Synthesis and characterization of a DNA analogue stabilized by mercapto C-nucleoside induced disulfide bonding

Article abstract of DOI:10.1016/j.bmcl.2004.03.015

A redox-active nucleobase analogue of a nucleotide was synthesized and incorporated into DNA using phosphoramidite chemistry. An analogue-containing oligonucleotide in the absence of a reducing reagent formed a stable duplex with a substantially higher me

Full text of DOI:10.1016/j.bmcl.2004.03.015

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2459–2462
Synthesis and characterization of a DNA analogue stabilized by
mercapto C-nucleoside induced disulfide bonding
Akihiko Hatano,a,* Seiji Makitab and Masayuki Kiriharaa
aDepartment of Materials Science, Shizuoka Institute of Science and Technology, 2200-2 Toyosawa, Fukuroi, Shizuoka 437-8555,
Japan
bResearch Center for Molecular-scale Nanoscience, Institute for Molecular Science, 38 Myodaiji, Okazaki 444-8585, Japan
Received 10 September 2003; revised 4 March 2004; accepted 4 March 2004
Abstract—A redox-active nucleobase analogue of a nucleotide was synthesized and incorporated into DNA using phosphoramidite
chemistry. An analogue-containing oligonucleotide in the absence of a reducing reagent formed a stable duplex with a substantially
higher melting temperature compared to that of a standard DNA duplex of the same length.
Ó 2004 Elsevier Ltd. All rights reserved.
The strategy of replacing the natural bases of DNA with
analogues has allowed the possibility of adding new
functions to the molecule. Efforts to expand the genetic
alphabet have been made with respect to artificial gene
control as well as the development of functionalized
biopolymers.1 Recently, investigations into duplex for-
mation, which depend on metal-mediated base pairing2
and interbase hydrophobic interactions,3 were reported.
Figure 1. Schematic representation of a DNA duplex induced by the
formation of disulfide bonding.
We are interested in increasing the information content
of DNA using unnatural nucleic acids, where the pairing
of the base analogues is driven by reversible covalent
bonding instead of hydrogen bonding. Thiol groups can
be easily oxidized into disulfides, and disulfide bonds
can be broken into thiol groups by reducing agents.
There are several papers in the literature describing the
application of disulfide cross-linked oligonucleotides for
supplying a conformational lock.4 However, there are
very few reports describing nonnatural DNA molecules
constructed in which base pairing is controlled by the
redox environment.5 Among nonnatural DNA mole-
cules, C-nucleotides are very interesting for their variety
of biological activity. C-nucleosides such as showdo-
mycine,6 have a broad spectrum of biological activity
and have stimulated considerable interest as potential
antitumor, anti-bacterial and anti-cancer agents.7 We
report here the synthesis of a novel thiophenol-bearing
b-C-nucleoside 3 and the influence of disulfide base
pairing on the thermal stability of the DNA duplex (Fig.
1).
The synthetic route to mercaptophenyl b-C-nucleoside 3
is shown in Scheme 1. We first tried the coupling reac-
tion of alkylation using the benzyl protected thiophenol
(1.5 equiv) and 3,5-ditoluoyl-1-a=b-methoxy-2-deoxy-D-
ribose8 1 (1 equiv) in the presence of a Lewis acid
(2 equiv SnCl4) in CH2Cl2 at )15 °C under an Ar
atmosphere. Although the yield of the Friedel–Crafts
alkylation was high (ꢀ65%), the S-benzyl group of the
product could not be deprotected with strong acids,
Birch reduction, etc. The effects of another protective
groups for thiophenol were investigated (Table 1). Each
thiophenol protected by S-p-nitrobenzyl, S-acetoami-
demethyl, S-cyanoethyl and S-benzoyl group did not
react with compound 1 under the same condition (ben-
zyl protected thiophenol). The p-methoxybenzyl pro-
tected thiophenol produced 9b and 9a. The free thiol
group attacked the anomeric carbon (10-position) of
compound 1 for cleavage of the protective group by the
Keywords: DNA; Disulfide base pair; C-nucleotides; Stable duplex.
