Synthesis of 8-oxo-dGTP and its β,γ-CH2-, β,γ-CHF-, and β,γ-CF2- analogues

Article abstract of DOI:10.1016/j.tetlet.2021.152890

Three novel bisphosphonate analogues of 8-oxo-dGTP 3 in which the bridging β,γ-oxygen is replaced by a methylene, fluoromethylene or difluoromethylene group (4–6, respectively) have been synthesized from 8-oxo-dGMP 2 by reaction of its morpholine 5′-phosphoramidate 14 or preferably, its N-methylimidazole 5′-phosphoramidate 15 with tri-n-butylammonium salts of the appropriate bisphosphonic acids, 11–13. The latter method also provides a convenient new route to 3. Analogues 4–6 may be useful as mechanistic probes for the role of 3 in abnormal DNA replication and repair.

Full text of DOI:10.1016/j.tetlet.2021.152890

Tetrahedron Letters 67 (2021) 152890
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of 8-oxo-dGTP and its b,c-CH2-, b,c-CHF-, and b,c-CF2-
analogues
⇑
Yiying Zheng, Pouya Haratipour, Boris A. Kashemirov, Charles E. McKenna
Department of Chemistry, University of Southern California, Los Angeles, CA 90089, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Three novel bisphosphonate analogues of 8-oxo-dGTP 3 in which the bridging b,c-oxygen is replaced by a
Received 9 November 2020
Revised 21 January 2021
Accepted 27 January 2021
Available online 4 February 2021
methylene, fluoromethylene or difluoromethylene group (4–6, respectively) have been synthesized from
8-oxo-dGMP 2 by reaction of its morpholine 50-phosphoramidate 14 or preferably, its N-methylimidazole
50-phosphoramidate 15 with tri-n-butylammonium salts of the appropriate bisphosphonic acids, 11–13.
The latter method also provides a convenient new route to 3. Analogues 4–6 may be useful as mechanistic
probes for the role of 3 in abnormal DNA replication and repair.
Keywords:
8-Oxo-dGTP
Ó 2021 Elsevier Ltd. All rights reserved.
b,c
b,c
b,c
-Methylene 8-oxo-dGTP
-Monofluoromethylene 8-oxo-dGTP
-Difluoromethylene 8-oxo-dGTP
DNA polymerases
Introduction
reference nucleotide, 3. The toolkit comprises three b,
bridged 8-oxo-dGTP analogues: b, -methylene- 4, b, -monofluo-
romethylene- 5, and b, -difluoromethylene-8-oxo-dGTP 6 (Fig. 1).
A similar toolkit based on the natural nucleotides has been used
to study leaving group effects on the nucleotidyl transfer kinetic
mechanisms and fidelity of pols [14–18]and other biocatalysts
c-CXY
c
c
Oxidative DNA damage due to reactive oxygen species (ROS)
has been implicated in the pathogenesis of a wide variety of dis-
eases, including cancer [1], neurodegenerative and neurodevelop-
mental disorders [2], inflammatory disorders [3] and aging [4]. 8-
Oxo-20-deoxyguanosine (8-oxo-dG, 1) is a harbinger of oxidative
DNA damage [5] and has been implicated in carcinogenesis by
inducing mutations [6] as well as by abnormal epigenetic modula-
tion of gene expression [7]. The mutagenicity of 1 is attributed to a
conformational shift of the N9-C10 glycosidic bond from anti to syn,
causing it to mimic a syn thymidine [8]. As a result of A:8-oxo-G
(A:8OG) Hoogsteen base mispairing, replicative DNA polymerases
(pols) often insert dATP opposite 1 instead of dCTP [9,10]. Pol b,
c
[19]. As the b,c-bridge atom X and Y substituents become more
electronegative, the pKa4 of the corresponding bisphosphonate
leaving group decreases [20,21], stabilizing the conjugate base
anion. If the rate-determining step (RDS) involves PAO bond break-
ing, then a Brønsted plot of the log of the catalytic rate constant
(kpol) versus pKa4 is predicted to be linear (linear free energy rela-
tionship, LFER) with a negative slope reflecting the sensitivity of
the TS to anion stabilization [22]. The bisphosphonates selected
provide a range of pKa4 of 2.75 units, centered on the pKa4 of the
leaving group in 3, pyrophosphoric acid [20].
pol
g, REV1, pol n and pol j have all been implicated [11] in the
incorporation of 8-oxo-dGMP 2 into DNA from 8-oxo-20-deoxygua-
nosine-50-triphosphate (8-oxo-dGTP, 3), present as an ROS in the
cellular nucleotide pool [12]. Wilson and co-workers recently
described the crystal structure of a pol b DNA complex in which
the adenine of a DNA (syn)8OG:A base pair was replaced at the pri-
mer terminus by a cytosine [13]. It is apparent that complementary
information about the functional mechanism and transition state
(TS) is desirable.
Results and discussion
Early oxidative methods [12,23] to prepare 3 itself directly from
dGTP in low or unstated yield were not reproduced by others [24],
as confirmed by own work (data not shown). Direct oxidative meth-
ods have a further limitation in that they do not give convenient
access to 8-[17O]- or 8-[18O]-oxo-guanosine derivatives [25]. Einolf
described an 8-step synthesis of 3 beginning from dG 7 involving
several protection/deprotection steps, culminating in separation
of the final compound from mono- and diphosphate 8-oxo-dG
byproducts [26]. Nampalli and Kumar [24] subsequently outlined
Herein, we report the synthesis of a small toolkit of 8-oxo-dGTP
bisphosphonate probes, including an alternate preparation of the
⇑
Corresponding author.
E-mail address: mckenna@usc.edu (C.E. McKenna).
https://doi.org/10.1016/j.tetlet.2021.152890
0040-4039/Ó 2021 Elsevier Ltd. All rights reserved.

