The Synthesis of 2′-O-[(Triisopropylsilyl)oxy] methyl (TOM) Phosphoramidites of Methylated Ribonucleosides (m1G, m2G, m22G, m1I, m3U, m4C, m6A, m62A) for Use in Automated RNA Solid-Phase Synthesis

Article abstract of DOI:10.1007/s00706-003-0592-1

The straightforward synthesis of eight methylated ribonucleoside phosphoramidites is described. These building blocks allow for incorporation of the naturally occuring nucleosides 1-methylguanosine (m¹G), N ²-methylguanosine (m²G), N²N ²-dimethylguanosine (m²₂G), 1-methylinosine (m¹I), 3-methyluridine (m³U), N⁴- methylcytidine (m⁴C), N⁶-methyladenosine (m⁶A), and N⁶,N⁶-dimethyladenosine (m⁶₂A) into oligoribonucleotides by automated RNA solid-phase synthesis. In all cases, the ribose 2′-hydroxyl group of the building blocks is masked by the recently introduced [(triisopropylsilyl)oxy]methyl (TOM) group.

Full text of DOI:10.1007/s00706-003-0592-1

Monatshefte f€ur Chemie 134, 851–873 (2003)
DOI 10.1007/s00706-003-0592-1
The Synthesis of 20-O-[(Triisopropylsilyl)oxy]
methyl (TOM) Phosphoramidites
of Methylated Ribonucleosides (m1G, m2G,
m2 G, m1I, m3U, m4C, m6A, m6 A) for Use
in Automated RNA Solid-Phase Synthesis
2
2
1
1
2;a
¨
Claudia Hobartner , Christoph Kreutz , Elke Flecker ,
Elke Ottenschlager , Werner Pils , Karl Grubmayr ,
2;b
2;c
2
¨
and Ronald Micura1;Ã
1
Institute of Organic Chemistry, University of Innsbruck, Austria
2
Institute of Chemistry, University of Linz, Austria
Received January 14, 2003; accepted January 20, 2003
Published online May 2, 2003 # Springer-Verlag 2003
Summary. The straightforward synthesis of eight methylated ribonucleoside phosphoramidites is
described. These building blocks allow for incorporation of the naturally occuring nucleosides
1-methylguanosine (m1G), N2-methylguanosine (m2G), N2,N2-dimethylguanosine ðm2 GÞ, 1-methyl-
2
inosine (m1I), 3-methyluridine (m3U), N4-methylcytidine (m4C), N6-methyladenosine (m6A), and
N6,N6-dimethyladenosine ðm6 AÞ into oligoribonucleotides by automated RNA solid-phase synthesis.
2
In all cases, the ribose 20-hydroxyl group of the building blocks is masked by the recently introduced
[(triisopropylsilyl)oxy]methyl (TOM) group.
Keywords. Phosphoramidites; TOM-chemistry; RNA solid-phase synthesis; Methylation; Modified
nucleosides.
Introduction
The rapid growth of demand for chemically synthesized RNAs is unequivocally
associated with the biological phenomenon of RNA interference (RNAi) [1]. There-
by, double-stranded RNA effects the silencing of genes which are homologous in
sequence to either of the RNA strands in the duplex [2]. The phenomenon results
a
Present address: Voestalpine, Voest-Alpine Strasse 3, A-4031 Linz
b
Present address: Baxter AG Austria, Lange Allee 51, A-1221 Vienna
c
Present address: DSM Fine Chemicals Austria, St. Peter Strasse 25, A-4021 Linz
Ã
Corresponding author. E-mail: ronald.micura@uibk.ac.at

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from degradation of the corresponding mRNA and can also be induced efficiently
by very short duplex RNA of 21- to 23-base pairs, so-called small interfering RNAs
(siRNA) [3]. siRNA is the upcoming gene silencing methodology and the key com-
ponents of this new technology are short chemically synthesized oligoribonucleo-
tides [4].
Most approaches for the chemical synthesis of RNA oligonucleotides have fo-
cused on retaining the established DNA protecting group concept of the acid-labile
Fig. 1. Scheme of the automated RNA solid-phase synthesis using N-acetyl-20-O-[(triisopropyl-
silyl)oxy]methyl (TOM) protected phosphoramidites; synthesis cycle: (1) removal of the 50-O-
DMT group: 4% dichloroacetic acid in 1,2-dichloroethane, 90 s; (2) activation and coupling:
0.25 M benzylthiotetrazole (65 eq)=0.1 M cyanoethyl phosphoramidite (6 eq) in acetonitrile, 90 s;
(3) capping: Ac2O=2,6-lutidine=THF (1=1=8, v=v) and N-methylimidazole=THF (16=84, v=v),
60 s; (4) oxidation: I2=H2O=pyridine=THF (3=2=20=75, w=w), 45 s; oligonucleotide deprotection:
(1) 10 M MeNH2 in EtOH=H2O (1=1), 1–24 h, 25–33ꢀC; (2) 1 M Bu4NF Á 3H2O in THF,
1–50 h, 25ꢀC; (3) 1 M Tris Á HCl, H2O, pH 7.4; 4,40-dimethoxytrityl (DMT), 2-cyanoethyl (CE),
ꢀ,ꢀ,ꢀ-tris(hydroxymethyl)methylamin (Tris)

RNA Solid-Phase Synthesis
853
4,40-dimethoxytrityl group (DMT) at the ribose 50-hydroxyl group combined with
masking of the nucleophilic exocyclic amino groups of adenine, cytosine, and
guanine by base-labile acyl protecting groups [5]. From the large number of docu-
mented ribose 20-O protecting groups, the fluoride-labile tert-butyldimethylsilyl
(TBDMS) group has found the widest application [6]. In 1998, the [(triisopropyl-
silyl)oxy]methyl (TOM) protection of the ribose 20-OH was introduced by S. Pitsch
et al. [7–9]. The high performance of this protecting group in automated RNA
solid-phase synthesis has soon resulted in wide acceptance for the ‘TOM-
chemistry’ (Fig. 1) [4].
Here, we describe the synthesis and characterization of eight 20-O-TOM nucle-
oside phosphoramidite building blocks that are methylated at the nucleobases ac-
cording to naturally occuring methylation patterns. These building blocks allow for
the incorporation of 1-methylguanosine (m1G), N2-methylguanosine (m2G), N2,N2-
dimethylguanosine ðm2 GÞ, 1-methylinosine (m1I), 3-methyluridine (m3U), N4-
2
methylcytidine (m4C), N6-methyladenosine (m6A), and N6,N6-dimethyladenosine
ðm6 AÞ into oligoribonucleotides by standard automated RNA solid-phase synthesis
2
(Fig. 2).
Following nucleosides with 20-O methylation, nucleosides that are methylated
at their nucleobases account for the second largest number of naturally occuring
nucleoside modifications. They are encountered in all major RNA species, such as
tRNA, rRNA, snRNA, and mRNA [10]. The function of the majority of nucleoside
modifications is far from being well understood. Straightforward synthetic proce-
dures that guarantee the unlimited availability of modified nucleotide building
blocks for their use in RNA solid-phase synthesis enable detailed investigation of
structure-function relations of unmodified versus modified oligoribonucleotides.
Studies of these kind contribute to rationalizing the impact of RNA modifications
Fig. 2. Selection of naturally occuring, methylated ribonucleosides that were elaborated to 20-O-
TOM protected phosphoramidite building blocks for RNA solid-phase synthesis; 1-methylguanosine
(m1G), N2-methylguanosine (m2G), N2,N2-dimethylguanosine ðm2 GÞ, 1-methylinosine (m1I), 3-
2
methyluridine (m3U), N4-methylcytidine (m4C), N6-methyladenosine (m6A), N6,N6-dimethyladeno-
sine ðm6 AÞ
2

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on important cellular processes such as mRNA and rRNA maturation, ribosome
assembly, rRNA processing, translation of the genetic code, recognition of tRNAs,
RNA folding, and many more [11].
Results and Discussion
General Strategy for the Synthesis of Nucleobase-Methylated
Ribonucleotide Phosphoramidites
Starting from the inexpensive ribonucleosides adenosine, cytidine, guanosine, uri-
dine, and inosine, the synthesis of the corresponding methylated nucleoside phos-
phoramidites was carried out by chemical manipulation at the nucleobase first,
introducing the methyl and the base protecting groups, followed by stepwise in-
troduction of the 50-O-DMT group, the 20-O-TOM group, and finally the 30-O-(2-
cyanoethyl diisopropylphosphoramidite) (CEP) moiety (Fig. 3). Only in the case of
m4C we prefered an alternative route starting from the prefunctionalized derivative
50-O-DMT-20-O-TOM-uridine with subsequent transformation of the nucleobase to
obtain higher overall yields.
50-O-Tritylation
The conditions we prefered for tritylation of the 50-OH were 1.1–1.6 equivalents of
DMT-Cl added in portions to a 0.2–0.8 M solution of the corresponding methylated
nucleoside in anhydrous pyridine. By-products such as 20-O and 30-O tritylated
Fig. 3. Synthetic concept for the preparation of methylated 20-O-TOM protected nucleoside phos-
phoramidites; 2-(4-nitrophenyl)ethyl (NPE), 2-cyanoethyl diisopropylphosphoramidite (CEP)

RNA Solid-Phase Synthesis
855
Fig. 4. Alkylation of 50-O-tritylated nucleoside derivatives with [(triisopropylsilyl)oxy]methylchlo-
ride (TOM-Cl) via cyclic 20,30-O-di-tert-butylstannylidene intermediates according to a procedure by
S. Pitsch [9]
regioisomers, and double tritylated nucleotides were separated by column chroma-
tography on silica gel. 50-O tritylation yields ranged from 50–90%.
20-O-Alkylation
The alkylation reagent [(triisopropylsilyl)oxy]methylchloride (TOM-Cl) was syn-
thesized as described by S. Pitsch et al. [9]. Alkylation of the 20,30-diol moieties of
the tritylated ribonucleosides was achieved via the corresponding cyclic 20,30-O-di-
tert-butylstannylidene derivatives formed in situ in the presence of ethyldiisopro-
pylamine and tert-Bu2SnCl2 in 1,2-dichloroethane at 70ꢀC (Fig. 4). Subsequent
treatment with 1.1–1.5 equivalents of TOM-Cl at room temperature afforded mix-
tures of 20-O- and 30-O-alkylated regioisomers in a ratio between 5:4 and 9:1. The
desired 20-O-alkylated derivatives were easily isolated in pure form by chromatog-
raphy on silica gel as the first-eluting isomers. Alkylation yields for the mixture of
isomers ranged up to 70%.
In general, transformation of the methylated nucleosides into their 20-O-TOM
derivatives is more robust against further nucleobase alkylation with TOM-Cl than
the same reaction employed to the four standard ribonucleosides. In case of the
m2 G, m6 A, m3U, and m1I precursors, even a larger excess of TOM-Cl is appli-
2
2
cable. Moreover, as mentioned by S. Pitsch et al. [9], the use of Bu2SnCl2 instead
of tert-Bu2SnCl2 results in lower yields (ꢁ10%). We can confirm this, especially, if
addition of TOM-Cl is performed at low temperatures.
30-O-Phosphitylation
The 50-O-DMT-20-O-TOM protected intermediates were converted into the phos-
phoramidite building blocks with 1.5–2.3 equivalents of 2-cyanoethyl diisopro-
pylphosphoramidochloridite preferably in the presence of a ten-fold excess of
ethyldimethylamine. Product yields usually exceeded 80%.
Synthesis of the Individual Building Blocks
Building Block of 1-Methylguanosine (m1G)
Guanosine was methylated with high regioselectivity at the amido nitrogen by
treatment with 1 equivalent of NaH in DMSO followed by additon of 1 equivalent
of methyl iodide (Fig. 5). Evaporation of the reaction mixture and subsequent

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Fig. 5. Synthesis of the m1G phosphoramidite 5; (a) 1.0 eq NaH, DMSO, rt, 2 h; (b) 1.0 eq MeI, rt,
5 h; (c) Ac2O=DMF=pyridine (1=1=1), 140ꢀC, 10 h (1: 85% over (a)–(c)); (d) 1 M NaOH in
THF=MeOH=H2O (5=4=2), rt, 15 min (2: 81%); (e) 1.2 eq dimethylformamide dimethylacetal,
pyridine=DMSO (5=1), rt, 2 h, then 1.4 eq DMT-Cl, overnight (3: 45%); (f) 4.0 eq ethyldiisopro-
pylamine, 1.2 eq tert-Bu2SnCl2, ClCH2CH2Cl, 70ꢀC, then 1.2 eq TOM-Cl, rt, 1 h, separation of
20-O-isomer by chromatography (4: 45%); (g) 10 eq ethyldimethylamine, 1.5 eq 2-cyanoethyl
diisopropylphosphoramidochloridite, CH2Cl2, rt, 2 h (5: 85%)
refluxing of the crude product with acetic anhydride in pyridine=DMF at 140ꢀC
bath temperature resulted after adsorption on silica gel and chromatography in 1.
Selective hydrolysis of the O-acetyl groups was optimized to obtain N2-acetyl-1-
methyl guanosine 2. Because of significant insolubility of 2 in pyridine, the intro-
duction of the trityl group according to the general procedure described gave only
poor yields (20%) and had to be improved. Therefore, 2 was reacted with N,N-
dimethylformamide dimethylacetal to transiently form the corresponding nucleo-
side 20,30-O-acetal [12]. Solubility was enhanced and 50-O tritylation yield
increased to 45%. Derivative 3 was then alkylated (4) and phosphitylated (5) as
described in the general section.
Building Blocks of N2-Methylguanosine (m2G)
and N2,N2-Dimethylguanosine ðm2 GÞ
2
Guanosine was transformed into 20,30,50-O-triacetyl-O6-[2-(4-nitrophenyl)ethyl]-
guanosine by a method of Pfleiderer (compare Fig. 6) [13, 14]. Further synthesis
of m2G and m2 G was achieved by a modified procedure of Eritja et al. [15].
2
Transformation of the exocyclic amino group proceeded under Schiemann condi-
tions via diazotation and fluoride displacement with sodium nitrite and tetra-
fluoroboric acid in acetone=water at À 20ꢀC. After neutralization of the reaction
mixture, extraction with CH2Cl2 resulted in sufficiently pure 2-fluoro nucleoside
to be further treated with 8 M CH3NH2 in ethanol. Substitution of the 2-fluoro
group and simultaneous cleavage of the O-acetyl groups gave O6-NPE-m2G (6)
after column chromatography.

