Synthesis of Selectively 15N-Labeled 2′ -O-{[(Triisopropylsilyl)oxy]methyl}(=tom)-Protected Ribonucleoside Phosphoramidites and Their Incorporation into a Bistable 32Mer RNA Sequence

Article abstract of DOI:10.1002/hlca.200390330

We present optimized reaction conditions for the conversion of 2′-O-{[(Triisopropylsilyl)oxy]methyl}(=tom) protected uridine and adenosine nucleosides into the corresponding protected (3- ¹⁵N)-labeled uridine and cytidine and (1-¹⁵N)-labeled adenosine and guanosine nucleosides 4, 6, 12, and 18, respectively (Schemes 1-4). On a DNA synthesizer, the resulting ¹⁵N-labeled 2′-O-tom-protected phosphoramidite building blocks 19-22 were efficiently incorporated into five selected positions of a bistable 32mer RNA sequence 23 (known to adopt two different structures) (Fig. 1). By 2D-HSQC and HNN-COSY experiments in H₂O/D₂O 9:1, the ¹⁵N-signals of all base-paired ¹⁵N-labeled nucleotides could be identified and attributed to one of the two coexisting structures of 23.

Full text of DOI:10.1002/hlca.200390330

Helvetica Chimica Acta Vol. 86 (2003)
3955
Synthesis of Selectively 15N-Labeled 2'-O-
{[(Triisopropylsilyl)oxy]methyl}(ˆtom)-Protected Ribonucleoside
Phosphoramidites and Their Incorporation into a Bistable 32Mer RNA
Sequence
by Philipp Wenter and Stefan Pitsch*
¬
¬
Laboratory of Nucleic Acid Chemistry, Ecole Polytechnique Federale de Lausanne (EPFL), EPFL-BCH,
CH-1015 Lausanne
Duilio Arigoni zum 75. Geburtstag gewidmet
We present optimized reaction conditions for the conversion of 2'-O-{[(triisopropylsilyl)oxy]methyl}-
(ˆtom) protected uridine and adenosine nucleosides into the corresponding protected (3-15N)-labeled uridine
and cytidine and (1-15N)-labeled adenosine and guanosine nucleosides 4, 6, 12, and 18, respectively (Schemes
1
4). On a DNA synthesizer, the resulting 15N-labeled 2'-O-tom-protected phosphoramidite building blocks
19 22 were efficiently incorporated into five selected positions of a bistable 32mer RNA sequence 23 (known to
adopt two different structures) (Fig. 1). By 2D-HSQC and HNN-COSY experiments in H2O/D2O 9 :1, the 15N-
signals of all base-paired 15N-labeled nucleotides could be identified and attributed to one of the two coexisting
structures of 23.
1. Introduction. We are interested in a detailed and quantitative investigation of
the RNA folding mechanism, including thermodynamic and kinetic aspects. On the
secondary-structure level, folding events lead to changes in the H-bond-mediated
Watson Crick base-pairing pattern. These base-pair contacts can be easily monitored
1
by H-NMR spectroscopy in H2O because, upon base-pair formation, the HÀN(3) of
uridines (paired with adenosines) and the HÀN(1) of guanosines (paired with
cytosines) are protected from fast exchange with the solvent, and are, therefore,
detectable. The observation of these characteristic NH¥¥¥ N signals (−imino-proton×
signals [1]), appearing at d 9 15, allows a sensitive characterization of base-pair
existence and dynamics [2]. However, due to signal overlap, detailed information from
these NH¥¥¥ N signals can often be obtained for only relatively short RNA sequences
(< 50mers). This serious limitation can be overcome by the introduction of 15N-labeled
nucleotides, allowing the application of heteronuclear NMR experiments that make use
of the 1J(H,N) scalar coupling and the two-bond −trans×-H-bond scalar coupling
2hJ(N,N) [3][4]. For the study of folding and refolding processes of rather large systems,
such as ribozymes or tRNAs, we are planning to introduce such labeled nucleotides
only at representative base pairs within defined secondary-structure motifs. Further-
more, it is sufficient for such NMR studies, to label only the N(3) position of the
pyrimidine and the N(1) position of the purine nucleotides, respectively.
Site-specifically isotope-labeled RNA sequences were prepared by transcription/
ligation strategies [5] or by automated chemical synthesis [6]. Whereas enzymatic
methods are ideal for the preparation of large sequences containing stretches of labeled

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Helvetica Chimica Acta Vol. 86 (2003)
nucleotides, chemical synthesis offers the unique possibility to introduce labeled
nucleotides anywhere in the sequence, without constraints of position or labeling
pattern. We recently introduced a reliable RNA-synthesis method based on {2'-O-
[(triisopropylsilyl)oxy]methyl}(ˆtom)-protected ribonucleoside phosphoramidites
that allows the preparation of RNA sequences under DNA-coupling conditions [7
9]. In the above-mentioned context, we report here the synthesis of four phosphor-
amidite analogues, each containing a single 15N-label (N(3) of uridine/cytosine and
N(1) of guanosine/adenosine), their incorporation into a known, bistable 32mer RNA
sequence [10]1), and preliminary NMR experiments.
2. Results. 2.1. Synthesis of 15N-Labeled, 2'-O-tom-Protected Phosphoramidites.
Several methods for the selective introduction of 15N-labels into position N(3) of
pyrimidine and position N(1) of purine nucleosides, and some examples for the
incorporation of such labeled ribonucleosides into RNA sequences have been reported
already [12 14]. In all these approaches, first the 15N-labeled nucleotides were
prepared by nucleobase-transformation reactions or nucleoside formation with
appropriately labeled nucleobases, followed by introduction of the nucleobase-
protecting groups, the 5'-O-(4,4'-dimethoxytrityl) (ˆ(MeO)2Tr) group, the 2'-O-(tert-
butyl)dimethylsilyl (ˆtbdms) group, and finally the 3'-(2-cyanoethyl diisopropylphos-
phoramidite) moiety. This strategy includes many steps and the isolation/purification of
very polar intermediates. In our approach, we decided to start from 2'-O-tom-protected
ribonucleosides and to carry out the introduction of the 15N-labels in the presence of
this lipophilic 2'-O-protecting group. Furthermore, we decided to use cheap 15NH4Cl as
the sole 15N-source and to choose/optimize reaction conditions carefully, to avoid the
use of large excesses of reagents.
The easily accessible 2'-O-tom-protected uridine nucleoside 1 [9] was chosen as
starting material for the synthesis of the 15N-labeled pyrimidine nucleosides 4 and 6.
Treatment of 1 with AcOH/H2O ( ! removal of the (MeO)2Tr group), followed by
acetylation of the 3'- and 5'-OHgroups with Ac O in pyridine gave the diacetylated
2
uridine derivative 2, which was isolated by chromatography (silica gel) and consec-
utively treated with NH4NO3/(CF3CO)2O in CH2Cl2 under strictly anhydrous
conditions according to Ariza et al. [13] (Scheme 1). After fast and careful extraction,
followed by filtration on silica gel, the resulting N-nitro derivative 3 was treated
immediately with 1.3 equiv. of 15NH4Cl in the presence of KOHand Et N in MeCN/
3
H2O, again following reported conditions [13]2). During this reaction, partial loss of the
acetyl groups was observed; therefore, after extraction, the mixture of differently
acetylated, 15N-labeled, 2'-O-tom-protected uridines was subjected directly to complete
deacetylation with NaOH in THF/MeOH/H2O. After extraction and without further
purification, the 5'-O-(MeO)2Tr group was introduced under standard conditions with
1
)
)
Bistable RNA sequences adopt two coexisting secondary structures (for other examples, see [11]).
This clean and efficient reaction sequence allows the straightforward introduction of 15N at position N(3)
of uridine and N(1) of inosine derivatives. Efficient formation of the N-nitro derivative proceeds only
under strictly anhydrous conditions and in the absence of light; the N-nitro derivatives cannot be stored for
a prolonged period of time and should be subjected immediately to the next step.
2

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Scheme 1
a) 1. AcOH, H2O, 258; 2. Ac2O, N,N-dimethylpyridin-4-amine (DMAP), pyridine, 258. b) NH4NO3,
(CF3CO)2O, CH2Cl2, 48. c) 1. 15NH4Cl, KOH, Et3N, MeCN, H2O, 258; 2. NaOH, THF, MeOH, H2O, 48;
3. (MeO)2TrCl, pyridine, 258.
(MeO)2TrCl in pyridine. The fully protected, 15N-labeled uridine building block 4 was
finally isolated by chromatography (silica gel).
The 15N-labeled, fully protected cytosine derivative 6 was prepared from the
corresponding uridine nucleoside 4 (Scheme 1) by first acetylating the 3'-OHgroup
with Ac2O in pyridine, followed by extractive workup and treatment of the 3'-O-
i
acetylated intermediate with (ClC6H4O)P(O)Cl2, 1H-1,2,4-triazole, and Pr2NEt in
MeCN ( ! formation of the 4-triazolyl derivative)3), followed by NH3 in dioxane/H2O
(Scheme 2). After extraction and treatment with NaOH in MeOH/THF/H2O, the
cytidine nucleoside 5 was isolated by chromatography (silica gel). It was finally
transformed into its N4-acetylated, 15N-labeled derivative 6 by selective N-acetylation
with Ac2O in DMF according to Bhat et al. [17]4) and isolated in a good yield by
chromatography (silica gel).
Scheme 2
a) 1. Ac2O, DMAP, pyridine, 258; 2. 1H-1,2,4-triazole, 4-chlorophenyl phosphorodichloridate, iPr2NEt, MeCN,
258; 3. NH3, dioxane, MeCN, H2O, 258; 4. NaOH, THF, MeOH, H2O, 48. b) Ac2O, DMF, 258.
The two 15N-labeled purine nucleosides 12 and 18 were prepared from the readily
available 2'-O-tom-protected adenosine nucleoside 7 [9]. First, the N-acetyl protecting
group and the 5'-O-(MeO)2Tr group of 7 were removed with MeNH2 in H2O/THF and
AcOH/H2O, respectively (Scheme 3). After precipitation from CH2Cl2/hexane, the
3
)
In preliminary studies, we found that these reaction conditions resulted in a much cleaner and faster
product formation than those of the similar reaction performed in pyridine, but without Pr2NEt [15], or
i
those involving activation with TsCl/(PhO)2P(O)OH[16].
This reaction proceeds very cleanly (without acetylation of the 3'-OHgroup), provided that pure starting
material is used.
4
)

