8-Methylguanosine: A Powerful Z-DNA Stabilizer

Article abstract of DOI:10.1021/ja036233i

Various modified guanine derivatives were synthesized and introduced into G₄ of d(CGCGCG)₂ to evaluate their capacity to stabilize Z-form DNA. It was found that the incorporation of 8-methylguanosine (m ⁸rG) in oligonucleo

Full text of DOI:10.1021/ja036233i

Published on Web 10/14/2003
8-Methylguanosine: A Powerful Z-DNA Stabilizer
Yan Xu,† Reiko Ikeda,† and Hiroshi Sugiyama*,†,‡
Contribution from the Institute of Biomaterials and Bioengineering, Tokyo Medical and
Dental UniVersity, Chiyoda, Tokyo 101-0062, Japan, and Department of Chemistry,
Graduate School of Science, Kyoto UniVersity, Sakyo, Kyoto 606-8501, Japan
Received May 20, 2003; E-mail: hs@kuchem.kyoto-u.ac.jp
Abstract: Various modified guanine derivatives were synthesized and introduced into G4 of d(CGCGCG)2
to evaluate their capacity to stabilize Z-form DNA. It was found that the incorporation of 8-methylguanosine
(m8rG) in oligonucleotides stabilizes the Z form more dramatically than does the incorporation of 8-methyl-
2′-deoxyguanosine (m8G). This enhancement is ascribed to a reduction in the entropic penalty, which arises
from the introduction of hydrophilic groups in solvent-exposed regions. The incorporation of m8rG into DNA
sequences markedly stabilizes the Z form even in the absence of NaCl. The Z-DNA stabilizer allows
oligonucleotides with a wide range of sequences to be converted to the Z form. It could be a powerful tool
for examining the molecular basis of many types of Z-form-specific reactions at the molecular level under
physiological salt conditions.
Introduction
the possible biological roles of Z-DNA have drawn much current
interest.6c,7
DNA is polymorphic and exists in a variety of distinct
conformations.1 Duplex DNA can adopt a variety of sequence-
dependent secondary structures, which range from the canonical
right-handed B form to the left-handed Z form.2 Triplex and
tetraplex structures also exist.2b-d All of these unique conforma-
tions are assumed to play important functional roles in gene
expression by altering DNA-protein interactions.3 Although
Z-form DNA is one of the characteristic and significant local
structures of DNA and has been extensively investigated in
relation to transcription,3a the methylation of cytosine,4 and the
level of DNA supercoiling,5 the biological function of Z-form
DNA has not been well established. However, Rich and
colleagues recently discovered that double-stranded RNA ad-
enosine deaminase (ADAR1),6 the tumor-associated protein
DLM-1,7 and the E3L protein of vaccina virus specifically bind
to Z-form DNA.8 Moreover, Liu and colleagues presented the
first evidence that Z-DNA-forming sequences are required for
chromatin-dependent activation of CSF1 promoter.3d Therefore,
Although numerous studies have been made of Z-form
DNA, its chemical properties are not well understood, presum-
ably because of the difficulty in obtaining stable Z-form oligo-
nucleotides under physiological salt conditions. We have
demonstrated previously that the incorporation of a methyl group
at the guanine C8 position (m8G) stabilizes the Z form of
oligonucleotides.9 For example, the concentration of NaCl
required to induce a B-Z transition is significantly reduced,
with the midpoint NaCl concentrations for d(CGCGCG)2 and
d(CGCm8GCG)2 being 2600 and 30 mM, respectively. The
development of a Z-stabilizing monomeric unit, the Z stabilizer,
has allowed us to understand the solution structure of Z-DNA9
and to identify the Z-form-specific C2′R-hydroxylation of the
IU-containing Z-form d(CGCGIUGCG)/d(Cm8GCACm8GCG)
duplex under UV irradiation.10
Although m8G is a useful Z stabilizer, the strength of
stabilization in an oligomer containing an AT base pair is
inadequate. For example, the midpoint NaCl concentration for
the B-Z transition of d(CGCGIUGCG)/d(Cm8GCACm8GCG)
is 800 mM, which is substantially higher than physiological salt
† Tokyo Medical and Dental University.
‡ Kyoto University.
(1) Cozzarelli, N. R.; Wang, J. C. DNA Topology and Its Biological Effects;
Cold Spring Harbor Laboratory Press: New York, 1990.
(2) (a) Wang, A. H.-J.; Quigley, G. J.; Kolpak, F. J.; Crawford, J. L.; van
Boom, J. H.; van der Marel, G. A.; Rich, A. Nature 1979, 282, 680-686.
(b) Sinden, R. R. DNA Structure and Function; Academic Press: New
York, 1994. (c) Niedle, S. DNA Structure and Recognition; IRL Press:
Oxford, 1994. (d) Travers, A. A. Annu. ReV. Biochem. 1989, 58, 427-
452.
(6) (a) Herbert, A.; Rich, A. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 12132-
12136. (b) Brown, B. A., II; Lowenhaupt, K.; Wilbert, C. M.; Hanlon, E.
