Synthesis of the anticodon hairpin (t)RNA(f)(Met) containing N-{[9-(β- D-ribofuranosyl)-9H-purin-6-yl]carbamoyl}-l-threonine (= N6-{{[(1S,2R)-1- carboxy-2-hydroxypropyl]amino}carbonyl}adenosine, t6A)

Article abstract of DOI:10.1002/(SICI)1522-2675(20000119)83:1<152::AID-HLCA152>3.0.CO;2-1

As part of our studies on the structure of yeast ¹RNA(f)(Met), we investigated the incorporation of N-{[9-(β-D-ribofuranosyl)-9H-purin-6- yl]carbamoyl}-L-threonine (t⁶A) in the loop of a RNA 17-mer hairpin. The carboxylic function of the L-threonine moiety of t⁶A was protected with a 2- (4-nitrophenyl)ethyl group, and a (tert-butyl)dimethylsilyl group was used for the protection of its secondary OH group. The 2'-OH function of the standard ribonucleotide building blocks was protected with a [(triisopropylsilyl)oxy]methyl group. Removal of the base-labile protecting groups of the final RNA with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and then with MeNH₂ was done under carefully controlled conditions to prevent hydrolysis of the carbamate function, leading to loss of the L-threonine moiety.

Full text of DOI:10.1002/(SICI)1522-2675(20000119)83:1<152::AID-HLCA152>3.0.CO;2-1

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Helvetica Chimica Acta ± Vol. 83 (2000)
Synthesis of the Anticodon Hairpin tRNAfMet Containing N-{[9-(b-d-
Ribofuranosyl)-9H-purin-6-yl]carbamoyl}-l-threonine (ˆ N6-{{[(1S,2R)-1-
Carboxy-2-hydroxypropyl]amino}carbonyl}adenosine, t6A)
by Valerie Boudoua), James Langridgeb), Arthur Van Aerschota), Chris Hendrixa), Alan Millarb), Patrick
Weissc), and Piet Herdewijna)*
a) Laboratory of Medicinal Chemistry, Rega Institute for Medical Research, K.U. Leuven, Minderbroederstraat
10, B-3000 Leuven (Fax. ‡32-16-337340; e-mail: Piet.Herdewijn@rega.kuleuven.ac.be)
b) Micromass UK Ltd. Floats Road, Wythenshawe, Manchester M23 9LZ, UK
c) Xeragon AG, Technoparkstrasse 1, CH-8005 Zürich
Dedicated to Prof. Dr. Frank Seela on the occasion of his 60th birthday
As part of our studies on the structure of yeast tRNAfMet, we investigated the incorporation of N-{[9-(b-d-
ribofuranosyl)-9H-purin-6-yl]carbamoyl}-l-threonine (t6A) in the loop of a RNA 17-mer hairpin. The
carboxylic function of the l-threonine moiety of t6A was protected with a 2-(4-nitrophenyl)ethyl group, and a
(tert-butyl)dimethylsilyl group was used for the protection of its secondary OH group. The 2'-OH function of the
standard ribonucleotide building blocks was protected with a [(triisopropylsilyl)oxy]methyl group. Removal of
the base-labile protecting groups of the final RNA with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and then
with MeNH2 was done under carefully controlled conditions to prevent hydrolysis of the carbamate function,
leading to loss of the l-threonine moiety.
t
Introduction. ± RNAs contain a whole series of modified nucleotides which
influence their structure and function [1]. The exact influence of the modified
t
t
nucleotide on RNA structure and on the recognition by RNA synthetase is largely
unknown. This situation is changing slowly because of both the progress in synthetic
methodologies in obtaining RNA and the availability of high-resolution NMR
instruments.
As a part of our studies on the structure of yeast tRNAfMet, we plan to determine the
t
solution conformation of the anticodon hairpin of this yeast RNA. The nucleotide
sequence of Saccharomyces cerevisiae RNAfMet in the cloverleaf form is depicted in
t
Fig. 1. [2] This tRNAMet was purified from bakerꢀs yeast, and it can be converted to the
formylated methionyl-tRNAf by extracts of E. coli [2]. The same anticodon hairpin is
t
also found in the initiator RNA of Torulopsis utilis [3], a yeast belonging to Fungi
imperfecti.
