Oligonucleotide incorporation of 8-Thio-2′-deoxyguanosine

Article abstract of DOI:10.1021/ol0484097

(Chemical Equation Presented) 8-Thio-2′-deoxyguanosine (SdG) is a useful analogue of the abundant promutagen 8-oxo-2′-deoxyguanosine (OdG). Its synthesis and DNA incorporation using standard phosphoramidite chemistry is reported. To prevent oxidation duri

Full text of DOI:10.1021/ol0484097

ORGANIC
LETTERS
2004
Vol. 6, No. 21
3817-3820
Oligonucleotide Incorporation of
8-Thio-2 -deoxyguanosine
′
Michelle L. Hamm,* Rushina Cholera, Courtney L. Hoey, and Timothy J. Gill
Department of Chemistry, UniVersity of Richmond, Gottwald N-113,
Richmond, Virginia 23173
mhamm@richmond.edu
Received August 11, 2004
ABSTRACT
8-Thio-2
′
-deoxyguanosine (SdG) is a useful analogue of the abundant promutagen 8-oxo-2
′
-deoxyguanosine (OdG). Its synthesis and DNA
incorporation using standard phosphoramidite chemistry is reported. To prevent oxidation during DNA synthesis, the sulfur was protected as
a 2-(trimethylsilyl)ethyl sulfide. Subsequent treatment with TBAF yielded the desired 8-thiocarbonyl functionality. Melting studies with SdG
revealed almost equal stabilities of SdG:dC and SdG:dA base pairs, lending insight into the base-pairing preferences of OdG.
8-Oxo-2′-deoxyguanosine (OdG) is an abundant DNA lesion
produced by the reaction of 2′-deoxyguanosine (dG) with
radical oxygen species.1 OdG leads to dG to T transversions,2
and its presence has been linked to aging3 and diseases such
as cancer.4 OdG differs from dG at only the N7 and C8
positions (Scheme 1); thus, these positions must be key in
determining the deleterious effects of OdG. For example,
both these positions are thought to play a critical role in the
promutagenicity of OdG. When synthesizing DNA, poly-
merases incorporate only 2′-deoxycytidine (dC) opposite dG
but can incorporate either dC or 2′-deoxyadenosine (dA)
opposite OdG.5 This is because while dG only forms base
pairs to dC, OdG forms stable base pairs to both dC and dA
(Scheme 2).6 When base pairing to dA, OdG adopts a syn
conformation about the glycosidic bond and uses the N7-
hydrogen and C6-oxygen of the Hoogsteen edge to contact
dA.7 When base pairing to dC, however, OdG is in the anti
base conformation and normal Watson-Crick pairing is
utilized.
Interestingly, melting studies have shown that despite
having one less hydrogen bond, OdG(syn):dA base pairs are
only slightly less stable than OdG(anti):dC base pairs. This
similarity is thought to be in part due to the C8-oxygen
which, because of its steric bulk, causes OdG to prefer the
syn conformation,8 thereby destabilizing OdG(anti):dC base
pairs.
Due to both its promutagenicity and abundance, cells have
evolved specific repair enzymes that recognize and remove
OdG from DNA. Since dG is not a substrate for these
enzymes, they likely recognize OdG at either the C8-oxygen
or N7-hydrogen. More recent crystallographic studies have
implicated the N7-hydrogen and not the C8-oxygen as the
(1) Kasai, H.; Nishimura, S. in OxidatiVe Stress: Oxidants and Anti-
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Carcinogenesis 1995, 16, 2779-2784.
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N. Proc. Natl. Acad. U.S.A. 1990, 87, 4533-4537.
(4) Floyd, R. A. Carcinogenesis 1990, 11, 1447-1450.
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434.
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10.1021/ol0484097 CCC: $27.50
© 2004 American Chemical Society
Published on Web 09/22/2004