B.; Rich, A. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 13532-13536. (c)
Schade, M.; Turner, C. J.; Kuhne, R.; Schmieder, P.; Lowenhaupt, K.;
Herbert, A.; Rich, A. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12465-12470.
(d) Herbert, A.; Spitner, J.; Lowenhaupt, K.; Rich, A. Proc. Natl. Acad.
Sci. U.S.A. 1995, 92, 7550-7554. (e) Schwartz, T.; Rould, M. A.;
Lowenhaupt, K.; Herbert, A.; Rich, A. Science 1999, 284, 1841-1845.
(7) Schwartz, T.; Behlke, J.; Lowenhaupt, K.; Heinemann, U.; Rich, A. Nat.
Struct. Biol. 2001, 8, 761-765.
(3) (a) Rich, A.; Zhang, S. Nat. ReV. Genet. 2003, 4, 566-572. (b) Wo¨lfl, S.;
Martines, C.; Rich, A.; Majzoub, J. A. Proc. Natl. Acad. Sci. U.S.A. 1996,
93, 3664-3668. (c) Herbert, A.; Rich, A. J. Biol. Chem. 1996, 271, 11595-
11598. (d) Liu, R.; Liu, H.; Chen, X.; Kirby, M.; Brown, P. O.; Zhao, K.
Cell 2001, 106, 309-318.
(8) Kim, Y, G.; Muralinath, M.; Brandt, T.; Pearcy, M.; Hauns, K.; Lowenhaupt,
K.; Jacobs, B. L.; Rich, A. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 6974-
6979.
(4) Zacharias, W.; O’Connor, T. R.; Larson, J. E. Biochemistry 1988, 27, 2970-
2978.
(9) Sugiyama, H.; Kawai, K.; Matsunaga, A.; Fujimoto, K.; Saito, I.; Robinson,
H.; Wang, A. H.-J. Nucleic Acids Res. 1996, 24, 1272-1278.
(10) (a) Kawai, K.; Saito, I.; Sugiyama, H. J. Am. Chem. Soc. 1999, 121, 1391-
1392. (b) Oyoshi, T.; Kawai, K.; Sugiyama, H. J. Am. Chem. Soc. 2003,
125, 1526-1531.
(5) (a) Sheridan, S. D.; Benham, C. J.; Hatfield, G. W. J. Biol. Chem. 1999,
274, 8169-8174. (b) Schroth G. P.; Chou, P.-J.; Ho, P. S. J. Biol. Chem.
1992, 267, 11846-11855. (c) Jaworski, A.; Higgins, N. P.; Wells, R. D.;
Zacharias, W. J. Biol. Chem. 1991, 266, 2576-2581.
9
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J. AM. CHEM. SOC. 2003, 125, 13519-13524
13519

A R T I C L E S
Xu et al.
Scheme 1
mM, although it is lower than that for the unmodified duplex
(2600 mM) (Table 1). The proportions of Z, B, and SS for these
hexamers as a function of temperature are shown in Figure
2a-g. Thermodynamic parameters were obtained by the van’t
Hoff plot for B-Z transition (Figure 2h), and the results are
summarized in Table 1. Although methyl substitution results
in a favorable enthalpic effect (13.4 and 14.2 kcal/mol vs 11.7
kcal/mol) on the stability of Z-DNA, an unfavorable entropic
effect is also induced by the CH2 group at the 6′ position and
the methyl group at the C8 position (44.6 and 43.9 vs 42.3
cal mol-1 K-1). Inspection of the molecular model of
d(CGCm8cGCG)2 suggests that the C6′ position of the m8cG
residue is exposed to solvent (Figure 3a), which causes a
large entropical penalty (2.3 cal mol-1 K-1), as observed in
the m8G residue (1.6 cal mol-1 K-1), relative to that of
d(CGCGCG)2. Accordingly, 2′-deoxycarbaguanosine(cG)-in-
corporated d(CGCcGCG)2 did not undergo transition to the Z
form at all, even in the presence of 5 M NaCl (Table 1 and
Figure 1c). These results suggest the intriguing possibility that
the introduction of a hydrophilic group to the solvent-exposed
site stabilizes the Z form (Figure 3b). In fact, incorporation of
8-methylguanosine (m8rG) possessing a C2′ hydroxyl group and
an O4′ oxygen into the hexamer greatly stabilized the Z form:
d(CGCm8rGCG)2 showed a CD spectrum typical of the Z form,
even in the absence of NaCl (Figure 1b). The thermodynamic
parameters of the B-Z transition clearly indicate that a reduction
in entropy (-1.9 cal mol-1 K-1) largely contributes to the
increased stabilization by m8rG as compared to that by m8G,
which arises from the introduction of hydrophilic groups in
solvent-exposed regions. Furthermore, at physiological salt
concentrations, the reduction in the entropic penalty is more
clearly observed (Table 1). In fact, incorporation of guanosine
(rG) possessing a hydrophilic group itself stabilized the Z form;12
the midpoint for d(CGCrGCG)2 containing an rG was 800 mM
(Table 1). In addition to hydrophilic stabilization, the preferred
C3′ endo conformation of ribose also contributes to the dramatic
stabilization of the Z form by m8rG.
