Synthesis and incorporation of C(5′)-ethynylated uracil-derived phosphoramidites into RNA

Article abstract of DOI:10.1002/hlca.200490271

The D-allo- and L-talo-hept-6-ynofuranosyluracil-derived phosphoramidites 11A and 11T were prepared in 9-10% yield over eight steps from the previously described propargylic alcohols 1A and 1T, respectively. The corresponding nucleotides were incorporated into rU₁₄ by standard solid-phase synthesis. While the duplex consisting of rU₁₄ with one L-talo-hept-6-ynofuranosyluracil in the middle of the strand and rA₁₄ (I-V) had the same melting point as the reference duplex rU₁₄- rA₁₄ (I-II), the duplex with one D-allo-hept-6-ynofurano-syluracil in the middle of rU₁₄ and rA₁₄ (I-III) melted 1.5° lower than the reference duplex. The duplex I-VI consisting of rU₁₄ with six L-talo-hept-6-ynofuranosyluracils distributed over the entire strand and rA₁₄ showed a melting point that is 11° lower than the reference duplex. The corresponding duplex I-IV of rU14 possessing six D-allo-hept-6-ynofuranosyluracils and rA₁₄ showed a melting point which is more than 20° below the one of the reference duplex. These results are in qualitative agreement with the predictions based on the conformational analysis of the nucleosides and the interference of the ethynyl moiety with the hydration of the oligonucleotides.

Full text of DOI:10.1002/hlca.200490271

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Helvetica Chimica Acta ± Vol. 87 (2004)
Synthesis and Incorporation of C(5')-Ethynylated Uracil-Derived
Phosphoramidites into RNA
by Simon Eppacher, Markus Christen, and Andrea Vasella*
Laboratorium für Organische Chemie, ETH-Hönggerberg, HCI, CH-8093Zürich
The d-allo- and l-talo-hept-6-ynofuranosyluracil-derived phosphoramidites 11A and 11Twere prepared in
9 ± 10% yield over eight steps from the previously described propargylic alcohols 1A and 1T, respectively. The
corresponding nucleotides were incorporated into rU14 by standard solid-phase synthesis. While the duplex
consisting of rU14 with one l-talo-hept-6-ynofuranosyluracil in the middle of the strand and rA14 (I ´ V) had the
same melting point as the reference duplex rU14 ´ rA14 (I ´ II), the duplex with one d-allo-hept-6-ynofurano-
syluracil in the middle of rU14 and rA14 (I ´ III) melted 1.58 lower than the reference duplex. The duplex I ´ VI
consisting of rU14 with six l-talo-hept-6-ynofuranosyluracils distributed over the entire strand and rA14 showed a
melting point that is 118 lower than the reference duplex. The corresponding duplex I ´ IV of rU14 possessing six
d-allo-hept-6-ynofuranosyluracils and rA14 showed a melting point which is more than 208 below the one of the
reference duplex. These results are in qualitative agreement with the predictions based on the conformational
analysis of the nucleosides and the interference of the ethynyl moiety with the hydration of the oligonucleotides.
Introduction. ± We are exploring the chemistry of ethynylene-linked oligonucleo-
tide analogues1) to further test our hypothesis that the structural differentiation
between backbone and nucleobases is not required for duplex formation [3]. In this
context, we prepared the monomers 1A and 1T [4]. Their availability leads to the
question about the influence of the ethynyl substituents at C(5') on the conformation
and pairing of RNA analogues derived from these monomers (Fig. 1). Information
about the influence of substituents at C(5') in DNA and RNA analogues on pairing is of
interest in the context of antisense or antigene therapy [5 ± 14] and in view of preparing
RNA analogues possessing new functional properties [15 ± 18]. Only a few nucleosides
modified at C(5')2) were incorporated into RNA or DNA, and had either little effect
on duplex stability [35][36] or destabilized it [18][37][38] (compare [28][39][40]).
Results and Discussion. ± 1. Conformational Analysis and Synthesis of the
Phosphoramidites. An evaluation of the influence of the C(5')-ethynyl substituent on
duplex formation for RNA analogues derived from 1A and 1T must take into account
the distribution of conformers resulting from rotation about the C(4')ÀC(5') bond, the
steric interaction of the ethynyl group with the nucleobases and the backbone, and the
influence of the ethynyl group on solvation. To evaluate the population of the three
staggered conformations resulting from rotation about the C(4')ÀC(5') bond, we
indicate a gauche (g) interaction of two substituents by a slash (C/O) and an
antiperiplanar (t) arrangement between H and O by a double slash (H//O), using the
1
)
)
See [1][2] and earlier papers of this series.
For pertinent examples, see [19 ± 34].
2
ꢀ 2004 Verlag Helvetica Chimica Acta AG, Zürich

Helvetica Chimica Acta ± Vol. 87 (2004)
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Fig. 1. Modified rUn oligonucleotides derived from b-d-allo- and a-l-talo-hept-6-ynofuranosyluracil 1A and 1T
following interaction energies [41 ± 44]: (O/Csp3) ˆ 0.45 kcal/mol; (Csp3/Csp) ˆ 0.2 kcal/
mol; O/O ˆ 0.45 kcal/mol. The value for O/Csp was estimated by considering that the
value for O/Csp3 is half the one for Csp3/Csp3 . In a first approximation, we assumed O/Csp
to be half the one for Csp3/Csp, i.e., 0.1 kcal/mol.
A H//O interaction of À 0.85 kcal/mol was deduced from the 57:29 :14 distribution
of the gg/gt/tg conformers resulting from rotation about the C(4')ÀC(5') bond of the b-
d-ribofuranosyl-nucleosides [45]. On the basis of these values, one predicts the
distribution of the conformers of b-d-allo-hept-6-ynofuranosyl- and a-l-talo-hept-6-
ynofuranosyl-nucleosides shown in Fig. 2. The preferred conformation differs for the
three nucleoside types. The b-d-ribofuranosyl-nucleosides prefer the gg, the b-d-allo-
hept-6-ynofuranosyl-nucleosides the gt-gg, and the a-l-talo-hept-6-ynofuranosyl-nu-
cleosides the gg-gt conformation. The results are strongly influenced by the value for
H//O, although, qualitatively, they are not affected even by considerable deviations
from it. Thus, assuming a value for H//O of À 0.45 instead of À 0.85 kcal/mol leads to a
gg-tg/gt-gg/tg-gt conformer ratio for the d-allo-nucleosides of 29 :48 :23, and to a gg-gt/
gt-tg/tg-gg ratio for the l-talo nucleosides of 65 :21:14. The endocyclic orientation of
the ethynyl group in the (preferred) gt-gg conformer of a d-allo-nucleoside and in the
(disfavoured) tg-gg conformation of the l-talo-isomer may lead to a destabilizing steric
interaction with the uracil moiety. This is suggested by the C(5') ´´´ HÀC(6) distance of
3.52 Š in C(5')-ethynylated derivatives, the C(3') endo-conformation of the furanosyl
unit and an anti-conformation of the nucleobases, as it is found in the A form of RNA
duplexes.
Inspection of an RNA strand in a A-helix [46] shows that a C(5')-substituent must
lead to a 1,5-interaction independently of the configuration at C(5') (Fig. 1). A b-d-
allo-hept-6-ynofuranosyl-nucleoside leads to a 1,5-interaction between the ethynyl
group and PÀOÀ. An a-l-talo-hept-6-ynofuranosyl-nucleoside leads to a 1,5-inter-
action between the ethynyl group and PÀOC(3'). Considering the small A value of the
ethynyl group (0.18 kcal/mol [47]) and the long PÀO bond, one expects that these 1,5-
diaxial interactions will result in only minor destabilizations of the duplex. This

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Helvetica Chimica Acta ± Vol. 87 (2004)
Fig. 2. Calculated population of the conformations obtained by rotation about the C(5')ÀC(4') bond of b-d-ribo-,
b-d-allo-hept-6-yno-, and a-l-talo-hept-6-ynofuranosyl-nucleosides
expectation is supported by the observation that Eschenmoserꢁs p-RNA forms highly
stable duplexes in spite of unavoidable 1,5-interactions [48 ± 50].
An evaluation of the influence of the ethynyl group must also take the influence of
the ethynyl group on the first hydration shell into account. H2O Molecules of the first
hydration shell are grouped into three classes [46] with decreasing binding affinity for
phosphate, phosphodiester/sugar, and base. The ethynyl group of the b-d-allo-hept-6-
ynofuranosyl-nucleosides points to the phosphate hydration site and should consid-
erably disturb the hydration shell of the anionic centre. The ethynyl group of the a-l-
talo-hept-6-ynofuranosyl-nucleosides points to the less-important phosphodiester/
sugar hydration site and should result in a smaller destabilization of hydration.
The gg-tg conformation of b-d-allo-hept-6-ynofuranosyl-nucleosides required for
duplex formation is disfavoured and leads to a destabilizing interaction of the ethynyl
group with the first hydration shell. The gg-gt conformation of the a-l-talo-hept-6-
ynofuranosyl-nucleosides required for duplex formation is preferred and leads to a
destabilizing interaction with the less-important second hydration shell. Thus, duplexes
containing b-d-allo-nucleotides are expected to be less stable then natural duplexes and
also less stable than duplexes containing a-l-talo-nucleotides.

