random pools of TNA sequences would provide some
evidence for the plausibility of such a proposal. The ability
to select functional TNA molecules requires the availability
of the TNA building blocks, the nucleoside-3′-triphosphates
(tNTPs), and their ability to function with modern protein-
based polymerization catalysts. Recently, we and others have
observed that certain DNA polymerases can incorporate tTTP
or tUTP to extend a DNA or RNA primer in response to a
dA or A template.5a-c Synthetic studies on these materials
include the preparation of the 3′-O-DMT 2′-phosphoramidites
necessary for the chemical synthesis of TNA oligomers.1
Here we describe the synthesis of the 3′-tNTP analogues and
their substrate activity.
In the described syntheses of the TNA phosphoramidites,2a
a DMT group was used as a temporary protecting group for
the secondary 3′-OH, but since the remaining 2′-OH is also
a secondary hydroxyl, this protection reaction typically
resulted in a mixture of regioisomeric products. In most
cases, the two DMT isomers (2′- and 3′-) could be chro-
matographically resolved, but in the case of R-L-threofura-
nosyl cytosine, the 3′-isomer was preferentially obtained.2b
The 3′-DMT isomers were subsequently converted to the
corresponding 2-phosphoramidites for use in the synthesis
of TNA sequences. Here we explore whether the late-stage
intermediate side products, the 2′-DMT isomers (1, Scheme
1), can be used to prepare the 3′-triphosphates (3).
the reaction sequence (2 f 3, Scheme 1) do not damage
the 3′-triphosphate. Using these late-stage intermediates for
T, G, and R-L-threofuranosyl 2,6-diaminopurine (DAP), we
first removed the nucleobase protecting groups in 8 M
methylamine-ethanol/12 M methylamine-water 1:1. We
then prepared the desired triphosphates using Eckstein’s
method and subsequently removed the 2′-DMT group under
mild acidic conditions with no observable cleavage of the
triphosphate (3, Scheme 1).
The triphosphate products were purified by anion exchange
HPLC chromatography (DEAE Sephadex), and after the
fractions containing product were pooled and reduced in
volume, desalting was performed using a 20 × 200 mm
column of polydivinyl-benzene eluting with aqueous triethyl-
ammonium acetate. The products were lyophilized to dryness
and stored at -20 °C.
This strategy failed in the case of tCTP, where the 3′-
DMT protected nucleoside (4, Scheme 2) was the major
Scheme 2. Synthesis of R-L-Threofuranosyl Cytosine
Triphosphate
Scheme 1. Synthesis of R-L-Threofuranosyl Nucleoside
Triphosphates from 2′-O-DMT Nucleosides
product2b and the presence of the 2′-regioisomer was
negligible. We therefore prepared the 3′-DMT derivative of
the nucleobase-protected R-L-threofuranosyl cytosine and
then acylated the 2′-hydroxyl group (f 5). Removal of the
DMT group generated the 2′-acetate (6). This derivative was
used to prepare the corresponding triphosphate using
Eckstein’s method (tTTP has also been prepared5b using
POCl3), after which concentrated aqueous ammonia was
used to deprotect the nucleobase and generate the final
product 8, which could be purified and desalted as described
above.
Syntheses of nucleoside-5′-triphosphates using POCl3 in
trimethyl phosphate rely upon the greater reactivity and lesser
steric hindrance of the primary 5-hydroxyl. That approach
was potentially problematic for the TNA building blocks with
two secondary hydroxyls. Additionally, the acid-labile DMT
group would be lost during such procedures. Eckstein’s
method6 (Scheme 1) can be more generally used, but it
requires protection of the nontarget hydroxyl groups.
For three of the tNTPs, this strategy could work using the
2′-DMT-protected TNA derivatives, providing that the acid
conditions necessary for DMT removal after completion of
To probe the activity of these derivatives with a selected
DNA polymerase, we prepared a primer sequence and a
series of five templates. Four of the templates were used for
single-nucleotide incorporation studies of each of the four
tNTPs, and the fifth template was used with all four tNTPs
(Figure 1). To date, the polymerase that most effectively uses
the tNTPs as substrates is the thermophilic Therminator DNA
polymerase, a site-specifically engineered (A485L) exonu-
clease-deficient form of “9°N” DNA polymerase. Incubation
of a radiolabeled DNA primer with each appropriate template
(5) (a) Chaput, J. C.; Szostak, J. W. J. Am. Chem. Soc. 2003, 125, 9274-
9275. (b) Kempeneers, V.; Vastmans, K, Rozenski, J.; Herdewijn, P. Nucleic
Acids Res. 2003, 31, 6221-6226. (c) Horhota, A.; Zou, K.; Ichida, J. K.;
Biao, Y.; McLaughlin, L. W.; Szostak, J. W.; Chaput, J. C. J. Am. Chem.
Soc. 2005, submitted.
(6) Ludwig, J.; Eckstein, F. J. Org. Chem. 1989, 54, 631-635.
1486
Org. Lett., Vol. 7, No. 8, 2005