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Helvetica Chimica Acta Vol. 86 (2003)
Scheme 3
a) 1. MeNH2, TH F, HO, 258; 2. AcOH, H2O, 258; 3. Ac2O, DMAP, pyridine, 258; 4. NaNO2, AcOH, H2O, 258.
2
b) NH4NO3, (CF3CO)2O, CH2Cl2, À 208. c) 15NH4Cl, KOH, Et3N, MeCN, H2O, 258. d) 3-Nitro-1H-1,2,4-
triazole, I2, PPh3, iPr2NEt, toluene, 958. e) 1. NH4Cl, tBuOK, Et3N, DMSO, 508; 2. BzCl, DMAP, pyridine, 258;
3. NaOH, THF, MeOH, H2O, 48; 4. (MeO)2TrCl, pyridine, 258.
intermediate 2'-O-tom-protected adenosine was converted with Ac2O in pyridine into
the corresponding 3',5'-di-O-acetyl derivative, which, after evaporation and extraction,
was deaminated with NaNO2 in AcOH/H2O 7:3. The resulting inosine derivative 8 was
isolated by crystallization. In analogy to the preparation of the 15N-labeled uridine
nucleoside 3 (Scheme 1), but at lower temperature, 8 was treated under strictly
anhydrous conditions with NH4NO3/(CF3CO)2O in CH2Cl2 [13]. As crude product, the
resulting N-nitro derivative 9 was treated with 1.3 equiv. of 15NH4Cl in the presence of
KOHand Et N in MeCN/H2O [13]2). This reaction proceeded much faster than with
3
the analogous uridine nucleoside 3, and without concomitant loss of acetyl groups.
After extraction, but without further purification, the thus-obtained 15N-labeled
inosine derivative 10 was treated with I2, PPh3, iPr2NEt, and 3-nitro-1H-1,2,4-triazole in
toluene, in analogy to Lin and Robins [18]5). The nitro-triazole derivative 11 was
5
)
These very efficient reaction conditions were originally developed for the introduction of imidazole and
cyclic secondary amines at C(6) of purine nucleosides [18]; we found, however, that these conditions
allowed also smooth preparation of the corresponding 3-nitro-1H-1,2,4-triazole derivative, which, for
substitution reactions with NH3, is the superior leaving group [19]. Our first, unsuccessful or inefficient
attempts to prepare similarly activated purine derivatives from 8 included the following conditions: 1H-
1,2,4-triazole/POCl3/iPr2NEt in MeCN [20] (very slow reaction); 1H-1,2,4-triazole/(ClC6H4O)P(O)Cl2/
iPr2NEt in pyridine [15] (low yield, significant side reactions); 1H-tetrazole/TsCl/(PhO)2P(O)OH/iPr2NEt
in pyridine [21] (incomplete conversion); 3-nitro-1-[(2,4,6-triisopropylphenyl)sulfonyl]-1H-1,2,4-triazole/
(PhO)2P(O)OH/iPr2NEt in pyridine [22] (fast formation of N(1)- and O6-sulfonylated products (1:1),
followed by slow conversion of the latter into the corresponding 3-nitro-1H-1,2,4-triazole derivative); 3-
nitro-1H-1,2,4-triazole/TsCl/(PhO)2P(O)OHin pyridine [19] (incomplete conversion).

Helvetica Chimica Acta Vol. 86 (2003)
3959
isolated in a good yield of 67% (based on 8) after evaporation and chromatography
(silica gel). Treatment of this activated purine nucleoside under carefully optimized
conditions with NH4Cl, tBuOK, Et3N in DMSO6), followed by precipitation with H2O,
resulted in quantitative isolation of the 3',5'-di-O-acetylated, 2'-O-tom-protected
adenosine. Without intermediate purification, this nucleoside was first transformed to
its N6-benzoyl derivative7) with BzCl in pyridine, then deacetylated with NaOHin
THF/MeOH/H2O, and finally converted to its 5'-O-(MeO)2Tr derivative 12 with
(MeO)2TrCl in pyridine. The fully protected, 15N-labeled adenosine building block 12
was isolated in a good yield by chromatography (silica gel).
The synthesis of the 15N-labeled guanosine nucleoside 18 was carried out from
inosine intermediate 8 (Scheme 3). In analogy to the preparation of 11, 8 was first
treated with I2, PPh3, iPr2NEt, and 3-nitro-1H-1,2,4-triazole in toluene (Scheme 4); the
resulting 3-nitro-1H-1,2,4-triazole derivative 13 was isolated by chromatography (silica
gel) and subsequently treated with 3 equiv. of 15NH4Cl in the presence of tBuOK/Et3N
in DMSO. The so-obtained 15N-labeled adenosine derivative 14 was precipitated with
H2O. According to TLC analysis, a partially deacetylated derivative was formed during
this step (ca. 5%); therefore, crude 14 was reacetylated with Ac2O in pyridine, and
finally purified by chromatography (silica gel). Transformation into the corresponding
N(1)-oxide 15 was achieved with 3-chloroperbenzoic acid in CH2Cl2 according to
MacCoss et al. [23]. The product was isolated in a yield of 79% by chromatography
(silica gel); additionally, 18% of unreacted 14 was recovered. Following (with some
adaptations) the well-known method of Goswami and Jones [14], the N-oxide 15 was
treated first with BrCN in MeOH; a DMF solution of the resulting dihydrooxadiazole
derivative was then treated consecutively with Et3N and MeI to yield a 1-methoxy-N6-
cyano-substituted derivative; the latter was treated first with NaOHin THF/H 2O and,
finally, after neutralizing with aq. HCl solution and addition of EtOH, kept at 608. In
the course of this Dimroth rearrangement, the 2-amino-N6-methoxy-substituted purine
derivative 16 was formed almost quantitatively, together with small amounts of still
partially acetylated derivatives8). After extraction, this product mixture was directly
subjected to an exhaustive acetylation with Ac2O in pyridine at 1008. Under these
conditions, a 1:1 mixture of the corresponding N2,N6,3'-O,5'-O-tetraacetyl and
N2,N2,N6,3'-O,5'-O-pentaacetyl derivatives was formed, which was filtered through
silica gel and then partially deacetylated with NaOH in THF/MeOH/H2O to yield the
N2-acetyl-N6-methoxy-2'-O-tom-substituted purine nucleoside. The latter was treated
as crude product with Raney-Ni in THF/MeOH/H2O, resulting in cleavage of the NÀO
bond and clean formation of 179). Without purification, this purine-2,6-diamine
nucleoside was first deaminated with NaNO2 in AcOH/H2O and then dimethoxytrityl-
ated with (MeO)2TrCl in pyridine; finally, the 15N-labeled, fully protected guanosine
6
)
)
Conditions developed for the preparation of the analogous compound 14 (Scheme 4) with 15NH4Cl.
For routine RNA synthesis, we are using N6-acetylated adenosine building blocks [8][9]. In the here
presented reaction sequence, however, it was more convenient to prepare the N6-benzoyl derivative.
This reaction sequence (formation of N(1)-oxide, Dimroth rearrangement) was also successfully applied to
corresponding transformations starting from 2'-O-tom-protected adenosine or 5'-O-(MeO)2Tr-2'-O-tom-
protected adenosine; in the context of the preceding and subsequent reactions, however, it was more
convenient to use the diacetylated adenosine derivative 14.
7
8
)
9
)
Unfortunately, a significant part of the product remains adsorbed on the Raney-Ni.

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Helvetica Chimica Acta Vol. 86 (2003)
Scheme 4
a) 3-Nitro-1H-1,2,4-triazole, I2, PPh3, iPr2NEt, toluene, 958. b) 1. 15NH4Cl, tBuOK, Et3N, DMSO, 508; 2. Ac2O,
DMAP, pyridine, 258. c) 3-Chloroperbenzoic acid, CH2Cl2, 258 (indicated yield based on consumption). d)
1. BrCN, MeOH, 258; 2. Et3N, DMF; 3. MeI, DMF, 258; 4. NaOH, THF, H2O, 258; 5. aq. HCl soln. ( ! pH7),
EtOH, 608. e) 1. Ac2O, pyridine, 1008; 2. NaOH, THF, MeOH, H2O, 48; 3. Raney-Ni, THF, MeOH, H2O, 808. f)
1. NaNO2, AcOH, H2O, 258; 2. (MeO)2TrCl, pyridine, 258.
nucleoside 18 was isolated in a yield of 33% (based on 15, ten consecutive steps) after
chromatography (silica gel).
All compounds presented in Schemes 1 4 were characterized by 1H-NMR,
13C-NMR, and MS (see Exper. Part). In the MS of all compounds 1 18, the m/z
value of the most-intense signal was in agreement with the calculated value (with the
exception of 3, which decomposed during analysis). The chemical-shift values d of the
fully protected, 15N-labeled nucleosides 4, 6, 12, and 18 were identical to the reported
values of the unlabeled analogues, both in 1H- and 13C-NMR analyses [7][9].
Nucleosides 4, 6, 12, and 18 (precursors of phosphoramidites 19 22, see below) were
additionally characterized by H-decoupled 15N-NMR spectroscopy, and each showed
one signal, corresponding to the 15N-label (d 133.1 (4), 208.1 (6), 227.2 (12), and 129.4
(18)). Many of the 15N-containing compounds exhibited additional J(H,N) couplings
(1H-NMR), and all showed additional J(C,N) couplings (13C-NMR). The following
1
values were recorded: J(H,N) ˆ 88.6 91.1 Hz for pyrimidine and purine nucleosides
4, 10, 14, 15, and 18, 3J(HÀC(5),N(3)) ˆ 2.1 Hz for uridine derivative 4, and
2J(HÀC(2),N(1)) ˆ 7.5, 16.1, and 15.9 Hz, for the inosine and adenosine derivatives
10, 11, and 12 respectively. The coupling constants J(C,N) of the 15N-labeled
nucleosides 4 6, 10 12, and 14 18 as derived from 13C-NMR spectra are summarized
in the Table.

Helvetica Chimica Acta Vol. 86 (2003)
3961
Table 1. Observed Couplings (J(C,N)) between Nucleobase 13C-and 15N-Atoms, as Obtained from 13C-NMR
Spectra of 15N-Containing Nucleosides (see Exper. Part)
J(C,N) [Hz]
C(2)
C(4)
C(5)
C(6)
4 (3-15N)
5 (3-15N)
17.0
7.4
7.7
8.4
2.5
3.0
2.5
8.4
6.3
7.4
5.6
4.2
1.0
7.0
3.2
1.5
3.3
1.3
2.0
1.1
8.3
6 (3-15N)
10 (1-15N)
11 (1-15N)
12 (1-15N)
14 (N6-15N)
15 (N6-15N)
16 (3-15N)
17 (3-15N)
18 (3-15N)
9.8
7.0
5.2
20.7
22.5
2.4
8.5
6.7
14.2
5.0
7.0
10.5
According to our standard procedure, the four 15N-labeled, 2'-O-tom-protected
nucleosides 4, 6, 12, and 18 were converted with 2-cyanoethyl diisopropylphosphor-
amidochloridite/iPr2NEt into the corresponding phosphoramidite building blocks 19
22 (Scheme 5), which were isolated in good yields by chromatography (silica gel).
Scheme 5
i
a) 2-Cyanoethyl diisopropylphosphoramidochloridite, Pr2NEt, CH2Cl2, 258.
MALDI-MS Analysis of the four 15N-labeled phosphoramidites 19 22 showed the
1
expected m/z values. The chemical-shift values of their H- and 31P-NMR spectra
corresponded well with the ones found for their unlabeled analogues [6][8]. The
1
1
additional J(H,N) coupling constants, detected by H-NMR, included J ˆ 83.8 and
82.5 Hz for 19 and 22, respectively, 2J ˆ 10.1 Hz for 21, and 3J ˆ 2.7 Hz for 19.
2.2. Synthesis of a 32Mer RNA Sequence. As a first example for our planned NMR
studies, we chose to prepare the bistable 32mer RNA sequence 23 and to incorporate
the 15N-labeled nucleoside building blocks 19 22 at the positions U28, C9, C29, A21,
and G20 (counted from the 5'-end). The same bistable RNA sequence (not containing