* Corresponding author. Tel.: +81-538-450177; fax: +81-538-450110;
e-mail: a-hatano@ms.sist.ac.jp
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.03.015

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A. Hatano et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2459–2462
showed two doublet signals; d 7.23 and 7.25 ppm. The
coupling constant (J) values were 12.2 and 11.8 Hz,
respectively, and both signals were coupled. Thus the
phenyl group at the position para to the S–S moiety of
the diphenyldisulfide attacks at the 10b position on the
ribose, so that the thiol group is located in the p-position
towards the C-nucleoside 3. The mercaptophenyl b-C-
nucleoside 3 was further S-benzoylated (71%), 50-O-di-
methoxytritylated (71%) and 30-O-phosphitylated (89%)
to give 2-cyanoethyl phosphoramidite 7.14 Having
obtained compound 7, the oligonucleotides containing
the mercaptophenyl b-C-nucleoside were readily pre-
pared by standard automated DNA synthetic methods.
The S-benzoyl group was cleaved by a 27% ammonia
solution at 55 °C for 2 h with the cleavage of oligonu-
cleotide from the solid phase.
Nucleoside 3 was allowed to dimerize (in 1%-H2O2,
MeOH) and then analyzed by TLC. The Rf value (0.14)
of the dimer 4 is lower than the Rf value (0.34) of
monomer 3 (CHCl3–MeOH 4:1). After 30 min, the spot
corresponding to monomer 3 disappeared on the TLC,
and solvent was then removed under reduced pressure.
The crude product was chromatographed and recrys-
1
tallized from H2O to afford 4.15 The H NMR spectra
support the formation of a disulfide dimer by the
downfield shift observed for the phenyl protons (two
doublets) of the base analogue in the dimer 4 (d 0.13,
0.20 ppm, respectively, compared with those of the
monomer). The chemical shifts of the phenyl protons
can be rationalized in terms of expanding the p-conju-
gation from the analogue base unit to the disulfide
bonding. The electron impact (EI) mass spectrum also
provided clear evidence for the formation of dimer 4 by
oxidation (m=z 450).
Scheme 1. Synthetic route of target mercapto-nucleoside 3 and 7. (i)
Diphenyldisulfide, SnCl4 (2 equiv) in CH2Cl2 at )15 °C; (ii) 10%-
TsOH, TFA in CH2Cl2 at 0 °C; (iii) LiAlH4 in THF at 4 °C; (iv) 1%-
H2O2 in MeOH at rt; (v) BzCl, DIPEA in THF at 4 °C; (vi) DMTrCl in
pyridine at rt; (vii) DIPEA, 2-cyanoethyl-N,N0-diisopropyl-chloro-
phosphoramidite in CH2Cl2 at rt.
Lewis acid. Therefore, as the starting material for the
synthesis of mercaptophenyl b-C-nucleoside 3, the
readily available diphenyldisulfide was used. The diph-
enyldisulfide was coupled with compound 1 in the
presence of SnCl4 (2 equiv). The b-anomer 2 was sepa-
rated by column chromatography and recrystallization
to obtain the production 18% yield (a-anomaer
6.4%).9;10 It was found that 2a was converted to 2b in
the presence of 10%-TsOH in TFA and CH2Cl2 (1:4) at
0 °C to afford the b isomer (2b 21%) and a-isomer (2a
11%).11;12 The three protected groups, two toluoyl
groups and a mercaptophenyl group, were removed in
one step using LiAlH4 (37%).13 The stereochemistry at
We tested the stability and pairing ability of mercapto-
nucleoside 3 (S) in the non-self-complementary 15 mer
duplexes by thermal denaturation (Fig. 2).16 The ther-
mal denaturation experiments of the corresponding
duplex were determined by UV-melting curve analysis
and the corresponding Tm-data are summarized in Table
2.17 In the presence of mercaptoethanol, the melting
temperature of duplex IIIÆIV (33 °C) was similar to that
of the mismatch duplex IÆIV (32 °C). Thus, under
reducing conditions, the presence of the two base ana-
logues in IIIÆIV destabilizes the duplex structure to
approximately the same extent as a mismatch base
pair. The presence of mercaptoethanol had little influ-
ence on the melting temperature of duplexes IÆIV and
IÆVI.
1
the anomeric position of 3 was identified by H NMR,
1H–1H COSY and 1H NOE experiments. The b anomer
had a 10-resonance which appeared as a nearly evenly
spaced doublet of doublets (J ¼ 5:4 and 10.7 Hz). Sim-
ilar coupling constants have been reported for related
b-C-nucleosides.12 Irradiation of the 10H gave a NOE on
40H (5.1%) and 20Ha (3.0%). Similarly irradiation on
20Hb gave a NOE on 30H (7.1%), and irradiation on
20Ha yielded enhancements on 10H (9.1%).
