Site-specific incorporation of 5-methylaminomethyl-2-thiouridine and 2-thiouridine(s) into RNA sequences

Article abstract of DOI:10.1016/j.tetlet.2011.12.079

We present a reproducible protocol for the site-specific incorporation of 5-methylaminomethyl-2-thiouridine (mnm⁵s²U) into a model RNA fragment and, together with 2-methyladenosine (m²A), into the native sequence of the Es

Full text of DOI:10.1016/j.tetlet.2011.12.079

Tetrahedron Letters 53 (2012) 1214–1217
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Site-specific incorporation of 5-methylaminomethyl-2-thiouridine and
2-thiouridine(s) into RNA sequences
Grazyna Leszczynska a, Jakub Pie˛ta a, Piotr Leonczak a, Agnieszka Tomaszewska b, Andrzej Malkiewicz a,
⇑
a Institute of Organic Chemistry, Technical University of Lodz, 90-924 Lodz, Zeromskiego 116, Poland
b Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, 90-363 Lodz, Sienkiewicza 112, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
We present a reproducible protocol for the site-specific incorporation of 5-methylaminomethyl-2-thio-
uridine (mnm5s2U) into a model RNA fragment and, together with 2-methyladenosine (m2A), into the
native sequence of the Escherichia coli tRNAGlu2 anticodon arm (E. coli ASLGlu2). This approach is also uti-
lized for the synthesis of oligomers modified with multiple 2-thiouridines.
Received 7 October 2011
Revised 9 December 2011
Accepted 20 December 2011
Available online 29 December 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
tRNAs modified components
Solid-phase synthesis
Oligoribonucleotides
2-Thiouridine (s2U)
5-Methylaminomethyl-2-thiouridine
(mnm5s2U)
2-Methyladenosine (m2A)
2-Thiouridine (s2U), its 20-O-methyl analog (s2Um), and vari-
ously 5-substituted 2-thiouridines (s2U⁄) are components of cyto-
solic and mitochondrial tRNAs sequences1 and control
fundamental cellular processes,2 while their absence results in
serious diseases.3 The s2U/s2U⁄ modified tRNA fragments have
been utilized, among others, in mutually related model studies
on the impact of post-transcriptional modifications on RNA confor-
mation/tertiary structure dynamics,4 and on the effectiveness of
homo- (RNA–RNA) and heterotropic (RNA–protein) biopolymer
interactions with other partners of the ribosomal decoding
machinery,5,6 or of the HIV-1 RNA reverse transcription process.7,8
The superior hybridization and base discrimination abilities of
oligoribonucleotides modified with s2U/s2Um compared to non-
modified oligomers9,10 has encouraged their use in ‘microarray’ con-
struction and probing, in this way, the RNA 2-D structure,11 and also
for the development of potential therapies based on selective gene
silencing.12
the type of 5-substituents.13 Numerous protocols for the incor-
poration of s2U/s2U⁄ into oligomers by solid-phase synthesis
have been reported,10,11,14,16–20 however none of them offers a
general approach.
