Cross-linked DNA: Propargylated ribonucleosides as "click" ligation sites for bifunctional azides

Article abstract of DOI:10.1021/jo300421p

2′-O or 3′-O-propargylated adenosines and ribothymidines were used as click targets for cross-linking of oligonucleotides with aliphatic and aromatic azides. The cross-link generates a sugar modification at the 2′-O-ligation site. Inexpensive ribonucleosi

Full text of DOI:10.1021/jo300421p

Note
pubs.acs.org/joc
Cross-Linked DNA: Propargylated Ribonucleosides as “Click” Ligation
Sites for Bifunctional Azides
Suresh S. Pujari and Frank Seela*
Laboratory of Bioorganic Chemistry and Chemical Biology, Center for Nanotechnology, Heisenbergstraße 11, 48149 Munster,
̈
Germany
Laboratorium fur Organische und Bioorganische Chemie, Institut fur Chemie, Universitat Osnabruck, Barbarastraße 7, 49069
̈
̈
̈
̈
Osnabruck, Germany
̈
S
* Supporting Information
ABSTRACT: 2′-O or 3′-O-propargylated adenosines and ribothymidines were used as
click targets for cross-linking of oligonucleotides with aliphatic and aromatic azides. The
cross-link generates a sugar modification at the 2′-O-ligation site. Inexpensive
ribonucleosides were used as starting materials. Cross-linking of oligonucleotides was
performed at internal or terminal positions. Hybridization of homodimers with two
complementary single strands resulted in stable ligated DNA duplexes.
ormation of cross-links in DNA can arrest cell replication,
thereby causing the death of the cell, a property that is
The 2′- or 3′-O-propargylated adenine and ribothymidine
nucleosides were synthesized.10 The bis-azides 5 and 6, one
being aliphatic and the other being aromatic, were prepared as
cross-linkers.11 As model compounds for the “bis-click”
reaction at the sugar moiety, “simple” nucleosides were used.
Consequently, the reaction was performed with the 2′-O-
propargylated derivatives, namely, nucleoside 1 was reacted
with bis-azide 6 and nucleoside 3 with bis-azide 5 (Scheme 1).
The obtained cross-linked nucleosides 7 (48%) and 8 (45%)
F
extensively exploited in cancer chemotherapy and genetic
engineering.1 For that purpose, a wide spectrum of cross-
linking agents has been reported in literature, among them are
cis-platin, nitrogen mustards, and natural compounds such as
psoralenes, nitrous acid, etc.1,2 Out of various techniques for
cross-linking,3 the Cu(I)-catalyzed azide−alkyne cycloaddition
“click chemistry” (CuAAC) can offer exciting prospects because
of its simplicity and bio-orthogonal nature.4
1
were characterized. For H NMR, 13C NMR, DEPT-135, and
1H−13C gated-decoupled NMR spectra, as well as mass spectra,
see Experimental Section and Supporting Information.
The bis-headed nucleosides 7 and 8 show relationship to
other bis-headed nucleosides synthesized in our laboratory.5,6,8f
They are good models for the study of intramolecular base
stacking, which comes by virtue of their flexibility. Moreover, as
these molecules contain two nucleoside functionalities, they can
act as bis-substrates or bis-inhibitors of nucleoside converting
enzymes, which find applications in drug discovery and
medicinal chemistry.12 To convert the bis-headed adenosine
derivative 8 into inosine derivative 9, adenosine deaminase
(ADA) was used (Scheme 1).13 The reaction was followed UV
spectroscopically (Figure S1, Supporting Information). The
inosine derivative 9 accessible in preparative scale was obtained
in 75% yield. We anticipate that related bis-headed adenosines
can be converted to the corresponding inosine derivatives as
well.