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To synthesise the required d-allo-configured phosphoramidite 11A, we transformed
the propargylic alcohol 1A [4] into the 2-nitrobenzoate 2A (Scheme). De-isopropyl-
idenation of 2A in CF3COOH/H2O 1:1 gave the diol 3A, which was converted to the
tribenzoyl derivative 4A (78%) and the dibenzoate 5A (5%). The selective removal of
the 2-nitrobenzoyl group of 5A with Zn and NH4Cl in MeCN/H2O 1:1 [51] to provide
the propargylic alcohol 7A proceeded well on a small scale, as suggested by TLC and a
1
crude H-NMR spectrum (Table 1, Entry 3). Performing the reaction on a gram scale
led, however, to considerable amounts of the 2-aminobenzoate 6A. Using activated Zn
powder and NH4OAc did not significantly affect the yield and the product distribution
(Entry 4). Zn/CF3COOH or Zn/CH3COOH (Entries 6 and 7) led to 6A as the main
product. Starting from 1 ± 5 g of 4A, the propargylic alcohol 7A was finally obtained in
reproducible yields of 60 ± 85% by portionwise addition of a Zn/NH4Cl mixture
(Entries 11 and 12). The propargylic alcohol 7A was converted to the phosporamidites
11A according to a procedure described by Pitsch et al. [52][53]. Thus, 7A was
dimethoxytritylated [54], and the crude product was O-debenzoylated under standard
conditions to give the diol 8A in 59% yield. The 2'-O-[(triisopropylsilyloxy)methyl]
(ˆ TOM) protecting group was introduced by stannylation with Bu2SnCl2 and
(i-Pr)2NEt, followed by treatment with TOMCl at 808, leading to the desired 2'-O-
alkylated nucleoside 9A as the major product (33%) that was separated by
chromatography from the 3'-O-alkylated isomer 10A (16%) and from starting material
8A (28%). The alcohol 9A was transformed into the phosphoramidites 11A (81%;
3:2 mixture of diastereoisomers) according to standard procedures [55].
Table 1. Conditions for the De(2-nitrobenzoylation) of 4A (Entries 1 ± 10: ca. 20-mg batch, Entries 11 and 12:
1 ± 5-g batch)
Entry
Reagents (each 5 equiv.)
Temp. and time
Solvent
Yield of 7A
1
2
3
4
5
6
7
8
9
Zn/NH4Cl (in 1 portion)
Zn/NH4Cl (in 2 portions)
Zn/NH4Cl (in 1 portion)
Zn/NH4OAc (in 1 portion)
Zn/NH4Cl (in 1 portion)
Zn/AcOH (in 1 portion)
Zn/CF3CO2H (in 1 portion)
Zn/NH4Cl (in 1 portion)b)
Zn/NH4Cl (in 1 portion)c)
Zn/NH4Cl (in 1 portion)d)
Zn/NH4Cl (in 5 portions)
Zn/NH4Cl (in 5 portions)
238, 8 h
238, 8 h
238, 8 h
238, 8 h
08, 5 h
238, 7 h
238, 7 h
238, 7 h
238, 7 h
238, 7 h
238, 7 h
238, 7 h
THF/H2O 1 : 1
THF/H2O 1 : 1
ca. 90%
ca. 90%
ca. 90%
ca. 90%
MeCN/H2O 1 : 1
MeCN/H2O 1 : 1
MeCN/H2O 1 : 1
MeCN/H2O 1 : 1
MeCN/H2O 1 : 1
MeCN/H2O 1 : 1
MeCN/H2O 1 : 1
MeCN/H2O 1 : 1
MeCN/H2O 1 : 1
MeCN/H2O 1 : 1
a
)
)
)
a
a
50%
60%
60%
60%
85%
10
11
12
a) Formation of 6A and 7A (not quantified); additional products in Entry 5. b) Excess NH4Cl. c) Zn treated
with 1m NaOH. d) Zn treated with 1m HCl.
Similarly, the a-l-talo-hept-6-ynofuranosyl-uracil 1T was transformed via the
intermediates 2T± 4T (13% of 5T were obtained as side product) to the dimethoxy-
tritylated diol 7T (72% overall yield). The TOM protecting group was introduced
similarly to the d-allo-series, providing mostly the O(2')-protected alcohol 9T (32%)

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Scheme
a) 2-Nitrobenzoyl chloride, DMAP, pyridine, CH2Cl2; 78% of 2A; 93 % of 2T. b) H2O/CF3COOH 1:1; quant.
c) BzCl, 4-pyrrolidinopyridine, pyridine/CH2Cl2 1 :1; 78% of 4A and 5% of 5A; 78% of 4T and 13% of 5T. d)
Zn powder, NH4Cl, MeCN/H2O 1 :1; 62% of 7A (see also Table 1); > 98% of 7T. e) DMTCl, AgNO3, sym-
collidine, CH2Cl2, then NaOH, THF/MeOH/H2O 5 :4 :1; 59% of 8A; 57% of 8T. f) Bu2SnCl2, (i-Pr)2NEt,
(CH2Cl)2, then TOMCl; 33% of 9A and 16% of 10A; 32% of 9T and 10% of 10T. g) 2-Cyanoethyl
diisopropylphosphoramidochloridite, (i-Pr)2NEt, CH2Cl2; 81% of 11A; 76% of 11T. DMAP ˆ 4-(Dimethyl-
amino)pyridine, DMTˆ 4,4'-dimethoxytrityl; TIPS ˆ triisopropylsilyl; TOM ˆ [(triisopropylsilyl)oxy]methyl.
besides its O(3')-protected isomer 10T (10%). The alcohol 9T was transformed to the
phosphoramidites 11T (76%; 3:2 mixture of diastereoisomers), similarly as described
for the synthesis of the d-allo-isomer 2.
The signals for HÀC(2'), HÀC(3'), and HÀC(5') geminal to BzO groups are
shifted downfield by 1.2 ± 1.5 ppm as compared to those of HÀC signals geminal to OH
groups (Tables 4 and 6 in the Exper. Part). The HÀC(1') and HÀC(4') signals are
shifted downfield by 0.2 ± 0.6 ppm upon benzoylation of the cis HOÀC(2') and

Helvetica Chimica Acta ± Vol. 87 (2004)
3009
HOÀC(3'). The upfield shift for HÀC(2') and HÀC(3') of 3A and 3T (4.24 ±
4.36 ppm), and of HÀC(5') of 7A and 7T (4.83± 4.86 ppm) show that the 2-
nitrobenzoyl and the Bz group did not migrate. J(1',2')/J(3',4') ˆ 1.0 ± 1.06 suggests a ca.
1:1 equilibrium of the (N)- and (S)-conformers of the isopropylidene acetals 2A and
2T, whereas the (N)-conformer is slightly preferred in the diols 3A and 3T (J(1',2')/
J(3',4') ˆ 0.73± 0.8). An increasing preference for the ( S)-conformation is observed in
the d-allo-series 8A, 4A/5A/9A/11A, 6A, and 7A/10A (J(1',2')/J(3',4') ˆ 1.5, 2.1 ± 2.3,
3.0, and 3.6 ± 4.2, resp.) and in the l-talo-series 4T/8T/11T, 7T/9T, and 10T (J(1',2')/
J(3',4') ˆ 2.9 ± 3.0, 3.9 ± 4.3, and 6.2, resp.). The l-talo-derivatives show stronger
preference for the (S)-conformation than the corresponding d-allo-isomers with the
exception of 7A/7T. J(4',5') for the l-talo derivatives 2T± 4Tand 8T± 10T is 1.6 ± 2.8 Hz
larger than J(4',5') for the corresponding d-allo-configured products, evidencing a
stronger preference for the antiperiplanar orientation of HÀC(4') and HÀC(5') in
these l-talo-isomers [56]. However, J(4',5') of 7T and 11T is similar to J(4',5') of 7A
and 11A (DJ ꢀ 0.3Hz). The value of J(4',5') of the l-talo-isomers 2T± 4T and 7T± 11T
(3.0 ± 6.5 Hz) indicates a higher population of the gt-tg conformation than expected by
the above calculation (9%).
The structure of the regioisomers 9A/10A and 9T/10T is revealed by the coupling
constants of HÀC(2') and HÀC(3'); the assignment was confirmed by H/D exchange
and selective irradiation experiments. The large J(2,OH) value of 9A and 9T (7.8 ±
8.0 Hz) and the small J(3,OH) value of 10A and 10T (1.6 ± 2.8 Hz) evidence a
intramolecular H-bond to the TOMO substituent (cf. [57][58]) and corroborate the
(S)-conformation with a pseudoequatorial O-substituent at C(2') and a pseudoaxial O-
substituent at C(3').
2. Solid-Phase Synthesis of C(5')-Ethynylated Ribonucleic Acids. For the pairing
studies, we chose the tetradecameric rU14 and rA14 strands. To study the influence of the
ethynyl group, we incorporated one modified unit derived from 11A or 11T in the
middle of the strand. In addition to the singly modified strands, we synthesized rU14
strands with six modifications distributed over the entire strand. The solid-phase
synthesis of the oligomers I ± VI (Table 2) were carried out on a 1.3-mmol scale
essentially according to the protocol developed for the synthesis of pyranosyl-RNA (p-
RNA) [59]. The standard phosphoramidites were protected with a DMT group at
OÀC(5'), a TOM group at OÀC(2'), a cyanoethyl group at OÀP, and an Ac group at
N6 of adenosine. Coupling was performed with 6 equiv. of phosphoramidite in the
presence of 80 equiv. of 1-(benzylsulfanyl)-1H-tetrazole. Considering the slower
detritylation of secondary alcohols, the detritylation of the hept-6-ynofuranosyl units
incorporated into the oligonucleotides was performed with 6% Cl2CHCOOH in
(CH2Cl)2 for 2.5 min. The time for coupling the phosphoramidites to the resulting
secondary propargyl alcohols was doubled from 5 to 10 min. These conditions led to
coupling yields of 90 ± 95%. The base- and phosphate-protecting groups, and the solid
support were cleaved off with 10m MeNH2 in EtOH/H2O 1:1 at 238 within 2 h.
Following this, the TOM and Et3Si protecting groups were removed by treatment with
1m Bu4NF (TBAF) ´ 3H 2O in THF 3). The products were treated with Tris ´ HCl buffer
(pH 7.4), desalted by chromatography on a Sephadex G-10 column, and purified by
3
)
N-Methylpyrrolidinone was added to dissolve rA14 (I) in THF.