Both loop structures contain the modified-adenosine nucleoside t6A (ˆ N-{[9-(b-d-
ribofuranosyl)-9H-purin-6-yl]carbamoyl}-l-threonine in the position next to the
anticodon, t6A representing an adenosine nucleoside substituted at the N6-position
with a l-threonine moiety via a carbonyl function [4]. Besides the occurrence in the
t
above mentioned RNA, a N-(9H-purin-6-ylcarbamoyl)-l-threonine ribonucleotide
t
has also been discovered in several other RNAs [5 ± 10] and has been isolated
from human urine [11][12]. The enzymatic synthesis of N-(9H-purin-6-ylcarbamoyl)-l-
threonine riboside has been investigated [13][14]. The structure of N-(9H-purin-6-

Helvetica Chimica Acta ± Vol. 83 (2000)
153
Fig. 1. Nucleotide sequence of yeast RNAfMet showing the hairpin structure with t6A in the position next to the
t
anticodon
ylcarbamoyl)-l-threonine riboside was confirmed by X-ray diffraction [15][16]. It was
t
observed that RNAs with codons starting with adenine mostly contain t6A next to
the 3'-side of the anticodon [6], and t6A is thought to stabilize U ´ A and U ´ G
anticodon ´ codon base pairs being formed adjacent to the 5'-side of the modified
nucleoside [17]. It was observed that t6A-deficient tRNA is significantly less efficient in
binding to ribosomes as compared to normal tRNA, and that t6A is required for proper
codon ´ anticodon interaction [18]. Another study points to the importance of t6A in the
recognition of the anticodon loop of tRNA1Ile by isoleucyl-tRNA synthetase from E. coli
[19]. NMR Studies point to the importance of t6A as a binding site for magnesium ion
t
in RNA [20][21].
The synthetic challenge for the incorporation of t6A into RNA is situated in
selecting an appropriate way to synthesize the modified nucleosides and to introduce
protecting groups which are stable during RNA synthesis and can be removed easily at
the end of the synthesis.
Results and Discussion. ± Synthesis. The naturally occurring nucleoside N-{[(9-(b-
d-ribofuranosyl)-9H-purin-6-yl]carbamoyl}-l-threonine (t6A) has been synthesized
before [22 ± 26]. Chheda and Hong described the displacement of the ethoxy group of
ethyl [9-(2,3,5-tri-O-acetyl-b-d-ribofuranosyl)-9H-purin-6-yl]carbamate with l-threo-
nine followed by deprotection of the acetyl groups with NH3/MeOH [22][25]. The
same reaction scheme was used later by Martin and Schlimme [26]. The alternative of
using an isocyanate intermediate of protected l-threonine was less successful [22]. As
the a-amino group of l-threonine is more nucleophilic than the 6-amino group of the
adenine base, we investigated the inverted approach and converted the protected
adenosine to a N6-isocyanate derivative.

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The selection of the protecting groups for l-threonine is based on the following
considerations: they should a) be stable during oligonucleotide synthesis, b) be easily
removable after oligonucleotide synthesis with standard reagents, c) in the case of the
carboxylic-acid protecting group, be removable before the ammonia-deprotection step
of the oligonucleotide thus avoiding amide formation, and d) be removable without
racemization of the l-amino acid. Therefore, we selected the (tert-butyl)dimethylsilyl
group (tBuMe2SiCl) for protecting the secondary OH function of l-threonine and the
2-(4-nitrophenyl)ethyl group as a protecting group for the carboxylic acid. The latter
protecting group can be introduced and removed without causing racemizing side
reactions [27]. l-Threonine (1) was converted into its 2-(4-nitrophenyl)ethyl ester and
isolated as p-toluenesulfonate salt 2 (Scheme 1) [27]. Protection of the secondary OH
group with (tert-butyl)dimethylsilyl chloride in pyridine catalyzed by 1H-imidazole
yielded the protected l-threonine 3.
Scheme 1. Synthesis of Protected l-Threonine
The OH groups of adenosine were first protected as acetates ( ! 5; Scheme 2) [26].
The 6-NH2 function of the protected adenosine 5 was reacted wtih triphosgene under
reflux. The isocyanate 6 was not isolated but was reacted directly with the protected l-
threonine 3 to give 7 in low yield (19%). The acetyl groups of 7 could selectively be
removed with methanolic ammonia solution at room temperature without con-
comitant conversion of the 2-(4-nitrophenyl)ethyl ester to an amide function ( ! 8).
Subsequent protection of the primary OH group with 4,4'-dimethoxytrityl chloride
((MeO)2TrCl) in pyridine ( ! 9; 94% yield), followed by reaction with tBuMe2SiCl in
THF in the presence of AgNO3 and pyridine gave the 2'-O-silylated nucleoside
derivative 10 (44% yield), besides the undesired 3'-O-silylated compound (30% yield).
The modified building block 11 for oligonucleotide synthesis was obtained by
phosphitylation of the 3'-OH group of according to 10 standard procedures as
described before [28].