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)20
and 20-O-methyl-2-thiouridine(s) (0.02 M solution of iodine in
pyridine-H2O).14
5-Methylaminomethyl-2-thiouridine (mnm5s2U) reveals signifi-
cant sensitivity to the oxidation step. The mnm5s2U modified se-
quence of Escherichia coli ASLLys was prepared using a 1 M solution
of tert-butyl hydroperoxide as oxidant in acetonitrile.18 We were
not able to repeat this synthesis, however, and a similar observation
was reported by another laboratory.21
In this communication, we present a reliable approach for the
incorporation of mnm5s2U into synthetic RNA sequences. The util-
ity of this protocol has been confirmed by the synthesis of model
oligomers modified with mnm5s2U, with site-specifically located
Polymer-supported synthesis of oligoribonucleotides by phos-
phoramidite chemistry necessitates a P(III) to P(V) oxidation
step. Under typical oxidizing conditions, 2-thiouridine and its
5-substituted derivatives undergo oxidation into the 2-oxo-
analogs and/or oxidative desulfurization into the respective 1-
s2U unit(s) and the native sequence of E. coli ASLGlu2 (mnm5s2U34
,
m2A37). It is noteworthy, that the mnm5s2U is located only in the
tRNA sequences from prokaryotes. Thus, the synthetic E. coli ASL-
Glu2
b-
D
-ribofuranosyl-1H-pyrimidin-4-one.13–15 The scope and nature
and ASLLys fragments containing mnm5s2U34 could be em-
of these processes depend not only on the oxidant, but also on
ployed in the automated system for the selection of potential
therapeutics against antibiotic-resistant E. coli strains.22
Considering the previously cited literature,13,14,20 the stability of
a monomeric unit derived from mnm5s2U (1) in a 0.02 M solution
⇑
Corresponding author. Tel.: +48 42 631 31 50; fax: +48 42 636 55 30.
E-mail address: ajmalk@p.lodz.pl (A. Malkiewicz).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.12.079

G. Leszczynska et al. / Tetrahedron Letters 53 (2012) 1214–1217
1215
Scheme 1. The products of oxidation and/or oxidative desulfurization of mnm5s2U derivative 1.
Table 1
Sensitivity of 1 toward oxidants (8 equiv) under various reaction conditions (concentration, solvent, time of reaction)
Entry
Conditions
Time (min)
Producta (%)
Recovery of 1 (%)
2
3
1
2
3
4
5
6
0.25 M t-BuOOH in toluene
0.5 M t-BuOOH in toluene
1 M t-BuOOH in toluene
0.05 M t-BuOOH in MeCN
0.02 M I2 in THF/H2O/pyridine 7/1/2 (v/v/v)
0.02 M I2 in MeCN/H2O/pyridine 7/1/2 (v/v/v)
40
10
2
2
2
2
5
5
nd
50
70
2
2
2
25
nd
nd
90
85
84
60
40
20
2
a
Yield of isolated product. nd = not detected.
Table 2
MALDI-TOF data for the synthesized oligoribonucleotides and yield of the products synthesized on a 0.2 lmol scale
Oligonucleotide Sequences
Calcd. [MꢀH]ꢀ
Found
Yieldb [OD260
]
a
50-UUUUUUUU-30
2385.2
2707.2
2739.2
3668.4
5422.8
2385.8
2709.1
2739.7
3671.0
5422.9
7.8
7.9
5.2
4.2
4.0
50-UUUUUUUs2UU-30
50-Us2UUUs2UUUs2UU-30
50-UUUUUUUmnm5s2UUUUU-30
50-CCGCCCUmnm5s2UUCm2ACGGCGG-30
a
Reference sequence.
b
Yield of oligoribonucleotide was determined by measurement of the UV absorbance at 260 nm, using a 1 cm path length quartz cuvette.
Figure 1. Structures of the fully protected 30-O-phosphoramidites of s2U (4), mnm5s2U (5) and m2A (6).
of iodine in THF/H2O/pyridine and, alternatively, in acetonitrile/
H2O/pyridine, as well as in the presence of various concentrations
of tert-butyl hydroperoxide in acetonitrile or toluene, was tested
(Scheme 1), with the results listed in Table 1. Compound 1 was ob-
tained as a side-product during the preparation of fully protected
mnm5s2U 30-O-phosphoramidite.23 Under oxidizing conditions,
derivative 1 undergoes transformation into 2-oxo analog 3 and/
or the product of oxidative desulfurization 2 (Scheme 1). Both
compounds 2 and 3 were synthesized independently, and their
structures were confirmed by standard methods.24,25
desulfurization 2, and its yield was notably higher in more polar
solvents (Table 1, entries 5 and 6).