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Helvetica Chimica Acta ± Vol. 87 (2004)
chromatography on a SAX anion exchange column at pH 11.5 with a NaCl gradient.
The chromatograms showed a composition in agreement with the detritylation assay.
Finally, the products were desalted on a RP C-18 column and analyzed by MALDI-
TOF mass spectroscopy [60] (Table 2).
Table 2. Synthesis and Characterization of the Oligonucleotides I ± VI
Sequence
(X ˆ 11A, Yˆ 11B)
Yield
Mol. formula
[M À H]À
calc.
b
c
)
)
obs.a)
I
II
III
IV
V
5'-r(AAAAAAAAAAAAAA)-3'
5'-r(UUUUUUUUUUUUUU)-3'
5'-r(UUUUUXUUUUUUUU)-3'
5'-r(UXUXUXUXUXUXUU)-3'
5'-r(UUUUUYUUUUUUUU)-3'
5'-r(UYUYUYUYUYUYUU)-3'
99%
88%
84%
64%
85%
67%
14%
5%d)
8%d)
13%
16%
8%
C140H169N70O82P13
C126H155N28O110P13
C128H155N28O110P13
C138H155N28O110P13
C128H155N28O110P13
C138H155N28O110P13
4547.1
4224.8
4248.7
4368.9
4224.8
4368.9
4547.9
4224.7
4248.7
4368.9
4223.5
4367.3
VI
a) By MALDI-TOF mass spectrometry according to [60]. b) Calculated from the yield of the coupling steps, as
determined by the detritylation assay. c) Yield after purification. d) Losses during purification.
3. Pairing Studies. The melting points of the duplexes consisting of oligonucleotide I
and the complementary oligonucleotides II ± VI were measured at single strand
concentrations of 5 mmol at pH 7 in an aqueous Na2HPO4/NaH2PO4 buffer and at a
0.15m NaCl concentration (Table 3). While the duplex I ´ V substituted with one a-l-
talo-hept-6-ynofuranosyluracil showed the same melting point as the unmodified
reference duplex I ´ II (208), the duplex I ´ III with one b-d-allo-hept-6-ynofuranosylur-
acil showed a melting point that is 1.58 lower than that of the reference I ´ II (Fig. 3,a).
Table 3. Melting Points Tm [8C] of I ´ II, I ´ III, I ´ IV, I ´ V, and I ´ VI Obtained from Temperature-Dependent UV
Spectroscopy
Entry
1
Duplex
Sequence (X ˆ 11A, Yˆ 11B)
Tm
Tm À Tm(I ´ II)
I ´ II
5'-(AAAAAAAAAAAAAA)-3'
3'-(UUUUUUUUUUUUUU)-5'
5'-(AAAAAAAAAAAAAA)-3'
3'-(UUUUUUUUXUUUUU)-5'
5'-(AAAAAAAAAAAAAA)-3'
3'-(UUXUXUXUXUXUXU)-5'
5'-(AAAAAAAAAAAAAA)-3'
3'-(UUUUUUUUYUUUUU)-5'
5'-(AAAAAAAAAAAAAA)-3'
3'-(UUYUYUYUYUYUYU)-5'
208
2
3
4
5
I ´ III
I ´ IV
I ´ V
18.58
< 08
208
À 1.58
>À 208
08
I ´ VI
98
À 118
The duplex I ´ VI substituted with six a-l-talo-hept-6-ynofuranosyluracils showed a
melting point of ca. 98 (Fig. 3,b). The partially sigmoidal curve allows only to derive an
approximate melting point. The corresponding duplex I ´ IV substituted with six b-d-
allo-hept-6-ynofuranosyluracils showed only the upper half of the sigmoidal curve. The
melting point of this duplex is estimated to be below 08.
We also determined the melting behaviour of the single strands. While the
absorption spectra of rU14 (II) shows only a minor dependence on the temperature, the

Helvetica Chimica Acta ± Vol. 87 (2004)
3011
Fig. 3. a) Temperature-dependent UV spectra (ꢂmelting curvesꢁ) of the non-modified and modified rA14 ´ rU14
duplexes I ´ II, I ´ III, and I ´ V, and b) of the non-modified and modified rA14 ´ rU14 duplexes I ´ II, I ´ IV,
and I ´ VI
absorption spectra of rA14 (I) depends considerably on the temperature. This explains
the additional rise of the hyperchromicity after the melting point.
To see whether the modified duplexes possess the same conformation as the
reference duplex I ´ II, we measured temperature dependent CD spectra (Fig. 4). As
the UV spectra, the CD spectra of the rA14 strand show a considerable dependence on
the temperature while the CD spectra of the rU14 strand show almost no temperature
dependence. One also observes a much larger ellipticity for rA14 than for rU14. The CD
spectra of rA14 (I), the duplex rA14 ´ rU14 (I ´ II), and the duplex with one b-d-allo-hept-
6-ynofuranosyluracil (I ´ III) are similar to each other and differ only by the change of
ellipticity between the melting-point temperatures, suggesting that all duplexes adopt
the same conformation.

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Fig. 4. CD Spectra of rA14 (I), the duplex rA14 ´ rU14 (I ´ II), and the modified duplex I ´ III
According to the temperature-dependent UVand CD spectra, incorporation of one
a-l-talo-hept-6-ynofuranosyluracil does not destabilize the resulting duplex, while
incorporation of one b-d-allo-hept-6-ynofuranosyluracil caused a slight destabilization.
The incorporation of six hept-6-ynofuranosyluracils, however, strongly destabilizes the
corresponding duplexes, and this much more so upon incorporating b-d-allo- than a-l-
talo-residues, in agreement with the results of the conformational analysis and the
relative importance of the different hydration sites.
We thank Prof. S. Pitsch for advice and help in the synthesis of the oligonucleotides, Mr. D. Huber for help
in the synthesis, Dr. B. Bernet for checking the Exper. Part, and the ETH-Zürich, and F. Hoffmann-La Roche
AG, Basel, for generous support.
Experimental Part
General. See [2]. Ion-exchange HPLC: Macherey-Nagel Nucleogel-SAX 1000 ± 8/77 (anal.) and 1000 ± 8/46
(prep.); eluent A, 10 mm Na2HPO4 in H2O (pH 11.5); eluent B, 10 mm Na2HPO4/1m NaCl in H2O (pH 11.5;
detection at 260 nm, elution at 238. MS: fast-atom-bombardment (FAB; 3-nitrobenzyl alcohol (NOBA)), or
matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF; 0.05m 1H-indole-3-
acrylic acid (IAA) in THF or 0.05m a-cyano-4-hydroxycinnamic acid (CCA) in MeCN/EtOH/H2O), and high-
resolution MALDI (HR-MALDI; 0.05m 2,5-dihydroxybenzoic acid (DHB) in THF). All measurements with
oligonucleotides were made in a phosphate buffer (10 mm aq. NaH2PO4 buffer containing 150 mm NaCl at
pH 7.0) with a total oligonucleotide concentration of 5 mm. Concentrations of oligonucleotide solns. were
calculated from the UV absorbance of the soln. at 260 nm and pH 7 using the following molar extinction
coefficients: e(r(A)) ˆ 15100, e(r(U)) ˆ 9900.
1-[6,7-Dideoxy-2,3-O-isopropylidene-5-O-(2-nitrobenzoyl)-7-C-(triethylsilyl)-b-d-allo-hept-6-ynofuranosyl]-
uracil (2A). A soln. of pyridine (1.7 ml, 21.1 mmol), DMAP (0.15 g, 1.2 mmol), and 1A [4] (2.9 g, 6.9 mmol) in
CH2Cl2 (70 ml) was treated dropwise with 2-nitrobenzoyl chloride (1.04 ml, 7.9 mmol), stirred at reflux for 24 h,
diluted with CH2Cl2 (30 ml), washed with sat. aq. NH4Cl soln., dried (Na2SO4), and evaporated. FC (AcOEt/
hexane 1 :2 ! 1:1) gave 2A (3.06 g 78%). Colourless foam. Rf (AcOEt/hexane 1:1) 0.32. [a]2D5 ˆ À60.2 (c ˆ
0.82, CHCl3). UV (MeOH): 258 (12200). IR (CHCl3): 3387w, 2958w, 2876w, 2200w, 1755m, 1728m, 1695s, 1645w,
1537m, 1457w, 1385w, 1351w, 1251w, 1156w, 1093m, 1070m, 1017w, 972w, 857w. 1H-NMR (300 MHz, CDCl3): see
Table 4; additionally, 8.65 (br. s, NH); 7.95 ± 7.90 (m, 1 arom. H); 7.70 ± 7.63( m, 3arom. H); 1.59, 1.37 (2 s,
Me2C); 1.00 (t, J ˆ 7.8, (MeCH2)3Si); 0.64 (q, J ˆ 7.8, (MeCH2)3Si). 13C-NMR (75 MHz, CDCl3): see Table 5;
additionally, 162.9 (s, OCˆO); 148.7 (s, C(2) of 2-NO2Bz); 133.3 (d, C(5) of 2-NO2Bz); 132.8 (d, C(4) of 2-
NO2Bz); 130.6 (d, C(6) of 2-NO2Bz); 126.5 (s, C(1) of 2-NO2Bz); 124.2 (d, C(3) of 2-NO2Bz); 115.4 (s, Me2C);