Helvetica Chimica Acta ± Vol. 83 (2000)
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Scheme 2. Synthesis of the Protected t6A Phosphoramidites and Structure of the Protected Regular Nucleoside
Phosphoramidites Used for Oligonucleotide Synthesis

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Oligoribonucleotide Synthesis and Characterization. Oligoribonucleotide synthesis
is currently carried out with a 2'-O-(tert-butyl)dimethylsilyl protecting group. Due to
the bulkiness of this protecting group, coupling times are rather long and coupling
yields are lower than in oligodeoxyribonucleotide synthesis. Therefore, we used the
recently developed [(triisopropylsilyl)oxy]methyl ((iPr)3SiOCH2; TOM) group to
protect the 2'-OH function of the standard nucleosides (see amidites 12a ± d), allowing
an efficient chemical synthesis of RNA. The (iPr)3SiOCH2 protecting group allowed us
to carry out RNA synthesis with short coupling times (2 min) and high coupling yields
(> 99%). The building blocks were used in 0.1m concentrations and were activated with
1-(benzylthio)-1H-tetrazole. Thus, a 17-mer corresponding to the anticodon loop
of RNAfMet was synthesized in which the adenine base at the 11th position was
t
replaced by the N-{[9-(b-d-ribofuranosyl)-9H-purin-6-yl]carbamoyl}(5'-CAGGGCU-
CAUt6AACCCUG-3'). After ꢁ(MeO)2Tr-offꢀ synthesis, the solid-phase material was
treated with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in THF to remove the 2-(4-
nitrophenyl)ethyl protecting groups, and then with MeNH2 in EtOH/H2O. The
(iPr)3SiOCH2 groups were removed with Bu4NF in THF. The oligoribonucleotide was
desalted on a Sephadex (G10) column, purified on a Dionex-Nucleopac-PA-100
column, and desalted again. The deprotection with DBU was necessary to obtain the
correct final material; when this deprotection step was skipped and the protected
oligoribonucleotide directly treated with MeNH2, the methyl carboxamide of the t6A
insert was obtained. More vigorous deprotection conditions led to loss of the whole l-
threonine moiety by hydrolysis of the carbamate function.
The modified RNA 17-mer was desalted with cation-exchange beads before it was
submitted to analysis by mass spectrometry. Fig. 2,a, shows the ESI-MS with several
peaks corresponding to multiply charged ions of the sample in the charge states [M
4 H]4 to [M 11 H]11 . The Max Ent 1 processed spectrum (Fig. 2,b) indicates the
experimental monoisotopic mass to be 5527.91 Da, confirming the identity of the RNA
17-mer (calc. 5527.80 Da).
Before starting NMR experiments, we determined the concentration-dependent Tm
of the synthesized oligonucleotide. As can be seen in Fig. 3, the thermal stability of the
oligomer did not change with increasing concentration of the oligonucleotide,
indicating formation of a stable hairpin structure. Both Tm in 0.1m NaCl (648) and
the shape of the curve were independent of oligonucleotide concentration.
Conclusion. ± The modified nucleoside. N-{[9-(b-d-ribofuranosyl)-9H-purin-6-
yl]carbamoyl}-l-threonine (t6A) was successfully incorporated into the loop of a
hairpin RNA. The (tert-butyl)dimethylsilyl group was used for the protection of the
secondary OH group of the modified nucleosides. The carboxy group was protected
with a 2-(4-nitrophenyl)ethyl group. The 2'-O-{[(triisopropylsilyl)oxy]methyl}-protect-
ed regular nucleoside phosphoramidites were used as building blocks for RNA
synthesis. The integrity of the final hairpin was established by ESI-MS. Thermal-
stability studies indicated the stability of the RNA hairpin, which is now used for
structural studies.
This research was supported by a grant for the K.U. Leuven (GOA 97/11). Arthur Van Aerschot is a
research associate of the Flemish Fund of Scientific Research. We are grateful to Dr. Jan Claereboudt,

Helvetica Chimica Acta ± Vol. 83 (2000)
157
Fig. 2. a) ESI-Mass spectrum of the RNA 17-mer corresponding to the anticodon loop of tRNAfMet in which the
adenine base at position 11 is replaced by N-{[9-(b-d-ribofuranosyl)-9H-purin-6-yl]carbamoyl}-l-threonine
(t6A) and b) its Max Ent 1 processed spectrum, indicating the experimental monoisotopic mass. See Exper. Part
for details.