RNA Solid-Phase Synthesis
857
Fig. 6. Synthesis of the m2G and m2 G phosphoramidites 9 and 13; (a) 100 eq HBF , 2.5 eq NaNO ,
2
4
2
acetone=water, À 20ꢀC to rt, 3 h; (b) 8 M CH3NH2, ethanol, 7 h (6: 47% over (a) and (b)); (c)
(CH3)2NH in ethanol=water, rt, 3 h (10: 51% over (a) and (c)); (d) 1.1 eq DMT-Cl, 0.35 eq DMAP
(0.1 eq for 11), pyridine, rt, overnight (7: 70%, 11: 83%); (e) 4.0 eq ethyldiisopropylamine, 1.2 eq
tert-Bu2SnCl2, ClCH2CH2Cl, 70ꢀC, then 1.1 eq TOM-Cl, rt, 3 h; separation of 20-O-isomer by
chromatography (8: 35%, 12: 33%); (f) 10 eq ethyldimethylamine, 1.5 eq 2-cyanoethyl diisopro-
pylphosphoramidochloridite (1.1 eq for 13), CH2Cl2, rt, 2 h (9: 72%, 13: 87%)
Likewise, the 2-fluoro nucleoside intermediate was converted into O6-NPE-
m2 G (10) by a 1:1 mixture of 33% (CH ) NH in ethanol and 40% (CH ) NH in
water. It has to be pinpointed that treatment with 33% (CH3)2NH in ethanol alone
2
3 2
3 2
gave 50-O-acetyl-O6-NPE-m2 G exclusively. From 6 and 10, the corresponding
2
phosphoramidite building blocks 9 and 13 were synthesized in good overall yields
as described in the general section above.
Building Blocks of 1-Methylinosine (m1I) and 3-Methyluridine (m3U)
In comparable manner to the preparation of 1-methylguanosine, methylation of
inosine at nitrogen-1 and methylation of uridine at nitrogen-3 was achieved regio-
selectively by stepwise treatment with NaH=DMSO and methyl iodide (Fig. 7).
Subsequent evaporation of the reaction mixture allowed direct tritylation without
prior purification to furnish 14 and 17 in fair to good yields. The tritylated deriv-
atives were alkylated (15, 18) and phosphitylated (16, 19) as described in the
general section.
Building Block of N4-Methylcytidine (m4C)
The synthesis of the m4C phosphoramidite has been accomplished by two different
routes. In analogy to a procedure by Verdine [16], 20,30,50-O-triacetyluridine was

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trisylated at O4. Subsequent substitution with CH3NH2 gave m4C ready to be
tritylated, tomylated, and phosphitylated. However, better overall yields were
obtained using the prefunctionalized 50-O-DMT-20-O-TOM-uridine derivative [9]
Fig. 7. Synthesis of the m1I and m3U phosphoramidites 16 and 19; (a) 1.0 eq NaH, DMSO, rt, 2 h;
(b) 1.0 eq MeI, rt, 4 h; (c) 1.1 eq DMT-Cl (1.6 eq for 17), pyridine, rt, overnight (14: 47%, 17: 55%
over (a)–(c)); (d) 4.0 eq ethyldiisopropylamine, 1.2 eq tert-Bu2SnCl2, ClCH2CH2Cl, 70ꢀC, then 1.1
eq TOM-Cl, rt, 1 h, separation of 20-O-isomer by chromatography (15: 45%, 18: 47%); (e) 10 eq
ethyldimethylamine, 2.3 eq 2-cyanoethyl diisopropylphosphoramidochloridite (1.5 eq for 19),
CH2Cl2, rt, 2 h (16: 92%, 19: 89%)
Fig. 8. Synthesis of the m4C phosphoramidite 22; (a) 1.1 eq Ac2O, 0.1 eq DMAP, pyridine, 0ꢀC to rt,
1 h (20: 96%); (b) 1.5 eq TRIS-Cl, 10 eq NEt3, 0.1 eq DMAP, rt, 1 h; (c) 8 M CH3NH2 in ethanol,
rt, overnight; (21: 81% over (b) and (c)); (d) 10 eq ethyldimethylamine, 1.5 eq 2-cyanoethyl
diisopropylphosphoramidochloridite, CH2Cl2, rt, 2 h (22: 64%); 2,4,6-triisopropylbenzenesulfonyl
(TRIS)

RNA Solid-Phase Synthesis
859
as starting material (Fig. 8). After 30-O-acetylation, 20 was trisylated and directly
converted into 50-O-DMT-20-O-TOM-N4-methylcytidine (21) upon treatment with
8 M CH3NH2 in ethanol. Phosphitylation to 22 was then accomplished according to
the general procedure.
Building Blocks of N6-Methyladenosine (m6A)
and N6,N6-Dimethyladenosine ðm6 AÞ
2
Inosine was used as starting material for the N6-methylated adenosine building
blocks (Fig. 9). After O-acetylation and reaction with chloromethylenedimethyl-
iminiumchloride (Vilsmeier reagent) 20,30,50-O-triacetyl-6-chloroinosine (23) was
obtained in good yields. Substitution of the 6-chloro group with 8 M CH3NH2 in
ethanol and simultaneous cleavage of the O-acetyl groups gave m6A. After evap-
oration of the solvents the crude product was tritylated without prior purification to
furnish 24.
Likewise, the 6-chloro nucleoside intermediate was converted into m6 A by
2
reaction with (CH3)2NH in ethanol=water. After evaporation of the solvents the
crude product was tritylated without prior purification and gave 27 in good yields.
Fig. 9. Synthesis of the m6A and m6 A phosphoramidites 26 and 29; (a) 10 eq Ac2O, pyridine, rt,
2
overnight; (b) 2 eq chloromethylenedimethyliminiumchloride, CHCl3, reflux, 4 h (23: 93% over (a)
and (b)); (c) CH3NH2 in ethanol=water, rt, 12 h; (d) (CH3)2NH in ethanol=water, rt, 9 h; (e) 1.6 eq
DMT-Cl (1.5 eq for 27), pyridine, rt, overnight (24: 74% over (c) and (e), 27: 61% over (d) and (e));
(f) 4.0 eq ethyldiisopropylamine, 1.2 eq tert-Bu2SnCl2, ClCH2CH2Cl, 70ꢀC, then 1.2 eq TOM-Cl (1.1
eq for 28), rt, 3 h, separation of 20-O-isomer by chromatography (25: 23%, 28: 20%); (g) 10 eq
ethyldimethylamine, 1.5 eq 2-cyanoethyl diisopropylphosphoramidochloridite, CH2Cl2, rt, 2 h (26:
86%, 29: 85%)

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From 24 and 27, the corresponding phosphoramidite building blocks 26 and 29
were conveniently synthesized as described in the general section.
Oligoribonucleotides
The incorporation of the m1G, m2G, m2 G, m1I, m3U, m4C, m6A, and m6 A phos-
2
2
phoramidites into oligoribonucleotides has already been documented by our group
[17–21]. All methylated building blocks mentioned here have been reproducibly
coupled under standard conditions with an average coupling yield between 97 and
99.5%. They are compatible to standard oligoribonucleotide two-step deprotection
with ethanolic=aqueous CH3NH2, followed by treatment with 1 M TBAF=THF.
Noteably, deprotection of the NPE-groups (m2G and m2 G) does not require an
2
additional step involving DBU in acetonitrile. The NPE-groups are simultaneously
cleaved during cleavage of the TOM protecting groups in 1 M TBAF=THF.
Conclusion
The straight-forward synthesis of the methylated phosphoramidite building blocks
for m1G, m2G, m2 G, m1I, m3U, m4C, m6A, and m6 A from standard ribonucleo-
2
2
sides generates the basis to study structural effects of these methylations on RNA.
In this sense, we have documented the impact of RNA methylations on duplex-
hairpin equilibria [17], the possible role of methylation in the ribosomal helix 45
with respect to secondary structure formation [18], and the stabilizing effect of a
methylated guanosine on codon-anticodon pairing by cyclic model compounds
[19–21]. Other studies on the structural impact of RNA methylations are currently
in progress in our laboratory. Due to the lack of an appropriate biosynthetic system
the chemical synthesis is the method of choice for site-specifically introducing
modified nucleosides into RNA oligoribonucleotides. Moreover, the novel 20-O-
TOM phosphoramidite building blocks presented in this paper should further
document the great flexibility of the still young ‘RNA-TOM-chemistry’ towards
applications involving modified nucleosides.
Experimental
1H, 13C, and 31P NMR spectra were recorded on a Bruker DRX 500MHz, Bruker DRX 300MHz, or
Varian Unity 500 MHz instrument. The chemical shifts are reported relative to TMS and referenced to
the residual proton signal of the deuterated solvents: CDCl3 (7.26 ppm), d6-DMSO (2.49 ppm) or d8-
1
toluene (7.08 ppm) for H NMR spectra; CDCl3 (77.0 ppm) or d6-DMSO (39.5 ppm) for 13C NMR
1
spectra. 31P shifts are relative to external 85% phosphoric acid. Multiplicity of H-NMR resonance
signals: doublet (d), triplet (t), and quartet (q); if no coupling constant is given, the abbreviations refer
to signal appearance and not to theoretical multiplicity. UV spectra were recorded on a Varian Cary
€
100. Mass spectra were obtained from the service facilities at ETH Zurich. Elemental analyses for all
20-O-TOM-nucleoside derivatives and the corresponding phosphoramidites were obtained from service
€
facilities (ETH Zurich); their values agreed favourably with the calculated ones.
Analytical thin-layer chromatography (TLC) was carried out on silica 60F-254 plates. Flash col-
umn chromatography was carried out with silica gel 60 (230–400 mesh). Packing of silica gel columns
was performed with 1% Et3N added to the first 100 cm3 of the corresponding starting eluent.
All reactions were carried out under Ar atmosphere. Workup implies partitioning of the reaction
mixture between CH2Cl2 and semi-saturated aqueous NaHCO3 solution, drying of the organic layer