Helvetica Chimica Acta ± Vol. 87 (2004)
3013
Table 4. Selected 1H-NMR Chemical Shifts [ppm] and Coupling Constants [Hz] of the d-allo-Hept-6-
ynofuranosyluridines 2A ± 11A in CDCl3 Solution
2A
3A
4A
5.89
5A
5.76
6A
5.67
7A
5.96
8A
9A
10A
11Aa)
HÀC(5)
HÀC(6)
HÀC(1')
HÀC(2')
HÀC(3')
HÀC(4')
HÀC(5')
J(5,6)
J(1',2')
J(2',3')
J(3',4')
J(4',5')
5.51
7.33
5.92
4.75
4.92
4.46
5.835.91
7.9
3.7
6.6
3.5
3.3
5.66
7.66
5.87
4.44
4.36
4.24
5.46
7.77
5.28
7.60
6.13 5.99
5.26
5.51/5.46
7.66
b
b
b
b
b
)
)
)
)
)
6.47
5.65
5.94
4.73
6.48
5.62
5.90
4.70
6.536.48
5.72
6.15
5.84
)
6.18/6.14
4.25c)
4.51
4.07
4.034.20/4.03
5.79
6.11
4.56
4.28
4.20
4.38/4.34
4.34
4.13/4.06
4.34
3.98
4.15
4.51d)
3.99
4.74
e
6.12
6.11
6.13 4.86
4.08
8.0
3.6
5.8
4.8
2.9
8.1
7.2
6.5
3.1
2.8
8.1
7.2
6.2
3.4
2.8
8.38.3 8.38.3 8.2
8.1/8.1
7.5
5.8
2.5
2.9
7.6
5.7
1.8
3.0
5.0
6.0
3.4
2.8
6.2
5.4
2.9
2.5
7.5
5.9
2.1
2.7
6.3/6.0
5.8/5.8
2.7/3.0
2.7/2.8
a) 3: 2 Mixture of diastereoisomers. b) Signal hidden by the signals of the aromatic H-atoms at 7.0 ± 8.0 ppm.
c) d(HOÀC(2')) ˆ 3.72 ppm,
J(2',OH) ˆ 8.0 Hz.
d) d(HOÀC(3')) ˆ 2.99 ppm,
J(3',OH) ˆ 2.8 Hz.
e) d(HOÀC(5')) ˆ 3.46 ppm, J(5',OH) ˆ 3.7 Hz.
Table 5. Selected 13C-NMR Chemical Shifts [ppm] of the d-allo-Hept-6-ynofuranosyluridines 2A ± 5A and 7A ±
11A in CDCl3 Solution
2A
3A
4A
5A
7A
8A
9A
10A
11Aa)
C(2)
C(4)
C(5)
C(6)
C(1')
C(2')
C(3')
C(4')
C(5')
C(6')
C(7')
163.8
150.1
102.7
164.4
151.8
102.9
162.0
149.7
103.6
163.7
150.7
103.7
162.3
150.0
103.6
164.0
151.8
102.5
140.1
89.7
75.3
71.0
86.385.8
65.5
103.4
91.6
163.3
150.6
102.6
140.5/140.3
86.8
82.5
70.5
85.9/85.7
65.8
103.2
91.6
162.6
150.4
102.5
162.8
150.2/150.0
102.5
140.3140.3148.7 9.7 13 140.3140.7
140.5
92.0
84.0
80.5
89.6
74.8
70.9
86.4
73.2
70.7
86.2
73.0
70.6
86.8
73.9
71.6
87.4
73.5
78.9
86.2/86.1
77.8
71.7/70.4
85.384.382.6
82.5
85.9
87.2
66.1
98.1
96.2
66.6
98.7
93.1
65.8
98.1
93.9
65.9
98.1
93.7
63.0
102.5
91.6
65.3
103.1
91.4
65.7/65.6
103.5/103.2
91.8/91.1
a) 3: 2 Mixture of diastereoisomers.
‡
27.1, 25.1 (2q, Me2C); 7.3( q, (MeCH2)3Si); 4.1 (t, (MeCH2)3Si). HR-MALDI-MS (DHB): 594.188 ([M ‡ Na] ,
‡
C27H33N3NaO9Si ; calc. 594.187). Anal calc. for C27H33N3O9Si (571.66): C 56.73, H 5.82, N 7.35; found: C 56.53,
H 5.82, N 7.32.
1-[6,7-Dideoxy-5-O-(2-nitrobenzoyl)-7-C-(triethylsilyl)-b-d-allo-hept-6-ynofuranosyl]uracil (3A). A sus-
pension of 2A (3.0 g, 5.2 mmol) in H2O (35 ml) was treated with CF3COOH (35 ml), stirred at 808 for 1 h,
diluted with H2O (180 ml), and extracted with CH2Cl2. The combined org. layers were washed with brine, dried
(Na2SO4), and evaporated to give crude 3A (3.1 g, quant.). Red foam. Rf (AcOEt/MeOH 20 :1) 0.56. [a]D25
ˆ
À43.9 (c ˆ 0.7, CHCl3). UV (MeOH): 259 (11500). IR (CHCl3): 3391w, 2958w, 2876w, 2185w, 1745m, 1693s,
1640w, 1537m, 1458w, 1350m, 1252m, 1114m, 1068m, 1005w, 964w, 847w. 1H-NMR (300 MHz, CDCl3): see
Table 4; additionally 9.73(br. s, NH); 7.97 ± 7.92 (m, 1 arom. H); 7.74 ± 7.66 (m, 3 arom. H); 3.9 ± 3.3 (br. s,
2 OH); 0.92 (t, J ˆ 8.1, (MeCH2)3Si); 0.58 (q, J ˆ 8.1, (MeCH2)3Si). 13C-NMR (75 MHz, CDCl3): see Table 5;
additionally, 162.9 (s, OCˆO); 148.7 (s, C(2) of 2-NO2Bz); 133.3 (d, C(5) of 2-NO2Bz); 132.6 (d, C(4) of 2-
NO2Bz); 130.2 (d, C(6) of 2-NO2Bz); 127.1 (s, C(1) of 2-NO2Bz); 124.5 (d, C(3) of 2-NO2Bz); 7.5 (q,
‡
‡
(MeCH2)3Si); 4.1 (t, (MeCH2)3Si). HR-MALDI-MS (DHB): 554.157 ([M ‡ Na] , C24H29N3NaO9Si ; calc.
554.157). Anal. calc. for C24H29N3O9Si (531.59): C 54.23, H 5.50, N 7.90; found: C 54.04, H 5.56, N 7.82.

3014
Helvetica Chimica Acta ± Vol. 87 (2004)
Benzoylation of 3A. A soln. of pyridine (40 ml), 4-pyrrolidinopyridine (0.62 g, 4.2 mmol), and 3A (2.2 g,
4.15 mmol) in CH2Cl2 (40 ml) was treated dropwise with BzCl (2.4 ml, 21 mmol), stirred at 238 for 24 h, diluted
with CH2Cl2, washed with brine, dried (Na2SO4), and evaporated. FC (AcOEt/hexane 1:4 ! 1:2) gave 4A
(2.7 g, 78%) and 5A (164 mg, 5%).
3-Benzoyl-1-[2,3-di-O-benzoyl-6,7-dideoxy-5-O-(2-nitrobenzoyl)-7-C-(triethylsilyl)-b-d-allo-hept-6-yno-
furanosyl]uracil (4A). Colourless foam. Rf (AcOEt/hexane 1:1) 0.66. [a]2D5 ˆ À64.2 (c ˆ 1.1, CHCl3). UV
(MeOH): 254 (19200). IR (CHCl3): 3032w, 2958w, 2876w, 2160w, 1732s, 1718m, 1678s, 1601w, 1584w, 1537m,
1450m, 1375m, 1350m, 1316m, 1265s, 1122m, 1107m, 1069m, 1025w, 973w, 920w, 847w. 1H-NMR (300 MHz,
CDCl3): see Table 4; additionally, 8.31 ± 7.23 (m, 19 arom. H, HÀC(6)); 1.00 (t, J ˆ 8.1, (MeCH2)3Si); 0.65 (q, J ˆ
8.1, (MeCH2)3Si). 13C-NMR (75 MHz, CDCl3): see Table 5; additionally, 168.5 (s, NCˆO); 165.6, 165.4, 164.1
(3s, 3 OCˆO); 148.3( s, C(2) of 2-NO2Bz); 135.3, 134.1, 134.0 (3d, C(4) of 3 Bz); 133.4 (d, C(4) of 2-NO2Bz);
132.7 (d, C(5) of 2-NO2Bz); 131.5 (s, C(1) of BzN); 130.7 (d, C(2) and C(6) of BzN); 130.3 (d, C(6) of 2-
NO2Bz); 130.2, 130.1 (2d, C(2) and C(6) of 2 BzO); 129.35 (d, C(3) and C(5) of BzN); 129.30 (s, C(1) of BzO);
128.9, 128.7 (2d, C(3) and C(5) of 2 BzO); 128.4 (s, C(1) of BzO); 126.7 (s, C(1) of 2-NO2Bz); 124.4 (d, C(3 ) of
‡
2-NO2Bz); 7.4 (q, (MeCH2)3Si); 4.0 (t, (MeCH2)3Si). HR-MALDI-MS (DHB): 866.235 ([M ‡ Na] ,
‡
C45H41N3NaO12Si ; calc. 866.236). Anal. calc. for C45H41N3O12Si (843.92): C 64.05, H 4.90, N 4.98; found:
C 64.23, H 5.16, N 4.73.
1-[2,3-Di-O-benzoyl-6,7-dideoxy-5-O-(2-nitrobenzoyl)-7-C-(triethylsilyl)-b-d-allo-hept-6-ynofuranosyl]ur-
acil (5A). Colourless foam. Rf (AcOEt/hexane 1:1) 0.36. [a]2D5 ˆ À55.9 (c ˆ 0.8, CHCl3). UV (MeOH): 248
(20200). IR (CHCl3): 3384w, 3032w, 2959w, 2871w, 2160w, 1732s, 1698s, 1602w, 1584w, 1537m, 1452m, 1350m,
1316w, 1261s, 1122m, 1107m, 1068m, 1025w, 973w, 847w. 1H-NMR (300 MHz, CDCl3): see Table 4; additionally,
8.65 (br. s, NH); 8.10 ± 7.23( m, 14 arom. H, HÀC(6)); 1.00 (t, J ˆ 8.1, (MeCH2)3Si); 0.65 (q, J ˆ 8.1,
(MeCH2)3Si). 13C-NMR (75 MHz, CDCl3): see Table 5; additionally, 165.4, 165.3, 164.1 (3s, 3 OCˆO); 148.3( s,
C(2) of 2-NO2BzO); 134.0, 133.9 (2d, C(4) of 2 BzO); 133.5 (d, C(4) of 2-NO2BzO); 132.6 (d, C(5) of 2-
NO2BzO); 130.4 (d, C(6) of 2-NO2BzO); 130.2 (d, C(2) and C(6) of 2 BzO); 128.9 (s, C(1) of BzO); 128.8, 128.7
(2d, C(3) and C(5) of 2 BzO); 128.5 (s, C(1) of BzO); 126.8 (s, C(1) of 2-NO2BzO); 124.4 (d, C(3 ) of 2-
NO2BzO); 7.3( q, (MeCH2)3Si); 4.0 (t, (MeCH2)3Si).
Partial Deprotection of 4A. a) A soln. of 4A (1.0 g, 1.19 mmol) in H2O (15 ml) and MeCN (15 ml) was
treated at 238 with four batches of NH4Cl (0.25 g, 4.7 mmol) and Zn powder (0.16 g, 2.4 mmol) in intervals of
20 min. The suspension was stirred for 7 h and filtered through silica gel (elution with AcOEt). The filtrate was
washed with sat. aq. NaHCO3 soln. and brine, dried (Na2SO4), and evaporated. FC (toluene/MeOH 100 :1 !
10 :1) gave 7A (1.8 g, 62%).
b) As in a), but heating after 3h to 80 8 and stirring at 808 for 2 h. FC (toluene/MeOH 100 :1) gave 6A (4%)
and crude 7A (ca. 24%) beside several decomposition products.
3-Benzoyl-1-[5-O-(2-aminobenzoyl)2,3-di-O-benzoyl-6,7-dideoxy-7-C-(triethylsilyl)-b-d-talo-hept-6-yno-
furanosyl]uracil (6A). Colourless foam. Rf (toluene/MeOH 4 :1) 0.54. 1H-NMR (200 MHz, CDCl3): see Table 4;
additionally, 8.07 ± 7.98 (m, 3arom. H,); 7.92 ± 7.76 ( m, 2 arom. H); 7.61 ± 7.22 (m, 11 arom. H, HÀC(6)); 6.84 ±
6.71 (m, 2 arom. H); 5.79 (br. s, exchanged with CD3OD, NH2); 0.95 (t, J ˆ 8.0, (MeCH2)3Si); 0.60 (q, J ˆ 8.0,
‡
‡
(MeCH2)3Si). HR-MALDI-MS (DHB): 836.259 ([M ‡ Na] , C45H43N3NaO16Si ; calc. 836.261).
3-Benzoyl-1-[2,3-di-O-benzoyl-6,7-dideoxy-7-C-(triethylsilyl)-b-d-allo-hept-6-ynofuranosyl]uracil (7A).
Colourless foam. Rf (AcOEt/hexane 1 :2) 0.38. [a]2D5 ˆ À76.4 (c ˆ 1.1, CHCl3). UV (MeOH): 252 (19100). IR
(CHCl3): 3350w (br.), 3035w, 2957w, 2876w, 2280w, 1755m, 1730s, 1711m, 1675s, 1601w, 1450m, 1378m, 1316w,
1
1270s, 1126m, 1092m, 1070m, 1024w, 974w, 921w. H-NMR (300 MHz, CDCl3): see Table 4; additionally, 8.80
(br. s, NH); 8.08 ± 7.21 (m, 15 arom. H, HÀC(6)); 0.94 (t, J ˆ 7.8, (MeCH2)3Si); 0.59 (q, J ˆ 7.8, (MeCH2)3Si).
13C-NMR (75 MHz, CDCl3): see Table 5; additionally, 168.7 (s, NCˆO); 165.8, 165.3(2 s, 2 OCˆO); 135.3 (d,
C(4) of BzN); 134.0, 133.9 (2d, C(4) of 2 BzO); 131.6 (s, C(1) of BzN); 130.8 (d, C(2) and C(6) of BzN); 130.12,
130.09 (2d, C(2) and C(6) of 2 BzO); 129.4 (d, C(3) and C(5) of BzN); 129.3 (s, C(1) of BzO); 128.9, 128.7 (2d,
C(3) and C(5) of 2 BzO); 128.6 (s, C(1) of BzO); 7.4 (q, (MeCH2)3Si); 4.1 (t, (MeCH2)3Si). HR-MALDI-MS
‡
‡
(DHB): 717.224 ([M ‡ Na] , C38H38N2NaO9Si ; calc. 717.224). Anal. calc. for C38H38N2O9Si (694.81): C 65.69,
H 5.51, N 4.03; found: C 65.51, H 5.63, N 4.16.
1-[6,7-Dideoxy-5-O-(4,4'-dimethoxytrityl)-7-C-(triethylsilyl)-b-d-allo-hept-6-ynofuranosyl]uracil (8A). A
soln. of 4,4'-dimethoxytrityl chloride (DMTCl; 1.0 g, 2.95 mmol) in CH2Cl2 (11 ml) was treated with sym-
collidine (2.4 ml), stirred at 238 for 15 min, treated with AgNO3 (0.5 g, 2.95 mmol), and stirred for 1 h. An
aliquot (8.2 ml, 1.80 mmol of the reagent) of this suspension was filtered into a soln. of 7A (500 mg, 0.72 mmol)
in CH2Cl2 (5 ml). The mixture was stirred at 238 for 30 min and filtered through silica gel (elution with AcOEt/
Et3N 99 :1). The filtrate was evaporated. A soln. of the residue in THF/MeOH/H2O 5 :4 :1 (50 ml) was cooled to

