Guanosine analog with respect to Z-DNA stabilization: Nucleotide with combined C8-bromo and C2′-ethynyl modifications

Article abstract of DOI:10.1002/ejoc.200700991

The stabilization of left-handed Z-DNA double strands by chemical modifications is especially of interest regarding investigations of its biological role. Incorporation of modified nucleotide building blocks in DNA allows recognition studies under physiological conditions without the need for a Z-DNA inducing environment. Approaches to enforce the Z-DNA double helix using guanosine derivatives are the introduction of sterically demanding groups at guanine C8 or of 2′-ribosyl substitutions determining the ribosyl conformation. 8-Bromo-2′-ethynyl-arabino-deoxyguanosine was synthesized as phosphoramidite building block. Incorporated in suitable oligonucleotides the potential to induce the Z-form DNA was investigated by CD spectroscopy. With a nucleotide containing the 8-bromo and the 2′-ethynyl group a synergistic effect of the two modifications was expected to provide stronger stabilization of Z-DNA. Wiley-VCH Verlag GmbH & Co. KGaA, 2008.

Full text of DOI:10.1002/ejoc.200700991

FULL PAPER
DOI: 10.1002/ejoc.200700991
Guanosine Analog with Respect to Z-DNA Stabilization: Nucleotide with
Combined C8-Bromo and C2Ј-Ethynyl Modifications
André Nadler[a] and Ulf Diederichsen*[a]
Keywords: Circular dichroism / Conformation / Nucleotide modification / Oligonucleotides / Z-DNA
The stabilization of left-handed Z-DNA double strands by
chemical modifications is especially of interest regarding in-
vestigations of its biological role. Incorporation of modified
nucleotide building blocks in DNA allows recognition studies
under physiological conditions without the need for a Z-DNA
inducing environment. Approaches to enforce the Z-DNA
double helix using guanosine derivatives are the introduction
of sterically demanding groups at guanine C8 or of 2Ј-ribosyl
substitutions determining the ribosyl conformation. 8-Bromo-
2Ј-ethynyl-arabino-deoxyguanosine was synthesized as
phosphoramidite building block. Incorporated in suitable oli-
gonucleotides the potential to induce the Z-form DNA was
investigated by CD spectroscopy. With a nucleotide contain-
ing the 8-bromo and the 2Ј-ethynyl group a synergistic effect
of the two modifications was expected to provide stronger
stabilization of Z-DNA.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2008)
proach for Z-DNA stabilization takes advantage of an
arabino-configured ribosyl modification of deoxyguanosine
introducing an ethynyl group at C2Ј (dGEt).[10] Oligomer
d(CGEtCGEtCG)2 exhibits a left-handed helical structure
even under salt free conditions,[10] whereas its natural coun-
terpart d(CGCGCG)2 requires 4  NaCl concentration to
adopt Z-form DNA.[11] The ethynyl substitution affects the
DNA topology by inducing a C2Ј-exo sugar conformation
which is closely related to the C3Ј-endo conformation re-
quired for Z-DNA. So far, 8-methylguanosine is the most
powerful Z-DNA stabilizing nucleotide combining the
steric demand of the methyl group at C8 with an 2Ј-OH
group in the solvent exposed area of the Z-DNA double
helix.[12] Furthermore, the ribosyl conformation of the
modified nucleotide is affected by the 2Ј-OH group towards
Z-form stabilization.
Introduction
The potential of DNA to adopt various double-strand
topologies besides B-DNA is crucial for protein and small
molecule recognition.[1] Effective DNA binding often de-
pends on the respective oligonucleotide conformation which
is influenced by the environment and can also be induced
by an interacting small molecule or protein.[2] Especially Z-
DNA as the only left-handed double helix is of interest
since only few small molecules[3] or proteins[4,5] are known
for specific Z-DNA binding. The transient stability of Z-
DNA conformation might be an explanation for the small
number of structurally characterized Z-DNA complexes.
The low population of the higher-energy Z-DNA confor-
mation is caused by the required syn-conformation for ev-
ery other purinyl nucleotide which is sterically disfavored
compared to the usual anti-conformation.[6] Further, phos-
phodiester distances in the backbone are lowered by incor-
poration of syn-nucleotides thereby enhancing the electro-
static repulsion. For studying the impact of the Z-DNA
topology on specific recognition phenomena and regulative
processes, double strands stabilized in Z-DNA conforma-
tion are desirable.
