Synthesis and enzymatic incorporation of α-l-threofuranosyl adenine triphosphate (tATP)

Article abstract of DOI:10.1016/j.bmcl.2012.12.080

Threose nucleic acid (TNA) is an artificial genetic polymer in which the natural ribose sugar found in RNA has been replaced with an unnatural threose sugar. TNA can be synthesized enzymatically using Therminator DNA polymerase to copy DNA templates into

Full text of DOI:10.1016/j.bmcl.2012.12.080

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1447–1449
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Bioorganic & Medicinal Chemistry Letters
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Synthesis and enzymatic incorporation of
adenine triphosphate (tATP)
a-L-threofuranosyl
⇑
Su Zhang, John C. Chaput
Center for Evolutionary Medicine and Informatics in the Biodesign Institute, and Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-5301, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Threose nucleic acid (TNA) is an artificial genetic polymer in which the natural ribose sugar found in RNA
has been replaced with an unnatural threose sugar. TNA can be synthesized enzymatically using Thermi-
nator DNA polymerase to copy DNA templates into TNA. Here, we expand the substrate repertoire of
Therminator DNA polymerase to include threofuranosyl adenine 30-triphsophate (tATP). We chemically
synthesized tATP by two different methods from the 20-O-acetyl derivative. Enzyme-mediated polymer-
ization reveals that tATP functions as an efficient substrate for Therminator DNA polymerase, indicating
that tATP can replace the diaminopurine analogue (tDTP) in TNA transcription reactions.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 24 October 2012
Revised 13 December 2012
Accepted 17 December 2012
Available online 5 January 2013
Keywords:
Threose nucleic acid (TNA)
Synthetic genetics
(30-20)
a-
L-Threose nucleic acid (TNA) is a polymer in which the
diaminopurine (tDTP) analogue of adenine in place of threofurano-
syl adenine 30 triphosphate due to the increased stability of the
tD-dT base pair.12,13 Here, we report the synthesis and enzymatic
incorporation of tATP into TNA using Therminator DNA
polymerase.
natural five-carbon ribose sugar found in RNA is replaced with an
unnatural four-carbon threose sugar and phosphodiester linkages
occur at the 20 and 30 vicinal positions in a diaxial orientation.1,2
This substitution shortens the repeat of the backbone unit by one
bond as compared to natural DNA and RNA. Despite this difference,
TNA can form stable Watson–Crick antiparallel duplexes with
complementary strands of DNA, RNA, and TNA.1,3 This observation
refutes the long held belief that nucleic acid polymers should have
six atoms in their backbone repeat unit to recognize DNA and
RNA.4 The NMR structure of a TNA duplex reveals a right-handed
structure with a helical geometry similar to A-form DNA and RNA.5
TNA has received considerable interest as a possible RNA pro-
genitor in the evolution of life due to its ability to exchange genetic
information with RNA and the chemical simplicity of threose rela-
tive to ribose.6 Threose is found in model pre-biotic reactions7 and
on the surface of meteorites indicating that the sugar can form
spontaneously in the absence of life.8 To explore the functional
properties of TNA, we and others have pursued enzymatic condi-
tions that would allow for the synthesis of TNA molecules
in vitro by extending a DNA primer annealed to a DNA template.9
These studies led to the unexpected discovery that Therminator
DNA polymerase can function as an efficient DNA-dependent
TNA polymerase.10 Under optimal conditions, Therminator can
faithfully copy long DNA templates into TNA. Using a self-priming
DNA library that establishes a genotype–phenotype link between
new synthesized TNA molecules and their encoding DNA tem-
plates, we evolved a TNA aptamer that binds to human thrombin
with high affinity and high specificity.11 This example used the
Previously, DMT-protected intermediates obtained from the
syntheses of TNA phosphoramidites were used to prepare TNA
triphosphates.14 This intermediate was a logical starting point for
the synthesis of TNA triphosphates, because DMT protection of
the free nucleoside gives a mixture of 20- and 30-O-DMT regioisom-
ers that can be separated by silica gel chromatography. Nucleo-
sides with the DMT group at the 30 position were used to
construct DMT protected phosphoramidites, while nucleosides
with the DMT group at the 20 position are used to construct TNA
nucleoside triphosphates. This approach worked well for tGTP,
tDTP, and tTTP, but an additional protection–deprotection strategy
was needed for tCTP as tritylation of the threofuranosyl cytosine
derivative gave exclusively the 30-O-DMT product.14
From our earlier synthesis of TNA phosphoramidites, we knew
that adenosine followed the same regiochemistry as cytosine
during DMT protection.1,11 We therefore chose to follow the pro-
tection–deprotection strategy previously developed for cytidine
by treating the 30-O-DMT compound 1 with acetic anhydride to
protect the 20-OH as an acetyl group 2 in a 94% yield (Scheme 1).
We then removed the DMT group with mild acid to give 3 in a
90% yield and used the Ludwig–Eckstein method to covert the
30-OH to the desired triphosphate 4.15 After phosphorylation, the
20-O-acetyl and N-6 benzoyl protecting groups were removed with
NH4OH. The crude product was purified by anion exchange
chromatography and reverse-phase HPLC to afford the triethylam-
monium salt, which was converted to sodium salt with an overall
yield of 20% from 3.
⇑
Corresponding author.
E-mail address: john.chaput@asu.edu (J.C. Chaput).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.080

