Two-step synthesis of 5′-deoxy-5′-thioguanosine-5′-monophosphorothioate and its incorporation efficiency into 5′-terminus of RNA for preparation of thiol-functionalized RNA

Article abstract of DOI:10.1016/j.tetlet.2010.04.124

Several 5′-modifications of RNA molecules have been shown to have broad applications in studying RNA structures, mapping RNA-protein interactions, and in vitro selection of catalytic RNAs. While phosphorothioate modification is one of the most popular methods for functionalizing the 5′-terminus of RNA by a transcription or kinase reaction, conjugation of terminal phosphorothioates with fluorophores has been achieved only with a low efficiency. To overcome this limitation, we have developed a two-step synthetic method for 5′-deoxy-5′-thioguanosine-5′-monophosphorothioate by combining two known reactions and measured its incorporation efficiency into the 5′-terminus of RNA by in vitro transcription using T7 RNA polymerase that requires guanosine to efficiently initiate transcription, followed by treatment of alkaline phosphatase, yielding a terminal sulfhydryl group at the 5′-termini of RNA molecules. Since the sulfhydryl group can be used as an alternative to phosphorothioates, our method may provide a useful route to efficiently introduce reporters, such as fluorophores, into the 5′-terminus of RNA via a stable thio-linker, or to tether the oligomer to a solid support.

Full text of DOI:10.1016/j.tetlet.2010.04.124

Tetrahedron Letters 51 (2010) 3446–3448
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Two-step synthesis of 50-deoxy-50-thioguanosine-50-monophosphorothioate
and its incorporation efficiency into 50-terminus of RNA for preparation
of thiol-functionalized RNA
Il-Hyun Kim a, , Seonmi Shin a, , Yong-Joo Jeong b, Sang Soo Hah a,
*
a Department of Chemistry and Research Institute for Basic Sciences, Kyung Hee University 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea
b Department of Bio and Nanochemistry, Kookmin University, 861-1 Jeongneung-dong, Seongbuk-gu, Seoul 136-702, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Several 50-modifications of RNA molecules have been shown to have broad applications in studying RNA
structures, mapping RNA–protein interactions, and in vitro selection of catalytic RNAs. While phosphoro-
thioate modification is one of the most popular methods for functionalizing the 50-terminus of RNA by a
transcription or kinase reaction, conjugation of terminal phosphorothioates with fluorophores has been
achieved only with a low efficiency. To overcome this limitation, we have developed a two-step synthetic
method for 50-deoxy-50-thioguanosine-50-monophosphorothioate by combining two known reactions
and measured its incorporation efficiency into the 50-terminus of RNA by in vitro transcription using
T7 RNA polymerase that requires guanosine to efficiently initiate transcription, followed by treatment
of alkaline phosphatase, yielding a terminal sulfhydryl group at the 50-termini of RNA molecules. Since
the sulfhydryl group can be used as an alternative to phosphorothioates, our method may provide a use-
ful route to efficiently introduce reporters, such as fluorophores, into the 50-terminus of RNA via a stable
thio-linker, or to tether the oligomer to a solid support.
Article history:
Received 30 March 2010
Revised 24 April 2010
Accepted 28 April 2010
Available online 17 May 2010
Keywords:
GSMP
Thiol-functionalized RNA
Alkaline phosphatase
Ó 2010 Elsevier Ltd. All rights reserved.
Although the modern biological systems are based on DNA gen-
omes and protein enzymes, RNA plays important roles in many
fundamental cellular processes including regulation of protein bio-
synthesis, RNA splicing, and retroviral replication, with remarkable
features: (1) it contains only four different ‘building blocks’ that
share similar chemical properties, (2) it can fold into various ter-
tiary structures that are highly tolerant of sequence variations,
and (3) it is easily soluble in water.1 In addition, the discovery of
the self-splicing pre-rRNA in Tetrahymena and the cleavage of tRNA
precursor by the RNA component within a ribonucleoprotein com-
plex demonstrated the existence of catalytic RNAs, the ribozymes.2
From this point of view, site-specific substitution and derivatiza-
tion of RNA can provide powerful tools for studying RNA structure
and function.3 Although the conventional solid-phase synthetic
method can be used to introduce functional groups at any specific
position of oligonucleotides shorter than approximately 40 nucle-
otides (nt),4 there are a limited number of methodologies for
site-specific modification and substitution of larger RNA
molecules.
