Efficient synthesis of 8-oxo-dGTP: A mutagenic nucleotide

Article abstract of DOI:10.1016/S0960-894X(00)00310-3

An efficient synthesis of mutagenic and oxidative DNA damage product, 8-oxo-dGTP (4) has been achieved in high yield, along with a serendipitous generation of 8-dimsyl-dG (2). In combination with dPTP (5), 8-oxo-dGTP (4) can be formulated into a kit for investigating DNA random mutagenesis. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00310-3

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1677±1679
Ecient Synthesis of 8-Oxo-dGTP: A Mutagenic Nucleotide
Satyam Nampalli and Shiv Kumar*
Amersham Pharmacia Biotech, 800 Centennial Ave, Piscataway, NJ 08855, USA
Received 24 March 2000; accepted 23 May 2000
AbstractÐAn ecient synthesis of mutagenic and oxidative DNA damage product, 8-oxo-dGTP (4) has been achieved in high
yield, along with a serendipitous generation of 8-dimsyl-dG (2). In combination with dPTP (5), 8-oxo-dGTP (4) can be formulated
into a kit for investigating DNA random mutagenesis. # 2000 Elsevier Science Ltd. All rights reserved.
Results and Discussion
8-Oxoguanosine, an oxidative DNA damage product, is
believed to play a signi®cant role in aging and cancer.1 In
the cellular nucleotide pool, 8-oxo-20-deoxyguanosine-50-
triphosphate is generated by the oxidation of 20-deoxy-
guanosine during normal cellular metabolism.2 When
added to PCR reactions, 8-oxo-dGTP 4 (Fig. 1) is
incorporated opposite adenine and cytosine bases with
almost equal eciency (Fig. 2), which results in two
transversion mutations, A!C and T!G. Another
nucleotide analogue, dPTP (5), can be incorporated in
place of TTP and dCTP causing any of the four possible
transition mutations. In PCR based random mutagenesis3
of DNA, utilization of these two nucleotide analoguesÐ8-
oxo-dGTP (4) and dPTP (5)Ðnot only helps understand
protein structure/function, but also sets the requirement
for some knowledge of the most important regions of the
gene to study. While there was easy access to the synthe-
sis of dPTP4 (5), for it to be part of the mutagenesis kit
there existed no reliable/reproducible literature method
for the synthesis of 8-oxo-dGTP (4). A direct oxidative
method5 on dGTP using hydrogen peroxide/ascorbic acid
in phosphate bu€er produces poor yields, and in our
hands it could not be reproduced. Recently, Guengerich
et al.6 reported a cumbersome 7-step synthesis of 8-oxo-
dGTP starting from 20-deoxy-dG. This method involved
unnecessary protection/de-protection strategy, in addition
to multistage puri®cation of the ®nal compound with no
mention of yields. Herein, we report on a short, ecient,
and high-yield synthesis of 8-oxo-dGTP (4), mechanism
of formation along with the unexpected generation of 8-
dimsyl-20-deoxyguanosine (5).
In an attempt to achieve the synthesis of 8-oxo-dGTP (4)
in commercial quantities for the formulation of muta-
genesis kit, 8-benzyloxy-20-deoxyguanosine7 3 (Scheme
1) was envisaged to be the suitably protected starting
material derivable from 8-bromo-20-deoxyguanosine7 (1).
Initial experiments to convert 8-bromo-20-deoxyguano-
sine (1) to 8-benzyloxy-20-deoxyguanosine (3) under the
Na/BnOH/DMSO (1/3), 65 ꢀC, 16 h heating conditions
resulted in a serendipitous formation of 8-dimsyl-dG 2,8
(major) and the desired 8-bezyloxy-dG 3 (minor). It was
expected that changing the ratio of the solvents BnOH/
DMSO from 1/3 to 3/2 would result in a product outcome
in favor of the desired 8-benzyloxy-dG (3) exclusively or
as the major compound. True to the expectation, exclusive
formation of 8-bezyloxy-dG (3) occurred in an isolated
yield of 80% by addition of 8-bromo-dG (1) to the
clearly dissolved solution of sodium metal in BnOH/
DMSO (3/2) mixture, at room temperature and heating
at 65 ꢀC for 16 h. Note that dissolution of sodium metal
in BnOH takes about 3 h at rt. DMSO was used not
only to help dissolve the solid 8-bromo-dG, but also for
its powerful solvating e€ect of cations (Na+) with con-
sequently enhanced reactivity9 of the counter ions
(BnO ), for nucleophilic displacement. It is extremely
*Corresponding author. Tel.: +1-732-980-2878; fax: +1-732-457-
8353; e-mail: shiv.kumar@am.apbiotech.com
Figure 1.
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00310-3

