Nucleotides: Part LXXII. Synthesis of N-methylated ribonucleosides for RNA synthesis

Article abstract of DOI:10.1002/hlca.200390205

The synthesis of various N-methylated nucleosides (m⁶A, m ³C, m⁴C, m³U) is described. These minor nucleosides can be obtained by simple methylation with diazomethane of [2-(4-nitrophenyl)ethoxy]carbonyl(npeoc)-protected nucleosides. These methylated compounds are easily further derivatized to fit into the scheme of the [2-(dansyl)ethoxy]carbonyl (dnseoc) approach for RNA synthesis (dansyl = [5-(dimethylamino)naphthalen-1-yl]sulfonyl). Various oligoribonucleotides containing N⁶-methyladenosine were synthesized, underlining the usefulness of the dnseoc approach, especially for the synthesis of natural tRNA-derived oligoribonucleotide sequences.

Full text of DOI:10.1002/hlca.200390205

Helvetica Chimica Acta Vol. 86 (2003)
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Nucleotides
Part LXXII1)
Synthesis of N-Methylated Ribonucleosides for RNA Synthesis
by Markus Beier and Wolfgang Pfleiderer*)
Fachbereich Chemie, Universit‰t Konstanz, Postfach 5560, D-78457 Konstanz
The synthesis of various N-methylated nucleosides (m6A, m3C, m4C, m3U) is described. These minor
nucleosides can be obtained by simple methylation with diazomethane of [2-(4-nitrophenyl)ethoxy]carbo-
nyl(npeoc)-protected nucleosides. These methylated compounds are easily further derivatized to fit into the
scheme of the [2-(dansyl)ethoxy]carbonyl (dnseoc) approach for RNA synthesis (dansyl ˆ [5-(dimethylami-
no)naphthalen-1-yl]sulfonyl). Various oligoribonucleotides containing N6-methyladenosine were synthesized,
underlining the usefulness of the dnseoc approach, especially for the synthesis of natural tRNA-derived
oligoribonucleotide sequences.
1. Introduction. In addition to the standard ribonucleosides adenosine, guanosine,
cytidine, and uridine, more than 100 so-called minor nucleosides have, so far, been
found in nature, especially as components of tRNAs. These represent modified
derivatives of the usual purine and pyridmidine bases and of 2'-O-methylribose.
Despite their low abundance, most of them play important roles in various biological
mechanisms. Among those minor nucleosides, the ones that show one or more N-
methylations at the heterocyclic nucleobase can be grouped together in comparison to
the standard nucleosides. Isolation from natural RNA led to the characterization of,
e.g., 1-methyl- (m1A), 2-methyl- (m2A), and N6-methyladenosine (m6A), N3-methyl-
(m3C), N4-methyl (m4C) [2], and 5-methylcytidine (m5C), N1-methyl- (m1G), N2-
methyl- (m2G), N2,N2-dimethyl- (m2 G), and N7-methylguanosine (m7G), as well as
2
N3-methyluridine (m3U) [3 5] (Fig. 1).
These N-methylated nucleosides play an important role, e.g., in the investigation of
certain receptors (m6A) [6], mechanisms of splicing (m2 G) [7], regulation of genes
2
(m5C) [8], or in studies of the HIV problem (m4C) [9]. Therefore, it would be
beneficial to have easy synthetic access to these minor nucleosides that would allow
their incorporation in oligoribonucleotides via commonly used phosphoramidite
chemistry.
Performing RNA synthesis via phosphoramidite chemistry applying N-methylated
ribonucleosides usually implies the introduction of the Me group in an early stage of the
synthetic scheme. Therefore, a large number of extra synthetic steps have to be
undertaken to finally reach the N-methylated phosphoramidites. Because of that, it
would be desirable to introduce the Me groups at a later stage to avoid additional
1
)
Part LXXI: [1].

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Fig. 1. Methylated minor nucleosides isolated from natural RNA
synthetic steps. For RNA synthesis, we followed the successful dnseoc strategy
described by Bergmann and Pfleiderer [10] (Fig. 2). Within this strategy, the NH2
functions of the heterocyclic bases are protected by the [2-(4-nitrophenyl)ethoxy]car-
bonyl group (npeoc) [11], which is cleavable by b-elimination with a non-nucleophilic
base, e.g., DBU (ˆ1,8-diazabicyclo[5.4.0]undec-7-ene), in an aprotic solvent. Fur-
thermore it was shown that it is of great advantage to protect also the lactam function of
guanosine by applying the 2-(4-nitrophenyl)ethyl group (npe), which follows the same
cleavage pattern. Compared to other strategies for RNA synthesis, the 5'-position is
protected within the dnseoc approach by the highly base-labile (2-dansylethoxy)car-
bonyl group (dnseoc) (dansyl ˆ [5-(dimethylamino)naphthalen-1-yl]sulfonyl). The 2'-
position is blocked by the tetrahydro-4-methoxy-2H-pyran-4-yl group (mthp) [12] and
the phosphate also by the 2-(4-nitrophenyl)ethyl group (npe). Since the temporary
base-labile 5'-dnseoc and the permanent 2'-mthp protecting groups are strictly
orthogonal, no loss of the 2'-protection has to be considered during machine-aided
synthesis, compared to other strategies. Furthermore, combining this protection
scheme with a DBU-stable linker system (LCAMA-CPG) [13], the removal of all
protecting groups except the 2'-O-mthp group can be performed in one step with the
oligomer still attached to the solid support. Therefore, all protecting-group residues can
simply be washed away, making a time-consuming HPLC purification unnecessary.
Ammonia treatment cleaves the oligomer from the support, and, in the final
deprotection step, the removal of the 2'-O-mthp acetal function can be achieved by
mildly acidic treatment [14], leading to RNA oligomers of high purity without any
significant tendency to undergo strand scission.
2. Synthesis.
The purpose of these investigations was to make some of the
naturally occurring N-methylated minor nucleosides easily available for phosphor-
amidite chemistry, starting from appropriately protected monomeric building blocks
applied already in the dnseoc strategy for RNA synthesis. Generally, heterocyclic bases
are known to be N-methylated either by direct alkylation with diazomethane [15],