Helvetica Chimica Acta ± Vol. 87 (2004)
3015
08, treated dropwise with an aq. 10m NaOH soln. (0.72 ml, 7.2 mmol), stirred at 08, treated with sat. aq. NH4Cl
soln., and extracted with AcOEt. The combined org. layers were dried (Na2SO4), filtered, and evaporated. FC
(hexane/AcOEt/Et3N 1:1 :0.01 ! 1:2 :0.01) gave 8A (0.29 g, 59%). Colourless foam. Rf (AcOEt/hexane 1 :1)
0.12. [a]2D5 ˆ À59.5 (c ˆ 0.9, CHCl3). UV (MeOH): 257 (14500). IR (CHCl3): 3391w, 3005w, 2957m, 2875w,
2838w, 2280w, 1691s, 1608m, 1509s, 1463m, 1395w, 1300m, 1252s, 1110w, 1092w, 1070w, 1036m, 975w, 910w, 829m.
1H-NMR (300 MHz, CDCl3): see Table 4; additionally, 7.51 ± 7.17 (m, 9 arom. H); 6.82 (d, J ˆ 9.0, 4 arom. H);
3.78, 3.77 (2s, 2 MeO); 0.88 (t, J ˆ 7.8, (MeCH2)3Si); 0.46 (q, J ˆ 7.8, (MeCH2)3Si). 13C-NMR (75 MHz, CDCl3):
see Table 5; additionally, 159.1, 159.07 (2s, C(4) of 2 MeOC6H4); 145.4 (s, C(1) of Ph); 135.9, 135.6 (2s, C(1) of
2 MeOC6H4); 130.9 (d, C(2) and C(6) of 2 MeOC6H4); 128.6 (d, C(2) and C(6) of Ph); 128.0 (d, C(3) and C(5)
of Ph); 127.2 (d, C(4) of Ph); 113.4, 113.3 (2d, C(3) and C(5) of 2 MeOC6H4); 88.4 (s, Ar3C); 55.3( q, 2 MeO);
7.4 (q, (MeCH2)3Si); 4.0 (t, (MeCH2)3Si). Anal. calc. for C38H44N2O8Si (684.86): C 66.64, H 6.48, N 4.09; found:
C 66.50, H 6.54, N 4.12.
(Silyloxy)methylation of 8A. A soln. of 8A (0.68 g, 0.99 mmol) and (i-Pr)2NEt (0.85 ml, 4.97 mmol) in
(CH2Cl)2 (4 ml) was treated with Bu2SnCl2 (0.33 g, 1.09 mmol), stirred for 90 min at 238, treated with
(triisopropylsilyl)oxymethyl chloride (TOMCl; 0.26 g, 1.19 mmol), heated to 808, stirred for 70 min, diluted
with sat. aq. NaHCO3 soln., and extracted with CH2Cl2. The org. phase was washed with brine, dried (Na2SO4),
and evaporated. FC (hexane/AcOEt/Et3N 4 :1:0.01 ! 2 :1:0.01) gave 9A (284 mg, 33%), 10A (122 mg, 16%),
and 8A (189 mg, 28%).
1-(6,7-Dideoxy-5-O-(4,4'-dimethoxytrityl)-7-C-(triethylsilyl)-2-O-{[(triisopropylsilyl)oxy]methyl}-b-d-allo-
hept-6-ynofuranosyl)uracil (9A). Colourless foam. Rf (AcOEt/hexane 1:2) 0.29. [a]2D5 ˆ À36.8 (c ˆ 0.9, CHCl3).
UV (MeOH): 257 (12900). IR (CHCl3): 3391w, 3005w, 2954m, 2870m, 2180w, 1693s, 1608w, 1509m, 1461m,
1386w, 1255m, 1079m, 1036m, 1015m, 882w, 827w. 1H-NMR (300 MHz, CDCl3): see Table 4; additionally, 8.08
(br. s, NH); 7.51 ± 7.21 (m, 9 arom. H); 6.81 ± 6.76 (m, 4 arom. H); 5.20, 4.93(2 d, J ˆ 4.7, OCH2O); 3.79 (s, 2
MeO); 1.13± 1.08 ( m, (Me2CH)3Si); 0.88 (t, J ˆ 7.5, (MeCH2)3Si); 0.45 (q, J ˆ 7.5, (MeCH2)3Si). 13C-NMR
(75 MHz, CDCl3): see Table 5; additionally 159.2, 159.1 (2s, C(4) of 2 MeOC6H4); 145.4 (s, C(1) of Ph); 135.8,
135.4 (2s, C(1) of 2 MeOC6H4); 131.0 (d, C(2) and C(6) of 2 MeOC6H4); 128.6 (d, C(2) and C(6) of Ph); 128.0
(d, C(3) and C(5) of Ph); 127.3 (d, C(4) of Ph); 113.4, 113.3 (2d, C(3) and C(5) of 2 MeOC6H4); 91.1 (t,
OCH2O); 88.6 (s, Ar3C); 55.3( q, 2 MeO); 17.85 (q, (Me2CH)3Si); 11.9 (d, (Me2CH)3Si); 7.4 (q, (MeCH2)3Si);
4.0 (t, (MeCH2)3Si).
1-(6,7-Dideoxy-5-O-(4,4'-dimethoxytrityl)-7-C-(triethylsilyl)-3-O-{[(triisopropylsilyl)oxy]methyl}-b-d-allo-
hept-6-ynofuranosyl)uracil (10A). Colourless foam. Rf (AcOEt/hexane 1:2) 0.14. 1H-NMR (300 MHz, CDCl3):
see Table 4; additionally, 7.51 ± 7.21 (m, 9 arom. H, HÀC(6)); 6.85 ± 6.75 (m, 4 arom. H); 5.14, 4.94 (2d, J ˆ 4.8,
OCH2O); 3.792, 3.790 (2s, 2 MeO); 1.15 ± 1.08 (m, (Me2CH)3Si); 0.87 (t, J ˆ 7.5, (MeCH2)3Si); 0.45 (q, J ˆ 7.5,
(MeCH2)3Si). 13C-NMR (75 MHz, CDCl3): see Table 5; additionally, 158.9, 158.7 (2s, C(4) of 2 MeOC6H4);
145.1 (s, C(1) of Ph); 135.5, 135.0 (2s, C(1) of 2 MeOC6H4); 130.7, 130.69 (2d, C(2) and C(6) of 2 MeOC6H4);
128.3( d, C(2) and C(6) of Ph); 127.8 (d, C(3) and C(5) of Ph); 127.1 (d, C(4) of Ph); 113.2, 113.1 (2d, C(3) and
C(5) of 2 MeOC6H4); 91.2 (t, OCH2O); 88.5 (s, Ar3C); 55.24, 55.21 (2q, 2 MeO); 17.8 (q, (Me2CH)3Si); 11.9 (d,
(Me2CH)3Si); 7.4 (q, (MeCH2)3Si); 4.0 (t, (MeCH2)3Si).
1-(6,7-Dideoxy-5-O-(4,4'-dimethoxytrityl)-7-C-(triethylsilyl)-2-O-{[(triisopropylsilyl)oxy]methyl}-b-d-allo-
hept-6-ynofuranosyl)uracil 3-[(2-Cyanoethyl) (Diisopropyl)phosphoramidite] (11A). A soln. of 9A (165 mg,
0.184 mmol) and (i-Pr)2NEt (0.08 ml, 0.47 mmol) in CH2Cl2 (1.2 ml) was treated dropwise with 2-cyanoethyl
(diisopropyl)phosphoramidochloridite (0.051 ml, 0.23mmol) and stirred at 23 8 for 24 h. Evaporation and FC
(hexane/AcOEt (1% Et3N) 3:1 ! 1:1) gave 11A (160 mg, 81%). Colourless foam. Rf (AcOEt/hexane 1:2) 0.39
and 0.34 (2 diastereoisomers). 1H-NMR (500 MHz, CDCl3; 3:2 mixture of diastereoisomers): see Table 4;
additionally, 7.50 ± 7.20 (m, 9 arom. H); 6.83± 6.79 ( m, 4 arom. H); 5.00 (d, J ˆ 5.1, 0.6 H), 4.96 (d, J ˆ 5.1, 0.4 H),
4.94 (d, J ˆ 5.2, 0.6 H), 4.85 (d, J ˆ 5.2, 0.4 H) (OCH2O); 3.93 ± 3.80 (m, NCCH2CH2O); 3.792, 3.790, 3.785,
3.782 (4s, 2 MeO); 3.69 ± 3.62 (m, NCCH2CH2O); 3.59 ± 3.50 (m, (Me2CH)2N); 2.67 ± 2.61 (m, NCCH); 2.35 (t,
J ˆ 6.7, NCCH); 1.20 ± 1.00 (m, (Me2CH)2N, (Me2CH)3Si); 0.88 (t, J ˆ 8.1, (MeCH2)3Si); 0.46 (q, J ˆ 8.1,
(MeCH2)3Si). 13C-NMR (75 MHz, CDCl3; 3:2 mixture of diastereoisomers): see Table 5; additionally, 158.75,
158.72 (2s, C(4) of 2 MeOC6H4); 145.14, 145.11 (2s, C(1) of Ph); 135.8, 135.7, 135.5, 135.3 (4s, C(1) of
2 MeOC6H4); 130.67 (2 C), 130.66, 130.62 (3d, C(2) and C(6) of 2 MeOC6H4); 128.45, 128.36 (2d, C(2) and C(6)
of Ph); 127.75, 127.71 (2d, C(3) and C(5) of Ph); 126.95 (d, C(4) of Ph); 117.71, 117.20 (2s, CN); 113.10 (2 C),
5
5
113.03, 112.99 (3d, C(3) and C(5) of 2 MeOC6H4); 89.48 (dt, J(C,P) ˆ 4.5, OCH2O); 89.04 (dt, J(C,P) ˆ 2.8,
OCH2O); 88.14, 88.05 (2s, Ar3C); 59.0, 57.9 (2dt, 2J(C,P) ˆ 17, NCCH2CH2O); 55.21, 55.20 (2q, 2 MeO); 43.40,
43.10 (2dd, 2J(C,P) ˆ 13, (Me2CH)2N); 24.61, 24.55 (2 C), 24.48 (3q, (Me2CH)2N); 20.40, 19.97 (2dt, 3J(C,P) ˆ
5.7, NCCH2CH2O); 17.80, 17.79, 17.74, 17.73(4 q, (Me2CH)3Si); 11.93, 11.89 (2d, (Me2CH)3Si); 7.41, 7.37 (2q,

