming one specific cross-linked duplex, which could
be isolated in good yield. Furthermore, the structure of the formed cross-linked duplexes could be unambiguously
characterized.
’ INTRODUCTION
drugs can often be attributed to enhanced ICL repair.23 These
repair pathways find their evolutionary origin in the necessity to
counteract the threat posed by endogenous and exogenous cross-
linking compounds, and failure to repair these lesions can lead to
inherited disorders such as Fanconi anemia.23 Thus, although
DNA repair is essential for a healthy cell, repair enzymes
counteract the efficacy of a number of important antitumor
agents whose therapeutic potential rests on the formation of
ICLs. Therefore, selective inhibition of DNA repair in cancer-
ous cells may greatly improve antitumor therapy, making
DNA repair enzymes a promising target for drug design.
In this context, understanding of the mechanisms of repair
enzymes should significantly increase our understanding of
the molecular basis of human disease. Significant progress has
been made to elucidate the diverse DNA repair pathways, but
the exact mechanisms underlying the repair of ICLs still
remain poorly understood.26 A major factor limiting this
research is the availability of short, well-defined, cross-linked
oligodeoxynucleotides (ODNs) as substrates for these repair
enzymes.26 Interstrand cross-linked ODNs can be gene-
rated by treating dsDNA with bifunctional alkylating agents.
The high specificity with which oligonucleotides recognize
nucleic acids has led to considerable interest in the design of modi-
fied oligonucleotides.1 These customized nucleic acid derivatives
can be used for a range of therapeutic and analytical purposes,
including the treatment of diseases (antisense,2-4 siRNA5-8), the
regulation of gene expression (antigene,9-11 decoy DNA12,13), the
investigation of RNA tertiary structures,14-16 and the study of
DNA damage and the resulting repair processes.17-21
DNA is not indefinitely stable, and numerous sources of DNA-
damaging agents of endogenous and exogenous origin contribute
further to the instability of DNA.22 DNA interstrand cross-links
(ICLs) are among the most cytotoxic lesions known, the covalent
bond between the two DNA strands prohibiting strand separa-
tion and thereby blocking vital aspects of DNA metabolism. ICLs
are mostly formed by reaction of cellular DNA with bifunctional
electrophilic compounds that are either formed endogenously
(e.g., lipid peroxidation) or present through exogenous exposure.23
Additionally, generation of an abasic site in the DNA upon
depurination can also give rise to ICL formation.20,24
The cytotoxic properties of such ICL-forming agents are
exploited by cancer chemotherapeutics, where their toxicity
ultimately leads to apoptosis of the malignant cells.25 Develop-
ment of resistance of the tumor cells to treatment with antitumor
Received: June 2, 2010
Published: December 16, 2010
r
2010 American Chemical Society
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Figure 1. Building blocks for the synthesis of furan-modified ODNs. Selective in situ oxidation is followed by instantaneous interstrand cross-link
formation.
However, as most alkylating agents offer little to no sequence
selectivity and produce a mixture of mono adducts and intra-
and interstrand cross-links, only very low yields of the desired
ICL are obtained (typically <10%).27 The selectivity problem
can be circumvented by inducing the ICL at a specific position
in the duplex. In a first approach, a cross-linked dinucleo-
tide can be incorporated in the DNA duplex.18,28-31 This
approach is, however, somewhat limited due to the need for up
to three different orthogonal protecting groups on the dimer
phosphoramidite. In an alternative approach, oligonucleotides
are equipped with a reactive functionality, allowing cross-
link formation after hybridization with their complementary
strand.12,24,32-42 Depending on the specific chemistry em-
ployed, only one or both strands of the duplex contain a
modified nucleoside. These modified residues can either be
intrinsically reactive or contain a latent functionality that is
activated by means of a triggering signal.
SN2 substitution with an in situ-generated azide nucleophile,
resulting in the formation of the desired 20-azido-uridine 8,45
which could be coupled to activated ester 5 in a Staudinger-
Vilarrasa coupling.46 Protection of the 50-hydroxyl group and
conversion of the nucleoside to phosphoramidite 10 were
performed by standard methods.
The 20-ureido-modified uridine 13 was synthesized by cou-
pling of 20-amino-uridine 11 to the commercially available
furfurylisocyanate 12. In this way, a similar three-atom linker
between the 20-N and the furan moiety was obtained. The
20-amino-uridine was obtained by reduction of 20-azido-uridine 8;
a Staudinger reduction47 was used rather than hydrogenation
with Pd/C48 to avoid reduction of the uracil base.49,50 DMTr
protection of the 20-ureido-uridine 13 and subsequent conver-
sion to the desired phosphoramidite yielded the necessary
building block for incorporation into ODN by automated
DNA synthesis.
ODN Synthesis. ODN synthesis was carried out on an auto-
mated DNA synthesizer, introducing a manual coupling step for
the modified phosphoramidites to prevent loss of precious
modified phosphoramidite in the tubing of the DNA synthesizer.
Additionally, dicyanoimidazole (DCI) was used instead of tetra-
zole as a more potent activator for the manual couplings. Using
our previously established standard conditions51 for the intro-
duction of the 20-modified uridines 10 and 14, initially a poor
coupling efficiency was observed for the 20-amido derivative 10,
and no incorporation at all was observed for the 20-ureido
derivative 14. We speculated this to be due to the presence of
a nucleophile at the 20-position that was able to attack the
activated phosphoramidite (Scheme 2). A similar explanation
has been put forward by Al-Rawi and co-workers when encoun-
tering problems with a 20-amido-derivatized phosphoramidite.52
If this holds true, then coupling efficiency should improve upon
activating the phosphoramidite once the growing ODN chain is
present. Indeed, by mixing the activator and the phosphoramidite
on the synthesis column, satisfying coupling efficiency was
observed for the 20-amido- as well as the 20-ureido-furan-mod-
ified nucleosides. The synthesized DMTr-protected ODNs were
deprotected and purified on a SEP-PAK column and character-
ized by reversed-phase high-performance liquid chromatography
(RP-HPLC) and electrospray ionization mass spectrometry
(ESI-MS) (see Supporting Information).