In order to further investigate the synergistic effect of
two nucleotide modifications with respect to the induction
of Z-DNA conformation, a nucleotide with a bromo sub-
stituent at C8 and the ethynyl group at C2Ј was prepared
and introduced in appropriate DNA oligomers to induce Z-
DNA (Figure 1).
It was expected that the combination of these approaches
for Z-DNA stabilization should provide nucleotides with in-
creased efficiency based on a large nucleobase substituent
and the preference for the deoxyribosyl C2Ј-exo conforma-
tion. This hypothesis was validated comparing oligonucleo-
tides containing 8-bromo-2Ј-ethynyl-arabino-deoxyguano-
sine modifications with the respective 8-bromo-deoxyguano-
sine (dGBr) oligonucleotides. The synthesis of 8-bromo-2Ј-
ethynyl-arabino-deoxyguanosine phosphoramidite (1) is de-
scribed and the potential of the artificial nucleoside for Z-
DNA induction was investigated by CD spectroscopy.
Typical nucleoside modifications favoring the syn-confor-
mation are the introduction of substituents like a methyl
group[7] and a bromo substituent at C8 of the purine[8] or
CH3 at C5 of cytosine, respectively.[9] These substituents are
sterically demanding and thereby competing with the nu-
cleobase for avoiding backbone interactions. A second ap-
[a] Institut für Organische und Biomolekulare Chemie, Georg
August Universität Göttingen,
Tammannstr. 2, 37077 Göttingen, Germany
Fax: +49-551-392944
E-mail: udieder@gwdg.de
1544
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 1544–1549

Nucleotide Modifications
lylation to nucleoside 5 was accomplished using TBAF in
toluene/AcOH. The temporary DMT-protecting group re-
quired at the 5Ј-OH for solid phase synthesis was attached
in good yield. The phosphoramidite building block 1 was
synthesized using standard procedures.[13] The 8-bromo-2Ј-
deoxyguanosine phosphoramidite required for comparison
of Z-form DNA induction was synthesized according to lit-
erature.[14]
Compound 1 (dG*) was incorporated in the oligo-
nucleotides d(CGCG*CG) (7), d(GCG*CGC) (8),
d(CG*CATG*TG) (9), and d(CTACG*TAG) (10). Oligo-
mers 7, 8, and 10 are self-complementary providing two
Figure 1. Guanosine phosphoramidite 1 for solid phase synthesis dG* modifications in the respective double strands whereas
containing the 8-bromo and 2Ј-ethynyl modifications.
oligomer 9 requires a counter strand d(CACATGCG) (11)
for duplex formation. The regular oligonucleotides d(CG)3
(12), d(GC)3 (13), and d(CTACGTAG) (14) as well as the 8-
bromo-deoxyguanosine modified oligomers d(CGCGBrCG)
Results and Discussion
(15) and d(GCGBrCGC) (16) were synthesized to evaluate
the synergistic effect of the bromo-ethynyl modification
with respect to Z-DNA induction.
The synthesis of 1 started from guanosine following the
procedure of Buff and Hunziker to obtain the 2Ј-ethynyl-
nucleoside 2.[10] Protection of the 5Ј- and 3Ј-OH with the
The modified oligomers 7 and 8 are derived from DNA
1,1,3,3-tetraisopropyldisiloxane-1,3-diyl (TIPDS) group double strands [d(CG)3]2 (12) and [d(GC)3]2 (13) containing
and of the nucleobase N2-amino group with the dimethyl- the alternation of guanosine and cytidine nucleosides that
aminomethylene group was followed by oxidation of the 2Ј- is necessary for facile induction of Z-DNA. The C/G-alter-
OH functionality with Dess–Martin periodinane. Lithium nating sequences serve as reference since they crystallize in
acetylide addition to the ketone was succeeded by radical left-handed Z-DNA conformation and are known to form
deoxygenation providing the 2Ј-ethynyl-nucleoside
2
the Z-DNA at high salt concentrations.[6] The double helix
(Scheme 1). For introduction of bromine at C8 of the 2Ј- conformation of modified oligomers is conveniently ana-
ethynyl-nucleoside 2 a mild protocol was applied using N- lyzed by CD spectroscopy deriving the helical sense and
bromosuccinimide (NBS) at 0 °C in MeCN/H2O, 4:1 using spectra for pure B-DNA and Z-DNA for compari-
(Scheme 1). The desired bromonucleoside 3 was obtained son.[15] The transition from B to Z-DNA is accompanied
in 76% yield. Nevertheless, separation from a cyclic byprod- by a complete inversion of the CD spectrum: A positive
uct 4 was required that was obtained in 10% yield likely Cotton effect around 280 nm for B-DNA is replaced by a
due to bromine addition at the alkyne followed by nucleo- more intense negative band at 295 nm in the Z-form. Simi-
philic attack of the guanine N1. Changing to more reactive larly, the negative Cotton effect around 250 nm detected
reaction conditions (3 h and 10 equiv. NBS) the cyclic prod- with B-DNA converts into a positive signal at 260 nm with
uct 4 can be obtained predominantly (50%). Complete desi- Z-DNA.