Y. Zheng, P. Haratipour, B.A. Kashemirov et al.
Tetrahedron Letters 67 (2021) 152890
Fig. 1. Structures of 8-oxo-dGTP 3, b,c-CH2- 4, b,c-CHF-5, and b,c-CF2-8-oxo-dGTP 6.
a gram-scale synthesis of 3 from 8-bromo-dG [27,28] 8 in 36% over-
all yield via conversion to the 8-benzyloxy derivative 9 using
sodium/benzyl alcohol in dimethyl sulfoxide, followed by treat-
ment with phosphorus oxychloride (POCl3) [29] in trimethyl phos-
phate and reaction with bis(tri-n-butylammonium) pyrophosphate.
The final product was obtained by hydrolysis of the resulting cyclic
triphosphate intermediate in aqueous triethylammonium bicar-
bonate (TEAB) at pH 7.5 (Schemes 1 and 2) [24].
avoided by using anhydrous N,N-dimethylformamide (DMF) in
place of DMSO as the solvent (Scheme 1). However, in our hands,
phosphorylation of 1 or 9 on a small scale using the literature
one-pot-three-step procedure [24,30,31] (Scheme 2) gave much
lower yields than anticipated.
We therefore examined an alternative synthesis of 3 starting
from 8-oxo-dGMP 2 (prepared in 33% yield by monophosphoryla-
tion of 1 with POCl3 in PO(OMe)3 [29] followed by aqueous workup
with 0.5 M TEAB) after activation by morpholine [32] or N-
methylimidazole [33,34] to facilitate coupling with pyrophosphate
8-Benzyloxy-dG 9 can be prepared from 7 in 78% yield [27,28],
and we found that formation of an 8-dimsyl-dG byproduct can be
Scheme 1. Synthesis of 8-oxo-dG 1 [24,27,28].
Scheme 2. ‘One-pot’ phosphorylation [30,31] synthesis of 8-oxo-dGTP 3 from 8-oxo-dG 1 [24]; (TEAB: triethylammonium bicarbonate).
2

