Incorporation of positively charged deoxynucleic S-methylthiourea linkages into oligodeoxyribonucleotides

Article abstract of DOI:10.1016/S0960-894X(01)00455-3

Oligodeoxyribonucleic acids (15- and 18-mers) containing both negatively charged phosphate and positively charged S-methyl thiourea internucleoside linkages (DNmt/DNA chimera) have been synthesized. DNA binding characteristics and nuclease resistance of DNmt/DNA chimera have been evaluated.

Full text of DOI:10.1016/S0960-894X(01)00455-3

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2423–2427
Incorporation of Positively Charged Deoxynucleic
S-Methylthiourea Linkages into Oligodeoxyribonucleotides
Hemavathi Challa and Thomas C. Bruice*
Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106, USA
Received 8 May 2001; accepted 21 June 2001
Abstract—Oligodeoxyribonucleic acids (15- and 18-mers) containing both negatively charged phosphate and positively charged S-
methyl thiourea internucleoside linkages (DNmt/DNA chimera) have been synthesized. DNA binding characteristics and nuclease
resistance of DNmt/DNA chimera have been evaluated. # 2001 Elsevier Science Ltd. All rights reserved.
The ability to introduce reporter groups into oligonu-
cleotides confers additional functionality and unique
properties to the oligonucleotides.1,2 These functional-
ized oligonucleotides can be used to study the structure
and hybridization properties of nucleic acids. Alter-
natively, they can be used to alter the solubility and
stability of the nucleic acids by choosing an appropriate
reporter group. While using covalently attached repor-
ter groups to study the hybridization properties of
nucleic acids, it is desirable not to change the natural
hybridization properties of the nucleic acid under study.
Thus, the presence of the reporter group on the back-
bone is less likely to disrupt the structure, unless the
probe is designed to achieve this. Of the several mod-
ified DNA/RNA backbones, three have been employed
for the attachment of reporter groups, viz., phosphoro-
thioates,3 phosphoramidates,4 and phosphotriesters.5
The attachment of reporter groups to these backbones is
limited by stereoisomeric complexity during synthesis.
To overcome this drawback, we incorporated neutral
thiourea linkages into otherwise negatively charged
DNA. The thio functionality now allows for introduc-
tion of any reporter group as a post-synthetic modifica-
tion devoid of stereoisomers. Preliminary study
involving the incorporation of S-methylated thiourea
into DNA to produce DNmt/DNA chimera (Fig. 1) is
reported. The hybridization properties of the DNmt/
DNA chimera with complementary DNA were eval-
uated using spectroscopic techniques. The stability of
the DNmt/DNA chimera towards nucleolytic cleavage
was also investigated.
To facilitate solid-phase synthesis of oligonucleotide
chimera containing both the standard phosphate and S-
methyl thiourea linkages,6 phosphoramidite 6 was syn-
thesized (Scheme 1). The synthesis of 6 was accom-
plished via the dimer 5, which involves coupling of a 30-
isothiocyanate 37 with a 50-amino group of 50-amino-50-
deoxythymidine 4. Dimer 58 was activated for use in
solid-phase synthesis by phosphitylation using [chloro-
(diisopropylamino)-b-cyanoethoxyphosphine] to give the
desired phosphoramidite 6.9 Both 3 and 4 were readily
obtained starting from thymidine. Isothiocyanate 3 was
synthesized in six steps involving selective hydroxyl
protection, formation of 30-azidothymidine through a
2,30-anhydrothymidine intermediate, hydrogenation of
*Corresponding author. Tel.: +1-805-893-2044; fax: +1-805-893-
2229; e-mail: tcbruice@bioorganic.ucsb.edu
Figure 1. Structures of DNA (A), DNmt (B), and DNmt/DNA chi-
mera (C).
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00455-3

2424
H. Challa, T. C. Bruice /Bioorg. Med. Chem. Lett. 11 (2001) 2423–2427
the 30-azide, and, finally reacting the corresponding 30-
amino - 50 - O - (diphenylmethoxy)trityldideoxythymidine
210 with thiopyridone at room temperature in pyridine.