Scheme 1. Synthesis of 8-bromo-2Ј-ethynyl-arabino-deoxyguanosine phosphoramidite (1).
Eur. J. Org. Chem. 2008, 1544–1549
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1545

A. Nadler, U. Diederichsen
FULL PAPER
First indication for the capability of 1 (dG*) to induce
Oligonucleotide d(CG)3 (12) has a higher Z-DNA form-
the Z-conformation was obtained by comparison of oligo- ing capability compared to oligomer d(GC)3 (13) due to
mer 7 containing only one modification with the natural stabilizing stacking and sugar interactions based on nucleo-
counterpart 12 and the oligonucleotide containing only the tide dimers required in Z-DNA double strands.[16] There-
8-bromo-2Ј-deoxyguanosine modification (dGBr) (Fig- fore, it was expected that the induction of Z-DNA confor-
ure 2). At 100 m NaCl concentration the self pairing mation would also be stronger in oligomer 7 compared to
oligomer 12 formed a B-DNA-like duplex, whereas for the the double strand of the oligomer with inversed sequence
modified hexamers 7 and 15 a left-handed helix was indi- 8. From the comparison of CD spectra of oligomers 8, 13,
cated. The modified nucleoside 1 (dG*) had the expected and 16 at 100 m NaCl concentration (Figure 4) it can be
Z-form inducing effect on double-stranded DNA providing concluded that the guanosine modification 1 is at least as
an increased Z-DNA helix propensity with the double effective with respect to Z-DNA induction in oligomer 8
modification dG* incorporated instead of dGBr.
compared to duplex 7. The strong Z-form induction of dG*
in d(GC)3 oligomer 8 is quite remarkable since one 8-bro-
moguanosine modification (dGBr) was not sufficient for Z-
DNA induction in this sequence.
Figure 2. CD spectra of oligomers d(CGCG*CG) (7),
d(CGCGBrCG) (15), and of oligomer d(CG)3 (12) all at 100 m
NaCl concentration in 10 m phosphate buffer, pH7.
Figure 4. CD spectra of oligomers d(GCG*CGC) (8),
d(GCGBrCGC) (16), and of oligomer d(GC)3 (13) all at 100 m
NaCl concentration in 10 m phosphate buffer, pH7.
The double strand stability of oligomer 7 (Tm = 28 °C,
8% hyperchromicity, 100 m NaCl, 6 µ) determined by
temperature dependent UV spectroscopy indicated that two
artificial nucleosides 1 (dG*) significantly effected the du-
plex stability since the natural counterpart 12 had a stability
of Tm = 44 °C (3% hyperchromicity, 100 m NaCl, 6 µ).
The thermal stability of the duplex formed by oligomer 7
was also followed by CD spectroscopy measured at various
temperatures without NaCl addition (Figure 3). A left-han-
ded helix was found at temperatures between 5 and 20 °C.
The helix propensity decreased with rising temperature in-
dicating a single strand without conformational preference
at higher temperatures.
A salt dependency of the Z-form stabilization was ob-
served for oligomer 8 (Figure 5) showing a decreasing helix
propensity at 50 m NaCl concentration compared to
oligomer 8 at 100 m NaCl. Without NaCl a complete loss
of double helix formation was observed. The CD spectra of
oligomer 8 without NaCl addition were also measured at
various temperatures; in contrast to DNA 7 even at low
temperatures only non-structured oligonucleotides have
been detected (data not shown).