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Helvetica Chimica Acta Vol. 86 (2003)
Fig. 1. Representation of the two coexisting structures (S1) and (S2) adopted by the RNA sequence 23 (according
to [9]). The encircled and numbered nucleosides derive from the 15N-labeled phosphoramidites 19 22
(numbering from 5' ! 3' end). In 150 mm NaCl and 50 mm sodium phosphate (pH7.4) at 23 8, an equilibrium
ratio 23(S1)/23(S2) of 7:3 was determined (see below, Fig. 3).
any 15N-labels) was first designed, prepared, and characterized by Hˆbartner and
Micura [10] and is known to adopt the two different folding motifs shown in Fig. 110).
The sequence 23 was prepared on a 10-mm scale from conventional 2'-O-tom-
protected ribonucleoside phosphoramidites and the 15N-labeled analogues 19 22
according to our standard protocols [9]. The anion-exchange (ˆAE) HPLC trace of
the crude product was in agreement with good coupling efficiences and showed a well-
separated, chromatographically homogeneous main product (Fig. 2,a). After purifi-
cation by prep. AE-HPLC and desalting (for details, see Exper. Part), 30 mg
(2.7 mmole; 27% yield, based on the solid support) of chromatographically pure
‡
‡
RNA sequence 23 (NH4 form, containing 1 mol-% of Et3NH , according to 1H-NMR
(Fig. 2,c)) were obtained. The LC-ESI-MS (neg. mode) of the purified sequence 23
showed the correct mass and indicated a high degree of purity (Fig. 2,b; for details see
1
Exper. Part). The H-NMR spectrum (400 MHz, D2O; Fig. 2,c) demonstrated the
spectral complexity of this 32mer RNA sequence.
2.3. NMR Experiments. The subsequently described NMR experiments with RNA
sequence 23 were carried out at c ˆ 2 mm in H2O/D2O 9 :1, at 150 mm NaCl, and 50 mm
sodium phosphate (pH7.0) at 23 811). Spectra were recorded at 600 MHz (Bruker
instrument), and a combined flip-back [24]/WATERGATE [25] pulse sequence was
used for water suppression. In Fig. 3, the −imino-proton× regions (d 9 15) of spectra
1
obtained from three different 1D experiments are shown. The conventional H-NMR
spectrum, recorded without 15N-decoupling (Fig. 3,a), shows the expected complex
10
)
)
Partial assignment was carried out by comparative NH¥¥¥ N 1H-NMR spectroscopy of small reference
hairpins [10].
Preliminary measurements at other temperatures showed a strong temperature dependence of the
equilibrium distribution between 23(S1) and 23(S2). At higher temperatures, 23(S2), and at lower
temperatures, 23(S1) was favored.
11

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3963
Fig. 2. Characterization of RNA sequence 23 (prepared according to [8]): a) AE-HPLC trace of the crude
product (detection at 260 nm, 15 70% B in 30 min); b) deconvoluted LC-ESI-MS (neg. mode) of the purified
product (M (calc.) 10129.2 amu; for details, see Exper. Part); c) 1H-NMR spectrum (400 MHz, D2O, c ˆ 2 mm)
‡
‡
of purified 23 (NH4 form, containing 1 mol-% of residual Et3NH ions)
pattern of signals, each corresponding to a proton involved in a individual base pair of
the two coexisting structures. Most of the signals appear in a narrow range between d
12.0 13.5, typical for the −imino-protons× of G ¥ C base pairs [1]. Upon broad-band 15N-
decoupling (Fig. 3,b), the ds at d 14.2, 12.1, and 11.9 (J(H,N) ꢀ 90 Hz) change to ss,
resulting in a spectrum which qualitatively corresponds to the spectrum of the
analogous, unlabeled, bistable RNA sequence reported by Hˆbartner and Micura
[10]12). As expected from the decoupling experiment, only the 3 ds of Fig. 3,a, appear
in the (1H-decoupled) 1D-HSQC spectrum (Fig. 3,c). These initial 1D-NMR experi-
ments allowed, in a straightforward way, to isolate −imino-protons× of base-paired, 15N-
15N
labeled uridines and guanosines and showed the occurrence of one A ¥ U (d 14.2) and
12
)
The d values observed for RNA sequence 23 and the unlabeled analogue [10] are almost identical.
However, the ratio 23(S1)/23(S2) was 7:3 in our, and 1:1 in the reported measurement. This difference
likely originates from the medium (150 mm NaCl and 50 mm sodium phosphate vs. 25 mm sodium arsenate
[10]).

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Helvetica Chimica Acta Vol. 86 (2003)
Fig. 3. −Imino-proton× region of 1H-NMR spectra (600 Hz, H2O/D2O 9 :1, for conditions, see text): a) 15N-
decoupled, b) 15N-broad-band-decoupled, and c) 1D-HSQC spectrum, showing the −imino-proton× signals of the
indicated base pairs
15N
two C ¥ G base pairs (d 12.1 and 11.9). Based on the two secondary structures of 23
(Fig. 1), the signal appearing at d 14.3 was assigned to base pair U28 ¥ A21-23(S1). The
other two signals, appearing in the HSQC spectrum at d 12.1 and 11.9, originate both
from G20; they were assigned to G20 ¥ C9-23(S2) and G20 ¥ C29-23(S1), respectively,
according to their integral ratio.
Two relevant parts of the 2D-HSQC spectrum of the RNA sequence 23, displaying
the heteronuclear 1H,15N-correlation, are shown in Fig. 4. The atom 15N(3) of the base
15
15N
pair U28 ¥ A21-23(S1) appears at d 163, whereas 15N(1) of both the base pairs NG20 ¥
15
C29-23(S1) and NG20 ¥ C9-23(S2) appear at the same d value of 146 (Fig. 4,a).
Furthermore, the two-bond scalar coupling between HÀC(2) and N(1) of A21
(2J(H,N) ˆ 16 Hz) resulted in two resolved cross-peaks at d(H/N) 7.82/222.1 and d(H/
N) 7.76/222.3, corresponding to both structures 23(S1) and 23(S2), respectively
(Fig. 4,b). By an additional HNN-COSY experiment (see below, Fig. 5), the former
cross-peak could be assigned to 15NA21-23(S1) in its base-paired form.
The main purpose of HNN-COSY experiments is the correlation of the H-bond-
donor with the H-bond-acceptor N-atom within a base pair, provided that both are 15N-
labeled [3]. The HNN-COSY experiment with our RNA sequence 23 was carried out
with a relaxation-optimized N,N-transfer time of 20 ms and a H,N-transfer time of
5 ms (Fig. 5). It provided a correlation for the three doubly 15N-labeled base pairs
A21 ¥ U28-23(S1), G20 ¥ C29-23(S1), and G20 ¥ C9-23(S1), and furthermore, the d
values for the acceptor nucleosides A21, C29, and C9 could be assigned. In contrast to

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3965
Fig. 4. Selected regions of a 2D-HSQC spectrum (600 MHz, H2O/D2O 9 :1, for conditions, see text): a)
HÀN(3),N(3) and HÀN(1),N(1) cross-peaks of the base-paired, 15N-labeled nucleotides U28-(S1) and G20-
(S1,S2); b) resolved HÀC(2),N(1) cross-peaks of the 15N-labeled nucleotide A21-(S1) and A21-(S2) (forming
base pairs in both structures)
Fig. 5. Selected regions of a HNN-COSY plot (600 MHz, N,N-transfer time 20 ms, H,N-transfer time 5 ms; H2O/
D2O 9 :1, for conditions, see text), showing correlation between 15N-resonances of doubly 15N-labeled base pairs

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G20, which displays the same chemical shift for both structures, its two pairing partners
show a detectable difference and appear at d 198.0 (C9-23(S2)) and d 198.5 (C29-
23(S1)).
3. Discussion.
NMR Analysis of larger RNA sequences (consisting of > 50
nucleotides) suffers from bad signal separation in the H- as well as in the 15N-
dimension, and assignment of these signals is often difficult. For investigations with
RNA sequences where a complete structure elucidation is not required, the site-specific
introduction of labeled nucleotides can overcome this limitation in a straightforward
way.
1
In this context, we prepared the 15N-labeled, 2'-O-tom-protected ribonucleoside
phosphoramidite building blocks 19 22, which are precursors of selectively 15N-
labeled RNA sequences. The excellent stability of the 2'-O-tom protecting group
towards a variety of different reaction conditions allowed us to perform all reactions in
its presence, and we never detected any trace of 2'-O-deprotected by-products. As a
consequence of its very lipophilic character, all 2'-O-tom-protected intermediates were
well-soluble in organic solvents, extractable, and easy to purify by chromatography
(silica gel). By carefully optimizing existing reaction conditions and developing new
ones, we were able to use the relatively cheap 15NH4Cl (our only 15N-source) in small
quantities (1.3 and 3.0 equiv. for the pyrimidine and the purine nucleosides, resp.). Our
convergent strategy allowed to reduce the number of steps and necessary purifications
to a minimum. The here presented route can also be applied, in principle, to the
straightforward synthesis of 2'-O-tom-protected phosphoramidites of other 15N-labeled
ribonucleosides, such as (1-15N)inosine from 10 and (1-15N)purine-2,6-diamine
ribonucleoside from 17, respectively.
As a model system for initial NMR experiments, we have prepared the bistable
RNA sequence 23, containing five 15N-labeled nucleotides. In the 1H-NMR spectrum,
the −imino-proton× signals of the G ¥ C base pairs (forming the stem regions of the two
coexisting hairpin structures 23(S1) and 23(S2)), are crowded in a narrow d range
(11.9 13.2 ppm), and some signal overlap occurs. The corresponding 1D-HSQC
spectrum, however, shows only the three resonances of the 15N-labeled, base-paired
uridines and guanosines; therefore, an unambiguous assignment of these signals to U28
(forming an A ¥ U base pair in 23(S1)) and G20 (forming C ¥ G base pairs in 23(S1) and
23(S2)) was possible. At the same time, it provided clear evidence that G20 is involved
in the formation of two different base pairs, and U28 in only one; this is consistent with
the two coexisting secondary structures, which were proposed upon comparison with
spectra of reference compounds [9]. From the 2D-HSQC experiments, 15N-chemical-
shift values the for base-paired, labeled guanosines and uridines were obtained, but
unfortunately, the 15N-signals of G20, involved in two different base pairs, were
isochronous13). The coupling between 15N(1) and HÀC(2) of A21, however, resulted in
cross-peaks with different d values, both in the 1H- and the 15N-dimension. As expected,
13
)
A synthetic approach to overcome such limitations would be the introduction of labeled base pairs in such
a way that, in one structure, both the donor and acceptor base are 15N-labeled, whereas, in the second
structure, only the donor base carries a 15N-label. This labeling pattern would allow to detect both 15N-
resonances in the HSQC experiment, and to assign them by a HNN-COSY experiment.

