Synthesis of activated 3′-amino-3′-deoxy-2-thio-thymidine, a superior substrate for the nonenzymatic copying of nucleic acid templates

Article abstract of DOI:10.1039/c5cc10317g

We present a scalable synthesis of 3′-amino-3′-deoxy-2-thio-thymidine-5′-phosphoro-2-methylimidazolide, an activated monomer that can copy adenosine residues in nucleic acid templates rapidly without a polymerase. The sulfur atom substitution enhances the

Full text of DOI:10.1039/c5cc10317g

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Synthesis of activated 30-amino-30-deoxy-2-
thio-thymidine, a superior substrate for the
Cite this:DOI: 10.1039/c5cc10317g
nonenzymatic copying of nucleic acid templates†
Enver Cagri Izgu,ab Seung Soo Ohab and Jack W. Szostak*abc
Received 15th December 2015,
Accepted 26th January 2016
DOI: 10.1039/c5cc10317g
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We present a scalable synthesis of 30-amino-30-deoxy-2-thio-thymidine-
50-phosphoro-2-methylimidazolide, an activated monomer that can
copy adenosine residues in nucleic acid templates rapidly without a
polymerase. The sulfur atom substitution enhances the rate of
template copying by 5-fold compared with the 30-amino-30-deoxy-T
monomer, while the 30-amino monomers exhibit a 2- to 30-fold
enhancement compared with their ribonucleotide counterparts.
Nonenzymatic template-directed replication of nucleic acids has
been hypothesized to be the mechanism of information transfer
in primitive cells prior to the advent of ribozyme polymerases.1
Early efforts involving high-energy nucleotide monomers such as
50-phosphoro-2-methylimidazolides (or 2-MeImpNs) (Fig. 1, top)
showed that RNA templates consisting of C residues can be
copied by 2-MeImpG in hours to days in the presence of divalent
cations (typically Mg2+).2 However, no enzyme-free process has
yet been discovered that enables the rapid and efficient copying
of mixed-sequence RNA templates with activated ribonucleotide
monomers. This problem has stimulated interest in alternative
nucleic acids that might exhibit faster replication chemistry;
such polymers are of interest both with respect to the origin of
life and in the context of designing artificial life forms based on
non-biological chemistry. The most promising non-biological
nucleic acids are the phosphoramidate polymers, which are
assembled from nucleotides with an amino group on the sugar
Fig. 1 Template-directed polymerization of RNA (top) and 3-NP-DNA
(bottom). NB denotes nucleobase.
instead of the less nucleophilic hydroxyl. N30-P50-linked phos- attractive alternative genetic model because it adopts a helical
phoramidate DNA3,4 (30-NP-DNA, Fig. 1, bottom) stands out as an geometry that is similar to that of A-form RNA.5 A-form geometry
is the preferred conformation for the nonenzymatic template-
directed oligomerization of activated ribonucleotides6,7 most
a Howard Hughes Medical Institute, Department of Molecular Biology and Center
likely because the A-form double helix of RNA brings the 30-OH
for Computational and Integrative Biology, Massachusetts General Hospital,
group of the primer in line with the leaving group of the incoming
185 Cambridge Street, Boston, Massachusetts 02114, USA.
monomer. We have previously shown8 that activated 30-amino-
E-mail: szostak@molbio.mgh.harvard.edu
b Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur,
20,30-dideoxynucleotide monophosphates (30-NH2-2-MeImpddNs)
(Fig. 1, bottom) rapidly polymerize on short, homopolymeric DNA,
RNA, and locked nucleic acid (LNA) templates. We also found9
that replacing 30-amino-T with 30-amino-2-thio-T enhances the
rate and fidelity of 30-NP-DNA synthesis in the copying of DNA,
RNA and 30-NP-DNA templates. However, further progress in the
Boston, Massachusetts 02115, USA
c Department of Chemistry and Chemical Biology, Harvard University,
12 Oxford St., Cambridge, Massachusetts 02138, USA
† Electronic supplementary information (ESI) available. CCDC 1440493. For ESI
and crystallographic data in CIF or other electronic format see DOI: 10.1039/
c5cc10317g
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development and quantitative analysis of this system has been
Our successful strategy to access 30-NH2-2-MeImpdds2T
hindered by lack of access to the critical substrate, the 50-phosphoro- (Scheme 1) commenced with the 50-tosylation12 of 30-azido-30-
imidazolide of 30-amino-2-thio-T (30-NH2-2-MeImp-dds2T), which deoxythymidine (AZT) 1.13 Intramolecular displacement of the
could previously be synthesized only in small quantities because of 50-tosylate by the C2-oxyanion formed in the presence of a
the expensive starting material and the very low yield of the desired Brønsted base (e.g., DBU) yielded the 2,50-O-anhydro-cyclo-
product.