RNA Solid-Phase Synthesis
861
(MgSO4 or Na2SO4), and evaporation. NaH from paraffine suspensions was washed three times with
hexane and dried under vacuum.
Chemical reagents and solvents were purchased from commercial suppliers and used without
further purification. Solvents for reactions were dried overnight over freshly activated molecular sieves
˚
(4A).
N2,20,30,50-O-Tetraacetyl-1-methylguanosine (1, C19H23N5O9)
Guanosine (3g, 10.6mmol) was dissolved in 20cm3 of anhydrous DMSO and treated with 254 mg of
NaH (10.6mmol). The mixture was stirred until H2 evolution ceased. Methyliodide (1.5g, 10.6mmol)
dissolved in 1 cm3 of DMSO was added dropwise. The mixture was stirred for 5 h at room temperature,
evaporated to dryness under reduced pressure at 80ꢀC and dissolved in pyridine=DMF=acetic anhy-
dride (50 cm3 each). The mixture was heated to 140ꢀC for 5 to 10 h and evaporated again. The resulting
solid was dissolved in 200 cm3 of MeOH and 50g of silica gel were added. MeOH was evaporated, the
silica gel coated by the crude product was dried overnight under reduced pressure, and applied to
column chromatography (silica gel, CH2Cl2:CH3OH¼ 40:1 to 25:1) resulting in 1 (4.2g, 85%). TLC
(silica gel, CH2Cl2:CH3OH¼ 25:1): Rf ¼ 0.5; 1H NMR (500MHz, CDCl3, 25ꢀC): ꢁ ¼ 2.07, 2.08, 2.13,
2.33 (4s, COCH3), 3.60 (s, 1-CH3), 4.40–4.46 (m, H1–C(50), H–C(40)), 4.53 (dd, J ¼ 5.2, 11.0Hz, H2–
C(50)), 5.67 (t, H–C(30)), 5.91 (t, H–C(20)), 5.99 (d, J ¼ 5.2 Hz, H–C(10)), 7.80 (s, H–C(8)), 8.76 (br s,
13
NH) ppm; C NMR (125MHz, CDCl3, 25ꢀC): ꢁ ¼ 20.37, 20.54, 20.79, 23.78 (COCH3), 31.90
(1-CH3), 63.19 (C(50)), 70.72 (C(30)), 72.95 (C(20)), 80.23 (C(40)), 87.00 (C(10)), 122.90, 138.80
(C(8)), 145.73, 146.90, 157.29, 169.34, 169.62, 170.10, 171.04 ppm.
N2-Acetyl-1-methylguanosine (2, C13H17N5O6)
Compound 1 (2.0g, 4.3 mmol) was dissolved in 24cm3 of THF=MeOH=H2O (5=4=2). Aqueous
NaOH (2cm3, 10 M) was added under vigorous stirring. The reaction was monitored by TLC
(CHCl3:MeOH ¼ 1:1) and was completed typically after 15min. The mixture was quenched with
ꢁ2 cm3 of acetic acid to give a final pH of 6.5. Product 2 (1.18 g, 81%) precipitated as pale yellow
solid, was collected by filtration and washed two times with 10 cm3 of cold THF=MeOH=H2O
1
( À 20ꢀC). TLC (silica gel, CHCl3:CH3OH¼ 8:5): Rf ¼ 0.5; H NMR (300MHz, d6-DMSO, 27ꢀC):
ꢁ ¼ 2.13 (s, COCH3), 3.41 (s, 1-CH3), 3.63 (dd, J ¼ 3.9, 11.9Hz, H1–C(50)), 3.53 (dd, J ¼ 4.0, 11.9Hz,
H2–C(50)), 3.91 (q, H–C(40)), 4.12 (t, H–C(30)), 4.45 (t, H–C(20)), 4.99 (br s, HO–C(50)), 5.15, 5.44
(2br s, HO–C(20), HO–C(30)), 5.79 (d, J ¼ 5.8 Hz, H–C(10)), 8.31 (s, H–C(8)) ppm; 13C NMR
(75 MHz, d6-DMSO, 27ꢀC): ꢁ ¼ 23.8 (COCH3), 31.9 (1-CH3), 62.1 (C(50)), 71.2 (C(30)), 74.9
(C(20)), 86.5 (C(40)), 87.8 (C(10)), 139.8 (C(8)), 122.0, 147.3, 148.0, 157.7, 171.0ppm; UV (H2O):
ꢂ(") ¼ 202 (max, 22400), 254 (max, 9880), 260 (8860) nm (molÀ 1 dm3 cmÀ 1).
N2-Acetyl-50-O-(4,40-dimethoxytrityl)-1-methylguanosine (3, C34H35N5O8)
Method A: A suspension of 2.0 g of 2 (5.89mmol) in 14 cm3 of DMSO and 25cm3 of pyridine was
heated to 60ꢀC and treated with 2.19g of 4,40-dimethoxytritylchloride (6.48 mmol) in three portions
over a period of 3 h. Stirring was continued overnight. Unreacted educt was recovered by filtration of
the reaction mixture. Addition of MeOH, evaporation to dryness, workup, and column chromatography
(silica gel, CH2Cl2:CH3OH¼ 20:1) yielded 0.76g of 3 as pale yellow foam (20%). The educt recov-
ered can be directly used for repeated tritylation.
Method B: To a suspension of 200 mg of 2 (0.59 mmol) in 3 cm3 of pyridine, 84.3mg of N,N-
dimethylformamide dimethylacetal (0.71 mmol) were added. The mixture was stirred for 2 h, heated to
50ꢀC, treated with 0.1 cm3 of DMSO, and concentrated to a volume of 1.5cm3. Addition of 3 cm3 of
pyridine=DMSO (5=1) resulted in a clear solution and 280 mg of 4,40-dimethoxytritylchloride

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862
(0.83 mmol) were added in three portions over a period of 24h. Addition of 1 cm3 of MeOH, evap-
oration of pyridine, workup, and column chromatography (silica gel, CH2Cl2:CH3OH¼ 20:1; 1%
Et3N) yielded 171 mg of 3 as pale yellow foam (45%). TLC (silica gel, CH2Cl2:CH3OH¼ 10:1):
1
Rf ¼ 0.4; H NMR (500 MHz, CDCl3, 25ꢀC): ꢁ ¼ 1.84 (COCH3), 3.22, 3.42 (2d, H2–C(50)), 3.45
(1-CH3), 3.71, 3.72 (2s, 2 OCH3), 4.29 (t, H–C(40)), 4.42 (t, H–C(30)), 4.88 (t, H–C(20)), 5.86 (d,
J ¼ 6.1 Hz, H–C(10)), 6.25 (br q, NH), 6.72–6.80 (m, 4H, trityl-H), 7.10–7.42 (m, 9H, trityl-H), 7.85 (s,
H–C(8)) ppm; 13C NMR (125 MHz, CDCl3, 25ꢀC): 23.62 (COCH3), 31.30 (1-CH3), 55.20 (2 CH3O),
63.68 (C(50)), 71.91 (C(30)), 74.88 (C(20)), 85.27 (C(40)), 86.40, 89.58 (C(10)), 113.19 (trityl-C), 121.2,
126.96, 127.90, 128.03, 129.99, 130.05 (trityl-C), 135.55, 135.63, 138.93 (C(8)), 144.66, 146.05,
146.43, 157.35, 158.54, 170.25 ppm; UV (CHCl3): ꢂ(") ¼ 260 (11500), 277 (max, 11000) nm
(molÀ 1 dm3 cmÀ 1); FAB-MS: m=z ¼ 642.2 (100, [M þ H]þ ), 303.1 (39, [(MeO)2Tr]þ ).
N2-Acetyl-50-O-(4,40-dimethoxytrityl)-1-methyl-20-O-[[(triisopropylsilyl)oxy]methyl]
guanosine (4, C44H57N5O9Si)
To a stirred solution of 345 mg of 3 (0.54 mmol) and 278 mg of ethyldiisopropylamine (2.15 mmol) in
3 cm3 of 1,2-dichloroethane, 198 mg of di-tert-butyltindichloride (0.65mmol) were added. The mixture
was heated to 70ꢀC for 15 min, allowed to cool to rt again, and treated with 134mg of [(triisopro-
pylsilyl)oxy]methylchloride (0.6mmol). Stirring was continued for 60 min, followed by addition of
0.2 cm3 of MeOH and workup. Column chromatography (silica gel, CH2Cl2:CH3OH¼ 70:1 to 50:1)
afforded 200 mg of 4 as white, solid foam (45%). The regioselectivity of 20-O-alkylated over 30-O-
alkylated product was approximately 7:2. TLC (silica gel, CH2Cl2:CH3OH¼ 20:1): Rf ¼ 0.4; 1H NMR
(500 MHz, CDCl3, 25ꢀC): ꢁ ¼ 0.98–1.07 (m, iPr3Si), 1.42 (s, COCH3), 3.00 (d, J ¼ 1.5 Hz, HO–C(30)),
3.17 (dd, J ¼ 3.2, 10.5Hz, H1–C(50)), 3.52 (s, 1-CH3), 3.55 (dd, J ¼ 1.8, 10.5Hz, H2–C(50)), 3.78, 3.79
(2s, 2 OCH3), 4.25 (m, H–C(40)), 4.52 (m, H–C(30)), 4.94 (q, H–C(20)), 4.90, 5.11 (2d, J ¼ 4.7 Hz,
OCH2O), 5.93 (d, J ¼ 7.3 Hz, H–C(10)), 6.80 (m, 4H, trityl-H), 6.98 (br s, NH), 7.17–7.54 (m, 9H,
13
trityl-H), 7.83 (s, H–C(8)) ppm; C NMR (125MHz, CDCl3, 25ꢀC): ꢁ ¼ 11.80 ((CH3)2CH), 17.75
((CH3)2CH), 22.89 (COCH3), 31.79 (1-CH3), 55.28 (2 CH3O), 63.78 (C(50)), 70.90 (C(30)), 81.96
(C(20)), 84.37 (C(40)), 86.37 (C(10)), 86.50, 91.07 (OCH2O), 113.26, 113.32 (trityl-C), 123.05, 127.16,
128.01, 128.08, 129.97, 130.12 (trityl-C), 135.68, 135.89, 139.58 (C(8)), 145.12, 146.24, 157.30,
158.72, 169.68 ppm; UV (CHCl3): ꢂ(") ¼ 260 (12600), 276 (max, 11200) nm (molÀ 1 dm3 cmÀ 1);
FAB-MS: m=z ¼ 828.2 (83, [Mþ H]þ ), 303.1 (100, [(MeO)2Tr]þ ).
N2-Acetyl-50-O-(4,40-dimethoxytrityl)-1-methyl-20-O-[[(triisopropylsilyl)oxy]methyl]
guanosine 30-(2-cyanoethyl diisopropylphosphoramidite) (5, C53H74N7O10PSi)
A solution of 183 mg of 4 (0.22 mmol) in 2 cm3 of CH2Cl2 was treated consecutively with 160 mg of
ethyldimethylamine (2.2mmol) and 79 mg of 2-cyanoethyl diisopropylphosphoramidochloridite
(0.33 mmol). The mixture was stirred for 2 h, quenched with 0.3cm3 of MeOH, and evaporated to
dryness. Workup and column chromatography (silica gel, CH2Cl2:CH3OH ¼ 60:1 ( þ 2% Et3N))
afforded 193 mg of 5 as white, solid foam (85%, 1:1 mixture of diastereoisomers). TLC (silica gel,
CH2Cl2:CH3OH ¼ 30:1): Rf ¼ 0.6; 1H NMR (500MHz, CDCl3, 25ꢀC): ꢁ ¼ 0.86–1.19 (m, 42H, iPr3Si,
24H, ((CH3)2CH)2N), 1.44, 1.66 (br s, 6H, COCH3), 2.27, 2.69 (2m, 4H, CH2CN), 3.21 (m, 2H, H1–
C(50)), 3.50–3.55 (m, 4H, ((CH3)2CH)2N), 3.51, 3.52 (2s, 6H, 1-CH3), 3.53, 3.59 (m, 2H, POCH2),
3.51, 3.62 (m, 2H, H2–C(50)), 3.77, 3.78 (2s, 12H, OCH3), 3.90, 3.97 (2m, 2H, POCH2), 4.27, 4.34 (2q,
2H, H–C(40)), 4.52, 4.57 (2m, 2H, H–C(30)), 4.89–4.96 (4d, 4H, J ¼ 5.2Hz, OCH2O), 5.00, 5.06 (t, q,
2H, H–C(20)), 5.89, 5.99 (2d, 2H, J ¼ 7.1 Hz, H–C(10)), 6.76–6.82, 7.20–7.31, 7.35–7.53 (m,
31
26H, trityl-H), 7.82, 7.84 (2s, 2H, H–C(8)) ppm; P NMR (200MHz, CDCl3, 27ꢀC): ꢁ ¼
149.98, 150.50 ppm; UV (MeOH): ꢂ(") ¼ 260 (13300), 274 (max, 12800), 281 (sh, 12000) nm
(molÀ 1 dm3 cmÀ 1); FAB-MS: m=z ¼ 1028.7 (44, [Mþ H]þ ), 821.5 (100), 303.1 (35, [(MeO)2Tr]þ ).