Nucleoside 1 remained unchanged for a relatively long time in a
0.25 M solution of tert-butyl hydroperoxide in toluene, but at high-
er oxidant concentrations the yield of 2 increased (Table 1, entries
1–3). It is noteworthy that even a dilute solution of tert-butyl
hydroperoxide in acetonitrile gave 3 as the sole product with a
notable yield of 25% (Table 1, entry 4).
The results of these model studies favor a dilute solution of tert-
butyl hydroperoxide in toluene as the most suitable oxidant for so-
lid-phase supported incorporation of mnm5s2U into oligoribonu-
cleotides using phosphoramidite methodology.
Treatment of 1 with 0.02 M I2 in acetonitrile/H2O/pyridine or in
THF/H2O/pyridine solution gave only the product of oxidative

1216
G. Leszczynska et al. / Tetrahedron Letters 53 (2012) 1214–1217
Some aspects of the mechanism of s2U/s2U⁄ oxidation/desulfur-
E. coli tRNAGlu2 anticodon arm bearing mnm5s2U and m2A have been
elaborated. The same procedure can be used for the synthesis of
tRNA fragments modified with mnm5s2U in the presence of t6A, as
well as for the incorporation of several 2-thiouridines into oligoribo-
nucleotide sequences.
ization have been studied, but only the pathway involving selective
desulfurization of s2U upon treatment with 2-phenylsulfonyloxaz-
iridine has been investigated in detail and discussed so far.15 Based
on literature and the results of model studies, the degrees of sensi-
tivity of s2U/s2U⁄ to oxidizers could be arranged in the ascending or-
der as follows: m5s2U < mcm5s2U 6 s2U ꢁ mnm5s2U < mo5s2U.
This ranking may reflect the tendency of s2U derivatives toward
enolization. A shift toward the enol form has been recorded for
mo5U26 and mo5s2U,27 while mnm5s2U crystallizes as a zwitter-
ion.28 Protonation of the side-chain amine function of mnm5s2U un-
der neutral conditions was postulated, and, based on pKa < 8
estimated for mnm5s2U, it can be expected that a considerable frac-
tion of this nucleoside is ionized under physiological conditions.29
Blocking the side-chain amine function of 1 with a strong elec-
tron-withdrawing CF3C(O)-group can induce a charge distribution
in the heterocyclic moiety similar to that originating from the
above-mentioned protonation process. This effect can be responsi-
ble for the enhanced tendency of the mnm5s2U-derived monomeric
unit to enolization, and consequently to oxidation/oxidative desul-
furization processes during the P(III) to P(V) oxidation step. The
substantial reduction of tert-butyl hydroperoxide degradation to
the active oxygen species in toluene compared to CH2Cl2,30 and
the diminished tendency of 1 to ionization in a non-polar aprotic
solvent would be expected to decrease the sensitivity of nucleoside
1 to oxidation.
Acknowledgment
This work was supported by Grant 1306/B/H03/2011/40 from
the National Science Centre, Poland.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2011.12.079.
References and notes
1. Jühling, F.; Mörl, M.; Hartmann, R. K.; Sprinzl, M.; Stadler, P. F.; Pütz, J. Nucleic
Acids Res. 2009, 37, D159.
2. Agris, P. F. EMBO Rep. 2008, 9, 629.
3. Suzuki, T.; Suzuki, T.; Wada, T.; Saigo, K.; Watanabe, K. EMBO J. 2002, 21, 6581.
4. Motorin, Y.; Helm, M. Biochemistry 2010, 49, 4934.
5. Agris, P. F.; Vendeix, F. A. P.; Graham, W. D. J. Mol. Biol. 2007, 366, 1.
6. Giegé, R.; Lapointe, J. In Grosjean, H., Ed.; DNA and RNA Modification Enzymes:
Structure, Mechanism, Function and Evolution; Landes Bioscience: Austin, TX,
2009; p 475.
