2′-Spiro ribo- and arabinonucleosides: Synthesis, molecular modelling and incorporation into oligodeoxynucleotides

Article abstract of DOI:10.1039/b306354b

We have synthesized four conformationally restricted bicyclic 2′-spiro nucleosides via 2′-C-allyl nucleosides as key intermediates. The ribo-configured 2′-spironucleosides 9b and 14b were obtained by a convergent strategy starting from 2-ketofuranose 1 whereas the arabino-configured 2′-spironucleosides 21 and 27 were obtained by a linear strategy with a 2′-ketouridine derivative as starting material. The furanose ring of 9b/14b adopts N-type conformations whereas the furanose ring of 21/27 exists as an N?S equilibrium. These compounds snowed no anti-HIV-1 activity or cytotoxicity. Incorporation of the four 2′-spironucleosides (as monomers X4 and X5) into oligodeoxynucleotides was accomplished using the phosphoramidite approach on an automated DNA synthesizer. Irrespective of monomeric configuration, hybridization studies revealed that these 2′-spironucleotide monomers (X4 and X5) induce decreased duplex thermostabilities compared with the corresponding DNA:DNA and DNA:RNA duplexes. Molecular modelling indicated that steric constraints are a possible reason for the lowered binding affinities of the modified oligodeoxynucleotides towards complementary single-stranded DNA and single-stranded RNA complements.

Full text of DOI:10.1039/b306354b

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2Ј-Spiro ribo- and arabinonucleosides: synthesis, molecular
modelling and incorporation into oligodeoxynucleotides†
B. Ravindra Babu,a Lise Keinicke,b Michael Petersen,a Claus Nielsenc and Jesper Wengel*a
a Nucleic Acid Center, Department of Chemistry, University of Southern Denmark,
DK-5230 Odense M, Denmark. E-mail: jwe@chem.sdu.dk; Fax: ϩ45 66158780;
Tel: ϩ45 65502510
b Department of Chemistry, University of Copenhagen, Universitetsparken 5, Denmark,
DK-2100 Copenhagen, Denmark
c Retrovirus Laboratory, Department of Virology, Statens Serum Institut,
DK-2300 Copenhagen, Denmark
Received 6th June 2003, Accepted 13th August 2003
First published as an Advance Article on the web 12th September 2003
We have synthesized four conformationally restricted bicyclic 2Ј-spiro nucleosides via 2Ј-C-allyl nucleosides as key
intermediates. The ribo-configured 2Ј-spironucleosides 9b and 14b were obtained by a convergent strategy starting
from 2-ketofuranose 1 whereas the arabino-configured 2Ј-spironucleosides 21 and 27 were obtained by a linear
strategy with a 2Ј-ketouridine derivative as starting material. The furanose ring of 9b/14b adopts N-type
conformations whereas the furanose ring of 21/27 exists as an N S equilibrium. These compounds showed no
anti-HIV-1 activity or cytotoxicity. Incorporation of the four 2Ј-spironucleosides (as monomers X4 and X5) into
oligodeoxynucleotides was accomplished using the phosphoramidite approach on an automated DNA synthesizer.
Irrespective of monomeric configuration, hybridization studies revealed that these 2Ј-spironucleotide monomers
(X4 and X5) induce decreased duplex thermostabilities compared with the corresponding DNA:DNA and
DNA:RNA duplexes. Molecular modelling indicated that steric constraints are a possible reason for the lowered
binding affinities of the modified oligodeoxynucleotides towards complementary single-stranded DNA and
single-stranded RNA complements.
X5-Ribo and X4-Ribo and S-type (C2Ј-endo) for the arabino-
configured nucleosides 21 and 27 and nucleotide monomers
X5-Ara and X4-Ara (Fig. 1).
