Synthesis and properties of oligonucleotides containing 8-bromo-2′-deoxyguanosine

Article abstract of DOI:10.1081/NCN-100002085

The preparation of oligonucleotides containing 8-bromo-2′-deoxyguanosine is described. Substitution of G by 8-bromoguanine on an alternating CG decamer stabilizes the Z-form in such a way that the B-form was not observed. Melting temperatures showed that duplexes in which 8-bromo-2′-deoxyguanosine paired with natural bases were much less stable.

Full text of DOI:10.1081/NCN-100002085

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SYNTHESIS AND PROPERTIES OF OLIGONUCLEOTIDES
CONTAINING 8-BROMO-2′-DEOXYGUANOSINE
Carme Fàbrega a , Maria J. Macías a & Ramon Eritja b
a European Molecular Biology Laboratory , Meyerhofstrasse I, Heidelberg, D-69117,
Germany
b Centro de Investigación y Desarrollo , C.S.I.C. Jordi Girona 18-26, Barcelona, E-08034,
Spain
Published online: 17 Aug 2006.
To cite this article: Carme Fàbrega , Maria J. Macías & Ramon Eritja (2001) SYNTHESIS AND PROPERTIES OF
OLIGONUCLEOTIDES CONTAINING 8-BROMO-2′-DEOXYGUANOSINE, Nucleosides, Nucleotides and Nucleic Acids, 20:3,
251-260, DOI: 10.1081/NCN-100002085
To link to this article: http://dx.doi.org/10.1081/NCN-100002085
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(3), 251–260 (2001)
SYNTHESIS AND PROPERTIES OF
OLIGONUCLEOTIDES CONTAINING
8-BROMO-2ꢀ-DEOXYGUANOSINE
Carme Fa`brega, Maria J. Mac´ıas, and Ramon Eritja∗
European Molecular Biology Laboratory, Meyerhofstrasse I, D-69117
Heidelberg, Germany
ABSTRACT
The preparation of oligonucleotides containing 8-bromo-2ꢀ-deoxyguanosine is
described. Substitution of G by 8-bromoguanine on an alternating CG decamer
stabilizes the Z-form in such a way that the B-form was not observed. Melt-
ing temperatures showed that duplexes in which 8-bromo-2ꢀ-deoxyguanosine
paired with natural bases were much less stable.
INTRODUCTION
Chemically modified oligonucleotides are widely used in the study of nucleic
acid structure and protein-nucleic acid interactions (1,2). Specifically, the intro-
duction of brominated and iodinated nucleosides in defined positions is applied in
the following areas: (a) photo-cross-linking with nucleic acid-binding proteins, in
which the halogen is excised selectively to generate the nucleobase radicals (3),
(b) in X-ray diffraction experiments, in which the higher electron density of the
bromine and iodine is used to solve the phase problem (4), (c) mutagenesis studies,
in which changes in pKa and/or tautomerism induced by the halogen alter base
pairing properties and polymerase fidelity (5).
In the recent years interest has focused on purine nucleosides carrying bulky
substituents at the 8-position to favor the syn conformation at the N-glycoside bond
∗Current address: Centro de Investigacio´n y Desarrollo, C.S.I.C. Jordi Girona 18-26, E-08034
Barcelona, Spain.
251
ꢁ
C
Copyright 2001 by Marcel Dekker, Inc.
www.dekker.com