nucleoside, and subsequent ring-opening in refluxing ethanol
Here, we present a concise and scalable synthesis of 30-amino-2- afforded the 2-ethoxythymidine 3 in 59% overall yield from 1.
thio-T and the corresponding 50-phosphoro-imidazolide, 30-NH2-2- After converting 3 into the acetate 4, we were able to incorporate
MeImpdds2T. We also present the first quantitative investigation the sulfur atom into the nucleobase using H2S in the presence of
of template-copying kinetics using this activated nucleotide. Our tetramethylguanidine (TMG)14 (see Fig. S1 in the ESI† for details
results show that nonenzymatic 30-NP-DNA synthesis using the of the reaction setup). 1H NMR analysis of an aliquot of the crude
2-thio modified 30-amino-T phosphoro-imidazolide monomer is reaction mixture after 1 hour revealed that 4 was fully consumed,
significantly faster than with unmodified 30-amino-T, and is while two new species were formed: 30-amino-2-ethoxythymidine
also considerably faster than RNA synthesis with activated U and 5 and 30-amino-2-thio-thymidine 6, in a molar ratio of 2 : 1 (5/6).
2-thio-U.
The relative abundance of 6 continued to increase as the reaction
Our earlier attempts to establish a concise synthetic route to progressed [up to ca. 1 : 3 (5/6) after 6 hours]. We did not observe
30-NH2-2-MeImpdds2T involved regioselective thio-carbonylation10 (by either ESI or 1H NMR analysis) the formation of any 30-azido-
of 4-O-protected 30-azido-30-deoxy-thymidine. However, these 2-thio-thymidine, suggesting that the incorporation of sulfur was
substrates afforded only deglycosylation products using Lawesson’s slower than the reduction of the 30-azide, and that 6 was likely
reagent, an observation later reported11 for 4-O-mesityl-30,50-O- formed from 5. The structure of the 2-thio nucleoside 6 was
1
di(TBS)-thymidine. We then studied nucleophilic ring opening confirmed by both H–1H gCOSY NMR spectroscopy and X-ray
of 2-thio-2,30-cyclonucleosides by azides at the 30-position, but crystallography (see the ESI†). We then converted 6 into the
observed little to no conversion (by 1H NMR analysis) of the phosphoroimidazolide precursor 7 via 50-deacetylation and Fmoc
substrates in the presence of either excess LiN3 or TMSN3 protection of the 30-amine. A one-flask 50-O-phosphorylation and
accompanied by various Lewis acids [e.g., Hg(OAc)2, Er(OTf)3 2-methyl-imidazolide synthesis, followed by the removal of Fmoc,
and Yb(O-iPr)3].
provided 30-NH2-2-MeImpdds2T in 26% overall yield from 7.
With 30-NH2-2-MeImpdds2T in hand we proceeded to carry
out nonenzymatic primer extension experiments to quantitatively
interrogate the effect of 2-thio substitution on RNA template-copying
rates (Fig. 2). We used a primer/template complex composed of a
DNA primer strand ending in a 30-NH2-G and a complementary
RNA template strand containing a 50-C2A4 overhang. pKa of the
protonated 30-amine of 30-amino-20,30-dideoxy-2-thio-thymidine
is 7.5 (see the ESI†), similar to that reported for 30-amino-20,30-
dideoxy-T (7.7).15 30-NH2-2-MeImpddNs tends to undergo intra-
molecular cyclization due to the proximity of the primary 30-amine
group to the phosphorus electrophile.8 Because the half-life (t1/2) of
30-NH2-2-MeImpddT is 1.2 h8 and that of 30-NH2-2-MeImpdds2T is
1.3 h (see the ESI† for details) under optimized primer extension
conditions [100 mM 1-(2-hydroxyethyl)-imidazole, pH 7.5, 4 1C],
we tracked primer extension only up to a maximum of 1 h. We
determined observed rate constants kobs for the first step of the
primer extension by following the loss of unreacted primer over
time (Fig. 2).
