Synthesis of DNA oligonucleotides containing 5-(mercaptomethyl)-2′-deoxyuridine moieties

Article abstract of DOI:10.1016/j.bmc.2006.05.054

Recently thiolated oligonucleotides have attracted significant interest due to their ability to efficiently undergo stable bond formation with gold nanoparticles and surfaces to form DNA conjugates. In this respect we became interested in the synthesis of

Full text of DOI:10.1016/j.bmc.2006.05.054

Bioorganic & Medicinal Chemistry 14 (2006) 6235–6238
Synthesis of DNA oligonucleotides containing
5-(mercaptomethyl)-20-deoxyuridine moieties
Benjamin Bornemann and Andreas Marx*
Fachbereich Chemie, Universita¨t Konstanz, Universita¨tsstrasse 10, 78457 Konstanz, Germany
Received 3 February 2006; revised 23 March 2006; accepted 30 May 2006
Available online 13 June 2006
Abstract—Recently thiolated oligonucleotides have attracted significant interest due to their ability to efficiently undergo stable
bond formation with gold nanoparticles and surfaces to form DNA conjugates. In this respect we became interested in the synthesis
of oligonucleotides that bear short thioalkyl functions located at the nucleobase. Here we present a strategy for the synthesis of
DNA oligonucleotides that bear 5-(mercaptomethyl)-20-deoxyuridine moieties. The building blocks were synthesized in a straight-
forward manner from thymidine. Only moderate changes of standard protocols for automated DNA synthesis are required for the
generation of modified oligonucleotides containing the thiolated building blocks.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
oligonucleotides that bear 5-(mercaptomethyl)-20-deox-
yuridine moieties (Fig. 1). We intended to develop a
strategy that allows positioning of the thiolated building
blocks at the 30- or 50-terminal nucleotide or at internal
locations to ensure maximum flexibility.
Efficient chemical modification of DNA and RNA oli-
gonucleotides is the basis for numerous subsequent stud-
ies like conjugation of reporter molecules, investigation
of biological systems or immobilization on surfaces.1,2
Among the numerous functionalities conjugated to
DNA, thiol moieties are of particular interest, for exam-
ple, for conjugation of reporter groups, immobilization
of DNA oligonucleotides on gold surfaces or nanoparti-
cles.1–7 In most current studies thioalkyl functions are
coupled to the 50- or 30-end of the oligonucleotides,
respectively, employing thioalkylated phosphoramidites
or solid supports.8,9 Recently, oligonucleotides that were
thioalkylated at the 30-terminus were employed in stud-
ies dedicated to directly measure electrical transport
through single DNA molecules.10,11 To fulfill these
tasks, respective oligonucleotide duplexes were equipped
with thioalkyl functions and employed to form double
stranded DNA (dsDNA) bridges between two gold
contacts. In this respect we became interested in the
synthesis of oligonucleotides that bear short thioalkyl
functions in close proximity to the p-system of the
nucleobase to facilitate electrical transport through
dsDNA. Thus, our first aim was the synthesis of DNA
Recently, a route for the synthesis of oligonucleotides
containing 5-(mercaptomethyl)-20-deoxyuridine moieties
was developed.12 The reported approach harbors several
disadvantages like the usage of organomercury species
and comparatively expensive starting materials. Here
we present an improved and concise strategy for the syn-
thesis of oligonucleotides that bear 5-(mercaptomethyl)-
20-deoxyuridine moieties in principle at any desired posi-
tion. The herein depicted approach relies on the selective
activation of the 5-methyl group of thymidines through
bromination and the subsequent nucleophilic replace-
ment of bromide by thioacetate.
SH
O
N
NH
O
O P O
O
O
O
SH
n
3' or 5' end
of DNA
O
O
Keywords: DNA; Thiol functions; Conjugation; Oligonucleotide;
Nucleoside; Phosphoramidite; Gold.
Figure 1. Thioalkyl functions conjugated to the 50- or 30-end of DNA
(left), 5-(mercaptomethyl)-20-deoxyuridine moieties incorporated into
DNA oligonucleotides.