RNA Solid-Phase Synthesis
863
N2-Methyl-O6-[2-(4-nitrophenyl)ethyl]guanosine (6, C19H22N6O7)
20,30,50-O-Triacetyl-O6-[2-(4-nitrophenyl)ethyl]guanosine [13, 14] (6.6g, 11.7 mmol) in 45cm3 of ac-
etone was cooled to À 20ꢀC and treated with 111 cm3 of 50% HBF4 (1.2mol). Under vigorous stirring
2.04g of NaNO2 (29.4mmol) in 28cm3 of water were added dropwise. The reaction mixture was
allowed to warm to room temperature, stirred for 3 h, and neutralized with 50% NaOH (or NaHCO3).
The reaction mixture was extracted two times with CH2Cl2, the organic layers were dried with Na2SO4
and evaporated to dryness. The crude material was dissolved in 8 M CH3NH2 in ethanol and stirred
for 7 h. The reaction mixture was evaporated to dryness. Column chromatography (silica gel,
CH2Cl2:MeOH ¼ 15:1) afforded 2.46 g of 6 (47%). TLC (silica gel, CHCl3:CH3OH¼ 10:1):
Rf ¼ 0.5; 1H NMR (500MHz, d6-DMSO, 50ꢀC): ꢁ ¼ 2.82 (d, J ¼ 4.8 Hz, N2-CH3), 3.26 (t, J ¼ 6.8 Hz,
CH2–C6H4–NO2), 3.55, 3.63 (2m, H2–C(50)), 3.90 (q, H–C(40)), 4.14 (q, H–C(30)), 4.57 (q, H–C(20)),
4.71 (t, J ¼ 6.8 Hz, O6–CH2), 4.84 (t, HO–C(50)), 4.99 (d, J ¼ 4.9 Hz, HO–C(30)), 5.23 (d, J ¼ 6.1 Hz,
HO–C(20), 5.79 (d, J ¼ 5.9 Hz, H–C(10)), 6.75 (br q, NH), 7.61 (d, J ¼ 8.5 Hz, 4-nitrophenyl
H–C(2)=H–C(6)), 8.02 (s, H–C(8)), 8.16 (d, J ¼ 8.5 Hz, 4-nitrophenyl H–C(3)=H–C(5)) ppm;
13C NMR (125 MHz, d6-DMSO, 40ꢀC): ꢁ ¼ 28.65 (N2–CH3), 34.82 (CH2–C6H4–NO2), 62.05 (C(50)),
65.79 (O6–CH2), 70.96 (C(30)), 73.56 (C(20)), 85.74 (C(40)), 87.49 (C(10)), 123.78 (4-nitrophenyl
C(3)=C(5)), 114.33, 130.63 (4-nitrophenyl C(2)=C(6)), 138.73, 138.79 (C(8)), 146.74, 147.08, 154.67,
159.76, 160.34 ppm.
50-O-(4,40-Dimethoxytrityl)-N2-methyl-O6-[2-(4-nitrophenyl)ethyl]guanosine
(7, C40H40N6O9)
Compound 6 (1.40 g, 3.14 mmol) was coevaporated three times with anhydrous pyridine to be finally
dissolved in 15 cm3. Then, 1.17g of 4,40-dimethoxytritylchloride (3.45 mmol) were added in three
portions over a period of 3 h. DMAP (0.14 g, 1.11mmol) was added and stirring was continued
overnight. Addition of MeOH, evaporation to dryness, workup, and column chromatography (silica
gel, CH2Cl2:CH3OH¼ 20:1) yielded 1.63g of 7 as pale yellow foam (70%). TLC (silica gel,
1
CH2Cl2:CH3OH ¼ 10:1): Rf ¼ 0.4; H NMR (500MHz, d6-DMSO, 60ꢀC): ꢁ ¼ 2.73 (d, J ¼ 4.8 Hz,
N2-CH3), 3.22 (d, J ¼ 4.7 Hz, H2–C(50)), 3.25 (t, J ¼ 6.7 Hz, CH2–C6H4–NO2), 3.71, 3.72 (2s,
2 OCH3), 4.01 (q, H–C(40)), 4.32 (q, H–C(30)), 4.64 (q, H–C(20)), 4.71 (t, J ¼ 6.7 Hz, O6–CH2),
4.97 (d, J ¼ 5.8 Hz, HO–C(30)), 5.28 (d, J ¼ 5.0 Hz, HO–C(20)), 5.82 (d, J ¼ 4.6 Hz, H–C(10)), 6.65
(br q, NH), 6.81 (m, 4H, trityl-H), 7.17–7.27, 7.33–7.37 (m, 9H, trityl-H), 7.60 (d, J ¼ 8.7 Hz, 4-
nitrophenyl H–C(2)=H–C(6)), 7.90 (s, H–C(8)), 8.15 (d, J ¼ 8.7 Hz, 4-nitrophenyl H–C(3)=H–C(5))
13
ppm; C NMR (125 MHz, d6-DMSO, 60ꢀC): ꢁ ¼ 28.54 (N2-CH3), 34.78 (CH2–C6H4–NO2), 55.45
(2 CH3O), 64.46 (C(50)), 65.81 (O6–CH2), 70.98 (C(30)), 73.26 (C(20)), 83.46 (C(40)), 85.98, 88.25
(C(10)), 113.56 (trityl-C), 114.49, 123.73 (4-nitrophenyl C(3)=C(5)), 126.98, 128.05, 128.17 (trityl-C),
130.03, 130.08, 130.58 (trityl-C, 4-nitrophenyl C(2)=C(6)), 136.08, 138.71 (C(8)), 145.22, 146.83,
147.06, 158.52, 158.55, 159.80, 160.40 ppm.
50-O-(4,40-Dimethoxytrityl)-N2-methyl-O6-[2-(4-nitrophenyl)ethyl]-20-O-
[[(triisopropylsilyl)oxy]methyl]guanosine (8, C50H62N6O10Si)
To a stirred solution of 1.63g of 7 (2.18 mmol) and 1.13g of ethyldiisopropylamine (8.71 mmol) in
20cm3 of anhydrous 1,2-dichloroethane, 0.79 g of di-tert-butyltindichloride (2.61 mmol) were added.
The mixture was heated to 70ꢀC for 15 min, allowed to cool to rt again and treated with 0.53 g of
[(triisopropylsilyl)oxy]methylchloride (2.40 mmol). Stirring was continued for 3 h, followed by addi-
tion of 0.5 cm3 of MeOH, evaporation to dryness and workup. Column chromatography (silica gel,
hexanes:EtOAc¼ 3:1 to 1:1) afforded 0.70 g of 8 as white solid foam (35%). The regioselectivity
of 20-O-alkylated over 30-O-alkylated product was approximately 3:2. TLC (silica gel,
1
i
hexanes:EtOAc¼ 1:1): Rf ¼ 0.4; H NMR (500MHz, CDCl3, 30ꢀC): ꢁ ¼ 0.98–1.10 (m, Pr3Si), 2.86

€
C. Hobartner et al.
864
(d, J ¼ 5.1 Hz, N2-CH3), 2.98 (d, J ¼ 4.1 Hz, HO–C(30)), 3.28 (t, J ¼ 6.9 Hz, CH2–C6H4–NO2), 3.36
(dd, J ¼ 4.5, 10.5Hz, H1–C(50)), 3.46 (dd, J ¼ 3.9, 10.5 Hz, H2–C(50)), 3.78, 3.79 (2s, 2 OCH3), 4.22
(q, H–C(40)), 4.58 (q, H–C(30)), 4.67 (br s, NH), 4.73 (t, J ¼ 6.9 Hz, O6–CH2), 4.95 (t, H–C(20)), 4.98,
5.13 (2d, J ¼ 4.8 Hz, OCH2O), 6.03 (d, J ¼ 5.4 Hz, H–C(10)), 6.79 (m, 4H, trityl-H), 7.19–7.32, 7.42–
7.43 (m, 9H, trityl-H), 7.47 (d, J ¼ 8.7 Hz, 4-nitrophenyl H–C(2)=H–C(6)), 7.71 (s, H–C(8)), 8.16 (d,
13
J ¼ 8.7 Hz, 4-nitrophenyl H–C(3)=H–C(5)) ppm; C NMR (75 MHz, CDCl3, 27ꢀC): ꢁ ¼ 11.85
((CH3)2CH), 17.74 ((CH3)2CH), 28.59 (N2-CH3), 35.24 (CH2–C6H4–NO2), 55.16 (2 CH3O), 63.55
(C(50)), 65.83 (O6–CH2), 70.99 (C(30)), 81.22 (C(20)), 83.74 (C(40)), 86.42, 86.70 (C(10)), 90.79
(OCH2O), 113.13 (trityl-C), 115.33, 123.68 (4-nitrophenyl C(3)=C(5)), 126.81, 127.79, 128.20 (tri-
tyl-C), 129.88, 130.08 (trityl-C, 4-nitrophenyl C(2)=C(6)), 135.77, 135.83, 138.02 (C(8)), 144.64,
146.11, 146.84, 154.10, 158.52, 159.64, 160.51ppm; UV (MeOH): ꢂ(") ¼ 260 (20100), 275 (max,
18300), 284 (max, 17900) nm (molÀ 1 dm3 cmÀ 1); MALDI-FTICR-MS: m=z ¼ 957.4189 (5,
[Mþ Na]þ ), 303.1399 (100, [(MeO)2Tr]þ ).
50-O-(4,40-Dimethoxytrityl)-N2-methyl-O6-[2-(4-nitrophenyl)ethyl]-20-O-
[[(triisopropylsilyl)oxy]methyl]guanosine 30-(2-cyanoethyl diisopropylphosphoramidite)
(9, C59H79N8O11PSi)
A solution of 137 mg of 8 (0.14 mmol) in 3 cm3 of anhydrous CH2Cl2 was treated consecutively with
103 mg of ethyldimethylamine (1.4mmol) and 50 mg of 2-cyanoethyl diisopropylphosphoramidochlo-
ridite (0.21 mmol). The mixture was stirred for 2 h, quenched with 0.15 cm3 of MeOH, and evaporated.
Workup and column chromatography (silica gel, hexane:EtOAc¼ 2:1 ( þ 1% Et3N)) afforded 120 mg
of 9 as white, solid foam (72%, 1:1 mixture of diastereoisomers). TLC (silica gel, hexane:
1
i
EtOAc) ¼ 1:1): Rf ¼ 0.5; H NMR (500 MHz, CDCl3, 25ꢀC): ꢁ ¼ 0.89–1.24 (m, 42H, Pr3Si, 24H,
((CH3)2CH)2N), 2.34, 2.65 (2m, 4H, CH2CN), 2.85 (2d, 6H, J ¼ 5.1 Hz, N2-CH3), 3.27 (t, 4H,
J ¼ 6.9 Hz, CH2–C6H4–NO2), 3.35–3.40 (m, 2H, H1–C(50)), 3.45–3.66 (m, 2H, H2–C(50), 2H,
POCH2, 4H, ((CH3)2CH)2N), 3.77, 3.78 (2s, 12H, OCH3), 3.82–3.96 (2m, 2H, POCH2), 4.31, 4.36
(2q, 2H, H–C(40)), 4.64 (br q, NH), 4.70 (m, 2H, H–C(30)), 4.73 (t, J ¼ 6.9 Hz, O6–CH2), 4.89–4.96
(m, 4H, OCH2O), 5.12 (t, 2H, H–C(20)), 6.00, 6.05 (2d, 2H, J ¼ 6.0 Hz, H–C(10)), 6.75–6.83, 7.18–
7.35, 7.40–7.44 (m, 26H, trityl-H), 7.48 (d, J ¼ 7.5 Hz, 4-nitrophenyl H–C(2)=H–C(6)), 7.69 (s, 2H,
H–C(8)), 8.15 (d, J ¼ 7.5 Hz, 4-nitrophenyl H–C(3)=H–C(5)) ppm; 31P NMR (200MHz, CDCl3,
27ꢀC): ꢁ ¼ 150.09; 150.63 ppm; UV (MeOH): ꢂ(") ¼ 260 (19800), 275 (max, 17100), 282 (max,
17500) nm (molÀ 1 dm3 cmÀ 1); MALDI-FTICR-MS: m=z ¼ 1157.5268 (8, [Mþ Na]þ ), 303.1395
(100, [(MeO)2Tr]þ ).
N2,N2-Dimethyl-O6-[2-(4-nitrophenyl)ethyl]guanosine (10, C20H24N6O7)
20,30,50-O-Triacetyl-O6-[2-(4-nitrophenyl)ethyl]guanosine [13, 14] (3.0g, 5.3 mmol) in 20cm3 of
acetone was cooled to À 20ꢀC and treated with 50cm3 of 50% HBF4 (0.55 mol). Under vigorous
stirring 0.92g of NaNO2 (13.4mmol) in 13cm3 of water were added dropwise. The reaction mixture
was allowed to warm to rt, stirred for 3 h, and neutralized with 50% NaOH (or NaHCO3). The reaction
mixture was extracted twice with CH2Cl2, the organic layers dried with Na2SO4 and evaporated to
dryness. The crude material was dissolved in a stirred solution of 33% (CH3)2NH in 20 cm3 of ethanol.
After 1 h a solution of 40% (CH3)2NH in 20cm3 of water was added and stirring was continued for
another 3 h. The reaction mixture was evaporated to dryness and coevaporated three times with
MeOH=toluene=CH2Cl2. Column chromatography (silica gel, CH2Cl2:MeOH¼ 15:1) afforded 1.24g
of 10 (51%). TLC (silica gel, CHCl3:CH3OH¼ 10:1): Rf ¼ 0.5; 1H NMR (500MHz, d6-DMSO, 25ꢀC):
ꢁ ¼ 3.12 (s, N2-(CH3)2), 3.27 (t, J ¼ 6.7 Hz, CH2–C6H4–NO2), 3.50, 3.61 (2m, H2–C(50)), 3.87 (q, H–
C(40)), 4.14 (q, H–C(30)), 4.59 (q, H–C(20)), 4.73 (t, J ¼ 6.7 Hz, O6–CH2), 4.90 (t, J ¼ 5.6 Hz, HO–
C(50)), 5.15 (d, J ¼ 4.9 Hz, HO–C(30)), 5.35 (d, J ¼ 6.2 Hz, HO–C(20), 5.80 (d, J ¼ 5.9 Hz, H–C(10)),