In the absence of a reducing reagent, the melting tem-
perature of the duplex IIIÆIV was 73 °C. The elevated
stabilization of the duplex in the absence of a reducing
reagent was significantly greater than for the natural
duplex IÆII (DTm 29 °C). This result suggests that the
matching S–S base pair was formed by air oxidation.
The melting temperature of duplex IIIÆIV was higher
than duplex IÆII since the disulfide base pair interacts
more strongly (i.e. covalently) in comparison to the
natural A–T base pair (i.e. hydrogen bonding). Duplex
VÆVI contains each S nucleoside at the penultimate base
The regioselectivity of the electrophilic substitution
reaction was identified by the multiplicity of the phenyl
protons of compound 3 in the 1H NMR spectrum. Two
protons of the phenyl group of the base analogue

A. Hatano et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2459–2462
2461
Table 1. Coupling reaction of the compound 1 and the protected thiophenol by Friedel–Crafts alkylation
Reactant
Products/%
a-form
b-form
Trace
No reaction
No reaction
No reaction
No reaction
Figure 2. Melting curves for duplexes induced by the formation of disulfide bonding. (A) S nucleoside at the center position. (B) S nucleoside at the
penultimate position. See conditions in Table 2.
position. This duplex also displayed a high melting
temperature in the absence of mercaptoethanol (70 °C).
The mismatch sequence IÆVI had a similar Tm to
VÆVI in the presence mercaptoethanol (37, 39 °C,
respectively).
In summary, we have demonstrated a synthetic route to
a novel mercapto C-nucleoside and characterized a
disulfide base pair incorporated into an oligonucleotide.
The disulfide base pair is formed inside the duplex and
significantly increases the thermal stability.

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A. Hatano et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2459–2462
Table 2. Sequence information and effects of Tm of DNA including
mercapto-nucleoside 3 (S)
127.8, 129.2, 129.5, 129.7, 129.7, 136.6, 136.9, 143.9, 144.2,
166.1, 166.4; FABMS m=e 571 (M+H)þ; Anal. Calcd for
C33H30O5S2: C, 69.45; H, 5.30; N, 0.00; S, 11.24. Found:
C, 69.45; H, 5.26; N, 0.00; S, 11.18. 2a: 1H NMR (CDCl3):
d 2.27 (1H, m), 2.39 (3H, s), 2.40 (3H, s), 2.91 (1H, m),
4.55 (2H, m), 4.66 (1H, m), 5.34 (1H, J ¼ 6:6 Hz, t), 5.58
(1H, m), 7.24–8.00 (17H, m). 13C NMR (CDCl3): d 21.6,
40.2, 64.4, 76.1, 79.6, 82.1, 126.1, 126.4, 126.8, 126.9,
127.3, 127.5, 128.7, 128.8, 128.8, 129.3, 129.4, 135.7, 136.7,
141.5, 143.5, 143.6, 165.6, 165.9; FABMS m=e 571
(M+H)þ.
DNA
Sequences of duplex
Additive Tm/°C
I
50-CACATTAATGTTGTA
30-GTGTAATTACAACAT
a
44
43
II
b
III
IV
50-CACATTASTGTTGTA
30-GTGTAATSACAACAT
a
73
33
b
I
IV
50-CACATTAATGTTGTA
30-GTGTAATSACAACAT
a
32
32
b
10. The recovery of the diphenyldisulfide was 70%.
11. Jiang, Y. L.; Stivers, J. T. Tetrahedron Lett. 2003, 44, 4051.
12. Ren, R. X.-F.; Chaudhuri, N. C.; Paris, P. L.; Rumney, S.,
IV; Kool, E. T. J. Am. Chem. Soc. 1996, 118, 7671.
13. LiAlH4 (0.524 g, 13.8 mmol) was carefully added to a
solution of the bis-toluoylester 2 (1.58 g, 2.76 mmol) in dry
THF (50 mL). The reaction mixture was stirred at 4 °C for
1 h and then quenched by a 1 N H2SO4 aqueous solution
(3 mL). The residue was poured into 50 mL of 1 N HCl,
and then extracted with 50 mL of CH2Cl2 (eight times),
dried over anhydrous MgSO4, and evaporated. The crude
product was chromatographed with CHCl3–MeOH (9:1)
and then recrystallized from MeOH to afford the mercap-
tophenyl nucleoside (3) as colorless needles (0.231 g, 37%).