Figure 3. CD spectra of oligomer d(CGCG*CG) (7) in 10 m
phosphate buffer, pH7 without NaCl at various temperatures: 5,
10, 15, 20, 30, 40, and 50 °C.
Figure 5. CD spectra of oligomer d(GCG*CGC) (8) at 0, 50 and
100 m NaCl concentration in 10 m phosphate buffer, pH7.
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© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 1544–1549

Nucleotide Modifications
DNA with a C/G-alternating sequence is destined for Z-
DNA formation under the appropriate conditions. Since it
is much more difficult to induce Z-form DNA with oligonu-
cleotides that also contain A-T base pairs, oligomer
d(CG*CATG*TG) (9) was investigated that together with
its counter strand d(CACATGCG) (11) still provides the
purine/pyrimidine alternation but includes three A-T base
pairs. It was expected that two modified nucleotides dG*
(1) might be sufficient to also facilitate the B to Z-DNA
transition in the A-T containing duplex 9 + 11. Neverthe-
less, it was not possible to induce Z-form DNA by NaCl
addition up to 4  concentration. The CD spectra measured
in aqueous buffer at pH7 at 300 m NaCl concentration
provided a negative Cotton effect around 288 nm at tem-
peratures below 5 °C that would be in line with a left-han-
ded helix (Figure 6). Anyhow, positive Cotton effects below
260 nm that are typical for Z-DNA were missing and at
higher temperatures the DNA double strand 9 + 11 was
likely to be right-handed but deviating from a B-DNA
double strand. The power of the guanosine analog 1 for Z-
DNA induction seems to be restricted to C/G-alternating
oligomers. Already by A/T incorporation the double helix
seems to be locked in a right-handed form that differs from
B-DNA.
Conclusions
The stabilization of DNA in its left-handed Z-form by
incorporation of modified nucleotides is of significance to
investigate the biological role of Z-DNA. Two approaches
to induce Z-DNA by incorporation of modified nucleotides
were combined in one derivative by synthesis of 8-bromo-
2Ј-ethynyl-arabino-deoxyguanosine phosphoramidite build-
ing block 1. The nucleoside carrying the bromine for steric
hindrance at C8 of the nucleobase and an ethynyl residue
at C2Ј to adjust the conformation of the ribosyl sugar unit
was incorporated as phosphoramidite into DNA sequences.
Within C/G-alternating oligomers that are likely to be
transformed to the Z-form DNA the guanosine derivative
with combined modifications strongly induced Z-DNA
transition. Compared to oligomers with an 8-bromogua-
nosine (dGBr) or
a 2Ј-ethynylguanosine modification
(dGEt)[10] only, a synergistic effect was detected for the com-
bined modifications (dG*). Nevertheless, the C/G alter-
nation seems to be a prerequisite for 8-bromo-2Ј-ethynyl
modification containing DNA double strands to adopt Z-
form DNA. A purine/pyrimidine alternating DNA double
strand including A-T base pairs was not converted into Z-
DNA.
Experimental Section
General Remarks: Infrared spectra were recorded with a Perkin–
1
Elmer type 1600 Series FT-IR infrared spectrophotometer. H and
13C NMR spectra were recorded with a Varian Unity 300 spec-
trometer. UV spectra were recorded with a Perkin–Elmer type
Lambda 10 UV/Vis spectrophotometer. ESI-MS data were ob-
tained with a Finnigan (type LGC or TSQ 7000) spectrometer, high
resolution spectra were obtained with a Bruker (type Apex-Q IV
7T). Flash chromatography was performed using Merck silica gel
60. HPLC purification of the oligonucleotides was carried out by
IBA using a DuPont Series 8800 Chromatograph and a Gemini 5µ
C18 150ϫ4.6 mm column. A 0–60 gradient of 50 m TEAA/
50 m TEAA 70% MeCN was utilized. CD spectra were recorded
with a Jasco J-810 spectropolarimeter. All samples were measured
in 10 m phosphate buffer at pH7.
Figure 6. CD spectra of an equimolar mixture of oligomers 9 and
11 in 10 m phosphate buffer, pH7 at 300 m NaCl concentration
with increasing temperature 0, 5, 10, 15, 20, and 30 °C.
