Chemical approach for the study of the 'kissing complex' of Moloney murine leukaemia virus

Article abstract of DOI:10.1002/hlca.200890133

The replicationof Moloney murine leukaemia virus relies onthe formation of a stable homodimeric 'kissing complex' of a GACG tetraloop interacting through only two C·G base pairs flanked of 5′-adjacent unpaired adenosines A9. PreviousNMRinvestigations of a model stem loop 1 has not permitted to reveal the origin of this interaction. Therefore, with the aim of deeper comprehension of the phenomena, the model sequence 10 was prepared where position 9 has been substituted for a nucleoside offering a wider π-stacking. In this context, the wyosine phosphoramidite building block 2 was prepared and incorporated by adapting the conditions of the automated synthesis and developing original templated enzymatic ligation. However, no 'kissing interaction' has been observed for this model sequence 10 due to steric hindrance as confirmed by computational simulation. Consequently, several other model sequences, 18, 23 - 26, containing modified nucleosides were prepared. Finally, the importance of the cross-loop H-bond between G8 and G11 nucleobases was revealed by preparing a 18mer RNA hairpin 27, where the guanosine G8 has been substituted for inosine. The latter, which does not possess a C³ amino function compared to guanosine, is unable to form any 'kissing complex' demonstrating the importance of this secondary interaction in the formation of the complex.

Full text of DOI:10.1002/hlca.200890133

Helvetica Chimica Acta – Vol. 91 (2008)
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Chemical Approach for the Study of the ꢀKissing Complexꢁ of Moloney murine
leukaemia Virus
by Se´bastien Porcher1)
Laboratory of Nucleic Acid Chemistry, EPFL-BCH, CH-1015 Lausanne
(e-mail: sebastien.porcher@hotmail.fr)
The replicationof Moloney murine leukaemia virus relies onthe formationof a stable homodimeric
ꢀkissing complexꢁ of a GACG tetraloop interacting through only two C · G base pairs flanked of 5’-
adjacent unpaired adenosines A9. Previous NMR investigations of a model stem loop 1 has not permitted
to reveal the origin of this interaction. Therefore, with the aim of deeper comprehension of the
phenomena, the model sequence 10 was prepared where position9 has beensubstituted for a nucleoside
offering a wider p-stacking. In this context, the wyosine phosphoramidite building block 2 was prepared
and incorporated by adapting the conditions of the automated synthesis and developing original
templated enzymatic ligation. However, no ꢀkissing interactionꢁ has been observed for this model
sequence 10 due to steric hindrance as confirmed by computational simulation. Consequently, several
other model sequences, 18, 23 – 26, containing modified nucleosides were prepared. Finally, the
importance of the cross-loop H-bond between G8 and G11 nucleobases was revealed by preparing a
18mer RNA hairpin 27, where the guanosine G8 has been substituted for inosine. The latter, which does
not possess a C3 amino function compared to guanosine, is unable to form any ꢀkissing complexꢁ
demonstrating the importance of this secondary interaction in the formation of the complex.
1. Introduction. – During the NMR investigation of a model stem loop 1 from
retroviral RNA of Moloney murine leukaemia virus, Kim and Tinoco Jr. have observed
that a highly conserved GACG tetraloop formed a stable homodimeric ꢀkissing
complexꢁ through the formationof only two C · G base pairs [1] ( Fig. 1). By mutagenic
Fig. 1. Model sequence designed by Kim and
Tinoco Jr. [1] for revealing the existence of the
ꢀkissing complexꢁinteraction and used in the present
work
1
ˆ
Present address: 7 rue du Tivoli, F-36000 Chateauroux.
)
ꢂ 2008 Verlag Helvetica Chimica Acta AG, Zürich

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Helvetica Chimica Acta – Vol. 91 (2008)
approach, it has been demonstrated that the unpaired adenosines 5’-adjacent to the G ·
C base pair play an essential role for complex formation. This configuration may figure
out the codon/anticodon interaction, where adjacent hypermodified purines stabilize
the mRNA/tRNA interaction. The isolation of key interactions contributing signifi-
cantly to the stabilization of the ꢀkissing complexꢁ may help to evaluate the importance
of 3’-end adjacent purines of the tRNA during the translation process. However, the
parameters generating the ꢀunexpected stabilityꢁ (Tm ¼ 488) of the ꢀkissing complexꢁ
remaindifficult to isolate.
The complex has been spectroscopically investigated showing changes in the
secondary structure and the tertiary folding of RNA sequence during the dimerization
process. Deeper investigations, highlighting the essential interactions, require the
preparation of modified RNA sequences. Whereas the mutagenic approach is widely
used for long RNA sequences, it appears less practical for short RNA sequences,
typically 20mers long [2]. For this latter application, the chemical preparation of
synthetic oligonucleotides is a particularly fast and efficient technique especially
because it allows the incorporation of non-canonical nucleosides into the sequence.
However, previously inthe context of preparationof fully modified tRNAs, several 2 ’-
O-TOM nucleobase modified phosphoramidites were synthesized [3]. The variety of
their structures and the singularity of their electronic properties offer the opportunity
to use them as tools for revealing the importance of the adenosines 5’-adjacent to the
G · C base pair inthe stabilizationof the kissing complex.
During their investigations, Kim and Tinoco Jr. have observed that these
nucleobases exerted a strong electrostatic interaction around the base pairs, which
canprobably be related to p-stacking interaction, particularly favorable in the AC · G’
edifice. Consequently, we may suppose that the incorporation of an adjacent nucleoside
offering a potential higher surface area [4] should produce a more stable ꢀkissing
complexꢁ. For this aim, the A9 of the 18mer RNA sequence was substituted for wyosine,
which offers a wider p-stacking by its tricyclic structure (Fig. 2). This natural
nucleoside is one of a wide variety of structurally related nucleosides which are
presented in Fig. 2. The importance of this type of nucleoside in reading frame
maintenance of the PheUUU codon reading has been poorly investigated. Most of these
nucleosides have been synthesized (imG [5], mimG [5], yW [6], OHyW [7], imG-14
[8], imG2 [5]), but, with the exception of 4-desmethylwyosine [9], never transformed
into phosphoramidite building blocks or incorporated into RNA sequences2).
Here, we report the optimized synthesis of 2’-O-[(triisopropylsilyl)oxy]methyl (2’-
O-TOM) protected wyosine and its incorporation into the self-associating RNA
sequence from Moloney murine leukaemia virus. Due to its lability towards acid (t1/2
¼
95 s at pH 1 and 258) [10], wyosine itself is not fully compatible with the automated
assembly of RNA sequences3) and can only be incorporated as the last nucleotide at
the 5’-end. Therefore, its presence at an interior position within a RNA sequence
requires the enzymatic ligation of the 5’-wyosine ending sequence with another strand
(Scheme 1).
2
)
)
No synthesis of the corresponding phosphoramidite building block has been reported.
Within each cycle of the assembly, a detritylation reaction under acidic conditions (e.g., 3%
3
CHCl2COOH inClCH CH2Cl [11]) is carried out.
2