Significant stabilization of the Z form by incorporation of
m8rG was observed in duplex c containing an AIU base pair,
which showed the typical Z form only in 5 mM sodium
cacodylate buffer. The CD spectra of duplex c under various
NaCl concentrations are shown in Figure 4a. Furthermore, the
effect of stabilization was confirmed in three AT-base-pair-
containing octanucleotides: the midpoint NaCl concentration
for duplex e was 200 mM, which is a more than 10-fold
reduction relative to the corresponding deoxyoctanucleotide d
(Table 2). The results indicate that the incorporation of m8rG
into just one strand is capable of stabilizing the Z-DNA. The
results of f and g clearly demonstrate that a single m8rGC base
pair is enough to stabilize Z-DNA.
To demonstrate the usefulness of m8rG as a Z-DNA stabilizer,
photoreaction of IU-containing Z-form deoxyoctanucleotide was
examined. Under 302 nm irradiation, duplex c in the presence
of 50 mM NaI underwent efficient Z-form-specific C2′R-
hydroxylation (>90%, based on consumed octamer) without the
halogen exchange reaction observed during the photoirradiation
of duplex a in 2 M NaCl (Figure 4b).10 Interestingly, the C2′R-
hydroxylated product (2′OH) stabilized the Z form: the midpoint
concentrations. Moreover, the synthesis of m8G monomer is
carried out by the addition of methyl radicals under acidic
conditions, during which depurination leads to a lowering of
the yield. The design of more powerful and chemically stable
new types of Z stabilizers is still desirable. Therefore, various
modified guanine units that do not undergo depurination under
acidic conditions were synthesized and introduced into G4 of
d(CGCGCG)2 to evaluate their capacity to stabilize Z-form DNA
(Scheme 1). It was found that incorporation of 8-methylgua-
nosine (m8rG) in DNA dramatically stabilizes the Z form even
in the absence of NaCl, and facilitates the B-Z transition even
for CA-containing sequences.
Results and Discussion
The syntheses of the 8-methyl-2′-deoxycarbaguanosine
(m8cG)- and m8rG-containing oligonucleotides were carried out
by phosphoramidite chemistry, beginning with 2-chloro-2′-deoxy-
carbainosine 1 and rG, respectively (Scheme 2). The monomeric
unit, m8cG, which possesses a cyclopentane ring in the sugar
moiety, favors the syn conformation to a greater extent than
does m8G, as suggested by the ab initio MO calculation and
experimentally confirmed by the nuclear Overhauser effect
(NOE) intensity between C1′H and 8CH3 (Figure 1S, Supporting
Information). The ab initio MO calculation at 6-31G* level
indicates that the stabilization energies of the syn conformations
of m8cG and m8G were 2.44 and 0.30 kcal/mol, respectively.
CD spectroscopy is one of the more convenient methods used
to study Z-DNA conformation.11 In B-DNA, a positive Cotton
effect appears at 280 nm, and a more intense negative band
appears at 295 nm in Z-DNA.11 Thus, the CD spectrum was
used to monitor the conformational state at various NaCl
concentrations, and the results are shown in Figure 1. Unexpect-
edly, when m8cG was incorporated, d(CGCm8cGCG)2 did not
convert as effectively to the Z form as did d(CGCm8GCG)2:
the midpoint NaCl concentration for d(CGCm8cGCG)2 was 1300
(11) (a) Pohl, F. M.; Jovin, T, M. J. Mol. Biol. 1972, 67, 375-396. (b) Behe,
M.; Felsenfeld, G. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 1619-1623. (c)
Soumpasis, D. M. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 5116-5120. (d)
Presler, R. S.; Chen, H. H.; Colombo, M. F.; Choe, Y.; Short, B. J., Jr.;
Rau, D. C. Biochemistry 1995, 34, 14400-14407. (e) Saenger, W.; Hunter,
W. N.; Kennard, O. Nature 1986, 324, 385-388.
(12) Stabilization of Z-form DNA by the O2′ hydroxyl group of ribose was
reported. Teng, M.; Liaw, Y.-C.; van der Marel, G. A.; van Boom, J. H.;
Wang, A. H.-J. Biochemistry 1989, 28, 4923-4928.
9
13520 J. AM. CHEM. SOC. VOL. 125, NO. 44, 2003

8-Methylguanosine: A Powerful Z-DNA Stabilizer
A R T I C L E S
Scheme 2 a
a (a) NH2NH2, CH3OH; (b) H2, PtO2; (c) pyridine, TMSCl, isobutyric anhydride; (d) H2SO4, FeSO4‚7H2O, tert-butylhydroperoxide; (e) pyridine, DMAP,
Et3N, DMTrCl; (f) NC(CH2)2OP(Cl)N(iPr)2, (iPr)2NH, CH2Cl2; (g) pyridine, TMSCl, isobutyric anhydride; (h) H2SO4, FeSO4‚7H2O, tert-butylhydroperoxide;
(i) pyridine, DMAP, Et3N, DMTrCl; (j) tert-butyldimethylsilyl chloride, dichloroethane, imidazole; (k) NC(CH2)2OP(Cl)N(iPr)2, (iPr)2NH, CH2Cl2.
