Base pair stability of 8-chloro- and 8-iodo-2′-deoxyguanosine opposite 2′-deoxycytidine: Implications regarding the bioactivity of 8-oxo-2′-deoxyguanosine

Article abstract of DOI:10.1021/ja052578k

8-Oxo-2′-deoxyguanosine (OdG) is an abundant and promutagenic damaged nucleotide that has been linked to aging and disease. To gain insight into the alternate base pairings of OdG, 8-chloro- and 8-iodo-2′-deoxyguanosine were incorporated into oligonucleot

Full text of DOI:10.1021/ja052578k

Published on Web 08/13/2005
Base Pair Stability of 8-Chloro- and 8-Iodo-2′-deoxyguanosine Opposite
2′-Deoxycytidine: Implications Regarding the Bioactivity of
8-Oxo-2′-deoxyguanosine
Michelle L. Hamm,* Sumika Rajguru, Anthony M. Downs, and Rushina Cholera
Department of Chemistry, UniVersity of Richmond, Gottwald B-100, Richmond, Virginia 23173
Received April 20, 2005; E-mail: mhamm@richmond.edu
Scheme 1
Reactive oxygen species, which are formed by solar radiation,
chemical carcinogens, and normal aerobic metabolism, produce a
number of complex lesions in DNA, the most common of which is
oxidation at the C8 position of 2′-deoxyguanosine (dG) to form
8-oxo-2′-deoxyguanosine (OdG).1 OdG is known to be mutagenic,1
and it has been linked to aging2 and several diseases, including
cancer.3
and iodine) at the C8 position. We then incorporated these analogues
into DNA and determined their relative base pair stabilities when
opposite dC.
Though OdG can exist in alternate tautomeric states, the 6,8-
diketo form predominates at physiological pH.4 In this form, N7
becomes a hydrogen bond donor; C8 becomes a hydrogen bond
acceptor, and the most stable conformation about the glycosidic
bond changes from anti (as in dG) to syn.5 When incorporated into
oligonucleotides, OdG retains its 6,8-diketo structure, but its
glycosidic bond conformation depends on the identity of its base
pair partner. Unlike unmodified dG, OdG can form stable base pairs
with both dC and dA. When pairing to dC, the unfavored anti
orientation is required,4a and the base pair is destabilized as
compared to a dG:dC base pair.6
Though 8-bromo-2′-deoxyguanosine (BrdG) has been known for
some time,9 efficient syntheses of 8-chloro-2′-deoxyguanosine
(CldG) and 8-iodo-2′-deoxyguanosine (IdG) derivatives have not
been reported. Previous methods of chlorination and iodination at
the C8 position of purines have included reactions with lithium
diisopropylamine and tosyl chloride10 and N-iodosuccinamide (NIS)
in DMSO,11 respectively. Unfortunately, none of these procedures
proved effective with dG. However, since the iodination procedure
was similar to the well-established route to BrdG9 (where dG is
reacted with N-bromosuccinamide in water), modifications of the
procedure were tested for the synthesis of CldG and IdG. After
much examination, good reactivity was found when THF was used
as the solvent and a protected dG derivative was used as the reagent.
Thus, the triisobutyrylated dG derivative 1 was reacted with
N-chlorosuccinamide (NCS) in THF for 2 days at room temperature
to yield the CldG derivative 2a, or NIS in THF for 3 days at 35 °C
to yield the IdG derivative 2b (Scheme 1). The triisobutyrylated
dG derivative was used not only because it was soluble in THF
but also because isobutyryl (iBu) groups are the standard protecting
group for the exocyclic amine during solid phase synthesis of
DNA. To further prepare CldG and IdG for DNA synthesis, the
isobutyryl ester protecting groups were selectively removed with
sodium methoxide before protection of the 5′-oxygen as a dimethoxy-
trityl (DMTr) ether. Finally, the 3′-oxygen was activated as a
phosphoramidite to produce the DNA synthesis-ready nucleosides
4a and 4b.
When paired to dA, however, OdG adopts the favored syn
conformation and uses its Hoogsteen edge to hydrogen bond with
the Watson-Crick face of dA.7 It has been shown that OdG(syn):
dA base pairs are only slightly less stable than OdG(anti):dC base
pairs, despite having one less hydrogen bond.6 Since the dual base
pairing ability of OdG is believed to be responsible for mutation
and possibly the link between OdG and aging and disease, it is of
great importance to fully understand the exact structural and
electronic properties of OdG that lead to the relatively similar
stabilities of OdG(anti):dC and OdG(syn):dA base pairs.