Helvetica Chimica Acta Vol. 86 (2003)
2535
Fig. 2. Protection scheme within the dnseoc approach
iodomethane [16][17], or dimethylsulfate [18], or by nucleophilic displacement of a
chloro [19], fluoro [9], or triazolyl group [20].
Using npe/npeoc-protected nucleosides, Sigmund [21] obtained N6-methyladeno-
sine by a Dimroth rearrangement from N1-methyl adenosine during deprotection of the
sugar moiety. Based on the findings of Cramer and Pfleiderer [22], who isolated N-
methylated nucleosides as by-products during the methylation of the 2'-O- and 3'-O
position of ribonucleosides with diazomethane omitting a Lewis catalyst, we
concentrated on the introduction of the Me groups by this procedure. Various N-
methylated ribonucleosides could be obtained by attaching the Me group to the
appropriately protected ribonucleosides on the latest stage of the synthesis to decrease
the overall number of steps during the conventional approach.
Initially employing diazomethane as a mild methylation agent for the heterocyclic-
moieties-protected ribonucleosides, 3',5'-O-tipds-N4-npeoc-cytidine (1; tips ˆ 1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl), 2'-O-mthp-uridine (2), and 2'-O-mthp-N2-npeoc-
O6-npe-guanosine (3) were subjected to diazomethane treatment (Scheme 1). Typi-
cally, 3 6 equiv. of diazomethane were employed in these reactions at 08. The uridine
and the cytidine derivatives showed relatively clean alkylations, giving only the
expected N3- and N4-methylated products. From 3',5'-O-tipds-N4-npeoc-cytidine (1),
two methylated products were obtained, the N3-methyl derivative 4 and the N4-methyl
derivative 5. Alkylation at the sterically less-hindered N3-position was clearly favored
over methylation at the N4-position. As expected, 2'-O-mthp-uridine (2) was alkylated
only at the N3-position. Although up to 14 equiv. of diazomethane were used in the
reaction of 2'-O-mthp-N2-npeoc-O6-npe-guanosine (3), no complete turnover was
observed. Instead a complex mixture that could hardly be separated by chromatog-
raphy was obtained. This mixture contained two methylated products and the educt.
The major methylated product was obtained almost pure by several chromatographic
purification steps. However, no clearcut assignment of the location of methylation was
possible by NMR. Elemental analysis established that only a single methylation had
taken place. From the similarity of the UV spectra of the isolated compound and the

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educt, we suggest that methylation had taken place at the N2-position. Alkylation at the
N1- or at the N7-position would have altered the chromophore of the aromatic moiety
more strongly, essentially resulting in a more-severe change of the UV spectrum of the
compound.
Scheme 1. Methylation of Cytidine and Uridine Nucleosides with Diazomethane
To explore in more detail at which stage the Me groups should be introduced, we
subjected various npe/npeoc-protected adenosine compounds 8 12 with different
substitution patterns to the diazomethane reaction (Scheme 2). Besides the common
mthp acetal for 2'-protection, the very acid-labile 2-methoxypropan-2-yl (ˆ1-methoxy-
1-methylethyl) group (mep) was tested. All reactions performed well, giving
exclusively the N6-methylated products 13 17 in good yields. This underlined the
finding of Cramer and Pfleiderer [22] that, omitting the Lewis catalyst, methylation
with diazomethane takes place only at the moderately nucleophilic amino position of
the aglycone, no matter whether or not the sugar OH groups are protected. Even the 2'-
O-mep protection proved to be orthogonal to the diazomethane reaction. However, in
the methylation of the 5'-O-dnseoc-protected derivative 12 with diazomethane,
unexpectedly, loss of the 5'-O-dnseoc protecting group was observed simulteanously
with N6-methylation. It seems that the basicity of diazomethane is strong enough to
promote b-elimination of the dnseoc group. Therefore, the introduction of the Me
group into the heterocylic bases with diazomethane has to be performed one step prior
to the introduction of the 5'-O-dnseoc group. Nevertheless, only one extra synthetic
step is necessary to make the minor N-methylated nucleosides available for RNA
synthesis, since all protecting groups (npeoc, npe, mthp, mep) employed in the dnseoc
strategy are stable during diazomethane methylation.
Interesting to note is that less selectivity was observed when the N6-benzoylated
adenosine compound 18 was submitted to the diazomethane reaction as compared to
the npeoc-protected analogues 8 12. Additional methylation was observed at the N1-
position and at the carbonyl function of the benzoyl moiety (Scheme 3).
To make the N-methylated nucleosides of uridine, cytidine, and adenosine available
for machine-aided RNA synthesis via the dnseoc strategy, the N-methylated
compounds were processed according to the usual protection/deprotection scheme
(Scheme 4). Starting from the 3',5'-O-tipds derivative 14, the 2'-O-mthp group was
introduced by means of 5,6-dihydro-4-methoxy-2H-pyran under acidic catalysis to give
15. Then, the cyclic silyl group was removed from 4, 5, and 15 in the usual manner by

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Scheme 2. N-Methylation of N6-npeoc-Protected Adenosine Derivatives
Scheme 3. Reaction of 3',5'-O-tipds-N6-benzoyladenosine (18) with Diazomethane
Bu4NF, resulting in formation of the 2'-O-mthp protected derivatives 22 24. In the
following step, the dnseoc group was selectively introduced at the 5'-O-position of 22
24 and 6 by means of the hydrochloride salt of 2-(dansyl)ethyl carbonochloridate
leading to 17 and 25 27. The intermediary adenosine derivative 17 was then converted
to the highly reactive diethylphosphoramidite 28 as well as the moderately reactive
diisopropylphosphoramidites 29 and 30 by standard procedures. For attachment of the
starting nucleoside to the solid support, the 5'-O-dnseoc-2'-O-mthp-N6-npeoc-adeno-
sine (17) was reacted with succinic anhydride to give the succinate 31, which was used to
derivatize the solid support 1400-ä LCAMA-CPG according to the reported
procedure [23].
Having synthesized all the necessary building blocks for machine-aided oligoribo-
nucleotide synthesis, N6-methylated adenosine was incorporated first in homomeric
(see P1 and P2) and then in mixed oligoribo sequences (see P3 P8; Table). The N6-
methyladenosine-containing ribonucleotides represent parts of the anticodon loop of
natural t-RNAs (RV 1460, RV 1180, RV 1662, RY 1440, RG 1140, RV 1180).

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Scheme 4. Synthesis of N-Methylated Ribonucleosides
Table. Oligoribonucleotide Sequences
RNA sequences
P1
P2
P3
P4
P5
P6
P7
P8
m6Am6Am6Am6Am6Am6Am6A
m6Am6Am6Am6Am6Am6Am6Am6Am6Am6A
CAC CUU GAC m6A
ACm6A AGC AGG CG
ACm6A AGG AGG GG
CUG UAm6A CCCACUU
UUCCm6A AGC UGA UU
AGA GCU CCU GCC UUA Cm6A
RNA Syntheses were performed on a ABI-392-DNA synthesizer according to a
standard protocol for RNA synthesis employing 5'-O-dnseoc-protected phosphorami-
dites. Instead of 1H-tetrazole, 0.5m pyridine hydrochloride in MeCN was used as the
activator of choice [24], resulting in a shortened coupling step of 280s without loss of
performance. Post-synthesis manipulations included overnight treatment with 1m DBU