Helvetica Chimica Acta – Vol. 91 (2008)
1221
Fig. 2. Structure, names, and symbols of wyosine derivatives (R ¼ ribose moiety or backbone) according
to http://medlib.med.utah.edu/RNAmods
Scheme 1. Retrosynthetic Approach for the Preparation of the Wyosine (Y, imG) Containing Sequence 10
and Relying on the Enzymatic Phosphorylation and Ligation of Chemically Prepared Oligonucleotides
(12 and 13)
2. Results and Discussion. – 2.1. Synthesis of Phosphoramidite Building Block 2. The
first attempt to introduce the 2’-O-TOM group under established conditions [11] into
the unprotected nucleotide wyosine [12][13] resulted in depurination and the
formation of several other unidentified products. Therefore, we decided to prepare
the phosphoramidites 2 from the 2’-O-TOM and 5’-O-dimethoxytrityl ((MeO)2Tr )
protected guanosine 3 [14] (Scheme 2).
Under the conditions reported for the formation of 4-desmethylwyosine from
guanosine (NaH/bromoacetone, DMF, À 158 [8]), partial decompositionof 3 was
observed. By employing milder conditions (K2CO3 instead of NaH, and addition of KI)
this problem was avoided, but now an efficient N(4)-alkylation of the product 4 was

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Helvetica Chimica Acta – Vol. 91 (2008)
1223
observed4). Finally, 5 was prepared intwo steps. N(1)-alkylationof 3 with K2CO3/
bromoacetone/KI in DMF at À 158, followed by work-up and extraction gave crude 4,
which then was transformed into the protected 4-desmethylwyosine 5 (56% yield) by
dehydrationand cyclizationwith 4-Š molecular sieves inCH 2Cl2 at 208. Removal of the
(MeO)2Tr group with CHCl2COOH inCH Cl2 gave 6 (85% yield). Treatment of this
2
intermediate with Ac2O inpyridine, followed by selective N(4)-deacetylationwith
MeOH/H2O/pyridine 1:1:1 resulted in the formation of the 3’,5’-di-O-acetylated
derivative 7 (91% yield). Efficient formation of the protected wyosine 8 (70% yield)
was achieved by N(5)-methylationwith CH I2/Et2ZninEt 2O at 48, according to [5].
2
The 5’-O-(tert-butyl)dimethylsilyl (5’-O-TBDMS) derivative 9 (89% yield) was
obtained after deacetylation of 8 with 10m NH3 inMeOH 5), concentration of the
reactionmixture and selective silylationwith TBDMS-Cl/imidazole inDMF/CH 2Cl2.
The corresponding fully protected wyosine phosphoramidite building block 2 (70%
yield) was obtained with 2-cyanoethyl diisopropylphosphoramidochloridite/iPr2NEt in
CH2Cl2 (Scheme 2).
2.2. Incorporation of Phosphoramidite 2 into RNA Sequences. Inpreliminary studies
with unprotected wyosine, its compatibility with RNA assembly and deprotection
conditions was investigated. In addition to the instability of wyosine towards acid [10]
5
and towards MeNH2 ), the nucleoside was found to be efficiently iodinated under the
standard oxidation conditions (I2/H2O/pyridine in THF [11])6). Oxidationwith
tBuOOH in MeCN according to [14], and a milder deprotection which involved
i
treatment of the solid support with Pr2NH inMeCN, followed by NH inMeOH,
3
however, were fully compatible with wyosine. Finally, employing these mild oxidation
conditions, the RNA sequence 13 (10mer) containing a 5’-terminal wyosine was
assembled from the phosphoramidite 2 and the 2’-O-TOM protected phosphoramidites
of uridine, N6-acetyladenosine, N4-acetylcytidine [11] and guanosine (without base
protecting group7)) [14]. After deprotectionwith NH 3 inMeOH and Bu NF · 3 H2O in
4
THF, the resulting product sequence was isolated in pure form by conventional anion
exchange (AE) chromatography according to [11] (Fig. 3,a). In Fig. 3,b, anESI mass
spectrum of the purified product 13 is shown. The other RNA and DNA sequences
were prepared under conventional conditions (see Exper. Part), and the 5’-terminal
wyosine containing sequence was enzymatically ligated to longer target sequences
(Schemes 3 and 4, Fig. 4).
2.3. Preparation of a 18mer Wyosine-Containing RNA Hairpin 10 and Dimerization
Studies. The 18mer RNA sequence 10, which contains a wyosine at position 9, was
prepared by ligation of the wyosine-containing 10mer fragment 11 and the unmodified
8mer RNA fragment 12. Ina first attempt, the 5 ’-OH group of the chemically prepared
RNA sequence 13 (Scheme 3) was phosphorylated with ATP inthe presence of the
4
)
)
HÀN(4) indesmethylwyosine has a p Ka value of 3.24 [13].
The attempted deacetylationof 8 with MeNH2 in EtOH resulted in ring-opening, presumably by
attack onC(9) [10].
Iodination is known to occur in a selective and efficient manner at position 7 and has been applied
for the preparationof wybutosine [7].
Complete deprotectionof the usually employed 2 ’-O-TOM protected N2-acetylguanosine
phosphoramidite requires stronger conditions.
5
6
7
)
)

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Helvetica Chimica Acta – Vol. 91 (2008)
Fig. 3. a) Ion-exchange HPLC trace (detection260 nm) of crude 13, and b) deconvulated ESI-MS (neg.
mode) analysis of the purified wyosine-containing sequence 13
Scheme 3. Description of the Preparation of Sequence 10 Relying on Direct Enzymatic T4 RNA Ligation
enzyme T4 PNK kinase. After thermal inactivation of the kinase, the phosphorylated
product sequence 11 was treated with various amounts of the complementary 8mer
RNA sequence 12 and ATP, in the presence of the enzyme T4 RNA ligase [15][16].
HPLC Analyses revealed the formation of a new product, but according to ESI-MS
analyses, it was not the desired hairpin sequence 10, but the cyclic homodimeric
sequence 10a (Scheme 3). This product is probably the result of autocomplexationof
the guanosine-rich RNA sequence 12, which prevents the required duplex formation
with the sequence 11. Therefore, the template-mediated ligationof 12 and 11 with ATP

Helvetica Chimica Acta – Vol. 91 (2008)
1225
Scheme 4. Description of the Preparation of Sequence 10 Relying on Modified Template Assisted T4
DNA Ligation
in the presence of the enzyme T4-DNA ligase8) (Scheme 4) was investigated. For this
reaction, a 16mer DNA template 14 was designed and prepared, which is fully
complementary to the 8mer RNA sequence 11, and which can form seven base pairs
with the 10mer RNA sequence 10. Since the tricyclic wyosine cannot form any
Watson – Crick interactions, an apurinic site derived from propane-1,3-diol 15 was
introduced at the opposing site of the template (Scheme 4 and Fig. 5). Incubation of the
two substrate sequences 11 and 12 (20 mm each) and the template 14 (30 mm) with ATP
(0.5 mm) and T4 DNA ligase (8 Weiss units) in an aqueous buffer (40 mm Tris · HCl,
2 mm MgCl2, 10 mm DTT, 0.5 mm ATP) at 378 resulted in clean and efficient (90%
conversion) formation of the product sequence 10 after 26 h (Fig. 4)9). According to
these conditions, a large-scale ligation was carried out, and 2.05 mg (18% yield) of the
RNA sequence 10 was isolated in pure form according to HPLC and ESI-MS (Fig. 4).
The wyosine containing 18mer RNA hairpin 10 was then analyzed by NMR imino
proton spectroscopy to detect the eventual formation of the corresponding ꢀkissingꢁ
complex (Fig. 6).
8
)
It is well-known that this DNA-processing enzyme catalyzes also the ligation of RNA sequences in
the presence of a DNA template [17]. It has been discovered that 2’-OMe-RNA templates or DNA/
2’-OMe-RNA hybride-templates are often superior and meanwhile, this reaction has been
optimized [18].
The first experiments were carried out according to the conventional one-pot protocol, by first
phosphorylating 13 in the presence of T4 PNK kinase (!11, followed by thermal inactivation (30
minat 60 8), cooling to 48, and addition of the other ligation components. However, under these
conditions, the kinase was not completely inactivated and a part of the product 13 was
phosphorylated. Inorder to completely avoid this side reaction, it was decided to isolate the
intermediate 11 by HPLC prior to carry out the ligationreaction.
9
)

