Synthesis of 5'-deoxy-5'-thioguanosine-5'-monophosphorothioate and its incorporation into RNA 5'-termini.

Article abstract of DOI:10.1021/ol006916s

[figure: see text] 5'-Deoxy-5'-thioguanosine-5'-monophosphorothioate (GSMP) was synthesized in four steps with 35% overall yield. GSMP serves as a good substrate for in vitro transcription with T7 RNA polymerase to yield 5'-GSMP-RNA, which was converted to 5'-HS-RNA by dephosphorylation with alkaline phosphatase. The thiol-reactive agents can be efficiently introduced into the 5'-terminus of RNA.

Full text of DOI:10.1021/ol006916s

ORGANIC
LETTERS
2
001
Vol. 3, No. 2
75-278
Synthesis of 5′-Deoxy-5′-thioguanosine-
′-monophosphorothioate and Its
2
5
Incorporation into RNA 5′-Termini
Biliang Zhang,* Zhiyong Cui, and Lele Sun
Program in Molecular Medicine, UniVersity of Massachusetts Medical School,
Worcester, Massachusetts 01605
biliang.zhang@umassmed.edu
Received November 22, 2000
ABSTRACT
5′-Deoxy-5′-thioguanosine-5′-monophosphorothioate (GSMP) was synthesized in four steps with 35% overall yield. GSMP serves as a good
substrate for in vitro transcription with T7 RNA polymerase to yield 5′-GSMP-RNA, which was converted to 5′-HS-RNA by dephosphorylation
with alkaline phosphatase. The thiol-reactive agents can be efficiently introduced into the 5′-terminus of RNA.
RNA molecules play critical roles in many cellular processes.
The development of methods for studying the molecular
details of the complex interactions and essential functions
of RNA in cellular metabolism is challenging. Site-specific
substitution and derivatization provide powerful tools for
studying RNA structures, mapping RNA-protein interac-
tions, and in vitro selection of catalytic RNAs. Phospho-
rothioate modification is one of the most popular methods
for functionalizing the 5′-terminus of RNA by a transcription
3
or kinase reaction. However, labeling efficiency of terminal
1
4
studying RNA structure and function. Although solid phase
phosphorothioate with fluorophores is low, and fluorophores
5
chemical synthesis can be used to introduce functional groups
at any specific position of oligonucleotides shorter than
are the most attractive probes for RNA structure. The
sulfhydryl group is a special reactive group that can be
2
6
approximately 40 nt, investigation of larger RNA molecules
incorporated into nucleic acids. The thiol-reactive functional
faces a limited number of methodologies for site-specific
modification and substitution. 5′-Modifications of RNA
molecules have been shown to have broad applications in
groups are primarily alkylating reagents, including iodo-
acetamides, maleimides, benzylic halides, and bromomethyl
ketones. The thiol group demonstrates a unique property;
7
that is, the thiol-disulfide exchange reaction. A pyridyl
disulfide group is the most popular type of thiol-disulfide
(
1) (a) Favre, A. Bioorganic Photochemistry: Photochemistry and the
Nucleic Acids; Morrison, H., Ed.; Wiley: New York, 1990; pp 379-425.
b) Uhlmann, E.; Peyman, A. Chem. ReV. 1990, 90, 543-584. (c)
(
Sontheimer, E. J.; Steitz, J. A. Science 1993, 262, 1989-1996. (d) Griffin,
E.; Qin, Z.; Michels, W.; Pyle, A. Chem. Biol. 1995, 2, 761-770. (e) Cech,
T. R.; Herschlag, D. Catalytic RNA; Eckstein, F., Lilley, D. M. J., Eds.;
Springer-Verlag: Berlin, 1996; Vol. 10, pp 1-17. (f) Dewey, T. M.;
Zyzniewski, M. C.; Eaton, B. E. Nuleosides Nucleotides 1996, 15, 1611-
(2) Gait, M. J.; Earnshaw, D. J.; Farrow, M. A.; Fogg, J. H.; Grenfell,
R. L.; Naryshkin, N. A.; Smith, T. V. RNA-Protein Interactions:
A
Practical Approach; Smith, C., Ed.; Oxford University Press: Oxford, 1998;
pp 1-36.