Figure 1. CD spectra of d(CGCm8cGCG)2 (a), d(CGCm8rGCG)2 (b), d(CGCcGCG)2 (c), and d(CGCrGCG)2 (d) in 5 mM Na cacodylate buffer, pH 7.0, at
10 °C at various NaCl concentrations (all oligomers, 0.15 mM base concentration).
Experimental Section
NaCl concentrations for a and b were 800 and 420 mM,
respectively (Table 2). It is interesting to note that a change in
the B-Z equilibrium induced by C2′R-hydroxylation might
represent a new type of damaging mechanism in 5-halouracil-
containing DNA under UV irradiation.
In conclusion, the present study demonstrates that the
incorporation of m8rG in oligonucleotides dramatically stabilizes
the Z form, mainly by a reduction in the entropic penalty, which
arises from the introduction of hydrophilic groups in solvent-
exposed regions. We have shown that m8rG is a much better Z
stabilizer than the previously used m8G, in that it allows
oligonucleotides with a wide range of sequences to be converted
to the Z form. Incorporation of the m8rG moiety into DNA
oligomers could be a powerful tool with which to examine the
molecular basis for many types of Z-form-specific reactions at
the molecular level under physiological salt conditions.
General. 2-Chloro-2′-deoxycarbaguanosine (1) was obtained from
Takeda Chemical Industries. 1H NMR (500 MHz, DMSO-d6): δ 8.04
(s, 1H, H-8), 4.91 (m, 1H, 1′), 4.07 (m, 1H, 3′), 3.46 (m, 2H, 5′), 2.33
(m, 1H, 6′), 2.15 (m, 1H, 2′), 2.01 (m, 2H, 2′′ and 4′), 1.65 (m, H, 6′′).
Reactions were monitored by thin-layer chromatography (TLC) using
0.25 mm silica gel 60 plates impregnated with 254 nm fluorescent
indicator (purchased from Merck). Column chromatography was
performed on Acros Chimica silica gel (0.2-0.5 mm, pore size 4 nm).
The NMR spectra were recorded on a JEOL JNM-A 500 magnetic
resonance spectrometer. Calf intestine alkaline phosphatase (AP) and
P1 nuclease were purchased from Boehringer Mannheim. The following
abbreviations apply to spin multiplicity: s (singlet), d (doublet), m
(multiplet), and b (broad). Fast atom bombardment mass spectra
(FABMS) were recorded on a JEOL-JMS-AX505H mass spectrometer.
Electrospray ionization mass spectra (ESI-MASS) were recorded on a
PE Sciex API 165 mass spectrometer.
9
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A R T I C L E S
Xu et al.
Table 1. Midpoint NaCl Concentrationsa and Thermodynamic
Parametersb for the B-Z Transition in Modified d(CGCGCG)2
Derivativesc
1 N H2SO4 was added an aqueous solution (50 mL) containing 1.3 mL
of 70% tert-butyl hydroperoxide (9.5 mmol) dropwise over a period
of 5 min. After being stirred at 0 °C for 60 min, the reaction mixture
was neutralized with saturated KOH solution. The supernatant obtained
by centrifugation resulting in a brownish solid was triturated three times
with 100 mL of methanol. The combined methanol solution was
concentrated, and the residue was subjected to silica gel column
chromatography. Elution with CH2Cl2/methanol (9:1) afforded 8-meth-
yl-N-isobutyryl-2′-deoxycarbaguanosine (5) as a white powder, yield
297K
NaCl
(mM)
∆G
∆H
(kcal mol-1
∆S
(cal mol-1 K-1
)
duplexes
(kcal mol-1
)
)
d(CGCGCG)2
2600 -0.86 ( 0.05 11.7 ( 1.0 42.3 ( 1.2
30 1.16 ( 0.10 14.2 ( 1.3 43.9 ( 1.5 (1.6)d
0.24 ( 0.04 10.1 ( 0.5 37.2 ( 0.9f
d(CGCm8GCG)2
d(CGCm8cGCG)2 1300 0.15 ( 0.03 13.4 ( 0.7 44.6 ( 1.7 (2.3)
d(CGCcGCG)2
>5000
nde
nd
nd
1
0.37 g (71%). H NMR (500 MHz, DMSO-d6): δ 4.90 (m, 1H, 1′),
d(CGCm8rGCG)2
0
1.32 ( 0.13 13.8 ( 0.8 42.0 ( 1.3 (-0.3)
0.42 ( 0.05 9.3 ( 0.6 33.1 ( 0.7f
4.10 (m, 1H, 3′), 3.55 (m, 2H, 5′), 2.76 (m, 1H, isobutyryl CH), 2.35
(m, 1H, 6′), 2.16 (m, 1H, 2′), 2.01 (m, 2H, 2′′ and 4′), 1.87 (s, 3H,
-8CH3), 1.61 (m, 1H, 6′′), 1.12 (d, 6H, J ) 6.5 Hz, 2CH3). ESMS,
m/e calcd for C16H23N5O4 (M + H) 350.3, found 350.5.
d(CGCrGCG)2
800 0.52 ( 0.04 13.0 ( 0.8 41.8 ( 0.9 (-0.5)
a Midpoint NaCl was determined from CD measurements at 10 °C at
various NaCl concentrations. The B-Z conformational transition was
analyzed by the data collected below 25 °C. b Thermodynamic parameters
were calculated by plotting ln(PZ/PB) versus 1/T in 3.0 M NaCl, 5 mM
Na-cacodylate buffer, pH 7.0. c All experiments were carried out using
0.15 mM hexanucleotide (base concentration) in 5 mM Na-cacodylate
buffer, pH 7.0. d The numbers in parentheses are the difference in ∆S rela-
tive to d(CGCGCG)2. e These data were not determined. f These data
were determined in the physiological salt concentrations (10 mM NaCl,
100 mM KCl).