mapping RNA–protein interactions, and in vitro selection of cata-
lytic RNAs, phosphorothioate modification is one of the most pop-
ular methods for functionalizing the 50-terminus of RNA by a
transcription or kinase reaction.5 Fluorophores are the most attrac-
tive probes for RNA structure, but conjugating terminal phosphoro-
thioates with fluorophores has been achieved with only a low
efficiency.6,7 The sulfhydryl group is another special reactive group
that can be incorporated into nucleic acids as an alternative to the
use of phosphorothioates,8 because the thiol-reactive functional
groups include haloacetamides, maleimides, benzylic halides, and
bromomethyl ketones,9 and the thiol group demonstrates a unique
property of the thiol-disulfide exchange reaction. A free thiol group
can be chemically introduced into the 50-termini of RNA using car-
bodiimide and cysteamine, but the phosphoramidate linkage is not
very stable.10
To overcome these limitations, we have developed a two-step
synthetic method for 50-deoxy-50-thioguanosine-50-monophosp-
horothioate (GSMP) and measured its incorporation efficiency into
the 50-terminus of RNA by in vitro transcription using T7 RNA poly-
merase that requires guanosine to efficiently initiate transcription,
followed by treatment of alkaline phosphatase for introduction of a
terminal sulfhydryl group into the 50-termini of RNA molecules.11
GSMP was synthesized via a two-step reaction by simply
combining two known reactions (Scheme 1).12,13 In brief, iodine
was added over 5 min to a magnetically stirred suspension of
While several 50-modifications of RNA molecules have been
shown to have broad applications in studying RNA structures,
* Corresponding author. Tel.: +82 2 961 2186; fax: +82 2 966 3701.
E-mail address: sshah@khu.ac.kr (S.S. Hah).
These authors contributed equally to this work.
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.04.124

I.-H. Kim et al. / Tetrahedron Letters 51 (2010) 3446–3448
3447
guanosine hydrate, triphenylphosphine, and imidazole in
N-methyl-2-pyrrolidinone at room temperature. After 3 h the solu-
tion was diluted with dichloromethane and water, yielding a white
crystalline solid 2.12a Iodinated guanosine 2 was then reacted with
trisodium thiophosphate in water for 3 days, yielding the desired
product 3.11,12b GSMP 3 was characterized by proton NMR and then
quantitatively tested as a substrate for in vitro transcription
(Scheme 2), since GSMP 3 had already been suggested that it could
be used to introduce a terminal sulfhydryl group into the 50-termini
of RNA molecules.11,14
A 97-mer single-stranded DNA containing a T7 promoter at the
50-end (50-CAG GAC TGC TCT CAC TCT CAC GCA CCA AGA AGC TGC
CAT TGA TCC CGC TGC TCA GCA GAT ACT CAG CGG CCC CCC CTA
TAG TGA GTC GTA TTA GTC C-30) was used as the template, and
the 71-nt 50-GSMP-RNA was synthesized by runoff transcription
in the presence of GSMP 3 with molar ratios of GSMP:GTP:ATP:CT-
P:UTP = 0:1:1:1:1, 4:1:1:1:1, 8:1:1:1:1, 10:1:1:1:1, 20:1:1:1:1, and
50:1:1:1:1 mM, respectively. Figure 1(a) shows that GSMP can be
used as a substrate for T7 RNA polymerase that requires guanosine
to efficiently initiate in vitro transcription, as reported,11,15
although a comparison of these incorporation efficiencies with
published reports using similar initiator nucleotides is complicated
by the facts that each laboratory prefers slightly different tran-
scription conditions and that the nucleotide concentrations fre-
quently vary among studies.
This result, however, clearly demonstrates that the in vitro tran-
scription can be used to incorporate a sulfhydryl moiety to 50-end
of RNA molecule, since an enzymatic step using alkaline phospha-
tase can be applied to convert 50-GSMP-RNA to 50-HS-RNA.14
Therefore, each of the 50-GSMP-RNA obtained was dephosphoryl-
ated, according to the literature,11 by incubation with 10 units of
alkaline phosphatase in buffer 3 (New England Biolab, MA) at
37 °C for 2 h and stopped by incubation with 10 lL of 200 mM
EGTA for 10 min at 65 °C. The resulting RNA was recovered by eth-
anol precipitation, and tested for quantitation of the thiol group
using thiol and sulfide quantitation kit (Molecular Probes, OR)
according to the manufacturer’s manual (see Table 1 for percent-
age of the sulfhydryl group existent at 50-end of RNA molecules).14
Scheme 1. Synthetic scheme for 50-deoxy-50-thioguanosine-50-monophosphorothioate
3 (GSMP). Reagents and conditions: (a) P(Ph)3, I2, imidazole, N-methyl-2-
pyrrolidinone; (b) trisodium thiophosphate, water.
Scheme 2. Schematic diagram for preparation of thiol-functionalized RNA at 50-end. Reagents and conditions: (a) T7 RNA polymerase, GSMP:GTP:ATP:CTP:UTP; (b) alkaline
phosphatase.