(3) (a) Burgin, A.; Pace, N. EMBO J. 1990, 9, 4111-4118. (b) Joseph,
S.; Noller, H. EMBO J. 1996, 15, 910-916. (c) Zhang, B.; Cech, T. R.
Nature 1997, 390, 96-100.
1
617. (g) Thomson, J. B.; Tuschl, T.; Eckstein, F. Catalytic RNA; Eckstein,
F., Lilley, D. M. J., Eds.; Springer-Verlag: Berlin, 1996; Vol. 10, pp 173-
1
1
1
96. (h) Allerson, C. R.; Chen, S. L.; Verdine, G. L. J. Am. Chem. Soc.
997, 119, 7423-7433. (i) Sontheimer, E. J.; Sun, S.; Piccirilli, J. A. Nature
997, 388, 801-805. (j) Strobel, S. A.; Shetty, K. Proc. Natl. Acad. Sci.
(4) Czworkowski, J.; Odom, O.; Hardesty, B. Biochemistry 1991, 30,
4821-4830.
(5) Qin, P. Z.; Pyle, A. M. Methods: Companion Methods Enzymol.
1999, 18, 60-70.
U.S.A. 1997, 94, 2903-2908.
1
0.1021/ol006916s CCC: $20.00 © 2001 American Chemical Society
Published on Web 12/29/2000

exchange functional group used in the construction of cross-
linkers or modification reagents. A pyridyl disulfide will
readily undergo an interchange reaction with a free sulfhydryl
to yield a single mixed disulfide product. Once a disulfide
linkage is formed, it may be cleaved using disulfide reducing
agents (DTT, etc.). Although 5′-(guanosine-5′-monophos-
phorothioate)-RNA (5′-GMPS-RNA) can react with pyridyl
yield12 and reacted with methyltriphenoxyphosphonium
1
3
iodide in THF to yield 2′,3′-isopropylidene-5′-deoxy-5′-
iodoguanosine (3) with 62% yield. The deprotection of 3
with 50% aqueous formic acid for 2.5 days and subsequent
reaction with trisodium thiophosphate yielded the desired
1
4
product 4 (68% yield from 3). GSMP 4 was characterized
by proton and phosphorus NMR and MS spectroscopy and
tested as a substrate for in vitro transcription.
disulfide to form a phosphorothioate sulfide compound (R-
8
SSPO
3
-RNA), a limitation of phosphorothioate sulfide
A 222-mer double-stranded (ds) DNA containing a T7
promoter was used as the template for in vitro transcription
(Scheme 2). Transcription reactions were carried out with
9
product is the low stability. The free thiol groups can be
introduced into the 5′-termini of RNA by chemically using
carbodiimide and cysteamine,1 but the phosphoramidate
linkage is also not very stable. However, we report herein
an enzymatic method for the introduction of 5′-terminal
sulfhydryl group at the 5′-termini of large RNA molecules.
0
Scheme 2. Schematic Diagram of Preparing Sulfhydryl
Incorporated RNA
The
5′-deoxy-5′-thioguanosine-5′-monophophorothioate
(GSMP) 4 was synthesized as a substrate for T7 RNA
polymerase that requires guanosine to efficiently initiate
transcription.11 The in vitro transcription was used to
incorporate a sulfhydryl moiety to 5′-end of RNA molecule.
5′-Deoxy-5′-thioguanosine-5′-monophosphorothioate was
synthesized shown on Scheme 1. Guanosine 1 was treated
Scheme 1. Synthesis of
′-Deoxy-5′-thioguanosine-5′-monophosphorothioate 4
a
5
2
0 units of T7 RNA polymerase in the presence of 1 mM
each GTP, ATP, CTP and UTP, 10 µg of DNA template,
3
2
1
1
0 µCi R- P-ATP, 4 mM spermidine, 0.05% Triton X-100,
2 mM MgCl , and 40 mM Tris buffer (pH 7.5) at 37 °C in
2
a total of 200 µL solution. The 196 nt 5′-GSMP-RNA was
synthesized by runoff transcription in the presence of GSMP
4
with a ratio of GSMP:GTP:ATP:CTP:UTP ) 8:1:1:1:1
(
7) Haugland, R. P. Handbook of Fluorescent Probes and Research
Chemicals, 6th ed.; Molecular Probes, Inc.: Eugene, OR, 1996; pp 48-
9.