3016
Helvetica Chimica Acta ± Vol. 87 (2004)
(MeCH2)3Si); 4.02, 3.96 (2t, (MeCH2)3Si). 31P-NMR (121 MHz, CDCl3; 3:2 mixture of diastereoisomers): 151.3
‡
‡
(0.4 P); 150.0 (0.6 P). FAB-MS (NOBA): 1071.6 (11, [M ‡ H] ), 303.1 (100, DMTr ).
1-[6,7-Dideoxy-2,3-O-isopropylidene-5-O-(2-nitrobenzoyl)-7-C-(triethylsilyl)-a-l-talo-hept-6-ynofuranosyl]-
uracil (2T). A soln. of pyridine (3.5 ml, 43.5 mmol), DMAP (0.30 g, 2.4 mmol), and 1T [4] (5.9 g, 14.0 mmol) in
CH2Cl2 (65 ml) was treated dropwise with 2-nitrobenzoyl chloride (2.12 ml, 16.1 mmol), stirred at 238 for 60 h,
diluted with CH2Cl2 (30 ml), washed with sat. aq. NH4Cl soln., dried (Na2SO4), and evaporated. FC (AcOEt/
hexane 2 :3 ! 1:1) gave 2T (7.42 g 93%). Colourless foam. Rf (AcOEt/hexane 1:1) 0.32. [a]2D5 ˆ ‡26.6 (c ˆ
0.49, CHCl3). UV (MeOH): 257 (11800). IR (CHCl3): 3375w, 2957w, 2850w, 2160w, 1750m, 1695s, 1630w, 1537m,
1
1457w, 1385w, 1351m, 1272m, 1150w, 1115w, 1092m, 1067m, 1005w, 961w, 859w. H-NMR (300 MHz, CDCl3):
see Table 6; additionally, 8.44 (br. s, NH); 7.96 ± 7.91 (m, 1 arom. H); 7.70 ± 7.61 (m, 3arom. H); 1.57, 1.36 (2 s,
Me2C); 1.01 (t, J ˆ 7.8, (MeCH2)3Si); 0.65 (q, J ˆ 7.8, (MeCH2)3Si). 13C-NMR (75 MHz, CDCl3): see Table 7;
additionally, 164.0 (s, OCˆO); 148.1 (s, C(2) of 2-NO2Bz); 133.4 (d, C(5) of 2-NO2Bz); 132.4 (d, C(4) of 2-
NO2Bz); 129.9 (d, C(6) of 2-NO2Bz); 127.2 (s, C(1) of 2-NO2Bz); 124.3( d, C(3) of 2-NO2Bz); 114.4 (s, Me2C);
‡
26.9, 25.0 (2q, Me2C); 7.3( q, (MeCH2)3Si); 4.1 (t, (MeCH2)3Si). HR-MALDI-MS (DHB): 594.188 ([M ‡ Na] ,
‡
C27H33N3NaO9Si ; calc. 594.189). Anal. calc. for C27H33N3O9Si (571.66): C 56.73, H 5.82, N 7.35; found: C 56.90,
H 5.88, N 7.31.
Table 6. Selected 1H-NMR Chemical Shifts [ppm] and Coupling Constants [Hz] of the l-talo-Hept-6-
ynofuranosyluridines 2T± 4T and 7T± 11T in CDCl3 Solution
2T
3T
4T
7T
8T
9T
10T
11Ta)
HÀC(5)
HÀC(6)
HÀC(1')
HÀC(2')
HÀC(3')
HÀC(4')
HÀC(5')
J(5,6)
J(1',2')
J(2',3')
J(3',4')
J(4',5')
5.64
7.47
5.72
4.94
4.98
4.48
5.89
8.0
2.2
6.6
2.2
6.5
5.65
7.63
5.84
4.36
4.36
4.26
5.95
8.2
5.94
)
5.95
)
6.47
5.74
5.86
4.49
5.65
7.66
5.75
4.29
4.48
3.64
4.24
8.1
6.2
5.7
2.2
4.7
5.64
7.737.55
6.09
4.10
4.64d)
3.64
4.29
8.4
7.8
5.4
1.8
4.4
5.66
5.61/5.60
b
b
7.66/7.62
6.44
5.74
5.97
4.69
6.034.83
8.1
7.2
6.1
2.5
5.0
5.88
6.08/6.12
4.33/4.38
4.68/4.61
3.94/3.91
4.28/4.22
8.2/8.2
4.14c)
4.57
3.43
4.31
8.1
8.1
6.2
.0 1.32.7/3
4.3
e
)
8.1
7.5
5.9
1.9
3.0
4.1
8.2/8.2
5.3/5.1
f
)
5.6
5.6
4.3/3.9
a) 3: 2 Mixture of diastereoisomers. b) Signal hidden by the signals of the aromatic H-atoms at 7.0 ± 8.0 ppm.
c) d(HOÀC(2')) ˆ 3.56 ppm,
J(2',OH) ˆ 7.8 Hz.
d) d(HOÀC(3')) ˆ 2.96 ppm,
J(3',OH) ˆ 1.6 Hz.
e) d(HOÀC(5')) ˆ 3.15 ppm, J(5',OH) ˆ 3.8 Hz. f) Not assigned.
Table 7. Selected 13C-NMR Chemical Shifts [ppm] of the l-talo-Hept-6-ynofuranosyluridines 2T± 4T and 7T±
11T in CDCl3 Solution
2T
3T
4T
7T
8T
9T
10T
11Ta)
C(2)
C(4)
C(5)
C(6)
C(1')
C(2')
C(3')
C(4')
C(5')
C(6')
C(7')
163.9
150.5
102.3
142.3140.4
95.5
84.7
81.8
87.9
66.4
164.3
151.8
102.8
162.1
149.8
103.7
162.3
150.0
103.5
163.8
151.8
102.7
163.2
150.6
103.2
163.3
151.0
103.2
140.1
/84.684.3
73.8
71.7/70.4
84.0
64.8
105.4
90.5
162.7
150.2/150.3
102.7/102.8
148.2
140.3140.5
140.2
140.1
89.2
75.1
70.7
84.6
65.4
99.4
92.6
87.0
73.7
71.372.4
82.5
65.2
87.0
73.9
70.9
85.6
63.1
88.385.2
75.3
70.4
86.1
64.7
105.2
90.4
86.8
82.9
77.8
77.2
84.9
64.6
106.3
91.2
85.6/86.2
65.1/64.8
105.5/105.0
90.4/90.9
99.1
92.6
98.9
93.8
103.8
90.4
a) 3: 2 Mixture of diastereoisomers.

