Synthesis, structure and imaging of oligodeoxyribonucleotides with tellurium-nucleobase derivatization

Article abstract of DOI:10.1093/nar/gkq1288

We report here the first synthesis of 5-phenyl-telluride-thymidine derivatives and the Te-phosphoramidite. We also report here the synthesis, structure and STM current-imaging studies of DNA oligonucleotides containing the nucleobases (thymine) derivatize

Full text of DOI:10.1093/nar/gkq1288

3962–3971 Nucleic Acids Research, 2011, Vol. 39, No. 9
doi:10.1093/nar/gkq1288
Published online 17 January 2011
Synthesis, structure and imaging of
oligodeoxyribonucleotides with
tellurium-nucleobase derivatization
Jia Sheng1, Abdalla E. A. Hassan1,2, Wen Zhang1, Jianfeng Zhou3, Bingqian Xu3,
Alexei S. Soares4 and Zhen Huang1,*
2
1Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA, Applied Nucleic Acids Research
3
Center, Zagazig University, Zagazig, Egypt, Faculty of Engineering and NanoSEC, University of Georgia,
4
Athens, GA 30602 and Department of Biology, Brookhaven National Laboratory, Upton, NY 11973, USA
Received October 20, 2010; Revised November 26, 2010; Accepted November 30, 2010
damaging and repairing (13–19) and DNA current/con-
ductivity imaging, including metallization and conjugating
with conductive nanoparticles or polymers through
scanning tunneling microscopy (STM) to image DNA
(20–24).
ABSTRACT
We report here the first synthesis of 5-phenyl–
telluride–thymidine derivatives and the Te-
phosphoramidite. We also report here the synthesis,
structure and STM current-imaging studies of DNA
oligonucleotides containing the nucleobases
(thymine) derivatized with 5-phenyl-telluride func-
tionality (5-Te). Our results show that the
5-Te-DNA is stable, and that the Te-DNA duplex
has the thermo-stability similar to the correspond-
ing native duplex. The crystal structure indicates
that the 5-Te-DNA duplex structure is virtually iden-
tical to the native one, and that the Te-modified T
and native A interact similarly to the native T and A
pair. Furthermore, while the corresponding native
showed weak signals, the DNA duplex modified
with electron-rich tellurium functionality showed
strong topographic and current peaks by STM
imaging, suggesting a potential strategy to directly
image DNA without structural perturbation.
Tellurium, which was recently introduced to the sugar
moiety of the nucleosides and nucleotides (25,26), is a
non-metal element with a much larger atomic size
˚
(atomic radius: 1.40 A) (27) in the same elemental family
˚
˚
˚
of oxygen (0.73 A), sulfur (1.02 A) and selenium (1.16 A)
(28). Tellurium has much higher metallic property and
stronger electron delocalizability than the other ones in
the same elemental family. Since DNA duplexes are
relatively electron deficient, we hypothesize that an
electron-rich tellurium atom will likely donate electron
and facilitate electron delocalization and DNA imaging,
when it is introduced into DNA duplexes via the
nucleobases.
Herein, we report the first synthesis of a tellurium-
nucleobase-derivatized nucleoside (Te-thymidine or TeT),
the Te-phosphoramidite and Te-DNAs. We found that the
TeT-DNA duplex has the thermo-stability similar to the
corresponding native duplex. Furthermore, our X-ray
crystal structure studies of the Te-derivatized DNAs
indicate that the Te-derivatized and native structures are
virtually identical, and that TeT and A interact as well as
the native T–A pair. Moreover, our STM imaging study
reveals that the Te-DNAs show strong characteristic
current peaks on graphite surface, suggesting a useful
DNA imaging strategy without perturbation of the local
and global structures.
INTRODUCTION
Well-behaved base pair recognitions and duplex structures
of DNAs and RNAs have stimulated extensive research
investigations, such as DNA nano-structure construction
and self-assembling (1–4), disease and pathogen detection
at single molecule level (5), oligonucleotide drug discovery
(6) and nanoelectronic device design based on DNA con-
ductivity and charge migration (7–12). Moreover,
chemical modifications of nucleobases have been widely
used to selectively tailor the chemical and biophysical
properties of DNAs and RNAs and to probe their
chemical and biochemical functions, including base
pairing specificity, polymerase recognition, DNA
MATERIALS AND METHODS
Synthesis of 5-TePh-thymidine phosphoramidite
30-O-tert-butyldimethylsilyl-50-O-(4,4-dimethoxytrityl)-20-
deoxy-5-iodouridine (2). Tert-butyldimethylsilyl chloride
*To whom correspondence should be addressed. Tel: +1 404 413 5535; Fax: +1 404 413 5535; Email: Huang@gsu.edu
ß The Author(s) 2011. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acids Research, 2011, Vol. 39, No. 9 3963
(TBDMSCl) (0.344 g, 2.3 mmol) was added to a mixture of
50-O-(4,4-dimethoxytrityl)-20-deoxy-5-iodouridine (1, 1 g,
1.52 mmol) and imidazole (0.3 g, 4.6 mmol) in anhydrous
DMF (10 ml). The mixture was stirred at room tempera-
ture for 1.5 h, and then quenched with MeOH. The
mixture was diluted with EtOAc (30 ml) and washed
with H2O (3 ꢀ 10 ml) and brine. The organic phase was
dried over anhydrous MgSO4 (s) and the solvents were
evaporated under reduced pressure. The residue was
purified by a flash silica gel column (eluent: 20% EtOAc
5 min. The mixture was stirred for 30 min, and then treated
with (Ph)2Te2 (0.25 g, 0.75 mmol). The mixture was further
stirred for 1 h at the same temperature (ꢁ78ꢂC). NaCl
(5 ml, saturated aqueous solution) was added, the
reaction flask was placed at room temperature and
allowed to warm up naturally to the room temperature.
