Chemical synthesis of an RNA sequence containing 2-thiocytidine (s 2C): The DY647 labelled anticodon stem and loop sequence of Staphylococcus aureus tRNAArg (s2C32, mnm 5U34, t6A

Article abstract of DOI:10.1016/j.tetlet.2011.06.078

Incorporation of 2-thiocytidine (s²C) into a DY647 labelled 17-mer RNA sequence corresponding to the anticodon stem loop of Staphylococcus aureus tRNA^Arg has been achieved by phosphoramidite chemistry. Key to the success was the use

Full text of DOI:10.1016/j.tetlet.2011.06.078

Tetrahedron Letters 52 (2011) 4443–4447
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Tetrahedron Letters
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Chemical synthesis of an RNA sequence containing 2-thiocytidine (s2C): the
DY647 labelled anticodon stem and loop sequence of Staphylococcus aureus
tRNAArg (s2C32, mnm5U34, t6A37)
a
a
b
a,
⇑
_
´
Grazyna Leszczynska , Jakub Pie˛ta , Brian Sproat , Andrzej Małkiewicz
a
_
´
´
Institute of Organic Chemistry, Technical University of Łódz, 90-924 Łódz, Zeromskiego 116, Poland
b Chemconsilium GCV, Jaarmarktstraat 48, 2221 Booischot, Belgium
a r t i c l e i n f o
a b s t r a c t
Incorporation of 2-thiocytidine (s2C) into a DY647 labelled 17-mer RNA sequence corresponding to the
anticodon stem loop of Staphylococcus aureus tRNAArg has been achieved by phosphoramidite chemistry.
Key to the success was the use of dilute iodine for the P(III) to P(V) oxidation step plus the choice of the
trimethylsilylethyl (tse) ester as protection for the threonyl carboxyl group of t6A. In trial experiments
with N4-benzoyl-2-thiocytidine, the use of dilute t-BuOOH in toluene as an alternative oxidising agent
caused substantial desulfurization.
Article history:
Received 19 April 2011
Revised 8 June 2011
Accepted 17 June 2011
Available online 25 June 2011
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
tRNA modified compounds
RNA fluorescent tag
Solid supported synthesis
2-Thiocytidine (s2C)
The majority of the 80 modified and hypermodified nucleosides
that occur in tRNA are located at positions 34 (‘wobble position’), 37
and 32 of the anticodon stem loop sequence (ASL) and they have a
strong influence on the various functions/activities of those tRNAs
in cells.1–4 Synthetic, hypermodified native tRNA fragments and/
or their analogues have been successfully utilized for model studies
on the complex mechanism of ribosomal protein biosynthesis inter
alia for the elucidation of structural constraints of hetero- and
homotropic interactions of RNA–protein and RNA–RNA.5–8 RNA
sequences modified with rare nucleosides and labelled with fluo-
rescent tags have been in common use for several years as a versa-
tile tool for the development of a new generation of anticancer/
antibacterial/antiviral therapies.9–11 Recently, both native and
fluorescent tag labelled human ASLLys,3 have been used to find
potent selective inhibitors of HIV-1 virus replication from an
antimetabolite library using a fully automated system.12
Allergy and Infectious Diseases (NIAID) list. Synthetic tRNA frag-
ments containing modifications specific to Prokaryotes and labelled
withfluorescentmarkers could be used for the selection ofinhibitors
of pathogenic bacteria, for example, S. aureus, replication from anti-
metabolite libraries.15
A fluorescent-labelled title sequence of S. aureus ASLArg fulfils
these conditions, however, incorporation of s2C into longer RNA se-
quences has not been reported as yet. Under the typical conditions
of PIII–PV oxidation (solid supported synthesis, phosphoramidite
methodology), 2-thiouridine and its native 5-substituted deriva-
tives undergo oxidation to the 2-oxo-analogues and/or oxidative
desulfurization into the respective 1-b-D-ribofuranosyl-1H-pyrimi-
din-4-one.16–18
Consequently, the synthesis of oligoribonucleotides modified
with 2-thiopyrimidine nucleosides using the phosphoramidite
method requires specific protocols for the oxidation step. Until
now the most effective oxidation systems have been elaborated
for incorporation of 5-methoxycarbonylmethyl-2-thiouridine (1 M
solution of cumene hydroperoxide in toluene)11 and 20-O-methyl-
2-thiouridine(s) (0.02 M solution of iodine in pyridine–H2O).16
The results of our model studies have shown that upon treatment
with a dilute solution of tert-butyl hydroperoxide in an aprotic sol-
vent, N4-benzoyl-2-thiocytidine undergoes transformations similar
to modified s2U, but it remains relatively intact in I2 solution.