5
(
8) (a) Lorsch, J. R.; Szostak, J. W. Nature 1994, 371, 31-36. (b)
Macosko, J. C.; Pio, M. S.; Tinoco, I.. Jr.; Shin, Y.-K. RNA 1999, 5, 1158-
1166.
(
9) (a) Goody, R. S.; Eckstein, F. J. Am. Chem. Sco. 1971, 93, 6252-
6
257. (b) Sengle, G.; Jenne, A.; Arora, P. S.; Seelig, B.; Nowick, J. S.;
Jaschke, A.; Famulok, M. Bioorg. Med. Chem. 2000, 8, 1317-1329.
(
10) (a) Chu, B. C. F.; Kramer, F. R.; Orgel, L. Nucleic Acids Res. 1986,
1
3
4, 5591-5603. (b) Chu, B. C. F.; Orgel, L. Nucleic Acids Res. 1988, 16,
671-3691.
(11) Milligan, J. F.; Uhlenbeck, O. C. Methods Enzymol. 1989, 180, 51-
6
2.
(12) Gibbs, D. E.; Verkade, J. G. Synth. Commun. 1976, 6, 563-573.
13) Dimitrijevich, S. D.; Verheyden, J. P. H.; Moffatt, J. G. J. Org.
Chem. 1979, 44, 400-406.
14) Hampton, A.; Brox, L. W.; Bayer, M. Biochemistry 1969, 8, 2303-
311. Preparation of compound 4. To a suspension of 5′-deoxy-5′-
a
4
(a) acetone, 70% HClO ; (b) methyltriphenoxy-phosphonium
(
iodide, THF; (c) (1) 50% HCOOH, (2) trisodium thiophosphate,
water, 3 days.
(
2
iodoguanosine (2.83 g, 7.2 mmol) in 140 mL of water was added trisodium
thiophosphate (4.8 g, 26 mmol). 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 100 mL of water and trisodium thiophosphate was
precipitated by addition of 200 mL of methanol. After removal of the
precipitate by filtration, the filtrate was evaporated and dissolved in a small
amount of water and subjected to reverse phase chromatography (C18). The
desired product was collected and dried by lyophilizer (1.9 g, 68% for two
with acetone and 70% perchloric acid at room temperature
for 70 min to give 2′,3′-isopropylideneguanosine 2 with 83%
(
6) (a) Fidanza, J. A.; Ozaki, H.; McLaughlin, L. W. Methods Mol. Biol.
1
3
994, 26, 121-43. (b) Musier-Forsyth, K.; Schimmel, P. Biochemistry 1994,
5, 1647-1650. (c) Sun, S.; Tang, X.-Q.; Merchant, A.; Anjaneyulu, P. S.
1
R.; Piccirilli, J. A. J. Org. Chem. 1996, 61, 5708-5709. (d) Sigurdsson, S.
steps). Rf ) 0.36 (i-PrOH/NH3/H2O ) 6:3:1); H NMR (400 MHz, DMSO-
Th.; Seeger, B.; Kutzke, U.; Eckstein, F. J. Org. Chem. 1996, 61, 6883-
d6 + D2O): δ 7.82 (s, 1H), 5.63 (d, J ) 5.9 Hz, 1H), 4.28 (dd, J ) 3.9 Hz
6
6
884. (e) Cohen, S. B.; Cech, T. R. J. Am. Chem. Soc. 1997, 119, 6259-
1H), 4.08 (m, 1H), 2.83 (m, 2H). 31P NMR (DMSO-d6 + D2O): δ 16.4
-
268.
ppm. MS (ESI) m/z found 378 [M - 1] (calcd C10H14N5O7PS, 379).
276
Org. Lett., Vol. 3, No. 2, 2001

mM. The body-labeled RNA was purified by denaturing 7.5
M urea/8% polyacrylamide gel electrophoresis. The 5′-
GSMP-RNA was dephosphorylated by incubation with 10
units of alkaline phosphatase in buffer 3 (New England
Biolab) at 37 °C for 2 h and stopped by incubation with 10
µL of 200 mM EGTA for 10 min at 65 °C. The RNA was
recovered by ethanol precipitation. The dephosphorylation
of 5′-GSMP-RNA can also be done by AgNO
plus DTT to produce 5′-HS-RNA (data not shown).