Water (10 ml) was added to the flask and EtOAc
(3 ꢀ 20 ml) was used to extract the crude product. The
organic phase was dried over MgSO4, and evaporated
under reduce pressure. The residue was purified by flash
silica gel chromatography (eluent: 40% EtOAc in hexane
containing 1% Et3N) to give 5 (106 mg, 64%) as a colorless
foam. Elution with 50% EtOAc in hexane gave 9 (33 mg,
1
in hexanes) to give 2 (1.15 g, 98%): H-NMR (CDCl3, d):
9.21 (1H, br, NH, exchangeable with D2O), 8.22 (1H, s,
H-6), 7.46–7.35 (9H, m, Ar), 6.89–6.86 (4H, m, Ar), 6.31
(1H, dd, H-10, J = 5.9, 7.5 Hz), 4.49 (1H, m, H-30), 4.15
(1H, m, H-40), 3.82 (6H, 2 s, CH3O), 3.45 (1H, dd, H-50a,
J = 2.7, 10.8 Hz), 3.32 (1H, dd, H-50b, J = 3.1, 10.8 Hz),
2.42 (1H, ddd, H-20a, J = 2.7, 5.8, 13.3 Hz), 2.20 (1H, m,
H-20b), 0.87 (9 H, 3 s, SiMe3), 0.05–0.002 (6H, 2s, SiMe2);
13C-NMR (CDCl3) d 160.41 (C4), 158.67 (Ar), 150.23
(C2), 144.37 (C-6), 144.33 (Ar), 135.54 (Ar), 135.46 (Ar),
130.13 (Ar), 130.08 (Ar), 128.10 (Ar), 127.06 (Ar), 113.39
(Ar), 87.29 (C40), 86.96 (Ar), 85.85 (C-10), 72.43 (C-30),
68.66 (C-5), 63.03 (H50), 55.27 (OMe), 42.00 (C20), 25.78
(CCH3), 17.97 (CCH3), ꢁ4.63 (SiCH3), ꢁ4.84 (SiCH3);
HRMS (ESI-TOF): molecular formula: C36H42N2O7Si;
[M-H]+: 769.1800 (Figure S5; calcd 769.1806)
1
29%) as a colorless foam. Spectral data for 7: H-NMR
(CDCl3, d): 8.64 (1H, br, NH, exchangeable with D2O),
7.82 (1H, s, H-6), 7.81–7.78 (2H, m, Ar), 7.33–7.24 (3H,
m, Ar), 6.25 (1H, dd, H-10, J = 5.6, 8.0 Hz), 4.31 (1H, m,
H-30), 3.92 (1H, m, H-40), 3.63–3.54 (2H, m, H-50a,b), 2.30
(1H, ddd, H-20a, J = 2.4, 5.6, 13.2 Hz), 1.94 (1H, m,
H-20b), 0.90 (9 H, s, SiCMe3), 0.88 (9 H, s, SiCMe3),
0.09–0.07 (12 H, 4s, SiMe2); 13C-NMR (DMDO-d6)
d 162.65 (C4), 150.21 (C2), 149.77 (C-6), 138.62 (Ar),
129.56 (Ar), 128.41 (Ar), 113.08 (C-5), 89.12 (Ar), 88.48
(C40), 85.85 (C-10), 72.57 (C-30), 63.16 (C-50), 42.04 (C20),
26.18 (CMe3), 26.08 (CMe3), 18.42 (CMe3), 18.42 (CMe3),
ꢁ4.48 (SiMe2), ꢁ4.66 (SiMe2); HRMS (ESI-TOF): mo-
lecular formula: C27H43N2O5Si2Te; [M-H+]ꢁ: 661.1775
(Figure S6; calcd 661.1773).
30,50-Di-O-tert-butyldimethylsilyl-20-deoxy-5-iodouridine
(4). TBDMSCl (1.28 g, 8.43 mmol) was added to a
mixture of 5-iodo-20-deoxyuridine (3, 1 g, 2.81 mmol) and
imidazole (1.3 g, 8.5 mmol) in anhydrous DMF (10 ml).
The mixture was stirred at room temperature for 2 h,
and then quenched with MeOH. The mixture was
diluted with EtOAc (30 ml) and washed with H2O
(3 ꢀ 10 ml) and brine. The organic phase was dried over
anhydrous MgSO4 (s) and the solvents were evaporated
under reduced pressure. The residue was purified by a
flash silica gel column (eluent: 30% EtOAc in hexanes)
30-O-tert-Butyldimethylsilyl-50-O-(4,4-dimethoxytrityl)-20-
deoxy-5-phenyltellurouridine (6). NaH 95% (19 mg,
0.67 mmol) was added portion wise to a solution of 2
(0.516 g, 0.67 mmol) in dry THF (2 ml) at room tempera-
ture in dry glove box. The mixture was stirred for 30 min
until complete cease of hydrogen gas evolution, then
cooled down to ꢁ78ꢂC and treated with n-BuLi (1.0 ml,
2.3 M solution in hexane; 2.3 mmol) dropwise over 10 min.
The mixture was stirred for 30 min, and then treated with
(Ph)2Te2 (0.75 g, 1.8 mmol). The mixture was further
stirred for 1 h at the same temperature (ꢁ78ꢂC). NaCl
(10 ml, saturated aqueous solution) was added, the
reaction flask was placed at room temperature and
allowed to warm up naturally to the room temperature.