Herein we report an efficient protocol for the synthesis of an s2C
modified RNA, in the presence of two other modifications (mnm5U,
t6A), and also labelled with a fluorescent tag (Fig. 1).
2-Thiocytidine (s2C) is specific to tRNA sequences from
Prokaryotes (Escherichia coli and Salmonella enterica serovar
Typhimurium).13 The high homology between E. coli ASL-tRNAArg,4
(s2C, mnm5U, t6A) and Staphylococcus aureus ASL-tRNAArg coding
sequences14 implies at least a similar set of modifications of the
anticodon loops.
It is noteworthy that antibiotics resistant S. aureus strains are
classified as high priority pathogens in the National Institute of
⇑
Corresponding author. Tel.: +48 42 631 31 50; fax: +48 42 636 55 30.
E-mail address: ajmalk@p.lodz.pl (A. Małkiewicz).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.06.078

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G. Leszczynska et al. / Tetrahedron Letters 52 (2011) 4443–4447
O
P
O
NHC(O)NHCHCOOH
CHOH
DY647
A27
U43
O
-
N
A
C
C
U
G
G
N
CH3
N
N
+
I-
N
N
Rb
G
C
t6A
s2C32
HO
A
DY647
O
Rb =
t6A37
U
NH2
O
mnm5U34
OH
HO
U
N
C
N
H
NH
N
S
N
Rb
mnm5U
O
Rb
s2C
Figure 1. Sequence of RNA modified with 2-thiocytidine (s2C), 5-methylaminomethyluridine (mnm5U) and N6-threonylcarbamoyladenosine (t6A).
NHCOPh
N
NCOPh
N
NHCOPh
N
NCOPh
N
oxidizing agent
SH
S
+
N
O
N
H
N
N
DMTrO
DMTrO
DMTrO
DMTrO
O
O
O
O
TBDMSO
OH
TBDMSO
TBDMSO
TBDMSO
OH
OH
OH
4
3
1
2
Scheme 1. Possible mechanism for the oxidation of 1.
Table 1
Stability of 1 under the oxidation conditions using iodine or tert-butyl hydroperoxide solution
Entry
Conditions
Time (min)
Equiva
Productb (%)
Recovery of 1 (%)
3
4
1
2
3
4
0.1 M t-BuOOH in toluene
0.25 M t-BuOOH in toluene
0.02 M I2 in THF/H2O/pyridine 90.5:0.45:9.05 (v/v/v)
0.1 M I2 in THF/H2O/pyridine 90.5:0.45:9.05 (v/v/v)
5
5
60
60
8
8
8
8
10
15
nd
nd
ndc
10
nd
nd
85
70
97
95
a
b
c
Equivalents of oxidising agent.
Yield of isolated product.
nd = not detected.
O
N
NHBz
OMe
B =
NH
N
N
COCF3
O
N
S
B
O
O
5
6
O
P
O
Si
OMe
O
N
Si
O
O
CN
O
N
OR
HN
N
H
N
N
N
7
R= -CH2CH2SiMe3
-CH2CH2-C6H4-NO2-p
8
Figure 2. Protecting groups selected for the 30-O-phosphoramidites of s2C (5), mnm5U (6) and t6A (7, 8).

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G. Leszczynska et al. / Tetrahedron Letters 52 (2011) 4443–4447
a
b
0.50
0.40
0.30
0.20
0.10
0.00
20.00
40.00
Minutes
60.00
c
d
300
250
200
150
100
50
0
000
2000
3000
4000
5000
6000
7000
m/z
Figure 3. (a) Preparative anion-exchange HPLC of crude, deprotected 17-mer 50-DY647-AUGGC-s2C-U-mnm5U-CU-t6A-AGCCAU-30. HPLC was performed at 38 °C using a
Waters AP-2 column packed with TSK SuperQÒ-5PW eluted with a linear gradient of NaBr (50 mM–0.65 M) in sterile 20 mM Na2HPO4–NaH2PO4 buffer solution (pH 7.5),
containing EDTA (50 lM) and 10% of CH3CN; flow 10 mL/min; (b) an analytical injection of the desalted and lyophilized oligoribonucleotide used for MALDI-TOF; HPLC was
performed using a Source™ 15Q 4.6/100PE column; flow 1 mL/min, under the same conditions as the preparative injection; (c) UV spectrum of purified, dye-labelled
oligoribonucleotide; the sample was dissolved in H2O; spectrum was measured in the range of 200–700 nm to confirm the integrity of the fluorescent dye structure (kmax
abs = 645 nm); (d) MALDI-TOF spectrum of 17-mer-ASL (experimental monoisotopic mass is 6068.208, the calculated monoisotopic mass for C169H212N60O124P16S2 is