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252
(6–8). These oligonucleotides may be used as model compounds to study DNA
fidelity and DNA repair. One of the most significant oxidative damage products
in DNA is 8-oxo-7,8-dihydro-2ꢀ-deoxyguanosine (8-oxoG) (9). This lesion is re-
paired by several enzymes, and it has been suggested that the higher population of
the syn conformation on 8-oxoG is the structural feature recognized by the repair
enzymes to distinguish 8-oxoG and dG (10). Bulky substituents on purine nucle-
osides at the 8-position shift the equilibrium to the syn conformation (11). On the
basis of these data, 8-bromo-2ꢀ-deoxyguanosine (B) was successfully used to probe
syn-anti conformational preferences on G-quartet structures (6). Moreover, bromi-
nation of poly (d(GC)) stabilizes the Z-DNA form (12). A modification of 38%
8-bromoguanine and 18% 5-bromocytosine is enough to render a stable Z-DNA
helix in physiological conditions.
Oligodeoxynucleotides containing 8-bromoguanine were prepared using the
isobutyryl group for the protection of the amino group at position 2 (6). In this
communication we report the synthesis of an 8-bromoguanine phosphoramidite
derivative protected with the dimethylformamidine (dmf) (13,14) group and its use
in the preparation of oligodeoxynucleotides carrying 8-bromoguanine. Moreover,
base-pairing properties of oligodeoxynucleotides containing 8-bromoguanine and
their ability to form Z-DNA are described.
RESULTS AND DISCUSSION
Preparation of the Protected Phosphoramidite
The synthesis of the 8-bromoguanine phosphoramidite building block is il-
lustrated in Scheme 1. 8-Bromo-2ꢀ-deoxyguanosine (1) was prepared from dG
as described elsewhere (15). Reaction of 8-bromo-2ꢀ-deoxyguanosine with N,N-
dimethylformamide dimethyl acetal in dimethylformamide (16) gave compound 2
which was used without purification in the following step. Compound 2 was treated
with dimethoxytrityl (DMT) chloride in pyridine for 5 hours at room temperature
yielding compound 3 in good yield (73% from 1). Reaction of compound 3 with 2-
cyanoethyl-N,N-diisopropyl chlorophosphine and N,N-diisopropylethylamine gave
the desired phosphoramidite 4 in 90% yield. Moreover, the DMT-nucleoside 3 was
converted to the 3ꢀ-O-hemisuccinate derivative 5, which was attached to amino-
❛
controlled pore glass (CPG, 500 A) supports following previously described proto-
cols (data not shown) (17,18).
One of the main differences between this and the previous preparation of
isobutyryl-protected 8-bromoguanine derivative (6), is that, bromination is per-
formed before the introduction of the DMT and the amino protecting group. In
this way some possible side reactions such as bromination of the DMT group and
premature cleavage of the acid labile DMT group are avoided, resulting in a much
higher yield. Also, the use of the more labile dmf group for the protection of the
amino group results in a more versatile building block.

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253
O
O
N
N
N
H
N
N
Br
N
H
Br
HO
N
NH2 HO
O
N
N
CHNMe2
Br2
O
dG
Me2N CH(OMe)2
OH
8-Br-dG
2
OH
1,
N(C3H7)2
O
Cl
P
O
N
N
N
H
OCH2CH2CN
CHNMe2
Br
N
N
N
H
DMT
pyr
DMT
O
N
Br
N
N
CHNMe2
O
O
DMT
N
O
N(C3H7)2
O
R = H, 3
4
R = OCH2CH2COOH, 5
P
OR
R = OCH2CH2CONH-CPG, 6
OCH2CH2CN
Scheme 1. Preparation of the phosphoramidite derivative of N-protected-8-bromo-2ꢀ-deoxyguanine.
Preparation of Oligonucleotides
Oligonucleotides A (5ꢀ TB 3ꢀ), B (5ꢀ CACAB 3ꢀ), C (5ꢀ GCAATGGABCCTC
TA 3ꢀ), D (5ꢀ CBCBCGCGCG 3ꢀ), E (5ꢀ 5B5BCGCGCG 3ꢀ) and F (5ꢀ 5G5GCGCG
CG 3ꢀ) where B = 8-bromoguanine and 5 = 5-bromocytosine were prepared on a
DNA synthesizer using both standard (benzoyl-dA and dC and isobutyryl-dG) and
t-butylphenoxyacetyl (TBPA)-protected (19) 2-cyanoethyl phosphoramidites toge-
ther with phosphoramidite 4 and the commercially available phosphoramidite of
5-bromo-2ꢀ-deoxycytidine (protected with the benzoyl group) and the appropriate
succinyl-CPG supports. During the syntheses, 0.02 M iodine solution (tetrahydro-
furane: water: pyridine 7:1:2) was used to prevent the formation of N-cyano nucle-
osides (20). t-Butylphenoxyacetyl anhydride was used instead of acetic anhydride
in the syntheses with TBPA-protected phosphoramidites to prevent acetylation of
TBPA-protected bases (19). After the assembly of the sequences, oligonucleotide-
supports were treated with concentrated (30%) aqueous ammonia solution. The
time needed for the removal of the dmf group on the dimer A was found to be less
than 1 hour at room temperature, because a single peak was observed on reverse
phase HPLC that had the expected molecular weight and the expected nucleo-
side composition after enzymatic digestion. Sequences carrying 5-bromocytosine
(E and F) were prepared using t-butylphenoxyacetyl (TBPA)-protected phospho-
ramidites and deprotected with concentrated ammonia at room temperature to avoid
the formation of 5-aminocytosine (21). Sequences B, C and D prepared with stan-
dard phosphoramidites were treated with concentrated ammonia overnight at 50◦C.
Special attention was paid to detect the decomposition of 8-bromoguanine to 8-
aminoguanine or 8-oxoguanine as it has been described for 5-bromo-pyrimidines
(21) and 8-bromo-dA (22) when using hot ammonia solutions for the removal of
protecting groups. In all cases, a major peak was observed that had the correct