Water (10 ml) was added to the flask and EtOAc
(3 ꢀ 20 ml) was used to extract the crude product. The
organic phase was dried over MgSO4 (s), and evaporated
under reduce pressure. The residue was purified by
flash silica gel chromatography (eluent: 40% EtOAc
in hexanes containing 1% Et3N) to give 6 (0.39 g, 68%)
as a colorless foam. Elution with 50% EtOAc in hex-
anes gave 8 (108 mg, 25%) as a colorless foam. Spectral
data for 6: 1H-NMR (CDCl3, d): 9.42 (1H, br, NH,
exchangeable with D2O), 7.92 (1H, s, H-6), 7.70–6.86
(18H, m, Ar), 6.29 (1H, dd, H-10, J = 6.4, 8.0 Hz), 4.27
(1H, m, H-30), 3.99 (1H, m, H-40), 3.81 (6H, 2 s, CH3O),
3.25 (2H, m, H-50a,b), 2.37 (1H, ddd, H-20a, J = 2.4, 5.6,
13.2 Hz), 2.08 (1H, m, H-20b), 0.87 (9 H, 3 s, SiMe3), 0.09–
0.05 (12 H, 4s, SiMe2); HRMS (ESI-TOF): molecular
formula: C42H48N2O7TeSi; [M+H+]+: 851.2357 (Figure
S1; calcd 851.2371).
1
to give 4 (1.34 g, 99%) as white foam: H-NMR (CDCl3,
d): 9.35 (1H, br, NH, exchangeable with D2O), 8.06 (1H, s,
H-6), 6.26 (1H, dd, H-10, J = 5.7, 7.9 Hz), 4.38 (1H, m,
H-30), 3.97 (1H, m, H-40), 3.86 (1H, dd, H-50a, J = 2.2,
11.5 Hz), 3.76 (1H, dd, H-50b, J = 2.4, 11.5 Hz), 2.30
(1H, ddd, H-20a, J = 2.3, 5.7, 13.1 Hz), 1.98 (1H, m,
H-20b), 0.93 (9 H, s, SiCMe3), 0.88 (9 H, s, SiCMe3),
0.14–0.06 (12 H, 4s, SiMe2); 13C-NMR (DMDO-d6)
d 160.50 (C4), 150.34 (C2), 144.51 (C-6), 88.49 (C40),
88.13 (C-10), 72.60 (C-30), 68.69 (C5), 63.14 (C-50), 41.70
(C20), 26.47 (CMe3), 25.89 (CMe3), 18.95 (CMe3), 18.12
(CMe3), ꢁ4.50 (SiMe2), ꢁ4.70 (SiMe2), ꢁ4.83 (SiMe2),
ꢁ4.96 (SiMe2); HRMS (ESI-TOF): molecular formula:
C21H40N2O5ISi2; [M+H+]+: 583.1525 (Figure S4; calcd
583.1521).
30,50-Di-O-tert-butyldimethylsilyl-20-deoxy-5-phenyltelluro-
uridine (5). NaH 95% (6 mg, 0.25 mmol) was added to a
solution of 4 (0.15 g, 0.25 mmol) in dry THF (1 ml) at room
temperature in dry glove box. The mixture was stirred for
30 min until complete cease of hydrogen gas evolution,
then cooled down to ꢁ78ꢂC. n-BuLi (0.36 ml, 2.3 M
solution in hexanes; 0.83 mmol) was added dropwise over

3964 Nucleic Acids Research, 2011, Vol. 39, No. 9
50-O-(4,4-dimethoxytrityl-20-deoxy-5-phenyltellurouridine
(7). TBAF (0.5 ml, 1 M solution in THF) was added to a
solution of 6 (0.26 g, 0.33 mmol) in THF (5 ml) at 0ꢂC. The
mixture was stirred for 3 h at room temperature. The
solvent was evaporated and the residue was partitioned
between EtOAc and H2O. The organic phase was dried
over anhydrous MgSO4 (s) before evaporation. The
residue was purified by silica gel column chromatography
(the silica gel was pre-equalized with 1% Et3N in CH2Cl2;
eluent: 4% MeOH in CH2Cl2) to give (0.2 g, 84%) of
seven as pale yellow foam (Figure S7). 1H-NMR
(CDCl3, d): 9.25 (1H, br, NH, exchangeable with D2O),
7.83 (1H, s, H-6), 7.62–7.01 (15 H, m, Ar), 6.86–6.82 (4H,
m, Ar), 6.23 (1H, dd, H-10, J = 6.4, 6.8 Hz), 4.34 (1H, m,
H-30), 3.98 (1H, m, H-40), 3.76 (6H, 2 s, CH3O), 3.29 (1H,
dd, H-50a, J = 4.4, 10.4 Hz), 3.18 (1H, dd, H-50b, J = 4.4,
10.8 Hz), 2.40 (1H, ddd, H-20a, J = 3.6, 6.0, 13.6 Hz), 2.16
(1H, m, H-20b); 13C-NMR (DMSO-d6) d 163.40 (C4),
159.33 (Ar), 151.06 (C2), 147.64 (C-6), 145.31 (Ar),
138.41 (Ar), 136.30 (Ar), 136.67 (Ar), 130.64 (Ar),
130.02 (Ar), 128.67 (Ar), 128.60 (Ar), 128.57 (Ar),
127.54 (Ar), 113.95 (C-5), 113.81 (Ar), 89.62 (Ar), 87.33
(Ar), 86.44 (C40), 85.72 (C-10), 72.82 (C-30), 64.26 (C-50),
55.81 (OMe), 41.35 (C20); HRMS (ESI-TOF): molecular
formula: C36H33N2O7Te; [M]+: 735.1342 (Figure S2; calcd
735.1350).