The 5′-GTP-RNA and 5′-HS-RNA were treated with
Biotin-PEG -iodoacetamide (5), Biotin-HPDP (6), and Bi-
otin-PEG -Maleimide (7) (Scheme 3) in 10 mM HEPES (pH
3 2
or HgCl
3
3
Figure 1. The autoradiogram of the streptavidin gel-shift analysis
of transcription products (5′-GTP-RNA and 5′-GSMP-RNA) fol-
lowing the incubation with 5, 6, and 7. Lane 1-3, 5′-GTP-RNA;
lane 4-10, 5′-HS-RNA.
Scheme 3. Structures of Thiol-Reactive Biotin Agents
8
, no biotinylated RNA was detected in the presence of
streptavidin (lane 1, 2, and 3, respectively). The retarded band
disappeared after treatment with DTT (lane 8), which
indicated the thiol-disulfide exchange reaction of 5′-HS-
RNA with Biotin-HPDP 7. The results suggested that the
biotin group was transferred to the terminal thiol of the 5′-
HS-RNA and not to other groups of RNA.
The overall yield (three steps) of biotinylated RNA is 57%
with 6 and 60% with 7 for GSMP (lane 6 and 9, respec-
tively). The average incorporation efficiency of GSMP with
6
and 7 is over 80% for each step with a ratio of GSMP:
GTP ) 8:1 for transcription. If the ratio of GSMP:NTP is
increased to 16:1, the incorporating yield will be significantly
increased (data not shown). The experiments demonstrated
that GSMP 4 can serve as a good initiator for T7 RNA
polymerase to introduce the sulfhydryl group into 5′-end of
RNA.
7.8), 300 mM NaCl, and 1 mM EDTA at room temperature
for 2 h. The reaction mixtures were extracted with phenol/
chloroform once and chloroform once and precipitated with
ethanol. The RNA pellets were resuspended in 20 µL of pure
water and stored at -20 °C. A 2 µL aliquot of RNAs was
incubated with 15 µg of streptavidin in the binding buffer
The 5′-deoxy-5′-thioribonucleotide-5′-triphosphate and 5′-
deoxy-5′-thio-2′-deoxyribonucleotide-5′-triphosphate used to
form the internal thiophosphate bridge are not substrates for
DNA-dependent RNA polymerase and DNA polymerase I
1
5
from E. coli, but weak competitive inhibitors. However,
our result is consistent with the observation that the 5′-
modified guanosines can serve as initiators for T7 RNA
polymerase transcript and supports that T7 RNA polymerase
does not interact with the 5′-phosphate of the initiating
(20 mM HEPES, pH 7.4, 5.0 mM EDTA and 1.0 M NaCl)
at room temperature for 20 min prior to mixing with 0.25
volumes of formamide loading buffer (90% formamide;
0.01% bromophenol blue and 0.025% xylene cyanol). The
1
6
biotinylated RNA products were resolved by electrophoresis
through 7.5 M urea/8% polyacrylamide gels. The biotinylated
RNA can form a complex with streptavidin and the mobility
of the 5′-biotin-RNA::streptavidin complex through the gel
will be retarded relative to unbiotinylated RNA. The fraction
of product formation relative to total RNA at each lane was
quantitated with a Molecular Dynamics PhosphorImager.
The streptavidin gel-shift results of 5′-HS-RNA with 5,
nucleotide.
Furthermore, the 5′-GMPS-RNA was also prepared by a
method similar to that described above with the modification
that guanosine-5′-monophosphorothioate (GMPS) 8 replaced
GSMP 4. To compare the transcriptional incorporation
efficiency of GSMP with GMPS, the 5′-GMPS-RNA and
5′-HS-RNA were coupled with 5 followed by analysis with
a streptavidin gel-shift assay because 5′-GMPS-RNA can
form stable biotin-RNA product with iodoacetamide 5
(Figure 2). In Figure 2, the overall yield of biotinylated RNA
with 5 is 55% for GSMP in three steps (lane 2), which is
6, and 7 are shown in Figure 1. When 5′-HS-RNA reacted
with 5, 6, and 7, the thiol-modified RNA molecules were
biotinylated. They showed a band-shift in the presence of
streptavidin upon gel electrophoresis that represented a
streptavidin::RNA complex (lanes 4, 6, and 9, respectively),
no retarded band was detected without streptavidin (lane 5,
(15) (a) Patel, B. K.; Eckstein, F. Tetrahedron Lett. 1997, 38, 1021-
024. (b) Scheit, K. H.; Stutz, A. J. Carbohydr. Nucleoside Nucleotide 1974,
1
1
, 485-490.