To widen the scope of this application to chemistry, chemical
biology, and nanotechnology, we recently reported on the “bis-
click” technology to cross-link DNA, which is site- and
template-independent.5,6 In all of these cases base modifications
were employed. Modification of the sugar moiety in nucleic
acids appears to be a highly enterprise work since the hydroxyl
groups of the sugar can be modified seemingly to any kind of
functionalities. We envisioned the ploy to modify the sugar as a
substrate unit to “bis-click” chemistry. Nucleobases,5−7 sugar
moieties,8 and phosphate backbones9 have been modified to
introduce cross-links by various methods but to the best of our
knowledge not by “click” cross-linking with bifunctional azides
on the sugar moiety. This manuscript reports on the viability of
the “bis-click” chemistry on the sugar moiety (Figure 1) and the
effect of cross-linking on duplex stability. For that, 2′/3′-O-
propargylated nucleosides 1−4 were prepared, employed in
solid-phase oligonucleotide synthesis, and used for cross-
linking. DNA ligation was performed using the bis-azides 5
and 6 (Figure 2).
Received: March 5, 2012
Published: April 5, 2012
© 2012 American Chemical Society
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Note
Figure 1. Schematic presentation of “bis-click” reaction on sugar modified DNA.
phosphoramidites of nucleosides 1−3 were synthesized.10
Then, a series of oligonucleotides containing the propargylated
nucleoside residues 1, 2, and 3 at various positions were
prepared on solid phase using the standard protocol of
phosphoramidite chemistry. The synthesized oligonucleotides
were characterized by MALDI-TOF mass spectra (experimental
part) and HPLC (Supporting Information).
With 2′- and 3′-O-propargylated oligonucleotides in hand, the
“bis-click” reaction was performed. For this, 5.0 A260 units of
ODN 10 dissolved in water was treated with Cu-TBTA, TCEP,
and NaHCO3. After that the oligonucleotides were clicked
together by adding the corresponding bis-azide 5 or 6.
Accordingly, a series of interstrand cross-linked oligonucleo-
tides (ICLs) were prepared (Table 1 and Supporting
Information, Figure S2). The “bis-click” reaction on ODNs is
outlined in Schemes 2 and 3. The composition of cross-linked
oligonucleotides was proven by MALDI-TOF spectra (Table 1)
and the purity by denaturing gel electrophoresis (PAGE) (for
details see Experimental Section).
Gel electrophoresis demonstrated that the cross-linked
oligonucleotides as a result of their increased number of
negative charges migrate significantly slower than the starting
materials (24-mer cross-linked oligonucleotides 21, 23 and 26,
27 compared to the 12-mer propargylated oligonucleotides 29
and 32). Figure 3 shows the PAGE results of alkynylated and
cross-linked oligonucleotides.
Figure 2. Alkynylated nucleosides and bis-azides used for “bis-click”
cross-linking.
To investigate the performance of the reaction on
oligonucleotides, the “bis-click” reaction was performed on
single-stranded oligonucleotides (ODNs). For this, the
Scheme 1. Cu(I)-Catalyzed “Bis-Click” Reaction to Cross-
Link 1 and 3 with Bis-azide 5 and 6 and Deamination of 8
Cross-linked oligonucleotides find applications in the
construction of nanostructures by which DNA machines such
as DNA tweezers, walker, and stepper are formed.14 Earlier
work from Endo et al. demonstrated the potential of sugar-
phosphate backbone cross-linked DNA.15 In their studies, the
cross-link uses the phosphodiester moiety as target site that was
converted in a phosphotriester by the cross-linking reagent.
However, phosphotriester cross-links make the backbone of
DNA labile against hydrolysis, remove negative charges, and
create chirality. Our cross-linked DNA is at least as stable as
canonical DNA, and introduction of the cross-link at the 2′-O-
position of the sugar moiety usually increases resistance of the
molecule against hydrolysis by nucleases.
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Note
a b
,
Table 1. Tm Values and Mass Spectrometric Data of Cross-Linked Oligonucleotides
a
T-T corresponds to a, A-A corresponds to 8, T*-T* corresponds to 7, A*-A* corresponds to b, T#-T# corresponds to c, and T§-T§ corresponds to
b
d. 16 corresponds to 5′-d(TAGGTCAATACT), and 17 corresponds to 3′-d(ATCCAGTTATGA). The structures of all cross-linked
oligonucleotides are shown in the Supporting Information, Figure S2. Measured at 260 nm in a 1 M NaCl solution containing 100 mM MgCl2 and
c
60 mM Na-cacodylate (pH 7.0) with 2.5 μM single-strand concentration.