Helvetica Chimica Acta ± Vol. 87 (2004)
3017
1-[6,7-Dideoxy-5-O-(2-nitrobenzoyl)-7-C-(triethylsilyl)-a-l-talo-hept-6-ynofuranosyl]uracil (3T). A sus-
pension of 2T (7.29 g, 12.75 mmol) in H2O (100 ml) was treated with CF3COOH (100 ml), stirred at 808 for
12 h, diluted with H2O (200 ml), and extracted with CH2Cl2. The combined org. layers were washed with brine,
dried (Na2SO4), and evaporated to give crude 3T (7.0 g, quant.). Red foam. Rf (AcOEt/MeOH 20 :1) 0.55.
[a]2D5 ˆ À43.9 (c ˆ 1.2, CHCl3). UV (CHCl3): 259 (11500). IR (CHCl3): 3391w, 2958w, 2876w, 2160w, 1746m,
1694s, 1537m, 1458w, 1395w, 1351m, 1280m, 1113w, 1069m, 1005w, 963w, 867w, 840w. 1H-NMR (300 MHz,
CDCl3): see Table 6; additionally, 9.73(br. s, NH); 7.95 ± 7.91 (m, 1 arom. H); 7.73± 7.60 ( m, 3arom. H); 3.24
(br. s, 2 OH); 0.99 (t, J ˆ 8.0, (MeCH2)3Si); 0.64 (q, J ˆ 8.0, (MeCH2)3Si). 13C-NMR (75 MHz, CDCl3): see
Table 7; additionally, 164.2 (s, OCˆO); 148.2 (s, C(2) of 2-NO2Bz); 133.6 (d, C(5) of 2-NO2Bz); 132.7 (d, C(4)
of 2-NO2Bz); 130.3 (d, C(6) of 2-NO2Bz); 126.9 (s, C(1) of 2-NO2Bz); 124.5 (d, C(3) of 2-NO2Bz); 7.5 (q,
‡
‡
(MeCH2)3Si); 4.1 (t, (MeCH2)3Si). HR-MALDI-MS (DHB): 554.156 ([M ‡ Na] , C24H29N3NaO9Si ; calc.
554.157). Anal. calc. for C24H29N3O9Si (531.59): C 54.23, H 5.50, N 7.90; found: C 54.22, H 5.50, N 7.70.
Benzoylation of 3T. A soln. of pyridine (75 ml), 4-pyrrolidinopyridine (1.90 g, 12.8 mmol), and 3T (6.8 g,
12.8 mmol) in CH2Cl2 (75 ml) was treated dropwise with BzCl (7.4 ml, 64.0 mmol), stirred at 238 for 36 h,
diluted with CH2Cl2, washed with brine, dried (Na2SO4), and evaporated. FC (AcOEt/hexane 1:4 ! 1:2) gave
4T (8.2 g, 78%) and 5T (1.28 g, 13%).
3-Benzoyl-1-[2,3-di-O-benzoyl-6,7-dideoxy-5-O-(2-nitrobenzoyl)-7-C-(triethylsilyl)-a-l-talo-hept-6-yno-
furanosyl]uracil (4T). Colourless foam. Rf (AcOEt/hexane 1:1) 0.66. [a]2D5 ˆ À64.2 (c ˆ 1.1, CHCl3). UV
(MeOH): 254 (19200). IR (CHCl3): 3034w, 2957w, 2876w, 2150w, 1752s, 1712m, 1678s, 1601w, 1584w, 1538m,
1451m, 1376m, 1351m, 1316m, 1268s, 1121m, 1091m, 1070m, 1024w, 973w. 1H-NMR (300 MHz, CDCl3): see
Table 6; additionally, 8.13± 7.23 ( m, 19 arom. H, HÀC(6)); 1.02 (t, J ˆ 7.8, (MeCH2)3Si); 0.68 (q, J ˆ 7.8,
(MeCH2)3Si). 13C-NMR (75 MHz, CDCl3): see Table 7; additionally, 168.6 (s, NCˆO); 165.7, 165.3(2 s, 2
OCˆO); 164.2 (s, OCˆO of 2-NO2BzO); 148.2 (s, C(2) of 2-NO2Bz); 139.6, 137.1 (2d, C(4) of 2 BzO); 135.4
(d, C(4) of BzN); 133.7 (d, C(4) of 2-NO2BzO); 132.7 (d, C(5) of 2-NO2BzO); 131.6 (s, C(1) of BzN); 130.9 (d,
C(2) and C(6) of BzN); 130.3 (d, C(6) of 2-NO2BzO); 130.2 (d, C(2) and C(6) of 2 BzO); 129.5 (d, C(3and
C(5) of BzN); 129.1 (s, C(1) of BzO); 128.9, 128.7 (2d, C(3) and C(5) of 2 BzO); 128.5 (s, C(1) of BzO); 126.8 (s,
C(1) of 2-NO2BzO); 124.5 (d, C(3) of 2-NO2BzO); 7.3( q, (MeCH2)3Si); 4.1 (t, (MeCH2)3Si). HR-MALDI-MS
‡
‡
(DHB): 866.235 ([M ‡ Na] , C45H41N3NaO12Si ; calc. 866.236). Anal. calc. for C45H41N3O12Si (843.92): C 64.05,
H 4.90, N 4.98; found: C 64.10, H 5.04, N 4.92.
1-[2,3-Di-O-benzoyl-6,7-dideoxy-5-O-(2-nitrobenzoyl)-7-C-(triethylsilyl)-a-l-talo-hept-6-ynofuranosyl]ur-
acil (5T). Rf (AcOEt/hexane 1:1) 0.36.
3-Benzoyl-1-[2,3-O-dibenzoyl-6,7-dideoxy-7-C-(triethylsilyl)-a-l-talo-hept-6-ynofuranosyl]uracil (7T). A
soln. of 4T (4.1 g, 4.86 mmol) in H2O (150 ml) and MeCN (150 ml) was treated at 238 with three batches of
NH4Cl (0.78 g, 14.6 mmol) and Zn powder (0.48 g, 7.3mmol) in intervals of 90 min, stirred for 6 h, and filtered
through silica gel (elution with AcOEt). The filtrate was washed with sat. aq. NaHCO3 soln. and brine, dried
(Na2SO4), and evaporated. FC (toluene/MeOH 100 :1 ! 10 :1) gave 7T (3.3 g, quant.). Colourless foam. Rf
(AcOEt/hexane 1:2) 0.38. [a]2D5 ˆ À45.8 (c ˆ 0.78, CHCl3). UV (MeOH): 254 (13400). IR (CHCl3): 3035w,
2958w, 2876w, 2150w, 1752m, 1730s, 1712m, 1676s, 1601w, 1452m, 1378m, 1316m, 1266s, 1126m, 1094m, 1071m,
1024w, 974w, 921w. 1H-NMR (300 MHz, CDCl3): see Table 6; additionally, 8.64 (br. s, NH); 8.03± 7.12 ( m,
15 arom. H, HÀC(6)); 1.01 (t, J ˆ 7.9, (MeCH2)3Si); 0.65 (q, J ˆ 7.9, (MeCH2)3Si). 13C-NMR (75 MHz, CDCl3):
see Table 7; additionally, 168.7 (s, NCˆO); 166.1, 165.8 (2s, 2 OCˆO); 135.4 (d, C(4) of BzN); 134.3, 134.1 (2d,
C(4) of 2 BzO); 131.5 (s, C(1) of BzN); 130.8 (d, C(2) and C(6) of BzN); 130.20, 130.15 (2d, C(2) and C(6) of
2 BzO); 129.4 (d, C(3) and C(5) of BzN); 129.0 (s, C(1) of BzO); 128.9, 128.7 (2d, C(3) and C(5) of 2 BzO);
‡
128.6 (s, C(1) of BzO); 7.5 (q, (MeCH2)3Si); 4.1 (t, (MeCH2)3Si). HR-MALDI-MS (DHB): 717.223([ M ‡ Na] ,
‡
C38H38N2NaO9Si ; calc. 717.224).
1-[6,7-Dideoxy-5-O-(4,4'-dimethoxytrityl)-7-C-(triethylsilyl)-a-l-talo-hept-6-ynofuranosyl]uracil (8T). A
soln. of DMTCl (6.0 g, 17.7 mmol) in CH2Cl2 (70 ml) was treated with sym-collidine (14.2 ml), stirred at 238
for 15 min, treated with AgNO3 (3.0 g, 17.7 mmol), and stirred for 1 h. An aliquot of this suspension (69.1 ml,
14.4 mmol of the reagent) was filtered into a soln. of 7T (4.0 g, 5.8 mmol) in CH2Cl2 (40 ml). The mixture was
stirred at 238 for 75 min and filtered through silica gel (elution with AcOEt/Et3N 99 :1). After evaporation, a
soln. of the residue in THF/MeOH/H2O 5 :4 :1 (400 ml) was cooled to 08, treated dropwise with 10m aq. NaOH
soln. (5.8 ml, 58.0 mmol), stirred at 08 for 2 h, treated with sat. aq. NH4Cl soln., and extracted with AcOEt/Et3N
99 :1. The org. phase was dried (Na2SO4), filtered, and evaporated. FC (hexane/AcOEt/Et3N 1:1 :0.01 !
1:2 :0.01) gave 8T (2.3g, 57%). Colourless foam. Rf (AcOEt/hexane 1 :1) 0.12. [a]2D5 ˆ ‡26.0 (c ˆ 0.8, CHCl3).
UV (MeOH): 260 (7380). IR (CHCl3): 3390w, 3006w, 2958m, 2875w, 2838w, 2125w, 1693s, 1608m, 1510m,
1463m, 1395w, 1280m, 1253s, 1116w, 1055w, 1037m, 965w, 890w, 829m. 1H-NMR (300 MHz, CDCl3): see Table 6;