7, and 10). When 5′-GTP-RNA was treated with 6, 7, and
(16) Martin, C. T.; Coleman, J. E. Biochemistry 1989, 28, 2760-2762.
Org. Lett., Vol. 3, No. 2, 2001
277

by denaturing 7.5 M urea/8% polyacrylamide gel electro-
phoresis, the 5′-Biotin-SSPO -RNA was completely decom-
3
posed (lane 5); but the 5′-Biotin-S-S-RNA remained stable
without any detectable decomposition under identical condi-
tions (lane 6). The 5′-Biotin-SSPO -RNA (lane 2) and 5′-
3
Biotin-SS-RNA (lane 4) are stable when they were incubated
with 50 mM Mg(II) at 37 °C for 2 h. Famulok et al.9b
3
reported that the phosphorothioate sulfide RNA (R-SSPO -
RNA) is slightly unstable compared to the alkyl-disulfide
RNA under certain condition (50 mM K-MOPS buffer, 200
mM NaCl, room temperature, and pH 7.4), but it is unstable
at pH > 7.5, temperature higher than 23 °C, or even low-
salt conditions. Their results suggested that the metal ions
can stabilize the phosphorothioate sulfide compounds.
We have also studied the derivatization of 5′-HS-RNA
with other different thiol-reactive functional agents: phen-
ylanaline-pyridyl disulfides, â-galactose-pyridyl disulfides,
and pyrene-maleimide. The coupling of 5′-thiol-RNA with
Figure 2. The autoradiogram of the streptavidin gel-shift assays
of 5′-GMPS-RNA and 5′-HS-RNA with 5.
almost the same overall yield (57%) as GMPS in two steps
(lane 5). Thus, GSMP can serve as good an initiator for T7
1
7
these reagents is almost quantitative.
RNA polymerase as GMPS.
In conclusion, the results demonstrate that GSMP can serve
as substrates for T7 RNA polymerase to introduce a
sulfhydryl group into the 5′-end of RNA. This is the first
report that a free thiol group can be incorporated into the
However, the major advantage of the 5′-HS-RNA gener-
ated from 5′-GSMP-RNA is that it provides a stable disulfide
linkage that is crucial for bioconjugation or immobilized
binding studies. We observed that the Biotin-linker-SSPO
3
-
5
′-terminus of RNA by in vitro transcription. This report
RNA is less stable than Biotin-SS-RNA (Figure 3). In Figure
provides a useful method to introduce reporters via a stable
thio-linkage, such as fluorophores, into the 5′-terminus of
RNA efficiently. This method may have potential applica-
tions in analysis and detection of RNA, mapping of RNA-
protein interactions, in vitro selection of novel catalytic
RNAs, and even gene array.
Acknowledgment. This research was partially supported
by a CFAR development grant of the University of Mas-
sachusetts Medical School. We thank R. Gottlieb for com-
ments on the manuscript.
Supporting Information Available: Experimental details
and spectral/analysis data. This material is available free of
charge via the Internet at http://pubs.acs.org.
Figure 3. The autoradiogram of the stability analysis of 5′-Biotin-
SSPO -RNA and 5′-Biotin-SS-RNA.
OL006916S
3
(17) The quantitative analysis for the coupling reactions of 5′-HS-RNA
with pyridyl disulfide reagents was determined using the concentration of
the pridine-2-thione released by measuring the absorbance at 343 nm;
quantification for pyrene-maleimide was determined by the Molecular
Dynamics PhosphorImager.
3
, the freshly made 5′-Biotin-SSPO
3
-RNA was detected by
streptavidin gel-shift assay (lane 1). After the purification
278
Org. Lett., Vol. 3, No. 2, 2001