Introduction
Conformational restriction of the furanose ring of nucleosides,
nucleotides and oligonucleotides has been intensively pursued
in recent years, stimulated by the potential use of these mole-
cules as therapeutic agents.1–4 Especially revealing with respect
to RNA-targeting has been the design and synthesis of oligo-
nucleotide analogues containing nucleotide monomers with
bicyclic furanose moieties.4–9 Alternatively, conformational
restriction of the furanose ring of nucleosides has been
achieved by the introduction of alkyl groups at the 2Ј-C- or 2Ј-
O-positions of the furanose ring leading to nucleosides10–12 and
oligonucleotides13–15 of therapeutic interest. An as yet relatively
unexplored strategy for conformational restriction is the
synthesis of spironucleosides. A 2Ј-spirocyclopropane derivative
of 2Ј-deoxyadenosine has been synthesized as a mechanistic
probe for ribonucleotide reductases,16 and 1Ј-spiro-17, 3-spiro-18
and 4Ј-spiro19 ribonucleoside derivatives have been prepared as
conformationally restricted derivatives. Herein we introduce
2Ј-spironucleoside derivatives (2Ј-C,2Ј-O-propano- and 2Ј-C,2Ј-
O-ethano nucleosides) as novel conformationally restricted
probes. The ribo-configured nucleosides 9b and 14b and the
arabino-configured nucleosides 21 and 27 were prepared
together with the corresponding phosphoramidite derivatives
to be used for incorporation of monomers X5-Ribo, X4-Ribo,
X5-Ara and X4-Ara into oligodeoxynucleotides. As a C-alkyl
group prefers a pseudoequatorial orientation and an O-alkyl
group an axial orientation, we anticipated preferred furanose
ring comformations to be N-type (C3Ј-endo) for the ribo-
configured nucleosides 9b and 14b and nucleotide monomers
Results and discussion
Synthesis
Recent reports20,21 describing the synthesis of 2Ј-C-alkyl ribo-
nucleosides inspired us to choose a short, flexible and conver-
gent approach towards the 2Ј-spiro ribonucleosides 9b and 14b
(Scheme 1). The first step, addition of an allyl group to the
β-face of the known 2-keto-tri-O-benzoyl-α--ribofuranose 122
required the use of a chemoselective organometallic reagent
able to add to ketone functionalities in the presence of esters.
Organocerium compounds are mild reagents that are known to
provide such selectivity.23 We preferred the allyl group because
it has the advantage of being a “hidden protecting group” as
the double bond can be oxidatively cleaved or hydrated regio-
selectively and thus easily converted into a 2-hydroxyethyl or a
3-hydroxypropyl group. The reaction of allylMgBr–CeCl3 with
the 2-ketofuranose 1 resulted in chemo- and stereoselective
addition of the allyl group. Due to in situ transesterification and
anomerization, an anomeric mixture of tri-O-benzoyl-2Ј-C-
allyl ribofuranoses was obtained that was benzoylated to afford
the anomeric mixture of tetra-O-benzoyl ribofuranose 2. At
this stage, the configuration at C2 was tentatively assigned as
shown in 2 based on the fact that the attack by the allyl group is
more likely to occur from the less hindered β-face of the furan-
ose ring. This hypothesis was later confirmed (see below).
Furanose 2 was stereoselectively coupled with persilylated
thymine using SnCl4 as Lewis acid which also resulted in partial
O2Ј-debenzoylation. Subsequent deprotection of this mixture
with saturated methanolic ammonia provided the β-configured
2Ј-C-allyl ribonucleoside 3 in 29% overall yield from ketofuran-
ose 1. Selective O5Ј-dimethoxytritylation followed by O3Ј-silyl-
† Electronic supplementary information (ESI) available: Copies of 13C
NMR spectra of compounds 2–4, 5a, 5b, 6a, 6b, 7a, 8a, 8b, 9a, 9b, 11a,
11b, 12a, 12b, 13a, 13b, 14a, 14b, 16, 19, 22 and 24, and 31P NMR
spectra of compounds 10, 15, 23 and 29. See http://www.rsc.org/supp-
data/ob/b3/b306354b/
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 5 1 4 – 3 5 2 6
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
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Fig. 1 Structures of the 2Ј-spironucleosides synthesized (9b, 14b, 21,
27) and of the 2Ј-spironucleotide monomers incorporated into
oligodeoxynucleotides (X5 and X4). Also shown is the two-state
Scheme 1 Reagents, conditions (and yields): i) a) allylMgBr, CeCl3,
THF, Ϫ78 ЊC, b) BzCl, DMAP, Et3N, CH2Cl2 (53%); ii) a) thymine,
SnCl4, BSA, CH3CN, b) saturated methanolic NH3 (54%); iii) DMTCl,
pyridine (86%); iv) TBDMSCl, imidazole, DMF (5a: 84%; 5b: 84%); v)
BH3 (7.8 M in 1,4-oxathiane), THF, aq. NaOH (2 M), H2O2 (6a: 55%;
6b: 39%); vi) MsCl, pyridine, CH2Cl2 (58%); vii) NaH, THF (8a: 87%;
13a: 93%; 13b: 77%); viii) a) MsCl, DMAP, Et3N, CH2Cl2, b) NaH,
THF (74%); ix) TBAF, THF (9a: 88%; 9b: 78%; 14a: 92%; 14b: 79%); x)
NC(CH2)2OP(Cl)N(i-Pr)2, EtN(i-Pr)2, CH2Cl2 (10: 76%; 15: 80%); xi) a)
OsO4 (2.5 M in n-butanol), NaIO4, THF, H2O, b) NaBH4, THF, H2O
(11a: 42%; 11b: 47%). xii) MsCl, DMAP, Et3N, CH2Cl2 (12a: 83%; 12b:
64%). DMT = 4,4Ј-dimethoxytrityl. T = thymin-1-yl.