Helvetica Chimica Acta Vol. 86 (2003)
2539
at room temperature for the removal of the npe and npeoc groups and treatment with
conc. NH3 solution (558) to cleave off the 2'-O-mthp-oligoribonucleotide from the solid
support, followed by analysis by reversed-phase and ion-exchange HPLC (Fig. 3).
Final removal of the 2'-O-mthp groups was accomplished by an overnight treatment
with 0.5m AcOH/NaOAc buffer pH 3.25. After neutralization, the free oligoribonu-
cleotides P1 P8 were isolated by precipitation with EtOH.
Fig. 3. HPLC of 2'-O-mthp-protected oligoribonucleotides
HPLC Analysis of all oligoribonucleotides showed high purity of the crude
products (Fig. 4). Enzymatic digestion of the free oligoribonucleotides with alkaline
phosphatase and snake-venom phosphodiesterase gave the expected ratios of the five
mononucleosides when analyzed by reversed-phase HPLC (Fig. 5).
Fig. 4. Reversed-phase HPLC of ribonucleotide P6
3. Conclusions. We were able to demonstrate an easy approach to the preparation
of the appropriately protected monomeric units of N6-methyladenosine, N3-methyl-
and N4-methylcytidine, and N3-methyluridine prone to RNA synthesis via the dnseoc
strategy. Various oligoribonucleotide sequences containing N6-methyl adenosine were
synthesized successfully. The applied blocking-group strategy allows the isolation of the
2'-O-mthp-oligoribonucleotides as stable storage forms that can easily be converted to
the free oligoribonucleotides by mild acid treatment. The dnseoc strategy for RNA
synthesis can be regarded as a powerful tool, especially for chemical synthesis of tRNA
sequences of biological interest containing minor nucleotides.

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Fig. 5. HPLC Analysis of enzymatic digestion of P6
Experimental Part
General. Products were dried under high vacuum. All solvents used were anh. grade. TLC: precoated silica-
gel thin-layer sheets 60 F 254 from Merck. Flash chromatography (FC): silica gel (Baker, 30 60mm); 0.2
0.3 bar. M.p.: Gallenkamp melting-point apparatus; no corrections. UV/VIS: Perkin-Elmer Lambda 5; lmax in
nm (log e). 1H-NMR: Bruker AC-250; d in ppm rel. to SiMe4 or CDCl3 ((D6)DMSO) as internal standard. 31P-
NMR: Jeol JMN-GX400.
1. Diazomethane. A 2-layer mixture of 1,2-dimethoxyethane (50ml) and 40% KOH soln. (33 ml) was
cooled to 08, and then N-methyl-N-nitrosourea (10g) was added carefully in small portions within 30min
keeping the temp. of the mixture below ‡ 18. After additional stirring for 20min at this temp., the org. phase
was separated and the aq. layer washed with 1,2-dimethoxyethane. The combined org. phase was dried for 4 h at
08 over KOH pellets. The solid was separated and the ca. 1m diazomethane soln. in 1,2-dimethoxyethane directly
used for the methylation reactions.
2. N3-Methyl-N4-{[2-(4-nitrophenyl)ethoxy]carbonyl}-2'-O-(tetrahydro-4-methoxy-2H-pyran-4-yl)-3',5'-O-
(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)cytidine (4) and N4-Methyl-N4-{[2-(4-nitrophenyl)ethoxy]carbonyl}-2'-
O-(tetrahydro-4-methoxy-2H-pyran-4-yl)-3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)cytidine (5). A soln.
of N4-{[2-(4-nitrophenyl)ethoxy]carbonyl}-2'-O-(tetrahydro-4-methoxy-2H-pyran-4-yl)-3',5'-O-(1,1,3,3-tetrai-
sopropyldisiloxane-1,3-diyl)cytidine (1; 3.97 g, 5 mmol) in toluene (30ml) was cooled to 0 8, and then 1m
CH2N2 in 1,2-dimethoxyethane (20ml) was added dropwise within 20min. The mixture was stirred for 9 h at
08, the ice-bath removed, and stirring at r.t. continued overnight. The mixture was evaporated and purification
achieved by CC (14 17% acetone/hexane): 2.2 g (55%) of 4 and 1.22 (30%) of 5.
Data of 4: Rf (hexane/acetone 3 :2) 0.69. UV (MeOH): 282 (4.38), 213 (sh, 4.25). 1H-NMR ((D6)DMSO):
8.15 (d, 2 Ho to NO2); 7.67 (d, HÀC(6)); 7.54 (d, 2 Hm to NO2); 6.18 (d, HÀC(5)); 5.64 (s, HÀC(1')); 4.48
(d, HÀC(2')); 4.32 (t, CH2CH2O); 4.18 (m, HÀC(3')); 4.16 (m, 1 HÀC(5')); 4.03 (m, HÀC(4')); 3.92
(m, 1 HÀC(5)); 3.66 3.47 (2m, CH2OCH2); 3.22 (2s, MeN, MeO); 3.08 (t, CH2CH2O); 1.75 1.83 (2m,
CH2CCH2); 0.92 1.05 (m, 4 Me2CH). Anal. calc. for C37H58N4O12Si2 (807.1): C 55.06, H 7.24, N 6.94; found:
C 54.92, H 7.22, N 6.84.
Data of 5: Rf (hexane/acetone 3 :2) 0.59. UV (MeOH): 284 (sh, 4.21), 251 (4.31), 203 (sh, 4.51). 1H-NMR
((D6)DMSO): 8.17 (d, 2 Ho to NO2); 8.10( d, HÀC(6)); 7.58 (d, 2 Hm, to NO2); 7.07 (d, HÀC(5)); 5.67
(s, HÀC(1')); 4.46 (t, CH2CH2O); 4.41 (m, HÀC(2')); 4.19 (m, HÀC(3'), 1 HÀC(5')); 4.09 (m, HÀC(4')); 3.93
(m, 1 HÀC(5')); 3.48, 3.67 (2m, CH2OCH2); 3.23 (s, MeN); 3.22 (s, MeO); 3.14 (t, CH2CH2O); 1.94, 1.77 (2m,
CH2CCH2); 1.91 1.05 (m, 4 Me2CH). Anal. calc. for C37H58N4O12Si2 (807.1): C 55.06, H 7.24, N 6.94; found:
C 54.91, H 7.22, N 6.98.
3. N3-Methyl-2'-O-(tetrahydro-4-methoxy-2H-pyran-4-yl)-uridine (6). According to Exper. 2, 2'-O-(tetra-
hydro-4-methoxy-2H-pyran-4-yl)uridine (2; 1.07 g, 3 mmol) in CH2Cl2 (30ml) and AcOEt (15 ml) was treated
with 1m CH2N2 (20ml). After evaporation of the mixture, the residue was suspended in AcOEt, whereby on
stirring, 0.6 g (54%) of crystalline 6 separated out and was collected by filtration. Purification of the filtrate by
CC (silica gel, toluene/AcOEt 5 :4 with 0 3.5% MeOH) gave additional 0.21 g (18%) of 6. Rf (CHCl3/MeOH
9 :1) 0.30. UV (MeOH): 260 (3.89), 207 (3.92). 1H-NMR ((D6)DMSO): 7.99 (d, HÀC(6)); 6.05 (d, HÀC(5));
5.86 (d, HÀC(1')); 5.21 (t, OHÀC(5')); 5.17 (d, OHÀC(3')); 4.33 (m, HÀC(2')); 3.97 (m, HÀC(3')); 3.91