At a 10 mM initial concentration of 30-NH2-2-MeImpddT
(Fig. 2, left), kobs of primer extension was 0.42 hꢀ1 (Table 1, entry 1).
Notably, 2-thio modification led to about a 5-fold rate enhancement
(Fig. 2, right), such that kobs for 10 mM 30-NH2-2-MeImpdds2T was
1.92 hꢀ1 (Table 1, entry 2). This increase likely results from the
additional stabilization induced into the primer/template duplex
afforded by the formation of a s2T:A base pair compared to a
canonical T:A base pair,16 as well as the more 30-endo-like sugar
puckering of 2-thio-nucleotide, which is the favoured sugar
conformation in nonenzymatic primer extension reactions.17 The
kobs values for reactions containing the activated ribonucleotides18
2-MeImpU, 2-MeImps2U and its ribo-T analog 2-MeImps2T were all
Scheme 1 Synthesis of 30-NH2-2-MeImpdds2T. Reaction conditions:
(a) TsCl, pyr, 0 to 20 1C, 6 h, 73%; (b) DBU, EtOH, 85 1C, 24 h, 81%;
(c) DMAP (cat.), Ac2O, 20 1C, 12 h, 495%; (d) H2S (gas), TMG, pyr, 0 to
20 1C, 16 h, 53%; (e) 7N NH3 in MeOH, 20 1C, 92%; (f) Fmoc-OSu, Na2CO3
(aq), pyr, 0 to 20 1C, 6 h, 78%; (g) 1. POCl3, 2,6-lutidine, PO(OMe)3, 3 Å MS,
0 to 20 1C, 2 h; 2. 2-Me-imidazole, 2 h, 30%; (h) piperidine, DMF, 0 1C, 0.5 h,
85%. X-ray structure: H: white, C: gray, O: red, N: blue, and S: yellow.
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(Table 1, entries 2 vs. 4) faster reaction. Further physical and
kinetic characterization will be required to distinguish the
contributions of enhanced monomer binding vs. enhanced
monomer reactivity for these observations.
In conclusion, we have developed a scalable synthesis of a
2-thio modified thymidine monomer, 30-NH2-2-MeImpdds2T.
Our synthetic route provided this highly reactive nucleotide in
sufficient amounts to perform quantitative measurements of
nonenzymatic RNA template-copying rates for the first time.
Our results show that 30-NH2-2-MeImpdds2T can polymerize on
a DNA/RNA primer/template complex significantly faster than
any other U or T monomer that has been reported thus far.
We thank Dr Shao-Liang Zheng (Harvard) for help with X-ray
data collection and structure determination, and Prof. Mohammad
Movassaghi (MIT) and Dr Justin Kim (MIT) for their guidance in
performing safe hydrogen sulfide experiments. We also thank
Dr Lijun Zhou and Ms Moriana Haj for technical assistance and
Dr Albert C. Fahrenbach, Dr Victor S. Lelyveld, Mr Tony Z. Jia,
and Mr Chun Pong Tam for helpful discussions. J. W. S. is an
Investigator of the Howard Hughes Medical Institute. This work
was supported in part by a grant (290363) from the Simons
Foundation to J. W. S.
Fig. 2 Kinetics of copying a r(A)4(C)2 template with 30-NH2-2-MeImpddT
(left) and 30-NH2-2-MeImpdds2T (right), in the presence of 100 mM
1-(2-hydroxyethyl)-imidazole, at pH 7.5 and 4 1C. Reactions were initiated
by addition of monomers and monitored by gel electrophoresis. The
triangle indicates the primer +4 product. (bottom) Natural log of the
fraction of the unreacted primer plotted against incubation time. Errors
were based on two experiments. Primer strand (DNA, 0.2 mM): 50-(FAM)-
AGC-GTG-ACT-GAC-TGG-(NH2)-30, obtained enzymatically in ca. 85%
purity based on LC-HRMS (see the ESI†). Primer concentration was
corrected for unreactive oligonucleotide impurity. Template strand (RNA,
1 mM): 50-CCAAAA-CCA-GUC-AGU-CAC-GCU-30 RNA.
Notes and references
1 M. P. Robertson and G. F. Joyce, The Origins of the RNA World,
CSHP Biol., 2012, 4, a003608.