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Helvetica Chimica Acta – Vol. 91 (2008)
Fig. 4. Ion-exchange HPLC trace (detection260 nm) of the template 14 assisted ligation reaction (T4
DNA ligase) of the wyosine-containing sequence 13 with the complementary strand 12 a) after 0 min and
b) after 24 hours. c) Plot against time of formation of product 10 and d) deconvulated ESI-MS (neg.
mode) analysis of the purified corresponding sequence.
Fig. 5. Design and structure of the ꢀapurinic site phosphoramiditeꢁ (propane-1,3-diol derivative) building
block 15
Surprisingly, no ꢀkissing interactionꢁ was observed even at lower temperature (158).
It has beenimmediately hypothesized that the extended p-system of wyosine was not
efficient since this nucleoside was incorporated adjacent to a C · G base pair instead of

Helvetica Chimica Acta – Vol. 91 (2008)
1227
Fig. 6. NMR Analysis of the imino H-atoms of a) the natural sequence 1 and b) the wyosine-containing
sequence 10 permitting the direct detection of the ꢀkissing complex interactionꢁ (scale inppm)
the naturally encountered A · U base pair. In order to evaluate the stacking energy
provided by wyosine, the four analogous substrate strands 16, 17, 19, 21 containing the
canonical nucleosides at the 5’-positionwere prepared and submitted to the same
ligation conditions. The course of these reactions was monitored by HPLC. The
efficiency of these reactions varied to a great extent. After 24 h reaction time, the
following conversion ratios were obtained as a function of the terminal nucleoside:
wyosine (11 ! 10, 90%)ꢀguanosine (16 ! 18, 48%) > adenosine (17 ! 1, 37%)
ꢀuridine (19 ! 20, 21%)ꢀcytidine (21 ! 22, 2%). These values correlated very well

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Helvetica Chimica Acta – Vol. 91 (2008)
with the DG values of stacking energies for canonical base pairs according to the
nearest neighbor model [19]. By linear extrapolation, the stacking energy for wyosine
to DGstacking ¼ À2.5 kcal molÀ1 (Fig. 7,b)10) canbe estimated. Therefore, the first
expectation on the favorable stacking stabilization provided by wyosine was confirmed.
Nevertheless, this result gave no information about the reason why the 18mer RNA
hairpin 10 offered no formation of a ꢀkissing complexꢁ. Further computational
investigations revealed that the steric hindrance of the wyosine nucleobase itself
prevented the connection of the two hairpins.
Fig. 7. a) Description of the template 12 assisted ligation reaction of specially designed RNA sequence
(X ¼ imG(Y), 11; A, 17; G, 16; U, 19; C, 21). b) Plot of the amount of the ligated products (1, 10, 18, 20,
22) relative to the sum of starting material 8mer 12 and 10mers of the respective (11, 16, 17, 19, 21) RNA
sequences.
In this extrapolation, the positive stacking energy of cytosine (DG ¼ þ0.4 kcal molÀ1) was
considered, but the intercept was not set to zero, assuming that a minimal energy maybe required
for structurationof the double helix [20].
10
)

Helvetica Chimica Acta – Vol. 91 (2008)
1229
To pursue the question about the ꢀunexpected stabilityꢁ of the ꢀkissing complexꢁ,
several analogues of the 18mer RNA sequence have been prepared by substituting the
A9 moiety for different modified nucleosides (Table). Therefore, nucleosides naturally
found at position 37 of the tRNA were incorporated: Guanosine 18, Methyl-1-
guanosine (m1G) 23, an dN6-methyladenosine 24 (m6A), but also some other
nucleosides found within the tRNA for which we have already prepared the 2’-O-
TOM phosphoramidites: inosine 25 and N2,N2-dimethylguanosine 26 (m2 G) [5]. Only
2
the adenosine derivatives provided sufficient p-stacking stabilisation energy, correlat-
ing well the fact that, in tRNAs, adenosine derivatives are adjacent to C · G base pairs,
whereas guanosine derivatives are found after G · C base pairs [21]. Thanks to the
NMR data accumulated by Kim and Tinoco Jr. attention was paid to the cross-loop
interactions, especially the one existing between the G8 and G11 nucleobases (Fig. 8).
Although this H-bond (NH2 ··· N7) may play a role inthe structurationand the
rigidificationof the loop, it could also provide anelectrophilic center. This delocaliza-
7
tionof the lone pair of N involved in the bonding with the adjacent H-atom could
stabilize significantly the G · C base pair, as already described for the interaction of the
guanine N7 N-atom with metallic cations (up to 70%) [22]. As a last example, the 18mer
RNA hairpin 27 was prepared, where the guanosine G8 was replaced by inosine. This
nucleoside does not possess, in contrast to guanine, a C3 amino function (Fig. 8). As
expected, this modified sequence did not form any ꢀkissing complexꢁ, demonstrating the
importance of this secondary H-bond in the formation of the complex. Unfortunately,
this observation led to the conclusion that this sequence was not suitable for modelling
of the codon/anticodon interaction.
Table. RNA Sequences Employed for Investigation of the ꢀKissing Complex Interactionꢁ
5’-GGUGGGAGXCGUCCCACC-3’
Xa)
Compound
m/z calc.
m/z foundb)
Interactionc)
A
1
5786
5854
5802
5816
5800
5787
5830
5786
5854
5802
5816
5800
5787
5830
Yes
No
No
No
Yes
No
No
imG (Y)
10
18
23
24
25
26
G
m1G
m6A
I
m2 G
2
a) Structure, name, and symbol of derivatives (R ¼ ribose moiety or backbone) according to http://
medlib.med.utah.edu/RNAmods. For clarity of the RNA sequence drawing, imG has been substituted for
the letter Y. b) LC/ESI-MS spectrometry (neg. mode) analysis. Detailed procedure in Exper. Part.
c) NMR Spectroscopy analysis. Detailed procedure in Exper. Part.
Conclusions. – The presented results prove the efficiency of the methodology,
combining chemical synthesis and template assisted enzymatic ligation, in the
preparationof modified oligonucleotides. This approach has allowed, for the first
time, the incorporation of the wyosine nucleoside and evaluation of the stacking
stabilizationprovided by its tricyclic moiety. Ultimately, all prepared modified RNA