Although considerable effort has been devoted to the synthesis
of cross-linked ODNs, few methods exist today that offer an
efficient, high-yielding, and selective method for the generation
of site-specifically cross-linked duplexes.
Here we present a cross-linking strategy inspired by the
natural toxicity of furan, where a furan unit is inserted at the
20-position of a uridine as a masked reactive γ-keto-enal. After
hybridization of an ODN containing a 20-furan-modified uridine
with its complement, selective in situ oxidation of the furan unit
to a reactive dicarbonyl moiety leads to fast and efficient
generation of an interstrand cross-linked duplex (Figure 1). In
continuation of our earlier preliminary studies,43 the furan
moiety was introduced into the oligonucleotides by incorpora-
tion of 20-modified uridine residues, presenting the furan moiety
through an amide (1) or ureide (2) linker. A detailed investiga-
tion of sequence selectivity and linker influence has been carried
out, and thorough structural characterization has now shed light
on the cross-linking mechanism.
’ RESULTS AND DISCUSSION
Synthesis of the Modified Nucleosides. Coupling of furan
onto uridine through an amide linker could be accomplished by
coupling activated ester 5 with 20-azido-uridine 8 (Scheme 1). A
three-carbon linker was selected in view of the commercial
availability of furan-2-acrylic acid 3. The necessary pentafluor-
ophenyl ester was synthesized by hydrogenation of 3, followed by
conversion of the obtained carboxylic acid 4 to the activated ester
with pentafluorophenol.
Duplex Stability and Cross-Linking Selectivity. In order
to assess the cross-linking selectivity of 20-amido- and 20-ureido-
furan-modified ODN, phosphoramidites 10 and 14 were in-
corporated in oligonucleotides ODN1 to ODN8 (Table 1),
where the bases flanking the modified residue are varied. They
were hybridized with different complementary DNA sequences,
The precursor 20-azido-uridine was synthesized by conversion
of uridine to the ring-closed intermediate 744 and subsequent
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Scheme 1. Synthesis of Furan-Modified 20-Amido- and 20-Ureido-uridine Phosphoramiditesa
a Reagents and conditions: (a) H2, Pd/C, NH4OH/MeOH, 18 h, rt, 73%; (b) DCC, C6F5OH, NEt3 THF, 15 h, rt, 88%; (c) (PhO)2CO, NaHCO3,
DMF, 16 h, 115 °C, 74%; (d) LiF, Me3SiN3, DMF/TMEDA, 63 h, 110 °C, 72%; (e) i. PPh3, 5, pyridine, 5 h, rt, ii. 13 h, 40 °C, 83%; (f) DMTrCI,
pyridine, 14 h, rt, 90%; (g) 2-cyanoethyl-N,N-diisopropylchlorophosphine, DIPEA, 2 h, 0 °C, 76%; (h) PPh3, MeOH, H2O, 3 h, 50 °C, 89%;
(i) 12, DMF, 1 h, 96%; (j) DMTrCI, pyridine,17 h, rt, 79%; (k) 2-cyanoethyl-N,N-diisopropylchlorophosphine, DIPEA, 2.5 h, 0 °C, 50%.
Scheme 2. Proposed Mechanism of Degradation of the
Table 1. Modified ODN Sequences and Their Complementsa
Modified Phosphoramidites in the Presence of DCI
complementary
modified sequence
sequence
ODN1:
50-CTG ACG G1G TGC-30
ODN2:
50-CTG ACG G2G TGC-30
ODN9a-d:
30-GAC TGC CNC ACG-50
ODN3:
50-CTG ACG T1T TGC-30
ODN4:
50-CTG ACG T2T TGC-30
ODN10a-d:
30-GAC TGC ANA ACG-50
ODN5:
50-CTG ACG C1C TGC-30
ODN6:
50-CTG ACG C2C TGC-30
ODN11a-d:
30-GAC TGC GNG ACG-50
ODN7:
50-CTG ACG A1A TGC-30
ODN8:
50-CTG ACG A2A TGC-30
a In the modified sequences: 1, 20-amidofuran-modified uridine; 2, 20-
ureidofuran-modified uridine. In the complements: a, N = A; b, N = C; c,
N = G; d, N = T.
ODN12a-d:
30-GAC TGC TNT ACG-50
varying the base opposite the modification. In this way, 32
duplexes were generated.
Duplex stability determination via UV melting temperatures
demonstrated that destabilization of the modified duplexes is
observed and ranges from -4.1 to -20.6 °C for 20-amido- and
from -3.7 to -14.1 °C for 20-ureido-furan-modified duplexes.
Interestingly, it can be noticed that duplexes incorporating 2
opposite A are considerably more stable than those incorporating
1 opposite A. The effect is more pronounced in the case of flank-
ing pyrimidines (with ODN4:ODN10a and ODN6:ODN11a
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Chart 1. Melting Temperatures of Modified versus Unmodified Duplexes
being 9-10 °C more stable than the corresponding amido
analogues) than in the case of flanking purines (with ODN2:
ODN9a and ODN8:ODN12a being 3-4 °C more stable than
the corresponding amido analogues) (Chart 1).
Characterization of Cross-Linked Species. The adducts
that form between the natural metabolite of furan, cis-2-butene-
dial, and the nucleobases of DNA are well described in the
literature. With dC showing the highest reactivity, the reaction
occurs according to the mechanism depicted in Scheme 3.53-58
Initial attack of the exocyclic amine is followed by a cyclative
rearrangement, resulting in bicyclic species 17. Through loss of
water, further aromatization can take place.
In order to characterize the nature of the covalent bond
formed within the duplexes, cross-linking reactions were re-
peated on a larger scale, and cross-linked ODNs were purified
by RP-HPLC and characterized by ESI-MS (see Supporting
Information). From these data, the cross-linked nature of the
isolated species can be confirmed, and bromination of purine
bases (a possible consequence of NBS treatment of oligonucleo-
tides) can be excluded.