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3967
the HNN-COSYexperiment provided correlated 15N-resonances for the doubly labeled
base pairs and allowed the assignment of the 15N-signals of cytidines C9 and C29, and of
adenosine A21.
Here, we present an application of site-specifically labeled RNA sequences as a tool
for assignment and probing for putative secondary-structure motifs. However, addi-
tional, more sophisticated NMR experiments, involving the 15N-dimension and
sensitive to dynamics, could be carried out with such RNA sequences, eventually
resulting in a detailed understanding of the dynamics of selected base pairs
(representative for certain secondary structures). Such preliminary experiments with
RNA sequence 23 revealed so far that the rate-constant k for the interconversion of the
two secondary structures of 23 is probably below 1 secÀ1 [26].
We are very grateful to Dr. Jens Dittmer and Prof. G. Bodenhausen (EPFL) for many helpful discussions
and continuous support in carrying out NMR experiments and we thank Prof. Ronald Micura (University of
Innsbruck) for his highly appreciated advice concerning bistable RNA sequences. This work was generously
supported by the EPFL and by the Swiss National Science Foundation (Grant Nr. 2000-06890).
Experimental Part
General. Reagents and solvents (highest purity) were from various suppliers and used without further
purification, unless otherwise stated. 15NH4Cl (15N > 99%) was from Spectra Stable Isotopes (Columbia, MD).
Unlabeled, 2'-O-tom-protected ribonucleoside phosphoramidites and supports were prepared according to [9].
Workup implies distribution of the reaction mixture between CH2Cl2 and sat. aq. NaHCO3 soln., drying of the
org. layer (MgSO4), and evaporation. TLC: precoated silica-gel 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): silica gel 60 (230 400 mesh) from Fluka. Anion-exchange (AE)
HPLC: DNAPAC PA-100 (9.0 Â 250 mm; Dionex), flow 2.5 ml/min; eluent A: 12 mm Tris ¥ H Cl (pH 7.4), 6m
urea; eluent B: 12 mm Tris ¥ HCl (pH 7.4), 0.5m NaClO4, 6m urea; detection at 260 nm, elution at 858. NMR
(Bruker instruments): chemical shift d in ppm rel. to external standards (1Hand 13C: Me4Si, 31P: 85% aq. H3PO4
soln., 15N: 20 mm 15NH4Cl in 10% aq. HCl soln.); coupling constants J in Hz; multiplicities (13C) according to
DEPT spectra. ESI-MS (pos. mode): SSQ 710 (Finnigan), measurements in MeCN/H2O/AcOH50 :50 :1.
MALDI-MS (pos. mode): Axima CFR Plus (Kratos/Shimadzu); matrix: 2,4,6-trihydroxyacetophenone,
diammonium citrate; m/z (rel. intensity in %). LC-ESI-MS (neg. mode): Q-Tof-Ultima (Micromass/Waters)
coupled to Cap-LC (Waters); injection: 2 ml aq. sample (c (RNA) ˆ 2.5 mm, c (EDTA) ˆ 1 mm); chromatog-
raphy on Xterra RP-C18 column (Waters, 5 mm, 0.32 Â 50 mm; flow 8 ml/min; eluent A: 25 mm aq. Me2NBu ¥
H2CO3 (pH8.4); eluent B: MeCN; elution at 608, sheath flow 25 ml/min (MeCN)); gradient A ! A/B 1:1
(15 min); deconvolution by MaxEnt1-software14).
3',5'-Di-O-acetyl-2'-O-{[(triisopropylsilyl)oxy]methyl}uridine (2). A soln. of 1 [9] (7.32 g, 10 mmol) in
AcOH/H2O 4 :1 (180 ml) was stirred for 30 min at r.t. After evaporation (under continuous addition of small
portions of H2O towards the end), followed by co-evaporation with toluene (2 Â 100 ml), the residue was
dissolved in pyridine (40 ml) and treated with DMAP (366 mg, 3 mmol) and Ac2O (2.83 ml, 30 mmol). After 1 h
at r.t., MeOH(5 ml) was added, and the mixture was evaporated. CC (SiO 2, (150 g), hexane/AcOEt 4 :1 ! 1:4)
1
gave 2 (4.47 g, 87%). Colorless foam. TLC (hexane/AcOEt 2 :3): Rf 0.45. H-NMR (400 MHz, CDCl3): 1.01
1.03 (m, iPr3Si); 2.12, 2.14 (2s, 2 MeCO); 4.31 4.36 (m, HÀC(4'), CH2(5')); 4.53 (t, J ˆ 5.7, HÀC(2')); 4.87, 4.95
(2d, J ˆ 5.1, OCH2O); 5.19 (dd, J ˆ 3.8, 5.7, HÀC(3')); 5.75 (d, J ˆ 8.3, HÀC(5)); 6.02 (d, J ˆ 6.0, HÀC(1')); 7.40
(d, J ˆ 8.3, HÀC(6)); 8.59 (br. s, HÀN(3)). 13C-NMR (100 MHz, CDCl3): 12.0 (d, Me2CH); 17.9 (q, Me2CH);
20.9 (q, MeCO); 63.6 (t, C(5')); 70.6 (d, C(3')); 75.8 (d, C(2')); 80.2 (d, C(4')); 88.4 (d, C(1')); 89.2 (t, OCH2O);
103.1 (d, C(5)); 140.0 (d, C(6)); 150.0 (s, C(2)); 162.6 (s, C(4)); 170.2 (s, MeCO). ESI-MS: 515.3 (100, [M ‡
‡
H] ).
3',5'-Di-O-acetyl-3-nitro-2'-O-{[(triisopropylsilyl)oxy]methyl}uridine (3). At 48 and under Ar, (CF3CO)2O
(6.55 g, 31.2 mmol) was added dropwise to a well-stirred suspension of finely powdered NH4NO3 (1.25 g,
14
)
Conditions adapted from [27].

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Helvetica Chimica Acta Vol. 86 (2003)
15.6 mmol; dried for 48 h at 608/0.05 mbar) in CH2Cl2 (20 ml). After 1 h at r.t., a clear soln. was obtained, to
which a soln. of 2 (4.03 g, 7.8 mmol; dried for 48 h at 608/0.05 mbar) in CH2Cl2 (20 ml) was added slowly. The
reaction was kept for 20 min at r.t. under exclusion of light. Workup at 48, followed by filtration through SiO2
(40 g, CH2Cl2 ! CH2Cl2/AcOEt 3 :2) gave 3 (4.01 g, 92%). Colorless foam (used immediately for the next step).
TLC (hexane/AcOEt 1 :1): Rf 0.71. 1H-NMR (400 MHz, CDCl3): 1.01 1.07 (m, iPr3Si); 2.12, 2.15 (2s, MeCO);
4.29 4.40 (m, HÀC(4'), CH2(5')); 4.55 (t, J ˆ 5.7, HÀC(2')); 4.86, 4.96 (2d, J ˆ 5.0, OCH2O); 5.19 (br. t, J ꢀ 4.4,
HÀC(3')); 5.88 (d, J ˆ 8.44, HÀC(5)); 5.99 (d, J ˆ 5.5, HÀC(1')); 7.47 (d, J ˆ 8.44, HÀC(6)). 13C-NMR
(100 MHz, CDCl3): 11.9 (d, Me2CH); 17.8 (q, Me2CH); 20.85, 20.88 (2q, MeCO); 63.3 (t, C(5')); 70.2 (d, C(3'));
75.9 (d, C(2')); 80.6 (d, C(4')); 88.2 (d, C(1')); 89.3 (t, OCH2O); 102.0 (d, C(5)); 139.4 (d, C(6)); 145.4 (s, C(2));
155.0 (s, C(4)); 170.1 (s, MeCO). ESI-MS: dec.
5'-O-(4,4'-Dimethoxytrityl)-2'-O-{[(triisopropylsilyl)oxy]methyl}(3-15N)uridine (4). Into a tightly stop-
pered, evacuated flask containing 15NH4Cl (0.552 g, 10.14 mmol) and KOH(0.525 g, 9.36 mmol), consecutively
H2O (20 ml), MeCN (20 ml), Et3N (1.43 ml, 10.14 mmol), and a soln. of 3 (4.01 g, 7.18 mmol) in MeCN (40 ml)
were added. The mixture was stirred for 7 d at r.t. and then extracted with CH2Cl2/H2O, and the residue obtained
after evaporation was treated at 48 with THF/MeOH 5 :4 (292.5 ml) and 2m aq. NaOH(32.5 ml). After 30 min
at 48, AcOH(3.72 ml) was added, and the mixture was concentrated to 100 ml. Workup and drying (14 h,
0.05 mbar) gave a colorless residue, which was dissolved in pyridine (32 ml) and treated with (MeO)2TrCl
(5.29 g, 15.6 mmol). Workup after 45 min at r.t. and CC (SiO2 (100 g), hexane/AcOEt 4 :1 ! 2 :3 (‡1% Et3N))
afforded 4 (3.20 g, 61%). Colorless, solid foam. TLC (hexane/AcOEt 1 :1): Rf 0.55. 1H-NMR (400 MHz,
CDCl3): 1.07 1.14 (m, iPr3Si); 3.17 (d, J ˆ 5.7, OHÀC(3')); 3.52 (br. d, J ꢀ 1, CH2(5')); 3.80 (s, 2 MeO); 4.11
(m, HÀC(4')); 4.26 (t, J ˆ 4.1, HÀC(2')); 4.46 (q, J ˆ 5.5, HÀC(3')); 5.03, 5.23 (2d, J ˆ 4.7, OCH2O); 5.29
(dd, J ˆ 9.5, J(H,N)ˆ 2.1, HÀC(5)); 6.03 (d, J ˆ 3.1, HÀC(1')); 6.84 (d, J ˆ 8.6, 4 arom. H); 7.24 7.39
(m, 9 arom. H); 7.94 (d, J ˆ 8.1, HÀC(6)); 8.34 (d, J(H,N) ˆ 90.6, HÀN(3)). 13C-NMR (100 MHz, CDCl3):
12.0 (d, Me2CH); 17.9 (q, Me2CH); 55.4 (q, MeO); 62.3 (t, C(5')); 69.5 (d, C(2')); 83.0 (d, C(3')); 83.8 (d, C(4'));
87.3 (s, arom. C); 88.0 (d, C(1')); 90.8 (t, OCH2O); 102.3 (d, J(C,N) ˆ 5.6, C(5)); 113.4 (d, arom. C); 127.3, 128.1,
128.3, 130.3 (4d, arom. C); 135.2, 135.5 (2s, arom. C); 140.3 (d, C(6)); 150.0 (s, J(C,N) ˆ 17.0, C(2)); 158.9
(s, arom. C); 162.9 (s, J(C,N) ˆ 8.4, C(4)). 15N-NMR (40 MHz, CDCl3): 133.1. ESI-MS: 756.30 (100, [M ‡
‡
Na] ).
5'-O-(4,4'-Dimethoxytrityl)-2×-O-{[(triisopropylsilyl)oxy]methyl}(3-15N)cytidine (5). A soln. of 4 (1.30 g,
1.77 mmol) in pyridine (7.1 ml) was treated with DMAP (43 mg, 0.35 mmol) and Ac2O (0.34 ml, 3.54 mmol).
Workup after 3 h at r.t., co-evaporation with toluene (2 Â 20 ml), extraction with CH2Cl2/10% aq. citric acid and
sat. aq. NaHCO3 soln., drying (MgSO4), and evaporation gave a yellow solid foam (1.25 g), which was carefully
dried (14 h at 608/0.05 mbar) and dissolved in MeCN (6 ml). Meanwhile, under Ar and at 48, 4-chlorophenyl
phosphorodichloridate (1.30 g, 5.31 mmol) was added dropwise to a suspension of finely powdered 1H-1,2,4-
i
triazole (2.13 g, 30.8 mmol; dried by sublimation) in dry MeCN (12 ml). After 15 min at 48, Pr2NEt (4.5 ml,
26.6 mmol) was added, and after 40 min at r.t., the mixture was again cooled to 48 and treated with the MeCN
soln. obtained before (6 ml, containing 1.25 g of the intermediate nucleoside). After 6 h at r.t., the soln. was
diluted with dioxane (18 ml), treated with 25% aq. NH3 soln. (27 ml) and stirred for another 3 h at r.t.
Extraction with CH2Cl2/10% aq. citric acid and sat. aq. NaHCO3 soln. gave a solid yellow foam (1.15 g), which
was dissolved in THF/MeOH 5 :4 (66 ml), cooled to 48, and treated with 2n NaOH(7.3 ml). After 30 min at 4 8,
the soln. was treated with AcOH(0.85 ml) and concentrated to 30 ml. Workup and CC (SiO 2 (15 g), CH2Cl2 !
CH2Cl2/MeOH19 :1) gave 5 (1.17 g, 90%). Colorless foam. TLC (AcOEt): Rf 0.40. 1H-NMR (400 MHz,
CDCl3): 1.05 1.14 (m, iPr3Si); 3.39 (d, J ˆ 7.9, OHÀC(3')); 3.48 (dd, J ˆ 11.0, 2.7, HÀC(5')); 3.57 (dd, J ˆ 11.0,
1.5, H'ÀC(5')); 3.79 (s, 2 MeO); 4.06 (td, J ˆ 6.2, 8.2, HÀC(4')); 4.22 (br. d, J ˆ 4.9, HÀC(2')); 4.37
(m, HÀC(3')); 5.12, 5.26 (2d, J ˆ 4.7, OCH2O); 5.1 (d, J ˆ 7.2, C(5)); 5.96 (br. s, HÀC(1')); 6.84 (d, J ˆ 8.7,
4 arom. H ); 7.21 7.43 (m, 9 arom. H); 8.08 (d, J ˆ 7.4, H ÀC(6)). 13C-NMR (100 MHz, CDCl3): 12.0
(d, Me2CH); 17.9 (q, Me2CH); 55.3 (q, MeO); 61.6 (t, C(5')); 68.2 (d, C(2')); 83.1 (d, C(3')); 84.0 (d, C(4'));
86.9 (s, arom. C); 89.7 (d, C(1')); 90.9 (t, OCH2O); 94.1 (s, arom. C); 113.3 (d, arom. C); 127.1, 128.0, 128.4
(3d, arom. C); 130.3 (d, J(C,N) ˆ 4.2, C(5)); 135.5, 135.7, 141.5 (3s, arom. C); 144.7 (d, C(6)); 155.7 (s, J(C,N) ˆ
‡
7.4, C(2)); 158.7 (s, arom. C); 165.9 (s, J(C,N) ˆ 6.3, C(4)). ESI-MS: 733.35 (100, [M ‡ 1] ).
N4-Acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-{[(triisopropylsilyl)oxy]methyl}(3-15N)cytidine (6). A soln. of 5
(1.15 g, 1.57 mmol) in DMF (7.5 ml) was treated with Ac2O (225 mg, 2.2 mmol) and stirred for 8 h at r.t.
Extraction with sat. aq. NaHCO3 soln. and AcOEt/hexane 1 :1, followed by CC (SiO2 (12 g) hexane/AcOEt
4 :1 ! 3 :7 (‡1% Et3N)) gave 6 (1.10 g, 90%). Colorless solid foam. TLC (hexane/AcOEt 3 :7): Rf 0.50.
1H-NMR (400 MHz, CDCl3): 1.07 1.14 (m, iPr3Si); 2.24 (s, MeCO); 3.35 (d, J ˆ 8.4, OHÀC(3')); 3.55 (dd, J ˆ
11.1, 2.3, HÀC(5')); 3.61 (dd, J ˆ 11.1, 2.3, H'ÀC(5')); 3.83 (s, 2 MeO); 4.11 (td, J ˆ 2.0, 8.7, HÀC(4')); 4.24