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Helvetica Chimica Acta – Vol. 91 (2008)
Fig. 8. Design of modified sequence 27 (Inosine at position 8 instead of G occurring in the natural
sequence 1) preventing formation of the ꢀkissing complex interactionꢁ
sequences give new insight into the interactions contributing to the formation of the
ꢀkissing complexꢁ. In this context, these studies have demonstrated that secondary H-
bonds (NH2 ··· N7) could contribute significantly to the stabilization of inter-strand
connections. Although these cross-strand bonds do not exist in the codon/anticodon
interaction, it can be expected that secondary H-bonds may play an important role in
the strength of mRNA/tRNA complex formation [23].
I am grateful to EPFL and to the Swiss National Science Foundation (Grant Nr. 2000-06890) for their
generous support. I would like to thank Prof. S. Pitsch, my supervisor of thesis, for introducing me to the
´
RNA world. I am grateful to Dr. Cyrille Denarie for his support inthe ligationexperiments, Prof. Ursula
Rçthlisberger and Dr. Christian Gossens for their support inthe computational simulationof the wyosine-
containing sequence. Dr. Philipp Wenter is also thanked for his continuous support in carrying out NMR
experiments.
Experimental Part
1. General. Reagents and solvents (highest purity) from various suppliers, used without further
purification; [(triisopropylsilyl)oxy]methyl chloride (¼ TOM-Cl) was prepared according to [5].
Workup implies distributionof the reactionmixture betweenCH 2Cl2 and sat. aq. NaHCO3 soln., drying
of the org. layer (MgSO4), and evaporation. TLC: precoated SiO2 plates from Merck, stained by dipping
into a soln. of anisaldehyde (10 ml), H2SO4 (10 ml), and AcOH (2 ml) in EtOH (180 ml) and subsequent
heating with a heat-gun. CC (column chromatography): SiO2 60 (230 – 400 mesh) from Fluka. The eluent
was systematically conditioned with few percent of Et3N (Fluka). ESI-MS (positive mode): SSQ 710
(Finnigan), measurements in MeCN/H2O/AcOH 50 :50 :1. Py ¼ pyridine, r.t. ¼ room temperature (ca.
208), DMAP ¼ N,N-dimethylpyridin-4-amine, TMS-Cl ¼ Me3Si-Cl, DME ¼ Dimethoxyethane.
The usual 2’-O-TOM protected RNA phosphoramidites according to [11] and DNA phosphor-
amidites from Glen Research; size exclusioncartridges: NAP-10 columns from Pharmacia, elution
according to the manufacturerꢁs instruction; Sepak cartridges from Waters. Sterile H2O from B. Braun
Medical AG. Anion exchange-HPLC: DNAPAC PA-100 (9.0 ꢁ 250 mm; Dionex), flow 2.5 ml/min;
eluent A: 12 mm Tris · HCl (pH 7.4), 6m urea; eluent B: 12 mm Tris · HCl (pH 7.4), 0.5m NaClO4, 6m
urea; detectionat 260 nm, elutionat 85 8. ESI-MS (pos. mode): SSQ 710 (Finnigan), measurements in
MeCN/H2O/AcOH 50 :50 :1. LC/ESI-MS (neg. mode): Q-Tof-Ultima (Micromass/Waters) coupled to
Cap-LC (Waters), injection: 2 ml aq. sample (c(RNA) ¼ 2.5 mm, c(EDTA) ¼ 1 mm); chromatography on