The observed molecular weight (MW) of the cross-linked
duplexes containing 20-amido- and the 20-ureido-furan-modified
building blocks is in accord with the general structure of
compound 17. The four diastereomers that can be formed can
account for the two peaks (each possibly representing two quasi-
enantiomeric structures) observed in RP-HPLC for the
20-amido-ODN (cf. Figure 3A, N = C). As for the 20-ureido-
furan-ODN, in some cases a smaller second peak was also
formed (see Supporting Information), both the main peak and
the smaller peak displaying a MW corresponding to general
structure 17. Thus, in addition to leading to a higher selec-
tivity, the more rigid 20-ureido linker also favors the formation
of one of the diastereomers.
For a more accurate characterization of the cross-linked
species and identification of the nature of the covalent bond
formed, enzymatic digestion of cross-linked duplexes, down to
the cross-linked dinucleotide, was carried out. In order to
unambiguously prove its structure, we independently prepared
a cross-linked dinucleoside via chemical synthesis for RP-HPLC
coinjection analysis.
Cross-linking experiments were performed by selective furan
oxidation using N-bromosuccinimide (NBS). Addition of 4 equiv
was necessary to ensure complete conversion of the modified
ODN. Reactions were followed by denaturing polyacrylamide gel
electrophoresis (PAGE) and RP-HPLC. PAGE experiments
show that, in the case of the amido-modified oligonucleotides
ODN1, 3, 5, and 7, cross-linking occurs when there is an adenine
or cytosine opposite the modified residue (Figure 2, panels
I-IV), as evidenced by the appearance of a new, slow-running
species.
In all cases, the signal of the modified strand has completely
disappeared in the RP-HPLC profile after addition of 4 equiv of
NBS. As can be observed in the control chromatograms
(Figure 3, left), upon NBS treatment the modified strands are
transformed into a mixture of degradation products, as indi-
cated by the formation of a very broad signal with low intensity.
In the case of adenosine as complementary base to the 20-amido-
furan-modified uridine, no substantial amount of cross-
linked product could be observed (Figure 3A, N = A). As a
suitable cross-linking complement is absent, the signal for the
unmodified ODN10a is still intact, and a broad smear can be
observed for the degradation products of the modified ODN.
However, with a cytosine opposite to the modified residue, two
new peaks (XL1 and XL2) are formed (Figure 3A, N = C), as
can also be observed on PAGE by the appearance of two closely
located spots. Further PAGE analysis at elevated temperatures
showed the originally formed species cross-linked to A not to
be stable. In the case of the 20-ureido-modified oligonucleo-
tides, we observed complete C-selectivity of the cross-linking
reaction (Figure 2, panels V-VIII). In this case, only one main
new peak (XL) appears in the RP-HPLC spectrum after
addition of 4 equiv of NBS (Figure 3B, N = C). With ODN6
and ODN8, a small second peak is also formed (see Supporting
Information).
Chemical Synthesis of Cross-Linked Dinucleosides. Be-
fore performing the cross-linking reaction of nucleoside 9 with
cytidine, the free hydroxyl groups of both compounds need to be
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Figure 2. Denaturing PAGE results of the cross-link reaction. Furan-containing ODN1-8 were annealed to complementary ODN9-12, after which
NBS was added in portions. ICL formation was analyzed by 20% denaturing PAGE. Ref A, modified ODN; Ref B, modified ODN þ 4 equiv of NBS.
acetylated to prevent reaction of the alcohol functionalities with
the oxidized furan moiety. Following protection, cross-linking
was performed by oxidation of the furan moiety in protected
nucleoside 19 with 1.1 equiv of NBS, after which 30,50-bis-acetyl-
protected cytidine 21 was added (Scheme 4).59 After straightfor-
ward deprotection with methanolic ammonia, the desired cross-
linked dinucleoside 23 was obtained in good yield. Dinucleoside
23 is, however, present as an inseparable diastereomeric mixture,
rendering NMR characterization complicated. To simplify iden-
tification, we attempted to convert dinucleoside 23 to the
aromatized nucleoside 24. Heating of dinucleoside 23 in water
or acetonitrile led to the formation of unwanted side products.
After evaluating several conditions, aromatization in 0.1 M HClaq
solution for 9 days was found to give the cleanest conversion
to compound 24 (Figure 4). The structure of this compound
could be unambiguously confirmed by NMR (see Supporting
Information).
In addition, this extra nitrogen can activate the keto function-
ality of the formed oxo-enal species 29 through hydrogen
bonding. As a consequence, next to the expected attack of the
exocyclic NH2 functionality according to pathway a, attack
through pathway b may become possible, allowing formation
of two regioisomeric aromatized species, 28 and 31, consistent
with the appearance of two signals of identical mass in the
ESI-MS spectrum (see Supporting Information).
As illustrated above, when the cross-linking reaction is per-
formed on the 20-ureido-furan-modified ODN, only one main
product with a mass corresponding to cross-linked compound
17 is formed. Most likely, when the nucleoside is incorporated
in an ODN duplex, it is forced into a specific conformation
that does not allow this H-bond formation, catalyzing these
side reactions. Additionally, as only one species is observed in
this case on RP-HPLC (cf. Figure 3B, N = C), cross-linking
reaction with these 20-ureido-furan-modified ODNs most likely
leads to the preferential formation of one diastereomer. These
results indicate that, upon incorporation of this nucleoside in
ODN, the linker is forced to adopt a specific conformation
in order to fit into the duplex, and this conformational predisposi-
tion has a significant influence on the selectivity of the reaction.
Aromatization of the Cross-Linked 20-Amido-furan-
Modified Duplex. To allow comparison of the chemically
synthesized, characterized dinucleotide 24 with a dinucleotide
obtained by enzymatic digestion of the 20-amido-modified
cross-linked duplex, the two diastereomeric cross-linked
duplexes (cf. Figure 3A, N = C) should be converted to the
aromatized duplex. In the first instance, the cross-linking
reaction was performed in a solution buffered at pH 5.
However, no different reactivity was observed compared to
Cross-linking of 20-ureido-furan-modified nucleoside 25 with
protected cytidine 21 was attempted under several reaction condi-
tions, but in each case a complex reaction mixture was obtained. The
reaction mixture was analyzed in detail by LC-MS, and the main
peaks could be assigned to unreacted starting material, overoxidized
nucleoside 26, two signals of which the MW corresponds to the mass
of cross-linked compound 27, and two signals of which the MW
corresponds to the mass of cross-linked, aromatized dinucleoside 28
(Scheme 5 and Supporting Information).