Helvetica Chimica Acta Vol. 86 (2003)
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(m, HÀC(4')); 3.30 3.80( m, CH2OCH2, 2 HÀC(5')); 3.15 (s, MeN); 2.91 (s, MeO); 1.55 1.80( m, CH2CCH2).
Anal. calc. for C16H24N2O8 (372.4): C 51.61, H 6.50, N 7.52; found: C 51.28, H 6.63, N 7.30.
4. N2-Methyl-N2-{[2-(4-nitrophenyl)ethoxy]carbonyl}-O6-[2-(4-nitrophenyl)ethyl](tetrahydro-4-methoxy-
2H-pyran-4-yl)guanosine (7). A soln. of 3 (1.7 g, 1.73 mmol) in a mixture of toluene (15 ml) and CH2Cl2 was
cooled to 08 and then, under stirring, 1m diazomethane in 1,2-dimethoxyethane (25 ml) was dropwise slowly
added within 20min. After stirring for 9 h at 0 8, the mixture was evaporated and the residue purified by CC
(silica gel (50g), toluene/AcOEt 5 :4 with 0 4% MeOH) to give, in the first fraction, 0.165 g (13%) of 7 as a
solid foam. The following fractions consisted of substance mixtures. 7: Rf (toluene/AcOEt/MeOH 5 :4 :1) 0.33.
UV (MeOH): 216 (4.49), 267 (5.00). 1H-NMR ((D6)DMSO): 8.60( s, HÀC(8)); 8.17 (d, 2 Ho to NO2); 8.05
(d, 2 Ho to NO2); 7.58 (d, 2 Hm to NO2); 7.44 (d, 2 Hm to NO2); 6.02 (d, HÀC(1')); 5.26 (d, OHÀC(3')); 5.09
(t, OHÀC(5')); 4.88 (m, HÀC(2')); 4.72 (t, CH2CH2O); 4.36 (t, CH2CH2O); 4.15 (m, HÀC(3')); 3.99
(m, HÀC(4')); 3.68 3.55 (m, 2 HÀC(5'), 1 H of CH2OCH2); 3.38 (m, 2 H of CH2OCH2); 3.27 (m, 1 H of
CH2OCH2, CH2CH2O, MeN); 3.18 (m, CH2CH2O); 2.56 (s, MeO); 1.76 1.43 (m, CH2CCH2). Anal. calc. for
C34H39N7O13 (753.7): C 54.18, H 5.21, N 13.01; found: C 53.95, H 5.33, N 12.17.
5. N6-Methyl-N6-{[2-(4-nitrophenyl)ethoxy]carbonyl}adenosine (13) [22]. Analogously to Exper. 2, with N6-
{[2-(4-nitrophenyl)ethoxy]carbonyl}adenosine (8; 2.31 g, 5 mmol) in anh. DMF (50ml) and 1 m CH2N2 (20ml).
Purification by CC (0 50% acetone/petroleum ether) gave 1.54 g (65%) of 13 ([22]: 72%).
6. N6-Methyl-N6-{[2-(4-nitrophenyl)ethoxy]carbonyl}-3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)ade-
nosine (14). 6.1. Analogously to Exper. 2, with N6-{[2-(4-nitrophenyl)ethoxy]carbonyl}-3',5'-O-(1,1,3,3-tetraiso-
propyldisiloxane-1,3-diyl)adenosine (9; 4.64 g, 6.61 mmol) in DMF (50ml) and 1 m CH2N2 (20ml). Purification
by CC (toluene/AcOEt 5 :4 with 0 1.5% MeOH) gave 2.56 g (54%) of 14.
6.2. After co-evaporation with anh. pyridine, 13 (1.46 g, 3.08 mmol) was dissolved in anh. pyridine (30 ml),
and 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (1.1 ml, 3.39 mmol) was added. After 3 d, the reaction was
quenched with MeOH, the mixture evaporated, and the residue dissolved in CH2Cl2 (50ml) and extracted with
NaHCO3 soln. (100 ml). The org. layer was dried (MgSO4) and purified by CC (toluene/AcOEt 5 :4 with 0
1.5% MeOH): 1.66 g (75%) of 14. Rf (toluene/AcOEt 1:1) 0.35. UV (MeOH): 272 (4.36), 212 (4.39). 1H-NMR
((D6)DMSO): 8.65 (s, HÀC(2)); 8.52 (s, HÀC(8)); 8.02 (d, 2 Ho to NO2); 7.26 (d, 2 Hm to NO2); 5.96
(s, HÀC(1')); 5.69 (d, OHÀC(2')); 4.75 (m, HÀC(3')); 4.60( t, HÀC(2')); 4.35 (m, CH2CH2CO); 4.04
(m, HÀC(4'), 2 HÀC(5')); 3.35 (s, MeN); 2.96 (t, CH2CH2O); 1.02 (m, 4 Me2CH). Anal. calc. for
C32H48N6O9Si2 (716.95): C 53.61, H 6.75, N 11.72; found: C 53.66, H 6.76, N 11.73.
7. N6-Methyl-N6-{[2-(4-nitrophenyl)ethoxy]carbonyl}-2'-O-(tetrahydro-4-methoxy-2H-pyran-4-yl)-3',5'-O-
(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)adenosine (15). 7.1. Analogously to Exper. 2, with 2'-O-mthp-N6-{[2-
(4-nitrophenyl)ethoxy]carbonyl}-3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)adenosine (10; 2.0 7 g,
2.53 mmol) in CH2Cl2 (40ml) and MeOH (2 ml) and 1 m CH2N2 (24 ml). Purification by CC (0 20%
acetone/petroleum ether) gave 1.58 g (75%) of 15.
7.2. To 14 (2.0g, 2.78 mmol) and TsOH ¥ H 2O (53 mg, 0.278 mmol) in anh. CH2Cl2 (20ml) was added 5,6-
dihydro-4-methoxy-2H-pyran (1.80g, 65%, 10.2 mmol) and the mixture was stirred for 7 h. The reaction was
quenched with sodium methoxide and the mixture extracted with CH2Cl2 (70ml) and sat. NaHCO 3 soln.
(100 ml). The org. layer was dried (MgSO4) and evaporated and the residue purified by CC (0 20% acetone/
petroleum ether): 1.87 g (81%) of 15. Rf (toluene/AcOEt/MeOH 5 :4 :1) 0.80. UV (MeOH): 272 (4.32), 212
(4.35). 1H-NMR ((D6)DMSO): 8.64 (s, HÀC(2)); 8.56 (s, HÀC(8)); 8.02 (d, 2 Ho to NO2); 7.26 (d, 2 Hm to
NO2); 6.10( s, HÀC(1')); 4.90( m, HÀC(3'), HÀC(2')); 4.35 (m, CH2CH2O); 4.05 (m, HÀC(4'), 2 HÀC(5'));
3.80 3.30 (2 m, CH2OCH2); 3.35 (s, MeN); 3.16 (s, MeO); 2.96 (t, CH2CH2O); 1.78 (m, CH2CCH2); 1.02
(m, 4 Me2CH). Anal. calc. for C38H58N6O11Si2 (831.1): C 54.92, H 7.03, N 10.11; found: C 55.13, H 7.17, N 10.50.
8. 2'-O-1-Methoxy-1-methylethyl)-N6-methyl-N6-{[2-(4-nitrophenyl)ethoxy]carbonyl}-3',5'-O-(1,1,3,3-tetrai-
sopropyldisiloxane-1,3-diyl)adenosine (16). Analogously to Exper. 2, with 2'-O-1-methoxy-1-methylethyl)-N6-
{[2-(4-nitrophenyl)ethoxy]carbonyl}-3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)adenosine (11; 2.63 g,
3.39 mmol) in DMF (50ml) and 1 m CH2N2 (14 ml). Purification by CC (0 50% AcOEt/toluene) gave 2.27 g
(85%) of 16. Rf (toluene/AcOEt 1:1) 0.43. UV (MeOH): 326 (sh, 3.34), 272 (4.33), 212 (4.37). 1H-NMR
((D6)DMSO): 8.64 (s, HÀC(2)); 8.57 (s, HÀC(8)); 8.01 (d, 2 Ho to NO2); 7.25 (d, 2 Hm to NO2); 6.05
(s, HÀC(1')); 4.85 (m, HÀC(2'), HÀC(3')); 4.36 (m, CH2CH2O); 3.96 (m, HÀC(4'), 2 HÀC(5')); 3.35
(s, MeN); 3.12 (s, MeO); 2.95 (t, CH2CH2O); 1.32 (2s, 2 Me); 1.0 2 m( , 4 Me2CH). Anal. calc. for
C36H56N6O10Si2 ¥ 0.25 toluene (812.1): C 55.83, H 7.20, N 10.35; found: C 56.20, H 7.41, N 10.69.
9. 5'-O-{{2-{[5-(Dimethylamino)naphthalen-1-yl]sulfonyl}ethoxy}carbonyl}-N6-methyl-N6-{[2-(4-nitro-
phenyl)ethoxy]carbonyl}2'-O-(tetrahydro-4-methoxy-2H-pyran-4-yl)adenosine (17). After co-evaporation with
anh. pyridine, N6-methyl-N6-{[2-(4-nitrophenyl)ethoxy]carbonyl}-2'-O-(tetrahydro-4-methoxy-2H-pyran-4-