8-Bromo-9-{2Ј-deoxy-3Ј,5Ј-O-(tetraisopropyldisiloxan-1,3-diyl)-N2-
[(dimethylamino)methylene]-2ЈC-[(trimethylsilyl)ethynyl]-β-D-
arabinofuranosyl}guanine (3): N-Bromosuccinimide (1.81 g,
9.63 mmol) was added in portions at 0 °C to a stirred solution of
2 (2.27 g, 3.43 mmol) in MeCN/H2O, 4:1 (80 mL). The reaction
was quenched by addition of saturated Na2S2O3 solution (20 mL)
after 45 min. The solvent was removed in vacuo and the residue
Inducing the Z-DNA conformation is even more difficult
in case of the self pairing oligomer d(CTACG*TAG) (10)
which besides containing four A-T base pairs has no strict
purine/pyrimidine alternation. Therefore, it was not surpris- was dissolved in ethyl acetate and washed with water (2ϫ50 mL).
The organic layer was dried with Na2SO4 and the solvent evapo-
rated. The residue was purified by flash chromatography using the
eluent CH2Cl2/MeOH, 92:8 to yield a yellowish oil (1.92 g, 76%).
Rf(CH2Cl2/MeOH, 9:1) = 0.54. UV/Vis: λmax = 236.5, 281.0,
ing that transition from B- to Z-DNA was not ac-
complished at 300 m or even at 4  NaCl concentration
(spectra not shown). As already observed for the double
strand formed by oligomers 9 + 11, the incorporation of
the guanosine modification dG* (1) in self pairing DNA 10
provided a right-handed helix. The guanosine modification
dG* (1) incorporated in a DNA oligomer that has not a
typical Z-DNA forming sequence is not strong enough to
induce a left-handed helix but leads to a defined right-han-
ded B-DNA like double strand.
307.5 nm. IR (KBr): ν = 2946, 2895, 2180, 1694, 1632, 1535, 1348,
˜
1290, 1249, 1206, 1068, 1036, 958, 885, 761, 701 cm–1. 1H NMR
(300 MHz, [D6]DMSO, 100 °C): δ = 11.06 (br. s, 1 H, NH), 8.43–
8.50 (m, 1 H, N=CH), 8.58 (d, 3J = 9.0 Hz, 1 H, H1Ј), 5.03–5.11
(m, 1 H, H3Ј), 4.19 (dd, 2J = 12.3, 3J = 6.3 Hz, 1 H, H5Ј), 4.08
3
(dd, 2J = 12.3, J = 3.3 Hz, 1 H, H5Ј), 3.85 (mz, 1 H, H4Ј), 3.74 (t,
3J = 8.7 Hz, 1 H, H2Ј), 3.15 (s, 3 H, NCH3), 3.05 (s, 3 H, NCH3),
Eur. J. Org. Chem. 2008, 1544–1549
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1547

A. Nadler, U. Diederichsen
1.01–1.11 (m, 28 H, iPr-H), –0.09 [s, 9 H, Si(CH3)3] ppm. 13C NMR 4 H, DMT-H), 6.44 (d, 3J = 8.7 Hz, 1 H, 3Ј-OH), 5.84 (d, 3J =
FULL PAPER
(75 MHz, [D6]DMSO, 100 °C): δ = 157.2 (Ar-C), 156.7 (Ar-C),
155.6 (Ar-C), 150.9 (Ar-C), 100.5 (CϵC), 85.1 (CϵC), 82.5 (C4Ј),
6.0 Hz, 1 H, H1Ј), 4.67–4.76 (m, 1 H, H3Ј), 3.95 (mz, 1 H, H4Ј),
3.71 (s, 6 H, OCH3), 3.70 (s, 6 H, OCH3), 3.56–3.67 (m, 2 H, H5Ј),
77.4 (C1Ј), 44.2 (C2Ј), 39.3 (NCH3), 34.1 (NCH3), 16.7, 16.6, 16.6, 3.20 (mz, 1 H, H2Ј), 2.98 (s, 3 H, NCH3), 2.93 (s, 3 H, NCH3), 2.86
16.5, 16.5, 16.4, 16.3, 16.3 (iPr-CH), 12.3, 12.1, 12.0, 11.7 (iPr-CH), (d, 4J = 2.4 Hz, 1 H, ϵCH) ppm. 13C NMR (75 MHz, [D6]DMSO,
–1.4 [Si(CH3)3] ppm. ESI: m/z (rel.%) = 739 (100) [M + H]+, 741
(90) [M + H]+. HR-ESI-MS: m/z [M + H]+ = calcd. 739.24849 for
C30H51BrN6O5Si3, found 727.24820.