Helvetica Chimica Acta Vol. 86 (2003)
3969
(d, J ˆ 4.9, HÀC(2')); 4.40 (dt, J ˆ 8.4, 5.0, HÀC(3')); 5.16, 5.30 (2d, J ˆ 4.7, OCH2O); 5.98 (s, HÀC(1')); 6.88
(d, J ˆ 8.8, 4 arom. H); 7.08 (d, J ˆ 7.4, HÀC(5)); 7.28 7.44 (m, 9 arom. H); 8.49 (d, J ˆ 7.4, HÀC(6)); 9.11 (br.
s, NHÀC(4)). 13C-NMR (100 MHz, CDCl3): 12.0 (d, Me2CH); 17.9 (q, Me2CH); 25.1 (q, MeCO); 55.4
(q, MeO); 61.3 (t, C(5')); 67.9 (d, C(2')); 83.5 (d, C(3')); 83.6 (d, C(4')); 87.2 (s, arom. C); 90.2 (d, C(1')); 90.9
(t, OCH2O); 96.5 (d, J(C,N) ˆ 1.0, C(5)); 113.4 (d, arom. C); 127.3, 128.2, 128.4, 130.3 (4d, arom. C); 135.7, 144.5
(2s, arom. C); 145.0 (d, C(6)); 155.4 (s, J(C,N) ˆ 7.7, C(2)); 158.8 (s, arom. C); 162.6 (s, J ˆ 7.4, C(4)); 170.0
‡
(s, MeCO). 15N-NMR (40 MHz, CDCl3): 208.1. ESI-MS: 775.3 (100, [M ‡ H] ).
3',5'-Di-O-acetyl-2'-O-{[(triisopropylsilyl)oxy]methyl}inosine (8). A soln. of 7 [9] (28.16 g, 35.29 mmol) in
41% aq. MeNH2 soln./THF 5 :4 (765 ml) was left for 20 min at r.t. and concentrated to 400 ml. After workup,
the residue was dissolved in AcOH/H2O 4 :1 (650 ml) and stirred for 30 min at r.t. The solvent was evaporated
(under addition of a small amount of H2O towards the end of the evaporation) and the residue co-evaporated
with toluene (3 Â 200 ml). To a well-stirred soln. of this residue in CH2Cl2 (200 ml), hexane (400 ml) was added
slowly. Filtration after 30 min gave a white solid, which was dissolved in pyridine (100 ml). After evaporation,
the residue was again dissolved in pyridine (175 ml) and treated with DMAP (0.43 g, 3.5 mmol) and Ac2O
(8.33 ml, 88.2 mmol). After 30 min at r.t., MeOH(15 ml) was added, the solvent evaporated, and the residue co-
evaporated with toluene (3 Â 100 ml) and dissolved in AcOH/H2O 7:3 (560 ml). At r.t., this soln. was treated
with five portions of NaNO2 (5 Â 40 g, 2.9 mol), which were added within 2.5 h. H2O (100 ml) was added and the
mixture extracted with CHCl3 (500 ml). The org. phase was extracted with H2O and sat. aq. NaHCO3 soln.,
dried (MgSO4), and evaporated. Recrystallization from AcOEt (400 ml) gave 8 (14.94 g, 78%). Colorless
crystals. M.p. 1908. TLC (CH2Cl2/MeOH9 :1): Rf 0.49. 1H-NMR (400 MHz, CDCl3): 0.93 (m, iPr3Si); 2.12, 2.17
(2s, 2 MeCO); 4.35 4.45 (m, HÀC(4'), CH2(5')); 4.86, 4.92 (2d, J ˆ 5.1, OCH2O); 5.07 (br. t, J ꢀ 5, HÀC(2'));
5.42 (dd, J ˆ 3.8, 5.1, HÀC(3')); 6.13 (d, J ˆ 5.9, HÀC(1')); 7.99 (s, HÀC(8)); 8.17 (s, HÀC(2)); 13.08
(s, HÀN(1)). 13C-NMR (100 MHz, CDCl3): 11.9 (d, Me2CH); 17.8 (q, Me2CH); 20.9 (q, MeCO); 63.5
(t, C(5')); 71.4 (d, C(3')); 76.8 (d, C(2')); 80.8 (d, C(4')); 87.6 (d, C(1')); 89.6 (t, OCH2O); 125.7 (s, C(5));
139.0 (d, C(8)); 145.2 (d, C(2)); 149.0 (s, C(4)); 159.3 (s, C(6)); 170.2, 170.5 (2s, MeCO). ESI-MS: 539.35 (100,
‡
[M ‡ H] ).
3',5'-Di-O-acetyl-1-nitro-2'-O-{[(triisopropylsilyl)oxy]methyl}inosine (9). At 48 and under Ar, (CF3CO)2O
(10.9 g, 52.0 mmol) was added dropwise to a well-stirred suspension of finely powdered NH4NO3 (2.08 g,
26 mmol; dried for 48 h at 608/0.05 mbar) in CH2Cl2 (35 ml). After 1 h at r.t., a clear soln. was obtained, to which
at À 208 a soln. of 8 (3.50 g, 78 mmol; dried for 48 h at 608/0.05 mbar) in CH2Cl2 (30 ml) was added slowly. The
reaction was kept for 20 min at À 208 under exclusion of light. Workup at 48 gave crude 9 (3.60 g) as a yellow oil,
which was used immediately for the next step. For characterization, 50 mg were subjected to CC (SiO2 (1 g),
CH2Cl2 ! CH2Cl2/MeOH19 :1): 35 mg ( ca. 70%) of pure 915). TLC (hexane/AcOEt 3 :7): Rf 0.81. 1H-NMR
(400 MHz, CDCl3): 0.88 1.09 (m, iPr3Si); 2.12, 2.17 (2s, 2 MeCO); 4.38 (br. t, J ꢀ 4, CH2(5')); 4.48 (dd, J ˆ 3.8,
7.3, HÀC(4')); 4.85, 4.91 (2d, J ˆ 5.1, OCH2O); 4.96 (t, J ˆ 5.7, HÀC(2')); 5.37 (dd, J ˆ 3.8, 5.0, HÀC(3')); 6.10
(d, J ˆ 6.0, HÀC(1')); 8.02 (s, HÀC(8)); 8.62 (s, HÀC(2)). 13C-NMR (100 MHz, CDCl3): 11.9 (d, Me2CH); 17.8
(q, Me2CH); 20.9 (q, MeCO); 63.4 (t, C(5')); 71.2 (d, C(3')); 76.8 (d, C(2')); 81.1 (d, C(4')); 87.7 (d, C(1')); 89.6
(t, OCH2O); 125.6 (s, C(5)); 140.1 (d, C(8)); 140.9 (d, C(2)); 145.4 (s, C(6)); 149.5 (s, C(4)), 170.1, 170.3
‡
(2s, MeCO). ESI-MS: 584.38 (100, [M ‡ H] ).
3',5'-Di-O-acetyl-2'-O-{[(triisopropylsilyl)oxy]methyl}(1-15N)inosine (10). Into a tightly stoppered, evac-
uated flask containing 15NH4Cl (0.46 g, 8.5 mmol) and KOH(0.43 g, 7.8 mmol), consecutively H 2O (16 ml),
MeCN (16 ml), Et3N (1.20 ml, 8.5 mmol), and a soln. of crude 9 (3.55 g, ca. 75 mmol) in MeCN (32 ml) were
added. The mixture was stirred for 6 h at r.t., extracted with CH2Cl2/H2O, and co-evaporated with toluene: crude
10 (3.40 g). Colorless solid foam. For characterization, a small amount was purified by prep. TLC. TLC (CH2Cl2/
1
MeOH9 :1): Rf 0.49. H-NMR (400 MHz, CDCl3): 0.87 1.06 (m, iPr3Si); 2.12, 2.17 (2s, 2 MeCO); 4.35 4.45
(m, HÀC(4'), CH2(5')); 4.86, 4.92 (2d, J ˆ 5.1, OCH2O); 5.07 (t, J ꢀ 5, HÀC(2')); 5.42 (dd, J ˆ 3.8, 5.1,
HÀC(3')); 6.13 (d, J ˆ 5.9, HÀC(1')); 7.99 (s, HÀC(8)); 8.17 (d, J(H,N) ˆ 7.5, HÀC(2)); 13.08 (d, J(H,N) ˆ
88.6, HÀN(1)). 13C-NMR (100 MHz, CDCl3): 11.9 (d, Me2CH); 17.8 (q, Me2CH); 20.9 (q, MeCO); 63.5
(t, C(5')); 71.4 (d, C(3')); 76.8 (d, C(2')); 80.8 (d, C(4')); 87.6 (4d, C(1')); 89.6 (t, OCH2O); 125.7 (s, J(C,N) ˆ 7.0,
C(5)); 139.0 (d, C(8)); 145.2 (d, J(C,N) ˆ 8.4, C(2)); 149.0 (s, C(4)); 159.3 (s, J(C,N) ˆ 9.8, C(6)), 170.2, 170.5
‡
(2s, MeCO). ESI-MS: 540.32 (100, [M ‡ H] ).
{3',5'-Di-O-acetyl-2'-O-{[(triisopropylsilyl)oxy]methyl}-b-d-ribofuranosyl}-6-(3-nitro-1H-1,2,4-triazol-1-
yl)(1-15N)purine (11). A suspension of crude 10 (3.35 g), 3-nitro-1,2,4-1H-triazole (2.60 g, 22.7 mmol), PPh3
15
)
During this CC, partial decomposition of 9 occurred (under formation of polar products).