8-Methyl-N2-isobutyryl-5′-O-(dimethoxytrityl)-2′-deoxycarba-
guanosine (6). To 0.3 g (0.9 mmol) of 5 dried three times by evap-
oration of pyridine (10 mL) and suspended in 8 mL of dry pyridine
were added 0.5 g (1.3 mmol) of 4,4′-dimethoxytrityl chloride, 0.2 mL
(1.3 mmol) of triethylamine, and 2.8 mg (0.03 mmol) of 4-dimeth-
lyaminopyridine. After it stood overnight, the reaction was cooled in
an ice bath, and 10 mL of 5% NaHCO3 was added. The mixture was
extracted twice with 60 mL of ethyl acetate. The organic layer was
evaporated to dryness, and the target compound was given by silica
2-Hydrazino-2′-deoxycarbaguanosine (2). The 2-chloro-2′-deoxy-
carbaguanosine (1) (3.0 g, 10.1 mmol) was added to methanol (30 mL).
To this solvent was added hydrazine hydrate (0.6 g, 12.3 mmol). The
mixture was refluxed gently for 24 h, and then solvent was removed
in a vacuum, and the residue was triturated with ethanol (100 mL).
The resulting white precipitate was filtered to give 2-hydrazine-2′-
1
gel column chromatography: 0.4 g, yield was 67%. H NMR (500
MHz, DMSO-d6): δ 7.20-7.39 (m, 9H, ph), 6.89 (m, 4H, ph), 4.96
(m, 1H, 1′), 4.16 (m, 1H, 3′), 3.74 (s, 6H, 2CH3), 3.18 (m, 2H, 5′),
3.02 (m, 1H 6′), 2.77 (m, 1H, isobutyryl CH), 2.14 (m, 1H, 2′), 2.05
(m, 2H, 2′′ and 4′), 1.88 (s, 3H, -8CH3), 1.62 (m, 1H, 6′′), 1.10 (d,
6H, J ) 6.5 Hz, 2CH3). ESMS, m/e calcd for C37H41N5O6 (M + H)
652.0, found 652.1.
1
deoxycarbainosine (2.1 g), yield 74%. H NMR (500 MHz, DMSO-
d6): δ 8.24 (s, NH), 4.85 (m, 1H, 1′), 4.07 (m, 1H, 3′), 3.46 (m, 2H,
5′), 2.27 (m, 1H, 6′), 2.14 (m, 1H, 2′), 1.98 (m, 2H, 2′′ and 4′), 1.64
(m, 1H, 6′′). FABMS, m/e calcd for C11H16N6O3 (M + H) 281.0, found
281.1.
N2-Isobutyrylguanosine (9). To 7 g (25 mmol) of guanosine dried
three times by evaporation of pyridine (100 mL) and suspended in 140
mL of dry pyridine was added 17.5 mL (125 mmol) of trimethylchlo-
rosilane. After the solution was stirred 2 h, 21 mL (125 mmol) of
isobutyric anhydride was added, and the mixture was stirred for 4 h at
room temperature under N2. The reaction was cooled in an ice bath,
and 35 mL of water was added. After 15 min, 35 mL of 29% aqueous
ammonia was added, and the reaction was stirred for 15 min. The
solution was then evaporated to near dryness, and the residue was
dissolved in 500 mL of water. The mixture was extracted twice with
200 mL of CH2Cl2. The organic layer was evaporated to drynesss, and
the target compound was given by silica gel column chromatography:
3.6 g, yield was 39%. 1H NMR (500 MHz, DMSO-d6): δ 8.24 (s, 1H,
H-8), 5.79 (m, 1H, 1′), 4.42 (m, 1H, 2′), 4.08 (m, 1H, 3′), 3.81 (m, 1H,
4′), 3.54 (m, 2H, â5′), 2.77 (m, 1H, isobutyryl CH), 1.12 (d, 6H J )
7.0 Hz, 2CH3). ESMS, m/e calcd for C15H21N5O6 (M + H) 354.3, found
354.5.
2′-Deoxycarbaguanosine (3). The 2-hydrazino-2′-deoxycarbainosine
(1.0 g, 3.6 mmol) was dissolved in a solution which was a mixture of
methanol (40 mL) and distilled water (10 mL). To this mixture was
added PtO2 (0.3 g). The mixture was hydrogenated for 40 h at 55 °C
under H2. After completion of the hydrogenation, the catalyst was
removed using a filter aid and washed with a methanol-water mixture.
The filtrate was concentrated to give the 2′-deoxycarbaguanosine (0.7
g) as a white powder, which was recrystallized from water (yield 70%).