35 °C): δ = 157.9 (Ar-C or N=CH), 157.9 (Ar-C or N=CH), 156.3
(Ar-C or N=CH), 149.5 (Ar-C), 144.8 (Ar-C), 136.0 (Ar-C), 135.4
(Ar-C), 129.6 (Ar-C), 129.4 (Ar-C), 127.6 (Ar-C), 127.5 (Ar-C),
123.8 (Ar-C), 112.9 (Ar-C), 112.9 (Ar-C), 85.4 (CϵC), 84.7 (C1Ј),
83.6 (C4Ј), 79.2 (CϵC), 74.8 (C3Ј), 63.6 (C5Ј), 54.9 (OCH3), 54.9
(OCH3), 42.6 (C2Ј), 40.7 (NCH3) 34.5 (NCH3) ppm. ESI-MS: m/z
(%) = 727 (100) [M + H]+, 729 (90) [M + H]+. HR-ESI-MS: m/z
[M + H]+ = calcd. 727.18742 for C36H35BrN6O6, found 727.18749.
Analytical Data of Cyclic Byproduct 4: In addition to 3 the annu-
lated nucleoside 4 was isolated as yellowish oil (280 mg, 10%).
Rf(CH2Cl2/MeOH, 9:1) = 0.50. 1H NMR (300 MHz, [D6]DMSO,
3
100 °C): δ = 8.73 (s, 1 H, N=CH), 6.26 (d, J = 6.2 Hz, 1 H, H1Ј),
2
2
2
2
4.85 (dd, J = 6.0, J = 6.3 Hz, 1 H, H2Ј), 4.38 (dd, J = 6.8, J =
2
3
5.3 Hz, 1 H, H3Ј), 4.13–4.17 (m, 1 H, H4Ј), 3.80 (dd, J = 12.3, J
= 3.7 Hz, 1 H, H5Ј), 3.26 (s, 3 H, NCH3), 3.04 (s, 3 H, NCH3), 2.98
(dd, 2J = 12.3, 3J = 6.7 Hz, 1 H, H5Ј), 0.98–1.07 (m, 28 H, iPr-
H), 0.09 [s, 9 H, Si(CH3)3] ppm. 13C NMR (75 MHz, [D6]DMSO,
100 °C): δ = 160.1 (Ar-C or N=CH), 159.9 (Ar-C or N=CH), 153.5
(Ar-C or N=CH), 138.7 (C=C), 132.5 (C=C), 119.1 (C8), 117.1
(C4), 86.0 (C4Ј), 84.5 (C1Ј), 74.4 (C3Ј), 62.8 (C5Ј), 53.4 (C2Ј), 40.7
(NCH3), 34.6 (NCH3), 16.7, 16.7, 16.5, 16.4, 16.4, 16.3, 16.3, 16.2
(iPr-CH3), 12.6, 12.0, 11.9, 11.8 (iPr-CH), –1.3 [Si(CH3)3] ppm.
ESI-MS: m/z (rel.%) = 817 (50) [M + H]+, 819 (90) [M + H]+, 821
(50) [M + H]+. HR-ESI-MS: m/z = [M + H]+ = calcd. 817.15900
for C30H51BrN6O5Si3, found 817.15890.