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Helvetica Chimica Acta Vol. 86 (2003)
(4.10 g, 14.9 mmol), and I2 (3.46 g, 13.6 mmol) in toluene (130 ml) was heated to 958 and treated with iPr2NEt
(5.60 ml, 32.5 mmol). After stirring for 50 min at 958, the solvent was evaporated, and CC (SiO2 (100 g), hexane/
AcOEt 4 :1 ! 2 :3) gave 11 (2.34 g, 67% based on 8) as a yellow foam. TLC (AcOEt/hexane 1:1): Rf 0.49.
1H-NMR (400 MHz, CDCl3): 0.88 0.91 (m, iPr3Si); 2.13, 2.19 (2s, 2 MeCO); 4.45 (br. dd, J ꢀ 4, 8, CH2(5')); 4.51
(m, HÀC(4')); 4.88, 4.94 (2d, J ˆ 4.9, OCH2O); 5.21 (br. t, J ꢀ 5, HÀC(2')); 5.47 (dd, J ˆ 3.9, 5.2, HÀC(3')); 6.30
(d, J ˆ 5.7, HÀC(1')); 8.45 (s, HÀC(8)); 9.0 (d, J(H,N) ˆ 16.1, HÀC(2)); 9.82 (s, HÀC(triazole)). 13C-NMR
(100 MHz, CDCl3): 11.9 (d, Me2CH); 17.8 (q, Me2CH); 20.9 (q, MeCO); 63.3 (t, C(5')); 71.2 (d, C(3')); 76.8
(d, C(2')); 81.2 (d, C(4')); 88.2 (d, C(1')); 89.7 (t, OCH2O); 123.9 (s, C(4)); 143.9 (s, J(C,N) ˆ 7.0, C(6)); 145.8
(d, C(8)); 147.4 (d, C(triazole)); 152.6 (d, J(C,N) ˆ 2.5, C(2)); 154.4 (s, J(C,N) ˆ 3.2, C(5)); 164.1 (s, C(tri-
‡
azole)); 170.1, 170.4 (2s, MeCO). ESI-MS: 636.33 (100, [M ‡ H] ).
N6-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-{[(triisopropylsilyl)oxy]methyl}(1-15N)adenosine
(12).
Through the septum of an evacuated flask containing NH4Cl (730 mg, 13.5 mmol), DMSO (21 ml) was added,
t
followed by a soln. of BuOK (1.45 g, 13.0 mmol) and Et3N (1.90 ml, 13.5 mmol) in DMSO (22 ml). The soln.
was stirred for 10 min at r.t. and cooled to À 2008 (liq. N2). The flask was then quickly opened, solid 11 (1.75 g,
2.7 mmol) was added to the frozen soln., and the flask was placed in an autoclave. After 14 h at 508, the mixture
was added slowly to well-stirred H2O (1 l) of 48. After 1 h at 48, the suspension was filtered over Celite, and the
solid was washed with H2O. The filter was then placed on top of a flask and rinsed with acetone (5 Â 50 ml).
Evaporation of the filtrate and workup gave a white solid, which was dissolved in pyridine (10 ml). After
evaporation, the residue was again dissolved in pyridine (11 ml) and treated with DMAP (200 mg, 1.6 mmol)
and BzCl (2.28 g, 16.2 mmol) for 14 h at r.t. After workup and co-evaporation with toluene (2 Â 10 ml), the
residue was dissolved in CH2Cl2 and extracted with 10% aq. citric acid soln., followed by sat. aq. NaHCO3 soln.
The solid obtained after drying (MgSO4) and evaporation was dissolved in THF/MeOH 5 :4 (100 ml) and
treated at 48 with 2n NaOH(11 ml). After 30 min at 4 8, AcOH(1.40 ml) was added, and the mixture was
concentrated to 40 ml. The solid obtained after workup was dried (14 h, 0.05 mbar), dissolved in pyridine
(14 ml), treated with (MeO)2TrCl (1.83 g, 5.4 mmol), and kept for 30 min at r.t. Workup and CC (SiO2 (50 g),
hexane/AcOEt 4 :1 ! 3 :7) gave 12 (1.63 g, 70%). Colorless foam. TLC (AcOEt/hexane 7:3): Rf 0.60. 1H-NMR
(400 MHz, CDCl3): 1.05 1.15 (m, iPr3Si); 3.12 (d, J ˆ 3.2, OHÀC(3')); 3.42 (dd, J ˆ 3.8, 10.9, HÀC(5')); 3.53
(dd, J ˆ 3.8, 10.9, H'ÀC(5')); 3.73 (s, 2 MeO); 4.33 (dd, J ˆ 3.7, 7.4, HÀC(4')); 4.60 (br. dd, J ꢀ 3, 7, HÀC(3'));
5.00 (t, J ˆ 5.2, HÀC(2')); 5.01, 5.18 (2d, J ˆ 4.7, OCH2O); 6.27 (d, J ˆ 5.3, HÀC(1')); 6.81 6.83 (m, 4 arom. H);
7.22 7.65 (m, 12 arom. H); 8.05 (d, J ˆ 7.6, 2 arom. H); 8.23 (s, HÀC(8)); 8.74 (d, J(H,N) ˆ 15.9, HÀC(2)); 9.04
(br. s, NHÀC(6)). 13C-NMR (100 MHz, CDCl3): 11.9 (d, Me2CH); 17.9 (q, Me2CH); 55.4 (q, MeO); 63.4
(t, C(5')); 71.1 (d, C(2')); 82.3 (d, C(3')); 84.5 (d, C(4')); 86.7 (s, arom. C); 87.3 (d, C(1')); 91.0 (t, OCH2O); 113.3
(d, arom. C); 123.6 (s, J(C,N) ˆ 1.5, C(5)); 127.1, 128.0, 128.3 (3d, arom. C); 129.0, 130.2, 132.9 (3d, arom. C),
133.8, 135.8 (2s, arom. C); 142.0 (d, C(8)); 144.6 (s, arom. C); 149.6 (s, J(C,N) ˆ 5.2, C(6)); 151.8 (s, J(C,N) ˆ
2.4, C(4)); 152.8 (d, J(C,N) ˆ 3.0, C(2)); 158.7 (s, arom. C), 164.7 (s, PhCO). 15N-NMR (40 MHz, CDCl3): 227.2.
‡
ESI-MS: 861.27 (100, [M ‡ H] ).
9-[3',5'-Di-O-acetyl-2'-O-{[(triisopropylsilyl)oxy]methyl}-b-d-ribofuranosyl]-6-(3-nitro-1H-1,2,4-triazol-1-
yl)purine (13). A suspension of 8 (6.53 g, 12.1 mmol), 3-nitro-1,2,4-1H-triazole (4.85 g, 42.5 mmol), PPh3
(7.64 g, 29.1 mmol), and I2 (6.47 g, 25.5 mmol) in toluene (240 ml) was heated to 958 and treated with iPr2NEt
(10.4 ml, 60.7 mmol). After stirring for 50 min at 958, the solvent was evaporated, and CC (SiO2 (160 g), hexane/
AcOEt 4 :2 ! 2 :3) gave 13 (6.92 g, 90%). Yellow foam. TLC (AcOEt/hexane 1 :1): Rf 0.49. 1H-NMR
(400 MHz, CDCl3): 0.88 0.91 (m, iPr3Si); 2.13, 2.19 (2s, 2 MeCO); 4.45 (br. dd, J ꢀ 4, 8, CH2(5')); 4.51
(m, HÀC(4')); 4.88, 4.94 (2d, J ˆ 4.9, OCH2O); 5.21 (br. t, J ꢀ 5, HÀC(2')); 5.47 (dd, J ˆ 3.9, 5.2, HÀC(3')); 6.30
(d, J ˆ 5.7, HÀC(1')); 8.45 (s, HÀC(8)); 9.0 (s, HÀC(2)); 9.82 (s, HÀC(triazole)). 13C-NMR (100 MHz, CDCl3):
11.9 (d, Me2CH); 17.8 (q, Me2CH); 20.9 (q, MeCO); 63.3 (t, C(5')); 71.2 (d, C(3')); 76.8 (d, C(2')); 81.2
(d, C(4')); 88.2 (d, C(1')); 89.7 (t, OCH2O); 123.9 (s, C(4)); 143.9 (s, C(6)); 145.8 (d, C(8)); 147.4 (d, C(tria-
zole)); 152.6 (d, C(2)); 154.4 (s, C(5)); 164.1 (s, C(triazole)); 170.1, 170.4 (2s, MeCO). ESI-MS: 635.38 (100,
‡
[M ‡ H] ).
3',5'-Di-O-acetyl-2'-O-{[(triisopropylsilyl)oxy]methyl}(N6-15N)adenosine (14). Through the septum of an
evacuated flask containing 15NH4Cl (1.78 g, 32.8 mmol), DMSO (29 ml) was added, followed by a soln. of
tBuOK (3.43 g, 30.6 mmol) and Et3N (4.60 ml, 32.8 mmol) in DMSO (28 ml). The soln. was stirred for 10 min at
r.t. and cooled to À 2008 (liq. N2). The flask was then quickly opened, solid 13 (6.93 g, 10.92 mmol) was added to
the frozen soln., and the flask was placed in an autoclave. After 14 h at 508, the mixture was added slowly to well-
stirred H2O (2.5 l) of 48. After 1 h at 48, the suspension was filtered over Celite, and the solid was washed with
H2O. The filter was then placed on top of a flask and rinsed with acetone (5 Â 100 ml). Evaporation of the
filtrate and workup gave a white solid, which was dissolved in pyridine (10 ml). After evaporation, the residue