1H NMR (CD3OD): d 1.90 (1H, m), 2.15 (1H, J ¼ 2:4, 5.1,
5.1 Hz, ddd), 3.65 (2H, m), 3.91 (1H, J ¼ 3:9, 10.7 Hz, td),
4.29 (1H, br), 5.05 (1H, J ¼ 5:4, 10.7 Hz, dd), 7.23 (1H,
J ¼ 12:2 Hz, d), 7.25 (1H, J ¼ 11:8 Hz, d); 13C NMR
(CD3OD): d 44.9, 64.1, 74.5, 81.3, 89.2, 128.0, 129.9, 130.5
132.2, 140.4; EIMS m=e 226 [M]þ; Anal. Calcd for
C11H14O3S: C, 58.38; H, 6.24; N, 0.00; S, 14.17. Found:
C, 58.25; H, 6.18; N, 0.00; S, 14.18. e260: 7080 cmꢁ1 Mꢁ1 in
100 mM NaCl and 10 mM Na-phosphate, pH 7.0.
V
VI
50-CSCATTAATGTTGTA
30-GSGTAATTACAACAT
a
70
39
b
I
VI
50-CACATTAATGTTGTA
30-GSGTAATTACAACAT
a
37
38
b
[Mercaptoethanol]: a ¼ 0 lM, b ¼ 75 lM. [DNA] ¼ 15 lM/base pair in
10 mM Na–phosphate buffer, 100 mM NaCl, pH 7.0, 1 °C/min.
References and notes
1. (a) Switzer, C.; Moroney, S. E.; Benner, S. A. J. Am.
Chem. Soc. 1989, 111, 8322; (b) Piccirilli, J. A.; Krauch,
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Bain, J. D.; Switzer, C.; Chamberlin, A. R.; Benner, S. A.
Nature 1992, 356, 537.
2. (a) Ericson, A.; McCary, J. L.; Coleman, R. S.; Elmroth,
S. K. C. J. Am. Chem. Soc. 1998, 120, 12680; (b) Tanaka,
K.; Shionoya, M. J. Org. Chem. 1999, 64, 5002; (c)
Meggers, E.; Holland, P. L.; Tolman, W. B.; Romesberg,
F. E.; Schultz, P. G. J. Am. Chem. Soc. 2000, 122, 10714;
(d) Tanaka, K.; Yamada, Y.; Shionoya, M. J. Am. Chem.
Soc. 2002, 124, 8802; (e) Zimmermann, N.; Meggers, E.;
Schultz, P. G. J. Am. Chem. Soc. 2002, 124, 13684; (f)
Tanaka, K.; Tengeiji, A.; Kato, T.; Toyama, N.; Shio-
noya, M. Science 2003, 299, 1212.
3. (a) Schweitzer, B. A.; Kool, E. T. J. Org. Chem. 1994, 59,
7228; (b) Schweitzer, B. A.; Kool, E. T. J. Am. Chem. Soc.
1995, 117, 7241; (c) Kool, E. T. Biopolymer 1998, 48, 3; (d)
McMinn, D. L.; Ogawa, A. K.; Wu, Y.; Liu, J.; Schultz, P.
G.; Romesberg, F. E. J. Am. Chem. Soc. 1999, 121, 11585;
(e) Parsch, J.; Engels, J. W. J. Am. Chem. Soc. 2002, 124,
5664; (f) Matsuda, S.; Henry, A. A.; Schultz, P. G.;
Romesberg, F. E. J. Am. Chem. Soc. 2003, 125, 6134.
4. (a) Ferentz, A. E.; Verdine, G. L. J. Am. Chem. Soc. 1991,
113, 4000; (b) Ferentz, A. E.; Keating, T. A.; Verdine,
G. L. J. Am. Chem. Soc. 1993, 115, 9006; (c) Wang, H.;
Osborne, S. E.; Zuiderweg, E. R. P.; Glick, G. D. J. Am.
14. Compound 6 (270 mg, 0.426 mmol) was dissolved in 3 mL
of dry CH2Cl2 and purged with Ar for 2 min. To the
stirred solution was added N,N0-diisopropylethylamine
(110 mg,
0.852 mmol)
and
2-cyanoethyl-N,N0-di-
isopropylchlorophosphoramidite (121 mg, 0.511 mmol).
The reaction mixture was stirred under Ar while protected
from light for 1 h. The reaction mixture was added to
30 mL of CH2Cl2 and washed twice with 30 mL of H2O.