8-Bromo-9-[2Ј-deoxy-2ЈC-ethynyl-3ЈO-[cyanoethyl(diisopropyl-
amino)phosphanyl]-5ЈO-dimethoxytrityl-β-D
-arabinofuranosyl]-N2-
[(dimethylamino)methylene]guanine (1): Cyanoethyl-N,N-diisopro-
pylphosphoramidite chloride (40.0 mg, 0.21 mmol) was added to a
solution of 6 (100 mg, 0.14 mmol) and DIPEA (53 mg, 0.41 mmol)
in dry CH2Cl2 (1.50 mL) under an argon atmosphere. The reaction
mixture was stirred for 6 h and the reaction quenched by addition
of a saturated NaHCO3 solution (5 mL). The resulting mixture was
extracted with CH2Cl2 (3ϫ10 mL) and the combined organic lay-
ers were dried with Na2SO4. The solvent was evaporated and the
residue purified by flash chromatography using the eluent CH2Cl2/
acetone/Et3N, 1:1:0.01. The raw phosphoramidite was dissolved in
a minimum amount of CHCl3 and the solution was added dropwise
to pentane (100 mL) at –17 °C. The resulting precipitate was col-
lected and dried in vacuo to give 1 (59 mg, 0.06 mmol, 46%) as a
8-Bromo-9-(2Ј-deoxy-2ЈC-ethynyl-β-D
-arabinofuranosyl)-N2-[(di-
methylamino)methylene]guanine (5): TBAF ϫ 3 H2O (2.20 g,
6.98 mmol) and AcOH (0.5 mL) were added to a solution of 3
(1.64 g, 2.26 mmol) in toluene (50 mL). The reaction mixture was
stirred at room temp. for 3 h. The solvent was evaporated and the
residue purified by flash chromatography using the eluent CHCl3/
MeOH, 9:1 to afford 5 (800 mg, 1.89 mmol, 84%) as a colorless
oil. Rf(CHCl3/MeOH, 9:1) = 0.30. UV/Vis: λmax = 235.5, 307.0 nm.
colorless powder. Rf(CH2Cl2/MeOH, 9:1) = 0.44. UV/Vis: λmax
=
234.5, 282.5, 310.0 nm. IR (KBr): ν = 2966, 2245, 1695, 1632, 1532,
˜
1423, 1347, 1286, 1250, 1178, 1115, 1031, 980, 830, 703 cm–1.
1H NMR (300 MHz, [D6]DMSO, 35 °C): δ = 11.95 (br. s, 1 H,
NH), 8.17 (s, 0.5 H, N=CH), 8.13 (s, 0.5 H, N=CH), 7.28–7.33 (m,
2 H, Ph-H), 7.14–7.22 (m, 7 H, DMT-H), 6.68–6.78 (m, 4 H, DMT-
H), 6.50 (d, 3J = 4.2 Hz, 0.5 H, H1Ј), 6.48 (d, 3J = 4.2 Hz, H1Ј,
0. H), 4.96–5.11 (m, 1 H, H3Ј), 4.11 (mz, 1 H, H4Ј), 3.87 (mz, 1 H,
H2Ј), 3.70–3.78, (m, 1 H, H5Ј), 3.72 (s, 6 H, OMe), 3.70 (s, 6 H,
OMe), 3.71 (s, 6 H, OMe), 3.43–3.66 (m, 4 H, iPr-CH, CH2CN),
IR (KBr): ν = 3130 (br), 2946, 2897, 2175, 1756, 1682, 1505, 1465,
˜
1385, 1251, 1199, 1115, 1008, 957, 921, 814, 786, 636 cm–1
.
1H NMR (300 MHz, [D6]DMSO, 35 °C): δ = 11.44 (br. s, 1 H,
NH), 8.58 (s, 1 H, N=CH), 6.37 (d, 3J = 8.4 Hz, 1 H, H1Ј), 5.81
(br. s, 1 H, 3Ј-OH), 4.80–4.75 (m, 2 H, H3Ј, 5Ј-OH), 3.78–3.66 (m,
2
3
2
3
3
4
3.33 (dd, J = 9.6, J = 1.8 Hz, 0.5 H, H5Ј), 3.27 (dd, J = 9.6, J
3 H, H4Ј, H5Ј), 3.59 (dd, J = 9.3, J = 2.7 Hz, 1 H, H2Ј), 3.17 (s,
3 H, NCH3), 3.06 (s, 3 H, NCH3), 2.88 (d, 4J = 2.4 Hz, 1 H, CϵC-
H) ppm. 13C NMR (75 MHz, [D6]DMSO, 35 °C): δ = 158.0 (Ar-C
or N=CH), 156.9 (Ar-C or N=CH), 156.3 (Ar-C or N=CH), 150.8,
(C4), 121.2 (C8), 120.1 (C5), 85.4 (C4Ј), 84.3 (C1Ј), 79.3 (CϵC),
75.5 (CϵC), 74.0 (C3Ј), 60.7 (C5Ј), 42.6 (C2Ј), 40.8 (NCH3), 34.6
(NCH3) ppm. ESI-MS: m/z (%) = 447.0, 449.0 (10) [M + Na]+,
870.9, 872.9, 874.8 (100) [2M + Na]+. HR-ESI-MS: m/z [M + H]+
= calcd. 425.05674 for C15H18BrN6O4, found 425.05705.