In contrast to the slow and difficult dehydration of 23 to 24,
aromatization of 27 to 28 occurs concurrently with the cross-link
formation. Presumably, the extra nitrogen in the 20-ureido-modified
nucleoside can catalyze the dehydration reaction, which could
account for the spontaneous aromatization (see Figure 5A).
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Figure 3. RP-HPLC of the cross-link reaction mixtures with (A) ODN3:ODN10 (50-CTG ACG T1T TGC-30:30-GAC TGC ANA ACG-50)
and (B) ODN4:ODN10 (50-CTG ACG T2T TGC-30:30-GAC TGC ANA ACG-50), before and after addition of 4 equiv of NBS. All ODN sequences
are mixed in a 1:1 ratio. XL represents the cross-linked product; depending on the cross-linking efficiency, the nonconsumed unmodified strand
can still be observed.
Scheme 3. Mechanism of Adduct Formation between dC and
an Oxidized Furan Derivative
reaction at pH 7. Since the aromatization reaction can also
Figure 4. Aromatization of diastereomeric compound 23 as followed by
be induced by heating, the two presumed diastereomeric
RP-HPLC.
duplexes were incubated at 50 °C (Figure 6). The reaction
was followed by HPLC, and, depending on the sequence,
was performed with snake venom phosphodiesterase (SVPD).
Unlike exonuclease III, this enzyme was able to surpass the cross-
link to produce the cross-linked dinucleotide (see Figure 7).60 After
removal of the phosphate groups with alkaline phosphatase, the
reaction mixture containing the desired dinucleotide can be ana-
lyzed by coinjection with cross-linked dinucleoside 24 (Scheme 4),
confirming the identity of the cross-link structure (Figure 8).
aromatization was finished after 1-3 days. The aromatized
cross-linked duplex (XLar) was purified by RP-HPLC and
analyzed by ESI-MS, which confirmed its aromatized structure
(see Supporting Information).
Confirmation of the Cross-Link Structure by Enzymatic
Digestion and Coinjection. Enzymatic digestion of the aromatized
duplex XLar, obtained after cross-linking ODN3 with ODN10b,
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Scheme 4. Chemical Synthesis of Cross-Linked Dinucleoside Derived from 20-Amido-Modified 9 and dCa
a Reagents and conditions: (a) Ac2O, DMAP, NEt3, ACN, 2 h, rt, 67%; (b) Ac2O, DMAP, NEt3, ACN, overnight, rt, 68%; (c) NBS, pyridine, THF/
acetone/H2O 5/4/2, overnight, rt; (d) NH3/MeOH, overnight, rt, 80% (over two steps); (e) 0.1 M HCl, 9 days, rt, quantitative.
Scheme 5. Chemical Synthesis of the Putative Cross-Linked Dinucleoside Derived from 20-Ureido-Modified 13 and dCa
a Reagents and conditions: (a) Ac2O, DMAP, NEt3, ACN, 1 h, rt, 67%; (b) 21, NBS, pyridine, THF/acetone/H2O 5/4/2, overnight, rt.
Figure 5. (A) Catalysis of the spontaneous aromatization reaction through intramolecular H-bonding. (B) Hydrogen bonding in the oxidized
nucleoside allows alternative attack according to pathway b.
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Table 2. Yields of Cross-Linked Duplexesa
yield based on
HPLC (%)
isolated
yield (%)
modified sequence
XL1
33
XL2
27
XL1 XL2 XLar
ODN1:
50-CTG ACG G1G TGC-30
ODN2
50-CTG ACG G2G TGC-30
ODN3:
50-CTG ACG T1T TGC-30
ODN4:
50-CTG ACG T2T TGC-30
ODN5:
50-CTG ACG C1C TGC-30
ODN6:
50-CTG ACG C2C TGC-30
ODN7:
50-CTG ACG A1A TGC-30
ODN8:
50-CTG ACG A2A TGC-30
12
21
28
16
18
20
19
29
12
25
32
27
26
18
55
30
37
29
26
41
39
25
Figure 6. Conversion of the diastereomeric cross-linked duplexes (XL1
and XL2) from ODN3-ODN10b to the aromatized duplex (XLar) by
prolonged heating at 50 °C.
31
10
23
7
17
8
7
5
a Complementary sequences in all cases feature C as the residue
opposing the modified building block. Isolated yields refer to RP-
HPLC purified material.
oxidation methodology for the production of considerable
quantities of site-specifically cross-linked duplexes.
Additionally, the stability of the formed cross-linked species
(XLar in the case of 20-amido-furan-modified duplexes and XL in
the case of 20-ureido-furan-modified duplexes) was assessed by
measurement of melting temperatures. In all cases, the melting
temperature was greatly increased by introduction of the cross-
link in the duplex (ΔTm = þ38 to þ58.3 °C, see Supporting
Information).
Figure 7. Enzymatic degradation of the cross-linked duplex.
’ CONCLUSION
Furan-modified nucleosides were synthesized for incorpora-
tion into ODNs, where the furan unit is attached to the
20-position of the sugar through an amide or ureide linker.
Conditions for incorporation into ODNs were optimized, and
the desired ODNs were obtained pure and in good yield by
simple SEP-PAK purification. Cross-linking reactions can be
performed by converting the stable furan functionality into a
reactive dicarbonyl moiety by selective oxidation with NBS.
The reaction proceeds instantaneously and in good yield,
especially considering that, in contrast to most literature
precedents, reaction mixtures have been RP-HPLC purified
and cross-linked duplexes have been isolated and quantified.
The selectivity of the cross-linking reactions was determined
by varying the bases flanking and opposite the modified residue.
Cross-linking does not appear to be influenced by the
sequence in which the modified uridine was incorporated
(bases flanking the modification). The 20-amido-modified
ODN showed cross-linking selectivity for adenine and cyto-
sine bases. As cross-links to adenine were not stable at elevated
temperatures, C-selectivity can be obtained in this case by
simple heating. The 20-ureido-modified ODN showed a clear
selectivity for cross-link formation with cytosine.