Helvetica Chimica Acta Vol. 86 (2003)
3971
was dissolved in pyridine (55 ml) and treated with DMAP (27 mg, 0.22 mmol) and Ac2O (0.21 ml, 2.2 mmol).
After 30 min at r.t., MeOH(5 ml) was added, and the solvent was evaporated. CC (SiO (140 g), CH2Cl2 !
2
CH2Cl2/MeOH93 :7) gave 14 (4.99 g, 85%). Colorless foam. TLC (CH2Cl2/MeOH9 :1): Rf 0.57. 1H-NMR
(400 MHz, CDCl3): 0.88 1.01 (m, iPr3Si); 2.11, 2.16 (2s, 2 MeCO); 4.37 4.48 (m, HÀC(4'), CH2(5')); 4.84, 4.90
(2d, J ˆ 4.8, OCH2O); 5.21 (t, J ˆ 5.76, HÀC(2')); 5.48 (dd, J ˆ 3.2, 5.3, HÀC(3')); 5.76 (d, J(H,N) ˆ 89.6,
NH2(6)); 6.12 (d, J ˆ 5.7, HÀC(1')); 7.93 (s, HÀC(8)); 8.34 (s, HÀC(2)). 13C-NMR (100 MHz, CDCl3): 11.9
(d, Me2CH); 17.7 (q, Me2CH); 20.9 (q, MeCO); 63.6 (t, C(5')); 71.6 (d, C(3')); 76.4 (d, C(2')); 80.7 (d, C(4'));
87.6 (d, C(1')); 89.6 (t, OCH2O); 120.4 (s, J(C,N) ˆ 3.3, C(5)); 139.5 (d, C(8)); 150.0 (s, C(4)); 153.3
(d, J(C,N) ˆ 2.5, C(2)); 155.6 (s, J(C,N) ˆ 20.7, C(6)); 170.2, 170.5 (2s, MeCO). MALDI-MS: 638.60 (100,
‡
[M ‡ H] ).
3',5'-Di-O-acetyl-2'-O-{[(triisopropylsilyl)oxy]methyl}(N6-15N)adenosine 1-Oxide (15). A suspension of
70% 3-chloroperbenzoic acid (5.50 g, 22.3 mmol) and anh. MgSO4 (5 g) in CH2Cl2 (55 ml) was stirred for 1 h at
r.t. and then filtered into a flask containing solid 14 (3.00 g, 5.57 mmol). The soln. was stirred for 14 h at r.t. and
then diluted with CHCl3 (150 ml) and 5% aq. NaI soln. (100 ml). After stirring for 10 min, solid Na2S3O3 was
added until the red color (I2) disappeared. The org. phase was extracted with sat. aq. NaHCO3 soln. and
evaporated. CC (SiO2 (75 g), CH2Cl2 ! CH2Cl2/MeOH93 :7) gave 15 (2.43 g, 79%) as a pink foam and 14
1
(0.54 g, 18%) as a colorless foam. 15: TLC (CH2Cl2/MeOH9 :1): Rf 0.46. H-NMR (400 MHz, CDCl3): 0.88
1.01 (m, iPr3Si); 2.10, 2.16 (2s, 2 MeCO); 4.34 4.44 (m, HÀC(4'), CH2(5')); 4.84, 4.90 (2d, J ˆ 4.8, OCH2O);
5.13 (t, J ˆ 5.4, HÀC(2')); 5.40 (dd, J ˆ 3.5, 5.2, HÀC(3')); 6.11 (d, J ˆ 6.1, HÀC(1')); 7.50 (br. d, J(H,N) ˆ 91.0,
NH2ÀC(6)); 8.04 (s, HÀC(8)); 8.69 (s, HÀC(2)). 13C-NMR (100 MHz, CDCl3): 11.8 (d, Me2CH); 17.7
(q, Me2CH); 20.9 (q, MeCO); 63.4 (t, C(5')); 71.3 (d, C(3')); 76.5 (d, C(2')); 80.8 (d, C(4')); 87.7 (d, C(1'));
89.6 (t, OCH2O); 120.0 (s, J(C,N) ˆ 1.3, C(5)); 142.1 (d, C(8)); 142.4 (s, C(4)); 144.2 (d, C(2)); 148.5
‡
(s, J(C,N) ˆ 22.5, C(6)); 170.1, 170.4 (2s, MeCO). MALDI-MS: 638.60 (100, [M ‡ H] ).
N6-Methoxy-9-[2'-O-{[(triisopropylsilyl)oxy]methyl}-b-d-ribofuranosyl](1-15N)purine-2,6-diamine (16). To
a soln. of 15 (2.43 g, 4.39 mmol) in MeOH(110 ml), BrCN (558 mg, 5.27 mmol) was added, and the mixture was
stirred for 3 h at r.t. After removing the solvent, the residue (pink foam) was dried (2 h, 0.05 mbar), redissolved
in DMF (17 ml), and treated with Et3N (1.54 ml, 10.97 mmol; freshly filtered over Al2O3) for 45 min at r.t. Then
MeI (1.49 g, 10.53 mmol) was added, and the soln. was stirred for 4 h at r.t. After evaporation, the residue was
dried (14 h, 0.05 mbar), resuspended in THF (36 ml), and treated with 0.5m aq. NaOH(36 ml) for 40 min at r.t.
Then the pHwas adjusted to 7.4 with 1m aq. HCl, EtOH (85 ml) was added, and the mixture was stirred for 6 h at
608. After concentration to 50 ml, workup, and drying (14 h, 0.05 mbar), crude 16 (2.4 g) was obtained as yellow
foam. For characterization, a small amount was purified by prep. TLC. TLC (CH2Cl2/MeOH9 :1): Rf 0.40.
1H-NMR (400 MHz, CDCl3): 0.88 1.06 (m, iPr3Si); 3.11 (s, OHÀC(3')); 3.74 (m, CH2(5')); 3.93 (s, MeO); 3.96
(br. d, J ˆ 12.4, HÀC(4')); 4.52 (d, J ˆ 5.13, HÀC(3')); 4.81, 5.02 (2d, J ˆ 5.1, OCH2O); 4.92 (dd, J ˆ 4.7, 7.5,
HÀC(2')); 5.01 (br. d, J ˆ 5.3, OHÀC(5')); 5.80 (d, J ˆ 8.0, HÀC(1')); 7.01 (br. s, NH2(2)); 7.57 (s, HÀC(8));
9.26 (br. s, NHÀC(6)). 13C-NMR (100 MHz, CDCl3): 11.9 (d, Me2CH); 17.8 (q, Me2CH); 63.7 (t, C(5')); 65.0
(q, MeO); 72.3 (d, C(3')); 81.2 (d, C(2')); 87.9 (d, C(4')); 89.6 (d, C(1')); 90.6 (t, OCH2O); 114.4 (s, J(C,N) ˆ 2.0,
C(5)); 139.1 (d, C(8)); 149.3 (s, C(4)); 156.5 (s, J ˆ 8.5, C(2)); 159.1 (s, J(C,N) ˆ 5.0, C(6)). ESI-MS: 500.35
‡
(100, [M ‡ H] ).
N2-Acetyl-9-[2'-O-{[(triisopropylsilyl)oxy]methyl}-b-d-ribofuranosyl](1-15N)purine-2,6-diamine (17). A
soln. of crude 16 (2.4 g) was dissolved in Ac2O/pyridine 1:2 (22 ml) and heated for 3 h at 1008. Evaporation
and co-evaporation with toluene (2 Â 50 ml) gave a residue, which was dissolved in CH2Cl2 (40 ml), adsorbed on
SiO2 (12 g), and filtered through a pad of SiO2 (12 g) (hexane/AcOEt 1:1 ! 9 :1, then AcOEt/MeOH99 :1).
The 1:1 mixture of peracetylated intermediates (2.11 g, TLC (hexane/AcOEt 1:9): Rf 0.49 and 0.66) was
dissolved in THF/MeOH 5 :4 (165 ml), cooled to 48 and treated with 2m aq. NaOH(18 ml). After 10 min at 4 8,
AcOH(2.1 ml) was added and the soln. concentrated to 80 ml. After workup, the N2-acetylated intermediate
was dissolved in THF/H2O 1:1 (27 ml) and treated under H2 (balloon) with a suspension of Raney-Ni (5.3 g,
prepared according to [28]) in MeOH(9 ml). After 1 h at 80 8, the hot soln. was filtered, the residue was rinsed
with hot MeOH/THF 1 :1 (5 Â 20 ml), and the combined filtrates were evaporated. Workup gave crude 17
(1.2 g). Colorless foam. For characterization, a small amount was purified by prep. TLC. TLC (CH2Cl2/acetone
1:1): Rf 0.51. 1H-NMR (400 MHz, CDCl3): 0.90 1.09 (m, iPr3Si); 2.43 (s, MeCO); 3.18 (s, OHÀC(3')); 3.77
(m, CH2(5')); 3.96 (br. d, J ˆ 2.4, HÀC(4')); 4.55 (d, J ˆ 4.4, HÀC(3')); 4.86, 5.04 (2d, J ˆ 4.8, OCH2O); 4.90
(dd, J ˆ 4.9, 6.9, HÀC(2')); 5.60 (br. s, OHÀC(5')); 5.90 (d, J ˆ 7.0, HÀC(1')); 6.21 (br. s, NH2ÀC(6)); 7.74
(s, HÀC(8)); 8.55 (br. s, NHÀC(2)). 13C-NMR (100 MHz, CDCl3): 11.9 (d, Me2CH); 17.8, (q, Me2CH); 25.2
(q, MeCO); 63.1 (t, C(5')); 71.8 (d, C(3')); 81.5 (d, C(2')); 87.2 (d, C(4')); 89.0 (d, C(1')); 90.7 (t, OCH2O); 117.9