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FABREGA, MACIAS, AND ERITJA
254
Figure 1. Reversed phase chromatography analysis of the mixture obtained after phosphodiesterase
and alkaline phosphatase degradation of purified pentadecamer C (5ꢀ GCA ATG GAB CCT CTA 3ꢀ).
nucleoside composition (see Fig. 1) and the expected molecular weight. The higher
stability of 8-bromoguanine to nucleophiles was also evident from the treatment
of dimer A with lithium azide. This treatment was performed in order to obtain
a dimer containing the photoreactive 8-azido-dG (data not shown). When support
carrying sequence A was treated with 0.16 M lithium azide in DMF overnight at
50◦C, followed by ammonia deprotection (1 h, R.T.) the dimer containing unaltered
8-bromo-G was isolated. The same experiment with a similar dimer but containing
8-bromo-A instead of 8-bromo-G gave total conversion of 8-bromo-A dimer to the
dimer containing 8-azido-A (23). Therefore, 8-bromoguanine oligonucleotides can
be prepared without detectable degradation using the standard ammonia deprotec-
tion conditions (60◦C, overnight).
Melting Experiments
Duplexes having 8-bromoguanine base pairs with the four natural bases
were analyzed. Melting temperatures (Tm) are shown in Table 1. Guanine and
Table 1. Melting Temperatures (Tm,◦C) of 8-bromoguanine
(B) and 8-oxo-G Duplexes (0.1 M sodium phosphate pH 7.0)
5ꢀGCA ATG GAX GCC TCT A 3ꢀ
3ꢀCGT TAC CTY CGG AGA T 5ꢀ
X = G
X = B
X = 8-oxo-G
Y = C
Y = A
Y = G
Y = T
62
55
55
54
56
49
n.c.#
56
60
55
54
50
#Curve with low hyperchromicity and not cooperative