*
Corresponding author. Tel.: +49 0 7531 88 5139; fax: +49 0 7531 88
5140; e-mail: Andreas.Marx@uni-konstanz.de
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.05.054

6236
B. Bornemann, A. Marx / Bioorg. Med. Chem. 14 (2006) 6235–6238
2. Results and discussion
2.2. Synthesis of modified oligonucleotides
2.1. Nucleoside synthesis
Next modified nucleoside 4 was converted into building
blocks suited for automated DNA synthesis (Scheme 2).
Our synthesis of suitable building blocks for the synthe-
sis of modified DNA oligonucleotides started with the
30,50-bis-O-(tert-butyl dimethylsilyl) protected thymi-
dine derivative 113 that was easily accessible from thymi-
dine in high yields (Scheme 1).
Thus, 4 was 50-O-protected with 4,40-dimethoxytrityl
(DMT) employing standard conditions to yield 5 in very
good yields. In order to obtain oligonucleotide strands 7
that bear modified thymidine residues at the 30-terminal
position, 5 was coupled to solid support to afford 6.
Employing solid support 6 oligonucleotides 7 were syn-
thesized using standard b-cyanoethyl phosphoramidites
and coupling conditions. However, the deprotection
protocol had to be adjusted: After completion of the
synthesis, solid supports were first treated with 1,8-diaz-
abicyclo[5.4.0]undec-7-ene (DBU) and afterwards with
concentrated ammonia in the presence of 1,4-dithiothre-
itol (DTT). In order to synthesize oligonucleotides that
bear the 5-thiolated thymidine moiety at the 50-terminal
position or at an internal position, phosphoramidite 8
was synthesized by phosphitylation of 5 in good yields.
Employing 8 oligonucleotides 9 were synthesized that
contain the modified building block at the 50-terminal
position or within the DNA strand. Table 1 lists the
synthesized oligonucleotides and provides the analytical
Radical bromination in the benzylic position was
achieved with N-bromo succinimide (NBS) and 2,20-
azo-bis-isobutyronitrile (AIBN) at elevated tempera-
ture. The resulting bromide 2 turned out to be rather
labile and was thus used crude for further transforma-
tion after filtration of the reaction mixture. Substitution
of bromide with potassium thioacetate afforded the pro-
tected 5-(mercaptomethyl)-20-deoxyuridine analog 3.
Selective 30,50-bis-O-desilylation was achieved smoothly
to yield 4 under acidic conditions employing a 4:1 mix-
ture of acetic acid (75%) and THF. Noteworthy, at-
tempts to synthesize 4 directly from thymidine without
any protection of the nucleoside failed as well as cleav-
age of the silyl ethers of 3 employing tetrabutyl ammo-
nium fluoride (TBAF).
O
N
O
O
N
O
O
N
O
N
NH
S
NH
S
NH
Br
NH
TBSO
TBSO
HO
TBSO
O
O
O
O
O
O
O
O
a
b
c
OTBS
OTBS
OH
OTBS
1
2
3
4
Scheme 1. Synthesis of 5-(mercaptomethyl)-20-deoxyuridine. Reagents and conditions: (a) NBS, AIBN, CCl4, reflux, 2.5 h; (b) potassium thioacetate,
DMF, 1.5 h, (39%, over two steps); (c) AcOH, THF, 24 h (73%).
O
O
N
SH
O
N
O
O
N
O
O
N
S
NH
NH
S
NH
S
NH
O
P
DMTO
O
O
HO
DMTO
O
O
O
O
O
O
O
O
c
b
a
O
O
O
OH
OH
OH
N
H
O
SH
O
N
O
S
O
NH
NH
O
DMTO
O
N
O
O
c
O
d
O
O
O
P
CN
O
P
O
N(i-Pr)2
O
Scheme 2. Synthesis of DNA oligonucleotides containing 5-(mercaptomethyl)-20-deoxyuridines. Reagents and conditions: (a) DMT-Cl, DMAP,
pyridine, 0 ꢁC, 24 h, (90%); (b) EDC, DMAP, succinylated LCAA-CPG, pyridine; then 4-nitrophenol; then piperidine, then acetic anhydride/
pyridine/THF (cap A) and 1-methylimidazole/THF (cap B); (c) i—oligonucleotide synthesis, ii—DBU, then 33% NH4OH, 0.2 M DTT; (d)
[(iPr2N)(NCCH2CH2O)P]Cl, NEt3, CH2Cl2, rt, 4 h, (66%).