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Helvetica Chimica Acta Vol. 86 (2003)
yl)adenosine (22; 1.05 g, 1.78 mmol) was dissolved in anh. pyridine (20 ml) and cooled to 08. Then 2-{[5-
(dimethylamino)naphthalen-1-yl]sulfonyl}ethyl carbonochloridate hydrochloride (0.742 g) was added. The
reaction was quenched after 75 min with MeOH and the mixture evaporated. The residue was extracted with
CH2Cl2, the org. phase washed with sat. NaHCO3 soln. (100 ml), dried (MgSO4), and evaporated, and the
residue purified by CC (toluene/AcOEt 5 :4 with 0 4% MeOH): 0.86 g (54%) of 17. Rf (toluene/AcOEt/
MeOH 5 :4 :1) 0.51. UV (MeOH): 343 (3.67), 261 (4.48), 213 (4.82). 1H-NMR ((D6)DMSO): 8.73 (2s, HÀC(2),
HÀC(8)); 8.51 (d, HÀC(2)(npht)); 8.15 (m, HÀC(4)(npht), HÀC(8)(npht); 8.04 (d, 2 Ho to NO2); 7.64
(m, HÀC(3)(npht), HÀC(7)(npht)); 7.34 (d, 2 Hm to NO2); 7.24 (d, HÀC(6)(npht)); 6.13 (d, HÀC(1')); 5.52
(m, OHÀC(3')); 5.05 (m, HÀC(2')); 4.35 (2t, 2 CH2CH2O); 4.20( m, HÀC(3'), HÀC(4')); 3.83 (t, CH2SO2);
3.20 3.70( m, 2 HÀC(5'), CH2OCH2); 3.37 (s, MeN); 2.95 (t, CH2CH2O); 2.79 (s, Me2N); 2.48 (s, MeO); 1.60
(2m, CH2CCH2). Anal. calc. for C41H47N7O14S (893.9): C 55.09, H 5.30, N 10.97; found: C 55.48, H 5.54, N 10.21.
10. N6-Benzoyl-N6-methyl-3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)adenosine (19), N6-Benzoyl-N1-
methyl-3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)adenosine (20), and N6-[Methoxy(phenyl)methylene]-
3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)purine (21). Analogously to Exper. 2, with N6-benzoyl-3',5'-O-
(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)adenosine (18; 4.0g, 6.51 mmol) in CH 2Cl2 (40ml) with 1m CH2N2
(20ml). Purification by CC (14 50% acetone/petroleum ether) gave 1.37 g (33%) of 19, 0.44 g (11%) of 20,
and 0.91 g (22%) of 21.
Data of 19: Rf (petroleum ether/AcOEt 3 :2) 0.63. UV (MeOH): 280 (4.10), 206 (4.40). 1H-NMR
((D6)DMSO): 8.50(2 s, HÀC(2), HÀC(8)); 7.31 (m, 3 H, bz); 7.20( m, 2 H, bz); 5.92 (s, HÀC(1')); 5.66
(d, OHÀC(2')); 4.67 (m, HÀC(3')); 4.53 (m, HÀC(2')); 3.99 4.04 (m, 1 HÀC(5'), HÀC(4')); 3.91
(m, 1 HÀC(5')); 3.65 (s, MeN); 0.94 1.02 (m, 4 Me2CH). Anal. calc. for C30H45N5O6Si2 (627.9): C 57.39,
H 7.22, N 11.15; found: C 57.36, H 7.13, N 11.05.
Data of 20: Rf (petroleum ether/AcOEt 3 :2) 0.42. UV (MeOH): 312 (4.08), 286 (3.99), 236 (4.23), 204
(4.48). 1H-NMR ((D6)DMSO): 8.37 (s, HÀC(2)); 7.97 (s, HÀC(8)); 7.92 (d, 2 H, bz); 7.49 (m, 1 H, bz); 7.41
(m, 2 H, bz); 5.77 (s, HÀC(1')); 5.60( d, OHÀC(2')); 4.59 (m, HÀC(3')); 4.46 (m, HÀC(2')); 3.90 4.01
(m, HÀC(4'), 2 HÀC(5')); 3.66 (s, MeN); 0.95 1.03 (m, Me2CH). Anal. calc. for C30H45N5O6Si2 (627.9):
C 57.39, H 7.22, N 11.15; found: C 57.21, H 7.25, N 10.93.
Data of 21: Rf (petroleum ether/AcOEt 3 :2) 0.57. UV (MeOH): 266 (4.02), 239 (4.10), 207 (4.41).
1H-NMR ((D6)DMSO): 8.49 (s, HÀC(2)); 8.35 (s, HÀC(8)); 7.23 7.36 (m, 5 H, bz); 5.90( s, HÀC(1')); 5.66
(d, OHÀC(2')); 4.69 (m, HÀC(3')); 4.56 (m, HÀC(2')); 4.04 (s, MeO); 4.01 (m, 1 HÀC(5'), HÀC(4')); 3.90
(m, 1 HÀC(5')); 0.98 (m, 4 Me2CH). Anal. calc. for C30H45N5O6Si2 (627.9): C 57.39, H 7.22, N 11.15; found:
C 57.63, H 7.42, N 10.41.
11. N6-Methyl-N6-{[2-(4-nitrophenyl)ethoxy]carbonyl}-2'-O-(tetrahydro-4-methoxy-2H-pyran-4-yl)adeno-
sine (22). To N6-methyl-N6-{[2-(4-nitrophenyl)ethoxy]carbonyl}-3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-
diyl)adenosine (15; 1.82 g, 2.19 mmol) in anh. THF (25 ml) was added Bu4NF (2.0g, 6.34 mmol). After
10min, the solvent was evaporated, the residue dissolved in CH 2Cl2 (80ml), and the org. soln. washed with sat.
NaHCO3 soln. (100 ml), dried (MgSO4), and adsorbed on silica gel. Purification by CC (toluene/AcOEt 5 :4
with 0 4% MeOH) gave 1.17 g (91%) of 22. Rf (toluene/AcOEt/MeOH 5 :4 :1) 0.22. UV (MeOH): 272 (4.37),
211 (4.39). 1H-NMR ((D6)DMSO): 8.77 (2s, HÀC(2), HÀC(8)); 8.05 (d, 2 Ho to NO2); 7.35 (d, 2 Hm to NO2);
6.13 (d, HÀC(1')); 5.32 (d, OHÀC(3')); 5.23 (t, OHÀC(5')); 4.90( m, HÀC(2')); 4.36 (t, CH2CH2O); 4.16
(m, HÀC(3')); 4.02 (m, HÀC(4')); 3.75 3.15 (3m, 2 HÀC(5'), CH2OCH2); 3.38 (s, MeN); 2.97 (t, CH2CH2O);
2.44 (s, MeO); 1.60( m, CH2CCH2). Anal. calc. for C26H32N6O10 ¥ 0.5 H2O (597.6): C 52.26, H 5.57, N 14.06;
found: C 52.20, H 5.67, N 13.40.
12. N3-Methyl-N4-{[2-(4-nitrophenyl)ethoxy]carbonyl}-2'-O-(tetrahydro-4-methoxy-2H-pyran-4-yl)cytidine
(23). A soln. of N3-methyl-N4-{[2-(4-nitrophenyl)ethoxy]carbonyl}-2'-O-(tetrahydro-4-methoxy-2H-pyran-4-
yl)-3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)cytidine (4; 2 g, 2.48 mmol) in anh. THF (30ml) and
Bu4NF (1.95 g, 6.19 mmol) was stirred for 10min. Then the solvent was evaporated, the residue dissolved in
CH2Cl2 (100 ml), the org. soln. washed with sat. NaHCO3 soln. (100 ml), dried (MgSO4), and evaporated, and
the residue purified by CC (toluene/AcOEt 5 :4) with 0 4% MeOH): 1.03 g (74%) of 23. Rf (toluene/AcOEt/
MeOH 5 :4 :1) 0.35. UV (MeOH): 280 (4.36). 1H-NMR ((D6)DMSO): 8.16 (d, 2 Ho to NO2); 7.85 (d, HÀC(6));
7.56 (d, 2 Hm to NO2): 6.26 (d, HÀC(5)); 6.01 (s, HÀC(1')); 5.19 (m, OHÀC(5'), OHÀC(3')); 4.33
(m, CH2CH2O, HÀC(2')): 3.95 (m, HÀC(3')); 3.90( m, 1 HÀC(5')); 3.70 3.30 ( m, CH2OCH2, HÀC(4'),
1 HÀC(5')); 3.22 (s, MeN); 3.08 (t, CH2CH2O); 2.89 (s, MeO); 1.80 1.60 ( m, CH2CCH2). Anal. calc. for
C25H32N4O11 ¥ 0.5 H2O (573.6): C 52.35, H 5.80, N 9.77; found: C 52.31, H 5.81, N 9.70.
13. N4-Methyl-N4-{[2-(4-nitrophenyl)ethoxy]carbonyl}-2'-O-(tetrahydro-4-methoxy-2H-pyran-4-yl)cytidine
(24). Analogously to Exper. 11 with N4-methyl-N4-{[2-(4-nitrophenyl)ethoxy]carbonyl}-2'-O-(tetrahydro-4-