Figure 8. Coinjection experiments. (A) Reference sample: injection of
A, C, T, and G in the presence of crude SVPD. (B) Reaction mixture
after overnight reaction with SVPD and 1 h reaction with alkaline
phosphatase. (C) Coinjection of the digestion mixture containing 32
and synthesized dinucleoside 24.
Cross-Linking Yield and Stability. The yield of the cross-
linking reactions was calculated by determination of the HPLC
peak area. Additionally, isolated yields were determined in the
case of XLar of the 20-amido-furan-modified duplexes and XL of
the 20-ureido-furan-modified duplexes (see Table 2).
As is obvious from Table 2, average yields of 20% are con-
sistently obtained, underscoring the efficiency of the furan
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Furthermore, we unambiguously proved the structure of the
formed cross-link species by coinjection of the enzymatic diges-
tion reaction mixture with the synthesized dinucleoside. Isolated
yields are such that substantial amounts of site-specifically cross-
linked duplexes can be obtained.
The current study illustrates the important influence of
small structural changes on cross-link selectivity. The 20-ureido-
furan-modified uridine described here is an ideal complement
to our toolbox of furan-modified building blocks, featuring an
unprecedented cross-link selectivity.
overnight at 40 °C. The reaction was quenched with H2O (0.4 mL) and
concentrated in vacuo. The majority of the formed triphenylphosphine
oxide could easily be separated from the reaction mixture by trituration in
toluene (15 mL). The resulting brownish foam was concentrated onto silica
and purified by flash chromatography with DCM:MeOH 94:6. The desired
product was obtained as a white foam (0.451 g, 1.23 mmol) in 83% yield. Rf
= 0.25 (DCM:MeOH 9:1). 1H NMR (300 MHz, MeOD): 7.95 (d, J = 8.1
Hz, 1H), 7.26 (dd, J = 1.9 and 0.8 Hz, 1H), 6.21 (dd, J = 1.9 and 3.1 Hz,
1H), 6.01 (d, J = 8.5 Hz, 1H), 5.95 (dd, J = 3.1 and 0.8 Hz, 1H), 5.69
(d, J = 8.1 Hz, 1H), 4.59 (dd, J = 8.5 and 5.6 Hz, 1H), 4.18 (dd, J = 5.6
and 1.3 Hz, 1H), 4.02 (m, 1H), 3.72 (m, 2H), 2.87 (m, 2H), 2.54 (m, 2H).
13C NMR (75 MHz, MeOD): δ 177.8, 168.6, 158.2, 155.3, 145.1,
144.9, 113.7, 108.8, 105.8, 91.2, 90.5, 74.8, 65.8, 59.3, 37.6, 27.4. ESI-MS:
calcd for C16H19N3O7, 365.1; found, 366.1 [MþH]þ. Anal. Calcd for
C16H19N3O7: C, 52.60; H, 5.24; N, 11.50. Found: C, 52.06; H, 5.45; N,
11.92.
’ EXPERIMENTAL SECTION
Materials and Methods for Chemical Synthesis. All solvents
and chemical reagents were purchased from Sigma-Aldrich. 1H and 13
C
20-Deoxy-20-[3-(2-furyl)propanamido)]-50-O-(4,40-dimethoxytrityl)-
uridine 30-(2-Cyanoethyl N,N-Diisopropylphosphoramidite), 10. Com-
pound 9 (0.199 g, 0.55 mmol) was dried by co-evaporation with pyridine
(3 ꢀ 5 mL) and then dissolved in 5.5 mL of pyridine and cooled to 0 °C.
DMTrCl (0.264 g, 0.78 mmol) was added, and the reaction was stirred
overnight at room temperature. The reaction was checked by TLC and
cooled to 0 °C, and then another portion of DMTrCl (0.186 g, 0.55 mmol)
was added. The reaction was allowed to stir at room temperature for 24 h,
after which the reaction was cooled to 0 °C and quenched with MeOH
(0.25 mL). The reaction mixture was diluted with EtOAc (20 mL) and
washed with water (15 mL), NaHCO3aq,sat (15 mL), and NaClaq,sat
(15 mL). The combined aqueous phases were again extracted with 45
mL of EtOAc, after which the organic phases were combined and dried on
Na2SO4. The filtrate was concentrated by rotary evaporation and purified by
flash chromatography with isooctane:EtOAc:TEA 80:19:1. The product
was obtained as an off-white foam (0.3295 g, 0.49 mmol) in 89% yield. Rf =
0.55 (DCM:MeOH 10:1). 1H NMR (MeOD, 300 MHz): 7.83 (d, J = 8.1
Hz, 1H), 7.23-7.44 (m, 10H), 6.90 (d, J = 8.7 Hz, 4H), 6.25 (m, 1H), 6.07
(d, J = 8.5 Hz, 1H), 6.01 (d, J = 3.0 Hz, 1H), 5.29 (d, J = 8.1 Hz, 1H), 4.88
(m, 1H), 4.33 (d, J= 5.3 Hz, 1H), 4.11 (m, 1H), 3.78 (s, 6H), 3.42 (m, 2H),
2.94 (m, 2H), 2.60 (m, 2H). 13C NMR (75 MHz, MeOD): 175.5, 166.1,
160.4, 155.7, 152.7, 145.8, 142.4, 136.9, 136.6, 131.4, 129.5, 129.0, 128.1,
114.3, 111.2, 106.3, 103.0, 88.5, 87.5, 87.3, 72.4, 65.1, 57.0, 55.8, 35.2, 25.0.
ESI-MS: calcd for C37H37N3O9, 667.3; found, 303.4 [DMTr]þ, 684.6
[MþNH4]þ. Anal. Calcd for C37H37N3O9: C, 66.56; H, 5.59; N, 6.29.
Found: C, 66.66; H, 5.69; N, 6.62.
NMR spectra were recorded on a Bruker Avance 300 or a Bruker DRX
500 spectrometer operating at room temperature. Chemical shifts are
reported in parts per million (δ) relative to the residual solvent peak.
