Biologically active oligodeoxyribonucleotides. Part 12:1 N2-methylation of 2'-deoxyguanosines enhances stability of parallel G-quadruplex and anti-HIV-1 activity

Article abstract of DOI:10.1016/S0960-894X(00)00432-7

2'-Deoxyguanosine residues of a 3',5'-end-modified hexadeoxyribonucleotide (R-95288) with anti-HIV-1 activity were substituted with N²-methyl-2'-deoxyguanosine (m²dG). These modified oligodeoxyribonucleotides (ODNs) showed a 2-fold higher activity than R-95288. Also, the CD spectra of these ODNs indicated that the m²dG modification stabilized the tertiary structure of the G-quadruplex. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00432-7

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2213±2216
Biologically Active Oligodeoxyribonucleotides. Part 12:1
N2-Methylation of 20-Deoxyguanosines Enhances Stability
of Parallel G-Quadruplex and Anti-HIV-1 Activity
Makoto Koizumi,a,* Keika Akahori,a Toshinori Ohmine,b Shinya Tsutsumi,c
Junko Sone,c Toshiyuki Kosaka,c Masakatsu Kaneko,a Satoshi Kimurad,e
and Kaoru Shimadad,f
aExploratory Chemistry Research Laboratories, Sankyo Co., Ltd, Tokyo 140-8710, Japan
bBiological Research Laboratories, Sankyo Co., Ltd, Tokyo 140-8710, Japan
cBiomedical Research Laboratories, Sankyo Co., Ltd, Tokyo 140-8710, Japan
dDepartment of Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo 108, Japan
eDepartment of Infection Control and Prevention, The University of Tokyo Hospital, Tokyo 113, Japan
fTokyo Senbai Hospital, Tokyo 108, Japan
Received 5 June 2000; accepted 29 July 2000
AbstractÐ20-Deoxyguanosine residues of a 30,50-end-modi®ed hexadeoxyribonucleotide (R-95288) with anti-HIV-1 activity were
substituted with N2-methyl-20-deoxyguanosine (m2dG). These modi®ed oligodeoxyribonucleotides (ODNs) showed a 2-fold higher
activity than R-95288. Also, the CD spectra of these ODNs indicated that the m2dG modi®cation stabilized the tertiary structure of
the G-quadruplex. # 2000 Elsevier Science Ltd. All rights reserved.
Oligodeoxyribonucleotides (ODNs) for drug candi-
dates, which can bind to nucleic acids, proteins and
small organic compounds that are disease-related, are
called antisense nucleic acids or aptamers.2 We have
found that a hexadeoxyribonucleotide (50 TGGGAG)
with a 3,4-dibenzyloxybenzyl (3,4-DBB) group at the 50
end had high anti-HIV-1 activity and low cytotoxicity.3
Modi®cation of the 30 end with a 2-hydroxyethyl phos-
phate group enhanced the anti-HIV-1 activity as well as
stability of the ODN in human plasma.4 This modi®ed
ODN (R-95288; 1) also showed in vivo anti-viral activity
using hu-PBL-SCID/beige mice as an animal model for
HIV-1 infection.5 This modi®ed ODN most likely binds
to the gp120 protein of the HIV-1 virus, inhibits absorp-
tion and entry of the virus, and acts as an aptamer.6
Moreover, this kind of short, guanine-rich ODN forms
a parallel-quadruplex based on anti-conformational
G-quartet (Fig. 1(A,B)).1,3,4,7
In this paper, we describe the N2-methylation of 20-
deoxyguanosine residues in R-95288 (1), stability of the
G-quartet by these ODNs and their anti-HIV-1 activity.
Although one of the protons of an exocyclic amine group
of 20-deoxyguanosine contributes to the hydrogen bonding
in the G-quartet, the other is available for modi®cation.
Once a 20-deoxyguanosine residue in the G-quadruplex
is modi®ed, this is the same as all other 20-deoxyguano-
sine residues in the G-quartet plate being modi®ed (Fig.