RNA Solid-Phase Synthesis
865
7.60 (d, J ¼ 8.7 Hz, 4-nitrophenyl H–C(2)=H–C(6)), 8.08 (s, H–C(8)), 8.16 (d, J ¼ 8.7 Hz, 4-nitrophe-
13
nyl H–C(3)=H–C(5)) ppm; C NMR (125MHz, d6-DMSO, 25ꢀC): ꢁ ¼ 34.77 (CH2–C6H4–NO2),
37.53 (N2-(CH3)2), 61.99 (C(50)), 65.81 (O6–CH2), 70.89 (C(30)), 73.41 (C(20)), 85.58 (C(40)), 87.40
(C(10)), 113.69, 123.85 (4-nitrophenyl C(3)=C(5)), 130.64 (4-nitrophenyl C(2)=C(6)), 139.29, 139.31
(C(8)), 146.68, 147.12, 154.71, 158.87, 159.86 ppm.
50-O-(4,40-Dimethoxytrityl)-N2,N2-dimethyl-O6-[2-(4-nitrophenyl)ethyl]guanosine
(11, C41H42N6O9)
Compound 10 (500 mg, 1.09 mmol) was coevaporated three times with anhydrous pyridine to be finally
dissolved in 4 cm3. Then, 410 mg of 4,40-dimethoxytritylchloride (1.21 mmol) were added in three
portions over a period of 3 h. DMAP (10 mg, 0.08mmol) was added and stirring was continued
overnight. Addition of MeOH, evaporation to dryness, workup, and column chromatography (silica
gel, CH2Cl2:CH3OH¼ 35:1) yielded 690mg of 11 as pale yellow foam (83%). TLC (silica gel,
1
CH2Cl2:CH3OH ¼ 20:1): Rf ¼ 0.4; H NMR (500MHz, CDCl3, 30ꢀC): ꢁ ¼ 3.04 (s, HO–C(30)), 3.18
(s, N2-(CH3)2), 3.24 (dd, J ¼ 3.5, 10.4Hz, H1–C(50)), 3.31 (t, J ¼ 6.8 Hz, CH2–C6H4–NO2), 3.41 (dd,
J ¼ 3.8, 10.4 Hz, H2–C(50)), 3.76, 3.77 (2s, 2 OCH3), 4.39 (d, H–C(30)), 4.44 (t, H–C(40)), 4.68 (t,
H–C(20)), 4.78 (t, J ¼ 6.8 Hz, O6–CH2), 5.83 (d, J ¼ 6.6 Hz, H–C(10)), 6.73–6.79 (m, 4H, trityl-H),
7.16–7.22, 7.26–7.30 (m, 9H, trityl-H), 7.49 (d, J ¼ 8.9 Hz, 4-nitrophenyl H–C(2)=H–C(6)), 7.84 (s,
H–C(8)), 8.16 (d, J ¼ 8.9 Hz, 4-nitrophenyl H–C(3)=H–C(5)) ppm; 13C NMR (125 MHz, CDCl3,
30ꢀC): ꢁ ¼ 35.16 (CH2–C6H4–NO2), 37.78 (N2-(CH3)2), 55.10 (2 CH3O), 63.73 (C(50)), 65.89
(O6–CH2), 72.84 (C(30)), 76.21 (C(20)), 86.34 (C(40), 86.38, 90.41 (C(10)), 113.06 (trityl-C), 114.10,
123.70 (4-nitrophenyl C(2)=C(6)), 126.77, 127.76, 127.97 (trityl-C), 129.82, 129.83, 129.89, 129.93
(trityl-C and 4-nitrophenyl C(3)=C(5)), 135.38, 135.55, 136.83 (C(8)), 144.27, 145.90, 146.81, 153.22,
158.45, 160.10 ppm; UV (MeOH): ꢂ(") ¼ 259 (max, 12400), 260 (12300), 275 (sh, 9400), 281
(sh, 9100) nm (molÀ 1 dm3 cmÀ 1); MALDI-FTICR-MS: m=z ¼ 785.2855 (6, [Mþ Na]þ ), 303.1
(100, [(MeO)2Tr]þ ).
50-O-(4,40-Dimethoxytrityl)-N2,N2-dimethyl-O6-[2-(4-nitrophenyl)ethyl]-20-O-
[[(triisopropylsilyl)oxy]methyl]guanosine (12, C51H64N6O10Si)
To a stirred solution of 540 mg of 11 (0.71 mmol) and 366 mg of ethyldiisopropylamine (2.84 mmol) in
6 cm3 of anhydrous 1,2-dichloroethane, 258 mg of di-tert-butyltindichloride (0.85 mmol) were added.
The mixture was heated to 70ꢀC for 15 min, allowed to cool to rt again and treated with 173 mg of
[(triisopropylsilyl)oxy]methylchloride (0.8mmol). Stirring was continued for 3 h, followed by addition
of 0.5 cm3 of MeOH, evaporation to dryness and workup. Column chromatography (silica gel,
hexane:EtOAc¼ 3:1 to 1:1) afforded 222 mg of 12 as white solid foam (33%). The regioselectivity
of 20-O-alkylated over 30-O-alkylated product was approximately 5:3. TLC (silica gel, hexane:
1
i
EtOAc¼ 1:1): Rf ¼ 0.4; H NMR (500MHz, CDCl3, 30ꢀC): ꢁ ¼ 1.02–1.12 (m, Pr3Si), 2.99 (d,
J ¼ 4.7, HO–C(30)), 3.10 (s, N2-(CH3)2), 3.30 (t, J ¼ 6.9 Hz, CH2–C6H4–NO2), 3.46 (d, H2–C(50)),
3.78 (s, 2 OCH3), 4.20 (q, H–C(40)), 4.57 (q, H–C(30)), 4.76 (t, J ¼ 6.9 Hz, O6–CH2), 4.92 (t, H–C(20)),
4.99, 5.14 (2d, J ¼ 4.8 Hz, OCH2O), 6.05 (d, J ¼ 4.9 Hz, H–C(10)), 6.78 (m, 4H, trityl-H), 7.19–7.33,
7.38–7.42 (m, 9H, trityl-H), 7.48 (d, J ¼ 8.4 Hz, 4-nitrophenyl C(2)=C(6)), 7.71 (s, H–C(8)), 8.17 (d,
13
J ¼ 8.4 Hz, 4-nitrophenyl H–C(3)=H–C(5)) ppm; C NMR (125 MHz, CDCl3, 30ꢀC): ꢁ ¼ 11.78
((CH3)2CH), 17.66 ((CH3)2CH), 35.23 (CH2–C6H4–NO2), 37.33 (N2-(CH3)2), 55.08 (2 CH3O),
63.59 (C(50)), 65.57 (O6–CH2), 70.80 (C(30)), 81.27 (C(20)), 83.49 (C(40)), 86.34, 86.75 (C(10)),
90.72 (OCH2O), 113.06 (trityl-C), 114.16, 123.65 (4-nitrophenyl C(3)=C(5)), 126.71, 127.71,
128.11 (trityl-C), 129.81, 129.99 (trityl-C, 4-nitrophenyl C(2)=C(6)), 135.70, 135.79, 137.74 (C(8)),
144.56, 146.11, 146.79, 154.13, 158.43, 159.04, 159.86 ppm; UV (MeOH): ꢂ(") ¼ 260 (18100) nm
(molÀ 1 dm3 cmÀ 1); MALDI-MS: m=z ¼ 949.2 (10, [Mþ H]þ ), 303.1 (100, [(MeO)2Tr]þ ).

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866
50-O-(4,40-Dimethoxytrityl)-N2,N2-dimethyl-O6-[2-(4-nitrophenyl)ethyl]-20-O-
[[(triisopropylsilyl)oxy]methyl]guanosine 30-(2-cyanoethyl diisopropylphosphoramidite)
(13, C60H81N8O11PSi)
A solution of 444 mg of 12 (0.47 mmol) in 4 cm3 of anhydrous CH2Cl2 was treated consecutively with
343 mg of ethyldimethylamine (4.7mmol) and 116mg of 2-cyanoethyl diisopropylphosphoramido-
chloridite (0.49 mmol). The mixture was stirred for 2 h, quenched with 0.15cm3 of MeOH, and evap-
orated to dryness. Workup and column chromatography (silica gel, hexane:EtOAc¼ 5:2 ( þ 2% Et3N))
afforded 469 mg of 13 as white, solid foam (87%, 1:1 mixture of diastereoisomers). TLC (silica gel,
1
i
hexane:EtOAc¼ 1:1): Rf ¼ 0.7; H NMR (500MHz, CDCl3, 26ꢀC): ꢁ ¼ 0.86–1.28 (m, 42H, Pr3Si,
24H, ((CH3)2CH)2N), 2.32, 2.64 (2m, 4H, CH2CN), 3.06, 3.07 (2s, 12H, N2-(CH3)2), 3.29 (t, 4H,
J ¼ 6.4 Hz, CH2–C6H4–NO2), 3.35–3.47 (m, 4H, H2–C(50)), 3.48–3.65 (m, 4H, ((CH3)2CH)2N, 2H,
POCH2), 3.77, 3.78 (2s, 12H, OCH3), 3.84–3.96 (2m, 2H, POCH2), 4.30, 4.33 (2q, 2H, H–C(40)),
4.62 (m, 2H, H–C(30)), 4.75 (t, J ¼ 6.4 Hz, O6–CH2), 4.88–4.97 (q, 4H, J ꢁ 6 Hz, OCH2O), 5.06 (m,
2H, H–C(20)), 6.05, 6.09 (2d, 2H, J ¼ 5.9 Hz, H–C(10)), 6.75–6.79, 7.20–7.40 (m, 26H, trityl-H), 7.48
(2d, J ꢁ 8 Hz, 4-nitrophenyl H–C(2)=H–C(6)), 7.70 (s, 2H, H–C(8)), 8.16 (d, J ꢁ 8 Hz, 4-nitrophenyl
31
H–C(3)=H–C(5)) ppm; P NMR (200 MHz, CDCl3, 27ꢀC): ꢁ ¼ 150.22, 150.59 ppm; UV (MeOH):
ꢂ(") ¼ 260 (max, 26400), 281 (sh, 18200) nm (molÀ 1 dm3 cmÀ 1); MALDI-FTICR-MS: m=z ¼
1171.5763 (6, [Mþ Na]þ ), 303.1412 (100, [(MeO)2Tr]þ ).
50-O-(4,40-Dimethoxytrityl)-1-methylinosine (14, C32H32N4O7)
Inosine (276 mg, 1.0mmol) was dissolved in 2.7 cm3 of anhydrous DMSO and treated with 25mg of
NaH (1.0mmol). The mixture was stirred until H2 evolution ceased followed by dropwise addition of
142 mg of methyliodide (1.0mmol). The mixture was stirred for 4 h at rt and evaporated to dryness.
The pasty solid was coevaporated with MeOH and three times with anhydrous pyridine to be finally
dissolved in 1.5 cm3. Then, 360 mg of 4,40-dimethoxytritylchloride (1.06 mmol) were added in three
portions over a period of 3 h. Stirring was continued for one more hour at 60ꢀC. Addition of MeOH,
evaporation to dryness, workup, and column chromatography (silica gel, CH2Cl2:CH3OH¼ 25:1 to
20:1) yielded 270 mg of 14 as pale yellow foam (47%). TLC (silica gel, CH2Cl2:CH3OH¼ 15:1):
Rf ¼ 0.4; 1H NMR (500 MHz, d6-DMSO, 25ꢀC): ꢁ ¼ 3.17 (m, H1–C(50)), 3.23 (m, H2–C(50)), 3.49 (s,
1-CH3), 3.71, 3.72 (2s, 2 OCH3), 4.06 (q, H–C(40)), 4.20 (q, H–C(30)), 4.56 (q, H–C(20)), 5.22 (d,
J ¼ 5.8 Hz, HO–C(30)), 5.56 (d, J ¼ 5.7 Hz, HO–C(20)), 5.88 (d, J ¼ 4.7 Hz, H–C(10)), 6.83 (m, 4H,
trityl-H), 7.16–7.27, 7.33–7.36 (m, 9H, trityl-H), 8.20 (s, H–C(8)), 8.32 (s, H–C(2)) ppm; MALDI-
FTICR-MS: m=z ¼ 607.2150 (8, [Mþ Na]þ ), 303.1379 (100, [(MeO)2Tr]þ ).
50-O-(4,40-Dimethoxytrityl)-1-methyl-20-O-[[(triisopropylsilyl)oxy]methyl]inosine
(15, C42H54N4O8Si)
To a stirred solution of 250 mg of 14 (0.43 mmol) and 220 mg of ethyldiisopropylamine (1.7mmol) in
3 cm3 of 1,2-dichloroethane, 156 mg of di-tert-butyltindichloride (0.51mmol) were added. The mixture
was heated to 70ꢀC for 15min, allowed to cool to rt again and treated with 105 mg of [(triisopropyl-
silyl)oxy]methylchloride (0.47 mmol). Stirring was continued for 3 h, followed by addition of
0.5 cm3 of MeOH, evaporation to dryness and workup. Column chromatography (silica gel,
CH2Cl2: MeOH ¼ 45:1 to 30:1) afforded 148 mg of 15 as white, solid foam (45%). The regioselectiv-
ity of 20-O-alkylated over 30-O-alkylated product was approximately 5:4. TLC (silica gel,
1
i
CH2Cl2:CH3OH ¼ 20:1): Rf ¼ 0.5; H NMR (500MHz, CDCl3, 25ꢀC): ꢁ ¼ 1.00–1.12 (m, Pr3Si);
2.99 (d, J ¼ 4.7 Hz, HO–C(30)), 3.39 (dd, J ¼ 4.2, 10.4Hz, H1–C(50)), 3.43 (dd, J ¼ 3.4, 10.4 Hz,
H2–C(50)), 3.61 (s, 1-CH3), 3.78 (s, 2 OCH3), 4.29 (q, H–C(40)), 4.53 (q, H–C(30)), 4.82 (t, H–
C(20)), 4.94, 5.13 (2d, J ¼ 4.8 Hz, OCH2O), 6.11 (d, J ¼ 4.9 Hz, H–C(10)), 6.81 (m, 4H, trityl-H),
7.18–7.35, 7.41–7.47 (m, 9H, trityl-H), 7.83 (s, H–C(8)), 7.93 (s, H–C(2)) ppm; 13C NMR (75 MHz,