Scheme 1. Structures dG, OdG, and SdG
mode of detection,9 though other biochemical studies suggest
In a similar fashion, the study of an analogue of OdG
where the 8-oxo moiety has been replaced with sulfur may
be a useful tool with which to better understand the specific
properties and interactions of the C8-oxygen that dictate the
activity of OdG in cells. For example, if steric bulk off of
C8 is important in the similar stabilities of OdG(syn):dA and
OdG(anti):dC base pairs, then 8-thio-2′-deoxyguanosine
(SdG; Scheme 1), which has greater bulk off C8, should form
base pairs to dC and dA with even more similar stabilities.
Additionally, since SdG differs from OdG at only the C8-
position, biochemical studies with the aforementioned repair
enzymes should lend insight into necessity of the 8-oxo
moiety.
the C8-oxygen may play some role.10 To address these
discrepancies and gain more insight into the exact role of
the C8-oxygen in OdG mutagenicity and repair, we set about
creating an analogue of OdG that would differ from it at
only the C8 position.
Scheme 2. Base Pairing of OdG
The main concern when incorporating any sulfur-contain-
ing analogue into DNA is to prevent oxidation of the sulfur
during the iodine oxidation or ammonium hydroxide depro-
tection steps of DNA synthesis. To prevent this, previous
syntheses of DNA containing sulfur analogues have usually
involved protection of the sulfur as a sulfide. For example,
6-thio-2′-deoxyguanosine, 4-thio-2′-deoxyuridine, 4-thio-2′-
deoxythymidine, 4-thiouridine, and 6-thioinosine are all
protected as â-cyanoethyl sulfides during DNA synthesis.15
Though such a protection scheme may well work for SdG,
this route would require the starting material 2-cyano-
ethanethiol which is highly toxic and not commercially
available. For synthetic ease, we chose to protect the sulfur
moiety as a 2-(trimethylsilyl)ethyl sulfide and use tetra-
butylammonium fluoride (TBAF) treatment as a means of
sulfur deprotection.
To this end, replacement of oxygen with sulfur has been
used extensively to probe the role of oxygen in biomolecular
recognition and catalysis. This is because though sulfur has
similar bonding properties to oxygen, it differs significantly
in its affinity toward metals,11 ability to hydrogen bond,12
and its atomic radius and bond length. For example, metal
replacement studies with 3′-thio-3′-deoxyuridine have sug-
gested a direct metal interaction to the active site 3′-oxygen
during the cleavage reaction of the Tetrahymena ribozyme,13
while stability studies of G-tetrads using 6-thio-2′-deoxy-
guanosine have demonstrated the importance of a 6-oxo
moiety to tetrad formation.14
Though there is no published synthesis to 8-(2-(tri-
methylsilyl)ethyl)thio-2′-deoxyguanosine (TMSE-SdG), 8-(2-
Scheme 3. Synthesis of SdG
(9) (a) Bruner, S. D.; Norman, D. P. G.; Verdine, G. L. Nature 2000,
403, 859-866. (b) Fromme, J. C.; Verdine, G. L. J. Biol. Chem. 2003,
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Arai, T.; Nishimura, S.; Aburatani, H.; Lindahl, T.; Ide, H. J. Biol. Chem.
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3818
Org. Lett., Vol. 6, No. 21, 2004

Scheme 4. Synthesis of the Fully Protected Phosphoramidite Derivative of SdG
(trimethylsilyl)ethyl)thio-2′-deoxyadenosine has been re-
The synthesis to the phosphoramidite derivative of 2 was
continued by protecting the exocyclic amine as an isobutyryl
(iBu) amide (Scheme 4) using the well-known transient
protection procedure.18 The 5′-hydroxyl was then protected
as a dimethoxytrityl (DMTr) ether to produce 5. Finally, the
3′-hydroxyl was activated as a phosphoramidite to create the
DNA synthesis ready 8-thio-2′-deoxyguanosine derivative
6.
Amidite 6 was then utilized in DNA synthesis using a
DNA synthesizer and standard procedures. Coupling yields
were over 95% according to HPLC or gel purification run
after ammonium hydroxide deprotection. Three different
oligonucleotides (2, 7, and 25 nucleotides long; Scheme 5)
were synthesized; their sequences were 5′-dSiSGT-3′ (7a),
5′-dACTSiSGTCA-3′ (7b), and 5′-dCATCGATACGA-
TCTSiSGCCTCTCTCTC-3′ (7c), respectively, where SiSG is
the TMSE-protected SdG derivative. The oligonucleotides
ported previously.16 In a reaction analogous to the adenosine
derivative, TMSE-SdG (2) was synthesized through treatment
of 8-bromo-2′-deoxyguanosine17 (1) with commercially
available 2-(trimethylsilyl)ethanethiol (Scheme 3). To con-
firm that no decomposition of the sulfur would occur during
solid-phase DNA synthesis, compound 2 was incubated
separately for 1 h at room temperature in the DNA synthesis
oxidizing reagent (0.02 M I2) or 20 h in aqueous ammonia
at 55 °C. After both incubations no decomposition was
observed, indicating that the trimethylsilylethyl (TMSE)
protection scheme is suitable for solid-phase phosphoramidite
chemistry. Further, to prove TBAF treatment would be an
appropriate means for sulfur deprotection, we treated nucleo-
side 2 with 1.0 M TBAF in tetrahydrofuran (THF) for 30
min. After HPLC purification, pure SdG (3; Scheme 3) was
obtained.
Scheme 5. DNA Incorporation and Sulfur Deprotection of SdG
Org. Lett., Vol. 6, No. 21, 2004
3819