Scheme 2. CuAAC “Bis-Click” Cross-Linking Reaction of 2′-O-Propargylated ODN 10 with Bis-azides 5 and 6
Scheme 3. CuAAC “Bis-Click” Cross-Linking Reaction of 3′-O-Propargylated ODN 13 with Bis-azides 5 and 6
Table 1 shows that the cross-linked homodimers hybridize
well with the complementary strands 5′-d(TAG GTC AAT
ACT) (ODN 16) and 3′-d(ATC CAG TTA TGA) (ODN 17)
to form double helices cross-linked by sugar linkages. The
introduction at the 2′-O-position keeps the duplex DNA intact
and has only a minor effect on duplex stability. This
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column; gradient system I). The purified “trityl-on” oligonucleotides
were treated with 2.5% Cl2CHCOOH/CH2Cl2 for 5 min at 0 °C to
remove the dimethoxytrityl residues. The detritylated oligomers were
purified by reversed-phase HPLC (gradient II). The oligomers were
desalted on a short column using distilled water for elution of salt,
while the oligonucleotides were eluted with H2O/MeOH (2:3). Then,
the solvent was evaporated using a SpeedVac evaporator to yield
colorless solids that were frozen at −24 °C. The molecular masses of
the oligonucleotides were determined by MALDI-TOF mass
spectrometry in the linear negative mode (experimental section).
Reversed-phase HPLC was carried out on a 250 mm × 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 50−10% A in B, flow rate 0.8 mL min−1;
(II) 0−25 min 0−20% A in B, flow rate 0.8 mL min−1; (III) 0−15 min
0−20% A in B, 15−18 min 20−40% A in B, flow rate 0.8 mL min−1.
Denaturing polyacrylamide gel electrophoresis (PAGE) was
performed on oligonucleotides. PAGE analysis was carried out on a
20% polyacrylamide gel (acrylamide/bisacrylamide 19:1 with 7 M
urea; 10 × 10 cm in 0.1 M tris-borate-EDTA (TBE) buffer containing
20 mM MgCl2, pH 8.4). A 30 min prerun was performed in TBE
buffer at rt. From a stock solution of 40 A260 units in 200 μL, a portion
of 2 μL was added to 5 μL of distilled water. Then, 10 μL of gel
loading buffer (TBE) containing formamide (1:1) was added, and the
oligonucleotide solution was loaded onto the gel. Electrophoresis was
performed at rt for 3.5 h at a constant field strength of 12 V/cm. The
gel was stained with 0.02% methylene blue for 20 min and was then
incubated in water for 1 h to remove excess dye; the electrophero-
grams were then scanned.
Melting curves were measured with a UV−vis spectrophotometer
equipped with a thermoelectrical controller with a heating rate of 1
°C/min. ESI-TOF mass spectra of the nucleosides were measured with
a Micro-TOF spectrometer. Molecular masses of oligonucleotides
were determined by MALDI-TOF mass spectrometry in the linear
negative mode with 3-hydroxypicolinic acid (3-HPA) as a matrix.
The formed duplexes with their respective Tm values and the
MALDI-TOF analysis of the corresponding modified single strands are
as follows: 5′-d(TAG GTC AA1 ACT) (28), MW calcd 3696, MW
found 3697, 47.5 °C for duplex 28·17; 5′-d(1AG GTC AAT ACT)
(29), MW calcd 3696, MW found 3699, 50.0 °C for duplex 29·17; 3′-
d(A1C CAG TTA TGA) (30), MW calcd 3696, MW found 3697,
50.0 °C for duplex 16·30; 5′-d(T3G GTC AAT ACT) (31), MW calcd
3696, MW found 3697, 48.5 °C for duplex 17·31; 5′-d(TAG GTC
3AT ACT) (10), MW calcd 3696, MW found 3697, 48.0 °C for
duplex 10·17; 3′-d(ATC C3G TTA TGA) (32), MW calcd 3696, MW
found 3700, 46.5 °C for duplex 16·32; 5′-d(TAG GTC AA2 ACT)
(13), MW calcd 3696, MW found 3697, 45.0 °C for duplex 13·17. The
Tm values were 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
single-strand concentration.