Figure 1. (a) Image of T7 RNA polymerase transcripts from the template to compare the in vitro transcription efficiency. Lane 1, ssDNA ladder (IDT, IA); lane 2, 50-GTP-RNA
resulting from the transcript without GSMP; lanes 3–7, mixtures of 50-GTP-RNA and 50-GSMP-RNA resulting from 4, 8, 10, 20, and 50 equiv of GSMP, respectively. The image
of the RNA products was obtained using Gel Doc 2000 Gel Documentation System (Bio-Rad, CA), following denaturing 7.5 M urea/8% polyacrylamide gel electrophoresis and
EtBr staining; (b) Quantitation of the sulfhydryl group existent at 50-end of RNA molecule, resulting from alkaline phosphatase treatment to convert 50-GSMP-RNA to 50-HS-
RNA.

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I.-H. Kim et al. / Tetrahedron Letters 51 (2010) 3446–3448
4. Gait, M. J.; Earnshaw, D. J.; Farrow, M. A.; Fogg, J. H.; Grenfell, R. L.; Naryshkin,
Table 1
N. A.; Smith, T. V. In RNA–Protein Interaction: A Practical Approach; Smith, C., Ed.;
Oxford University Press: Oxford, 1998; pp 1–36.
Percentage of the sulfhydryl group existent at 5’-end of RNA molecule, resulting from
alkaline phosphatase treatment to convert 50-GSMP-RNA to 50-HS-RNA
5. (a) Zhang, B.; Cech, T. R. Nature 1997, 390, 96–100; (b) Joseph, S.; Noller, H. F.
EMBO J. 1996, 15, 910–916; (c) Burgin, A. B.; Pace, N. R. EMBO J. 1990, 9, 4111–
4118.
6. Czworkowski, J.; Odom, O. W.; Hardesty, B. Biochemistry 1991, 30, 4821–4830.
7. Qin, P. Z.; Pyle, A. M. Methods 1999, 18, 60–70.
8. (a) Cohen, S. B.; Cech, T. R. J. Am. Chem. Soc. 1997, 119, 6259–6268; (b)
Sigurdsson, S. Th.; Seeger, B.; Kutzke, U.; Eckstein, F. J. Org. Chem. 1996, 61,
6883–6884; (c) Sun, S.; Tang, X.-Q.; Merchant, A.; Anjaneyulu, P. S. R.; Piccirilli,
J. A. J. Org. Chem. 1996, 61, 5708–5709; (d) Musier-Forsyth, K.; Schimmel, P.
Biochemistry 1994, 35, 1647–1650; (e) Fidanza, J. A.; Ozaki, H.; McLaughlin, L.
W. Methods Mol. Biol. 1994, 26, 121–143.
[GSMP]:[GTP]:[ATP]:[CTP]:[UTP]
Percentage quantity of thiol at 50 end
4:1:1:1:1
8:1:1:1:1
10:1:1:1:1
20:1:1:1:1
50:1:1:1:1
38.9 (3.44
66.0 (3.10
81.5 (3.45
94.6 (2.80
100 (2.26
l
l
l
l
g)
g)
g)
g)
l
g)
The total amount of each alkaline phosphatase-treated RNA used for percentage
measurement of the sulfhydryl group existent at 5’-end of RNA molecule is pre-
sented in the parenthesis.
9. Haugland, R. P. Handbook of Fluorescent Probes and Research Chemicals, 6th ed.;
Molecular Probes: Eugene, OR, 1996. pp 48–59.
10. (a) Chu, B. C. F.; Orgel, L. Nucleic Acids Res. 1988, 16, 3671–3691; (b) Chu, B. C.
F.; Kramer, F. R.; Orgel, L. Nucleic Acids Res. 1986, 14, 5591–5603.
11. It should be noted that it was reported that GSMP could be prepared via a
4-step synthesis and used to introduce the sulfhydryl group at the 50-terminus
of RNA (Zhang, L.; Sun, L.; Cui, Z.; Gottlieb, R. L.; Zhang, B. Bioconjugate Chem.
2001, 12, 939–948).
12. (a) McGee, D. P. C.; Martin, J. C. Can. J. Chem. 1986, 64, 1885–1889; (b)
Hampton, A.; Brox, L. W.; Bayer, M. Biochemistry 1969, 8, 2303–2311.
13. General methods: Unless otherwise noted, reagents were obtained from
commercial suppliers and were used without further purification. Depc-
treated deionized water was used whenever necessary. 1H NMR spectra were
carried out on Bruker 400 MHz spectrometer and TMS was used as an internal
reference for 1H. All experiments were performed in duplicate.