Helvetica Chimica Acta Vol. 86 (2003)
2543
methoxy-2H-pyran-4-yl)-3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)cytidine (5; 1 g, 1.24 mmol) and
Bu4NF (0.98 g, 3.1 mmol) in anh. THF (20 ml). Purification by CC (toluene/AcOEt 5 :4) with 0 4% MeOH)
gave 0.35 g (50%) of 24. Rf (toluene/AcOEt/MeOH 5 :4 :1) 0.25. UV (MeOH): 279 (4.16), 254 (4.29), 204
(4.40). 1H-NMR ((D6)DMSO): 8.23 (d, HÀC(6)); 8.18 (d, 2 Ho to NO2); 7.60( d, 2 Hm to NO2): 7.09
(d, HÀC(5)); 6.09 (s, HÀC(1')); 5.20( t, OHÀC(5')); 5.16 (d, OHÀC(3')); 4.47 (t, CH2CH2O); 4.32
(m, HÀC(2')); 3.98 (m, HÀC(3')); 3.92 (m, 1 HÀC(5')); 3.70 3.25 ( m, CH2OCH2, HÀC(4'), 1 HÀC(5'));
3.21 (s, MeN); 3.14 (t, CH2CH2O); 2.81 (s, MeO); 1.85 1.60( m, CH2CCH2). Anal. calc. for C25H32N4O11 ¥ 0 .5
H2O (573.6): C 52.35, H 5.80, N 9.77; found: C 52.50, H 5.80, N 9.80.
14. 5'-O-{{2-{[5-(Dimethylamino)naphthalen-1-yl]sulfonyl}ethoxy}carbonyl}-N3-methyl-N4-{[2-(4-nitro-
phenyl)ethoxy]carbonyl}-2'-O-(tetrahydro-4-methoxy-2H-pyran-4-yl)cytidine (25). Analogously to Exper. 9,
with 23 (0.94 g, 1.41 mmol) and 2-{[5-(dimethylamino)naphthalen-1-yl]sulfonyl}ethyl carbonochloridate hydro-
chloride (0.756 g, 1.2 mmol) in anh. pyridine (20 ml) and anh. MeCN (10 ml) for 1 h at 08. Purification by CC
(toluene/AcOEt 5 :4 with 0 1% MeOH) gave 0.48 g (34%) of 25. Rf (toluene/AcOEt/MeOH 5 :4 :1) 0.56. UV
1
(MeOH): 344 (3.64), 282 (sh, 4.35), 262 (4.45), 214 (4.74). H-NMR ((D6)DMSO): 8.52 (d, HÀC(2)(npht));
8.21 8.09 (m, HÀC(4)(npht), HÀC(8)(npht), 2 Ho to NO2); 7.70 7.51 ( m, HÀC(3)(npht), HÀC(7)(npht),
HÀC(6), 2 Hm to NO2); 7.26 (d, HÀC(6)(npht)); 6.25 (d, HÀC(5)); 5.94 (d, HÀC(1')); 5.38 (d, OHÀC(3'));
4.40 4.27 ( m, 2 CH2CH2O, HÀC(2')); 4.15 (m, HÀC(3'), HÀC(4')); 3.95 3.83 (m, CH2SO2, 2 HÀC(5'));
3.80 3.30 ( m, CH2OCH2); 3.24 (s, MeN); 3.04 (t, CH2CH2O); 2.93 (s, MeO); 2.80( s, Me2N); 1.80 1.60
(m, CH2CCH2). Anal. calc. for C40H47N5O15S (869.9): C 55.23, H 5.44, N 8.05; found: C 55.38, H 5.61, N 7.70.
15. 5'-O-{{2-{[5-(Dimethylamino)naphthalen-1-yl]sulfonyl}ethoxy}carbonyl}-N4-methyl-N4-{[2-(4-nitro-
phenyl)ethoxy]carbonyl}-2'-O-(tetrahydro-4-methoxy-2H-pyran-4-yl)cytidine (26). Analogously to Exper. 9,
with 24 (0.5 g, 1.34 mmol) and 2-{[5-(dimethylamino)naphthalen-1-yl]sulfonyl}ethyl carbonochloridate hydro-
chloride (0.265 g, 0.70 mmol) in anh. pyridine (10 ml) and anh. MeCN (5 ml) for 3 h at 08. Purification by CC
(toluene/AcOEt 5 :4 with 0 3.5% MeOH) gave 0.13 g (26%) of 24. Rf (toluene/AcOEt/MeOH 5 :4 :1) 0.34.
UV (MeOH): 342 (3.67), 288 (sh, 4.17), 253 (4.54), 213 (4.83). 1H-NMR ((D6)DMSO): 8.55
(d, HÀC(2)(npht)); 8.15 (m, HÀC(4)(npht), HÀC(8)(npht), 2 Ho to NO2); 7.92 (d, HÀC(6)); 7.67 7.55
(m, HÀC(3)(npht), HÀC(7)(npht), 2 Hm to NO2); 7.25 (d, HÀC(6)(npht)); 7.07 (d, HÀC(5)); 6.00