Helvetica Chimica Acta – Vol. 91 (2008)
1231
Xterra RP-C18 column( Waters, 5 mm, 0.32 ꢁ 50 mm; flow 8 ml/min, eluent A: 25 mm aq. Me2NBu · H2CO3
(pH 8.4); eluent B: MeCN, elutionat 60 8, sheath flow 25 ml/min(MeCN)); gradient A ! A/B 1:1
(15 min.); deconvolution by MaxEnt1 software. Ligation buffer for T4 DNA ligase (Labforce) mediated
ligation40 m m Tris · HCl (Sigma), 2 mm MgCl2 (Fluka), 10 mm DTT (1,4-Dithio-dl-threitol) (Fluka),
0.5 mm ATP (Fluka). Same buffer employed for T4 polynucleotide kinase mediated phosphorylation
(NewEnglandBiolabs). NMR-spectroscopy: Bruker 400 MHz (1H: 400 MHz, 13C: 100 MHz, 31P:
162 MHz). Chemical shift d in ppm, relative to external standards (1H an d13C: Me4Si, 31P: 85% aq.
H3PO4); coupling constants J inHz; multiplicities ( 13C) according to DEPT-spectra. NMR Experiments
were carried out ona Bruker AV 600 MHz spectrometer equipped with a 5 mm TX1-HCN cryogenic
probe with z-gradients. The RNA samples (amounts determined spectrophotometrically) were dissolved
in0.3 ml potassium arsenate buffer (25 m m, pH 7.0) inH 2O/D2O 9 :1. Restricted volume Shigemi tubes
were used for all experiments. 1D 1H-NMR Spectra were recorded by combining a water flip-back pulse
and 3-9-19 WATERGATE for suppression of the water signal.
2. Preparation of the Wyosine Phosphoramidite Building Block. N4-Desmethyl-5’-O-(4,4’-dimethoxy-
trityl)-2’-O-{[(triisopropylsilyl)oxy]methyl}wyosine (¼ 3-[5-O-[Bis(4-methoxyphenyl)phenylmethyl]-2-
O-({[tris(1-methylethyl)silyl]oxy}methyl)-b-d-ribofuranosyl]-3,5-dihydro-6-methyl-9H-imidazo[1,2-
a]purin-9-one; 5). A soln. of 3 (1.00 g, 1.3 mmol) prepared according to [14] in DMF (12 ml) was treated
with K2CO3 (197 mg, 1.4 mmol) and stirred at r.t. for 1 h. This suspension was cooled to À 158 and
successively treated with KI (107 mg, 0.6 mmol) and bromoacetone (178 mg, 1.3 mmol), and stirred for
8 h. The mixture was poured ona cold 10% soln. of AlCl 3 (150 ml) and AcOEt (150 ml). The aq. layer
was extracted twice with each 150 ml AcOEt, and the combined org. layers were dried over MgSO4.
After evaporationof the solvent, the yellow oily residue 4 was redissolved inDMF (10 ml) and stirred
overnight on K2CO3 (197 mg, 1.4 mmol) or 4-Š molecular sieve. Workup and CC (SiO2 (30 g); CH2Cl2/
acetone 0 :1 ! 2 :8) gave 5 (588 mg, 56%) as greenoil and 3 (199 mg, 20%). Rf (CH2Cl2/MeOH 9 :1)
0.53. 1H-NMR (400 MHz, CDCl3): 1.04 – 1.11 (m, iPr3Si); 2.26 (s, MeÀC(6)); 3.15 (br. s, HOÀC(3’)); 3.35
(dd, J ¼ 3.4, 10.3, HÀC(5’)); 3.51 (dd, J ¼ 3.2, 10.3, H’ÀC(5’)); 3.77, 3.79 (2s, 2 MeO); 4.29 (dd, J ¼ 2.6,
3.2, HÀC(4’)); 4.64 (dd, J ¼ 2.2, 4.4, HÀC(3’)); 4.95 (d, J ¼ 4.7, 1 H, OCH2O); 5.08 (t, J ¼ 5.7, HÀC(2’));
5.13 (d, J ¼ 4.7, 1 H, OCH2O); 6.02 (d, J ¼ 6.2, HÀC(1’)); 6.80 – 6.82 (m, 4 arom. H); 7.26 – 7.49 (m, 9
arom. H, HÀC(7)); 7.81 (br. s, HÀC(2)), 10.11 (br. s, HÀN(5)). 13C-NMR (100 MHz, CDCl3): 11.3 (q,
MeÀC(6)); 12.2 (d, Me2CH); 18.2 (q, Me2CH); 55.7 (q, 2 MeO); 64.0 (t, C(5’)); 71.7 (d, C(4’)); 81.7 (d,
C(2’)); 84.4 (d, C(3’)); 86.7 (s, arom. C); 86.9 (d, C(1’)); 91.4 (t, OCH2O); 104.6 (d, C(7)); 113.4 (d, arom.
C); 117.7 (s, C(9a)); 127.3, 128.3, 128.8, 130.7 (4d, arom. C); 136.1 (s, arom. C); 137.9 (d, C(2)); 145.1 (s,
C(6)); 145.9 (s, C(4a)); 150.2 (s, C(3a)); 152.4 (s, C(9)); 158.9 (s, arom. C). ESI-MS: 810.30 (100, [M þ
H]þ).
N4-Desmethyl-2’-O-{[(triisopropylsilyl)oxy]methyl}wyosine
(¼ 3,5-Dihydro-6-methyl-3-[2-O-
({[tris(1-methylethyl)silyl]oxy}methyl)-b-d-ribofuranosyl]-9H-imidazo[1,2-a]purin-9-one; 6). A soln. of
5 (823 mg, 1.0 mmol) inCH 2Cl2 (10 ml) was treated with CHCl2COOH (0.4 ml, 6.0 mmol) and stirred at
r.t. After 15 min, the mixture was quenched with MeOH (2.6 ml) on a 10% cold soln. of NH4HCO3
(50 ml) and CH2Cl2 (50 ml). The aq. layer was extracted twice with each 150 ml of AcOEt, and the
combined org. layers were washed with brine. CC (SiO2 (15 g); hexane/AcOEt 1:1 ! 0 :1) afforded 6
(431 mg, 85%). Light greenfoam. TLC (CH 2Cl2/MeOH 9 :1): Rf 0.50. 1H-NMR (400 MHz, CDCl3):
i
1.01 – 1.07 (m, Pr3Si); 2.30 (br. s, MeÀC(6)); 3.05 – 3.60 (br. s, HOÀC(3’), HOÀC(5’)11)); 3.83 (d, J ¼
11.7, HÀC(5’)); 4.01 (d, J ¼ 12.5, HꢁÀC(5’)); 4.29 (br. s, HÀC(4’)); 4.57 (d, J ¼ 4.2, 1 H, OCH2O); 4.88 –
4.95 (m, HÀC(2’), OCH2O); 5.08 (d, J ¼ 4.6, HÀC(3’)); 5.89 (d, J ¼ 7.3, HÀC(1’)); 7.28 (s, HÀC(7));
7.76 (br. s, HÀC(2)); 11.50 (br. s, HÀN(5)11). 13C-NMR (100 MHz, CDCl3): 11.3 (q, MeÀC(6)); 12.2 (d,
Me2CH); 18.1 (q, Me2CH); 63.7 (t, C(5’)); 72.2 (d, C(3’)); 81.2 (d, C(2’)); 87.2 (d, C(4’)); 89.8 (d, C(1’));
90.9 (t, OCH2O); 104.5 (d, C(7)); 118.1 (s, C(9a)); 126.5 (s, C(6)); 139.4 (d, C(2)); 145.4 (s, C(4a)); 148.6
(s, C(3a)); 152.1 (s, C(9)). ESI-MS: 508.32 (100, [M þ H]þ)11).
3’,5’-Di-O-acetyl-N4-desmethyl-2’-O-{[(triisopropylsilyl)oxy]methyl}wyosine (¼ 3-[3,5-Di-O-acetyl-
2-O-({[tris(1-methylethyl)silyl]oxy}methyl)-b-d-ribofuranosyl]-3,5-dihydro-6-methyl-9H-imidazo[1,2-a]-
purin-9-one; 7). A soln. of 6 (360 mg, 0.7 mmol) inpyridine (5 ml) was treated with Ac 2O (0.4 ml,
11
)
Confirmed by H2O exchange with D2O.