To this end, it has been proposed that the instability of OdG:dC
base pairs relative to dG:dC base pairs may be due, at least in part,
to the steric bulk of the 8-oxygen. It has been reasoned that when
in the anti conformation the 8-oxygen is in steric clash with the
connected deoxyribose sugar, thus destabilizing the base pair
overall.4 However, this rationale has its critics,8 and the question
remains somewhat controversial. To address the effect of C8 steric
bulk on the stability of dG:dC base pairs, we synthesized analogues
of dG that contained halogens of increasing size (chlorine, bromine,
The two phosphoramidite derivatives were then utilized in DNA
synthesis using a DNA synthesizer and all standard procedures,
except that with the iodinated derivative, ammonium hydroxide
deprotection was carried out at room temperature for 22 h (to lessen
deiodination). Two 11 nucleotide long oligonucleotides were
synthesized, each with the sequence 5′-dCCATCXCTACC-3′, but
where X was CldG (5b) or IdG (5d). To complete the analysis,
9
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10.1021/ja052578k CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Melting Temperatures (Tm) of DNA Duplexes (°C)a
In conclusion, these data stand in strong agreement with the
premise that steric bulk of C8 destabilizes dG:dC base pairs by
destabilizing the anti conformation of dG. Accordingly, it stands
to reason that the steric bulk of the C8-oxygen plays some role in
the destabilization of OdG:dC base pairs and thus the overall similar
stabilities of OdG:dC and OdG:dA base pairs. It is interesting to
note that the melting temperature with OdG (5e) varied from the
trends for the halogens (Figure 1). These differences may be due,
at least in part, to the increased nonpolarity and polarizability of
the halogenated bases since both properties are known to increase
helix stability.13
X
)
dG
X
)
OdG
X
)
CldG
X
)
BrdG
X ) IdG
57.5 ( 0.5
52.7 ( 0.5
51.1 ( 0.3
49.6 ( 0.4
47.6 ( 0.4
a Conditions: 1 M NaCl, 0.1 mM EDTA, and 100 mM sodium phosphate,
pH 7.0. Average Tm values ( standard deviation were calculated from three
or more melts.
Finally, although methods were developed for the syntheses of
oligonucleotides containing CldG and IdG because of their rel-
evance to OdG, they are likely to find much wider application since
halogenated nucleotides are commonly used in nucleic acid research.
For example, halogenated nucleotides have found a great deal of
use in crystallographic15 and photo-cross-linking experiments16 and
as convertible nucleosides17 and structural probes.18
Figure 1. Graphs of melting temperature versus atomic radius (left) and
bond length (right) at C8 with 5a-d (circles) and 5e (squares).
Acknowledgment. The authors wish to thank Paul Nyffeler and
Stuart Clough for careful review of the manuscript. This work was
partially supported by Research Corporation and the NSF-CAREER
and MRI programs. M.H. is a Camille and Henry Dreyfus Start-up
Awardee.
Table 2. Chemical Shifts of Nuclei of dG, CldG, BrdG, and IdGa
C4′
C1
′
C3
′
C2
′
2′-H
dG
87.5
87.8
87.8
88.0
82.5
83.9
85.0
87.3
70.6
70.9
70.9
71.2
39.5
36.6
36.4
36.7
2.50
3.09
3.16
3.18
CldG
BrdG
IdG
Supporting Information Available: Experimental procedures for
the synthesis and purification of 2a,b, 3a,b, 4a,b, 5b, 5d, and 6a,b. 1H
and 13C for 2a,b, 3a,b, and 6a,b. 31P NMR for 4a,b. HPLC traces for
5b and 5d. Oligonucleotide digestion and analysis, and melting and
NMR studies. Raw and/or complete data for Tables 1 and 2 and Fig-
ure 1. This material is available free of charge via the Internet at
http://pubs.acs.org.
a Conditions: 0.04 M in DMSO-d6. All shifts are relative to TMS.
oligonucleotides, where X was dG (5a), BrdG (5c), or OdG (5e),
were purchased. The oligonucleotides were then purified by gel
electrophoresis and reverse-phase HPLC before their purity and
identity were confirmed by nuclease digest experiments (see
Supporting Information).
References
(1) Grollman, A. P.; Moriya, M. Trends Genet. 1993, 9, 246-249 and
references therein.
(2) Ames, B. N.; Shigenaga, M. K.; Hagen, T. M. Proc. Natl. Acad. Sci.
U.S.A. 1993, 90, 7915-7922.
(3) Floyd, R. A. Carcinogenesis 1990, 11, 1447-1450.