Figure 3. Denaturing polyacrylamide gel electrophoresis of alkynylated
and cross-linked oligonucleotides.
phenomenon is similar to helices ligated by their side chain at
the nucleobases.16 At the center of the duplex, cross-linking is
slightly destabilizing, while for cross-linking at the termini of
the helices stabilization was observed. So, cross-links introduced
into the sugar moiety do not perturb the helix structure. When
the sequential propargylation and cross-linking was performed
at the 3′-O-position, the resulting 2′-5′-oligonucleotides
(17·14·17, 17·15·17) are much more destabilized compared
to corresponding 3′-5′ connected counterparts (Table 1), which
results from the unusual phosphodiester linkage.
In conclusion, our “bis-click” protocol is efficient and
proceeds almost quantitatively with no side product formation
when identical oligonucleotide strands are used. In analogy to
nucleobase cross-linking,5,6 the method has the potential to
ligate nonidentical strands in a stepwise way. Our methodology
is applicable to nucleosides and oligonucleotides bearing
propargyl residues at the sugar moiety. The cross-link can be
introduced at any position of an oligonucleotide chain. A
diversity of bis-azides can be applied to cross-link oligonucleo-
tides. This protocol has the potential to construct oligonucleo-
tide−peptide, −carbohydrate, or −lipid conjugates by a
stepwise procedure. The method is applicable for the
construction of larger assemblies, which are accessible by a
combination of covalent cross-linking and noncovalent
assembly by hydrogen bonding.
EXPERIMENTAL SECTION
■
General Methods and Materials. All chemicals and solvents
were of laboratory grade as obtained from commercial suppliers and
were used without further purification. Thin-layer chromatography
(TLC) was performed on TLC aluminum sheets covered with silica
gel 60 F254 (0.2 mm). Flash column chromatography (FC): silica gel
60 (40−60 μM) at 0.4 bar. Adenosine deaminase (EC 3.5.4.4), Type V
from bovine spleen. Solution in 50% glycerol, 50 mM potassium
phosphate, pH 6.0, 160 units per mL. The adenosine deaminase was
diluted with 0.06 M Sørensen buffer (pH 7.0). A 40-fold diluted
solution (0.004 units per μL) was used. The substrate was dissolved in
the same buffer after the deaminase (1 μL, 0.004 units) was added.
The UV absorbance was detected and recorded at certain wavelengths
where the UV spectra have the maximal changes. UV spectra: λmax in
nm, ε in dm3 mol−1 cm−1. 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; δ values in ppm relative to Me4Si as internal standard. For
NMR spectra recorded in DMSO, the chemical shift of the solvent
peak was set to 2.50 ppm for 1H NMR and 39.50 ppm for 13C NMR.
The syntheses of oligonucleotides were performed on a DNA
synthesizer at a 1 μmol scale (trityl-on mode) using the
phosphoramidites S4−S6 and the standard phosphoramidite building
blocks following the synthesis protocol for 3′-O-(2-cyanoethyl)-
phosphoramidites. After cleavage from the solid support, the
oligonucleotides were deprotected in 25% aqueous ammonia solution
for 12−16 h at 60 °C. The purification of the “trityl-on”
oligonucleotides was carried out on reversed-phase HPLC (RP-18
Bis-ribothymidine Click Adduct 7. To a solution of compound 1
(70 mg, 0.23 mmol) and 6 (22 mg, 0.12 mmol) in THF/H2O/t-
BuOH (3:1:1, 3 mL) was added a freshly prepared 1 M solution of
sodium ascorbate (232 μL, 0.23 mmol) in water, followed by the
addition of copper(II) sulfate pentahydrate 7.5% in water (201 μL,
0.06 mmol), and the reaction mixture was stirred at room temperature
for 5 h. After completion of the reaction (monitored by TLC), the
reaction mixture was evaporated, and the residue was applied to flash
chromatography (FC) (silica gel, CH2Cl2/MeOH, 85:15). From the
main zone compound 7 (45 mg, 48%) was isolated as a colorless solid.