1232
Helvetica Chimica Acta – Vol. 91 (2008)
6.0 mmol) and stirred at r.t. for 2 h. After workup, the solvent was removed under reduced pressure and
the mixture was stirred for 2 h inpyridien/H 2O/MeOH 1:1:1 (6 ml). Workup, evaporation, and
coevaporation with benzene (2 ml) gave 7 (382 mg, 91%). Yellow foam. No further purificationwas
1
i
necessary. Rf (CH2Cl2/MeOH 9 :1) 0.42. H-NMR (400 MHz, CDCl3): 0.90 – 1.10 (m, Pr3Si); 2.09, 2.20
(2s, 2 MeCO); 2.38 (s, MeÀC(6)); 4.45 – 4.47 (m, CH2(5’)); 4.50 – 4.60 (m, HÀC(4’)); 4.84 (d, J ¼ 4.6, 1 H,
OCH2O); 4.89 (d, J ¼ 4.7, 1 H, OCH2O); 5.23 (t, J ¼ 5.6, HÀC(2’)); 5.53 (dd, J ¼ 3.3, 5.0, HÀC(3’)); 6.01
(d, J ¼ 6.3, HÀC(1’)); 7.38 (d, J ¼ 5.5, HÀC(7)); 7.74 (br. s, HÀC(2)); 9.77 (br. s, HÀN(5)). 13C-NMR
(100 MHz, CDCl3): 11.5 (q, MeÀC(6)); 12.1 (d, Me2CH); 18.0 (q, Me2CH); 21.2 (q, MeCO); 21.3 (q,
MeCO); 64.4 (t, C(5’)); 72.2 (d, C(3’)); 76.0 (d, C(2’)); 80.7 (d, C(4’)); 88.4 (d, C(1’)); 89.9 (t, OCH2O);
104.7 (d, C(7)); 118.0 (s, C(9a)); 125.6 (s, C(6)); 137.9 (d, C(2)); 146.1 (s, C(4a)); 149.9 (s, C(3a)); 152.3 (s,
C(9)); 170.6, 171.6 (2s, MeCO). ESI-MS: 592.80 (100, [M þ H]þ).
3’,5’-Di-O-acetyl-2’-O-{[(triisopropylsilyl)oxy]methyl}wyosine (¼ 3-[3,5-Di-O-acetyl-2-O-({[tris(1-
methylethyl)silyl]oxy}methyl)-b-d-ribofuranosyl]-3,4-dihydro-4,6-dimethyl-9H-imidazo[1,2-a]purin-9-
one; 8). The regioselective methylationof the tricyclic moiety was performed according to [5]. A light-
prevented soln. of CH2I2 (1.2 ml, 14.9 mmol) indry Et 2O (9 ml) was treated with a 1m soln. of Et2Znin
hexane (7.5 ml, 7.5 mmol) and stirred for 30 min at r.t. The clear soln. gave a cloudy white suspension
uponadditionof DME (0.8 ml, 7.5 mmol). After 30 min, the mixture was cooled to 4 8 and a soln. of
starting material 7 (0.424 g, 0.71 mmol) inCH 2Cl2 (1 ml) was quickly added. After 3 minof stirring, the
mixture was poured onanice cold 1 m aq. soln. of ammonium carbonate (50 ml) and CH2Cl2 (50 ml). The
aq. layer was extracted twice with each 150 ml of AcOEt, and the combined org. layers were washed with
(100 ml) of a 0.1m soln. of thiosulfate. Drying over MgSO4, evaporation, and CC (SiO2 (15 g); CH2Cl2/
acetone 99 :1 ! 95 :5 (þ1% Et3N)) gave 8 (300 mg, 70%). Yellow foam. Rf (CH2Cl2/MeOH 9 :1) 0.38.
1H-NMR (400 MHz, CDCl3): 1.00 – 1.08 (m, iPr3Si); 2.10, 2.23 (2s, 2 MeCO); 2.35 (s, MeÀC(6)); 4.20 (s,
MeN(4)); 4.23 (dd, J ¼ 2.6, 12.3, HÀC(5’)); 4.32 (dd, J ¼ 3.1, 12.1, H’ÀC(5’)); 4.51 (dd, J ¼ 2.2, 4.2,
HÀC(4’)); 4.90 (dd, J ¼ 4.6, 10.4, 1 H, OCH2O); 4.93 (dd, J ¼ 4.6, 10.4, 1 H, OCH2O); 4.97 (t, J ¼ 4.8,
HÀC(2’)); 5.42 (dd, J ¼ 2.1, 4.9, HÀC(3’)); 6.20 (d, J ¼ 6.8, HÀC(1’)); 7.47 (br. s, HÀC(7)); 7.92 (br. s,
HÀC(2)). 13C-NMR (100 MHz, CDCl3): 12.2 (d, Me2CH); 14.7 (q, MeÀC(6)); 18.2 (q, Me2CH); 21.1 (q,
MeCO); 21.2 (q, MeCO); 34.3 (q, MeN(4)); 63.8 (t, C(5’)); 71.6 (d, C(3’)); 72.1 (d, C(2’)); 77.2 (d, C(4’));
81.8 (d, C(1’)); 89.7 (t, OCH2O); 107.1 (d, C(7)); 117.3 (s, C(9a)); 134.2 (d, C(2)); 138.4 (s, C(6)); 140.1 (s,
C(3a)); 142.9 (s, C(4a)); 152.6 (s, C(9)); 170.4 (s, 2 MeCO). ESI-MS: 606.83 (100, [M þ H]þ).
5’-O-tert-Butyldimethylsilyl-2’-O-{[(triisopropylsilyl)oxy]methyl}wyosine (¼ 3-[5-O-[(1,1-Dimethyl-
ethyl)dimethylsilyl]-2-O-({[tris(1-methylethyl)silyl]oxy}methyl)-b-d-ribofuranosyl]-3,4-dihydro-4,6-di-
methyl-9H-imidazo[1,2-a]purin-9-one; 9). A methanolic (0.5 ml) soln. of 8 (81 mg, 0.13 mmol) was
treated with a sat. soln. of NH3 in MeOH (2 ml) and stirred for 3 h at r.t. After evaporation to dryness, the
residue was dissolved inCH 2Cl2/DMF 2 :1 (1.1 ml) and treated with imidazole (26 mg, 0.32 mmol). After
5 min, the mixture was cooled to 48 and TBDMS-Cl (26 mg, 0.15 mmol) was added, followed by stirring
at 48 for 1 h. Workup and CC (SiO2 (2 g); CH2Cl2/acetone 1:1 ! 0 :1) afforded 9 (85 mg, 98%). Light
1
t
yellow oil. Rf (CH2Cl2/MeOH 1:9) 0.75. H-NMR (400 MHz, CDCl3): 0.25, 0.26 (2s, BuMe2Si); 1.00 –
i
t
1.29 (m, Pr3Si, BuMe2Si); 2.47 (br. s, MeÀC(6)); 2.60 (br. s, HOÀC(3’)); 3.98 (dd, J ¼ 1.6, 11.5,
HÀC(5’)); 4.06 (dd, J ¼ 1.9, 11.5, H’ÀC(5’)); 4.29 (s, MeN(4)); 4.48 (br. s, HÀC(4’)); 4.58 – 4.62 (m,
HÀC(2’), HÀC(3’)); 5.00 (d, J ¼ 5.1, 1 H, OCH2O); 5.28 (d, J ¼ 5.1, 1 H, OCH2O); 6.39 (d, J ¼ 6.3,
HÀC(1’)); 7.58 (br. s, HÀC(7)); 8.17 (br. s, HÀC(2)). 13C-NMR (100 MHz, CDCl3): À 5.6, À 5.5 (2q,
Me2Si); 11.9 (d, Me2CH); 14.3 (q, MeÀC(6)); 17.7 (q, Me2CH); 18.4 (s, Me3C); 26.0 (q, Me3C); 34.3 (q,
MeN(4)); 63.8 (t, C(5’)); 71.2 (d, C(3’)); 84.9 (d, C(2’)); 86.2 (d, C(4’)); 86.9 (d, C(1’)); 90.9 (t, OCH2O);
106.6 (d, C(7)); 116.6 (s, C(9a)); 134.7 (d, C(2)); 137.9 (s, C(6)); 139.6 (s, C(3a)); 142.6 (s, C(4a)); 152.3 (s,
C(9)). ESI-MS: 636.37 (100, [M þ H]þ).
5’-O-tert-Butyldimethylsilyl-2’-O-{[(triisopropylsilyl)oxy]methyl}wyosine 3’-(2-Cyanoethyl Diiso-
propylphosphoramidite (¼ 3-[3-O-{[Bis(1-methylethyl)amino](2-cyanoethoxy)phosphino}-5-O-[(1,1-di-
methylethyl)dimethylsilyl]-2-O-({[tris(1-methylethyl)silyl]oxy}methyl)-b-d-ribofuranosyl]-3,4-dihydro-
4,6-dimethyl-9H-imidazo[1,2-a]purin-9-one; 2). A soln. of 9 (83 mg, 0.13 mmol) inCH 2Cl2 (1.0 ml) was
i
treated consecutively with Pr2NEt (0.06 ml, 0.32 mmol) and 2-cyanoethyl diisopropylphosphoramido-
chloridite (40 mg, 0.16 mmol). After stirring for 14 h at r.t., the mixture was subjected to CC (SiO2 (3 g);
hexane/AcOEt 4 :1 ! 1:4 (þ 3% Et3N)): 2 (75 mg, 70%; 1:1 mixture of diastereoisomers). Colorless