B. Bornemann, A. Marx / Bioorg. Med. Chem. 14 (2006) 6235–6238
Table 1. Synthesized DNA oligonucleotides
6237
Sequence
Calcd mass [MꢀH]ꢀ
3619.4
6244.1
6244.1
6548.3
Found mass [MꢀH]ꢀ
3619.6
6243.7
6246.8
6551.1
S1
S2
S3
S4
50-TTT TTT TTT TTT*-30
50-CGT TGG TCC TGA AGG AGG AT*-30
50-CGT TGG TCC T*GA AGG AGG AT-30
50-T*CG TTG GTC CTG AAG GAG GAT-30
T*, 5-(mercaptomethyl)-20-deoxyuridine.
data obtained by electrospray ionization mass spectro-
meter (ESI-MS).
2.13 mmol) in dry CCl4 (20 ml) under argon atmosphere
NBS (567 mg, 3.18 mmol) and AIBN (35 mg,
0.21 mmol) were added and heated at reflux for 2.5 h.
The brownish reaction mixture was then filtered through
a sintered glass frit (D4) to remove the succinimide and
evaporated to dryness. Compound 2 was used immedi-
ately without further purification.
3. Conclusions
Acetylated 5-(mercaptomethyl)-20-deoxyuridine 4 was
synthesized in good yields in a straightforward synthesis
from thymidine. Compound 4 was subsequently convert-
ed into suitable building blocks for the automated synthe-
sis of DNA oligonucleotides that contain the modified
residues at desired positions. Through only moderate
changes of standard protocols for automated DNA syn-
thesis the generation of modified oligonucleotides con-
taining the thiolated building blocks was feasible.
Currently, we are investigating the properties of the syn-
thesized oligonucleotides for immobilization on gold
surfaces.
4.1.2.
S-Acetyl-30,50-bis-O-(tert-butyldimethylsilyl)-5-
(mercaptomethyl)-20-deoxyuridine (3). To a solution of
crude 2 in dry DMF (20 ml) under argon atmosphere
potassium thioacetate (1.00 g, 8.75 mmol) was added
and heated to 75 ꢁC for 1.5 h. The reaction mixture
was diluted with CH2Cl2 (100 ml) and washed with
water (3· 50 ml). The organic phase was dried with mag-
nesium sulfate and evaporated to dryness. The product 3
was purified by column chromatography (petrol ether–
ethyl acetate, 3:1) to a yield of 444 mg (39%, over two
steps). 1H NMR (250 MHz, CDCl3): d 0.08 (s, 6H,
Si(CH3)2), 0.12 (s, 6H, Si(CH3)2), 0.89 (s, 9H,
SiC(CH3)3), 0.92 (s, 9H, SiC(CH3)3), 1.92–2.02 (m, 1H,
H20A), 2.22–2.23 (m, 1H, H20B), 2.30 (s, 3H, (CO)CH3),
3.75 (s, 2H, CH25), 3.79–3.84 (m, 2H, H50A and H50B),
3.92–3.98 (m, 1H, H40), 4.41–4.46 (m, 1H, H30), 6.28
(dd, 1H, J = 6.4 Hz and 6.4 Hz, H10), 7,75 (s, 1H, H6),
9.70 (br s, 1H, NH); MS (MALDI-TOF, DHB): Calcd
for C24H44N2O6SSi2 [M+Na]+: 567.24, C24H44N2O6SSi2
[M+K]+: 583.22. Found: 567.1, 583.1.
4. Experimental
4.1. General
All temperatures quoted are uncorrected. All reagents
are commercially available and used without further
purification. Solvents are purchased over molecular
sieves (Fluka) and used directly without further purifica-
tion unless otherwise noted. All reactions were conduct-
ed under rigorous exclusion of air and moisture.