1678
S. Nampalli, S. Kumar / Bioorg. Med. Chem. Lett. 10 (2000) 1677±1679
dicult to get the 8-bromo-dG (1) dissolved in BnOH
without using DMSO.
Having obtained the desired 8-benzyloxy-dG 3 (Scheme 2)
in an excellent yield,10 it was phosphorylated11 at 0±5 ꢀC
with POCl3 in (EtO)3PO, followed by simultaneous addi-
tion of tri-n-butylamine and bis-tri-n-butylammonium-
pyrophosphate. This treatment directly produced 8-oxo-
dGTP (4) in 45% isolated yield12 after being stirred with
1.0 M TEAB (triethylammoniumbicarbonate) and RP-
HPLC puri®cation, without requiring removal of benzyl
group under catalytic hydrogenolysis conditions.
Mechanistically, in situ generated HCl from the reaction of
3 with POCl3 is believed to have catalyzed the cleavage of
benzyl iminol ether in 50-phosphorodichloridate inter-
mediate 4a, giving rise to a benzylketoxonium intermediate
4b. Finally, cleavage of benzyl moiety from 4b occurs as
soon as chloride anion attacks the benzylic position to
give intermediate 4c, which then reacts with bis-tri-n-
Figure 2. The base pairing properties of dPTP and dGTP: dPTP can base
pair with dA or dGTP and 8-Oxo-dGTP can base pair with dA or dC.
Scheme 1.
Scheme 2.