appropriate nucleoside through a succinate linker. All
the oligonucleotides were prepared with DMTr-on form.
After synthesis, the DNA oligonucleotides were cleaved
from the solid support and fully deprotected by the treat-
ment of 0.05 M K2CO3 solution in methanol for 8 h at
room temperature. The 50-DMTr deprotection was per-
formed in a 3% trichloroacetic acid solution for 2 min,
followed by neutralization to pH 7.0 with a freshly made
aqueous solution of triethylamine (1.1 M) and extraction
by petroleum ether to remove DMTr-OH.
HPLC analysis and purification
The Te-DNA oligonucleotides were analyzed and purified
by reverse phase high performance liquid chromatography
(RP-HPLC) both DMTr-on and DMTr-off. Purification
was carried out using a 21.2 ꢀ 250 mm Zorbax, RX-C8
column at a flow rate of 10 ml/min. Buffer A consisted
of 20 mM triethylammonium acetate (TEAAc, pH 7.2),
while buffer B contained 50% aqueous acetonitrile and
10 mM TEAAc, pH 7.1. Similarly, analysis was performed
on a Zorbax SB-C18 column (4.6 ꢀ 250 mm) at a flow of
1.0 ml/min using the same buffer system. The DMTr-on
oligonucleotides were eluded with up to 100% buffer B in
20 min in a linear gradient, while the DMTr-off oligo-
nucleotides were eluted with up to 70% of buffer B in a
linear gradient in the same period of time. The collected
fractions were lyophilized; the purified compounds were
re-dissolved in water. The pH was adjusted to 7.0 after the
final purification of the Te-oligonucleotides without the
DMTr group.
1-[20-deoxy-30-O-(2-cyanoethyl-N,N-diisopropylamino)-
phosphoramidite-50-O-(4,4-dimethoxytrityl-ꢀ-D-erythro-
ribofuranosyl]-5-phenyltellurouridine
(10). Disopropylethylamine (44 ml, 0.25 mmol) and N,N-
diisopropylamino
cyanoethylphosphamidic
chloride
Thermodenaturization of duplex DNAs
(57 mg, 0.24 mmol) were added to a solution of 7 (0.15 g,
0.2 mmol) in CH2Cl2 (2 ml) at 0ꢂC. The mixture was
stirred for 2 h at room temperature, and then was slowly
poured into pentane (100 ml) under vigorous stirring. The
produced white precipitate was filtered out, dissolved in
CH2Cl2 (1 ml) and re-precipitated in pentane. The col-
lected fine white powder was dried under reduced
pressure to give 142 mg 10 (75% yield) as a mixture of
two diastereomers, which was directly used for solid
phase synthesis without further purification. 31P-NMR
(Figure S8, CDCl3, d): 149ꢃ3, 149ꢃ8: HRMS (ESI-TOF):
molecular formula: C45H51N4O8PTe; [M-H+]ꢁ: 935.257
(Figure S3; calcd 935.242).
Solutions of the duplex DNAs (2 mM) were prepared by
dissolving the DNAs in a buffer containing NaCl (50 mM),
sodium phosphate (10 mM, pH 7.2), EDTA (0.1 mM) and
MgCl2 (2 mM). The solutions were then heated to 90ꢂC for
3 min, cooled slowly to room temperature and stored at
4ꢂC overnight before measurement. Prior to thermal de-
naturation, argon was bubbled through the samples. Each
denaturizing curves were acquired at 260 nm by heating
and cooling for four times in a rate of 0.5ꢂC/min using
Cary-300 UV–visible spectrometer equipped with tempera-
ture controller system.
Crystallization
The purified oligonucleotides (1 mM) were heated to 70ꢂC
for 2 min, and cooled slowly to room temperature. The 20-
Se–dU was incorporated into the Te-DNA for the crystal-
lization facilitation. Both native buffer and Nucleic Acid
Mini Screen Kit (Hampton Research) were applied to
screen the crystallization conditions with l mM of DNA
sample at different temperatures using the hanging drop
method by vapor diffusion.
Synthesis of the 5-Te-functionalized DNA
All the DNA oligonucleotides were chemically synthesized
in a 1.0 mmol scale using an ABI392 or ABI3400 DNA/
RNA
Synthesizer.
The
ultra-mild
nucleoside
phosphoramidite reagents were used in this work (Glen
Research). The concentration of the Te-uridine
phosphoramidite was identical to that of the conventional
ones (0.1 M in acetonitrile). Coupling was carried out
using
a
5-(benzylmercapto)-1H-tetrazole (5-BMT)
Data collection
solution (0.25 M) in acetonitrile. The coupling time was
25 s for both native and modified samples.