The organic layer was dried over MgSO4, filtered and
evaporated. The crude compound was chromatographed
with 1% trimethylamine in hexane–EtOAc (3:1) to obtain
1
7 as a colorless solid (276 mg, 89%). H NMR (CDCl3): d
1.12–1.31 (14H, m), 1.65 (1H, br), 2.04 (1H, m), 2.38 (1H,
m), 2.46 (1H, J ¼ 6:3 Hz, t), 2.62 (1H, J ¼ 6:5 Hz, t), 3.31
(2H, m), 3.78 (6H, s), 4.27 (1H, br), 4.53 (1H, br), 5.22
(1H, J ¼ 4:6, 9.6 Hz, dd), 6.84 (4H, J ¼ 5:0, 9.0 Hz, dd),
7.19–7.66 (16H, m), 8.04 (2H, J ¼ 8:3 Hz, d); FABMS m=e
833 [M+H]þ; HRMS (FAB) calcd for C48H54N2O7PS
833.3389, found 833,3376.
€
Chem. Soc. 1994, 116, 5021; (d) Osborne, S. E.; Volker, J.;
Stevens, S. Y.; Breslauer, K. J.; Glick, G. D. J. Am. Chem.
Soc. 1996, 118, 11993.
5. (a) Coleman, R. S.; McCary, J. L.; Perez, R. J. Tetrahe-
dron Lett. 1999, 40, 13; (b) Coleman, R. S.; McCary, J. L.;
Perez, R. J. Tetrahedron 1999, 55, 12009.
6. (a) Barton, D. H. R.; Ramesh, M. J. Am. Chem. Soc. 1990,
112, 891; (b) Kang, S. O.; Lee, S. B. J. Chem. Soc., Chem.
Commun. 1995, 1017.
7. (a) Daves, G. D., Jr. Acc. Chem. Res. 1990, 23, 201; (b)
Shaban, M. A. E.; Nasr, A. Z. Adv. Heterocycl. Chem.
1997, 68, 223.
15. Selected data for dimer 4. 1H NMR (CD3OD): d 1.90 (1H,
m), 2.17 (1H, J ¼ 5:4, 13.2 Hz, dd), 3.64 (2H, J ¼ 4:9 Hz,
d), 3.92 (1H, br), 4.29 (1H, br), 5.09 (1H, J ¼ 5:4, 10.7 Hz,
dd), 7.36 (1H, J ¼ 8:3 Hz, d), 7.45 (1H, J ¼ 7:8 Hz, d);
EIMS m=e 450 [M]þ; HRMS (FAB) calcd for C22H27O6S2
451.1249, found 451.1251.
16. All Tm values of the duplexes (15 lM/base pair) were
measured in 100 mM NaCl and 10 mM Na-phosphate,
pH 7.0. The absorbances of the duplexes were monitored at
260 nm from 10 to 90 °C using a heating rate of 1 °C/min.
17. Maldi-Tof MS data for oligonucleotides and duplexes. III:
[(M)H)ꢁ] calcd 4537.8, found 4536.4. IV: [(M)H)ꢁ] calcd
4555.8, found 4552.8. IIIÆIV: [(M)H)ꢁ] calcd 9092.6,
found 9093.6. V: [(M)H)ꢁ] calcd 4537.8, found 4537.2.
VI: [(M)H)ꢁ] calcd 4555.8, found 4555.1. VÆVI: [(M)H)ꢁ]
calcd 9092.6, found 9091.2.
8. Hoffer, M. Chem. Ber. 1960, 93, 2777.
1
9. Selected data for compound 2. 2b: H NMR (CDCl3): d
2.20 (1H, m), 2.39 (3H, s), 2.43 (3H, s), 2.49 (1H, J ¼ 9:3,
14.1 Hz, dd), 4.52 (1H, m), 4.63 (2H, m), 5.21 (1H,
J ¼ 5:1, 11.2 Hz, dd), 5.59 (1H, J ¼ 6:4 Hz, d), 7.27 (9H,
m), 7.46 (4H, m), 7.91 (2H, J ¼ 8:3 Hz, d), 7.97 (2H,
J ¼ 8:3 Hz, d). 13C NMR (CDCl3): d 21.7, 21.7, 41.7, 64.7,
80.3, 83.0, 126.7, 126.9, 127.0, 127.2, 127.5, 127.6, 127.7,