7. Bilbille, Y.; Vendeix, F. A. P.; Guenther, R.; Malkiewicz, A.; Ariza, X.; Vilarrasa, J.;
Agris, P. F. Nucleic Acids Res. 2009, 37, 3342.
8. Graham, W. D.; Barley-Maloney, L.; Stark, C. J.; Kaur, A.; Stolyarchuk, K.; Sproat,
B.; Leszczynska, G.; Malkiewicz, A.; Safwat, N.; Mucha, P.; Guenther, R.; Agris, P.
F. J. Mol. Biol. 2011, 410, 698.
9. Kumar, R. K.; Davis, D. R. Nucleosides Nucleotides 1997, 16, 1469.
10. Okamoto, I.; Seio, K.; Sekine, M. Bioorg. Med. Chem. 2008, 16, 6034.
11. Carlucci, M.; Kierzek, E.; Olejnik, A.; Turner, D. H.; Kierzek, R. Biochemistry
2009, 48, 10882.
12. Sipa, K.; Sochacka, E.; Kazmierczak-Baranska, J.; Maszewska, M.; Janicka, M.;
Nowak, G.; Nawrot, B. RNA 2007, 13, 1301.
13. Sochacka, E. Nucleosides Nucleotides Nucleic Acids 2001, 20, 1871.
14. Okamoto, I.; Seio, K.; Sekine, M. Tetrahedron Lett. 2006, 47, 583.
15. Sochacka, E.; Fratczak, I. Tetrahedron Lett. 2004, 45, 6729.
16. Kumar, R. K.; Davis, D. R. J. Org. Chem. 1995, 60, 7726.
17. Diop-Frimpong, B.; Prakash, T. P.; Rajeev, K. G.; Manoharan, M.; Egli, M. Nucleic
Acids Res. 2005, 33, 5297.
18. (a) Davis, D. R.; Bajji, A. C. Methods Mol. Biol. 2005, 288, 187; (b) Sundaram, M.;
Crain, P. F.; Davis, D. R. J. Org. Chem. 2000, 65, 5609.
19. Hayakawa, Y.; Kataoka, M. J. Am. Chem. Soc. 1998, 120, 12395.
20. Eshete, M.; Marchbank, M. T.; Deutscher, S. L.; Sproat, B.; Leszczynska, G.;
Malkiewicz, A.; Agris, P. F. Protein J. 2007, 26, 61.
A solution of 0.25 M t-BuOOH in toluene was successfully ap-
plied to the solid-phase supported synthesis (phosphoramidite
methodology) of model oligoribonucleotides modified with
mnm5s2U or s2U unit(s) and double modified E. coli ASLGlu2, with
site-specifically located mnm5s2U34 and m2A37 (Table 2).
The 50-O-DMTr, 20-O-TBDMS protection strategy for the phos-
phoramidites of s2U (4), mnm5s2U (5) and m2A (6) was employed
(Fig. 1). Compounds s2U and mnm5s2U were synthesized according
to previously reported methods.31,32 2-Methyladenosine (m2A)
was prepared as follows: peracetylated guanosine was chlorinated
to give 2-amino-6-chloro-9-(20,30,50-tri-O-acetylribofuranosyl) pur-
ine,33 and this compound was converted into 2-iodoadenosine.34
Subsequently, 2-iodoadenosine was alkylated with trimethylalu-
minium in the presence of a palladium catalyst to give 2-methylad-
enosine (m2A).35 The side-chain amine function of m2A was
protected with a benzoyl group as previously described.36 Standard
procedures were employed for final protection of the 50-O- and 20-
O-functions with DMTr and TBDMS groups, respectively, as well as
for 30-O-phosphitylation.18,36,37 The analytical and spectral data of
monomer units 4–6 were identical with those reported in the
literature.36,38
21. Sproat, B. personal communication.
22. (a) Patent application: Guenther, R. H.; Newman, W. H.; Yenne, S. P.; Mitchell,
D.; Malkiewicz, A. WO/2008/064304A2, Chem. Abstr. 2008, 148, 577353s.; (b)
Patent application: Agris, P. F. US 2011/0098215A1, Chem. Abstr. 2713423.;
http://www.businesswire.com/news/home/20081008005360/en/Trana.