1(B)). Substitution at the 8 position of 20-deoxyguano-
sine residues in the anti-parallel G-quadruplex stabilizes
*Corresponding author. Tel.: +3-3492-3131; fax: +3-5436-8570; e-mail: koizum@shina.sankyo.co.jp
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00432-7

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M. Koizumi et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2213±2216
Figure 1. (A) Proposed parallel G-quadruplex structure of R-95288.4 Circles and p show 3,4-DBB groups and 2-hydroxyethyl phosphate groups,
respectively. Fifth 20-deoxyadenosine from the 50-end can also form the quartet structure.16,17 (B) G-quartet structure by 20-deoxyguanosine residues
or N2-methyl-20-deoxyguanosine residues.
the tertiary structure, as the 20-deoxyguanosine residues
are now ®xed in the syn conformation.8,9 However, the
actual e€ect of the N2-methylation of 20-deoxyguanosine
residues in the parallel G-quadruplex is not yet known.
A set of ODNs containing N2-methyl-20-deoxyguanosine
was synthesized on a DNA synthesizer by the phos-
phoramidite method. 50-O-(3,4-DBB)thymidine-30-O-
phosphoramidite13 and ethylene glycol-modi®ed con-
trolled pore glass (CPG)3 were used for the 50 and 30 end
modi®cation as reported previously. Phosphoramidite 5
was incorporated into the TGGGAG sequence instead
of 20-deoxyguanosine residues at various positions as
shown in the Table 1. After chain-elongation, the mod-
i®ed ODNs were cleaved from the CPG support and
deprotected with 28% aqueous ammonia solution. The
puri®cation of the ODNs was performed by C18 reversed
phase column chromatography.3 The structures of the
modi®ed ODNs were determined by negative MALDI
TOF mass spectroscopy (Table 1).
The synthesis of the N2-methyl-20-deoxyguanosine
(m2dG) unit to form modi®ed R-95288 is shown in the
Scheme 1. Selective methylation at the N-2 position was
performed according to Reese's method for N2-methyl-
guanosine synthesis.10,11 Mannich-like reaction of 30,50-
TIPDS-20-deoxyguanosine 2 by treatment with HCHO
and thiocresol gave compound 3. Reduction by NaBH4,
followed by protection of the O-6 position with N,N-
diphenylcarbamonyl chloride, gave N2-methylated 20-
deoxyguanosine 4.12 Subsequently, compound 4 was
deprotected by TBAF in THF, treated with 4,40-dime-
thoxytrityl chloride (DMTrCl) in pyridine and phos-
phitylated by 2-cyanoethyl N,N,N,N-tetraisopropyl-
phosphordiamidite in the presence of N,N-diisopropyl-
ammonium tetrazolide to give phosphoramidite 5.
Anti-HIV-1 assays were performed using ODNs 6±10
containing m2dG at various positions. The 50% inhibi-
tory concentration (IC50) of the cytopathic e€ect for MT-
4 cells infected by an HIV-1IIIB strain and the 50% cyto-
toxicity concentration (CC50) were determined according
to a procedure reported previously.13 We reported that
the IC50 of R-95288 (1) was 0.19 mg/mL (0.08 mM) and
its CC50 was up to 100 mg/mL (>40 mM).3 Relative IC50
values of m2dG-containing ODN 6±10 to R-95288 were
improved by approximately 2-fold (Table 1). These
Table 1. Anti-HIV-1 activity and characterization of m2dG-contain-
ing ODNs
MS [M±H]
b
Compoundsa
Relative IC50
Calcd
Found
1 (TGGGAG, R-95288)
6 (T(m2dG)GGAG)
7 (TG(m2dG)GAG)
8 (TGG(m2dG)AG)
9 (TGGGA(m2dG))
10 (T(m2dG)3AG)
11 (TGGG)
1.0
0.63
0.63
0.45
0.63
0.46
43
2296.47
2310.49
2310.49
2310.49
2310.49
2338.52
1654.36
1696.41
2296.30
2310.10
2310.21
2310.55
2310.14
2338.57
1654.54
1696.38
Scheme 1. (i) HCHO, AcOH, thiocresol, 70%; (ii) (a) NaBH4, DMSO;
(b) Ph2NC(ˆO)Cl, (iPr)2NEt; two steps 49%; (iii) (a) TBAF, THF; (b)
DMTrCl, pyridine; two steps 77%; (c) N,N-diisopropylammonium tet-
razolide, 2-cyanoethyl N,N,N,N-tertaisopropylphosphordiamidite,
THF, 91%.
