hammerhead ribozyme uses a 2′-OH of the active-site
cytidine optimally positioned to transesterify the adjacent 3′-
phosphodiester. Replacement of this functional group with
a 2′-SH results not in transesterification of the neighboring
phosphodiester, but rather in deglycosylation (attack at C1′)
of the active-site cytidine.7
Scheme 2. Syntheses of ddtNTPs
Previous reports suggest that ddtNTPs are unlikely to be
effective polymerase substrates (at least after the incorpora-
tion of a single chain-terminating monomer),4 and that thiols
in general are poor nucleophiles for phosphates.2-7 Although
some synthetic efforts have been described for these deriva-
tives,8,9 we decided to prepare samples of all four ddtNTPs
and provide some preliminary data for the activities of these
derivatives as potential polymerase substrates or inhibitors.
The 2′,3′-dideoxy-3′-thiopyrimidine nucleosides were pre-
pared from the relevant O2-C3′ anhydronucleosides es-
sentially as described10 (Scheme 1a). After removal of the
Scheme 1. Syntheses of ddtNTP Precursors
used in a second epimerization to form the 2′,3′-dideoxy-
3′-thiopurine nucleosides (Scheme 1b). These derivatives
were stored as the thioesters (disulfide formation was less
efficient for the purine derivatives).
Formation of the corresponding nucleoside triphosphates
used the procedure described by Eckstein12 and either the
dinucleoside disulfides (Scheme 2a) or the corresponding
thiobenzoates (Scheme 2b). We were attracted to the
possibility of long-term storage of the triphosphates as the
disulfide dimers, and for 2′,3′-dideoxy-3′-thiothymidine as
well as 2′,3′-dideoxy-3′-thiocytidine, formation of the bis-
(triphosphate) occurred with high yield (Scheme 2a). Al-
though the bis(triphosphates) were not pure since triphos-
phate formation was not quantitative at both O5′-oxygens,
they could be stored in this state. The desired 3′-thio ddNTPs
were obtained after treatment of the bis(triphosphates) with
TCEP and HPLC isolation. For the thiopurines it was more
effective to convert the thiobenzoates into the desired
triphosphates followed by treatment with ammonium hy-
droxide to generate the 3′-thio NTPs.
It seemed likely that DNA polymerases would elongate a
DNA primer and form the N + 1 product with ddtNTP
substrates, since the reaction chemistry is unchanged in the
first incorporation. However, formation of the N + 2 and
longer extension products requires that the thiol take part as
the substrate nucleophile in the formation of internucleotide
phosphorothiolate diesters (Figure 1). To test these possibili-
ties, we initially screened a number of DNA polymerases
(Pol I KF (exo-), Taq, Bst, Sequenase, Superscript reverse
transcriptase, Therminator and wt and several mutants of
Deep Vent (exo-) polymerase) for primer-extension with
ddtTTP. Of these, the Y410F mutant of Deep Vent exo-
polymerase appeared the best and was chosen for further
study. We then examined all four 2′,3′-dideoxy-3′-thio-
nucleoside triphosphates for substrate activity using a DNA
primer/template complex with each template containing a
stretch of four identical residues (T, C, A, or G) at its 5′-
S-benzoyl protecting group the nucleosides could be oxidized
to the disulfides and stored in this state (see Scheme 2a).
The disulfides also functioned to protect the thiol group
during subsequent formation of the bis(triphosphates). The
purine derivatives were not so easily obtained since formation
of the corresponding anhydro derivatives cannot occur. The
most efficient procedure to epimerize the C3′ carbon and
prepare it for a simple displacement reaction with thiol is
that described by Herdewijn11 in which formation of the 5′-
benzoate and 3′-triflate results in a displacement of the triflate
ester and migration of the benzoate ester to generate the 2′-
deoxy-xylo-nucleoside (Scheme 1b). After removal of the
benzoate ester and formation of the 5′-DMT ether and 3′-
mesyl ester, the sodium salt of thiobenzoic acid could be
(7) Hamm, M. L.; Schwans, J. P.; Piccirilli, J. A. J. Am. Chem. Soc.
2000, 122, 4223-4224.
(8) Yuzhakov, A. A.; Chidzhavadze, Z. G.; Bibilashvilli, R. Sh.;
Kraevskil, A. A.; Galegov, G. A.; Dorneeva, M. N.; Nosik, D. N.; Kilesso,
T. Yu. Bioorg. Khim. 1991, 17, 504-509.
(9) El-Babary, A. A.; Khodair, A. I.; Pedersen, E. B.; Nielsen, C.
Monatsh. Chem. 1994, 125, 1017-1025.
(10) Sabbagh, G.; Fettes, K. J.; Gosain, R.; O’Neil, I. A.; Cosstick, R.
Nucleic Acids Res. 2004, 32, 495-501.
(11) Herdewijn, P. J. Org. Chem. 1988, 53, 5050-5053.
(12) Ludwig, J.; Eckstein, F. J. Org. Chem. 1989, 54, 631-635.
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