TLC (silica gel, CH2Cl2/MeOH, 80:20): Rf 0.3. λmax (MeOH)/nm
260 (ε/dm3 mol−1 cm−1 19 000), 266 (20 200). 1H NMR (DMSO-d6,
300 MHz): δ 1.73 (s, 6H, 2 × CH3), 3.43−3.66 (m, 4H, 2 × C5′-H),
3.84 (d, J = 3.6 Hz, 2H, 2 × C4′-H), 4.04 (t, J = 5.1 Hz, 2H, 2 × C3′-
H), 4.14−4.15 (m, 2H, 2 × C2′-H), 4.58−4.69 (m, 4H, 2 × CH2),
5.18 (br s, 4H, 2 × C3′-OH, 2 × C5′-OH), 5.54 (s, 4H, 2 × CH2), 5.86
(d, J = 5.1 Hz, 2H, 2 × C1′-H), 7.28 (s, 4H, phenyl-H), 7.72 (s, 2H, 2
× triazole-H), 8.10 (s, 2H, 2 × C6-H), 11.32 (s, 2H, 2 × NH). 13C
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NMR (DMSO-d6, 75.4 MHz): δ 163.7 (C4), 150.6 (C2), 144.1
(triazole), 128.4 (C6), 124.2 (triazole), 109.3 (C5), 85.9 (C1′), 85.0
(C4′), 80.4 (C3′), 68.3 (C2′), 62.8 (C5′), 60.6 (CH2), 52.4 (CH2).
1H−13C Coupling constants in Hz (DMSO-d6, 75.4 MHz): 161.1 [1J
(C6, H-C6)], 8.0 [3J (C2, H-C6)], 5.2 [2J (C5, H-C6)], 9.6 [3J (C4,
H-C6)], 196.2 [1J (triazole, H-C5)], 9.6 [2J (triazole C4, H-C5)],
168.8 [1J (C1′, H-C1′)], 143.7 [1J (C2′, H-C2′)], 148.7 [1J (C3′, H-
C3′)], 146.6 [1J (C4′, H-C4′)], 140.8 [1J (C5′, H-C5′)]. ESI-TOF calcd
for C34H40N10O12Na (M + Na+) 803.2725; m/z found 803.2719.
Bis-adenosine Click Adduct 8. To a solution of compound 3
(150 mg, 0.49 mmol) and 5 (38 mg, 0.24 mmol) in THF/H2O/t-
BuOH (3:1:1, 3 mL) was added a freshly prepared 1 M solution of
sodium ascorbate (484 μL, 0.5 mmol) in water, followed by the
addition of copper(II) sulfate pentahydrate 7.5% in water (419 μL,
0.12 mmol), and the reaction mixture was stirred at room temperature
for 5 h. After completion of the reaction (monitored by TLC), the
reaction mixture was evaporated, and the residue was applied to flash
chromatography (FC) (silica gel, CH2Cl2/MeOH, 80:20). From the
main zone compound 8 (85 mg, 45%) was isolated as a colorless solid.