23. 50-O-(4,40-Dimethoxytrityl)-30-O-(tert-butyldimethylsilyl)-5-(N-
trifluoroacetyl)methylaminomethyl-2-thiouridine (1). Readily available 2-
thiouridine31 was converted into 20,30-O-isopropylidene-5-methylamino-
methyl-2-thiouridine via the 5-chloromethyluridine derivative, as described
The synthesis of the RNA sequences was conducted automati-
cally on a 0.2 lmol scale using commercial Pac(tac)-protected
previously.32a The heterobase side chain was efficiently protected with
a
phosphoramidites (ProligoÒ) and standard coupling chemistry with
the exception of t-BuOOH oxidation.39 Oligoribonucleotides were
deprotected according to a slightly modified Sproat protocol.40
The fully deprotected oligomers were precipitated and purified by
anion-exchange HPLC (see Supplementary data). The integrity of
the synthesized oligoribonucleotide sequences was confirmed by
analytical HPLC and MALDI-TOF mass spectrometry (Table 2).
In conclusion, under oxidizing conditions, the heterocyclic
moieties of 2-thiouridine derivatives undergo side-reactions, and
consequently the P(III) to P(V) oxidation is a critical step for the so-
lid-phase supported synthesis of oligoribonucleotides containing
s2U/s2U⁄. Protected 5-methylaminomethyl-2-thiouridine appeared
to be very sensitive to the standard oxidants used in the phospho-
ramidite methodology, however it remains intact in a dilute solution
of tert-butyl hydroperoxide in toluene. Based on these observations,
a reliable approach to the synthesis of model oligoribonucleotides
modified with mnm5s2U as well as the native sequence of the
trifluoroacetyl group and the 20,30-O-acetonide was removed under mild acidic
conditions to give an N-protected nucleoside. The 50-O-DMTr, 30-O-TBDMS and
50-O-DMTr, 20-O-TBDMS masked nucleosides were then synthesized according
to the standard procedure18 and separated by silica gel chromatography. 50-O-
DMTr, 30-O-TBDMS isomer: TLC, Rf = 0.71 (CH2Cl2/acetone 9:1 v/v); 1H NMR
(CDCl3, 250 MHz): d = ꢀ0.13 (s, 3H, SiCH3), 0.00 (s, 3H, SiCH3), 0.81 (s, 9H,
Si(CH3)3), 3.22 (s, 3H, NCH3), 3.45–3.59 (m, 4H, 5-CH2, H50, H5’’), 3.78 (s, 6H,
2xOCH3), 4.17–4.20 (m, 3H, H20, H30, H40), 6.66 (d, 1H, J = 2.5 Hz, H10), 6.80–
7.48 (m, 13H, Ar), 8.02 (s, 1H, H6); FAB MS m/z for C40F3H48N3O8SiS calcd 815,
found 814.3 [MꢀH]ꢀ. 50-O-DMTr, 20-O-TBDMS isomer: TLC Rf = 0.80 (CH2Cl2/
acetone 9:1 v/v); 1H NMR (CDCl3, 250 MHz): d = 0.12 (s, 3H, SiCH3), 0.14 (s, 3H,
SiCH3), 0.91 (s, 9H, Si(CH3)3), 3.21 (s, 3H, NCH3), 3.45–3.59 (m, 9H, 5-CH2,
2xOCH3, H50), 4.09–4.22 (m, 2H, H30, H40), 4.40–4.44 (m, 1H, H20), 6.75 (d, 1H,
J = 4.75 Hz, H10), 6.81–7.48 (m, 13H, Ar), 7.98 (s, 1H, H6).