Y. Zheng, P. Haratipour, B.A. Kashemirov et al.
Tetrahedron Letters 67 (2021) 152890
Scheme 3. Synthesis of 3–6 via the 50-morpholidate 14.
Scheme 4. Synthesis of 3–6 via the 50-N-methylimidazolide 15.
10, as a method likely to be adaptable to the synthesis of 4–6 from
the appropriate bisphosphonate tri-n-butylammonium salt 11–13
[20], prepared by treatment of commercially available methylene-
onances is seen with more electronegative substituents
(CF2 > CHF > CH2) on the bridging b-methylene group. There is
no effect on the P resonance which remains constant at about
a
bis(phosphonic acid) or its
a
-fluorinated derivatives [35] with tri-
ꢀ10 ppm.
n-butylamine in aq. ethanol. 8-Oxo-dGMP-morpholidate 14 gave 3
and the target bisphosphonate nucleotides 4–6, but the reactions
were sluggish, with very poor yields (Scheme 3).
Consistent with published data for the b,c-monofluoro and
b,
resonances at d ꢀ217.32 and d ꢀ120.95 ppm, respectively. The
31Pb, 31 and 19F peaks of 5 exhibit a slight broadening consistent
c
-difluoro analogues of dGTP [15], 5 and 6 exhibit 19F NMR
Better results were obtained with N-methylimidazole activation
[33,34]. Thus, 2 suspended in a mixture of triethylamine and
excess trifluoroacetic anhydride in acetonitrile was treated with
N-methylimidazole to give the corresponding 8-oxo-dGMP-N-
methylimidazolide 15, which was then added to tri-n-butylammo-
nium pyrophosphate 10 or the tri-n-butylammonium bisphospho-
nate 11, 12 or 13 in DMF (Scheme 4). Deactivated 8-oxo-dGMP 2
can be recovered during purification of the final products (charac-
terized by 1H, 31P and 19F NMR, LC-MS and HRMS) via SAX/C18
preparative HPLC.
P
c
with a small
Dd for the R v S diastereomers [15] (the individual
isomers should be accessible via chiral synthons derived from 12
[20]).sy).]
Conclusion
In summary, we examined several alternative approaches for
the synthesis of 8-oxo-dGTP 3 and three novel bisphosphonate
analogues: b,
c-methylene- 4, (R/S)-b,c-monofluoromethylene- 5,
In the coupling reactions to form 3–6, the reaction time was
much shorter with the N-methylimidazolides (2–3 h) compared
to the morpholidates (3–5 d) [30–32]. It is critical to thoroughly
dry the tri-n-butylammonium salt of the bisphosphonic acid by
repeated coevaporation with anhydrous DMF before use to achieve
optimal yields.
and b, -difluoromethylene-8-oxo-dGTP 6. Conjugation of the cor-
c
responding bisphosphonic acid n-butylammonium salts 11–13
with 8-oxo-dGMP-N-methylimidazolide 15 in anhydrous DMF is
a practical route to these compounds, albeit in low (not optimized)
yields. The availability of the resulting 8-oxo-dGTP analogue
toolkit provides a novel means to probe leaving group effects on
the binding and kinetic mechanisms of 3 interacting with nucleic
acid polymerases.
The proton-decoupled 31P NMR spectra of 3–6 are compared in
Fig. S44. As expected, a dramatic upfield shift of the P and Pb res-
c
3

Y. Zheng, P. Haratipour, B.A. Kashemirov et al.
Tetrahedron Letters 67 (2021) 152890
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Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgments
We thank Prof. N. A. Graham and A. Delfarah for assistance in
obtaining the high-resolution mass spectra. NIH grant
U19CA177547 partially supported this work. Pouya Haratipour
was a USC Dornsife Chemical Biology Trainee.
[17] V.K. Batra, K. Oertell, W.A. Beard, B.A. Kashemirov, C.E. McKenna, M.F.
Goodman, S.H. Wilson, Biochemistry 57 (2018) 3934.
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Appendix A. Supplementary data
Supplementary data (details of synthetic procedures with spec-
troscopic and other characterization data) to this article can be
found online at https://doi.org/10.1016/j.tetlet.2021.152890.
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