6069.1 Da).

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4446
Based on previously reported data that describe the sensitivity
pyridine. Based on the above-mentioned observations an efficient
approach for solid supported insertion of s2C, together with two
other modified units (mnm5U, t6A) into oligoribonucleotides la-
belled with the fluorescent tag at 50-end has been elaborated.
The synthesis described here would provide a practical and repro-
ductable methodology for preparation of interesting models for
studies on the structure–function relation of modified tRNA
components as well as a useful tool for the selection of pathogenic
bacteria replication inhibitors from antimetabolite libraries.
of 2-thiouridine derivatives towards various oxidants,16–18 we
have examined the stability of a monomeric unit derived from
s2C in both dilute solutions of tert-butyl hydroperoxide in an
aprotic solvent and in iodine solution in THF–H2O–pyridine. For
these studies N4-benzoyl-2-thiocytidine derivative 1 that was
isolated as the side product in the course of s2C fully protected
30-O-phosphoramidite synthesis was utilized.19
A five-minute treatment of 1 with either 0.1 M or 0.25 M tert-
butyl hydroperoxide in toluene gave rise to a measurable amount
of oxidative desulfurization product 3 and a mixture of 3 and cyti-
dine derivative 4, respectively (Scheme 1, Table 1). The structures
of nucleosides 3 and 4 were confirmed by NMR spectroscopy and
LSI mass spectrometry. Tautomeric equilibrium and participation
of intermediate 2 in both directions of the oxidation process can
be expected (Scheme 1).
Acknowledgements
This work was partially supported by Grants 0278/H03/2006/31
from the Ministry of Education (granted to A.M.) and 1306/B/H03/
2011/40 from the National Centre of Education (granted to G.L.),
Poland.
On the other hand compound 1 did not undergo the above-
mentioned side-reaction after 60 min treatment with 0.02 M iodine
solution in THF–H2O–pyridine (Table 1). Moreover, 1 remained
intact even in a 0.1 M iodine solution (Table 1). These results are in
complete contrast to those reported for 2-thiouridine which proved
to be very reactive in concentrated iodine solution.16 However, it
should be noted that in a series of seven 5-substituted 2-thiouridine
derivatives that are natural components of tRNAs and mt-tRNAs,
striking differences in sensitivity towards various oxidants have also
been observed.20
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2011.06.078.
References and notes
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2. Giegé, R. Nat. Struct. Mol. Biol. 2008, 15, 1007.
A 0.02 M iodine solution in THF–H2O–pyridine was successfully
applied to the solid supported synthesis of the triply-modified and
DY647 fluorescent labelled 17-mer (Fig. 1). The selected protecting
group strategy for the three modified nucleoside phosphoramidites
is shown in Figure 2.
3. Motorin, Y.; Helm, M. Biochemistry 2010, 49, 4934.
4. Phizicky, E. M.; Alfonzo, J. D. FEBS Lett. 2010, 584, 265.
5. Yarian, C.; Townsend, H.; Czestkowski, W.; Sochacka, E.; Malkiewicz, A. J.;
Guenther, R.; Miskiewicz, A.; Agris, P. F. J. Biol. Chem. 2002, 277, 16391.
6. Murphy, F. V., 4th; Ramakrishnan, V.; Malkiewicz, A.; Agris, P. F. Nat. Struct. Mol.
Biol. 2004, 11, 1186.
7. Phelps, S. S.; Malkiewicz, A.; Agris, P. F.; Joseph, S. J. Mol. Biol. 2004, 338, 439.
8. Ashraf, S. S.; Guenther, R. H.; Ansari, G.; Malkiewicz, A.; Sochacka, E.; Agris, P. F.
Cell Biochem. Biophys. 2000, 33, 241.