Elemental analysis was carried out by the microanalysis
facility of the University of Konstanz. NMR spectra
were recorded on Bruker AC 250 Cryospec (1H:
250 MHz), Jeol JNA-LA-400 (1H: 400 MHz, 13C:
100 MHz), and Bruker DRX 600 (1H: 600 MHz, 13C:
150 MHz). Chemical shifts are given in parts per million
(d) relative to the residual solvent signal. MALDI-TOF
mass spectra were recorded on a Kompact MALDI II
mass spectrometer (Kratos Analytical) in positive, linear
mode with delayed extraction MALDI source and a
nitrogen laser (337 nm). ESI-IT mass spectra were
recorded on a Bruker Daltonics esquire 3000+ in posi-
tive or negative mode with a flow rate of 3 ll/min.
DNA oligonucleotides were synthesized on an Ap-
plied-Biosystems 392 DNA/RNA-synthesizer employing
a standard phosphoramidite strategy. Flash chromato-
graphy: Merck silica gel G60 (230–400 mesh). Thin-
layer chromatography: Merck precoated plates (silica
gel 60 F254). Reversed-phase HPLC was performed on
a prominence-line HPLC (Shimadzu) with a Nucleosil-
100-5-(250/4)-C18-column from Macherey-Nagel and a
binary gradient system (TEAA-buffer (0.1 M), acetonitrile).
4.1.3. S-Acetyl-5-(mercaptomethyl)-20-deoxyuridine (4).
Compound 3 (440 mg, 0.81 mmol) was deprotected in a
mixture (20 ml) of 75% acetic acid and THF (4:1). The
reaction was monitored by TLC (CH2Cl2–methanol,
95:5). After complete conversion (24 h), the reaction
mixture was evaporated to dryness and purified by col-
umn chromatography (CH2Cl2–methanol, 95:5) to yield
1
187 mg (73%) of 4. H NMR (250 MHz, MeOH-d4) d
2.07–2.23 (m, 2H, H20A and H20B), 2.35 (s, 3H,
(CO)CH3), 3.71–3.86 (m, 4H, H50A, H50B and SCH2)
3.93 (dt, 1H, J = 3.7 Hz and 4.3 Hz, H40), 4.41 (dt,
1H, J = 2.7 Hz and 4.3 Hz, H30), 6.22 (dd, 1H,
J = 6.7 Hz and 6.7 Hz, H10), 8.00 (s, 1H, H6); Calcd
for C12H16N2O6S: C, 45.56; H, 5.10; N, 8.86. Found:
C, 45.33; H, 5.19; N, 8.87; MS (ESI-MS, MeOH): Calcd
for C12H16N2O6S [M+H]+: 316.07, C12H16N2O6S
[M+Na]+: 338.07. Found: 317.1, 338.1.
4.1.4. S-Acetyl-50-O-(dimethoxytrityl)-5-(mercaptometh-
yl)-20-deoxyuridine (5).
A solution of 4 (180 mg,
0.57 mmol) in dry pyridine (10 ml) under argon atmo-
sphere was cooled to 0 ꢁC, dimethoxytritylchloride
(212 mg, 0.63 mmol) and DMAP (7 mg, 0.06 mmol)
were added. The reaction was kept at 0 ꢁC for 24 h
and then quenched with 1 ml methanol. The solution
4.1.1. 5-Bromomethyl-30,50-bis-O-(tert-butyldimethylsi-
lyl)-20-deoxyuridine (2). To a solution of 113 (1.00 g,

6238
B. Bornemann, A. Marx / Bioorg. Med. Chem. 14 (2006) 6235–6238
was evaporated to dryness, dissolved in CH2Cl2 (10 ml),
and washed with saturated NaHCO3 solution. The
organic phase was dried with magnesium sulfate, evapo-
rated to dryness, and purified by column chromatogra-
phy (1% Et3N in ethyl acetate) to yield 317 mg (90%)
phosphoramidite 8 (0.1 M in acetonitrile). The synthesis
of oligonucleotides was carried out on a DNA-synthe-
sizer on 0.2 lmol scale applying commercially available
2-cyanoethylphosphoramidites.