Monomer 4 was obtained in three steps as previously
reported.11
K)+; calculated for {M+H}+ 5465.0]. Thus, DNmt/
DNA chimeras were successfully synthesized using
automated solid-phase synthesis methods.
Two 15-mer single base sequences, 1 and 2, and an 18-
mer mixed base sequence, 3, were used to evaluate the
hybridization properties of DNmt/DNA chimera (Fig.
2).13 Sequences 1 and 3 contain two S-methyl thiourea
linkages, one at each end of the sequence; while
sequence 2 has a single S-methyl thiourea linkage posi-
tioned at the center of the strand. All the three sequen-
ces were hybridized with their respective complementary
strands in a 1:1 mol ratio in phosphate buffer (10 mM
Na2HPO4, pH 7.1, 0/10/100 mM NaCl). The CD spec-
tra (Fig. 3)14 of the DNmt/DNA hybrids (1ꢀ 7 and 2ꢀ 7)
are very similar to the CD spectrum reported15 for
dT19ꢀ dA19 duplex indicating that both 1 and 2 form
duplexes. UV–thermal denaturation studies were per-
formed to observe the effect of the S-methyl thiourea
linkage on the formation of duplex. All of the tempera-
ture versus absorbance curves were sigmoidal, indicat-
ing that double helix formation is cooperative (Fig. 4).
It is observed that incorporation of one or two S-methyl
thiourea linkages (DNmt/DNA sequence 1 or 2) has no
effect on the hybridization properties with com-
Phosphoramidite 6 was used as a building block to
introduce thiourea linkages at desired positions in the
chimeric oligonucleotides.12 The chimeras were synthe-
sized with the 50-trityl group on to allow for HPLC
purification. The thiourea linkages in the oligonucleo-
tide were methylated while on solid support (CPG). In
order to insure methylation occurred only on the
thiourea linkages and not on the nucleotide bases, short
pentamer sequences (50-TT [T/A/C/G] TT-30) were syn-
thesized and treated under the employed methylation
conditions. Both HPLC and mass spectrometry (ESI)
indicated that the methylation condition used does not
methylate the nucleobases. The final detritylated and
HPLC purified oligonucleotides were analyzed by mass
spectrometry (ESI) to be the desired chimeric products:
1 [m/z=4484.0, 4506.0, 4522.0 (M+H/Na/K)+; calcu-
lated for {M+H}+ 4483.8], 2 [m/z=4492.0, 4514.0,
4530.0 (M+H/Na/K)+; calculated for {M+H}+
4491.8], 3 [m/z=5465.5, 5487.5, 5503.5 (M+H/Na/
Scheme 1. Preparation of dimer synthon (6) for DNmt/DNA synthesis. (a) Mmt-Cl, DMAP/TEA, anhyd pyridine, rt, 73%; (b) methanesulfonyl
chloride, TEA, dichloromethane, 82%; (c) phthalimide, DMF/H2O, ꢁ100 ꢂC, 86%; (d) LiN3/DMF, ꢁ100 ꢂC, 70%; (e) Pd/C, EtOH/H2O, 86%; (f)
thiocarbonylpyridone, dichloromethane, rt, 90%; (g) p-toluene-sulfonyl chloride, pyridine, 0 ꢂC–rt, 65%; (h) LiN3/DMF, ꢁ100 ꢂC; (i) Pd/C, EtOH/
H2O, 99%; (j) 3, DMAP, pyridine, 96%; (k) [chloro-(diisopropylamino)-b-cyanoethoxyphosphine], DIEA, dichloromethane.

H. Challa, T. C. Bruice /Bioorg. Med. Chem. Lett. 11 (2001) 2423–2427
2425
plementary DNA sequence 7 at all salt concentrations
(Table 1). A slight destabilization of the DNmt/
DNAꢀ DNA duplex occurs when compared with the
control DNAꢀ DNA duplex, as reported for the DNG/
DNA chimera.16 For the duplex formed with the mixed
base DNmt/DNA chimera, 3ꢀ 5, no apparent change in
Tm occurs between the control duplex (4ꢀ 5) and 3ꢀ 5
duplex at all (0, 10, and 100 mM) salt concentrations.