2 T. Inoue and L. E. Orgel, J. Am. Chem. Soc., 1981, 103, 7667.
3 W. S. Zielinski and L. E. Orgel, Nucleic Acids Res., 1985, 13, 2469.
4 M. Tohidi, W. S. Zielinski, C. H. Chen and L. E. Orgel, J. Mol. Evol.,
1987, 25, 97.
Table 1 Reaction kinetics measured for 10 mM T/U monomers at 4 1C
5 V. Tereshko, S. Gryaznov and M. Egli, J. Am. Chem. Soc., 1998,
120, 269.
Relative
kobs
¨
¨
6 M. Kurz, K. Gobel, C. Hartel and M. W. Gobel, Angew. Chem., Int.
Ed. Engl., 1997, 36, 842.
Entry
Template
Monomer
kobs (hꢀ1
)
7 I. A. Kozlov, P. K. Politis, A. V. Aerschot, R. Busson, P. Herdewijn and
L. E. Orgel, J. Am. Chem. Soc., 1999, 121, 2653.
8 S. Zhang, N. Zhang, J. C. Blain and J. W. Szostak, J. Am. Chem. Soc.,
2013, 135, 924.
9 S. Zhang, J. C. Blain, D. Zielinska, S. M. Gryaznov and J. W. Szostak,
Proc. Natl. Acad. Sci. U. S. A., 2013, 110, 17732.
1a
2a
3b
4b
5b
r(A)4(C)2
r(A)4(C)2
r(A)6
r(A)6
r(A)6
30-NH2-2-MeImpddT
30-NH2-2-MeImpdds2T
2-MeImpU
0.42 (1)
1.92 (2)
ND
0.064 (1)
0.22 (6)
7
30
—
1
2-MeImps2U
2-MeImps2T
3
10 Y. Masaki, R. Miyasaka, K. Hirai, H. Tsunoda, A. Ohkubo, K. Seio
and M. Sekine, Chem. Commun., 2012, 48, 7313.
a
In the presence of 100 mM 1-(2-hydroxyethyl)imidazole. b Data obtained
from ref. 18. Reactions performed with 200 mM MgCl2 at pH 7.0.
11 P. C. Tlatelpa and H. Huang, Tetrahedron Lett., 2014, 55, 4780–4784.
12 T.-S. Lin, Z.-Y. Shen, E. M. August, V. Brankovan, H. Yang,
I. Ghazzouli and W. H. Prusoff, J. Med. Chem., 1989, 32, 1891.
13 This compound is 200 times cheaper (per 1 gram) than the starting
material [50-O-(dimethoxytrityl)-N3/O4-(toluoyl)-2-thiothymidine] that
we used for the earlier synthetic route9.
lower than the values for the activated 30-amino nucleotides
described above (Table 1), even though these ribonucleotide
polymerizations were assayed in the presence of 200 mM Mg2+
to optimize the reactivity. The rate enhancement observed for
2-MeImps2T vs. 2-MeImps2U suggests that methylation at the
5-position of 2-thiouracil leads to stronger monomer–primer
stacking. Additionally, primer extension reactions with 30-NH2-
2-MeImpdds2T are 10-fold faster than with the corresponding
ribonucleotide, 2-MeImps2T, presumably due to the greater
nucleophilicity of the 30-amine. Remarkably, combining the effect
of the 30-amine and the 5-methyl groups results in an ca. 30-fold
14 A. Miazga, F. Hamy, S. Louvel, T. Klimkait, Z. Pietrusiewicz,
˜
˜
A. Kurzynska-Kokorniak, M. Figlerowicz, P. Winska and T. Kulikowski,
Antiviral Res., 2011, 92, 57.
15 E. Kervio, A. Hochgesand, U. E. Steiner and C. Richert, Proc. Natl.
Acad. Sci. U. S. A., 2010, 107, 12074.
16 A. T. Larsen, A. C. Fahrenbach, J. Sheng, J. Pian and J. W. Szostak,
Nucleic Acids Res., 2015, 43, 7675.
17 N. Zhang, S. Zhang and J. W. Szostak, J. Am. Chem. Soc., 2012,
134, 3691.
18 B. D. Heuberger, A. Pal, F. D. Frate, V. V. Topkar and J. W. Szostak,
J. Am. Chem. Soc., 2015, 137, 2769.
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