9. Ki, K. H.; Park, Y. S.; Lee, S. H.; Kim, N. Y.; Choi, B. M.; Noh, G. J. Korean J.
Anesthesiol. 2010, 59, 403.
10. Yu, W. H.; Høvik, H.; Olsen, I.; Chen, T. BMC Mol. Biol. 2011, 12, 3.
11. Eshete, M.; Marchbank, M. T.; Deutscher, S. L.; Sproat, B.; Leszczynska, G.;
Malkiewicz, A.; Agris, P. F. Protein J. 2007, 26, 61.
A readily accessible perbenzoylated 2-thiouridine and a combi-
nation of previously reported methods were used for the prepara-
tion of N4-benzoyl-2-thiocytidine.19 mnm5U was prepared and
protected with a trifluoroacetyl group on the secondary amine.21
To optimize the synthetic strategy for the ASL sequence labelled
with the fluorescent dye, two combinations of t6A amino acid res-
idue protection were tested. N6-Phenoxycarbonyl-20,30,50-tri-O-
acetyladenosine22 was condensed (CH2Cl2, rt, 24 h) with threonine
derivatives in which the hydroxy and carboxyl functions were pro-
tected with TBDMS and trimethylsilylethyl (tse)23,24 groups or
TBDMS and p-nitrophenylethyl (npe)25 groups, respectively, to give
fully protected t6A derivatives in good yields. The sugar moiety was
selectively deprotected by treatment with 8 M ethanolic ammonia
(rt, 24 h). All modified units were protected with DMTr and TBDMS
on the 50- and 20-hydroxy, respectively, and were then phosphity-
lated to give the fully protected 30-O-phosphoramidites,26 5–8
(Fig. 2).
12. Patent application: WO/2008/064304A2, 2008; Chem. Abstr. 2008, 148,
577353s.
13. Jühling, F.; Mörl, M.; Hartmann, R. K.; Sprinzl, M.; Stadler, P. F.; Pütz, J. Nucleic
Acids Res. 2009, 37, D159.
14. http://wishart.biology.ualberta.ca/BacMap/.
15. http://www.businesswire.com/news/home/20081008005360/en/Trana.
16. Okamoto, I.; Seio, K.; Sekine, M. Tetrahedron Lett. 2006, 47, 583.
17. Sochacka, E. Nucleosides, Nucleotides Nucleic Acids 2001, 20, 1871.
18. Sochacka, E.; Fratczak, I. Tetrahedron Lett. 2004, 45, 6729.
19. Compound 1 was prepared from 20,30,50-tri-O-benzoyl-2-thiouridine obtained
by glycosylation of silylated 2-thiouracil with the ribose derivative
[Vorbrüggen, H.; Strehlke, P. Chem. Ber. 1973, 106, 3039]. Perbenzoyl-2-
thiouridine was converted into the 4-(1,2,4-triazol-1-yl) derivative by reaction
with
a mixture of phosphoryl chloride, 1,2,4-triazole, and Et3N/CH3CN,
according to the modified Reese procedure [Joshi, B. U.; Reese, C. B.;
Varaprasad C. Nucleosides Nucleotides 1995, 14, 209]. Ammonolysis of the
triazoyl group with simultaneous deprotection of the benzoyl groups from the
sugar moiety was achieved by treatment with 8 M methanolic NH3. Next, the
exocyclic amine function of 2-thiocytidine was protected with benzoyl, and the
50- and 30-hydroxy groups with DMTr and TBDMS, respectively.
The synthesis of the title RNA sequence (Fig. 1) was conducted
manually on a 15 lmol scale, according to a slightly modified ver-
sion of Sproat’s protocol.27,28 The DY647 fluorescent marker has an
absorption maximum at 645 nm. Only the tse protected t6A was
compatible with the generating dye-labelled fully modified RNA
that maintained the correct UV–vis profile of the dye (Fig. 3c).
Deblocking of npe protected t6A-containing polymer-bound oligo-
mer required a 10% DBU solution in CH3CN (40 minutes at 45 °C),
conditions which led to substantial degradation of the dye, as
was confirmed by a distinctive change in the UV–vis spectrum of
the labelled RNA. Following deprotection and purification
460A260 units of the desired RNA were obtained. The homogeneity
and structure of the 17-mer were unequivocally confirmed by
HPLC (Fig. 3a,b) and MALDI-TOF mass spectrometry data (Fig. 3d).