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Helvetica Chimica Acta ± Vol. 83 (2000)
Fig. 3. Concentration-dependent Tm of the RNA hairpin with incorporated t6A
Micromass, Belgium, for all the efforts in providing us with the Q-TOF mass spectra. We thank Chantal
Biernaux for editorial help.
Experimental Part
General. Anh. solvents were obtained as follows: DMF was dried over molecular sieves; CH2Cl2 was stored
over P2O5, refluxed, and distilled; THF was stored over Na/benzophenone, refluxed, and distilled; pyridine was
stored over CaH2 refluxed, and distilled. Column chromatography (CC): Acros silica gel (0.060 ± 0.200 nm).
TLC: precoated Macherey-Nagel Alugram SIL G/UV254 plates. 1H- and 13C-NMR Spectra: Varian-Gemini-200
spectrometer; d in ppm rel. to SiMe4 as internal standard, J in Hz; the tyrosine numbering is used for the side
‡
chain at NH2 C(6) of adenosine in 7 ± 11. Liquid secondary-ion mass spectra (LSI-MS): Cs as primary-ion
beam Kratos-Concept-IH (Kratos; Manchester, UK); solns. in NBA (ˆ 3-nitrobenzyl alcohol), THGLY
(ˆthioglycerol), of NPOE (ˆ2-nitrophenyl octyl ether); acceleration of secondary ions at 6 ZV; scans at 10 s/
decade from m/z 1500 down to m/z 50. ESI-MS. Micromass-Q-TOF mass spectrometer (Whytenshawe,
Manchester, UK) fitted with a standard electrospray-ion source.
l-Threonine 2-(4-Nitrophenyl)ethyl Ester 4-Methylbenzenesulfonate Salt (2) [27]. l-Threonine (500 mg,
4.2 mmol), 2-(4-nitrophenyl)ethanol (npe-OH; 12.6 mmol, 2.11 g) and TsOH (12.6 mmol, 2.40 g) were heated
in toluene (100 ml) at 1058 for 17 h in a Dean-Stark apparatus. The soln. was cooled to r.t., and Et2O (25 ml) was
added. The oily residue was decanted, and the upper layer was removed to collect the oil. Precipitation of 2 was
realized by adding to the oil MeOH (25 ml) and Et2O (100 ml): 1.17 g (63%) of 2, identical to the compound
described in [27]. 1H-NMR ((D6)DMSO): 1.14 (d, J ˆ 6.6, Me(g)); 2.29 (s, MeC6H4); 3.10 (t, J ˆ 6.2,
CH2CH2O); 3.89 (d, J ˆ 4.0, CH(a)); 4.0 ± 4.1 (m, CH(b)); 4.45 (t, J ˆ 6.2, CH2CH2O); 5.6 (br. s, OH); 7.11
(d, J ˆ 8.2, 2 arom. H(Ts)); 7.48 (d, J ˆ 8.2, 2 arom. H(Ts)); 7.58 (d, J ˆ 8.8, 2 arom. H(npe)); 8.1 ± 8.2 (m, NH3,
2 arom. H(npe)). 13C-NMR ((D6)DMSO): 19.96 (Me(g)); 20.72 (MeC6H4); 33.83 (CH2CHO); 57.87
(CH2CH2O); 64.91 (CH(a)); 65.43 (CH(b)); 123.52 (arom. C(npe)); 125.65 (arom. C(Ts)); 128.14 (arom.
C(npe)); 130.38 (arom. C(Ts)); 137.73 (arom. C(Ts)); 146.32 (2 arom. C(Ts, npe)); 146.53 (arom. C(npe));
‡
168.23 (COO). LSI-MS (THGLY/NBA): 483 ([M ‡ K] ).
O-[(tert-Butyl)dimethylsilyl]-l-threonine 2-(4-Nitrophenyl)ethyl Ester (3). Compound
2 (500 mg,
1.15 mmol) was dissolved in dry pyridine (30 ml) and treated with one half of tBuMe2SiCl (260 mg, 3.45 mmol)
and 1H-imidazole (120 g, 3.45 mmol). After 10 min, the second half was added, and the reaction was allowed to
continue for 17 h at r.t. The mixture was diluted with CH2Cl2 (500 ml) and washed successively with sat.