(d, HÀC(1')); 5.36 (d, OHÀC(3')); 4.50 4.30 ( m, 2 CH2CH2O, HÀC(2')); 4.18 (m, HÀC(3'), HÀC(4'));
4.00 3.70 (m, CH2SO2, 2 HÀC(5')); 3.70 3.30 ( m, CH2OCH2), 3.24 (s, MeN); 3.13 (t, CH2CH2O); 2.86
(s, MeO); 2.80( s, Me2N); 1.80 1.60 ( m, CH2CCH2). Anal. calc. for C40H47N5O15S (869.9): C 55.23, H 5.44,
N 8.05; found: C 54.98, H 5.55, N 7.72.
16. 5'-O-{{2-{[5-(Dimethylamino)naphthalen-1-yl]sulfonyl}ethoxy}carbonyl}-N3-methyl-2'-O-(tetrahydro-
4-methoxy-2H-pyran-4-yl)uridine (27). Analogously to Exper. 9, with 6 (0.5 g, 1.34 mmol) and 2-{[5-(dimethyl-
amino)naphthalen-1-yl]sulfonyl}ethyl carbonochloridate hydrochloride (0.584 g, 1.54 mmol) in anh. pyridine
(10ml) for 1 h at 0 8. Purification by CC (toluene/AcOEt 5 :4 with 0 2.5% MeOH) gave 0.35 g (39%) of 27. Rf
(toluene/AcOEt/MeOH 5 :4 :1) 0.44. UV (MeOH): 343 (3.68), 255 (4.36), 212 (4.74). 1H-NMR ((D6)DMSO):
8.54 (d, HÀC(2)(npht)); 8.19 (m, HÀC(4)(npht), HÀC(8)(npht)); 7.69 (m, HÀC(3)(npht), HÀC(7)(npht),
HÀC(6)); 7.28 (d, HÀC(6)(npht)); 5.96 (d, HÀC(5)); 5.84 (d, HÀC(1')); 5.37 (d, OHÀC(3')); 4.38
(m, CH2CH2O, HÀC(2')); 4.15 (m, HÀC(3')); 4.00 3.82 (m, CH2SO2, HÀC(4')); 3.80 3.30 ( m, CH2OCH2,
2 HÀC(5')); 3.18 (s, MeN); 2.94 (s, MeO); 2.82 (s, Me2N); 1.82 1.55 (m, CH2CCH2). Anal. calc. for
C31H39N3O12S ¥ 1/4 toluene (700.8): C 56.13, H 5.90, N 6.00; found: C 56.16, H 5.90, N 6.01.
17. 5'-O-{{2-{[5-(Dimethylamino)naphthalen-1-yl]sulfonyl}ethoxy}carbonyl}-N6-methyl-N6-{[2-(4-nitro-
phenyl)ethoxy]carbonyl}-2'-O-(tetrahydro-4-methoxy-2H-pyran-4-yl)adenosine 3'-[2-(4-Cyanophenyl)ethyl Di-
ethylphosphoramidite] (28). Under exclusion of humidity, 17 (0.45 g, 0.5 mmol) was dissolved in anh. THF
(8 ml). Then, ethyldiisopropylamine (0.5 ml, 3 mmol) and 2-(4-cyanophenyl)ethyl diethylphosphoramidochlor-
idite (0.285 g, 1 mmol) were added, and the mixture was stirred for 1 h. The reaction was quenched with iPrOH
(0.2 ml), and after extraction with CH2Cl2 (30ml) and NaHCO 3 soln. (30ml), the org. layer was dried
(MgSO4), and evaporated. Purification by CC (40 50% AcOEt/toluene) gave 0.35 g (61%) of 28. Rf
(petroleum ether/AcOEt/Et3N 1:9 :1) 0.73. 1H-NMR ((D6)DMSO): 8.75 (m, HÀC(2), HÀC(8)); 8.50
(d, HÀC(2)(npht)); 8.20( m, HÀC(4)(npht), HÀC(8)(npht)); 8.03 (m, 2 Ho to NO2); 7.75 (m, 2 Ho to CN);
7.70( m, 2 Hm to NO2); 7.46 (m, HÀC(3)(npht), HÀC(7)(npht)); 7.36 (d, 2 Hm to CN); 7.26
(m, HÀC(6)(npht)); 6.10(2 d, HÀC(1')); 5.25 (2m, HÀC(2')); 4.35 (m, 3 ¥ CH2CH2O, HÀC(3'), HÀC(4'),
2 HÀC(5')); 3.82 (t, SO2CH2); 3.55 (m, CH2OCH2); 3.37 (s, MeN); 2.96 (m, 2 CH2CH2O, 2 CH2N); 2.79
(s, Me2N); 2.31 (2s, MeO); 1.66 (m, CH2CCH2); 1.02 (m, 2 MeCH2). 31P-NMR ((D6)DMSO): 149.91, 149.03
(2s). Anal. calc. for C54H64N9O15PS (1142.2): C 56.78, H 5.65, N 11.04; found: C 57.35, H 6.04, N 11.32.