RNA Solid-Phase Synthesis
867
CDCl3, 28ꢀC): ꢁ ¼ 12.25 ((CH3)2CH), 18.14 ((CH3)2CH), 34.46 (1-CH3), 55.61 (2 CH3O), 64.00
(C(50)), 71.52 (C(30)), 82.79 (C(20)), 84.78 (C(40) and trityl-C), 87.03 (C(10)), 91.27 (OCH2O),
113.57 (trityl-C), 125.45, 127.29, 128.23, 128.56, 130.48, 130.51 (trityl-C), 136.04, 136.15, 139.22
(C(8)), 144.97, 147.48 (C(2)), 148.09, 157.37, 159.00ppm; UV (MeOH): ꢂ(") ¼ 235 (max, 31700),
260 (plateau, 8800), 281 (sh, 6600) nm (molÀ 1 dm3 cmÀ 1); MALDI-FTICR-MS: m=z ¼ 793.3797 (42,
[Mþ Na]þ ), 303.1413 (100, [(MeO)2Tr]þ ).
50-O-(4,40-Dimethoxytrityl)-1-methyl-20-O-[[(triisopropylsilyl)oxy]methyl]inosine
30-(2-cyanoethyl diisopropylphosphoramidite) (16, C51H71N6O9PSi)
A solution of 300 mg of 15 (0.39 mmol) in 3 cm3 of anhydrous CH2Cl2 was treated consecutively with
286 mg of ethyldimethylamine (3.9mmol) and 208mg of 2-cyanoethyl diisopropylphosphoramido-
chloridite (0.88 mmol). The mixture was stirred for 2 h, quenched with 0.15cm3 of MeOH, and evap-
orated to dryness. Workup and column chromatography (silica gel, hexane:EtOAc¼ 7:3 ( þ 2% Et3N))
afforded 348 mg of 16 as white, solid foam (92%, 1:1 mixture of diastereoisomers). TLC (silica gel,
hexane:EtOAc (2% TEA) ¼ 4:1): Rf ¼ 0.5; 1H NMR (500MHz, CDCl3, 25ꢀC): ꢁ ¼ 0.86–1.19 (m, 42H,
iPr3Si, 24H, ((CH3)2CH)2N), 2.38, 2.65 (2m, 4H, CH2CN), 3.32–3.40 (m, 2H, H1–C(50)), 3.41–3.52
(m, 2H, H2–C(50)), 3.60, 3.61 (2s, 6H, 1-CH3), 3.60–3.70 (m, 2H, POCH2, 4H, ((CH3)2CH)2N), 3.78,
3.79 (2s, 12H, OCH3), 3.84–3.97 (m, 2H, POCH2), 4.35, 4.41 (2q, 2H, H–C(40)), 4.57–4.65 (m, 2H,
H–C(30)), 4.91–4.98 (4d, 4H, J ¼ 5.0 Hz, OCH2O), 5.00–5.04 (2t, 2H, H–C(20)), 6.05, 6.09 (2d, 2H,
J ¼ 6.1 Hz, H–C(10)), 6.75–6.83, 7.18–7.35, 7.40–7.44 (m, 26H, trityl-H), 7.78 (s, 2H, H–C(8), 7.92,
31
7.93 (2s, 2H, H–C(2)) ppm; P NMR (200MHz, CDCl3, 27ꢀC): ꢁ ¼ 150.06, 150.73ppm; UV
(MeOH): ꢂ(") ¼ 235 (max, 26000), 260 (plateau, 6900), 281 (sh, 5000) nm (molÀ 1 dm3 cmÀ 1);
MALDI-FTICR-MS: m=z ¼ 993.4972 (24, [Mþ Na]þ ), 303.1414 (100, [(MeO)2Tr]þ ).
50-O-(4,40-Dimethoxytrityl)-3-methyluridine (17, C31H32N2O8)
Uridine (10.0 g, 41mmol) was dissolved in 100cm3 of anhydrous DMSO and treated with 985mg of
NaH (41 mmol). The mixture was stirred until H2 evolution ceased. Methyliodide (5.8g, 41mmol) was
added dropwise. The mixture was stirred for 5 h at rt and evaporated to dryness. The pasty solid was
coevaporated with MeOH and three times with anhydrous pyridine to be finally dissolved in 50cm3.
Then, 22.2g of 4,40-dimethoxytritylchloride (66 mmol) were added in four portions over a period of
4 h. Stirring was continued for one more hour at 60ꢀC. Addition of MeOH, evaporation to dryness,
workup, and column chromatography (silica gel, CH2Cl2:CH3OH ¼ 30:1) yielded 12.7g of 17 as pale
1
yellow foam (55%). TLC (silica gel, CH2Cl2:CH3OH¼ 20:1): Rf ¼ 0.4; H NMR (500MHz, CDCl3,
30ꢀC): ꢁ ¼ 3.35 (s, 3-CH3), 3.40 (dd, J ¼ 3.4, 10.9Hz, H1–C(50)), 3.49 (dd, J ¼ 2.9, 10.9Hz, H2–
C(50)), 3.79 (s, 2 OCH3), 4.28 (q, H–C(40)); 4.34 (m, 2H, H–C(30) and H–C(20)), 5.56 (d, J ¼ 8.2 Hz,
H–C(5)); 5.80 (d, J ¼ 3.6 Hz, H–C(10)), 6.83 (m, 4H, trityl-H), 7.23–7.36 (m, 9H, trityl-H), 7.75 (d,
13
J ¼ 8.2 Hz, H–C(6)) ppm; C NMR (125MHz, CDCl3, 27ꢀC): ꢁ ¼ 27.47 (3-CH3), 55.16 (2 CH3O),
62.52 (C(50)), 71.25 (C(30)), 76.49 (C(20)), 84.92 (C(40)), 87.01, 92.06 (C(10)), 101.37 (C(5)), 113.20,
127.06, 127.89, 127.95, 129.97 (trityl-C), 135.06, 135.15, 137.30 (C(6)), 144.15, 152.12, 158.66,
162.53 ppm; UV (MeOH): ꢂ(") ¼ 233 (max, 24400), 260 (10500) nm (molÀ 1 dm3 cmÀ 1); MALDI-
FTICR-MS: m=z ¼ 583.2051 (3, [Mþ Na]þ ); 303.1388 (100, [(MeO)2Tr]þ ).
50-O-(4,40-Dimethoxytrityl)-3-methyl-20-O-[[(triisopropylsilyl)oxy]methyl]uridine
(18, C41H54N2O9Si)
To
a stirred solution of 3.5 g of 17 (6.25 mmol) and 3.23g of ethyldiisopropylamine
(25 mmol) in 30 cm3 of 1,2-dichloroethane, 2.28g of di-tert-butyltindichloride (7.5mmol) were

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C. Hobartner et al.
868
added. The mixture was heated to 70ꢀC for 15 min, allowed to cool to rt and treated with 1.53g of
[(triisopropylsilyl)oxy]methylchloride (6.88 mmol). Stirring was continued for 60min, followed by
addition of 2 cm3 of MeOH and workup. Column chromatography (silica gel, hexane:EtOAc¼ 4:1
to 1:1) afforded 2.18 g of 18 as white, solid foam (47%). The regioselectivity of 20-O-alkylated over 30-
O-alkylated product was approximately 3:2. TLC (silica gel, hexane:EtOAc¼ 2:1): Rf ¼ 0.5; 1H NMR
(500 MHz, CDCl3, 30ꢀC): ꢁ ¼ 1.07–1.27 (m, iPr3Si), 3.16 (d, J ¼ 6.3 Hz, HO–C(30)), 3.32 (s, 3-CH3),
3.51 (q, H2–C(50)), 3.80 (s, 2 OCH3), 4.10 (m, H–C(40)), 4.26 (m, H–C(20)), 4.44 (q, H–C(30)), 5.06,
5.24 (2d, J ¼ 4.7 Hz, OCH2O), 5.39 (d, J ¼ 8.2 Hz, H–C(5)), 6.03 (d, J ¼ 3.1 Hz, H–C(10)), 6.83 (m,
4H, trityl-H), 7.24–7.40 (m, 9H, trityl-H), 7.90 (d, J ¼ 8.2 Hz, H–C(6)) ppm; 13C NMR (75 MHz,
CDCl3, 27ꢀC): ꢁ ¼ 11.87 ((CH3)2CH), 17.76 ((CH3)2CH), 27.51 (3-CH3), 55.21 (2 CH3O), 62.07
(C(50)), 69.19 (C(30)), 82.84 (C(20)), 83.54 (C(40)), 87.05 (C(10)), 88.65, 90.58 (OCH2O), 101.58
(C(5)), 113.26, 113.27, 127.09, 127.94, 128.17, 130.11, 130.15 (trityl-C), 135.18, 135.42, 137.71
(C(6)), 144.36, 151.04, 158.69, 158.72, 162.80 ppm; UV (MeOH): ꢂ("))¼ 234 (max, 23600), 260
(10400) nm (molÀ 1 dm3 cmÀ 1); MALDI-FTICR: m=z ¼ 769.3489 (5, [Mþ Na]þ ); 303.1388 (100,
[(MeO)2Tr]þ ).
50-O-(4,40-Dimethoxytrityl)-3-methyl-20-O-[[(triisopropylsilyl)oxy]methyl]uridine
30-(2-cyanoethyl diisopropylphosphoramidite) (19, C50H71N4O10PSi)
A solution of 1.6 g of 18 (2.14 mmol) in 7 cm3 of CH2Cl2 was treated consecutively with 1.6 g of
ethyldimethylamine (21.5 mmol) and 760 mg of 2-cyanoethyl diisopropylphosphoramidochloridite
(3.21 mmol). The mixture was stirred for 2 h, quenched with 0.3cm3 of MeOH, and evaporated to
dryness. Workup and column chromatography (silica gel, hexane:EtOAc¼ 3:1 ( þ 2% Et3N)) afforded
1.80g of 19 as white, solid foam (89%, 1:1 mixture of diastereoisomers). TLC (silica gel,
1
i
hexane:EtOAc¼ 2:1): Rf ¼ 0.5; H NMR (500MHz, CDCl3, 25ꢀC): ꢁ ¼ 0.99–1.08 (m, Pr3Si),
1.12–1.26 (m, ((CH3)2CH)2N), 2.39, 2.64 (2m, 4H, CH2CN), 3.31 (2s, 6H, 3-CH3), 3.39 (m, 2H,
H1–C(50)), 3.53–3.60 (m, 2H, H2–C(50), 2H, POCH2, 4H, ((CH3)2CH)2N), 3.78, 3.79 (2s, 12H,
OCH3), 3.81–3.96 (m, 2H, POCH2), 4.24, 4.29 (2q, 2H, H–C(40)), 4.40–4.46 (m, 4H, H–C(20)),
H–C(30)), 4.99–5.07 (4d, 4H, J ¼ 5.0 Hz, OCH2O), 5.41, 5.46 (2d, J ¼ 8.1 Hz, H–C(5)), 6.16, 6.18
(2d, 2H, J ¼ 4.7 Hz, H–C(10)), 6.82–6.85, 7.26–7.37, 7.35–7.53 (m, 26H, trityl-H), 7.78, 7.81 (d,
J ¼ 8.1 Hz, H–C(6)) ppm; 31P NMR (200 MHz, CDCl3, 27ꢀC): ꢁ ¼ 149.90, 150.48 ppm; UV (MeOH):
ꢂ(") ¼ 260 (11100) nm (molÀ 1 dm3 cmÀ 1); MALDI-FTICR-MS: m=z ¼ 969.4564 (8, [Mþ Na]þ ),
303.1394 (100, [(MeO)2Tr]þ ).
30-O-Acetyl-50-O-(4,40-dimethoxytrityl)-20-O-[[(triisopropylsilyl)oxy]methyl]uridine
(20, C42H54N2O10Si)
A stirred solution of 430 mg of 50-O-(4,40-dimethoxytrityl)-20-O-[[(triisopropylsilyl)oxy]methyl]uri-
dine (0.59 mmol) [9] and 7 mg of DMAP (0.06 mmol) in 0.75 cm3 of anhydrous pyridine under Ar
atmosphere was cooled to 0ꢀC and treated with 0.06 cm3 of acetic anhydride (0.65 mmol). The mixture
was stirred for 1 h at room temperature, quenched with 0.1 cm3 of MeOH, and evaporated to dryness.
Workup and column chromatography (silica gel, hexane:EtOAc¼ 6:4 ( þ 1% Et3N)) afforded 440 mg
of 20 as white, solid foam (96%). TLC (silica gel, CH2Cl2:MeOH¼ 98:2): Rf ¼ 0.5; 1H NMR
i
(300 MHz, CDCl3, 27ꢀC): ꢁ ¼ 0.97–1.08 (m, Pr3Si), 2.10 (s, COCH3), 3.44 (dd, J ¼ 2.5, 11.0Hz,
H1–C(50)), 3.53 (dd, J ¼ 2.5, 11.0 Hz, H2–C(50)), 3.79 (s, 2 OCH3), 4.21 (q, H–C(40)), 4.56 (t, H–
C(20)), 4.93 (t, H–C(30)), 4.95 (s, OCH2O), 5.39 (d, J ¼ 8.2 Hz, H–C(5)), 6.13 (d, J ¼ 5.9 Hz, H–C(10)),
6.84 (m, 4H, trityl-H), 7.24–7.39 (m, 9H, trityl-H), 7.75 (s, H–C(6)), 8.54 (s, NH) ppm; 13C NMR
(75 MHz, CDCl3, 27ꢀC): ꢁ ¼ 11.86 ((CH3)2CH), 17.74 ((CH3)2CH), 27.77 (COCH3), 55.22 (2 CH3O),
62.67 (C(50)), 71.18 (C(30)), 77.20 (C(20)), 81.90 (C(40)), 86.64 (C(10)), 87.44, 89.39 (OCH2O), 102.65
(C(5)), 113.35, 127.20, 128.04, 128.14, 130.07, 130.12 (trityl-C), 135.04, 135.15, 140.05 (C(6)),