12 (T(m2dG)3)
2.0
aAll ODNs have a 3,4-DBB group and a 2-hydroxyethyl group at the
50 and 30 end, respectively.
bValue of IC50 (mM)/IC50 value (mM) of R-95288 (1).

M. Koizumi et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2213±2216
2215
Figure 2. CD spectra of R-95288 and compounds 6±10 in phosphate-bu€ered saline (10 mM phosphate (pH 7), 138 mM NaCl, and 2.7 mM KCl;
Sigma) at 20 ꢀC (black), 30 ꢀC (red), 40 ꢀC (green), 50 ꢀC (pink), 60 ꢀC (blue), 70 ꢀC (yellow), and 80 ꢀC (orange). Tm values, which are temperatures
at which the maximum wave length changed in the CD spectra, are shown in parentheses.
results indicate that the free proton of the exocyclic
amino group of 20-deoxyguanosine may not be involved
in the interaction with the targeted protein, gp120. In
particular, substitution at the fourth residue from the 50
end (compounds 8 and 10) seems to be e€ective for anti-
HIV-1 activity. All modi®ed compounds showed no
cytotoxicity up to 100 mg/mL (>40 mM).
quadruplex due to the poor stacking of the terminal
base. These results might be due to the improvement of
base stacking potential by methylation in the nucleo-
base.15 Interestingly, compound 10 with three con-
tiguous m2dGs dissociated the least, with little change,
even at 80 ꢀC. This stabilizing e€ect of a modi®cation
with three m2dGs corresponds to that of ®ve contiguous
guanosine residues in an ODN such as (3,4-DBB)-
T(G)5.3
Guanine-rich ODNs can form parallel or anti-parallel
quadruplex structures.1,3,4,7 9 We have found that
R-95288 also forms the parallel structure at less than
50 ꢀC (this temperature is called Tm).4 Oligonucleotides
containing N2-methylguanosine can form duplexes
identical to that of normal oligonucleotides.14 This
means that methylation at the N-2 position of guano-
sine does not inhibit formation of hydrogen bonds. To
investigate the e€ect of N2-methylation on the stability
of ODNs, we measured the CD spectra of these m2dG-
containing compounds in phosphate-bu€ered saline at
various temperatures.
On the basis of these results, the structure R-95288 (1)
was minimized down to three contiguous m2dGs, that is,
a TGGG sequence with three 20-deoxyguanosines (com-
pound 11). This was the minimum required structure but
had less anti-HIV-1 activity than R-95288.3 Compound
11 was substituted with three modi®ed nucleosides to
obtain compound 12. By comparing the IC50s, the
activity of compound 11 was 43 times lower than
R-95288. However, as predicted, the activity of
compound 12 with three contiguous m2dGs was nearly
identical to R-95288, due to the stabilization of the
G-quadruplex.
The CD spectra demonstrated that compounds 6±8, in
which one guanine residue of the internal contiguous
guanines was substituted with m2dG, formed the par-
allel G-quadruplex as well as did the parent com-
pound R-95288 and had higher stability than R-95288
(Fig. 2). Tm values (70 ꢀC) for compounds 7 and 8, in
which was substituted the second or the third 2-deoxy-
guanosine among the three contiguous 20-deoxyguano-
sine residues, were much higher than that of compound
6 (60 ꢀC), in which the ®rst guanosine residue was sub-
stituted with m2dG. On the other hand, terminal sub-
stitution with m2dG (compound 9) had no e€ect on the
In conclusion, we demonstrated that the N2-methylation
of 20-deoxyguanosine on the parallel G-quadruplex
enhanced the thermodynamic stability of the G-quartet,
and that a set of ODNs modi®ed with m2dG had
higher anti-HIV-1 activity than the parent R-95288,
due to stabilizing e€ects. N2-Alkylation of guanine
residues is a promising way to improve the stability
of the parallel G-quadruplex, as long as the modi®-
cation does not sterically hinder the formation of the
G-quadruplex.

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M. Koizumi et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2213±2216
Dougherty, J. P.; Terhorst, T.; Law, V. S.; Grin, L. C.;
References and Notes
Coutre, S.; Bischofberger, N. J. Med. Chem. 1998, 41, 2234.