Trichloroacetic acid (3%) in methylene chloride was
used for the 50-detritylation. Synthesis were performed
on control pore glass (CPG-500) immobilized with the
Glycerol (30%), PEG 400 or the perfluoropolyether was
used as a cryoprotectant during the crystal mounting, and
data collection was taken under nitrogen gas at 99ꢂK. The
Te-DNA-8-mer crystal data were collected at beam line

Nucleic Acids Research, 2011, Vol. 39, No. 9 3965
X12C in NSLS of Brookhaven National Laboratory. A
number of crystals were scanned to find the one with
strong anomalous scattering at the K-edge absorption of
selenium. The distance of the detector to the crystals was
Probe Inc.), and only the top of the tip was exposed as
reported by Lindsay (37).
˚
RESULTS AND DISCUSSION
set to 150 mm. The wavelengths of 0.9795 A was chosen
for data collection. The crystals were exposed for 10–15 s
per image with 1ꢂ oscillation, and a total of 180 images
were taken for each data set. All the data were processed
using HKL2000 and DENZO/SCALEPACK (29).
Synthesis of 5-TePh-thymidine phosphoramidite
Aryl-chalcogenides (S and Se), such as phenyl sulfenyl and
phenyl selenyl moieties, have been tethered to the
5-position of pyrimidine nucleosides and been
incorporated into DNA (38–41). Methods, which are
available in the literature for the introduction of the
alkyl or aryl chalcogenides (S and Se) at the 5-position
of pyrimidines, include the electrophillic addition–elimin-
ation on the C5–C6 double bond (42), the palladium-
catalyzed nucleophillic substitution of 5-mercururio de-
rivatives (39,43), and the lithiation of 5-halo derivatives
in the presence of the electrophilic species (44). However,
no literature report can be found on the corresponding
5-aryl- or alkyl-tellurides. Incorporation of the tellurium
functionality to the 5-position is a long-standing chal-
lenge. We have attempted, without success, to introduce
an alkyltelluro or aryltelluro moiety at the 5-position of
pyrimidines under several conditions. However, by
applying the lithium–halogen exchange strategy (45) on
a protected 5-iodo-20-deoxyurdine, we have successfully
incorporated the Te-functionality to the 5-position. We
found that the key steps are the transient protection of
5-iodo-20-deoxyuridine derivative 2 as a sodium salt, and
the treatment with n-BuLi and (PhTe)2. To the best of our
knowledge, we have established the first synthesis of the
5-Te-pyrimidine derivatives.
Structure determination and refinement
The crystal structure of Te-DNA-8-mer was solved by
molecular replacement with Phaser (30) using the auto-
mated search mode, followed by the refinement of
selenium and tellurium atom positions in both CNS (31)
and Refmac 5.0 (32). The refinement protocol includes
simulated annealing, positional refinement, restrained
B-factor refinement and bulk solvent correction. The
stereo-chemical topology and geometrical restrain param-
eters of DNA/RNA (33) have been applied. The
topologies and parameters for modified dU with
20-SeMe (UMS) and 5-tellurium (TTE) were constructed
and applied. After several cycles of refinement, a number
of highly ordered waters were added. Final, the
occupancies of selenium and tellurium were adjusted.
Cross-validation (34) with a 5–10% test set was monitored
during the refinement. The sA-weighted maps (35) of the
(2m|Fo| ꢁ D|Fc|) and the difference (m|Fo| ꢁ D|Fc|) density
maps were computed and used throughout the model
building.
The synthesis of the 5-TePh-deoxyuridine and
5-TePh-deoxyuridine phosphoramidite is showed in
Scheme 1, starting from the silylation of commercially
available 5-iodo-20-deoxyuridine derivative 1. After puri-
fication, this intermediate (2) was treated with NaH/n-
BuLi, followed by adding diphenylditelluride (PhTe)2.
This reaction generated an inseparable mixture of the
desired 5-PhTe derivative (6) and the tentative 6-PhTe de-
rivative (in 10 : 1 ratio, with 72% yield), along with the
reduced byproduct (8, in 25% yield). After several failed
trials to separate the isomers, such as re-crystallization
and very-slow column chromatography, we switched the
starting compound to the 30,50-di-O-silyl derivatives.
Unfortunately, this 5-isomer was still difficult to be
purified from the 6-regio-isomer. Later, various concen-
trations of the reactant (30-O-silyl-50-DMTr derivative 2)
were tested (Scheme 1). Finally, it was found that when
the concentration of the reactant was increased to as high
as 0.15 or 0.18 M in THF solution, the 6-TePh isomer
could indeed be completely eliminated. Desired
5-Te-isomer 6 was formed as the major product, while 8
was formed as a minor byproduct. Then the desilylation of
6 with TBAF/THF was carried out, generating 7 quanti-
tatively, which was finally converted into 5-phenyltelluro
thymdine (TeT) phosphoramidite (10) for solid phase syn-
thesis of the Te-DNA. The characteristic distributions of
the tellurium isotopes in mass spectra of 6, 7 and 10 were
observed and confirmed the incorporation of the
Te-functionality.