NaHCO3 soln. (2 Â 300 ml) and H2O (3 Â 300 ml). The org. layer was dried, evaporated, co-evaporated with
toluene and MeOH and purified by CC (silica gel, 0 ± 6% MeOH/CH2Cl2): 416 mg (95%) 3. Oil. 1H-NMR

Helvetica Chimica Acta ± Vol. 83 (2000)
159
((D6)DMSO): 0.17, 0.06 (2s, 2 MeSi); 0.75 (s, tBuSi); 1.11 (d, J ˆ 6.2, Me(g)); 1.6 (br. s, NH2); 3.06 (t, J ˆ 6.2,
CH2CH2O); 3.17 (d, J ˆ 2.4, CH(a)); 3.9 ± 4.4 (m, CH(b), CH2CH2O); 7.57 (d, J ˆ 8.8, 2 arom. H); 8.18 (d, J ˆ
8.8, 2 arom. H). 13C-NMR ((D6)DMSO): 5.44, 4.47 (2 MeSi); 17.59 (Me3CSi); 20.42 (Me(g)); 25.58
(Me3CSi); 34.14 (CH2CH2O); 59.99 (CH2CH2O); 64.21 (CH(a)); 69.74 (CH(b)); 123.61 (2 arom. C); 130.50
‡
(2 arom. C); 146.89 (2 arom. C); 174.39 (COO). LSI-MS (THGLY): 383 ([M ‡ H] ). Anal. calc. for
C18H30N2O5Si (382.5): C 56.52, H 7.90, N 7.32; found: C 56.81, H 7.72, N 7.18.
N6-{{{(1S,2R)-2-{[(tert-Butyl)dimethylsilyl]oxy}-1-{[2-(4-nitrophenyl)ethoxy]carbonyl}propyl}amino}car-
bonyl}adenosine 2',3',5'-Triacetate (7). A suspension of 5 [26] (2.40 g, 6.14 mmol) in dry toluene (300 ml) was
treated with triphosgene (3.64 g, 12.28 mmol), and the mixture was heated under reflux (1208) for 4 h to give
crude isocyanate 6. The soln. was evaporated and then dissolved in anh. CH2Cl2/toluene 1:1 (60 ml). A soln. of 3
(1.76 g, 4.60 mmol) in dry toluene (30 ml) was added dropwise to the soln. of 6, and the reaction was continued
for 17 h at 808. The brown mixture was evaporated and directly purified by CC (silica gel, 0 ± 5% MeOH/
CH2Cl2): 934 mg (19%) of pure 7. 1H-NMR ((D6)DMSO): 0.03, 0.07 (2s, 2 MeSi); 0.91 (s, tBuSi); 1.26 (d, J ˆ
6.2, Me(g)); 2.09, 2.12, 2.16 (3s, 3 MeCO); 3.03 (t, J ˆ 6.2, CH2CH2O); 4.3 ± 4.6 (m, CH(a), CH(b), CH2CH2O,
H
C(4'), 2 H C(5')); 5.68 (t, J ˆ 4.9, H C(3')); 6.06 (t, J ˆ 5.4, H C(2')); 6.24 (d, J ˆ 5.4, H C(1')); 7.30
(d, J ˆ 8.5, 2 arom. H (npe)); 7.92 (d, J ˆ 8.5, 2 arom. H (npe)); 8.32, 8.48 (2s, H C(2), H C(8)); 8.61 (br. s,
NH C(6)); 10.03 (d, J ˆ 9.0, NH C(a)). 13C-NMR ((D6)DMSO): 5.55, 4.43 (2 MeSi); 17.69 (Me3CSi);
20.31, 20.46, 20.64 (3 MeCO); 21.00 (Me(g)); 25.44 (Me3CSi); 34.66 (CH2CH2O); 59.58 (CH2CH2O); 63.04
(CH2(5')); 64.37 (CH(a)); 68.59 (CH(b)); 70.66 (CH(3')); 72.93 (CH(2')); 80.43 (CH(4')); 86.68 (CH(1'));
121.04 (C(5)); 123.50 (2 arom. C (npe)); 129.72 (2 arom. C (npe)); 141.89 (CH(8)); 145.56 (2 arom. C (npe));
150.42 (CH(2)); 151.45 (C(4), C(6)); 154.40 (NHCONH); 169.48, 169.69, 170.51 (3 MeCO); 170.99
‡
(COOCH2CH2). LSI-MS (THGLY): 802 ([M ‡ H] ). Anal. calc. for C35H47N7O13Si (801.9): C 52.42, H 5.91,
N 12.23; found: C 52.21, H 6.02, N 12.14.