In conclusion, by treatment with a dilute solution of tert-butyl
hydroperoxide in toluene, the N4-benzoyl-2-thiocytidine hetero-
base moiety undergoes oxidation or/and oxidative desulfurization,
but it remains intact in the 0.02 M iodine solution in THF–H2O–
20. Leszczyn
´ ska, G.; Pie˛ta, J.; Leonczak, P.; Małkiewicz, A. unpublished results.
21. Małkiewicz, A.; Sochacka, E. Tetrahedron Lett. 1983, 24, 5387.
´
22. Adamiak, R. W.; Stawinski, J. Tetrahedron Lett. 1977, 18, 1935.
23. Stuart, J. W.; Gdaniec, Z.; Guenther, R.; Marszalek, M.; Sochacka, E.;
Małkiewicz, A.; Agris, P. F. Biochemistry 2000, 39, 13396.
24. Sundaram, M.; Crain, P. F.; Davis, D. R. J. Org. Chem. 2000, 65, 5609.
25. Boudou, V.; Langridge, J.; Van Aerschot, A.; Hendix, C.; Millar, A.; Weiss, P.;
Herdewijn, P. Helv. Chim. Acta 2000, 83, 152.
26. Agris, P. F.; Malkiewicz, A.; Kraszewski, A.; Everett, K.; Nawrot, B.; Sochacka, E.;
Jankowska, J.; Guenther, R. Biochimie 1995, 77, 125.
27. Sproat, B. S. Methods Mol. Biol. 2005, 288, 17.
28. The commercially available monomeric units A, C, U and G were protected with
DMTr and TBDMS on the 50- and 20-hydroxy functions, respectively, and the
exocyclic amine functions of A,
C and G were masked with 4-tert-
butylphenoxyacetyl (tac) (ProligoÒ). The NuLight DY647 phosphoramidite
was purchased from Thermo ScientificÒ and dried under vacuum over P2O5.
The typical rU(tac)-succinyl-CPG (ProligoÒ) support was utilized. A, C, G and U
monomers were coupled in 5 molar excess for 8 min in the presence of a 0.3 M

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G. Leszczynska et al. / Tetrahedron Letters 52 (2011) 4443–4447
solution of 5-(benzylmercapto)-1H-tetrazole in CH3CN, whereas modified
units were used in 3 molar excess and coupled for 12 min. The DY647
monomer required a 15 min coupling time. The capping was performed with
tac anhydride (2 min) followed by the oxidation cycle using 0.02 M I2 solution
in THF–H2O–pyridine (90.5:0.45:9.05 v/v/v) for 2 min (8 equiv). The duration
of the oxidation steps was reduced to 70 s after incorporation of s2C. For the
first deprotection step the support-bound RNA was treated with Et3N–CH3CN
1:1 v/v for 20 min to deprotect the phosphate functions and avoid heterobase
alkylations by acrylonitrile via Michael-type addition [Capaldi, D. C.; Gaus, H.;
Krotz, A. H.; Arnold, J.; Carty, R. L.; Moore, M. N.; Scozzari, A. N.; Lowery, K.;
Cole, D. L.; Ravikumar, V. T. Org. Process Res. Dev. 2003, 7, 832–838]. Next,
treatment of the oligomer with 7 M ethanolic NH3 (8 h, 20 °C) resulted in
deprotection of the exocyclic amine functions and cleavage from the support.
The TBDMS protecting groups and the tse group were removed by treatment
with 1 M TBAF in 1-methyl-2-pyrrolidinone over 24 h at 20 °C. The reaction
was quenched by addition of 0.05 M Na2HPO4–NaH2PO4 buffer solution (pH
7.5) and the crude RNA was desalted on a column packed with SephadexÒ G-
25. The fully deprotected RNA was purified by preparative anion-exchange
HPLC (Waters AP-2 column packed with TSK SuperQÒ-5PW resin eluted with a
linear gradient of NaBr (50–650 mM) in sterile 20 mM Na2HPO4–NaH2PO4
buffer solution (pH 7.5), containing EDTA (50 lM) and 10% of CH3CN over
64 min). Fractions containing the desired product were desalted on a column
packed with SephadexÒ G-25.