The stability of the DNmt/DNAꢀ DNA duplexes (1ꢀ 7,
2ꢀ 7, and, 3ꢀ 5) increases with the increase in salt con-
centration (0–100 mM NaCl) as seen for DNAꢀ DNA
duplexes (6ꢀ 7 and 4ꢀ 5). This indicates that the incor-
poration of one or two S-methyl thiourea linkages in
place of the phosphodiester linkages of the normal
DNA does not affect the overall electrostatics of duplex
formation with complementary DNA strand.
The ÁG values (at 25 ꢂC) calculated using the van’t
Hoff enthalpy values for transitions involving DNmt/
DNAzꢀ DNA and DNAꢀ DNA duplexes, indicate that
the free energy for formation of DNmt/DNAꢀ DNA
duplex is about the same as for DNAꢀ DNA duplex.17ꢃ19
This may support that little structural difference exists
between DNA containing one or two S-methyl thiourea
linkages and DNA composed entirely of phosphodiester
linkages.
In order to investigate the sequence specificity of bind-
ing of DNmt/DNA chimera with complementary DNA,
duplexes were formed between DNmt/DNA chimera (1
and 2) and DNA sequences containing single base mis-
matches at 30-end (8), 50-end (9), or in the center (10).
The stability of the duplex was monitored by thermal
denaturation studies. In general (Table 2), the central
mismatch destabilizes the duplex (DNAꢀ DNA or
Figure 2. Sequences used for DNmt/DNA characterization. S-Methyl
thiourea linkage is indicated by ‘m’.
Table 1. Melting temperatures and thermodynamic parameters for
helix–coil transitions of DNmt/DNA chimeras with complementary
DNA
0 mM NaCl
10 mM NaCl
100 mM NaCl
Duplexa Tm
ꢃÁG25
Tm
ꢃÁG25
Tm
ꢃÁG25
kJ/mol
b
c
b
c
b
c
ꢂC
kJ/mol
ꢂC
kJ/mol
ꢂC
6ꢀ 7
1ꢀ 7
2ꢀ 7
4ꢀ 5
3ꢀ 5
19.8
17.6
17.8
31.7
32.1
27.3
24.6
25.0
38.6
38.8
23.6
20.3
20.2
36.0
36.1
30.8
28.3
28.0
43.5
43.0
33.4
30.0
29.9
47.0
46.1
38.4
35.7
36.1
50.9
49.6
Figure 3. CD spectra of duplexes formed by annealing 1:1 mol ratio of
1, 2, 3, 4, and 6 with their respective complementary strands (7 or 5) in
phosphate buffer (10 mM Na2HPO4, 100 mM NaCl, pH 7.1), at 15 ꢂC.
aAbsorbance was measured at 260 nm in phosphate buffer; the con-
centration of each strand was 6 mM.
bThe maximum of the derivative plot (Tmax) was multiplied by 0.971 to
obtain the Tm.18 The reported Tm values are an average of three
experiments (ꢅ0.2).
cThermodynamic parameters were calculated by the method of Gralla
and Crothers.17,19
Table 2. Melting temperatures for hybrids formed between DNmt/
DNA chimera and sequences containing single base-pair mismatches
Xꢀ Y Duplexa Tmb (ꢂC)
X=Y
7
8
9
10
6
1
2
33.4
30.0
29.9
28.7
26.5
24.4
30.0
28.5
26.2
24.2
19.8
22.2
aAbsorbance was measured at 260 nm; the concentration of each
strand was 6 mM in 10 mM Na2HPO4, 100 mM NaCl, pH 7.1.
bThe maximum of the derivative plot (Tmax) was multiplied by 0.971 to
obtain the Tm.18 The reported Tm values are an average of three
experiments (ꢅ0.2).
Figure 4. Thermal denaturation curves for 6ꢀ 7 (^), 1ꢀ 7 (*), 2ꢀ 7 (&),
4ꢀ 5 (ꢄ), and, 3ꢀ 5 (+) duplexes. Absorbance was measured at 260 nm;
the concentration of each strand was 6 mM in 10 mM Na2HPO4,
100 mM NaCl, pH 7.1.