A standard method
for 2-cyanoethylphosphoramidites was used, with the
exception that the coupling time from the modified
nucleotides was extended to 10 min. Yields for modified
oligonucleotides are comparable to those obtained for
unmodified oligonucleotides. The synthesized oligonu-
cleotide sequences are listed in Table 1. Following
deprotection strategy was used: The solid-phase support
was treated with 1 ml of 10% DBU in acetonitrile for
30 min followed by washing with 5 ml acetonitrile. The
deprotection with ammonium hydroxide (33%) occurred
in the presence of excess DTT (0.2 M) at 56 ꢁC for 16 h.
This deprotection procedure leads to the desired product
and a minor product with a mass 120 Da higher, pre-
sumably the DTT-adduct, which can be easily separated
by HPLC. The oligonucleotides were purified by RP-
HPLC with a binary gradient system (A: 0.1 M
TEAA-buffer and B: acetonitrile) and a gradient of 5–
20% B over 25 min. Integrities of all modified oligonu-
cleotides were confirmed by ESI MS.
1
of 5. H NMR (400 MHz, acetone-d6): d 2.06–2.09 (m,
2H, H20A and H20B), 2.20 (s, 3H, (CO)CH3), 3.29–
3.55 (m, 4H, H50A, H50B and SCH2), 3.79 (s, 6H,
OCH3), 4.06 (dt, 1H, J = 4.0 Hz and 4.4 Hz, H40), 4.51
(dt, 1H, J = 4.0 Hz and 6.0 Hz, H30), 6.29 (dd, 1H,
J = 6.6 Hz and 6.6 Hz, H10), 6.89 (d, 4H, J = 9.0 Hz,
Ar), 7.23 (t, 1H, J = 7.3 Hz, Ar), 7.32 (dd, 2H,
J = 7.3 Hz and 7.5 Hz, Ar), 7.38 (d, 2H, J = 9.0 Hz,
Ar), 7.39 (d, 2H, J = 9.0 Hz, Ar), 7.51 (d, 2H,
J = 8.5 Hz, Ar), 7.77 (s, 1H, H6), 10.25 (br s, 1H,
13
NH); C NMR (100 MHz, acetone-d6): d 42.0 (C20),
56.5 (OCH3), 65.8 (C50), 73.0 (C30), 86.6 (C10), 88.0
(C40), 88.3 (CAr3), 111.9 (C5), 115.0 (Ar), 127.2 (Ar),
128.7 (Ar), 129.8 (Ar), 130.1 (Ar), 130.9 (Ar), 132,1
(Ar), 132.1 (Ar), 137.8 (C6), 147.1 (C2) 160.8 (C4),
196.8 (CO); MS (ESI-MS, acetone): Calcd for
C33H34N2O8S
[M+H]+:
619.20,
C33H34N2O8S
[M+Na]+: 641.20, C33H34N2O8S [M+K]+: 657.18.
Found: 619.1, 641.1, 657.1.
4.1.5.
S-Acetyl-40-O-(2-cyanoethoxy)(diisopropylami-
Acknowledgments
no)phosphino-50-O-(dimethoxytrityl)-5-(mercaptometh-
yl)-20-deoxyuridine (6). To a solution of 5 (100 mg,
0.162 mmol) in dry CH2Cl2 (5 ml) under argon atmo-
sphere triethylamine (82 mg, 0.81 mmol) and 2-cyano-
ethyl-N,N-diisopropylchlorophosphoramidite (77 mg,
0.32 mmol) were added. The reaction was monitored
by TLC (petrol ether–ethyl acetate, 1:2). After complete
conversion (4 h), the reaction mixture was evaporated to
dryness and purified by column chromatography (1%
Et3N in petrol ether–ethyl acetate, 1:2) to yield 88 mg
(66%) of 6. 1H NMR (600 MHz, acetone-d6): d 1.18
(d, 6H, J = 7.5 Hz, NCH(CH3)), 1.20 (d, 6H,
J = 7.5 Hz, NCH(CH3)), 2.20 (s, 3H, (CO)CH3), 2.36–
2.48 (m, 2H, H20A and H20B), 2.64 (t, 2H, J = 5.9 Hz,
CH2CN), 3.44–3.48 (m, 2H, H50A and H50B), 3.62–
3.68 (m, 2H, CH25), 3.70–3.76 (m, 2H, N(CH)), 3.80
(s, 6H, OCH3), 4.17–4.22 (m, 1H, H40), 4.66–4.73 (m,
1H, H30), 6.29 (dd, 1H, J = 6.6 Hz and 6.6 Hz, H10),
6.91 (d, 4H, J = 9.0 Hz, Ar), 7.25 (t, 1H, J = 7.3 Hz,
Ar), 7.34 (dd, 2H, J = 7.7 Hz and 7.7 Hz, Ar), 7.41 (d,
4H, J = 8.9 Hz, Ar), 7.53 (d, 2H, J = 7.3 Hz, Ar),
7.80 (s, 1H, H6), 10.16 (br s, 1H, NH); 13C NMR
We gratefully acknowledge financial support by the
DFG within the framework of the SFB 513. The assis-
tance of Dr. Karl-Heinz Jung in manuscript preparation
is kindly acknowledged.