10. Bridson, P. K.; Reese, C. B. Bioorg. Chem. 1979, 8, 339.
11. Uchiyama, Y.; Miura, Y.; Inoue, H.; Ohtsuka, E.; Ueno,
Y.; Ikehara, M.; Iwai, S. J. Mol. Biol. 1994, 243, 782.
1. Part 11; Koizumi, M.; Koga, R.; Hotoda, H.; Ohmine, T.;
Furukawa, H.; Agatsuma, T.; Nishigaki, T.; Abe, K.; Kosaka,
T.; Tsusumi, S.; Sone, J.; Kaneko, M.; Kimura, S.; Shimada,
K. Bioorg. Med. Chem. 1998, 6, 2469.
12. Protection at the O-6 position was necessary for oligonu-
cleotide synthesis, because O-6 unprotected N2-methyl-20-
deoxyguanosine-30-O-phosphoramidite unit had low solubility
2. Osborne, S. E.; Ellington, A. D. Chem. Rev. 1997, 97, 349.
3. Hotoda, H.; Koizumi, M.; Koga, R.; Kaneko, M.;
Momota, K.; Ohmine, T.; Furukawa, H.; Agatsuma, T.;
Nishigaki, T.; Sone, J.; Tsusumi, S.; Kosaka, T.; Abe, K.;
Kimura, S.; Shimada, K. J. Med. Chem. 1998, 41, 3655.
4. Koizumi, M.; Koga, R.; Hotoda, H.; Momota, K.;
Ohmine, T.; Furukawa, H.; Agatsuma, T.; Nishigaki, T.; Abe,
K.; Kosaka, T.; Tsusumi, S.; Sone, J.; Kaneko, M.; Kimura,
S.; Shimada, K. Bioorg. Med. Chem. 1997, 5, 2235.
5. Agatsuma, T.; Abe, K.; Furukawa, H.; Koga, R.; Koizumi,
M.; Hotoda, H.; Kaneko, M. Antiviral Res. 1997, 34, 121.
6. Agatsuma, T.; Furukawa, H.; Hotoda, H.; Koizumi, M.;
Koga, R.; Kaneko, M. Antiviral Chem. Chemother. 1997, 8,
429.
1
in acetonitrile. H NMR (360 MHz, CDCl3) d ppm: 8.00 (s,
1H, H8), 7.47±7.22 (m, 10H, Ph), 6.27±6.24 (dd, 1H, H10), 5.88
(brs, 1H, NH), 4.76±4.69 (m, 1H, H30), 4.13±3.98 (m, 2H,
H50), 3.91±3.88 (m, 1H, H40), 3.04 (s, 3H, NHCH3), 2.68±2.58
(m, 2H, H20), 1.10±1.02 (m, 28H, CH(CH3)). FAB-MS m/z
719 (MH+).
13. Hotoda, H.; Momota, K.; Furukawa, H.; Nakamura, T.;
Kaneko, M.; Kimura, S.; Shimada, K. Nucleosides Nucleotides
1994, 13, 1375.
14. Rife, J. P.; Cheng, C. S.; Moore, P. B.; Strobe, S. A.
Nucleic Acids Res. 1998, 26, 3640.
15. Broom, A. D.; Schweizer, M. P.; Ts'O, P. O. P. J. Am.
Chem. Soc. 1967, 89, 3612.
16. Katahira, M.; Moriyama, K.; Kanagawa, M.; Saeki, J.;
Kim, M. H.; Nagaoka, M.; Uesugi, S.; Kono, T. Nucleic Acids
Symp. Ser. 1995, 34, 197.
7. Wyatt, J. R.; Vicker, T. A.; Roberson, J. L.; Buckheit, R.;
Klimkait, T.; DeBaets, E.; Davis, P. W.; Rayner, B.; Imbach,
J. L.; Ecker, D. J. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 1356.
8. Dias, E.; Battiste, J. L.; Williamson, J. R. J. Am. Chem.
Soc. 1994, 116, 4479.
17. Watanabe, T.; Hanzawa, H.; Haruyama, H.; Koizumi,
M.; Kaneko, M. Seikagaku 1996, 68, 941.
9. He, G.-X.; Krawczyk, S. H.; Swaminathan, S.; Shea, R. G.;