N6-{{{(1S,2R)-2-{[(tert-Butyl)dimethylsilyl]oxy}-1-{[2-(4-nitrophenyl)ethoxy]carbonyl}propyl}amino}car-
bonyl}adenosine (8). A methanolic NH3 soln. (100 ml) of 7 was stirred at r.t. for 3 h. Evaporation and
purification by CC (silica gel, 0 ± 10% MeOH/CH2Cl2) afforded pure
8
(598 mg, quant.). 1H-NMR
((D6)DMSO): 0.13, 0.01 (2s, 2 MeSi); 0.82 (s, tBuSi); 1.15 (d, J ˆ 6.2, Me(g)); 3.04 (t, J ˆ 6.0, CH2CH2O);
3.5 ± 3.8 (m, 2 H C(5')); 4.0 (m, H C(4')); 4.2 (m, H C(3')); 4.2 ± 4.5 (m, CH(a)), CH(b), CH2CH2O); 4.6
(m, H C(2')); 5.12 (t, J ˆ 5.5, OH C(5')); 5.22 (d, J ˆ 4.8, OH C(3')); 5.52 (d, J ˆ 5.6, OH C(2')); 6.01
(d, J ˆ 5.2, H C(1')); 7.48 (d, J ˆ 8.6, 2 arom. H (npe)); 8.00 (d, J ˆ 8.6, 2 arom. H (npe)); 8.36, 8.70 (2s,
H
C(2), H C(8)); 9.86 (d, J ˆ 9.2, NH C(a)); 9.93 (br. s, NH C(6)). 13C-NMR ((D6)DMSO): 5.68, 4.50
(2 MeSi); 17.44 (Me3CSi); 20.90 (Me(g)); 25.36 (Me3CSi); 33.98 (CH2CH2O); 59.03 (CH2CH2O); 61.24
(CH2(5')); 64.82 (CH(a)); 68.34 (CH(b)); 70.26 (CH(3')); 74.11 (CH(2')); 85.67 (CH(4')); 88.01 (CH(1'));
121.13 (C(5)); 123.37 (2 arom. C(npe)); 130.35 (2 arom. C(npe)); 142.52 (CH(8)); 146.68 (2 arom. C(npe));
‡
150.29 (C(4), C(6)); 154.06 (NHCONH); 170.84 (COOCH2CH2). LSI-MS (THGLY): 676 ([M ‡ H] ). Anal.
calc. for C29H41N7O10Si (675.8): C 51.54, H 6.12, N 14.51; found: C 51.32, H 6.17, N 14.23.
N6-{{{(1S,2R)-2-{[(tert-Butyl)dimethylsilyl]oxy}-1-{[2-(4-nitrophenyl)ethoxy]carbonyl}propyl}amino}car-
bonyl}-5'-O-(4,4'-dimethoxytrityl)adenosine (9). To a cooled (08) soln. of 8 (854 mg, 1.26 mmol) in dry pyridine
(15 ml), a soln. of 4,4'-dimethoxytrityl chloride (596 mg, 1.76 mmol) in dry CH2Cl2 (5 ml) was added dropwise.
After 15 min at 08, the mixture was allowed to warm to r.t. and the reaction continued for 17 h. MeOH (5 ml)
was added, the soln. evaporated, and the residue dissolved in CH2Cl2 (200 ml). The org. layer was washed
successively with sat. NaHCO3 soln. (2 Â 150 ml), brine (2 Â 150 ml), and H2O (2 Â 150 ml), and then dried,
evaporated, and co-evaporated. Purification by CC (silica gel, 0 ± 3% MeOH/CH2Cl2 1% Et3N) afforded 1.16 g
(94%) of 9. 1H-NMR ((D6)DMSO): 0.13, 0.01 (2s, 2 MeSi); 0.82 (s, tBuSi); 1.18 (d, J ˆ 5.8, Me(g)); 3.03
(t, J ˆ 6.2, CH2CH2O); 3.3 (m, 2 H C(5')); 3.70 (s, 2 MeO); 4.1 (m, H C(4')); 4.3 ± 4.5 (m, H C(3'), CH(a),
CH(b), CH2CH2O); 4.8 (m, H C(2')); 5.26 (d, J ˆ 5.8, OH C(3')); 5.61 (d, J ˆ 5.6, OH C(2')); 6.03 (d, J ˆ
4.0, H C(1')); 6.7 ± 7.4 (m, 13 H, (MeO)2Tr); 7.48 (d, J ˆ 8.8, 2 arom. H (npe)); 7.99 (d, J ˆ 8.8, 2 arom. H
(npe)); 8.25, 8.59 (2s, H C(2), H C(8)); 9.88 (d, J ˆ 9.2, NH C(a)); 9.98 (br. s, NH C(6)). 13C-NMR
((D6)DMSO): 5.68, 4.50 (2 MeSi); 17.44 (Me3CSi); 20.93 (Me(g)); 25.36 (Me3CSi); 34.01 (CH2CH2O);
55.08 (2 MeO); 59.02 (CH2CH2O); 63.76 (CH2(5')); 64.85 (CH(a)); 68.37 (CH(b)); 70.35 (CH(3')); 73.14
(CH(2')); 83.34 (CH(4')); 85.58 (CH(1')); 88.89 ((MeO)2Tr); 113.23, 126.77, 127.89, 129.89, 135.75, 158.24 (arom.