2426
H. Challa, T. C. Bruice /Bioorg. Med. Chem. Lett. 11 (2001) 2423–2427
DNmt/DNAꢀ DNA) to the maximum extent (9.2 ꢂC for
6ꢀ 10, 10.2 ꢂC for 1ꢀ 10, and 7.7 ꢂC for 2ꢀ 10). There is a
ÁTm of 1.3 ꢂC–2.0 ꢂC between the 30end and 50end mis-
match for duplexes 6ꢀ 10, 1ꢀ 10, and 2ꢀ 10. In all cases, the
30-end mismatch is more tolerated than the 50-end mis-
match. Thus, the DNmt/DNA chimeras clearly bind to
their complementary DNA with great sequence specifi-
city.
References and Notes
1. Goodchild, J. Bioconjugate Chem. 1990, 1, 165.
2. Manoharan, M. In Antisense Research and Applications;
Crooke, S. T., Lebleu, B., Eds.; CRC: Boca Raton, 1993; p
303.
3. Fidanza, J. A.; Ozaki, H.; McLaughlin, L. W. J. Am.
Chem. Soc. 1992, 114, 5509.
4. Jager, A.; Levy, M. J.; Hecht, S. M. Biochemistry 1988, 27,
7237.
Exonuclease I digests single-stranded DNA catalyzing
the hydrolysis of phosphosdiester linkages from the 30-
terminus to 50-terminus. Thus, it is assumed that upon
modifying the phosphodiester linkage at the 30-termi-
nus, the oligonucleotide could be resistant to exonu-
clease digestion. To investigate this, DNmt/DNA
oligonucleotides 1–3 were subjected to nucleolytic clea-
vage by exonuclease I and the hydrolyzate was analyzed
by RP-HPLC.20 Natural unmodified oligonucleotides of
the same sequence, 4 and 5, were used as controls.
Under the conditions employed, the control oligonu-
cleotides were readily hydrolyzed to shorter length pro-
ducts within 1 h of incubation; however, the DNmt/
DNA chimera 1 and 3 were unaltered even after 12 h of
incubation. The DNmt/DNA chimera 2 (rt 20.8 min),
which contains only one methyl thiourea linkage at the
center of the oligonucleotide, was partially hydrolyzed
after 1 h (rt 19.5 and 20.8 min) of incubation and
remained further unaltered even after 12 h (rt 19.5 and
20.8 min). These observations clearly indicate that the
DNmt/DNA oligonucleotides with positively charged
methyl thiourea linkages at 30-terminus, 1 and 3, are
totally stable to cleavage by nucleolytic enzyme exonu-
clease I.
5. Asseline, U.; Delarue, M.; Lancelot, G.; Toulme, F.;
Thuong, N. T.; Montenay-Garestier, T.; Helene, C. Proc.
Natl. Acad. Sci. U.S.A. 1984, 81, 3297.
6. Arya, D. P.; Bruice, T. C. J. Am. Chem. Soc. 1998, 120,
6619.
7. 3: To a solution of 1.10 g (2.14 mmol) of 30-amino-50-O-(4-
methoxyphenyl)-diphenylmethyl-30-deoxythymidine in 75 mL
of dichloromethane was added 0.51 g (2.20 mmol) of thio-
carbonylpyridone. The resulting solution was stirred at rt for
6 h. The reaction was monitored by TLC to completion and
the solvent was evaporated to dryness under reduced pressure.
The crude residue was subjected to column chromatography
using EtOAc/hexanes (1:1) solvent system containing 0.5%
Et3N to give the pure product as a brittle white foam. Yield
was 0.92 g (90%). Rf 0.38 (EtOAc/hexanes, 1:1); 1H NMR
(400 MHz, CDCl3) d 8.2 (s, br, 1H), 7.6 (s, 1H), 7.2–7.4 (m,
12H), 6.9 (d, 2H), 6.3 (t, 1H), 4.6 (q, 1H), 4.2 (m, 1H), 3.8 (s,
3H), 3.4–3.6 (2ꢄd, 2H), 2.5–2.7 (m, 2H), 1.5 (s, 3H); 13C NMR
(100 MHz, CDCl3) d 160.0, 150.0, 144.0, 136.1, 131.3, 129.0,
128.0, 114.2, 85.0, 84.0, 63.0, 56.1, 40.0, 12.7. m/z (HRESI)
578.1703, calcd for C31H29N3O5S (M+Na)+ 578.1726.