Helvetica Chimica Acta – Vol. 91 (2008)
1233
1
t
foam. Rf (hexane/AcOEt 3 :7) 0.50. H-NMR (400 MHz, CDCl3): 0.09 – 0.13 (m, BuSi); 0.91 – 1.00 (m,
iPr3Si); 1.17 – 1.25 (m, (Me2CH)2N); 2.34 (s, MeÀC(6)); 2.66 (br. d, J ¼ 5.1, CH2CN); 3.65 – 3.99 (m,
(MeCH)2N, CH2(5’), POCH2, 6 H); 4.15, 4.18 (2s, MeN(4)); 4.34 (br. s, HÀC(4’)); 4.53 – 4.61 (m,
HÀC(3’)); 4.75 – 4.81 (m, HÀC(2’)); 4.96 – 5.03 (m, OCH2O); 6.22 – 6.25 (m, HÀC(1’)); 7.46 (br.
s, HÀC(7)); 8.04 (s, HÀC(2)). 31P-NMR (162 MHz, CDCl3): 149.9, 151.6. MALDI-MS: 836.81 (100,
[M þ H]þ).
3. Preparation of RNA Sequences. RNA Sequence r(GGUGGGAG) (12). The assembly of the RNA
sequence was carried out on a Gene Assembler Plus (Pharmacia), from 55 mg solid support (loading
32 mmol/g) and 2’-O-TOM-protected ribonucleoside phosphoramidites according to [11]. After the
assembly, the solid support was treated with a mixture of 12m MeNH2 inH 2O an d 8m MeNH2 inEtOH
(1 ml) for 4 h at 358. The supernatant was removed by centrifugation and evaporated to dryness; the
residue was treated with a THF soln. (1 ml) of Bu4NF · 3 H2O (1m) for 14 h at 208, diluted with aq. Tris ·
HCl (1 ml, 1m, pH 7.4), and evaporated to a volume of 1 ml. The remaining soln. was applied on a NAP-
10 cartridge (Pharmacia) and eluted with H2O. The first 1.5 ml of soln. was purified by ion-exchange
HPLC: AE-HPLC (20 – 60% B in30 min) flow 2.5 ml/min; eluent A: 12 mm Tris · HCl (pH 7.4), 6m urea;
eluent B: 12 mm Tris · HCl (pH 7.4), 0.5m NaClO4, 6m urea; (detectionat 260 nm, elutionat 85 8): tR
11.0 min. The fraction containing pure product was diluted with 1m aq. Et3N · H2CO3 to a final 0.1m
concentration and applied to a Sepak cartridge (conditioned by washing with MeCN (10 ml) and 0.1m aq.
Et3N · H2CO3 (10 ml)). The cartridge was washed with 20 mm Et3N · H2CO3 (10 ml), and the RNA
sequence was eluted with MeCN/H2O 1:1 (4 ml). In order to completely remove the remaining Et3N ·
H2CO3, 0.5 ml H2O were added to the residue, followed by lyophilization(this procedure was carried
out twice). OD260 46 (¼0.48 mmol, 25%). MALDI-MS (neg mode): m/z ¼ 2645 (calc. 2645).
RNA Sequence r(imG-CGUCCCACC) (13). The RNA sequence was prepared as previously
described for the RNA sequence 12, but employing the 2’-O-TOM wyosine phosphoramidite building
block 2. Furthermore, a 2’-O-TOM H2NÀC(2) unprotected guanosine phosphoramidite building block,
prepared according to [14], was introduced by performing a double coupling cycle. For the wyosine
building block, the oxidation step has been adapted. The final oxidation was achieved with a 1.1m soln. of
tBuOOH inMeCN (flow 0.5 ml/minduring 18 min). The solid support was washed with iPr2NH/MeCN
1:9 for 20 min(flow-rate 2.5 ml/min). Cleavage from the solid support and deprotectionwas carried out
with 12m dry NH3 inMeOH (1 ml) for 14 h at 20 8. The supernatant was removed by centrifugation and
evaporated to dryness; the residue was treated with a THF soln. (1 ml) of Bu4NF · 3 H2O (1m) for 14 h at
208, diluted with aq. Tris · HCl (1 ml, 1M, pH 7.4), and concentrated to a volume of 1 ml. After desalting
ona NAP cartridge, the crude product was purified by ion-exchange HPLC: AE-HPLC (0 – 40% B in
40 min) flow 2.5 ml/min; eluent A: 12 mm Tris · HCl (pH 7.4), 6m urea; eluent B: 12 mm Tris · HCl
(pH 7.4), 0.5m NaClO4, 6m urea; detectionat 260 nm, elutionat 85 8: tR 30.0 min. OD260 61 (¼0.71 mmol,
37%). LC/ESI-MS: m/z ¼ 3147 (calc. 3147).
RNA Sequence 5’-Monophosphate-r(imG-CGUCCCACC) (11). The oligonucleotide was dissolved
to a final concentration of 20 mm for a volume of reactionof 60 ml. The phosphorylationreactionwas
performed according to the instructions from the supplier (Fermentas). The reactionwas performed for
1.5 h at 378. The crude mixture was purified by AE-HPLC. (0 – 40% B in40 min) flow 2.5 ml/min; eluent
A: 12 mm Tris · HCl (pH 7.4), 6m urea; eluent B: 12 mm Tris · HCl (pH 7.4), 0.5m NaClO4, 6m urea;
detectionat 260 nm, elutionat 85 8: tR 30 min. OD260 39 (¼0.46 mmol, 64%). LC/ESI-MS: m/z ¼ 3226
(calc. 3226).
Analytical HPLC for Kinetic Monitoring Control. AE-HPLC: (0 – 50% B in30 min) flow 1.0 ml/min;
eluent A: 12 mm Tris · HCl (pH 7.4), 6m urea; eluent B: 12 mm Tris · HCl (pH 7.4), 0.5m NaClO4, 6m urea;
detectionat 260 mn , elutionat 85
8. r(imG-CGUCCCACC): tR 20.3 min, 5’-monophosphate-r(im-
GCGUCCCACC): tR 21.8 min.
DNA Sequence d(GGUGGGACG-_-CUCCCA) (14). The assembly of the sequence was carried out
under standard conditions [11]. 2’-Deoxyphosphoramidites from Glen Research and propylphosphor-
amidite linker prepared according to [24] were used. AE-HPLC: (0 – 60% B in36 min) flow 2.5 ml/min;
eluent A: 12 mm Tris · HCl (pH 7.4), 6m urea; eluent B: 12 mm Tris · HCl (pH 7.4), 0.5m NaClO4, 6m urea;
detectionat 260 nm, elutionat 85 8: tR 25.0 min. OD260 69 (¼0.44 mmol, 23%). LC/ESI-MS: m/z ¼ 4731
(calc. 4731).