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Helvetica Chimica Acta Vol. 86 (2003)
18. 5'-O-{{2-{[5-(Dimethylamino)naphthalen-1-yl]sulfonyl}ethoxy}carbonyl}-N6-methyl-N6-{[2-(4-nitro-
phenyl)ethoxy]carbonyl}-2'-O-(tetrahydro-4-methoxy-2H-pyran-4-yl)-adenosine 3'-[2-(4-Cyanophenyl)ethyl
Diisopropylphosphoramidite] (29). Analogously to Exper. 17, with 17 (0.447 g, 0.5 mmol), ethyldiisopropyl-
amine (0.5 ml, 3 mmol), and 2-(4-cyanophenyl)ethyl diisopropylphosphoramidochloridite (0.313 g, 1 mmol).
Purification by CC (20 50% AcOEt/toluene) gave 0.29 g (49%) of 29. Rf (petroleum ether/AcOEt/Et3N
1 :9 :1) 0.76. 1H-NMR ((D6)DMSO): 8.75 (2s, HÀC(2), HÀC(8)); 8.51 (m, HÀC(2)(npht)); 8.16
(m, HÀC(4)(npht), HÀC(8)(npht)); 8.04 (d, 2 Ho to NO2); 7.77 (m, 2 Ho to CN); 7.62 (m, HÀC(3)(npht),
HÀC(7)(npht)); 7.46 (m, 2 Hm to NO2); 7.33 (d, 2 Hm to CN); 7.22 (m, HÀC(6)(npht)); 6.07 (2d, HÀC(1'));
5.25 (2m, HÀC(2')); 4.30( m, 3 CH2CH2O, HÀC(3'), HÀC(4'), 2 HÀC(5')); 3.80( m, SO2CH2); 3.40
(m, CH2OCH2); 3.38 (s, MeN); 2.97 (m, 2 CH2CH2O, 2 Me2CH); 2.79 (s, Me2N); 2.49 (s, MeO); 1.66
(m, CH2CCH2); 1.08 (m, 2 Me2CH). 31P-NMR ((D6)DMSO): 150.29, 148.65 (2s). Anal. calc. for C56H68N9O15PS
(1170.25): C 57.48, H 5.86, N 10.77; found: C 58.18, H 6.31, N 10.56.
19. 5'-O-{{2-{[5-(Dimethylamino)naphthalen-1-yl]sulfonyl}ethoxy}carbonyl}-N6-methyl-N6-{[2-(4-nitro-
phenyl)ethoxy]carbonyl}-2'-O-(tetrahydro-4-methoxy-2H-pyran-4-yl)adenosine 3'-[2-(4-Nitrophenyl)ethyl Di-
isopropylphosphoramidite] (30). Analogously to Exper. 17, with 17 (0.894 g, 1 mmol), ethyldiisopropylamine
(1 ml, 6 mmol), and [2-(4-nitrophenyl)ethyl diisopropylphosphoramidochloridite (0.7 g, 2.5 mmol). Purification
by CC 25 50% acetone/petroleum ether) gave 0.64 g (54%) of 30. Rf (petroleum ether/AcOEt/Et3N 1 :9 :1)
0.74. 1H-NMR ((D6)DMSO): 8.76 (2s, HÀC(2), HÀC(8)); 8.49 (m, HÀC(2)(npht)); 8.19 8.02
(m, HÀC(4)(npht), HÀC(8)(npht), 4 Ho to NO2); 7.65 7.49 (m, HÀC(3)(npht), HÀC(7)(npht), 2 Hm to
NO2); 7.35 (m, 2 Hm to NO2); 7.24 (m, HÀC(6)(npht)); 6.07 (2d, HÀC(1')); 5.28 (m, HÀC(2')); 4.35 4.12
(m, 3 CH2CH2O, HÀC(3'), HÀC(4')); 4.06 3.69 (m, 2 HÀC(5'), SO2CH2); 3.65 3.25 (m, 2 Me2CH, MeN,
CH2OCH2); 3.20 2.90( m, 2 CH2CH2O); 2.78 (s, Me2N); 2.31 (s, MeO); 1.75 1.25 (m, CH2CCH2); 1.19 1.06
(m, 12 H, 2 Me2CH). 31P-NMR ((D6)DMSO): 153.30, 151.64 (2s). Anal. calc. for C55H68N9O17PS (1190.2):
C 55.50, H 5.76, N 10.59; found: C 54.96, H 5.73, N 10.22.
20. 5'-O-{{2-{[5-(Dimethylamino)naphthalen-1-yl]sulfonyl}ethoxy}carbonyl}-N6-methyl-N6-{[2-(4-nitro-
phenyl)ethoxy]carbonyl}-2'-O-(tetrahydro-4-methoxy-2H-pyran-4-yl)adenosine 3'-(Hydrogen Butanedioate)
(31). A soln. of 17 (0.268 g, 0.3 mmol), N,N-dimethylpyridin-4-amine (DMAP; 48 mg, 0.39 mmol), and
succinic anhydride (60mg, 0.6 mmol) was stirred for 9.5 h in anh. CH 2Cl2 (30ml). The mixture was extracted
with CH2Cl2 (20ml) and sat. NaHCO 3 soln. (25 ml) and the org. layer washed with 10% citric acid soln., dried
(MgSO4), and evaporated: 0.29 g (97%) of 31. Anal. pure material was obtained by CC (33 66% acetone/
petroleum ether). Rf (toluene/AcOEt/MeOH 5 :4 :1) 0.27. UV (MeOH): 343 (3.63), 262 (4.47), 213 (4.80).
1H-NMR ((D6)DMSO): 12.32 (s, COOH); 8.76 (2s, HÀC(2), HÀC(8)); 8.52 (d, HÀC(2)(npht)); 8.18
(m, HÀC(4)(npht), HÀC(8)(npht)); 8.04 (d, 2 Ho to NO2); 7.62 (m, HÀC(3)(npht), HÀC(7)(npht))); 7.35
(d, 2 Hm to NO2); 7.25 (d, HÀC(6)(npht)); 6.17 (d, HÀC(1')); 5.34 (m, HÀC(2'), HÀC(3')); 4.38 4.23
(m, 2 CH2CH2O, 2 HÀC(5')); 3.83 (t, CH2SO2); 3.50 3.10(2 m, CH2OCH2); 3.38 (s, MeN); 2.96 (t, CH2CH2O);
2.79 (s, Me2N); 2.56 (2m, CH2CH2); 2.35 (s, MeO); 1.75 1.30( m, CH2CCH2). Anal. calc. for C45H51N7O17S
(994.0): C 54.38, H 5.17, N 9.86; found: C 54.31, H 5.36, N 9.50.
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Received January 29, 2003