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255
8-oxoguanine base pairs were included as reference. The oligonucleotide contain-
ing 8-oxoguanine were prepared as previously described (16). As expected in all
cases the most stable base pair is formed between the guanine derivative and cyto-
sine. The relative stability of the different guanine derivatives is G
C. A similar trend is observed with the G A mispairs. Duplex containing B
basepair did not exhibit the cooperative helix-to-coil transition and for this reason
Tm was not calculated. In contrast, G T and 8-oxo-d G T mispairs were less stable
than B T mispair. In general, melting temperatures of duplexes containing B were
ꢀ
C>8-oxo-G
ꢀ
C>
B
ꢀ
ꢀ
ꢀ
G
ꢀ
ꢀ
ꢀ
lower than 8-oxo-G and guanine duplexes. The degree of destabilization is similar
to the destabilization found in duplexes containing 8-methoxy-dA (7). This desta-
bilization has been assigned to a major content of the syn conformer that prevents
the normal Watson-Crick hydrogenbonding (7).
B to Z Transition Studies
Previously it was shown that bromination of poly (d(GC)) stabilizes the Z
form (12) but no data have appeared on the role of each individual reaction product:
5-bromocytosine and 8-bromoguanine. We addressed this question by preparing de-
camers containing alternating CG sequences in which two of the five residues were
replaced by the corresponding bromo derivative. The contribution of each bromo
derivative to the stabilization was estimated from the mid-point NaCl concentration
for the B-Z transition of different self-complementary decamers measured by cir-
cular dichroism. As seen in Table 2, the presence of 5-bromocytosine induced only
moderate stabilization of the Z-form (mid-point 1.2 M NaCl compared to a mid-
point of 2.2 M NaCl for d(CGCGCGCGCG)2. On the other hand, 8-bromoguanine
induces a large stabilization of the Z-form in such a way that even without NaCl
added the CD spectra of 8-bromoguanine decamers correspond to the Z-form
(see Fig. 2). The strong stabilization of the Z-form caused by 8-bromoguanine is
in agreement with the strong stabilization observed for 8-methylguanine (24) and
7-deaza-8-methylguanine (25,26). Incorporation of 8-bromoguanine, 8-methylgua-
nine or 7-deaza-8-methylguanine into oligonucleotides may be useful for the
Table 2. Midpoint NaCl Concentration in B->Z Transitions for 8-
bromoguanine (B) and 5-bromocytosine (5) oligodeoxynucleotides (50 mM
tris HCl pH 7.5)
Oligonucleotide
NaCl (M)a
d(CGCGCGCGCG)2
d(5G5GCGCGCG)2
d(CBCBCGCGCG)2
d(5B5BCGCGCG)2
2.2
1.2
no transition, Z-form even at 0 M NaCl
no transition, Z-form even at 0 M NaCl
aIn all cases the solutions were 50 mM Tris HCl buffer, pH 7.5

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256
Figure 2. CD spectra of decamers containing 5-bromocytosine and 8-bromoguanine in 50 mM Tris
HCl buffer, pH 7.5, (without NaCl added) at 20◦C.
preparation of DNA substrates to study Z-DNA binding proteins, (27,28) although
we believe 8-bromoguanine oligonucleotides are easier to prepare.
Conclusion
In conclusion, a new 8-bromo-dG phosphoramidite derivative is described.
The new derivative is easily prepared and can be removed in mild conditions.
Contrary to other brominated nucleosides (21,22), 8-bromo-dG was found to be
very stable to ammonia deprotection. Therefore, the use of more labile groups for
the natural bases is not required. Substitution of G by 8-bromo-G destabilizes the
duplex structure as expected from the higher content of the syn conformer, but it
also strongly stabilizes the Z-form. Thus, 8-bromo-dG may be a useful probe for
locating subtle differences in DNA polymerases (29) and DNA repair glycosylases
due to differences in conformational properties, and for producing substrates for
the study of Z-DNA binding proteins.
EXPERIMENTAL SECTION
General Methods
All reactions were carried out in oven-dried glassware, under a nitrogen or
argon atmosphere, unless specified otherwise. Before use, starting materials were
dried by evaporation with the dry solvent which was later used for the reaction.
Reagents for oligonucleotide synthesis were from Glen Research and PE Applied