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Helvetica Chimica Acta Vol. 86 (2003)
(s, J(C,N) ˆ 1.1, C(5)); 140.3 (d, C(8)); 149.6 (s, C(4)); 152.7 (s, J(C,N) ˆ 6.7, C(2)); 156.3 (s, J(C,N) ˆ 7.0, C(6));
‡
171.2 (s, MeCO). ESI-MS: 512.30 (100, [M ‡ H] ).
N2-Acetyl-5'-O-(4,4'-dimethoxytrityl)-2×-O-{[(triisopropylsilyl)oxy]methyl}(1-15N)guanosine (18). At r.t., a
soln. of crude 17 (1.2 g) in AcOH/H2O 7:3 (73 ml) was treated with 5 portions of NaNO2 (5 Â 5 g, 36 mmol),
which were added within 2 h. H2O (20 ml) was added and the mixture extracted with CHCl3 (150 ml). The org.
phase was extracted with H2O and sat. aq. NaHCO3 soln., dried (MgSO4), and evaporated. The residue was co-
evaporated with toluene (2 Â 20 ml), dried (14 h, 0.05 mbar), redissolved in pyridine (22 ml), and treated with
(MeO)2TrCl (1.78 g, 5.27 mmol). After 1 h at r.t., workup and CC (SiO2 (35 g), hexane/AcOEt 1:1 ! 1:9, then
AcOEt/MeOH99 :1) gave 18 (1.18 g, 33% from 15). Off-white foam. TLC (CH2Cl2/MeOH19 :1): Rf 0.55.
1H-NMR (400 MHz, CDCl3): 0.98 1.10 (m, iPr3Si); 1.46 (s, MeCO); 3.08 (d, J ˆ 3.2, OHÀC(3')); 3.12 (dd, J ˆ
2.9, 10.7, HÀC(5')); 3.53 (dd, J ꢀ 2, 10.6, H'ÀC(5')); 3.74, 3.78 (2s, 2 MeO); 4.22 (q, J ˆ 1.7, HÀC(4')); 4.60 (br.
dd, J ˆ 4.8, 1.6, HÀC(3')); 4.93, 5.11 (2d, J ˆ 4.6, OCH2O); 5.06 (dd, J ˆ 5.2, 6.9, HÀC(2')); 5.90 (d, J ˆ 7.3,
HÀC(1')); 6.76 6.78 (m, 4 arom. H); 7.18 7.54 (m, 9 arom. H); 7.81 (s, HÀC(8)); 7.84 (br. s, NHÀC(2)); 11.79
(d, J(H,N) ˆ 91.1, HÀN(1)). 13C-NMR (100 MHz, CDCl3): 11.9 (d, Me2CH); 17.9 (q, Me2CH); 23.6 (q, MeCO);
55.4 (q, MeO); 63.9 (t, C(5')); 70.9 (d, C(2')); 81.4 (d, C(3')); 84.5 (d, C(4')); 86.4 (d, C(1')); 86.8 (s, arom. C);
91.1 (t, OCH2O); 113.4 (d, arom. C); 122.6 (s, J(C,N) ˆ 8.3, C(5)); 127.3, 128.1, 128.2, 130.1 (4d, arom. C); 135.7,
136.2 (2s, arom. C); 139.3 (d, C(8)); 145.2 (s, arom. C); 146.9 (s, J(C,N) ˆ 14.2, C(2)); 148.3 (s, C(4)); 155.61
(s, J(C,N) ˆ 10.5, C(6)), 158.9 (s, arom. C); 171.5 (s, MeCO). 15N-NMR (40 MHz, CDCl3): 129.4. ESI-MS:
‡
815.29 (100, [M ‡ H] ).
5'-O-(4,4'-Dimethoxytrityl)-2'-O-{[(triisopropylsilyl)oxy]methyl}(3-15N)uridine 3'-(2-Cyanoethyl Diisopro-
pylphosphoramidite) (19). A soln. of 4 (1.20 g, 1.64 mmol) in CH2Cl2 (6.6 ml) was treated consecutively with
iPr2NEt (0.70 ml, 4.10 mmol) and cyanoethyl diisopropylphosphoramidochloridite (466 mg, 1.97 mmol). After
stirring for 14 h at r.t., the mixture was subjected to CC (SiO2 (25 g), hexane/AcOEt 9 :1 ! 3 :7 (‡2% Et3N)):
19 (1.53 g, 92%; 1:1 mixture of diastereoisomers). Colorless foam. TLC (hexane/AcOEt 1:1): Rf 0.65. 1H-NMR
(400 MHz, CDCl3): 1.02 1.05 (m, iPr3Si); 1.16 1.17 (m, (Me2CH)2N); 2.39 (t, J ˆ 6.4, 1 H, CH2CN); 2.64
(dt, J ˆ 2.7, 6.3, 1 H, CH2CN); 3.39 (dt, J ˆ 2.6, 9.5, HÀC(5')); 3.51 3.70 (m, 4 H (Me 2CH)2N, H'ÀC(5'),
POCH2); 3.79, 3.80 (2s, 2 MeO); 3.82 3.97 (m, 1 H, POCH2); 4.19, 4.27 (2q, J ˆ 2.4, HÀC(4')); 4.40 4.48
(m, HÀC(2'), HÀC(3')); 4.99 5.06 (m, OCH2O); 5.32, 5.36 (2dd, J ˆ 8.1, J(H,N) ˆ 2.7, HÀC(5)); 6.12 (d, J ˆ
4.7, 0.5 H, HÀC(1')); 6.13 (d, J ˆ 4.8, 0.5 H, HÀC(1')); 6.82 6.86 (m, 4 arom. H); 7.23 7.42 (m, 9 arom. H);
7.81, 7.88 (2d, J ˆ 8.1, HÀC(6)); 8.16 (br. d, J(H,N) ˆ 83.8, HÀN(3)). 31P-NMR (162 MHz, CDCl3): 150.8, 151.3.
‡
MALDI-MS: 934.87 (100, [M ‡ H] ).
N4-Acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-{[(triisopropylsilyl)oxy]methyl}(3-15N)cytidine 3'-(2-Cyanoethyl
i
Diisopropylphosphoramidite) (20). As described for 19, with 6 (1.20 g, 1.56 mmol), CH2Cl2 (6.3 ml), Pr2NEt
(0.60 ml, 3.90 mmol), and cyanoethyl diisopropylphosphoramidochloridite (444 mg, 1.88 mmol). CC (SiO2
(30 g), hexane/AcOEt 1:1 ! AcOEt (‡2% Et3N)) gave 20 (1.42 g, 94%; 1:1 mixture of diastereoisomers).
1
Colorless foam. TLC (hexane/AcOEt 3 :7): Rf 0.75. H-NMR (400 MHz, CDCl3): 0.98 1.08 (m, iPr3Si); 1.13,
1.16 (2d, J ˆ 6.7, (Me2CH)2N); 2.21, 2.22 (2s, MeCO); 2.38 (t, J ˆ 6.4, 1 H, CH2CN); 2.60 (q, J ˆ 6.1, 1 H,
CH2CN); 3.42 3.69 (m, 1 Hof POCH 2, (Me2CH)2N, HÀC(5')); 3.81, 3.82 (2s, 2 MeO); 3.92 (m, 1 H, POCH2);
4.27 4.42 (m, HÀC(2'), HÀC(4')); 4.51 (m, HÀC(3')); 5.15 5.22 (m, OCH2O); 6.15 (d, J ˆ 1.3, 0.5 H,
HÀC(1')); 6.16 (d, J ˆ 1.8, 0.5 H, HÀC(1')); 6.83 6.87 (m, 4 arom. H); 6.94, 7.01 (2d, J ˆ 7.4, HÀC(5)); 7.26
7.44 (m, 9 arom. H); 8.37, 8.48 (2d, J ˆ 7.5, HÀC(6)); 9.11, 9.18 (2 br. s, NHÀC(4)). 31P-NMR (162 MHz,
‡
CDCl3): 150.6, 151.9. MALDI-MS: 975.68 (100, [M ‡ H] ).
N6-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-{[(triisopropylsilyl)oxy]methyl}(1-15N)adenosine 3'-(2-Cya-
noethyl Diisopropylphosphoramidite) (21). As described for 19, with 12 (1.40 g, 1.20 mmol), CH2Cl2 (6.4 ml),
iPr2NEt (0.68 ml, 4.00 mmol), and cyanoethyl diisopropylphosphoramidochloridite (456 mg, 1.92 mmol). CC
(SiO2 (28 g), hexane/AcOEt 9 :1 ! 3 :7 (‡2% Et3N)) gave 21 (1.54 g, 90%; 1:1 mixture of diastereoisomers)).
1
Colorless foam. TLC (hexane/AcOEt 1:1): Rf 0.70. H-NMR (400 MHz, CDCl3): 0.86 0.93 (m, iPr3Si); 1.08,
1.18, 1.20 (3d, J ˆ 6.6, (Me2CH)2N); 2.38 (t, J ˆ 6.5, 1 H, CH2CN); 2.64 (dt, J ˆ 2.5, 6.4, 1 H, CH2CN); 3.34
(dt, J ˆ 4.3, 10.5, 1 H, POCH2); 3.51 3.72 (m, 1 Hof POCH 2, (MeCH)2N, HÀC(5')); 3.76, 3.77 (2s, 2 MeO);
3.83 3.99 (m, 1 H, POCH2); 4.38, 4.43 (2q, J ˆ 3.9, HÀC(4')); 4.67 4.75 (m, HÀC(3')); 4.95, 4.97, 5.01 (3d, J ˆ
5.0, OCH2O); 5.19, 5.21 (2t, J ˆ 5.3, HÀC(2')); 6.19, 6.21 (2d, J ˆ 5.5, HÀC(1')); 6.75 6.79 (m, 4 arom. H);
7.17 7.61 (m, 12 arom. H); 8.01 (d, J ˆ 7.3, 2 arom. H); 8.17, 8.19 (2s, HÀC(8)); 8.67, 8.71 (2d, J(H,N) ˆ 10.1,
HÀC(2)); 9.04 (br. s, NHÀC(6)). 31P-NMR (162 MHz, CDCl3) 150.8, 151.6. MALDI-MS: 1061.48 (100, [M ‡
‡
H] ).
N2-Acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-{[(triisopropylsilyl)oxy]methyl}(1-15N)guanosine 3'-(2-Cya-
noethyl Diisopropylphosphoramidite) (22). A as described for 19, with 18 (1.10 g, 1.35 mmol), CH2Cl2

Helvetica Chimica Acta Vol. 86 (2003)
3973
(5.5 ml), iPr2NEt (0.59 ml, 3.4 mmol), and cyanoethyl diisopropylphosphoramidochloridite (384 mg,
1.62 mmol). CC (SiO2 (25 g), hexane/AcOEt 1:1 ! AcOEt, then AcOEt/MeOH99 :1 ! 19 :1 (‡2% Et3N))
gave 22 (1.23 g, 90%; 1:1 mixture of diastereoisomers). Colorless foam. TLC (hexane/AcOEt 3 :7): Rf 0.55.
1H-NMR (400 MHz, CDCl3): 0.92 0.93 (m, iPr3Si); 1.02 1.19 (4d, J ˆ 6.8, (Me2CH)2N); 1.70, 1.82 (2s, MeCO);
2.17 2.35 (m, 1 H , CHCN); 2.69 2.82 (m, 1 H , CHCN); 3.22 (dd, J ˆ 3.6, 10.7, 0.5 H, HÀC(5')); 3.26 (dd, J ˆ
2
2
5.3, 10.6, 0.5 H, HÀC(5')); 3.46 3.63 (m, 3.5 H, (MeCH)2N, HÀC(5'), POCH2); 3.68 (m, 0.5 H, POCH2); 3.757,
3.760, 3.768, 3.773 (4s, 2 MeO); 3.92 4.05 (m, 1.5 H, POCH2); 4.23 (br. q, J ꢀ 2, 0.5 H, HÀC(4')); 4.33 (br. t, J ꢀ
3, 0.5 H, HÀC(4')); 4.53 (ddd, J ˆ 1.8, 4.7, 12.1, 0.5 H, HÀC(3')); 4.62 (m, 0.5 H, HÀC(3')); 4.92 (s, 1 H ,
OCH2O); 4.89, 4.99 (2d, J ˆ 5.3, 1 H, OCH2O); 5.02 (dd, J ˆ 5.0, 7.4, 0.5 H, HÀC(2')); 5.06 (t, J ˆ 5.8, 0.5 H,
HÀC(2')); 5.86 (d, J ˆ 5.5, 0.5 H, HÀC(1')); 5.97 (d, J ˆ 7.3, 0.5 H, HÀC(1')); 6.77 6.82 (m, 4 arom. H); 7.20
7.53 (m, 9 arom. H); 7.73, 7.80 (2s, HÀC(8')); 8.19, 8.51 (2 br. s, NHÀC(2)); 11.7 (br. d, J(H,N) ˆ 82.5,
HÀN(1)). 31P-NMR (162 MHz, CDCl3): 149.8, 150.4. MALDI-MS: 1015.46 (100, [M ‡ H] ).
‡
15
15
15
RNA Sequence r(GpGpCpGpUpUpUpUp(3- N)CpGpCpCpUpUpCpGpGpGpCp(1- N)Gp(1- N)ApUpUpUp-
15
15
UpUpA p(3- N)Up(3- N)CpGpCpU) (23). The assembly was carried out on a Gene Assembler Plus (Pharmacia),
from 320 mg of solid support (loading 32 mmol/g) and 2'-O-tom-protected ribonucleoside phosphoramidites
according to [9]. The solid support was subsequently removed from the cartridge and treated with a 1:1 mixture
of 12m MeNH2 in H2O and 8m MeNH2 in EtOH(4 ml) for 6 h at 35 8. By centrifugation, the supernatant soln.
was separated from the solid support and evaporated, and the residue was dissolved in 1m Bu4NF ¥ 3 H2O soln. in
THF (4 ml). After 14 h at 308, 1m Tris ¥ HCl buffer (pH 7.4, 4 ml) was added. The soln. was concentrated to 4 ml
and desalted on 4 NAP columns (Pharmacia) according to the manufacturer×s instructions. The crude RNA
sequence (Fig. 2,a) was purified by AE-HPLC (15 70% B in 60 min, 10 injections). The fractions containing
pure 23 were pooled ( ! 30 ml), treated with 1m aq. Et3N ¥ AcOH(pH7, 5 ml), and applied to 2 Sepak cartridges
(Waters): after elution of the salts with 0.1m aq. Et3N ¥ AcOH(pH7, 10 ml), followed by H 2O (20 ml), 23
‡
(Et3NH form) was eluted with MeCN/H2O 1:1 (5 ml). After evaporation, the residue was twice dissolved in
H2O (2 Â 1.5 ml) and then treated with (NH4)HCO3 (2 Â 8 mg). The mixture was evaporated, the residue
dissolved in H2O (2 ml), and the soln. desalted on two NAP columns according to the manufacturer×s
‡
instructions: aq. soln of 23 (30 mg (determined by UV spectroscopy, e (260 nm) 323800 l ¥ molÀ1 ¥ cmÀ1); NH4
form, containing 1 mol-% of Et3NH (according to 1H-NMR); 2.7 mmole, 27% yield (based on solid support)).
‡
AE-HPLC (15 70% B in 30 min): tR 25.2 min (Fig. 2,a). LC-ESI-MS (neg. mode): tR 11.5 min, 10129.4 amu
(after deconvolution, Fig. 2,b). 1H-NMR (400 MHz, D2O, c ˆ 2 mm): see Fig. 2,c.
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¡
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Received September 1, 2003