TLC (silica gel, CH2Cl2/MeOH, 60:40): Rf 0.3. λmax (MeOH)/nm
260 (ε/dm3 mol−1 cm−1 19 100). 1H NMR (DMSO-d6, 300 MHz): δ
3.56 (br s, 2H, 2 × C5′-H), 3.66−3.71 (m, 6H, 2 × CH2, 2 × C5′-H),
4.00−4.01 (m, 2H, 2 × C4′-H), 4.33−4.38 (m, 6H, 2 × CH2, 2 × C3′-
H), 4.50−4.54 (m, 2H, 2 × C2′-H), 4.60−4.69 (m, 4H, 2 × CH2),
5.37 (br s, 2H, 2 × C5′-OH), 5.46 (br s, 2H, 2 × C3′-OH), 6.00 (d, J =
6.0 Hz, 2H, 2 × C1′-H), 7.34 (br s, 4H, 2 × NH2), 7.77 (s, 2H, 2 ×
triazole), 8.12 (s, 2H, 2 × C8-H), 8.29 (s, 2H, 2 × C2-H). 13C NMR
(DMSO-d6, 75.4 MHz): δ 156.1 (C6), 152.5 (C2), 148.9 (C4), 143.4
(triazole), 139.7 (C8), 124.2 (triazole), 119.2 (C5), 86.4 (C4′), 86.0
(C1′), 80.3 (C3′), 69.0 (C2′), 68.4 (CH2), 62.8 (C5′), 61.5 (CH2),
49.1 (CH2). 1H−13C Coupling constants in Hz (DMSO-d6, 75.4
MHz): 11.3 [3J (C6, H-C2)], 199.2 [1J (C2, H-C2)], 216.5 [1J (C8, H-
C8)], 11.4 [3J (C5, H-C8)], 11.5 [3J (C4, H-C2)], 196.4 [1J (triazole,
H-C5)], 9.4 [2J (triazole C4, H-C5)], 165.2 [1J (C1′, H-C1′)], 144.3
[1J (C2′, H-C2′)], 149.5 [1J (C3′, H-C3′)], 148.5 [1J (C4′, H-C4′)],
141.9 [1J (C5′, H-C5′)]. ESI-TOF calcd for C30H38N16O9Na (M +
Na+) 789.2906; m/z found 789.2900.
General Procedure for Cross-Linking Oligonucleotides. To
the solution of oligonucleotide 10 (5.0 A260 units, 50 μmol) in 20 μL
of water were added a mixture of a CuSO4−TBTA ligand complex (50
μL of 20 mmol solution of CuSO4 in H2O/t-BuOH/DMSO (1:1:3)
and 50 μL of 20 mmol solution of TBTA in t-BuOH/DMSO (1:3),
tris(carboxyethyl)phosphine (TCEP; 50 μL of 20 mmol solution in
H2O), sodium bicarbonate (NaHCO3, 50 μL of 200 mmol solution in
H2O), and the bis-azide 5 or 6 (2.5 μL of 20 mmol respective azide
stock solution in THF/H2O (1:1) for azide 5 and in THF for azide 6)
and 30 μL of DMSO, and then the reaction mixture was stirred at
room temperature for 6 h. The reaction mixture was concentrated in a
Speed Vac, dissolved in 0.3 mL of bidistilled water, and centrifuged for
20 min at 14 000 rpm. The supernatant was collected and further
purified by reversed-phase HPLC, using the following gradient: 0−15
min 0−20% B in A, 15−18 min 20−40% B in A, 18−25 min 40−0% B
in A, flow rate 0.8 mL min−1; A: 0.1 M (Et3NH)OAc (pH 7.0)/MeCN
95:5; B: MeCN) to give the corresponding cross-linked oligonucleo-
tide 11 or 12 (2.5 A260 units, 50%). The other cross-linked
oligonucleotides were prepared accordingly in 50−60% yields.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis of phosphoramidites, HPLC purification profiles of
oligonucleotides, structures of oligonucleotides, H and 13C
1
NMR, DEPT-135, and 1H−13C gated decoupled spectra of the
nucleoside derivatives and click conjugates. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +49(0) 251 53406 500. Fax: +49(0) 251 53406 857.
E-mail: Frank.Seela@uni-osnabrueck.de.
■
Notes
The authors declare no competing financial interest.
Bis-inosine Click Adduct 9. Compound 8 (20 mg, 0.02 mmol)
was dissolved in Sørensen buffer (pH 7.0, 2 mL). To this solution was
added the enzyme adenosine deaminase (EC 3.5.4.4), type V from
bovine spleen, solution in 50% glycerol, 50 mM potassium phosphate,
pH 6.0, 160 units per mL, 40-fold diluted (0.004 units per μL) with
the same buffer (3 μL). The reaction was monitored by UV (for time
scan see Figure S1, Supporting Information). After completion the
reaction mixture was evaporated to dryness, and the residue was
dissolved in a mixture of MeOH and CH2Cl2 (v/v; 3:7). The
supernatant liquid obtained after centrifuging was applied to
preparative TLC on silica coated TLC plates. The solution was
divided into two parts, and each part was adsorbed in two different
TLC plates (20 cm × 10 cm) and run for separation. After separation
on TLC, the band with the compound was scratched, and the silica
was washed with a mixture of CH2Cl2/MeOH, (70:30) and dried
under reduced pressure to obtain 9 as colorless solid (15 mg, 75%).