24. 50-O-(4,40-Dimethoxytrityl)-30-O-(tert-butyldimethylsilyl)-1-(b-
5-(N-trifluoroacetyl)methylaminomethyl-4-pyrimidinone (2). Nucleoside 1 (50
mg, 0.061 mmol) was dissolved in a mixture of anhydrous CH2Cl2 (500 l) and
anhydrous pyridine (120 l) and then 0.2 M mCPBA in CH2Cl2 (602 l) was added.
The solution was stirred at room temperature for 4 h. After TLC analysis (Rf = 0.12;
CH2Cl2/acetone 9:1 v/v), the mixture was washed with 5% aq. NaHCO3 (500 l),
dried over MgSO4 and evaporated under reduced pressure. The crude product was
D-ribofuranosyl)-
l
l
l
l

G. Leszczynska et al. / Tetrahedron Letters 53 (2012) 1214–1217
1217
co-evaporated with anhydrous toluene and purified by silica gel column
34. Nair, V.; Young, D. A. J. Org. Chem. 1985, 50, 406.
35. Adah, S. A.; Nair, V. Tetrahedron 1997, 53, 6747.
36. Agris, P. F.; Malkiewicz, A.; Kraszewski, A.; Everett, K.; Nawrot, B.; Sochacka, E.;
Jankowska, J.; Guenther, R. Biochimie 1995, 77, 125.
37. Dahma, M. J.; Ogilvie, K. K. Methods Mol. Biol. 1993, 20, 81.
38. Malkiewicz, A.; Sochacka, E. Tetrahedron Lett. 1983, 24, 5391.
39. The oligoribonucleotides were synthesized on a Gene World DNA synthesizer
chromatography, using CHCl3 as the eluent. Yield 82%. 1H NMR (C6D6, 700 MHz):
d = 0.04 (s, 3H, SiCH3), 0.15 (s, 3H, SiCH3), 0.91 (s, 9H, Si(CH3)3), 2.93 (s, 3H, NCH3),
3.35 (s, 6H, 2xOCH3), 3.37 (dd, 1H, J = 3.5 Hz, 11.2 Hz, H5’’), 3.65 (dd, 1H, J = 3.5 Hz,
11.2 Hz, H50), 3.87 (d, 1H, J = 14.0 Hz, 5-CH2), 3.89 (d, 1H, J = 14.0 Hz, 5-CH2), 4.09–
4.11 (m, 1H, H40), 4.10 (dd, 1H, J = 3.5 Hz, 6.3 Hz, H30), 4.35–4.36 (m, 1H, H20), 5.14
(d, 1H, J = 6.3 Hz, H10), 6.85–7.65 (m, 13H, Ar), 7.81 (s, 1H, H6), 8.05 (br s, 1H, H2);
FAB MS m/z for C40F3H48N3O8Si calcd 783, found 784.5 [M+H]+; 782.3 [MꢀH]ꢀ.
25. 50-O-(4,40-Dimethoxytrityl)-30-O-(tert-butyldimethylsilyl)-5-(N-trifluoroacetyl)-
methylaminomethyluridine (3) was prepared according to the previously
described method36 and protected in the same way as the 2-thio analog 1.
TLC, Rf = 0.33 (CH2Cl2/acetone 9:1 v/v); 1H NMR (C6D6, 700 MHz): d = ꢀ0.09 (s,
3H, SiCH3); ꢀ0.01 (s, 3H, SiCH3), 0.77 (s, 9H, Si(CH3)3), 2.85 (s, 3H, NCH3), 3.34 (s,
6H, 2xOCH3), 3.44 (dd, 1H, J = 4.0 Hz, 11.25 Hz, H5’’), 3.47 (d, 1H, J = 14 Hz,
5-CH2), 3.63 (dd, 1H, J = 2.8 Hz, 11.25 Hz, H50), 3.74 (d, 1H, J = 14.0 Hz, 5-CH2),
4.13–4.15 (m, 1H, H40), 4.19 (dd, 1H, J = 4.2 Hz, 8.4 Hz, H30), 4.43–4.45 (m, 1H,
H20), 5.14 (d, 1H, J = 3.5 Hz, H10), 6.81–7.70 (m, 13H, Ar), 7.89 (s, 1H, H6); FAB
MS m/z for C40F3H48N3O9Si calcd 799, found 798.3 [MꢀH]ꢀ.