S. Nampalli, S. Kumar / Bioorg. Med. Chem. Lett. 10 (2000) 1677±1679
1679
butylammoniumpyrophosphate in the presence of tri-n-
butylamine a€ording 8-oxo-dGTP(4). The formation of
4 occurs via the usual cyclic intermediate 4d, after being
hydrolyzed by aqueous TEAB at pH 8. As enol ethers
are readily hydrolyzed by acids,13 we have experimented
and demonstrated that the iminol benzyl ether (3) col-
lapses to 8-oxo-dG (6)14 upon treatment with 1.0 N
HCl/MeOH.
by rotary evaporation from methanol under reduced pressure.
Silica gel column chromatography of the adsorbed solid elut-
ing with 5±10% MeOH in CHCl3 plus a few drops of NH4OH
eliminated the impurities. 8-Benzyloxy-dG 3 (2.5 g, 80%) was
isolated as a white solid eluting with 15±20% MeOH in CHCl3
plus a few drops of NH4OH. Compound 3 showed similar
spectral properties as reported in the literature.7
11. Kovacs, T.; Otvos, L. Tetrahedron Lett. 1988, 29, 4525.
12. 8-Oxo-dGTP (4). To a magnetically stirred and cooled (0±
5 ꢀC) solution of 8-benzyloxy-dG 3 (2.1 g, 5.62 mmol) in tri-
ethylphosphate (25 mL) under Argon atmosphere was added
POCl3 (1.29 g, 8.41 mmol, 1.5 equiv) drop-wise. After 2 h,
simultaneously were added 5 equiv each of 0.5 M DMF solu-
tion of bis-tri-n-butylammonium pyrophosphate (15.42 g,
28.10 mmol) and tri-n-butylamine (5.21 g, 28.10 mmol). The
cyclic phosphate formed was stirred at rt for 30 min, then
hydrolyzed with 1.0 M TEAB (ꢁ500 mL, pH 7.2) overnight.
TEAB and DMF were evaporated from the reaction mixture
under reduced pressure, the residue obtained was dissolved in
S.Q. (super quality) water, and ®ltered through 0.4 m ®lter-
ware. The ®ltrate containing the desired 8-oxo-dGTP (4) was
subjected to Sephadex column puri®cation using 0.05 to 1.0 M
TEAB. The fractions containing the desired compound were
pooled, evaporated, and the residue obtained was further
subjected to RP-HPLC, on a Delta Pak C18 preparative col-
umn (5Â30 cm), using gradient 0.1 M TEAB (bu€er A, pH
7.0) and 25% CH3CN in 0.1 M TEAB (bu€er B, pH 7.0) at
130 mL/min. Fractions containing the desired compound were
pooled, evaporated and lyophilized to obtain 8-oxo-dGTP 4
(2.24 g, 45.5%) as an amorphous powder. 31P NMR (D2O):
In summary, the synthesis of 8-benzyloxy-dG (3) could be
achieved in an improved yield (80%), and converted
directly to 8-oxo-dGTP (4) in high yield (45%), which helps
in studying the random DNA mutagenesis in formulation
with dPTP (5) analogue. In order to help explain the direct
generation of 8-oxo-dGTP (4) from 3 in the phosphoryla-
tion reaction, an HCl catalyzed mechanistic pathway for
the cleavage of benzyl group in 3 has been invoked.
References and Notes
1. Ames, B. N.; Gold, L. S. Mutation Res. 1991, 250, 3.
2. Mo, J.-Y.; Maki, H.; Sekiguchi, M. Proc. Natl. Acad. Sci.
USA 1992, 89, 11021.
3. Zoccolo, M.; Williams, D. M.; Brown, D. M.; Gherardi, E.
J. Mol. Biol. 1996, 255, 589.
4. Kong, P.; Lin, T.; Brown, D. M. Nucleic Acids Res. 1989,
17, 10373.
5. Purmal, A. A.; Kow, Y. W.; Wallace, S. S. Nucleic Acids
Res. 1994, 22, 3930.
6. Einolf, J. H.; Schnetz-Boutaud, N.; Guengerich, F. P. Bio-
chemistry 1998, 37, 13300.
7. Lin, T.-S.; Cheng, J-C.; Ishiguro, K.; Sartorelli, A. C. J.
Med. Chem. 1985, 28, 1194.
1
8.1 (d, g-P), 10.1 (d, a-P), 22.22 (t, b-P) ppm. H NMR
(D2O) d 6.13 (1H, dd, J=6.0 Hz, 9.0 Hz, 10-H), 4.22 (1H, m,
30-H), 4.05 (1H, m, 40-H), 3.18 (2H, m, 50-H2), 2.16 (1H, m, 20-
Ha), 2.85 (1H, m, 20-Hb), NH2, NH, 30-OH, and 50-OH, pro-
tons have been exchanged with D2O. UV (10 mM Tris, pH
6.9) lmax 247, 296 nm.
8. Nampalli, S.; Livshin, I.; Kumar, S. Nucleosides Nucleo-
tides 1999, 18, 697.
13. Chwang, W. K.; Kresge, A. J.; Wiseman, J. R. J. Am.
Chem. Soc. 1979, 101, 6972.
9. Langhals, E.; Langhals, H. Tetrahedron Lett. 1990, 31, 859.
10. 8-Benzyloxy-dG (3). Freshly cut Na metal (0.96 g, 41.74
mmol, 5 equiv) was dissolved in BnOH (30 mL) at rt by mag-
netic stirring under Argon atmosphere, solid 8-bromo-dG 1
(2.9 g, 8.3 mmol) was added followed by anhyd DMSO (20
mL) to ensure complete dissolution of the solid 8-bromo-dG.
The reaction mixture was heated at 65 ꢀC for 16 h, after cool-
ing, the reaction was poured into magnetically stirred ether
(800 mL) solvent for precipitation of the desired compound.
The precipitated solid was ®ltered and adsorbed onto silica gel
14. 8-Oxo-dG (6). To a stirred solution of 8-benzyloxy-dG 3
(50 mg) in MeOH (5 mL) at rt was added 1.0 M HCl (0.5 mL).
The reaction was stirred for 1 h. Removal of water and MeOH
under reduced pressure followed by trituration of the residue
from MeOH/ether a€orded quantitative yield of the known7
8-oxo-dG (6). 1H NMR (DMSO-d6) d 6.55 (2H, bs, D2O exch.
NH2), 6.07 (1H, dd, J=6.0 Hz, 9.0Hz, 10-H), 5.18 (1H, bs,
D2O exch. 30-OH), 4.86 (1H, bs, D2O exch. 50-OH), 4.60 (1H,
bs, 8-OH), 3.79 (1H, m, 30-H), 3.56 (1H, m, 40-H), 3.56 (2H, m,
50-H2), 2.97 (1H, m, 20-Ha 2.08 (1H, m, 20-Hb).