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257
Biosystems. Dry solvents were from SDS and Romil. HPLC grade solvents were
from Romil and E. Merck. Snake venom phosphodiesterase (from Crotalus duris-
sus) and alkaline phosphatase were from Boehringer Mannheim. The rest of the
reagents were from Aldrich and Fluka and were used without further purification.
Analytical TLC was run on aluminum sheets coated with silica gel 60 F254 from
Merck. Silica gel column chromatography was performed with Chromatogel 60 A
C.C. (40–60 microns, 230–400 mesh, SDS).
Instrumental
1H-NMR (250 MHz) 13C-NMR (63 MHz) and 31P-NMR (101 MHz) spectra
were recorded on a Bru¨ker AM-250. HPLC chromatography was performed on
an HPLC System Gold (Beckman) and a Waters instrument. Mass spectra were
obtained on a API III SCIEX-Perkin Elmer equipped with a triple quadrupole
detector.
5ꢀ-O-(Dimethoxytrityl)-N2-(N,N-dimethylaminomethyliden)-
8-bromo-2ꢀ-deoxyguanosine (3)
8-Bromo-2ꢀ-deoxyguanosine (15) (1.5 g, 4.3 mmol) was reacted with N,N-
dimethylformamide dimethyl acetal (1.72 ml, 15.9 mmol) in 20 cm3 of N,N-
dimethylformamide for 4 hours at room temperature giving quantitatively com-
pound 2, which was used without purification in the following step.
The residue of the previous reaction (compound 2) was dissolved in 20 ml
of pyridine and treated with dimethoxytrityl chloride (6.5 mmol) for 5 hours at
room temperature. Methanol (1 ml) was added to stop the reaction and the mixture
was concentrated to dryness. The residue was dissolved in dichloromethane and
washed with 1M aq. NaHCO3. The organic phase was dried and concentrated to
dryness. The residue was purified on silica gel eluted with 0-10% methanol gradient
in dichloromethane to afford compound 3 (2.2 g, 73% from 1) (Found: C, 57.7; H,
5.3; N, 12.1. C34H35N6O6 requires C, 58.0; H, 5.0; N, 11.9), Rf 0.10 (5% methanol
in chloroform); δH(CD3OD) 2.29–2.42 (2H, m), 2.98 (3H, s), 3.03 (3H, 1s), 3.16–
3.49 (2H, m), 3.69 (6H, s), 4.05 (1H, m), 4.81 (1H, m), 6.34 (1H, dd, J = 7.8 Hz,
J = 4.4 Hz), 6.64 (4H, d, J = 5.8 Hz), 6.70 (4H, d, J = 5.8 Hz), 7.05-7.42 (5H,
m), 8.34 (1H, s), 8.50 (1H, d, J = 4.2 Hz); δC(CD3OD) 38.3, 35.4, 41.6, 55.6, 65.0,
72.4, 87.0, 87.1, 87.4, 113.9, 121.6, 127.6, 129.1, 129.8, 130.9, 131.1, 138.0, 138.1,
144.6, 152.5, 158.1, 159.0, 159.3, 150.9.
3ꢀ-O-[(2-Cyanoethoxy)-(N,N-diisopropylamino)phosphinyl]-5ꢀ-O-
(dimethoxytrityl)-N2-[(N,N-dimethylamino)methyliden]-8-bromo-2ꢀ-
deoxyguanosine (4)
Compound 3 (0.50 g, 0.74 mmol) was dissolved in dry acetonitrile and
reacted with 0.20 ml (0.88 mmol) of (2-cyanoethoxy)-(N,N-diisopropylamino)