8. 5: To a solution of 0.30 g (0.55 mmol) of 30-isothiocyanato-
50-O-(4-methoxyphenyl)-diphenylmethyl-30-deoxythymidine (3)
in 30 mL of anhydrous pyridine was added 0.31 g (1.29 mmol)
of 50-amino-50-deoxythymidine (4) followed by 15.0 mg
(0.12 mmol) of (dimethylamino)pyridine. The resulting solu-
tion was stirred for 2 h at rt. The solvent was evaporated at
reduced pressure, and 30 mL of water was added to the residue
to precipitate the product. The product was extracted into
2ꢄ30 mL of CHCl3 and washed with water to remove the
excess amine. The combined organic extracts were dried over
anhydrous Na2SO4 and evaporated under reduced pressure to
give a white flaky product. Yield was 0.42 g (96%). Rf 0.43
(CH2Cl2/MeOH, 9:1); 1H NMR (400 MHz CDCl3) d 7.9 (s, br,
1H), 7.6 (s, 1H), 7.2–7.5 (m, 12H), 7.18 (s, 1H), 6.9 (d, 2H),
6.41 (m, 1H), 6.05 (m, 1H), 5.35 (m, 1H), 4.5 (m, 1H), 4.2 (m,
1H), 3.9 (m, 1H), 3.8 (s, 3H), 3.7 (m, 1H), 2.4–3.4 (m, 4H), 1.9
(s, br, 2H), 1.45 (s, 3H), 1.15 (s, 3H). m/z (HRESI) 819.2784,
calcd for C41H44N6O9S (M+Na)+ 819.2788.
9. 6: m/z (FAB) 997.0, calcd for C50H61N8O10SP (M+H)+
997.1.
10. Dempcy, R. O.; Almarsson, O.; Bruice, T. C. Proc. Natl.
Acad. Sci. U.S.A. 1994, 91, 7864.
11. Horwitz, J. P.; Tomson, A. J.; Urbanski, J. A.; Chua, J. J.
Org. Chem. 1962, 27, 3045.
12. Oligonucleotide synthesis and purification: All modified
oligonucleotides were synthesized on 1.3 mmol scale on a Phar-
macia Gene Assembler Plus DNA synthesizer. Standard DNA
synthesis conditions were employed, viz., CPG support and base
protected 50-O-(4,40-dimethoxytrityl)deoxyribonucleoside-3-[O-
(diisopropylamino)-b-cyanoethylphosphoramidite] monomers.
The phosphoramidite activated dimer 6 was used, at an extra
monomer position, in order to introduce a thiourea linkage
into the oligonucleotide. The standard synthesis cycle was
modified to perform an extended coupling (15 min) during the
coupling of the modified phosphoramidite dimer 6; a coupling
efficiency of >95% was observed for this step. The resin con-
taining the fully protected oligonucleotide was treated with
MeI/EtOH (1:1) for 1 h at rt in a peptide synthesis vessel to
In conclusion, a method for the incorporation of a
positively charged internucleoside linkage, S-methyl
thiourea, into otherwise negatively charged DNA has
been demonstrated. The insertion of the methyl thiourea
linkage was accomplished using the standard DNA
phosphoramidite chemistry and automated solid-phase
synthesis techniques. The binding of the DNmt/DNA
chimera to its complementary DNA strand occurs as
with the unmodified DNAꢀ DNA duplex exhibiting
similar stability trends with increasing salt conditions.
Also, a high degree of sequence specificity is displayed
during the binding of DNmt/DNA chimera to its com-
plementary DNA. Incorporation of positively charged
S-methyl thiourea linkages at the 30-terminus of the
DNA confers resistance towards digestion by exonu-
clease I. Thus, DNmt/DNA is a molecule with binding
characteristics similar to DNA but with enhanced
nuclease resistance. The nucleophilic nature of the thio
functionality in the molecule may enable one to
introduce fluorescent molecules into DNA by alkyla-
tion for developing molecular biology tools or insert
groups that may enhance cellular uptake of oligonu-
cleotides.