RNA Solid-Phase Synthesis
869
144.12, 150.21, 158.77, 162.68, 170.05 ppm; UV (MeOH): ꢂ("))¼ 235 (max, 23300), 260 (11300) nm
(molÀ 1 dm3 cmÀ 1).
50-O-(4,40-Dimethoxytrityl)-N4-methyl-20-O-[[(triisopropylsilyl)oxy]methyl]cytosine
(21, C41H55N3O8Si)
Method A: To a stirred solution of 105 mg of 20 (0.14 mmol) and 1.2 mg of DMAP (0.01 mmol) in
anhydrous 1.5 cm3 of 1,2-dichloroethane, 142 mg of triethylamine (1.4mmol) and subsequently, 62 mg
of triisopropylbenzenesulfonylchloride (0.21 mmol) were added. The solution was stirred for 1 h. After
workup the crude product was dissolved in 33% CH3NH2 in ethanol and stirred overnight at rt. The
solvents were evaporated and column chromatography (silica gel, CH2Cl2:MeOH ¼ 99:1 to 96:4))
afforded 85 mg of 21 as white solid foam (81%).
Method B: To a stirred solution of 490 mg of 50-O-(4,40-dimethoxytrityl)-N4-methylcytosine
(0.88 mmol) [16] and 455 mg of ethyldiisopropylamine (3.52 mmol) in 3.5cm3 of 1,2-dichloroethane,
320 mg of di-tert-butyltindichloride (1.06 mmol) were added. The mixture was heated to 70ꢀC for
15min, allowed to cool to rt again and treated with 235 mg of [(triisopropylsilyl)oxy] methylchloride
(1.06 mmol). Stirring was continued for 3 h, followed by addition of 0.5 cm3 of MeOH, evaporation to
dryness and workup. Column chromatography (silica gel, EtOAc) afforded 100 mg of 21 as white solid
foam (15%). The regioselectivity of 20-O-alkylated over 30-O-alkylated product was approximately 5:4.
1
i
TLC (silica gel, EtOAc): Rf ¼ 0.5; H NMR (300 MHz, d6-DMSO, 27ꢀC): ꢁ ¼ 0.92–1.13 (m, Pr3Si),
2.74 (d, J ¼ 4.5 Hz, N4-CH3), 3.21 (m, H2–C(50)), 3.73 (s, 2 OCH3), 3.94 (q, H–C(40)), 4.14–4.19 (q,
H–C(20), H–C(30)), 4.95–5.02 (m, OCH2O, HO–C(30)), 5.53 (d, J ¼ 7.5 Hz, H–C(5)), 5.93 (d,
J ¼ 3.5 Hz, H–C(10)), 6.88, 7.27–7.39 (d, 4H, m, 9H, trityl-H), 7.60 (d, J ¼ 7.5 Hz, H–C(6)), 7.67
(q, NH) ppm; 13C NMR (75 MHz, d6-DMSO, 27ꢀC): ꢁ ¼ 11.17 ((CH3)2CH), 17.59 ((CH3)2CH), 26.77
(N4-CH3), 54.96 (2 CH3O), 63.00 (C(50)), 68.72 (C(30)), 78.02 (C(20)), 82.46 (C(40)), 85.82, 87.59
(C(10)), 88.44 (OCH2O), 94.71 (C(5)), 113.14 (trityl-C), 117.63, 126.69, 127.68, 127.77, 129.67 (trityl-
C), 135.27, 135.39, 139.51 (C(6)), 144.59, 154.89, 158.07, 163.68 ppm; UV (MeOH): ꢂ(") ¼ 233
(max, 26400), 260 (10200), 273 (max, 11600) nm (molÀ 1 dm3 cmÀ 1); MALDI-TOF-MS:
m=z ¼ 746.0 (23, [M þ H]þ ), 303.1 (100, [(MeO)2Tr]þ ).
50-O-(4,40-Dimethoxytrityl)-N4-methyl-20-O-[[(triisopropylsilyl)oxy]methyl]cytosine
30-(2-cyanoethyl diisopropylphosphoramidite) (22, C50H72N5O9PSi)
A solution of 95mg of 21 (0.13 mmol) in 3 cm3 of CH2Cl2 was treated consecutively with 95 mg of
ethyldimethylamine (1.3mmol) and 47 mg of 2-cyanoethyl diisopropylphosphoramidochloridite
(0.20 mmol). The mixture was stirred for 1 h, quenched with 0.1cm3 of MeOH, and evaporated to
dryness. Workup and column chromatography (silica gel, hexane:EtOAc¼ 1:1 ( þ 1% Et3N)) afforded
78mg of 22 as white foam (64%, 1:1 mixture of diastereoisomers). TLC (silica gel, hexane:
1
EtOAc¼ 2:8): Rf ¼ 0.4; H NMR (500MHz, CDCl3, 26ꢀC, four species: mixture of 2 diastereomers
i
and 2 rotamers): ꢁ ¼ 0.92–1.27 (m, 42H, Pr3Si, 24H, ((CH3)2CH)2N), 2.36 (t, J ¼ 6.5 Hz, 2H,
CH2CN), 2.61 (m, 2H, CH2CN), 2.81, 2.99 (2br s, 3:1 (rotamers), 6H, N4-CH3), 3.34–3.37 (m, 2H,
H1–C(50)), 3.45–3.70 (m, 8H, ((CH3)2CH)2N, H2–C(50), POCH2), 3.78, 3.79 (2s, 12H, 2 OCH3), 3.82–
3.96 (m, 2H, POCH2), 4.12–4.30 (4 m, 2H, H–C(40), 2H, H–C(20)), 4.40, 4.49 (2m, 2H, H–C(30)), 4.81
(br, 2H, NH), 5.12–5.20 (m, 4H, OCH2O, 2H, H–C(5)), 6.12, 6.17 (2br s, 2H, H–C(10)), 6.80–6.84,
7.18–7.43 (m, 26H, trityl-H), 7.88, 7.97, 8.07, 8.11 (2d, 2br s, 3:3:1:1, 2H, H–C(6)) ppm; the addi-
tional splitting of some signals (N4-CH3, H–C(6)) is tentatively assigned to rotamers around the
glycosidic bond or around the C(4)-N4-bond; at 40ꢀC (CDCl3) the rotamer ratio changes to 4:1 (judged
by the signal of N4-CH3); at 60ꢀC (d8-toluene) only a single rotamer of each P-diastereomer is
31
observed; P NMR (200MHz, CDCl3, 27ꢀC): ꢁ ¼ 149.89, 150.58 ppm; UV (MeOH): ꢂ(") ¼ 233
(max, 24800), 260 (10100), 273 (max, 11300) nm (molÀ 1 dm3 cmÀ 1); MALDI-TOF-MS: m=z ¼
946.3 (10, [Mþ H]þ ), 303.1 (100, [(MeO)2Tr]þ ).

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20,30,50-O-Triacetyl-6-chloroinosine (23, C16H17N4O7Cl)
20,30,50-O-Triacetylinosine (5.41 g, 13.7mmol) in 70cm3 of CHCl3 was treated with 3.51 g of chlo-
romethylenedimethyliminiumchloride (27.4mmol) and refluxed for 4 h. The reaction mixture was
transferred into a separatory funnel and slowly dropped into 100cm3 of a stirred, semi-saturated
aqueous NaHCO3 solution. The organic layer was washed twice with water, dried with Na2SO4,
and evaporated to dryness. Column chromatography (silica gel, CH2Cl2:MeOH¼ 20:1) yielded
1
5.24g of 23 (93%). TLC (silica gel, CH2Cl2:CH3OH ¼ 20:1): Rf ¼ 0.7; H NMR (500MHz, CDCl3,
30ꢀC): ꢁ ¼ 2.10, 2.13, 2.17 (3s, COCH3), 4.40 (dd, J ¼ 4.4, 12.2Hz, H1–C(50)), 4.44–4.50 (m, H2–
C(50), H–C(40)), 5.65 (t, H–C(30)), 5.96 (t, H–C(20)), 6.24 (d, J ¼ 5.2 Hz, H–C(10)), 8.29 (s, H–C(8)),
13
8.79 (s, H–C(2)) ppm; C NMR (125 MHz, CDCl3, 30ꢀC): ꢁ ¼ 20.24, 20.40, 20.62 (COCH3), 62.79
(C(50)), 70.42 (C(30)), 73.05 (C(20)), 80.51 (C(40)), 86.81 (C(10)), 132.32, 143.40 (C(8)), 151.65,
151.65, 152.25 (C(2)), 169.19, 169.40, 170.10 ppm.
50-O-(4,40-Dimethoxytrityl)-N6-methyladenosine (24, C32H33N5O6)
Compound 23 (960 mg, 2.32 mmol) was dissolved in a mixture of 33% CH3NH2 in 8 cm3 of ethanol
and 40% (CH3)NH2 in 8 cm3 of water. The solution was stirred for 12h, then evaporated to dryness,
three times coevaporated with anhydrous pyridine to be finally dissolved in 5 cm3. Then, 1.30g of 4,40-
dimethoxytritylchloride (3.83 mmol) were added in three portions over a period of 6 h. Stirring was
continued overnight. Addition of MeOH, evaporation to dryness, workup, and column chromatography
(silica gel, CH2Cl2:CH3OH ¼ 50:1 to 10:1) yielded 1.0g of 24 as pale yellow foam (74%). TLC (silica
1
gel, CH2Cl2:CH3OH¼ 20:1): Rf ¼ 0.5; H NMR (300MHz, CDCl3, 27ꢀC): ꢁ ¼ 3.20 (br d, N 6-CH3),
3.26 (dd, J ¼ 3.3, 10.5Hz, H1–C(50)), 3.44 (dd, J ¼ 3.3, 10.5 Hz, H2–C(50)), 3.76 (s, 2 OCH3), 4.38–
4.42 (m, H–C(40), H–C(30)), 4.77 (t, H–C(20)), 5.97 (d, J ¼ 5.7 Hz, H–C(10)), 6.15 (q, NH), 6.73 (m,
4H, trityl-H), 7.16–7.30 (m, 9H, trityl-H), 8.02 (s, H–C(8)), 8.34 (H–C(2)) ppm; 13C NMR (75 MHz,
CDCl3, 27ꢀC): ꢁ ¼ 27.45 (br, N 6-CH3), 55.15 (2 CH3O), 63.60 (C(50)), 72.55 (C(30)), 75.95 (C(20)),
86.13, 86.49 (C(40)), 90.64 (C(10)), 113.11 (trityl-C), 120.22, 126.84, 127.78, 128.03, 129.93, 129.94
(trityl-C), 135.50, 135.63, 137.96 (C(8)), 144.33, 152.62 (C(2)), 155.56, 158.52 ppm; UV (MeOH):
ꢂ(") ¼ 234 (max, 20800), 260 (14500), 265 (max, 15600) nm (molÀ 1 dm3 cmÀ 1).
50-O-(4,40-Dimethoxytrityl)-N6-methyl-20-O-[[(triisopropylsilyl)oxy]methyl]adenosine
(25, C42H55N5O7Si)
To a stirred solution of 970 mg of 24 (1.66 mmol) and 858 mg of ethyldiisopropylamine (6.64 mmol) in
6 cm3 of 1,2-dichloroethane, 605 mg of di-tert-butyltindichloride (1.99mmol) were added. The mixture
was heated to 70ꢀC for 15min, allowed to cool to rt and treated with 443mg of [(triisopropylsilyl)
oxy]methylchloride (1.99 mmol). Stirring was continued for 60min, followed by addition of 0.4 cm3 of
MeOH and workup. Column chromatography (silica gel, hexane:EtOAc¼ 6:4 to 1:9) afforded 290 mg
of 25 (23%) as white, solid foam. The regioselectivity of 20-O-alkylated over 30-O-alkylated product
1
was approximately 5:4. TLC (silica gel, hexane:EtOAc¼ 1:9): Rf ¼ 0.6; H NMR (300 MHz, CDCl3,
27ꢀC): ꢁ ¼ 0.96–1.05 (m, iPr3Si), 3.06 (d, J ¼ 3.9 Hz, HO–C(30)), 3.19 (d, J ¼ 4.5 Hz, N 6-CH3), 3.38
(dd, J ¼ 4.2, 10.2Hz, H1–C(50)), 3.50 (dd, J ¼ 3.6, 10.2Hz, H2–C(50)), 3.78 (s, 2 OCH3), 4.26 (q, H–
C(40)), 4.52 (q, H–C(30)), 4.93 (t, H–C(20)), 4.98, 5.14 (2d, J ¼ 4.8Hz, OCH2O), 5.75 (br, NH), 6.14
(d, J ¼ 5.4 Hz, H–C(10)), 6.78 (m, 4H, trityl-H), 7.22–7.44 (m, 9H, trityl-H), 7.93 (s, H–C(8)), 8.33 (s,
13
H–C(2)) ppm; C NMR (75 MHz, CDCl3, 27ꢀC): ꢁ ¼ 11.80 ((CH3)2CH), 17.70 ((CH3)2CH); 27.70
(br s, N 6-CH3), 55.13 (2 CH3O), 63.37 (C(50)), 70.80 (C(30)), 81.80 (C(20)), 84.06 (C(40)), 86.48, 87.05
(C(10)), 90.72 (OCH2O), 113.10 (trityl-C), 120.41, 126.79, 127.76, 128.18, 130.05 (trityl-C), 135.77,
135.78, 138.55 (C(8)), 144.59, 153.27 (C(2)), 155.46, 158.50 ppm; UV (MeOH): ꢂ("))¼ 232 (max,
28700), 260 (17300), 265 (max, 18400) nm (molÀ 1 dm3 cmÀ 1); MALDI-TOF-MS: m=z ¼ 769.9 (20,
[Mþ H]þ ), 303.1 (100, [(MeO)2Tr]þ ).