1448
S. Zhang, J. C. Chaput / Bioorg. Med. Chem. Lett. 23 (2013) 1447–1449
NHBz
NHBz
NHBz
N
N
N
N
N
N
N
N
N
N
N
N
DMTO
DMTO
HO
O
Ac2O
O
CF3COOH
O
OH
OAc
OAc
1
2
3
O
O
4-
P2O7
ii.
iii.
O
P
I2/py/H2O
iv. Na2SO3
i.
Cl
NH2
NHBz
N
N
N
N
N
O
P
O-
O
P
O-
O
O
P
O-
O
O
P
O-
N
N
N
HO
P
O-
O
O
O
HO
O
P
O-
O
O
v. NH4OH
O
O
OH
OAc
4
Scheme 1. tATP synthesis using the Ludwig–Eckstein method.15
NHBz
N
N
N
N
HO
O
O
O
O
O
ii.
O
P
O-
O-
O
P
i. P2O74-, DMF/tributylamie
3
OAc
O
O
Cl
P
O
P
O-
O
NHBz
NH2
N
O
-O
N
N
N
N
O
P
O-
O
P
O-
O
P
O-
P
O
O
iii. I2/py/H2O
iv. NH4OH
N
N
N
O
P
O
HO
O
O
O
O
O
P
-O
O
OAc
OH
4
Scheme 2. tATP synthesis using the Huang method.16
Huang and co-workers have reported a simpler strategy for syn-
thesizing nucleoside triphosphates.16 To determine whether this
new synthetic approach could be used to improve the yield or re-
duce the time required to generate TNA triphosphates, we decided
to compare our previous method to Huang’s method. This strategy
proceeds via a cyclic metaphosphite intermediate introduced at
the 30-OH position of 3 (Scheme 2). In our case, we observed the
metaphosphite intermediate as the major product by 31P NMR;
however, TLC analysis indicated a significant amount of unreacted
starting material. No additional product was observed after pro-
longed reaction times or under modified reaction conditions. This
is not entirely unexpected as it is much easier to phosphitylate a
primary alcohol than a secondary alcohol. The intermediate was
oxidized, hydrolyzed, and treated with NH4OH to give the desired
tATP molecule 4. The crude tATP was ethanol precipitated, purified
by reverse-phase HPLC, and validated by HPLC and MS. The overall
reaction yield from 3 is ꢀ5% compared to the Ludwig–Eckstein
method which gave tATP 4 in ꢀ20% yield.
merase, we compared the efficiency of TNA synthesis for tNTP mix-
tures that contained either tATP or tDTP in addition to tCTP, tGTP,
and tTTP (Fig. 1, left panel). In both cases, the DNA primer was ex-
tended with 58 sequential TNA residues to yield the desired full-
length product. No significant difference was observed between
the two sets of TNA substrates (tA vs tD). Time course analysis indi-
cated that the full-length product is visible after 30 min and the
reaction appears to be complete after ꢀ2 h (Fig. 1, right panel).
The ability to synthesize long TNA polymers using all four nat-
ural genetic letters (A, C, T, and G) has a number of advantages.
First, substitution of diaminopurine for adenosine makes it easier
to predict secondary structures of in vitro selected TNA aptamers
as the 20 amino group of diaminopurine can form additional inter-
actions that are difficult to identify using existing computational
methods. Second, substitution of diaminopurine for adenosine
makes comparing DNA and TNA aptamers selected to the same tar-
get straightforward as the only structural difference between the
two sets of polymers is the sugar. Last, DNA templates containing
diaminopurine are less amenable to amplification using the
polymerase chain reaction (PCR), which can make these sequences
more difficult to construct.
Based on established methodology for the synthesis of TNA oli-
gonucleotides, we reasoned that tATP should be a reasonable sub-
stitute for tDTP in a polymerase reaction. We examined this
possibility by attempting to extend a P32-labeled DNA primer
annealed to a 90-nt DNA template. Using Therminator DNA poly-
In summary, we describe the chemical synthesis of tATP and
demonstrate the successful synthesis of TNA polymers using an

S. Zhang, J. C. Chaput / Bioorg. Med. Chem. Lett. 23 (2013) 1447–1449
1449
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.12.
080.
References and notes
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Figure 1. Primer extension assays analyzed by denaturing polyacrylamide gel
electrophoresis. For TNA synthesis, each reaction included 0.5 M primer-template
complex, 20 mM Tris–HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1%
Triton-X 100, 0.1 g/ L BSA, 1 mM DTT, 0.1 units/ L Therminator DNA polymerase,
1.25 mM MnCl2, and 100 M tNTPs and incubated at 55 °C. Left panel: Lane 1:
marker (M), primer only. Lane 2: (A) tATP reaction. Lane 3: (D) tDTP reaction. Lane
4: maker (M), reaction conducted with dNTPs and Klenowexoꢁ. Right panel: time
course study of Therminator-mediated TNA synthesis. Lane 1: maker (M), primer
only. Lane 2–7: 0.5, 1, 2, 4, 6, and 8 h, respectively.
l
l
l
l
l
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molecules that can fold into shapes with discrete secondary and
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Acknowledgments
The authors thank Dr. NourEddine Fahmi for helpful comments
and suggestions and the Biodesign Institute at Arizona State Uni-
versity for support.