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FABREGA, MACIAS, AND ERITJA
258
chlorophosphine and 0.40 ml (2.22 mmol) of ethyldiisopropylamine. After 1 hour
of magnetic stirring, the reaction was stopped by adding 1 ml of MeOH. The sol-
vent was evaporated and the residue was dissolved in DCM (20 ml) and washed
with 10% aq. NaHCO3 followed by saturated aq. NaCl. The organic phase was
dried and concentrated to dryness. The residue was purified on silica gel eluted
with dichloromethane/ ethyl acetate/triethylamine (45:45:10) to give compound 4
(0.55 g, 90%) (Found: C, 56.7; H, 5.7; N, 12.7. C43H52N8O7BrP requires C, 57.1;
H, 5.8; N, 12.4); Rf 0.8 (dichloromethane/ethyl acetate/triethylamine 45:45:10);
δp (CH3CN, external reference H3PO4) 149.2 and 149.3 (two diastereoisomers).
Oligonucleotide Synthesis
The following sequences have been synthesized: A (5ꢀ TB 3ꢀ), B (5ꢀ CACAB
3ꢀ), C (5ꢀ GCAATGGABCCTCTA 3ꢀ), D (5ꢀ CBCBCGCGCG 3ꢀ), E(5ꢀ 5B5BCGCG
CG 3ꢀ), and F (5ꢀ 5G5GCGCGCG 3ꢀ) being B = 8-bromo-G and 5 = 5-bromo-
cytosine. Oligonucleotides were prepared on an automatic DNA synthesizer using
commercially available 2-cyanoethyl phosphoramidites and the modified phos-
phoramidite. Oligonucleotide sequences B and C were prepared using using both
standard (benzoyl-dA and dC and isobutyryl-dG) and t-butylphenoxyacetyl
(TBPA)-protected (19) phosphoramidites. Oligonucleotide sequences E and F were
prepared using using t-Butylphenoxyacetyl (TBPA)-protected phosphoramidites as
decribed (23). During the syntheses, 0.02 M iodine solution (tetrahydrofurane: wa-
ter: pyridine 7:1:2) was used to prevent the formation of N-cyano nucleosides
(20). t-Butylphenoxyacetyl anhydride was used instead of acetic anhydride on the
syntheses with TBPA-protected phosphoramidites to prevent acetylation of TBPA-
protected bases (19).
Oligonuclcotide-supports were treated with 32% aqueous ammonia at 50 ◦C
for 16 h (when benzoyl and isobutyryl groups are used) and at room temperature
for 16 hr (when TBPA groups are used). Ammonia solutions were concentrated to
dryness and the products were purified either by cartridge purification (COP), by
reversed phase HPLC or by PAGE electrophoresis. All purified products presented
a major peak that was collected and analyzed by snake venom phosphodiesterase
and alkaline phosphatase digestion followed by HPLC analysis of the nucleosides
(HPLC conditions B, see Fig. 2). HPLC solutions are as follows. Solvent A: 100
mM triethylammonium acetate (pH 7.8) and solvent B: 50% ACN in 100 mM
triethylammonium acetate pH 7.8. For analytical runs the following conditions were
used. Column: Nucleosil 120C18, 250 × 4 mm, flow rate: 1 ml min−1. Conditions
A) a 40 min linear gradient from 0 to 100% B. Conditions B) a 20 min linear
gradient from 0 to 40% B. For semipreparative runs the following conditions were
used: Columns: Nucleosil 120C18, 250 × 10 mm. Flow rate: 3 ml min−1. A 20 min
linear gradient from 20-80% B (DMT-on), or a 30 min linear gradient from 0-60%
B (DMT-off).
Mass spectra (electrospray) of dimer A: 648.9 and 650.9 (expected for
C20H26N7O11PBr649.3, 651.3), pentamerB:1550.7(expectedforC48H60N21O26P5

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259
Br 1549.7, 1551.7), pentadecamer C: 4654.3 (expected for C146H183N58O87P14Br
4654.2, 4656.3), decamer D: 3186.3 (expected for C95H119N40O58P9Br2: 3187.6),
decamer E: 3344.6 (expected for C95H117N40O58P9Br4: 3345.6).
Melting and Circular Dichroism Experiments
Melting experiments of pentadecamers duplexes were performed as previ-
ously described (17). Duplex concentration was 4 µM.
Circular dichroism (CD) spectra were recorded on a Jasco J-710 spectropo-
larimeter with a Peltier temperature controller. CD measurements were taken at
20◦C with a 5 mm pathlength cell. Duplex concentration was 4 µM. UV melting
curves were performed with the samples from CD measurements. Melting temper-
atures of dodecamers were between 60 and 85◦C depending on the oligonucleotide
and the salt concentration.
ACKNOWLEDGMENTS
WethankMartenWiersmaforhistechnicalassistanceandthegroupofMattias
Mann for the mass spectra.
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Received September 4, 2000
Accepted November 22, 2000

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