ACKNOWLEDGMENTS
■
We thank Dr. S. Budow and Dr. P. Leonard for helpful
discussions and support while preparing the manuscript. We
also thank Mr. H. Mei for measuring the NMR spectra and Mr.
Nhat Quang Tran for the oligonucleotide syntheses. We would
like to thank Dr. H. Luftmann, Organisch-chemisches Institut,
Universitat Munster, Germany, for the measurement of the
̈
̈
MALDI spectra. Financial support by ChemBiotech, Munster,
̈
Germany, is highly appreciated.
REFERENCES
■
(1) (a) Rajski, S. R.; Williams, R. M. Chem. Rev. 1998, 98, 2723−
2795 and references therein. (b) Noll, D. M.; Mason, T. M.; Miller, P.
S. Chem. Rev. 2006, 106, 277−301 and references therein.
(2) (a) Cimino, G. D.; Gamper, H. B.; Isaacs, S. T.; Hearst, J. E.
Annu. Rev. Biochem. 1985, 54, 1151−1193. (b) Li, H.; Broughton-
Head, V. J.; Peng, G.; Powers, V. E. C.; Ovens, M. J.; Fox, K. R.;
Brown, T. Bioconjugate Chem. 2006, 17, 1561−1567. (c) Rink, S. M.;
Lipman, R.; Alley, S. C.; Hopkins, P. B.; Tomasz, M. Chem. Res.
Toxicol. 1996, 9, 382−389.
1
TLC (silica gel, CH2Cl2/MeOH, 70:30): Rf 0.25. H NMR (DMSO-
d6, 300 MHz): δ 3.48−3.57 (m, 2H, 2 × C5′-H), 3.58−3.74 (m, 6H, 2
× CH2, 2 × C5′-H), 3.96−3.99 (m, 2H, 2 × C4′-H), 4.35 (t, J = 3.3
Hz, 2H, 2 × C3′-H), 4.39−4.42 (m, 4H, J = 4.8 Hz, 2 × CH2), 4.50−
4.57 (m, 4H, 2 × CH2), 4.66−4.69 (m, 2H, CH2), 5.35 (br s, 2H, 2 ×
C5′-OH), 5.98 (d, J = 6.0 Hz, 2H, 2 × C1′-H), 7.81 (s, 2H, 2 ×
triazole), 8.05 (s, 2H, 2 × C8-H), 8.26 (s, 2H, 2 × C2-H). 13C NMR
(DMSO-d6, 75.4 MHz): δ 156.7 (C6), 148.0 (C4), 146.1 (C2), 143.4
(triazole), 138.5 (C8), 124.4 (C5), 124.3 (triazole), 86.2 (C4′), 85.6
(C1′), 80.8 (C3′), 68.8 (C2′), 68.4 (CH2), 62.8 (C5′), 61.1 (CH2),
49.0 (CH2). 1H−13C Coupling constants in Hz (DMSO-d6, 75.4
MHz): 6.9 [3J (C6, H-C2)], 205.8 [1J (C2, H-C2)], 215.2 [1J (C8, H-
C8)], 11.3 [3J (C5, H-C8)], 14.4 [3J (C4, H-C2)], 196.5 [1J (triazole,
H-C5)], 9.9 [2J (triazole C4, H-C5)], 167.1 [1J (C1′, H-C1′)], 147.9
[1J (C2′, H-C2′)], 147.6 [1J (C3′, H-C3′)], 148.4 [1J (C4′, H-C4′)],
143.0 [1J (C5′, H-C5′)]. ESI-TOF calcd for C30H38N16O9Na (M +
Na+) 791.2586; m/z found 791.2580.
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