1H NMR (500 MHz, DMSO-d6): δ 7.78 (s, 1H, H-8), 4.81 (m, 1H,
1′), 4.06 (m, 1H, 3′), 3.44 (m, 2H, 5′), 2.27 (m, 1H, 6′), 2.10 (m, 1H,
2′), 1.96 (m, 2H, 2′′ and 4′), 1.58 (m, 1H, 6′′). FABMS, m/e calcd for
C11H15N5O3 (M + H) 266.0, found 266.0.
N2-Isobutyryl-2′-deoxycarbaguanosine (4). To 0.6 g (2.2 mmol)
of 2′-deoxycarbaguanosine dried three times by evaporation of pyridine
(10 mL) and suspended in 8 mL of dry pyridine was added 3.2 mL
(25 mmol) of trimethylchlorosilane. After the solution was stirred for
30 min, 4.1 mL (25 mmol) of isobutyric anhydride was added, and the
mixture was stirred for 3 h at room temperature under N2. The reaction
was cooled in an ice bath, and 10 mL of water was added. After 5
min, 10 mL of 29% aqueous ammonia was added, and the reaction
was stirred for 15 min. The solution was then evaporated to near
dryness, and the residue was dissolved in 25 mL of water. The solution
was washed once with 25 mL of ethyl acetate/ether (1:1). The organic
layer was extracted with 12.5 mL of water, and the combined aqueous
layers were concentrated to about 15 mL. Crystallization occurred
quickly. The target compound (0.5 g) was given by concentration of
8-Methyl-N2-isobutyrylguanosine (10). To a solution of N 2-iso-
butyrylguanosine (1 g, 2.6 mmol) and FeSO4‚7H2O (6.7 g, 24.1 mmol)
in 160 mL of 1 N H2SO4 was added dropwise an aqueous solution
(100 mL) containing 2.6 mL of 70% tert-butyl hydroperoxide (9.5
mmol) over a period of 5 min. After being stirred at 0 °C for 2 h, the
reaction mixture was neutralized with saturated KOH solution. The
supernatant obtained by centrifugation resulting in a brownish solid
was triturated three times with 100 mL of methanol. The combined
methanol solution was concentrated, and the residue was subjected to
silica gel column chromatography. Elution with CH2Cl2/methanol
(9:1) afforded 8-methyl-N 2-isobutyrylguanosine (10) as a white pow-
1
der: yield 0.6 g (59%). H NMR (500 MHz, DMSO-d6): δ 5.81 (m,
1
1H, 1′), 4.62 (m, 1H, 2′), 4.12 (m, 1H, 3′), 3.82 (m, 1H, 4′), 3.60 (m,
2H, 5′), 2.78 (m, 1H, isobutyryl CH), 2.49 (s, 3H, -8CH3), 1.12 (d,
6H, J ) 7.0 Hz, 2CH3). ESMS, m/e calcd for C15H21N5O6 (M + H)
367.4, found 368.0.
8-Methyl-N2-isobutyryl-5′-O-(dimethoxytrityl)guanosine (11). To
0.5 g (2.4 mmol) of the 8-methyl-N 2-isobutyrylguanosine dried three
times by evaporation of pyridine (15 mL) and suspended in 15 mL of
the filtrate (yield 69%). H NMR (500 MHz, DMSO-d6): δ 8.12 (s,
1H, H-8), 4.92 (m, 1H, 1′), 4.08 (m, 1H, 3′), 3.45 (m, 2H, 5′), 2.77 (m,
1H, isobutyryl CH), 2.34 (m, 1H, 6′), 2.15 (m, 1H, 2′), 2.01 (m, 2H,
2′′ and 4′), 1.62 (m, 1H, 6′′), 1.12 (d, 6H, J ) 6.5 Hz, 2CH3). FABMS,
m/e calcd for C15H15N5O3 (M + H) 336.1, found 336.3.
8-Methyl-N2-isobutyryl-2′-deoxycarbaguanosine (5). To a solution
of 4 (0.5 g, 1.5 mmol) and FeSO4‚7H2O (3.4 g, 12 mmol) in 80 mL of
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13522 J. AM. CHEM. SOC. VOL. 125, NO. 44, 2003

8-Methylguanosine: A Powerful Z-DNA Stabilizer
A R T I C L E S
Figure 2. Proportions of Z, B, and SS conformations of (a) d(CGCGCG)2, (b) d(CGCm8cGCG)2, (c) d(CGCrGCG)2, (d) d(CGC8mGCG)2, and (e)
d(CGC8mrGCG)2. Sample solutions contained 0.15 mM hexanucleotide (base concentration) in 3.0 M NaCl, 5 mM Na cacodylate buffer, pH 7.0. (f) and (g)
are d(CGC8mGCG)2 and d(CGC8mrGCG)2 in the physiological salt concentrations (10 mM NaCl, 100 mM KCl). (h) van’t Hoff plot for the B-Z confor-
mational transition of these hexamers.