Mass analysis of Te–C bond breaking caused by X-ray
irradiation
In our work,
a few crystals were analyzed by
MALDI-TOF MS before X-ray irradiation. As a compari-
son, a few crystals were analyzed by MALDI-TOF MS
after X-ray irradiation. This Te-functionality is relatively
stable in the absence of X-ray irradiation, and no signifi-
cant decomposition was observed in 2 months under air.
The Te-DNA was also heated at 90ꢂC over 1 h without
significant decomposition.
STM experiment
In this work, highly oriented pyrolytic graphite (HOPG)
was used as a support substrate to study DNA molecules
(36). The HOPG was freshly cleaved with scotch tapes
prior to each experiment, and treated with UV Surface
Decontamination System (Novascan Technologies Inc.)
for 20 min before modifying the surface of the hydropho-
bic HOPG to the hydrophilic surface. The bare HOPG
surface did not show obvious structure changes even at
the atomic level. Both the native and TeT-DNA samples
(100 ml, 0.03 mM) were dissolved in PBS solution, and the
sample solutions were then loaded on the HOPG surface.
After 20 min, the DNA samples were scanned with STM
in the PBS buffer. To reduce the influence of the leakage
current, the newly cut Au STM tip was coated with
Apiezon W Wax (SPI Supplies Division of Structure

3966 Nucleic Acids Research, 2011, Vol. 39, No. 9
O
N
O
N
O
Ph Te
I
NH
NH
NH
O
O
O
N
R1O
R1O
R1O
b
O
O
O
+
R2O
R2O
1: R1 = DMTr, R2 = H
R2O
8: R1 = DMTr, R2 =TBDMS
9: R1 =R2 =TBDMS
5: R1 = TBDMS; R2 = TBDMS
6: R1 = DMTr, R2 = TBDMS
a
a
2: R1 = DMTr, R2 = TBDMS
3: R1 = H, R2 = H
4: R1 = TBDMS; R2 = TBDMS
c
7: R1 = DMTr, R2 = H
d
O
O
Ph Te
Ph Te
O
NH
NH
DNA
P
-O
O
O
O
N
N
DMTrO
O
O
e
O
P
O
P
O
DNA
OCH2CH2CN
N
-O
10
Scheme 1. Synthesis of the 5-phenyltelluro-20-deoxyuridine, its phosphoramidite, and the Te-DNAs. (A) TBDMS-Cl, Im., DMF, 4 h, room tem-
perature (RT); (B) (i) NaH, THF, 15 min, RT; (ii) n-BuLi, THF, 10 min, ꢁ78ꢂC; (iii) Ph2Te2, 1 h, ꢁ78ꢂC; (C) TBAF, THF, 4 h, RT;
(D) i-Pr2NP(Cl)CH2CH2CN, DIEA, CH2Cl2, 1 h, RT; (E) the solid-phase synthesis.
120
Table 1. MALDI-TOF MS of the Te-derivatized DNA
oligonucleotides
15.3 min
100
Entry
DNA sequences
Measured (calcd) m/z
[M]ꢁ: 2919.3 (2919.5)
[M]ꢁ: 2784.4 (2784.4)
[M]ꢁ: 3464.5 (3463.8)
[M+H+]+: 3218.0 (3218.7)
[M]ꢁ: 2599.4 (2599.3)
[M+H+]+: 2679.2 (2679.2)
80
60
40
20
0
1
2
3
4
5
6
ATGG(TeT)GCTC
C93H114N32O54P8Te
CTCCCA(TeT)CC
C89H113N27O53P8Te
CT(TeT)CTTGTCCG
C111H140N32O69P10Te
GCG(TeT)ATACGC
C102H125N38O58P9Te
GTG(TeT)ACAC
C83H101N30O46P7Te
G(20-Se-dU)G(TeT)ACAC
C83H101N30O46P7SeTe
0
10
20
30
Time
Figure 1. HPLC analysis of 5-TePh-9-mer: [50-DMTr-ATGG
(5-TePh-T)GCTC-30]. HPLC conditions: Welchrom C18-XB column
(4.6 ꢀ 250 mm, 5 m), 25ꢂC, 1 ml/min, gradient from buffer A to 90%
buffer B in 20 min. The retention time of the full-length sample is
15.3 min.
The synthesized Te-DNAs were then purified in a prepara-
tive RP-HPLC system in trityl-on form. After treated with
3% trichloroacedic acid for 2 min, the solution was
neutralized with triethylamine and extracted with petrol-
eum ether, followed by the second purification in trityl-off
form. All the Te-modified DNA oligonucleotides were
characterized by MALDI-TOF, and several Te-DNA
oligonucleotides were listed in Table 1.
Synthesis of the 5-Te-functionalized DNA
Considering that the Te-functionality is electron rich, its
oxidative removal might happen during solid-phase
synthesis and purification. Fortunately, this Te-
phosphoramidite is well compatible with the solid-phase
synthesis conditions, thereby being incorporated into
several designed DNA sequences. After K2CO3 (0.05 M
in MeOH) cleavage and deprotection, the crude
DNA solution was analyzed by analytical RP-HPLC.
As showed in Figure 1, the coupling yield is over 95%.