Acknowledgements
This work was supported by a grant from the National
Institutes for Health, NIH Grant 5R37DK09171-36.

H. Challa, T. C. Bruice /Bioorg. Med. Chem. Lett. 11 (2001) 2423–2427
2427
methylate the thiourea linkages. Cleavage and deprotection of
the oligonucleotides was achieved by subjecting the resin to a
solution of K2CO3 in methanol (0.05 M) for 2 h at rt. All oli-
gonucleotides were desalted on polypak catridges prior to
purification on RP-HPLC. A semipreparative C-8 RP column
(Altech, 7 m, 10ꢄ250 mm) with 0.1 M TEAA, pH 7.0 as solvent
A and CH3CN as solvent B was used for all HPLC purifica-
tions with a gradient of 0.2% B and 3 mL/min flow rate. The
HPLC-purified trityl-on oligonucleotides were then detrity-
lated using 1 mL of 80% acetic acid (1 h, rt), lyophilized,
redissolved in double distilled water, and further purified by
RP-HPLC.
13. TtT and TmT refer to thymidyl dinucleotide with a
thiourea linkage and methylated thiourea linkage, respectively.
All nucleotide solution concentrations were determined using
the extinction coefficients (per mole of nucleotide) calculated
according to nearest neighbor approximation for the DNA
(Gray, D. M.; Hung, S.-H.; Johnson, K. H. In Methods in
Enzymology; Academic: New York, 1995; Vol. 246, p 19). All
standard unmodified DNA oligomers were obtained from
Integrated DNA Technologies (Coralville, IA).
block. Absorbance measurements were made at 260 nm using
1 cm path length quartz cuvettes at a heating rate of 0.2 ꢂC/
min over the range of 8–70 ꢂC.
15. Hopkins, H. P.; Hamilton, D. D.; Wilson, W. D.; Zon, G.
J. Phys. Chem. 1993, 97, 6555.
16. Barawkar, D. A.; Bruice, T. C. Proc. Natl. Acad. Sci.
U.S.A. 1998, 95, 11047.
17. Breslauer, K. J. In Methods in Molecular Biology; Agra-
wal, S., Ed.; Humana: Totowa, 1995; Vol. 26, p 347.
18. Johnson, W. T.; Zhang, P.; Bergstrom, D. E. Nucleic
Acids Res. 1997, 25, 559.
19. Gralla, J.; Crothers, D. M. J. Mol. Biol. 1973, 78, 301.
20. Exonuclease I digestion studies: Oligonucleotides 1–4 and
6, with phosphodiester or S-methyl thiourea linkages, were
treated with exonuclease I (USB). A typical reaction mixture
.
contained the following in 200 mL: 67 mM Tris HCl (pH 8.5),
6.7 mM MgCl2, 20 mM 2-mercaptoethanol, ꢁ0.2 OD of oli-
gonucleotide, and ꢁ20 units of exonuclease I. The reactions
were incubated at 37 ꢂC. Aliquots (40 mL) were taken at dif-
ferent time intervals (0, 1, 3, 6, and 12 h), quenched by rapid
freezing in dry ice–2-propanol bath, and stored frozen until
HPLC analysis. Reaction mixtures were analyzed on C-8 RP-
HPLC column (Altech, 7 m, 4.6ꢄ250 mm) using a gradient of
0.5%/min of CH3CN in 0.1 M TEAA, pH 7.0, for 50 min at a
flow rate of 1 mL/min. Reactions without enzyme were run for
each oligonucleotide and analyzed by HPLC as controls.
14. Spectroscopy: CD spectra were obtained using AVIV cir-
cular dichroism spectrophotometer. Scans were run from
300 nm to 200 nm taking a measurement every 1 nm. Melting
curves were determined using a Cary 100 UV–vis spectro-
photometer equipped with a temperature programmable cell