(s, 6H, 2CH3), 3.16 (m, 2H, 5′), 2.71 (m, 1H, isobutyryl CH), 2.45 (s,
3H, -8CH3), 1.09 (d, 6H, J ) 7.0 Hz, 2CH3). ESMS, m/e calcd for
C36H39N5O8 (M + H) 670.2, found 670.3.
8-Methyl-N2-isobutyryl-5′-O-dimethoxytrityl-2′-O-tert-butyldi-
methylsilylguanosine (12). To a solution of 11 (1 g, 1.4 mmol) and
imidazole (0.2 g, 3.0 mmol) in dry 1,2-dichloroethane (15 mL) was
added tert-butyldimethylsilyl chloride (0.3 g, 2 mmol), and the mixture
was kept at 20 °C for 16 h. A mixture of CH2Cl2 (20 mL) and 10%
aqueous NaHCO3 (15 mL) was added, and the organic layer was washed
with water (15 mL), dried over Na2SO4, and evaporated in vacuo to
dryness. The residue was purified by column chromatography on silica
gel (hexane:EtOAc ) 97:3) to give 0.3 g of target compound, yield
was 31%. 1H NMR (500 MHz, DMSO-d6): δ 7.19-7.38 (m, 9H, ph),
6.81 (m, 4H, ph), 5.85 (m, 1H, 1′), 4.94 (m, 1H, 2′), 4.35 (m, 1H, 3′),
4.08 (m, 1H, 4′), 3.80 (s, 6H, 2CH3), 3.33 (m, 2H, 5′), 2.77 (m, 1H,
Figure 3. Molecular models of (a) d(CGCm8cGCG)2 and (b)
isobutyryl CH), 2.47 (s, 3H, -8CH3), 1.13 (d, 6H, J ) 7.0 Hz, 2CH3),
0.90 (s, 9H, iBu), 0.07 (s, 3H, -CH3), 0.01 (s, 3H, -CH3). ESMS, m/e
calcd for C42H53N5O8Si (M + H) 784.0, found 784.1.
d(CGCm8rGCG)2.
dry pyridine were added 0.7 g (3.7 mmol) of 4,4′-dimethoxytrityl
chloride, 0.3 mL (2.1 mmol) of triethylamine, and 4.2 mg (0.04 mmol)
of 4-dimethylaminopyridine. After it stood overnight, TLC (CH2Cl2:
CH3OH ) 97:3) showed complete reaction. The reaction was cooled
in an ice bath, and 50 mL of 5% NaHCO3 was added. The mixture
was extracted twice with 60 mL of CH2Cl2. The organic layer was
evaporated to dryness, and the target compound was given by silica
gel column chromatography: 0.5 g, yield was 61%. H NMR (500
MHz, DMSO-d6): δ 7.16-7.29 (m, 9H, ph), 6.77 (m, 4H, ph), 5.82
(m, 1H, 1′), 4.78 (m, 1H, 2′), 4.27 (m, 1H, 3′), 3.98 (m, 1H, 4′), 3.71
Preparation of the Amidite Monomers 7 and 13, and Solid-Phase
Oligonucleotide Synthesis. The dimethoxytritylated guanine derivative
6 or 12 (0.5 mmol) was treated with dry N,N-diisopropylethylamine
(1.5 mmol) and 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite
(0.75 mmol) in dry dichloromethane (5 mL) and stirred at room
temperature for 24 h. The reaction was added to CH2Cl2 (50 mL),
washed with 5% aqueous NaHCO3, and dried over Na2SO4. Fol-
lowing column chromatography with n-hexane:acetone:triethylamine
(49:49:2), an amount of 7 (0.7 mmol, 70%) or 13 (0.6 mmol, 84%) of
1
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J. AM. CHEM. SOC. VOL. 125, NO. 44, 2003 13523

A R T I C L E S
Xu et al.
NH4OH (28% in H2O) (1:1) was used as a fast cleavage and de-
protection reagent, with complete cleavage of modified oligonucleotides
from the solid support occurring in 10 min at room temperature, and
complete deprotection occurring in 10 min at 65 °C. Removal of the
TBDMS-protecting group was done with 1 M TBAF in THF solution
at room temperature. The reaction was quenched by addition of 0.4
mL of 1 M TEAA solution and was desalted on an ion exchange
cartridge (Poly-Pak II).14 The oligonucleotides were purified by reverse
phase HPLC. ESI-MASS (negative mode), (I): calculated, 1804.3;
found, 1804.4. (II): calculated, 1790.2; found, 1790.3. (III): calculated,
1822.0; found, 1822.1. (IV): calculated, 2454.5; found, 2454.7. (V):
calculated, 2484.5; found, 2484.6. The guanosine (rG)-containing
oligomer d(CGCrGCG) was purchased from JBioS. Concentrations and
compositions of oligomers were determined by complete digestion of
oligomers to mononucleosides using P1 nuclease and bacterial alkaline
phosphatase.