4
= 1.8 Hz, 0.5 H, H5Ј), 2.76 (d, J = 2.7 Hz, 0.5 H, ϵCH), 2.74 (d,
4J = 2.7 Hz, 0.5 H, ϵCH), 2.47–2.51 (m, 2 H, PCH2), 1.11 (d, J
3
= 7.2 Hz, 6 H, iPr-CH3) ppm. 31P NMR (122 MHz, [D6]DMSO,
35 °C): δ = 152.07 (br. s), 151.04 ppm. ESI-MS: m/z (%) = 927
(100) [M + H]+, 929 (90) [M + H]+. HR-ESI-MS: m/z [M + H]+
=
calcd. 927.29527 for C45H53BrN8O7P, found 927.29576.
DNA Oligomer 7: ESI-MS: m/z (%) = 1894.26 [M], 1916.23 [M –
H+Na], 1938.19 [M – 2H + 2Na]. Rt = 11.86 min; Tm = 28 °C, 8 %
hyperchromicity, 100 m NaCl, 6 µ.
8-Bromo-9-(2Ј-deoxy-2ЈC-ethynyl-5ЈO-dimethoxytrityl-β-D-arabino-
furanosyl)-N2-[(dimethylamino)methylene]guanine (6): DMTCl
(1.60 g, 4.73 mmol) was added to a solution of 5 (770 mg,
1.82 mmol) in dry pyridine under an argon atmosphere. The reac-
tion mixture was stirred for 18 h and then poured into a mixture
of CH2Cl2 (100 mL) and saturated NaHCO3 solution (100 mL).
The organic layer was dried with Na2SO4 and the solvent evapo-
rated. The residue was purified by flash chromatography using the
eluent CH2Cl2/MeOH/Et3N, 92:7:1 to afford 6 (998 mg, 1.37 mmol,
76%) as yellowish foam. Rf(CH2Cl2/MeOH, 9:1) = 0.40. UV/Vis:
DNA Oligomer 8: ESI-MS: m/z (%) = 1894.25 [M], 1916.24 [M –
H+Na], 1938.22 [M – 2H + 2Na], 1960.21 [M – 3H+3Na]. Rt =
12.15 min; Tm = 17 °C, 2% hyperchromicity, 100 m NaCl, 6 µ.
DNA Oligomer 9: ESI-MS: m/z = 2628.29 [M], 2650.27 [M – H +
Na], 2672.26 [M – 2H + 2Na]. Rt = 11.79 min; double-strand sta-
bility of oligomer 9 + 11: Tm = 15 °C, 8% hyperchromicity, 100 m
NaCl, 6 µ.
λmax = 235.0, 283.0, 310.0 nm. IR (KBr): ν = 3377 (br), 3001 (br),
˜
2930, 2045, 1685, 1632, 1534, 1422, 1346, 1249, 1176, 1114, 829,
DNA Oligomer 10: ESI-MS: m/z (%)= 2510.38 [M – H]–, 2532.36
1
704 cm–1. H NMR (300 MHz, [D6]DMSO, 35 °C): δ = 11.43 (br. [M – 2H + Na]–, 2554.34 [M – 3H + 2Na]–, 2576.32 [M – 4H +
s, 1 H, NH), 8.12 (br. s, 1 H, N=CH), 7.31 (dd, 3J = 7.8, 4J = 3Na]–, 2598.301 [M – 5H + 4Na]–, 2620.29 [M – 6H + 5Na]–. Rt
1.8 Hz, 2 H, Ph-H), 7.15–7.21 (m, 7 H, DMT-H), 6.72–6.80 (m,
1548
= 12.02 min; Tm = 28 °C, 8% hyperchromicity, 100 m NaCl, 6 µ.
www.eurjoc.org
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 1544–1549

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Acknowledgments
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Received: October 18, 2007
Published Online: February 5, 2008
Eur. J. Org. Chem. 2008, 1544–1549
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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