Multiplicities are reported as singlet (s), doublet (d), doublet of doublets
(dd), triplet (t), or multiplet (m). Purities of small compounds were
checked by RP-HPLC-MS or by RP-HPLC. For RP-HPLC-MS, an
Agilent 1100 series LC-MS system was used, equipped with a Phenom-
enex Luna C18 column (250 ꢀ 4.6 mm, 5 μm, at 35 °C). The used
solvent system was 5 mM NH4OAc in water (A) and MeCN (B). The
gradient went from 0 to 100% B in 15 min. For RP-HPLC, an Agilent 1100
system was used, equipped with a Phenomenex Luna C18 column (250 ꢀ
4.6 mm, 5 μm, at 50 °C), with 0.1 M TEAA (containing 8% MeOH) and
MeOH as mobile phase (linear gradient: 0-30% MeOH in 15 min,
30-100% MeOH in 3 min). HRMS spectra were recorded on a Micromass
Q-Tof 2 instrument (Z-spray electrospray quadrupole/orthogonal accel-
eration time-of-flight tandem MSWaters CapLC with PDA).
3-(Furan-2-yl)propanoic Acid, 4. Furan-2-acrylic acid (0.5 g, 3.62
mmol) was dissolved in MeOH (25 mL). A concentrated NH4OH
solution (270 μL) was added, followed by Pd/C (10%, 8.5 mg).
Hydrogenation was performed under a slight positive hydrogen pressure
for 18 h. Pd/C was removed by filtration through Celite. The product
was concentrated, and the resulting oil was dissolved in 5 mL of H2O.
The pH of this solution was adjusted to 2 with 2 N HCl, resulting in a
white precipitate. The desired product was extracted using ethyl acetate
(4 ꢀ 25 mL). The combined organic layers were dried on MgSO4 and
concentrated. The residue was purified by flash chromatography using
isooctane:EtOAc 4:1. The desired product was obtained as white
needles (0.369 g, 2.63 mmol) in 73% yield. See ref 61 for analytical data.
Pentafluorophenyl-3-(2-furyl) Propanoate, 5. Solid DCC (0.161 g,
0.780 mmol) was added in portions over a period of 15 min to a solution
of 4 (0.101 g, 0.724 mmol) with pentafluorophenol (0.146 g, 0.795
mmol) and anhydrous TEA (0.11 mL) in anhydrous THF (1.0 mL)
under an argon atmosphere at 0 °C. After overnight reaction at room
temperature, a white precipitate was formed. Following filtration of the
residue, the filtrate was concentrated and purified by flash chromatog-
raphy using isooctane:ethyl acetate 95:5. The product was obtained as
white needles (0.195 g, 0.64 mmol) in 88% yield. Rf = 0.75 (isooctane:
EtOAc 6:4). 1H NMR (500 MHz, CDCl3): δ 7.34 (m, 1H), 6.31 (m,
The DMTr-protected nucleoside (0.1643 g, 0.25 mmol) was evapo-
rated twice with dry toluene (2 ꢀ 1 mL) and further dried in vacuo. The
product was dissolved in DCM (2.5 mL) and cooled in an ice bath.
DIPEA (132 μL, 0.74 mmol) was added, followed by the addition of
2-cyanoethyl N,N-diisopropylchlorophosphoramidite (82 μL, 0.37 mmol).
The reaction mixture was stirred for 1 h at 0 °C and then quenched with
MeOH (0.2 mL). The reaction mixture was diluted with EtOAc
(40 mL), washed once with a saturated NaHCO3 aq solution and
three times with saturated NaClaq (3 ꢀ 40 mL), dried on Na2SO4,
filtered, and concentrated under vacuum. The product was further
purified by flash chromatography with EtOAc:isooctane:TEA 60:39:1,
resulting in an off-white foam (0.1631 g, 0.19 mmol) in 76% yield. Rf =
1
1H), 6.11 (m, 1H), 3.12 (t, J = 7.4 Hz, 2H), 3.03 (t, J = 7.4 Hz, 2H). 13
C
0.53, 0.57 (isooctane: EtOAc 8:2). The H NMR was complex and
NMR (CDCl3, 125 MHz): δ 168.5, 152.8, 142.1, 141.6, 140.6, 140.2,
138.9, 138.5, 136.9, 110.3, 105.9, 31.9, 23.3. ESI-MS: calcd for C13H7-
F5O3, 306.0; found, 329.2 [MþNa]þ. Anal. Calcd for C13H7F5O3: C,
51.00; H, 2.30. Found: C, 50.76; H, 2.44.
showed the presence of two diastereomers. 31P NMR (CDCl3, 121
MHz): δ 151.15, 149.45. ESI-MS: calcd for C46H54N5O10P, 867.4;
found, 303.3 [DMTr]þ, 867.1 [MþH]þ.
20-Amino-20-deoxyuridine, 11. Triphenylphosphine (0.434 g,
1.65 mmol) was added to a solution of nucleoside 8 (0.406 g, 1.51 mmol)
in MeOH:H2O 9:1 (16.5 mL). The reaction mixture was stirred for 3 h
at 50 °C and then concentrated by rotary evaporation. After recrystalli-
zation in toluene, the product was obtained as a yellowish powder
(0.3269 g, 1.34 mmol) in 89% yield. See ref 62 for analytical data.