CD Measurement and Thermodynamic Parameters Determina-
tion. CD spectra were measured with an AVIV model 62 DS/202 CD
spectrophotometer. CD spectra of oligonucleotide solutions (0.15 mM
base concentration in 5 mM sodium cacodylate buffer, pH 7.0, at 10
°C at various NaCl concentrations) were recorded using a 1 cm path-
length cell (Figure 1). The proportions of Z, B, and SS for these
oligomers at 3 M NaCl as a function of temperature are shown in Figure
2, as previously reported.9,15 To obtain the population of B- and Z-DNA
(PB and PZ), we analyzed the CD spectra taking into account the three
forms (B, Z, SS) present in solution in the temperature range covered
(0-60 °C). Hence, at each temperature, we assumed that15
295
∆ꢀ295 ) ∆ꢀB295PB + ∆ꢀZ295PZ + ∆ꢀSS
P
(1)
SS
Figure 4. (a) CD spectra for d(CGCGIUGCG)/d(Cm8rGCACm8rGCG)
in 5 mM Na-cacodylate buffer, pH 7.0, at 10 °C at various NaCl
concentrations. (b) HPLC profiles of UV-irradiated d(CGCGIUGCG)/
d(Cm8rGCACm8rGCG) in 50 mM NaI. The reaction mixture (100 µL)
containing octamer (0.5 mM total base concentration) was irradiated for 2
h at 0 °C with a monochromator (302 nm). HPLC analysis was carried out
on a Cosmosil 5C18-MS column; elution was with 0.05 M ammonium
formate containing 3-8% acetonitrile, in a linear gradient at a flow rate of
1.0 mL/min for 50 min, at 30 °C. 2′OH: C2′R-hydroxylated product
d(CGCrGUGCG).
1 ) PB + PZ + PSS
(2)
with the P’s being the molar fraction of the B, Z, and single strand
components. The ∆ꢀ’s, relative to the limit forms, have been estimated
from the CD signal at 295 nm. They have been considered independent
of the temperature and salt concentration, even though a small, but not
insignificant, dependence was observed. PSS at each temperature was
determined by a subsequent UV melting experiment (of the same
solution) monitoring the absorbance at the B-Z isosbestic point (270
nm).15 Therefore, eqs 1 and 2, relative to each CD spectrum at a given
temperature, were solved to provide estimates of PB and PZ. The B-Z
conformational transition was analyzed by using the data collected
below 25 °C. Thermodynamic parameters were calculated by plotting
ln(PZ/PB) versus 1/T: ln(PZ/PB) ) (∆H°/R)(1000/T) - ∆S°/R in 3.0
M NaCl or in 10 mM NaCl and 100 mM KCl, 5 mM sodium cacodylate
buffer, pH 7.0. The free energy change associated with the duplex
formation at 297 K, ∆G297K°, was calculated from the obtained ∆H°
and ∆S° values using the following equation: ∆G297K° ) ∆H° - T∆S°.
Table 2. Midpoint NaCl Concentrations for B-Z Transitions of
Various Deoxyoctanucleotides Derivativesa
duplexes
NaCl
a
b
c
d
e
f
d(CGCGIUGCG)/d(Cm8GCACm8GCG)
d(CGCrGUGCG)/d(Cm8GCACm8GCG)
d(CGCGIUCGC)/d(Cm8rGCACm8rGCG)
d(CACATGCG)/d(Cm8GCATm8GTG)
d(CACATGCG)/d(Cm8rGCATm8rGTG)
d(Cm8GCACm8GCG)/d(CGCGTG)
d(Cm8rGCACm8rGCG)/d(CGCGTG)
800
420
0
2450
200
1100
210
Supporting Information Available: Information on the
NOESY spectra of 8-methyl-2′-deoxycarbaguanosine (m8cG)
and 8-methyl-2′-deoxyguanosine (m8G) (PDF). This material
is available free of charge via the Internet at http://pubs.acs.org.
g
a Experiments were carried out as described in the footnote to Table 1.
the amidite was isolated. ESMS for 13, m/e calcd for C51H70N7O9PSi
(M + H) 984.2, found 985.1; ESMS for 7, m/e calcd for C46H58N7O7
(M + H) 852.0, found 852.1. The corresponding 2-cyanoethyl phos-
phoramidite 7 or 13 was used at 0.12 M in acetonitrile solution, and
coupling was allowed to proceed for 3 min, resulting in highly effi-
cient coupling (>95%). Oligonucleotides, (I) d(CGCm8cGCG), (II)
d(CGCcGCG),13 (III) d(CGCm8rGCG), (IV) d(Cm8rGCACm8rGCG),
and (V) d(Cm8rGCATm8rGTG), were synthesized. Methylamine/
JA036233I
(13) Xu, Y.; Kino, K.; Sugiyama, H. J. Biomol. Stuct. Dyn. 2002, 20, 437-
446.
(14) Wincott, F.; Direnzo, A.; Shaffer, C.; Grimm S.; Tracz, D.; Workman, C.;
Sweedler, D.; Gonzalez, C.; Scaring, S.; Usman, N. Nucleic Acids Res.
1994, 22, 2430-2431.
(15) (a) Xodo, L.-E.; Manzini, G.; Quadrifoglio, F.; van der Marel, G. A.; van
Boom, J. H. Biochemistry 1988, 27, 6327-6331. (b) Otokiti, E. O.; Sheardy,
R. D. Biochemistry 1997, 36, 11419-11427.
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13524 J. AM. CHEM. SOC. VOL. 125, NO. 44, 2003