C(MeO)2Tr)); 120.88 (C(5)); 123.40 (2 arom. C (npe)); 130.41 (2 arom. C (npe)); 143.13 (CH(8)); 145.13
(CH(2)); 146.35, 146.74 (2 arom. C (npe)); 150.50 (C(4), C(6)); 154.11 (NHCONH); 170.93 (COOCH2CH2).
‡
LSI-MS (THGLY) 1000 ([M ‡ Na] ). Anal. calc. for C50H59N7O12Si (978.1): C 61.40, H 6.08, N 10.02; found:
C 61.21, H 5.81, N 9.83.

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Helvetica Chimica Acta ± Vol. 83 (2000)
2'-O-[(tert-Butyl)dimethylsilyl]-N6-{{{(1S,2R)-2-{[(tert-butyl)dimethylsilyl]oxy}-1-{[2-(4-nitrophenyl)eth-
oxy]carbonyl}propyl}amino}carbonyl}-5'-O-(4,4'-dimethoxytrityl)adenosine (10). To
a soln. of 9 (1.10 g,
1.13 mmol) in dry THF (15 ml), AgNO3 (230 mg, 1.35 mmol) was added. The mixture was sonicated and
stirred 10 min, before tBuMe2SiCl (221 mg, 1.47 mmol) was added. After 17 h at r.t., TLC indicated incomplete
reaction. Thus, more AgNO3 (54 mg, 0.32 mmol), tBuMe2SiCl (51 mg, 0.34 mmol), and pyridine (100 ml) were
added. After 3 h more, the mixture was filtered through Celite into a sat. NaHCO3 soln. and extracted with
CH2Cl2 (300 ml). The org. layer was washed with sat. NaHCO3 soln. (2 Â 200 ml), brine (2 Â 200 ml), and H2O
(2  200 ml), dried, and evaporated. Purification by CC (silica gel, 0 ± 2% MeOH/CHCl3 ‡ 1% Et3N) followed
by purification with a Chromatotron ꢂ apparatus (4-mm layer; eluants: 35, 45, 55, 65, and 75% AcOEt/hexane)
1
afforded pure 10 (539 mg, 44%). H-NMR ((D6)DMSO): 0.14, 0.04, 0.01 (3s, 4 MeSi); 0.74, 0.79 (2s, 2
tBuSi); 1.16 (d, J ˆ 6.2, Me(g)); 3.02 (t, J ˆ 5.7, CH2CH2O); 3.3 (m, 2 H C(5')); 3.70 (s, 2 MeO); 4.1
(m, H C(4')); 4.2 ± 4.4 (m, H C(3'), CH(a), CH(b), CH2CH2O); 4.98 (t, J ˆ 5.0, H C(2')); 5.19 (d, J ˆ 5.4,
OH C(3')); 6.04 (d, J ˆ 4.8, H C(1')); 6.7 ± 7.5 (m, 15 arom. H, (MeO)2Tr, npe); 7.98 (d, J ˆ 8.8, 2 arom. H
(npe)); 8.25, 8.61 (2s, H C(2), H C(8)); 9.87 (d, J ˆ 8.8, NH C(a)); 10.02 (br. s, NH C(6)). 13C-NMR
((D6)DMSO): 5.71, 5.35, 4.83, 4.56 (4 MeSi); 17.41, 17.84 (2Me3CSi); 20.87 (Me(g)); 25.27, 25.58, 25.82
(2 Me3CSi); 33.99 (CH2CH2O); 55.05 (2 MeO); 58.99 (CH2CH2O); 63.7 (CH2(5')); 64.85 (CH(a)); 68.34
(CH(b)); 70.22 (CH(3')); 74.74 (CH(2')); 83.79 (CH(4')); 85.64 (CH(1')); 88.77 ((MeO)2Tr); 113.20, 126.77,
127.86, 129.89, 135.66, 158.27 (arom. C ((MeO)2Tr)); 120.94 (C(5)); 123.34 (2 arom. C (npe)); 130.38 (2 arom. C
(npe)); 143.16 (CH(8)); 145.19 (CH(2)); 146.32, 146.71 (2 arom. C (npe)); 150.29, 150.53 (C(4), C(6)); 154.06
‡
(NHCONH); 170.90 (COOCH2CH2): LSI-MS (THGLY): 1114 ([M ‡ Na] ). Anal. calc. for C56H73N7O12Si2
(1092.4): C 61.57, H 6.74, N 8.98; found: C 61.36, H 6.51, N 8.82.