Thermodenaturization study of oligonucleotide duplexes
containing the 5-Te derivatization
To evaluate the potential structure perturbation caused by
this bulky tellurium moiety (Ph–Te), we conducted the
UV–thermal denaturation studies. Our experimental

Nucleic Acids Research, 2011, Vol. 39, No. 9 3967
Table 2. UV-melting study of the Te-DNA duplexes
Table 3. X-ray data collection: the data collected at selenium K-edge
˚
Entry
DNA duplex
Tm (ꢂC)
Data collection
ꢁ = 0.9795 A
18.89–1.50 (1.55–1.50)
3659
94.4
7.5 (42.7)
14.9 (2.4)
8.63 (5.30)
0.93 (0.42)
˚
Resolution range, A (last shell)
Unique reflections
Completeness (%)
Rmerge (%)
1
2
3
4
50-ATGG(TeT)GCTC-30
30-TACCACGAG-50
40.3 0.2
39.8 0.3
44.1 0.3
44.0 0.2
50-ATGGTGCTC-30
30-TACCACGAG-50
hI/s(I)i
50-CT(TeT)CTTGTCCG-30
30-GAAGAACAGGC-50
50-CTTCTTGTCCG-30
30-GAAGAACAGGC-50
Redundancy
Reduced chi-squared
Rmerge = Æ|I ꢁ hIi|/ÆI.
the crystallization by using our 20-Se modification strategy
to facilitate the crystal growth (46–48). The modified
1.0
0.8
0.6
0.4
0.2
0.0
20-Se
DNA (50-G-
dU-G-TeT-ACAC-30) was designed,
synthesized and purified. Its crystallization conditions
were screened with the Hampton Mini-Screening kit. As
a result, the Te-DNA crystals were formed in all the 24
buffer conditions of the kit. Moreover, crystals with
high-diffraction quality and reasonable sizes were
generated in 2 days. Crystals, which formed in buffer #7
of the kit (10% v/v MPD, 40 mM sodium cacodylate pH
6.0, 12 mM spermine tetra–HCl, 80 mM potassium
chloride and 20 mM magnesium chloride), were shown
in Figure 3 as an example. By the crystal diffraction
screening, the crystals formed in buffer #7 were identified
Te-9mer
Native 9mer
10
20
30
40
50
60
70
Temperature (°C)
˚
to give the highest diffraction resolution (up to 1.50 A). In
Figure 2. Normalized thermal–denaturation curves of the native and
Te-modified DNA duplexes. The Te-DNA duplex: 50-ATGG(5-
TePh-T)GCTC-30 and 50-GAGCACCAT-30. The solid and dashed
curves represent the Te-modified and the corresponding native
duplexes with 40.3 0.2 and 39.8 0.3ꢂC as their melting tempera-
tures, respectively.
addition, several data sets with high diffraction resolution
were also collected for further refine purpose. The data
collection and refine parameters are listed in Tables 3
and 4.
Crystal structure of the Te-DNA 8-mer
This Te-DNA crystal has the same tetragonal space group
P43212 as the corresponding native one (49). The occu-
pancy of Se and Te are 1.0 and 0.35, respectively. The
refined B-factor of Se and Te are 28.55 and 23.64, respect-
ively, comparing with other atoms in the structure. The
˚
Te-DNA structure (1.50 A resolution) revealed that both
global and local structures of this Te-DNA are virtually
identical to the native one (Figure 4A and B), indicating
the Te-moiety does not cause significant perturbation. The
structure result is also consistent with our biophysical
study, suggesting that TeT and A (Figure 4B and C) pair
as well as native T and A. Furthermore, the Te-DNA is
stable under a higher temperature. As shown by Figure 5,
the Te-DNA was heated at 90ꢂC for 1 h without signifi-
cant decomposition (<2%). However, since the Te–C
bond is sensitive to X-ray irradiation, the occupancy of
tellurium (ꢄ40%) in the crystal structure is expected. The
low electron density of the phenyl group (ꢄ16% of
expected density) is consistent with cleavage of these two
Te–C bonds [Te–C5 and Te–C(Ph)] by the X-ray irradi-
ation. To verify this, the mass analysis of the Te-DNA
crystals was carried out. The Te-functionality is stable in
the absence of X-ray irradiation, our MS analysis
indicated no significant decomposition of the Te-DNA
crystals in the crystallization droplet after 3 weeks
(Figure 6A). However, after X-ray irradiation, the
MALDI-TOF-MS spectra (Figure 6B) showed the loss
Figure 3. Crystal photo of the Te-DNA octomer [50-G(20-SeMe-dU)
G(TeT)ACAC-3]2 crystallized in buffer #7 with 0.2 ꢀ 0.1 ꢀ 0.1 mm in
size.
results indicated that the Te-derivatized duplex structures
are as stable as the corresponding natives (Table 2 and
Figure 2). Clearly, the bulky tellurium substitution does
not significantly change the melting temperatures and alter
the duplex structure.