were then purified by preparative HPLC (7a) or gel purifica-
tion and preparative HPLC (7b,c) before treatment with 1.0
M TBAF in THF for 30 min to reveal the free sulfur and
produce oligonucleotides 8a-c.
Table 1. Melting Temperatures (Tm) of DNA Duplexes (°C)a
To further prove the 8-sulfur moiety was not compromised
during oligonucleotide synthesis, deprotection, or purifica-
tion, oligonucleotide 8b was digested to its individual
nucleotides and analyzed by analytical HPLC.19 As seen in
Figure 1A, only four peaks (corresponding to dC, T, dA,
Y ) dC
Y ) dA
X ) dG
X ) OdG
X ) SdG
57.6 ( 0.5
52.6 ( 0.5
46.5 ( 0.3
43.0 ( 0.2
48.0 ( 0.4
47.7 ( 0.3
a Conditions: 1 M NaCl, 0.1 mM EDTA, and 100 mM sodium phosphate
pH 7.0. Average Tm ( standard deviations were calculated from three or
more melts.
dC and dG:dA base pairs (Tm differences of 4.6 and 14.6
°C, respectively). Interestingly, we found SdG:dC and SdG:
dA base pairs to be of almost equal stability (Tm difference
of 1.2 °C). When compared to OdG, the similar stabilities
of the SdG base pairs appear to arise from additional
destabilization of SdG:dC since SdG:dA and OdG:dA base
pairs are of equal stability, while SdG:dC base pairs are much
less stable than OdG:dC base pairs (Tm difference of 6.1 °C).
Considering sulfur has a larger atomic radius and longer
bonding distance than oxygen, this finding is in agreement
with the theory that additional steric bulk off of dG-C8
destabilizes the anti base conformation,8 thereby destabilizing
base pairs to dC (Scheme 2). Other possible explanations
involving distortion of the purine base are not likely as crystal
structure studies with other sulfur-containing purines have
confirmed that the a thio substitution causes no significant
structure change.20 The finding that SdG:dA and OdG:dA
base pairs are of equal stability is also of interest since it
suggests that added steric bulk off of C8 does not play a
significant role in the stabilities of these base pairs.
In summary, we have developed a rapid and efficient
synthesis to oligonucleotides containing SdG using the
phosphoramidite method. Ready access to such products will
enable experiments that should lend further insight into the
exact role of the C8-oxygen in the bioactivity of OdG. We
are currently testing SdG with different repair enzymes to
determine whether the 8-oxo moiety is required for OdG
recognition.
Figure 1. Analytical HPLC analysis of (A) nuclease-digested 8b
and (B) mock nuclease-digested 3 (an authentic standard of SdG).
and SdG) were observed. To further characterize the sup-
posed SdG peak, we compared digested 8b to an authentic
sample of compound 3 (Figure 1B). The supposed SdG peak
and 3 had identical retention times (16.0 min) and coeluted
under the same reaction and chromatography conditions.
Furthermore, the supposed SdG peak is not dG or OdG which
have retention times of 6.2 and 7.1 min, respectively, when
treated under the same reaction and chromatography condi-
tions. It should be noted that longer TBAF deprotection
treatments (4 h) did result in some decomposition of SdG to
form dG (as characterized by HPLC and mass spectrometry),
presumably by oxidative desulfurization.
To gain insight into the base pairing preference of OdG,
we incorporated SdG into an 11 nucleotide long oligo-
nucleotide and tested its base-pairing stabilities opposite dC
and dA (Table 1). We also tested the base pairing of dG
and OdG in the same duplex and under the same conditions.
Similar to previous studies,6 we found OdG:dC and OdG:
dA base pairs to be of much more similar stability than dG:
Acknowledgment. We thank P. Nyffeler for many helpful
discussions and P. Nyffeler and E. Fenlon for reviewing the
manuscript. This work was partially supported by the Thomas
F. Jeffress and Kate Miller Jeffress Memorial Trust and the
NSF-CAREER program. Additionally, acknowledgment is
made to Donors of the American Chemical Society Petro-
leum Research Fund for partial support of this research.
M.L.H. is a Camille and Henry Dreyfus Start-up Awardee.
(14) Rao, T. S.; Durland, R. H.; Seth, D. M.; Myrick, M. A.; Bodepudi,
V.; Revankar, G. R. Biochemistry 1995, 34, 765-772.
(15) (a) Adams, C. J.; Murray, J. B.; Farrow, M. A.; Arnold, J. R. P.;
Stockley, P. G. Tetrahedron Lett. 1995, 36, 5421-5424. (b) Kadokura, M.;
Wada, T.; Seio, K.; Sekine, M. J. Org. Chem. 2000, 65, 5104-5113. (c)
Glen Research Catalog, Sterling, VA.
Supporting Information Available: Experimental pro-
cedures for the synthesis and purification of 2-6, 7a-c, and
8a-c, oligonucleotide digestion and analysis, and melting
1
studies; H and 13C for 2-5; 31P NMR for 6; HPLC traces
(16) Chambert, S.; Gautier-Luneau, I.; Fontecave, M.; Decout, J.-L. J.
Org. Chem. 2000, 65, 249-253.
for 7a-c and 8a-c; raw melting data for Table 1. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(17) Gannett, P. M.; Sura, T. P. Synth. Commun. 1993, 23, 1611-1615.
(18) Ti, G. S.; Gaffney, B. L.; Jones, R. A. J. Am. Chem. Soc. 1982,
104, 1316-1319.
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Cathcart, R. Anal. Biochem. 1987, 165, 442-447. (b) Kuimelis, R. G.;
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OL0484097
(20) Thewalt, U.; Bugg, C. E. J. Am. Chem. Soc. 1972, 94, 8892-8898.
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