2'-O-[(tert-Butyl)dimethylsilyl]-N6-{{{(1S,2R)-2-{[(tert-butyl)dimethylsilyl]oxy}-1-{[2-(4-nitrophenyl)eth-
oxy]carbonyl}propyl}amino}carbonyl}-5'-O-(4,4'-dimethoxytrityl)adenosine 3'-(2-Cyanoethyl Diisopropylphos-
phoramidite) (11). A soln. of 10 (0.40 mmol) in CH2Cl2 (5 ml) was phosphitylated with N,N-diisopropylethyl-
amine (3equiv.) and 2-cyanoethyl diisopropylphosphoramidochloridite (1.5 equiv.) The reaction was difficult to
follow as only a slight change of Rf was seen on TLC in several systems. Therefore, after 2 h at r.t., an extra equiv.
of both reagents was added, and after further 2 h, the mixture was quenched by addition of H2O. The mixture
was partitioned between CH2Cl2 (50 ml) and sat. aq. NaHCO3 soln. (30 ml), the org. phase washed with brine
(2Â 30 ml), dried (Na2SO4), and evaporated, and the residue purified by flash chromatography (silica gel (30 g),
hexane/acetone/Et3N 68 :30 :2) and precipitation in cold hexane: 0.29 mmol (72%) of 11. LSI-MS (NPOE):
‡
1293 ([M ‡ H] ; for C65H90N9O13PSi2, calc. 1291.593). 31P-NMR: (external ref. H3PO4): 149.75, 151.55.
Oligoribonucleotide Synthesis. The oligoribonucleotide containing the l-threonine-modified adenosine
nucleoside was prepared on a Pharmacia-Gene-Assembler-Special-DNA/RNA synthesizer at 1.5-mmol scale by
solid-phase 2-cyanoethyl phosphoramidite chemistry with the 2'-O-[(triisopropylsilyl)oxy]methyl (iPr3SiOCH2;
TOM) protecting group for the standard RNA monomers 12a ± d. The synthesis was performed ꢁ(MeO)2Tr-offꢀ
on LCAA CPG (500 Š) with standard RNA cycles, except for the shorter coupling times (2 min) for TOM
phosphoramidite chemistry. Coupling time for the modified phosphoramidite 11 and the subsequent TOM
amidite was 10 min. (Benzylthio)-1H-tetrazole (0.35m) was used as activator. Average coupling efficiency for
the synthesis was 99.3% as determined by the release of the 4,4-dimethoxytrityl cation. The solid-phase material
was treated with 10% DBU in THF (1 ml) for 90 min at 458 to remove the 2-(4-nitrophenyl)ethyl protecting
group. After lyophilization, the solid phase was reacted with 10m MeNH2 in 50% aq. EtOH (500 ml) at r.t. for
4.5 h. The oligoribonucleotide in MeNH2 soln. was decanted, the solid support washed with sterile H2O (2 Â
200 ml) and then the soln. evaporated in a Speedvac concentrator. Desilylation of the 2'-O-[(triisopropylsilyl)-
oxy]methyl protecting groups was achieved by dissolving the dried oligoribonucleotide in 1m Bu4NF in THF
(500 ml) at r.t. for 13.5 h. Tris buffer (pH 7.4, 500 ml) was added, and the oligoribonucleotide was desalted by CC
(Sephadex (G10) sterile H2O). The fully deprotected oligoribonucleotide was purified by a further CC (Dionex
Nucleopac PA-100 (4 Â 250 mm), 508, gradient 10 ! 500 mm sodium perchlorate (10 ± 35%) with in 30 min),
10 mm Tris, and 5m urea at pH 8.0). The desired product was eluted at 24 min at a flow rate of 1 ml/min. Then,
the oligoribonucleotide was again desalted by CC (Sephadex (G10)) and lyophilized on a Speedvac
concentrator.
For ESI-MS, the RNA sample was dissolved in H2O and desalted by means of Dowex-50W cation-exchange
beads. The supernatant was diluted by the addition of an equal volume of both MeCN and a 1% NH3 soln. The
Q-TOF mass spectrometer was operated in the negative mode with a source temp. of 808, a counter current gas
flow rate of 40 l/h and a potential of ca. 3200 V applied to the source needle (sample flow rate 7 ml/min). The
instrument was calibrated with a two-point calibration using the singly charged ion of the monomer and dimer of
raffinose.

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Received September 15, 1999