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50-O-(4,40-Dimethoxytrityl)-N6-methyl-20-O-[[(triisopropylsilyl)oxy]methyl]adenosine
30-(2-cyanoethyl diisopropylphosphoramidite) (26, C51H72N7O8PSi)
A solution of 200 mg of 25 (0.26 mmol) in 3 cm3 of CH2Cl2 was treated consecutively with 336 mg of
ethyldimethylamine (2.6mmol) and 92 mg of 2-cyanoethyl diisopropylphosphoramidochloridite
(0.39 mmol). The mixture was stirred for 2 h, quenched with 0.3cm3 of MeOH, and evaporated to
dryness. Workup and column chromatography (silica gel, hexane:EtOAc¼ 3:1 to 3:2 ( þ 1% Et3N))
afforded 216 mg of 26 as pale yellow, solid foam (86%, 1:1 mixture of diastereoisomers). TLC (silica
1
gel, hexane:EtOAc¼ 1:1): Rf ¼ 0.5; H NMR (500MHz, CDCl3, 26ꢀC): ꢁ ¼ 0.88–1.26 (m, 42H,
iPr3Si, 24H, ((CH3)2CH)2N), 2.37 (t, J ¼ 6.5 Hz, 2H, CH2CN), 2.65 (m, 2H, CH2CN), 3.20 (br s,
6H, N6-CH3), 3.30–3.33 (m, 2H, H1–C(50)), 3.49–3.70 (m, 4H, ((CH3)2CH)2N, 2H, H2–C(50), 2H,
POCH2), 3.77, 3.78 (2s, 12H, 2 OCH3), 3.86–3.97 (m, 2H, POCH2), 4.32, 4.37 (2q, 2H, H–C(40)),
4.67–4.75 (m, 2H, H–C(30)), 4.91–5.01 (4d, J ¼ 5.0 Hz, 4H, OCH2O), 5.16–5.21 (m, 2H, H–C(20)),
5.66 (br, 2H, NH), 6.11, 6.13 (2d, J ¼ 5.5 Hz, 2H, H–C(10)), 6.75–6.79, 7.17–7.41 (m, 26H, trityl-H),
31
7.90, 7.92 (2s, 2H, H–C(8)), 8.28, 8.30 (2s, 2H, H–C(2)) ppm; P NMR (200MHz, CDCl3, 27ꢀC):
ꢁ ¼ 149.89, 150.58 ppm; UV (MeOH): ꢂ(") ¼ 232 (max, 23200), 260 (15400), 265 (max, 16400) nm
(molÀ 1 dm3 cmÀ 1); MALDI-TOF-MS: 970.2 (45, [Mþ H]þ ), 303.1 (100, [(MeO)2Tr]þ ).
50-O-(4,40-Dimethoxytrityl)-N6,N6-dimethyladenosine (27, C33H35N5O6)
Compound 23 (950mg, 2.30 mmol) was dissolved in a solution of 33% (CH3)2NH in 6.0 cm3 of
ethanol. After 1 h a solution of 40% (CH3)2NH in 3.0cm3 of water was added and stirring was
continued for another 8 h. The reaction mixture was evaporated to dryness and coevaporated three
times with anhydrous pyridine to be finally dissolved in 2.3 cm3. Then, 1.16 g of 4,40-dimethoxytri-
tylchloride (3.41 mmol) were added in three portions over a period of 3 h. DMAP (45 mg, 0.36 mmol)
was added and stirring was continued overnight. Addition of MeOH, evaporation to dryness, workup,
and column chromatography (silica gel, CH2Cl2:CH3OH¼ 25:1) yielded 840 mg of 27 as white foam
1
(61%). TLC (silica gel, CH2Cl2:CH3OH¼ 15:1): Rf ¼ 0.4; H NMR (500MHz, CDCl3, 30ꢀC):
ꢁ ¼ 3.14 (s, OH), 3.20 (dd, J ¼ 3.6, 10.6Hz, H1–C(50)), 3.44 (dd, J ¼ 3.3, 10.6 Hz, H2–C(50)), 3.45–
3.65 (br s, N6-(CH3)2), 3.76, 3.77 (2s, 2 OCH3), 4.35 (d, H–C(40)), 4.47 (t, H–C(30)), 4.71 (t, H–C(20)),
5.91 (d, J ¼ 6.4 Hz, H–C(10)), 6.72–6.75 (m, 4H, trityl-H), 7.04–7.28 (m, 9H, trityl-H), 8.01 (s, H–
13
C(8)), 8.28 (H–C(2)) ppm; C NMR (75 MHz, CDCl3, 27ꢀC): ꢁ ¼ 38.53 (br s, N6-(CH3)2), 55.15 (2
CH3O), 63.64 (C(50)), 72.94 (C(30)), 76.33 (C(20)), 86.46 C(40)), 86.48, 91.00 (C(10)), 113.13 (trityl-C),
120.54, 126.82, 127.81, 128.03, 129.92, 129.94 (trityl-C), 135.50, 135.67, 136.17 (C(8)), 144.33,
149.42, 151.58 (C(2)), 151.59, 154.96, 158.51 ppm; UV (MeOH): ꢂ(") ¼ 260 (13400), 274 (max,
19400) nm (molÀ 1 dm3 cmÀ 1); MALDI-FTICR-MS: m=z ¼ 620.2482 (3, [Mþ Na]þ ), 303.1390
(100, [(MeO)2Tr]þ ).
50-O-(4,40-Dimethoxytrityl)-N6,N6-dimethyl-20-O-[[(triisopropylsilyl)oxy]methyl]adenosine
(28, C43H57N5O7Si)
To a stirred solution of 3.23g of 27 (5.41 mmol) and 2.79 g of ethyldiisopropylamine (21.6mmol) in
35cm3 of anhydrous 1,2-dichloroethane, 1.81 g of di-tert-butyltindichloride (5.95 mmol) were added.
The mixture was heated to 70ꢀC for 15 min, allowed to cool to rt and treated with 1.32 g of [(triiso-
propylsilyl)oxy]methylchloride (5.95 mmol). Stirring was continued for 3 h, followed by addition of
0.5 cm3 of MeOH, evaporation to dryness and workup. Column chromatography (silica gel, hexane:
EtOAc¼ 3:1 to 1:1) afforded 831 mg of 28 as white, solid foam (20%). The regioselectivity of 20-O-
alkylated over 30-O-alkylated product was approximately 5:4. TLC (silica gel, hexane:
1
i
EtOAc¼ 1:1): Rf ¼ 0.7; H NMR (300 MHz, CDCl3, 27ꢀC): ꢁ ¼ 1.00–1.11 (m, Pr3Si), 3.05 (d, J ¼
4.1 Hz, HO–C(30)), 3.36 (dd, J ¼ 4.3, 10.5Hz, H1–C(50)), 3.50 (dd, J ¼ 3.0, 10.5Hz, H2–C(50), 3.45–
3.58 (br s, N6-(CH3)2), 3.78, 3.79 (2s, 2 OCH3), 4.26 (q, H–C(40)), 4.48 (q, H–C(30)), 4.87 (t, H–C(20)),

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4.99, 5.15 (2d, J ¼ 4.7 Hz, OCH2O), 6.17 (d, J ¼ 5.2 Hz, H–C(10)), 6.78–6.82 (m, 4H, trityl-H),
7.18–7.32, 7.37–7.46 (m, 9H, trityl-H), 7.93 (s, H–C(8)), 8.27 (s, H–C(2)) ppm; 13C NMR
(125 MHz, CDCl3, 30ꢀC): ꢁ ¼ 11.77 ((CH3)2CH), 17.66, 17.71 ((CH3)2CH), 38.42 (br, N6-(CH3)2),
55.10 (2 CH3O), 63.36 (C(50)), 70.68 (C(30)), 81.85 (C(20)), 83.84 (C(40)), 86.38, 86.82 (C(10)), 90.70
(OCH2O), 113.07 (trityl-C), 120.63, 126.72, 127.73, 128.14, 130.01 (trityl-C), 135.78, 135.81, 136.78
(C(8)), 144.59, 150.35, 152.40 (C(2)), 154.87, 158.43 ppm; UV (MeOH): ꢂ(") ¼ 260 (12800), 274
(max, 18200) nm (molÀ 1 dm3 cmÀ 1); MALDI-FTICR-MS: m=z ¼ 806.3910 (9, [Mþ Na]þ ),
303.1392 (100, [(MeO)2Tr]þ ).
50-O-(4,40-Dimethoxytrityl)-N6,N6-dimethyl-20-O-[[(triisopropylsilyl)oxy]methyl]adenosine
30-(2-cyanoethyl diisopropylphosphoramidite) (29, C52H74N7O8PSi)
A solution of 713 mg of 28 (0.91 mmol) in 8.5cm3 of anhydrous CH2Cl2 was treated consecutively
with 697 mg of ethyldimethylamine (9.53 mmol) and 324 mg of 2-cyanoethyl diisopropylphosphor-
amidochloridite (1.37 mmol). The mixture was stirred for 2 h, quenched with 0.15 cm3 of MeOH, and
evaporated to dryness. Workup and column chromatography (silica gel, hexane:EtOAc¼ 5:2 ( þ 2%
Et3N)) afforded 780 mg of 29 as white, solid foam (85%, 1:1 mixture of diastereoisomers). TLC (silica
1
gel, hexane:EtOAc) ¼ 2:1): Rf ¼ 0.6; H NMR (500MHz, CDCl3, 28ꢀC): ꢁ ¼ 0.88–1.20 (m, 42H,
iPr3Si, 24H, ((CH3)2CH)2N), 2.37, 2.65 (2m, 4H, CH2CN), 3.30 (m, 2H, H1–C(50)), 3.45–3.68 (m,
2H, H2–C(50), 12H, N6-(CH3)2, 4H, ((CH3)2CH)2N, 2H, POCH2), 3.77, 3.78 (2s, 12H, 2 CH3O), 3.81–
3.98 (2m, 2H, POCH2), 4.32, 4.38 (2q, 2H, H–C(40)), 4.68, 4.73 (2m, 2H, H–C(30)), 4.92–5.01 (2m,
4H, J ꢁ 5 Hz, OCH2O), 5.16 (m, 2H, H–C(20)), 6.15 (2d, 2H, J ¼ 5.5 Hz, H–C(10)), 6.78, 7.20–7.40
(m, 26H, trityl-H), 7.88, 7.90 (2s, 2H, H–C(8)), 8.22, 8.24 (2s, 2H, H–C(2)) ppm; 31P NMR (200 MHz,
CDCl3, 27ꢀC): ꢁ ¼ 149.86, 150.54; UV (MeOH): ꢂ(") ¼ 232 (max, 23100), 260 (13700), 274 (max,
19700) nm (molÀ 1 dm3 cmÀ 1); MALDI-FTICR-MS: m=z ¼ 1006.4996 (12, [Mþ Na]þ ), 303.1394
(100, [(MeO)2Tr]þ ).
Acknowledgements
Financial support from the Austrian Science Fund (P15042) is gratefully acknowledged. Special thanks
from R. M. to Prof. S. Pitsch, ETH Lausanne, for many stimulating discussions.
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