Crystallization and data collection of Te-DNA
To further confirm that this 5-TePh modification does not
cause obvious structure perturbations, we have carried out

3968 Nucleic Acids Research, 2011, Vol. 39, No. 9
of 78 (the phenyl group) and 203.9 mass units (the
phenyltelluro group), indicating the partial cleavage of
both Te–C bonds by X-ray irradiation.
native DNA. To investigate this, we synthesized the
Te-DNA [9 nt, 50-ATGG(TeT)-GCTC-30, Figure 7],
which is complementary to a native DNA [54 nt, 50-(GA
GCACCAT)6-30] by six repeats, and conducted the STM
studies. Interestingly, our experiments revealed that the
Te-modified DNA duplex showed much stronger topo-
graphic and current peaks (Figure 7A and B) than the
corresponding native duplex (Figure 7C and D). Clearly,
both the topographic and current images of the Te-duplex
showed that the Te-derivatization allowed better
STM imaging study
Since tellurium atom has the metallic property, we expect
that the Te-modified DNA has higher visibility and con-
ductivity under STM imaging than the corresponding
Table 4. Refinement statistics in Te-8-mer
Refinement
˚
200
Resolution range, A (last shell)
Number of reflections
Rwork (%)
25–1.50 (1.55–1.5)
6859 (424)
19.3
Original
90°C for 1h
Rfree (%)
21.5
150
Number of atoms
Nucleic Acid (single)
Heavy atom
162
2 (Se, Te)
28
100
50
0
Water
RMSD
Bond length, A
Bond angle
˚
0.010
1.73
2
˚
Average B-factors, A
All atoms
Wilson plot
22.3
19.6
0
5
10
15
20
Overall anisotropic B-values
B11/B22/B33
Time (Min)
0.6/0.6/ꢁ1.2
Bulk solvent correction
˚
Solvent density, e/A
Figure 5. The Te-DNA thermal stability and HPLC analysis. Buffer A:
20 mM TEAAc; buffer B: 50% acetonitrile in buffer A. HPLC condi-
tions: Welchrom C18-XB column (4.6 ꢀ 250 mm, 5 m), 25ꢂC, 1 ml/min,
gradient from buffer A to 50% buffer B in 15 min. HPLC profiles of
Te-DNA [50-G(20-SeMe-dU)G(TeT)ACAC-30] thermal analysis: the
solid line (without heating), the dashed line (heated in 10 mM
Na-PO4 buffer, pH 7.3, at 90ꢂC for 1 h).
3
0.44
59.6
2
˚
B-factors, A
˚
Coordinates error (c.-v.), 5 A
˚
Esd. from Luzzatt plot, A
0.17
0.15
˚
Esd. from SIGMAA, A
A
B
C
2.87 Å
2.77 Å
2.37
2.10
Figure 4. The global and local structures of the Te-DNA duplex [50-G(20-SeMe-dU)G(TeT)ACAC-30]2. The red and yellow balls represent Te and Se
˚
atoms, respectively. (A) The Te-dsDNA structure (in cyan; 1.50 A resolution; PDB ID: 3FA1) is superimposed over the native one (in magenta; PDB
ID: 1DNS). (B) The local structure of the TeT/A base pair (in cyan) is superimposed over the native T/A base pair (in magenta). (C) The experi-
mental electron density (2|Fo| ꢁ |Fc| map) of the TeT/A base pair (s = 1.0). The green ball represents approximately one-sixth of phenyl group (one
carbon).

Nucleic Acids Research, 2011, Vol. 39, No. 9 3969
visualization of the 54-bp DNA duplex (calculated length:
18 nm) with ꢄ20 nm in measured length. Our result also
suggested that the native and Te-modified duplexes can
assemble with different orientations on the graphite
surface, indicated by the topographic and current images
of the native and Te-modified duplexes.
CONCLUSIONS
In summary, we have first incorporated the Ph–
Te-functionality (5-Te) to the 5-position of a pyrimidine,
and successfully synthesized the Te-nucleobase-modified
nucleoside, phosphoramidite and DNA oligonucleotides.
The Te-modified DNA is stable, and the native and
modified duplexes have the similar stability. Our biophys-
ical and structural studies indicate that the Te-modified T
and A interact as well as native T and A pair, and that the
structures of the Te-derivatized and native duplexes are
virtually identical. Furthermore, the Te-DNA duplex is
2679.2 [M+H+]+
2475.3 [M+H+-TePh]+
100
90
readily visible under STM, suggesting
a potential
B
80
70
60
50
40
30
20
10
0
A
strategy to directly image DNA without the structural
perturbation. This Te-modification will open a novel
research avenue for nucleic acids, including mechanism
and function studies, STM imaging and nano-electronic
materials.
2601.2 [M+H+-Ph]+
2679.2 [M+H+]+
2302
2605
ACCESSION NUMBER
3FA1.
1999
2302
2605
2908
Figure 6. MALDI-TOF mass spectra of the Te-DNA [50-G
(20-SeMe-dU)G(TeT)ACAC-30; molecular formula: C83H101N30O46P7
SeTe; F.W.: 2678.2]. (A) The crystal sample without X-ray irradiation;
(B) the crystal sample with X-ray irradiation.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
Figure 7. The STM images of the Te-modified DNA duplex [50-ATGG(TeT)-GCTC-30 and 50-(GAGCACCAT)6-30] and its counterpart native duplex
on HOPG. The arrows indicate the edges or current peaks of the measured molecules. (A) Topographic image of Te-duplex; (B) current image of
Te-duplex; (C) topographic image of native duplex; (D) current image of native duplex. Comparing to the Te-modified DNA duplex, the native
sample does not show obvious conductivity. The insets are 2D images corresponding to the 3D images. The sample bias: 0.50 V, the current set point:
100 pA.

3970 Nucleic Acids Research, 2011, Vol. 39, No. 9
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in Brookhaven National Laboratory for their help in
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FUNDING
Georgia Cancer Coalition Distinguished Cancer
Clinicians and Scientists Award and the US National
Science Foundation (MCB-0824837). Funding for open
access charge: US National Science Foundation (MCB-
0824837).
Conflict of interest statement. None declared.
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