on a 0.2 lmol scale using 0.1 M acetonitrile solutions of commercially available
5’-O-DMTr and 2’-O-TBDMS phosphoramidites of U, A, C and G, with tac
protection of the side-chain amine functions (ProligoÒ). Typical rU/rG(tac)-
succinyl-CPG (ProligoÒ) supports were utilized. The detritylation reagent was a
3% solution of dichloroacetic acid in methylene chloride. Modified units as well
as A, C, G and U monomers were coupled in 10 M excess for 10 min in the
presence of
a 0.25 M solution of 5-(benzylmercapto)-1H-tetrazole in
acetonitrile. Capping was performed with tac anhydride (35 s) followed by
oxidation using 0.25 M t-BuOOH solution in toluene for 100 s (8 equiv). The
oxidizing agent was prepared from a commercial 6 M solution of t-BuOOH in
decane (AldrichÒ) and anhydrous toluene.
26. Hillen, W.; Egert, E.; Lindner, H. J.; Gassen, H. G.; Vorbrüggen, H. J. Carbohydr.
Nucleos. Nucleot. 1978, 5, 23.
27. (a) Malkiewicz, A.; Nawrot, B. Z. Naturforsch. 1987, 42, 355; (b) Gałdecki, Z.;
Łuciak, B.; Malkiewicz, A.; Nawrot, B. 2nd Symposium of Bioorganic Chemistry
and Biophysics, Karpacz, Poland, 1989.
40. (a) Sproat, B. Methods Mol. Biol. 2005, 288, 17. (b) Upon completion of the
synthesis, the oligonucleotides were cleaved from the solid support and
deprotected by treatment with concentrated NH3: 8 M methylamine in EtOH
(1:1 v/v) for 30 min at 65 °C. The TBDMS protecting groups were removed by
treatment with Et3Nꢂ3HF in DMF for 2.5 h at 60 °C. The oligomers were then
precipitated in isopropyl trimethylsilyl ether. The fully deprotected RNAs were
28. Hillen, W.; Egert, E.; Lindner, H. J.; Gassen, H. G. FEBS Lett. 1978, 94, 361.
29. Takai, K.; Yokoyama, S. Nucleic Acids Res. 2003, 31, 6383.
purified by semipreparative anion-exchange HPLC (Source 15Q 4.6/100PEÒ
,
30. Hill, J. G.; Rossiter, B. E.; Sharpless, K. B. J. Org. Chem. 1983, 48, 3607.
31. Vorbrüggen, H.; Strehlke, P. Chem. Ber. 1973, 106, 3039.
32. (a) Ikeda, K.; Tanaka, S.; Mizuno, Y. Chem. Pharm. Bull. 1975, 23, 2958; (b)
Malkiewicz, A.; Nawrot, B.; Sochacka, E. Z. Naturforsch. 1987, 42b, 360.
33. Robins, M. J.; Uznan´ ski, B. Can. J. Chem. 1981, 59, 2601.
eluted with a linear gradient of NaClO4 (0.01–0.6 M) in sterile 50 mM Tris HCl
buffer at pH 7.6 containing 50 lM EDTA and 10% of acetonitrile for 55 min).
The desired products were desalted on a C18 cartridge (Sep-PakÒ, Waters) and
the integrity of the synthesized oligoribonucleotides was confirmed
unambiguously by analytical HPLC and MALDI-TOF mass spectrometry.