1234
Helvetica Chimica Acta – Vol. 91 (2008)
4. Ligation Experiments. RNA Sequence r(GGUGGGAG-imG-CGUCCCACC) (10). The pre-
viously prepared 8mer 12, 10mer 11, and 16mer template 14 were mixed to a final concentration of 20 mm
for the substrates and 30 mm for the template (1.5 equiv.). After the additionof PEG 6000 (3 ml of 50%
PEG stock soln.) and H2O (q.s.p. 54 ml), the mixture was heated at 958 for 4 min and subsequently cooled
downto 40 8 by steps of 0.18 per second and finally to 48 within1.5 min. After the additionof ligation
buffer (40mm Tris · HCl, 2 mm MgCl2, 10 mm DTT, 0.5 mm ATP) and 8 units of ribonuclease inhibitor
(Fermentas), the reaction was initiated by adding T4 DNA ligase (8 Weiss units), and kept at 378. For the
ligation kinetics, aliquots were taken and diluted in 1 ml of H2O and injected into an anal. AE-HPLC.
AE-HPLC: (0 – 60% B in36 min) flow 1.0 ml/min; eluent A: 12 mm Tris · HCl (pH 7.4), 6m urea; eluent
B: 12 mm Tris · HCl (pH 7.4), 0.5m NaClO4 , 6m urea; detectionat 260 mn , elutionat 85
8.
r(GGUGGGAG): tR 17.7 min( 12). 5’-monophosphate-r(imG-CGUCCCACC): tR 21.4 min( 11).
d(GGUGGGACG-_-CUCCCA): tR 24.9 min( 14). r(GGUGGGAG-imG-CGUCCCACC): tR 26.0 min
(10). Yield: 90%. LC/ESI-MS: m/z ¼ 5854 (calc. 5854).
Preparation of Unmodified RNA Sequences 5’-Monophosphate-r(X-CGUCCCACC) (16 – 19). The
RNA sequences were prepared under conditions as previously described for 12 from the conventional 2’-
O-TOM phosphoramidite building blocks and phosphorylating reagent [3-(4,4’-dimethoxytrityloxy)-2,2-
dicarboxyethyl]propyl(2-cyanoethyl)(N,N-diisopropyl)phosphoramidite (Glen Research). The crude
sequences were purified by AE-HPLC (0 – 50% B in30 min) flow 2.5 ml/min; eluent A: 12 mm Tris · HCl
(pH 7.4), 6m urea; eluent B: 12 mm Tris · HCl (pH 7.4), 0.5m NaClO4, 6m urea; detectionat 260 nm,
elutionat 85 8: tR 21 min. LC/ESI-MS: m/z for X ¼ A 16 (calc. 3159, found 3159), G 17 (calc. 3174, found
3174), U 19 (calc. 3135, found 3135), C 21 (calc. 3135, found 3135).
RNA Sequences r(GGUGGGAG-X-CGUCCCACC) (10). The previously prepared 8mer 12, 10mers
16, 17, 19, an d21, and 16mer template 14 were mixed to a final concentration of 20 mm for the substrates
and 30 mm for the template (1.5 equiv.) and ligated as previously described for sequence 10. For the
ligation kinetics, aliquots were taken and diluted in 1 ml of H2O and injected into an anal. AE-HPLC.
AE-HPLC: (0 – 60% B in36 min) flow 1.0 ml/min; eluent A: 12 mm Tris · HCl (pH 7.4), 6m urea; eluent
B: 12 mm Tris · HCl (pH 7.4), 0.5m NaClO4 , 6m urea; detectionat 260 mn , elutionat 85
8.
r(GGUGGGAG): tR 17.7 min( 12). 5’-monophosphate-r(X-CGUCCCACC): tR 21 min( 16, 17, 19, an d
21). d(GGUGGGACG-_-CUCCCA): tR 24.9 min( 14). r(GGUGGGAG-X-CGUCCCACC): tR 26 min
(X ¼ A 1 (37%); G 18 (48%); U 20 (21%); C 22 (2%)).
5. NMR Experiments. Preparative Ligation of RNA Sequence r(GGUGGGAG-imG-CGUCC-
CACC) (10). 160 Ligations of 60 ml batches each were performed, purified, and pooled as described
above. The Et3NHþ form of the sequence 10 was transformed into its Na salt by dissolving in H2O (2 ꢁ
1.5 ml), treatment with NaHCO3 (10 equiv. per phosphate group), and evaporating to dryness. The
residue was dissolved inH O (2 ml) and desalted on 2 NAP-columns according to the manufacturerꢁs
2
instructions: aq. soln. of RNA sequence. OD260 6.12 (¼0.03 mmol). 1H-NMR (400 MHz, D2O, c ¼ 100 mm).
Preparation of Modified RNA Sequences r(GGUGGGAG-X-CGUCCCACC) (18, 23 – 26). The
assembly of RNA sequences was carried out on a Gene Assembler Plus (Pharmacia), from 60 mg solid
support (loading 32 mmol/g) and 2’-O-TOM-protected ribonucleoside phosphoramidites according to
X ¼ G 18 [11]. The modified nucleotides were introduced conventionally from the 2’-O-TOM-protected
ribonucleoside phosphoramidites X ¼ m1G 23, m6A 24, I 25, m2 G 26, prepared according to [3]. After
2
the assembly, the solid supports were treated with a mixture of 12m MeNH2 inH 2O/8m MeNH2 inEtOH
(1 ml) for 4h at 358. Then, the same procedure as described for 12 was applied to the RNA sequences 18
and 23 – 26. The crude sequences were purified by AE-HPLC: (0 – 60% B in36 min) flow 2.5 ml/min;
eluent A: 12 mm Tris · HCl (pH 7.4), 6m urea; eluent B: 12 mm Tris · HCl (pH 7.4), 0.5m NaClO4, 6m urea;
detectionat 260 nm, elutionat 85 8: tR 26 min. LC/ESI-MS: m/z for X ¼ G 18 (calc. 5802, found 5802),
m1G 23 (calc. 5816, found 5816), m6A 24 (calc. 5800, found 5800), I 25 (calc. 5787, found 5787), m2 G 26
2
(calc. 5830, found 5830). 0.1 mmol of each sequence was isolated and submitted to NMR experiments.
Preparation of the Modified RNA Sequence r(GGUGGGA-I-ACGUCCCACC) (27). The RNA
sequence was prepared as previously described for RNA sequence 12. LC-MS (ESI): m/z ¼ 5787 (calc.
5787). 0.1 mmol of the sequence was isolated and submitted to NMR experiments.
Preparation of Samples for NMR Analysis of Sequences 1, 10, 18, and 23 – 27. The Et3NHþ form of the
sequences was transformed into their Na salt by dissolving in H2O (2 ꢁ 1.5 ml), treatment with NaHCO3

Helvetica Chimica Acta – Vol. 91 (2008)
1235
(Fluka) (10 equiv. per phosphate group), and evaporating to dryness. The residue was dissolved in H2O
(2 ml) and desalted on 2 NAP-columns according to the manufacturerꢁs instructions: aq. soln. of RNA
sequence. 1H-NMR (400 MHz, D2O, c ¼ 100 mm).
REFERENCES
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[2] S. Muller, J. Wolf, S. A. Ivanov, Curr. Org. Synth. 2004, 1, 293.
[3] S. Porcher, S. Pitsch, Helv. Chim. Acta 2005, 88, 2683.
[4] K. M. Guckian, B. A. Schweitzer, R. X.-F. Ren, C. J. Sheils, D. C. Tahmassebi, E. T. Kool, J. Am.
Chem. Soc. 2000, 122, 2213.
[5] H. Bazin, X.-X. Zhou, C. Glemarec, J. Chattopadhyaya, Tetrahedron Lett. 1987, 28, 3275.
[6] T. Itaya, M. Shimomichi, M. Ozasa, Tetrahedron Lett. 1988, 29, 4129.
[7] T. Itaya, T. Kanai, T. Iida, Tetrahedron Lett. 1997, 38, 1979.
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Received March 3, 2008