Figure 1b shows a saturation graph for the percent quantity of the
sulfhydryl group at 50-end of RNA molecule. When the ratio of
GSMP:GTP was 4:1, approximately 39% of the nascent transcript
were initiated with GSMP. The percent of transcripts initiated with
GSMP increased to 66%, 81%, 95%, and 100% as the GSMP:GTP ratio
was varied to 8:1, 10:1, 20:1, and 50:1, respectively, although 50
times excess of GSMP appeared to slightly inhibit transcription
by T7 RNA polymerase (Fig. 1a). These thiol-containing RNAs gen-
erated by the transcription/dephosphorylation reactions were suc-
cessfully conjugated to maleimide-activated nanoparticles (data
not shown).
GSMP synthesis: GSMP was synthesized via a two-step reaction (Scheme 1 and
see Ref.12). In brief, iodine (4.02 g, 15.8 mmol) was added over 5 min to a
magnetically stirred suspension of guanosine hydrate (1.5 g, 5 mmol),
triphenylphosphine (4.32 g, 16.5 mmol), and imidazole (2.25 g. 33.1 mmol) in
N-methyl-2-pyrrolidinone (20 ml) at room temperature. During the addition
complete dissolution occurred and the solution warmed to 60 °C. The
solution cooled back to room temperature and after 3 h was diluted with
To summarize, we report that GSMP, an initiator for the in vitro
transcription, was synthesized via
a two-step reaction by
combining two known reactions and used according to the litera-
ture11 as a substrate for T7 RNA polymerase, which was followed
by an additional step of dephosphorylation of 50-GSMP-RNA in
order to produce 50-HS-RNA. Our method may provide a useful
route to efficiently introduce reporters, such as fluorophores, into
the 50-terminus of RNA via a stable thio-linkage, or to tether the
oligomer to a solid support.
dichloromethane (200 ml) and water (60 ml).
A white crystalline solid
separated from solution and was collected by filtration to give 1.4 g (71.2%) of 2.
Trisodium thiophosphate (0.48 g, 2.6 mmol) was added to a suspension of
iodinated guanosine 2 (0.283 g, 0.72 mmol) in 1.4 ml of water. The reaction
mixture was stirred for 3 days at room temperature under argon atmosphere.
After filtration to remove any precipitate, the filtrate was evaporated under
reduced pressure. The residue was dissolved in 10 ml of water and precipitated
by addition of 20 ml of methanol. After removing the precipitate by filtration,
filtrate was evaporated and dissolved in small amount of water and applied to
reverse-phase chromatography. The desired product was collected and dried by
lyophilizer (68% yield from 2). Rf = 0.36 (i-PrOH/NH3/H2O = 6:3:1). 1H NMR
(400 MHz, DMSO-d6+D2O): d 7.82 (s, 1H), 5.63 (d, J = 5.9 Hz, 1H), 4.28 (dd,
J = 3.9 Hz, 1H), 4.08 (ddd, 2H), 2.83 (m, 2H).
Acknowledgement
This work was supported in part by Basic Science Program
through the National Research Foundation of Korea (KRF) funded
by the Ministry of Education, Science and Technology (MEST)
(No. 2010-0015218) and by the Kyung Hee University Research
Fund in 2010.
14. In vitro transcription and thiol quantitation: A 97-mer single-stranded DNA
containing a T7 promoter at the 50-end (50-CAG GAC TGC TCT CAC TCT CAC GCA
CCA AGA AGC TGC CAT TGA TCC CGC TGC TCA GCA GAT ACT CAG CGG CCC CCC
CTA TAG TGA GTC GTA TTA GTC C-30) was used as the template. Transcription
reactions were carried out with 50 units of T7 RNA polymerase in the presence
of 0.2 mM each GTP, ATP, CTP, and UTP, 12 lg of DNA template, 2 mM
spermidine, 10 mM dithiothreitol, 6 mM MgCl2, and 40 mM Tris buffer (pH 7.9)
at 37 °C in a total of 0.2 mL solution. The 71-nt 50-GSMP-RNA was synthesized
by runoff transcription in the presence of GSMP 3 with a ratio of GSMP:GTP:
ATP:CTP:UTP = 0:1:1:1:1, 4:1:1:1:1, 8:1:1:1:1, 10:1:1:1:1, 20:1:1:1:1, and
50:1:1:1:1 mM, respectively. Each of the GSMP-labeled RNAs was purified by
denaturing 7.5 M urea/8% polyacrylamide gel electrophoresis, and the
resulting 50-GSMP-RNA was dephosphorylated by incubation with 10 units
of alkaline phosphatase in buffer 3 (New England Biolab, MA) at 37 °C for 2 h
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and stopped by incubation with 10 ll of 200 mM EGTA for 10 min at 65 °C. The
RNA was recovered by ethanol precipitation, and tested for quantitation of the
thiol group using thiol and sulfide quantitation kit (Molecular Probes, OR)
according to the manufacturer’s manual.
15. Milligan, J. F.; Uhlenbeck, O. C. Methods Enzymol. 1989, 180, 51–62.