3018
Helvetica Chimica Acta ± Vol. 87 (2004)
additionally, 7.51 ± 7.23( m, 9 arom. H); 6.84 ± 6.80 (m, 4 arom. H); 3.80 (s, 2 MeO); 2.60 (s, 2 OH); 0.93( t, J ˆ
7.8, (MeCH2)3Si); 0.51 (q, J ˆ 7.8, (MeCH2)3Si). 13C-NMR (75 MHz, CDCl3): see Table 7; additionally, 159.1 (s,
C(4) of 2 MeOC6H4); 145.1 (s, C(1) of Ph); 136.0, 135.7 (2s, C(1) of 2 MeOC6H4); 130.9, 130.7 (2d, C(2) and
C(6) of 2 MeOC6H4); 128.6 (d, C(2) and C(6) of Ph); 128.1 (d, C(3) and C(5) of Ph); 127.4 (d, C(4) of Ph);
113.40, 113.35 (2d, C(3) and C(5) of 2 MeOC6H4); 88.6 (s, Ar3C); 55.3( q, 2 MeO); 7.4 (q, (MeCH2)3Si); 4.1 (t,
(MeCH2)3Si). Anal. calc. for C38H44N2O8Si (684.86): C 66.64, H 6.48, N 4.09; found: C 66.73, H 6.41, N 3.87.
(Silyloxy)methylation of 8T. A soln. of 7T (1.78 g, 2.6 mmol) and (i-Pr)2NEt (2.2 ml, 13.0 mmol) in
(CH2Cl)2 (12 ml) was treated with Bu2SnCl2 (0.95 g, 3.12 mmol), stirred for 90 min at 238, treated with TOMCl
(0.81 g, 3.64 mmol), heated to 808, stirred for 45 min, treated with sat. aq. NaHCO3 soln., and extracted with
CH2Cl2. The org. phase was washed with brine, dried (Na2SO4), and evaporated. FC (hexane/AcOEt/Et3N
4 :1:0.01 ! 2 :1 :0.01) gave 9T (716 mg, 32%) and 10T (235 mg, 10%).
1-(6,7-Dideoxy-5-O-(4,4'-dimethoxytrityl)-7-C-(triethylsilyl)-2-O-{[(triisopropylsilyl)oxy]methyl}-a-l-talo-
hept-6-ynofuranosyl)uracil (9T). Colourless foam. Rf (AcOEt/hexane 1:2) 0.29. [a]2D5 ˆ 58.8 (c ˆ 0.76, CHCl3).
UV (MeOH): 260 (18800). IR (CHCl3): 3392w, 3007w, 2957m, 2871m, 2140w, 1695s, 1608w, 1510m, 1463m,
1385w, 1295w, 1255m, 1111w, 1075m, 1046m, 1011m, 883w, 829w. 1H-NMR (300 MHz, CDCl3): see Table 6;
additionally, 8.02 (br. s, NH); 7.51 ± 7.21 (m, 9 arom. H); 6.85 ± 6.79 (m, 4 arom. H); 5.18, 4.86 (2d, J ˆ 4.9,
OCH2O); 3.799, 3.796 (2s, 2 MeO); 1.11 ± 1.07 (m, (Me2CH)3Si); 0.94 (t, J ˆ 7.8, (MeCH2)3Si); 0.54 (q, J ˆ 7.8,
(MeCH2)3Si). 13C-NMR (75 MHz, CDCl3): see Table 7; additionally, 159.0 (s, C(4) of 2 MeOC6H4); 145.1 (s,
C(1) of Ph); 136.0, 135.7 (2s, C(1) of 2 MeOC6H4); 130.8, 130.7 (2d, C(2) and C(6) of 2 MeOC6H4); 128.6 (d,
C(2) and C(6) of Ph); 128.0 (d, C(3) and C(5) of Ph); 127.3 (d, C(4) of Ph); 113.4, 113.3 (2d, C(3) and C(5) of
2 MeOC6H4); 90.0 (t, OCH2O); 88.4 (s, Ar3C); 55.4 (q, 2 MeO); 18.0 (q, (Me2CH)3Si); 12.1 (d, (Me2CH)3Si); 7.7
‡
‡
(q, (MeCH2)3Si); 4.3( t, (MeCH2)3Si). FAB-MS (NOBA): 870 (1, M ), 303 (100, DMTr ). Anal. calc. for
C48H66N2O9Si2 (871.22): C 66.17, H 7.64, N 3.22; found: C 65.93, H 7.60, N 3.23.
1-(6,7-Dideoxy-5-O-(4,4'-dimethoxytrityl)-7-C-(triethylsilyl)-3-O-{[(triisopropylsilyl)oxy]methyl}-a-l-talo-
hept-6-ynofuranosyl)uracil (10T). Colourless foam. Rf (AcOEt/hexane 1 :2) 0.14. 1H-NMR (300 MHz, CDCl3):
see Table 6; additionally, 7.51 ± 7.23( m, 9 arom. H); 6.86 ± 6.78 (m, 4 arom. H); 5.09, 4.95 (2d, J ˆ 4.7, OCH2O);
3.798, 3.796 (2s, 2 MeO); 1.14 ± 1.06 (m, (Me2CH)3Si); 0.97 (t, J ˆ 7.8, (MeCH2)3Si); 0.57 (q, J ˆ 7.8,
(MeCH2)3Si). 13C-NMR (75 MHz, CDCl3): see Table 7; additionally, 159.3( s, C(4) of 2 MeOC6H4); 145.1 (s,
C(1) of Ph); 136.0, 135.7 (2s, C(1) von 2 MeOC6H4); 130.8, 130.6 (2d, C(2) and C(6) of 2 MeOC6H4); 128.5 (d,
C(2) and C(6) of Ph); 128.2 (d, C(3) and C(5) of Ph); 127.5 (d, C(4) of Ph); 113.5 (d, C(3) and C(5) of
2 MeOC6H4); 89.7 (t, OCH2O); 88.8 (s, Ar3C); 55.4 (q, 2 MeO); 17.9 (q, (Me2CH)3Si); 12.0 (d, (Me2CH)3Si); 7.5
(q, (MeCH2)3Si); 4.2 (t, (MeCH2)3Si).
1-(6,7-Dideoxy-5-O-(4,4'-dimethoxytrityl)-7-C-(triethylsilyl)-2-O-{[(triisopropylsilyl)oxy]methyl}-a-l-talo-
hept-6-ynofuranosyl)uracil 3-[(2-Cyanoethyl) (Diisopropyl)phosphoramidite] (11T). A soln. of 9T (400 mg,
0.46 mmol) and (i-Pr)2NEt (0.24 ml, 1.38 mmol) in CH2Cl2 (2.5 ml) was treated dropwise with 2-cyanoethyl
(diisopropyl)phosphoramidochloridite (0.14 ml, 0.64 mmol) and stirred at 238 for 3h. Evaporation and FC
(hexane/AcOEt/Et3N 4 :1 :0.01 ! 1:1:0.01) gave 11T (374 mg, 76%). Rf (AcOEt/hexane 1:2) 0.48, 0.44 (2
diastereoisomers). [a]2D5 ˆ 5.9 (c ˆ 0.86, CHCl3). UV (MeOH): 259 (12300). IR (CHCl3): 3400w, 2960m, 2870m,
2250w, 1724m, 1695s, 1608w, 1510m, 1463m, 1382w, 1365w, 1301w, 1120w, 1041m, 981w, 829w. 1H-NMR
(500 MHz, CDCl3; 3:2 mixture of diastereoisomers): see Table 6; additionally, 7.50 ± 7.20 (m, 9 arom. H); 6.84 ±
6.79 (m, 4 arom. H); 5.00 (d, J ˆ 5.4, 0.4 H), 4.97 (d, J ˆ 5.5, 0.6 H), 4.85 (d, J ˆ 5.4, 0.4 H), 4.82 (d, J ˆ 5.4,
0.6 H) (OCH2O); 3.792, 3.790, 3.785, 3.782 (4s, 2 MeO); 3.67 ± 3.55 (m, NCCH2CH2O, (Me2CH)2N); 2.65 ± 2.52
(m, NCCH); 2.35 (m, NCCH); 1.21 ± 1.17 (m, (Me2CH)2N, (Me2CH)3Si); 1.04 ± 0.91 (m, (Me2CH)3Si,
(MeCH2)3Si); 0.58 ± 0.53( m, (MeCH2)3Si). 13C-NMR (75 MHz, CDCl3; 3:2 mixture of diastereoisomers): see
Table 7; additionally, 158.95 (2 C), 158.92, 158.86 (3s, C(4) of 2 MeOC6H4); 144.96, 144.82 (2s, C(1) of Ph);
135.66, 135.56, 135.39, 135.35 (4s, C(1) of 2 MeOC6H4); 130.85, 130.75 (2d, C(2) and C(6) of 2 MeOC6H4);
128.61 ± 127.16 (several d); 117.63, 117.47 (2s, CN); 113.15, 113.11, 113.05, 113.00 (4d, C(3) and C(5) of
2
2 MeOC6H4); 89.35, 89.30 (2t, OCH2O); 88.67 (s, Ar3C); 58.75, 57.90 (2dt, J(C,P) ˆ 17, NCCH2CH2O); 55.22,
55.20 (2q, 2 MeO); 43.52, 43.41 (2 C), 43.30 (3d, 2 (Me2CH)2N); 24.69 ± 24.50 (several q, (Me2CH)2N); 20.40
(dt, 3J(C,P) ˆ 6.3), 19.73 (dt, 3J(C,P) ˆ 7.5 ) ( NCCH2CH2O); 17.71, 17.68 (4 C), 17.67 (3q, (Me2CH)3Si); 11.90 (d,
(Me2CH)3Si); 7.40, 7.39 (2q, (MeCH2)3Si); 4.03, 3.97 (2t, (MeCH2)3Si). 31P-NMR (121 MHz, CDCl3 ;
‡
3:2 mixture of diastereoisomers): 151.3 (0.6 P), 150.0 (0.4 P). FAB-MS (NOBA): 1071 (10, [ M ‡ H] ), 303
‡
(100, DMTr ).
Oligonucleotide Synthesis. Oligonucleotides were synthesised on a Pharmacia Gene Assembler on a 1.3-
mmol scale. The commercial phoshophoramidites and the CPG solid supports were obtained from Xeragon.
Solvents and reagents were prepared essentially according to the protocol for the synthesis of p-RNA [59].

Helvetica Chimica Acta ± Vol. 87 (2004)
3019
Detritylation was accomplished within 2.5 min by 6% Cl2CHCOOH in (CH2Cl)2. Couplings (0.12 ml of 0.1m
phosphoramidite soln. in MeCN or in MeCN/THF 7:3for phosphoramidite 11A and 11T, resp., and 0.36 ml of
0.3m 1-(benzylsulfanyl)-1H-tetrazole in MeCN) were performed within 5 min (RNA-phosphoramidites coupled
on unmodified nucleosides), or within 10 min (modified phosphoramidites 11A and 11T, and RNA-
phosphoramidites coupled with modified nucleotides). Capping and oxidation was accomplished under
standard conditions (see user manual for Gene Assembler Plus).
Cleavage of Cyanoethyl and Nucleobase Protecting Groups with Concomitant Detachment from the CPG
Solid Support and Cleavage of the TOM and Et3Si Protecting Groups. After completion of the automated
synthesis, the CPG support carrying the oligonucleotide was dried in vacuo for 30 min, placed in Eppendorf
tubes (1.5 ml), and treated with 10m MeNH2 in H2O/EtOH 1 :1 (1 ml) for 2 h at 238, followed by centrifugation.
The supernatant was removed, and the CPG support was washed with H2O (3 Â 0.5 ml). The combined
washings and supernatant were evaporated with a SpeedVac evaporator. The residue was treated with 1m TBAF
in THF (1 ml; for II ± VI) or with 0.5m TBAF in THF/N-methylpyrrolidinone 1:1 (1 ml; for I) at 238 for 12 h.
The mixture was treated with 1m Tris ´ HCl (pH 7.4, 1 ml) and concentrated to 1 ml (SpeedVac evaporator). Salts
were removed by chromatography (Sephadex G-10 column, elution with sterile H2O, flow rate: 1 ml/min). The
UV absorbing fractions eluting between 12 ± 18 min were collected and evaporated.
HPLC Purification of the Oligonucleotides. Oligonucleotides were purified by ion-exchange HPLC.
Column: SAX 1000-8 (Macherey & Nagel); buffer A: 10 mm Na2HPO4 in H2O, pH 10.5; buffer B: 10 mm
Na2HPO4 in H2O, 1m NaCl, pH 10.5. Product fractions were collected in 0.1m aq. Et3NH ´ HCO3 buffer soln.
Desalting of the Oligonucleotides. The product fractions obtained by HPLC were combined, diluted with
0.1m aq. Et3NH ´ HCO3 buffer to twice the volume, and applied to a previously conditioned reverse-phase
SepPak cartridge (C18 Waters). Successive elution with 0.1m aq. Et3NH ´ HCO3 buffer soln., H2O, and MeCN/
H2O 1:1, followed by lyophillization of the MeCN/H2O fractions containing the products afforded the salt-free
oligonucleotides.
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Received September 23, 2004