20-Deoxy-20-[3-(2-furyl)propanamido)]uridine, 9. Nucleoside 8
(0.401 g, 1.49 mmol) was added to a mixture of activated ester 5
(0.455 g, 1.49 mmol) and triphenylphosphine (0.428 g, 1.63 mmol)
in pyridine (15 mL). After 4 h, the starting nucleoside was com-
pletely consumed, and the reaction mixture was further stirred
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Journal of the American Chemical Society
ARTICLE
20-Deoxy-20-[3-(2-furyl)-2-azapropanamido)]uridine, 13. Compound
11 (0.200 g, 0.82 mmol) was dissolved in dry DMF (8.2 mL) and cooled
in an ice bath. Furfurylisocyanate 12 (100 μL, 0.90 mmol) was added
dropwise, and the reaction was allowed to stir for 1 h. The reaction was
quenched with methanol and concentrated under vacuum. After flash
chromatography with DCM:MeOH 95:5, the product was obtained as an
off-white foam (0.290 g, 0.79 mmol) in 96% yield. Rf = 0.24 (DCM:MeOH
9:1). 1H NMR (MeOD, 300 MHz): δ 7.99 (d, J = 8.1 Hz, 1H), 7.37 (d, J =
1.5 Hz, 1H), 3.31 (m, 1H), 6.17 (d, J= 3.2 Hz, 1H), 5.98 (d, J=8.5Hz, 1H),
5.73 (d, J= 8.1 Hz, 1H), 4.46 (dd, J= 8.3 and 5.7 Hz, 1H), 4.22 (s, 2H), 4.20
(m, 1H), 4.02 (m, 1H), 3.74 (m, 2H). 13C NMR (MeOD, 75 MHz): δ
166.1, 160.0, 154.2, 152.8, 143.1, 142.8, 111.3, 107.4, 103.2, 88.6, 88.5,
72.3, 63.3, 57.4, 37.9. ESI-MS: calcd for C15H18N4O7, 366.1; found,
367.1 [MþH]þ, 389.4 [MþNa]þ. HRMS (þESI): calcd for
C15H19N4O7 (MþHþ), 367.1248; found, 367.1259; calcd for C15H18N4O7-
Na (MþNaþ), 389.1067; found, 389.1067; calcd for C15H18N4O7K
(MþKþ), 405.0807; found, 405.0804.
system equipped with a Phenomenex Clarity column (250 ꢀ 4.6 mm,
5 μm) at 50 °C, with 0.1 M TEAA (with 8% MeOH) and MeOH as
mobile phase (linear gradient: 0-30% MeOH in 15 min, 30-100%
MeOH in 3 min). The chromatograms were analyzed at 260 nm.
Enzymatic digestion reaction mixtures were analyzed on a Phenomenex
Luna C18 column (250 ꢀ 4.6 mm, 5 μm) using the same method. Mass
spectra of oligonucleotides were recorded on a quadrupole ion trap LC
mass spectrometer (Thermofinnigan, San Jose, CA) equipped with
electrospray ionization. Data were acquired in the negative ionization
mode from m/z = 100 to m/z = 1500. The mass spectra were
deconvoluted; i.e., the molecular weights of the compounds were
reconstructed from the spectra using the Agilent LC/MSD ChemSta-
tion software (version A.08014). Cross-linking yields based on HPLC
were calculated by comparing the peak area from the spectra before and
after addition of NBS, taking into account the extinction coefficients
calculated by the nearest-neighbor method. The extinction coefficient of
the cross-linked duplex is taken to be 0.9 times the sum of the extinction
coefficients of its composing sequences.
20-Deoxy-20-[3-(2-furyl)-2-azapropanamido)]-50-O-(4,40-dimethoxy-
trityl)uridine 30-(2-Cyanoethyl N,N-Diisopropylphosphoramidite),
14. Compound 12 (0.076 g, 0.207 mmol) was co-evaporated three
times with pyridine (3 ꢀ 1 mL) and then dissolved in pyridine (2 mL)
and cooled to 0 °C. DMTrCl (0.0990 g, 0.29 mmol) was added, and
the reaction was stirred overnight at room temperature. The reaction
was checked by TLC, and a fresh portion of DMTrCl (0.0350 g, 0.10
mmol) was added at 0 °C. The reaction was further stirred at room
temperature for an additional 4 h and then cooled in an ice bath
and quenched with MeOH (100 μL). The reaction mixture was diluted
with EtOAc (10 mL) and then washed with water (10 mL), saturated
NaHCO3 aq (10 mL), and saturated NaCl (10 mL). The combined
aqueous layers were extracted with 30 mL of EtOAc, and the
combined organic layers were dried on Na2SO4. Flash chromatogra-
phy with EtOAc:isooctane:TEA 70:29:1 resulted in an off-white foam
(0.110 g, 0.164 mmol) in 79% yield. Rf = 0.43 (DCM:MeOH 9:1). 1H
NMR (MeOD, 300 MHz): δ 7.77 (d, J = 8.1 Hz, 1H), 7.08-7.34 (m,
9H), 7.27 (m, 1H), 6.78 (d, J = 8.7 Hz, 4H), 6.20 (m, 1H), 6.09 (d, J =
3.2 Hz, 1H), 5.91 (d, J = 8.3 Hz, 1H), 5.25 (d, J = 8.1 Hz, 1H), 4.58
(dd, J = 5.7 and 8.3 Hz, 1H), 4.16 (m, 3H), 3.98 (m, 1H), 3.64 (s, 6H),
3.24 (m, 2H). 13C NMR (MeOD, 125 MHz): δ 160.0, 160.3, 160.1,
154.1, 152.7, 145.8, 143.0, 142.6, 136.9, 136.7, 131.4, 129.5, 129.0,
128.1, 114.3, 111.3, 107.4, 103.0, 88.4, 88.3, 87.2, 72.4, 65.2, 57.6,
55.7, 38.0. ESI-MS: calcd for C36H36N4O9, 668.2; found, 303.3
[DMTr]þ, 685.6 [MþNH4]þ. HRMS (þESI): calcd for C36H38N4-
O9Na (MþNaþ), 691.2374; found, 691.2375; calcd for C36H38N4-
O9K (MþKþ), 707.2114; found, 707.2125.
Synthesis of Modified DNA. All oligonucleotides were synthesized
DMT-on using an ABI 394 DNA synthesizer at 1 μmol scale. A standard
synthesis protocol was used except for coupling of the modified residues.
The synthesis column was removed from the DNA synthesizer for the
introduction of the modified residues. For these manual couplings, a 0.06
M solution of the phosphoramidite in acetonitrile and a 0.1 M solution of
DCI in acetonitrile were dried on molecular sieves for about 20 min. Small
portions of the phosphoramidite solution (0.4 mL) and the DCI solution
(0.5 mL) were then alternately pushed over the reaction column. The
column was flushed with acetonitrile (1 mL), and a mixture of Cap A (0.5
mL) and Cap B (0.5 mL) was pushed over the column, after which it was
again flushed with acetonitrile (1 mL). The column was reinstalled on the
DNA synthesizer, and automated synthesis was resumed.