(4) (a) Oda, Y.; Uesugi, S.; Ikehara, M.; Nishimura, S.; Kawase, Y.; Ishikawa,
H.; Inoue, H.; Ohtsuka, E. Nucleic Acids Res. 1991, 19, 1407-1412. (b)
Jang, Y. H.; Goddard, W. A.; Noyes, K. T.: Sowers, L. C.; Hwang, S.;
Chung, D. S. Chem. Res. Toxicol. 2002, 15, 1023-1035.
(5) Gannett, P. M.; Sura, T. P. Chem. Res. Toxicol. 1993, 6, 690-700.
(6) Plum, G. E.; Grollman, A. P.; Johnson, F.; Breslauer, K. J. Biochemistry
1995, 34, 16148-16160.
(7) Kouchakdjian, M.; Bodepudi, V.; Shibutani, S.; Eisenberg, M.; Johnson,
F.; Grollman, A. P.; Patel, D. J. Biochemistry 1991, 30, 1403-1412.
(8) Lipscomb, L. A.; Peek, M. E.; Morningstar, M. L.; Verghis, S. M.; Miller,
E. M.; Rich, A.; Essigmann, J. M.; Williams, L. D. Proc. Natl. Acad. Sci.
U.S.A. 1995 92, 719-723.
The five oligonucleotides were paired with complimentary DNA
strands and tested for their stabilities using melting studies (Table
1). Similar to previous work, OdG:dC6 and BrdG:dC12 base pairs
were found to be less stable than dG:dC base pairs. CldG:dC and
IdG:dC base pairs, which were also less stable than dG:dC base
pairs, had stabilities that directly associate to atomic radius and
bond length; the greater the atomic radius or bond length, the less
stable the analogue in a base pair opposite dC. Interestingly, the
correlation between melting temperature and either atomic radius
or bond length at C8 with 5a-d was entirely uniform with this
duplex (Figure 1). Though an 8-halogen also destabilized dG base
pairs to dA and T, as well as slightly stabilized dG base pairs to
dG, the uniform correlation was unique for base pairs to dC (see
Supporting Information).
To address whether steric bulk off of C8 may destabilize dG:dC
base pairs by destabilizing the anti conformation of dG, the
deprotected nucleosides dG, CldG, BrdG, and IdG were synthesized
(CldG (6a) and IdG (6b)) or purchased (dG and BrdG) and tested
for their inherent glycosidic bond conformation using NMR
spectroscopy. It is known that an anti to syn conformational change
results in a downfield shift of the C1′, C3′, C4′, and H2′ signals,
as well as an upfield shift of the C2′ signal.5,14 As seen in Table 2,
such shifts were observed for all three halogenated nucleosides as
compared to that of dG, indicating their preference for the syn
conformation. These results are similar to previous results for OdG5
and are consistent with the argument that steric bulk off of C8
destabilizes the anti conformation of dG.
(9) Gannett, P. M.; Sura, T. P. Synth. Commun. 1993, 23, 1611-1615.
(10) (a) Chen, L. S.; Bahr, M. H.; Sheppard, T. L. Bioorg. Med. Chem. 2003,
13, 1509-1512. (b) Hayakawa, H.; Tanaka, H.; Haraguchi, K.; Mayumi,
M.; Nakajima, M.; Sakamaki, Y.; Miyasaka, T. Nucleosides Nucleotides
1988, 7, 121-128.
(11) Lipkin, D.; Howard, F. B.; Nowotny, D.; Sano, M. J. Biol. Chem. 1963,
238, 2249-2251.
(12) Fabrega, C.; Macias, M. J.; Eritja, R. Nucleosides Nucleotides 2001, 20,
251-260.
(13) Rosemeyer, H.; Seela, F. J. Chem. Soc., Perkin Trans. 2 2002, 746-750
and references therein.
(14) (a) Greene, K. L.; Wang, Y, Live, D. J. Biomol. NMR 1995, 5, 333-338.
(b) Uesugi, S.; Ikehara, M. J. Am. Chem. Soc. 1977, 99, 3250-3253.
(15) Golden, B. L.; Gooding, A. R.; Podell, E. R.; Cech, T. R. RNA 1996, 2,
1295-1305.
(16) Willis, M. C.; Hicke, B. J.; Uhlenbeck, O. C.; Cech T. R.; Kock, T. H.
Science 1993, 262, 1255-1257.
(17) Harris, C. M.; Zhou, L.; Strand, E. A.; Harris, T. M. J. Am. Chem. Soc.
1991, 113, 4328-4329.
(18) Dias, E.; Battiste, J. L.; Williamson, J. R. J. Am. Chem. Soc. 1994, 116,
4479-4480.
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