References and notes
1. Bioconjugation Protocols (Methods in Molecular Biology
Series Vol. 283), Niemeyer, C. M., Ed.; Humana Press:
Totowa, 2004.
2. DNA and Aspects of Molecular Biology (Comprehensive
Natural Products Chemistry Vol. 7); Kool, E. T., Ed.;
Pergamon Press: Oxford, 2002.
3. Beaucage, S. L.. In DNA and Aspects of Molecular Biology
(Comprehensive Natural Products Chemistry); Kool, E. T.,
Ed.; Pergamon Press: Oxford, 2002; Vol. 7, pp 153–249.
4. Bain, C. D.; Whitesides, G. M. Angew. Chem., Int. Ed.
Engl. 1989, 28, 506.
5. Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff,
J. J. Nature 1996, 382, 607.
6. Alivisatos, A. P.; Johnsson, K. P.; Peng, X.; Wilson, T. E.;
Loweth, C. J.; Bruchez, M. P., Jr.; Schultz, P. G. Nature
1996, 382, 609.
7. Pale-Grosdemange, C.; Simon, E. S.; Prime, K. L.;
Whitesides, G. M. J. Am. Chem. Soc. 1991, 113, 12.
8. Connolly, B. A.; Rider, P. Nucl. Acids Res. 1985, 13, 4485.
9. Sinha, N. D.; Cook, R. M. Nucl. Acids Res. 1988, 16,
2659.
(150 MHz, acetone-d6):
d
20.6 (CH2CN), 24.4
(CH(CH3)2), 26.5 (5-CH2), 30.1 ((CO)CH3), 40.0 (C20),
43.9 (NCH), 44.0 (NCH), 55.0 (OCH3), 64.1 (C50),
74.0 (C30), 85.7 (C10), 86.1 (C40), 87.3 (CAr3), 110.9
(C5), 114.0 (Ar), 118.8 (CN), 127.6 (Ar), 128.7 (Ar),
129.1 (Ar), 131.1 (Ar), 136.6 (C6), 138.9 (Ar), 145.9
(Ar), 150.8 (C2), 159.7 (Ar), 162.9 (C4), 195.5 (CO);
MS (ESI-MS, acetone): Calcd for C42H51N4O9PS
[M+Na]+: 841.31, C42H51N4O9PS [M+K]+: 857.28.
Found: 842.0, 857.9.
10. Cohen, H.; Nogues, C.; Naaman, R.; Porath, D. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102, 11589.
11. Nogues, C.; Cohen, S. R.; Daube, S. S.; Naaman, R. Phys.
Chem. Chem. Phys. 2004, 6, 4459.
12. Goodwin, J. T.; Glick, G. D. Tetrahedron Lett. 1993, 34,
5549.
4.2. Synthesis of modified oligonucleotides
13. Negron, G.; Calderon, G.; Vazquez, F.; Lomas, L.;
Cardenas, J.; Marquez, C.; Gavino, R. Synth. Commun.
2002, 32, 1977.
For the synthesis of oligonucleotides S1 and S2 solid-
phase support 6 was used, in all other cases the modified