Oligonucleotides were cleaved from solid support and deprotected by
incubation at 55 °C overnight in concentrated aqueous ammonia. The
synthesized DMTr oligonucleotides were deprotected and purified on
SEP-PAK C18 cartridges (obtained from Waters).
NaCl was used from a 1 M stock solution. Phosphate buffer was
prepared in a 0.1 M stock solution (490 mg of NaH2PO4 H2O and
3
548 mg of Na3PO4 12H2O in 50 mL of H2O).
3
Thermal Denaturation Experiments. All UV experiments were
recorded on a Varian Cary 300 Bio instrument equipped with a six-cell
thermostatted cell holder. Melting curves were monitored at 260 nm
with a heating rate of 0.3 °C/min. The buffer contained 100 mM NaCl
and 10 mM phosphate buffer (pH 7). Oligonucleotide concentration
was 2 μM for each strand. Melting temperatures were calculated from
the first derivative of the heating curves using the Cary 300 Bio software.
Cross-Linking Reactions. The modified strands were mixed with
their complements in equimolar amounts at 0.02 mM concentration in
10 mM phosphate buffer (pH 7) and 100 mM NaCl before annealing
from 95 °C to room temperature. Temperatures during the whole
reaction were kept constant in an Eppendorf thermomixer comfort at
25 °C. A stock solution of NBS (1 equiv/2 μL) was freshly prepared, and
to start the reaction, 1 equiv of NBS was added. This was repeated every
15 min until complete disappearance of the modified oligonucleotide.
The reactions were monitored by RP-HPLC.
The DMTr-protected nucleoside (0.079 g, 0.118 mmol) was co-
evaporated twice with toluene (2 ꢀ 1 mL) and once with DCM (1 mL).
The product was then dissolved in DCM (1.2 mL) and cooled to 0 °C.
DIPEA (65 μL, 0.364 mmol) was added to the reaction, followed by
2-cyanoethyl N,N-diisopropylchlorophosphoramidite (40 μL, 0.179
mmol). The reaction mixture was stirred for 1.5 h at 0 °C and then
quenched with MeOH (100 μL) and diluted with EtOAc (20 mL).
The organic phase was washed once with saturated NaHCO3 aq
(20 mL) and three times with saturated NaClaq (3 ꢀ 20 mL), dried
on Na2SO4, and concentrated by rotary evaporation. The residue was
purified by flash chromatography using EtOAc:isooctane:TEA
55:44:1, and the pure product was obtained as an off-white foam
(0.035 g, 0.060 mmol) in 50% yield. Rf = 0.45 (isooctane:EtOAc 8:2).
The 1H NMR was complex and showed the presence of two
diastereomers. 31P NMR (CDCl3, 121 MHz): δ 151.44, 148.21.
ESI-MS: calcd for C45H53N6O10P, 868.4; found, 303.3 [DMTr]þ,
891.2 [MþNa]þ.
Aromatization of the 20-Amido-furan-Modified ODN. NBS and
salts were removed from the cross-linking reaction mixture using a
1 kDa cutoff filter (Pall Life Sciences). The reaction mixture was diluted
with 1 mL of H2O and centrifuged for 90 min at 7500 rpm. The residue
was twice again redissolved in 1 mL of H2O, and the same procedure was
repeated. The residue was then taken in H2O and incubated at 50 °C
(1-3 days) until complete conversion to the aromatized cross-linked
duplex. The reaction was followed by RP-HPLC.
Materials and Methods for Oligonucleotides. Reagents for
DNA synthesis were obtained from Glen Research. All RP-HPLC
experiments with oligonucleotides were recorded on an Agilent 1100
Gel Electrophoresis Experiments. A 20% polyacrylamide gel
(acrylamide:bisacrylamide 19:1) with 1x TBE buffer and 7 M urea was
805
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Journal of the American Chemical Society
ARTICLE
used for all analyses. The gel was prepared by dissolving 21.2 g of urea in 5 mL
of 10x TBE buffer and 5 mL of 40% acrylamide. The solution was
adjusted to 50 mL and slightly heated until urea was completely
dissolved. After the solution was cooled by refrigeration, ammonium
peroxodisulfate (165 μL, 100 mg/mL) was added, followed by N,N,N0,
N0-tetramethylethylenediamine. The solution was poured in between
two glass plates (20 cm ꢀ 22 cm) and allowed to polymerize for 30 min,
with 1x TBE used as the running buffer. The sample was mixed with
formamide loading buffer, and the gels were run overnight at a voltage
of 120 V. The temperature of the gel was stabilized with a Julabo F12
circulator at 25 °C. The power supply used for gel electrophoresis was a
Consort EV202. Gels were stained with GelRed (VWR), and pictures
were taken with an Autochemi imaging system (UVP).
Enzymatic Digestion. Crude SVPD was dissolved in H2O to a
concentration of 0.0003 U/μL and centrifuged to remove insoluble
contaminants. The supernatant was then used in the enzymatic degrada-
tion reactions.
The purified cross-linked aromatized duplex ODN3:ODN10b XLar
(0.4 nmol) was taken in 50 mM Tris-HCl buffer, pH 8, containig 10 mM
MgCl2 (100 μL). SVPD (13.2 μL, 0.0003 U/μL) was added to the
reaction mixture, and it was shaken overnight at 37 °C. The reaction was
checked by RP-HPLC, and then 0.6 U of alkaline phosphatase (from
bovine intestinal mucosa, Sigma Aldrich) was added and the reaction
mixture was further shaken at 37 °C for 1 h. The reaction was again
checked by RP-HPLC, after which the identity of the cross-linked
dinucleoside could be confirmed by coinjection with dinucleoside 24.
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Corresponding Author
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’ ACKNOWLEDGMENT
M.O.d.B. is indebted to the Agency for Innovation by Science
and Technology in Flanders (IWT) and the Special Research
Fund of Ghent University (BOF-GOA2007, BOF-BAS 01B04405).
We further thank the FWO Vlaanderen for financial support
(FWO-KAN 1.5.137.09N, FWO-KAN 1.5.186-03).
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