conformational equilibrium of
a
2Ј-deoxynucleoside and the
anticipated N-type and S-type furanose conformations of the ribo- and
arabino-configured 2Ј-spiro derivatives, respectively. For a detailed
description of furanose conformations and the pseudorotational cycle
the reader is referred to, e.g., ref. 1.
ation afforded the key intermediate 5a. For the synthesis of the
2Ј-spiro ribonucleosides containing an additional O2Ј–C2Ј-
linked five-membered ring, hydroboration of 5a followed by
alkaline hydrogen peroxide oxidation gave the anti-Marko-
vnikov product 6a exclusively in 55% yield. Attempts to cyclize
this compound using Mitsunobu conditions failed. However,
selective mesylation and subsequent treatment with sodium
hydride induced cyclization to afford the 2Ј-spiro ribonucleo-
side 8a. In order to obtain oligonucleotides (ONs) containing
the nucleotide monomer X5-Ribo, O3Ј-desilylation to give 9a
followed by phosphitylation afforded the phosphoramidite
building block 10 in 67% yield (Scheme 1).
give 8b) and, finally, desilylation afforded compound 9b in 19%
yield (from 3). Similarly, oxidative cleavage of the terminal
double bond of compound 5b, followed by reduction, mesyl-
ation, intramolecular cyclization and desilylation afforded
compound 14b in 15% yield (from 3). In addition, nucleoside
6b was desilylated to provide 1-[2-C-(3-hydroxypropyl)-β--
ribofuranosyl]thymine (16) in order to evaluate the effect of a
2Ј-C-hydroxypropyl group on biological activities.
For the synthesis of 2Ј-spiro ribonucleosides with an
additional O2Ј–C2Ј-linked four-membered ring, the terminal
double bond of compound 5a was oxidatively cleaved using a
catalytic amount of osmium tetraoxide and sodium periodate
as a co-oxidant and then reduced by treatment with sodium
borohydride resulting in the formation of the 2Ј-C-(2-hydroxy)-
ethyl ribonucleoside 11a. Selective mesylation followed by
base-induced ring closure afforded in 77% yield the 2Ј-spiro
ribonucleoside 13a. Desilylation of compound 13a followed by
3Ј-O-phosphitylation furnished the phosphoramidite building
block 15 in 74% yield (Scheme 1).
Efforts to obtain the free dihydroxy nucleosides 9b and 14b
for biological evaluation by detritylation of compounds 9a and
14a using either 10% dichloroacetic acid in dichloromethane or
90% acetic acid solution, in the presence of a DMT cation
scavenger (triethylsilane), proved futile as analysis of the reac-
tion mixture by analytical TLC showed the formation of several
inseparable products. Therefore, in order to obtain 9b and 14b,
compound 3 was O3Ј,O5Ј-disilylated to give nucleoside 5b.
Subsequent anti-Markovnikov hydration of the terminal double
bond (to give 6b), cyclization via the mesyl intermediate 7b (to
The synthesis of the epimeric 2Ј-spiro arabinonucleosides 21
and 27 was accomplished using a linear strategy (Scheme 2). In
general, addition reactions of alkyl groups to 2Ј-keto nucleo-
sides using organometallic reagents usually proceed from the
sterically less hindered α-face of the sugar ring.24,25 Accordingly,
CeIII-assisted Grignard addition of the allyl group to 3Ј,5Ј-
di-O-(tetraisopropylidinedisiloxane-1,3-diyl)-2Ј-ketouridine26
occurred stereoselectively from the α-face of the furanose ring
affording, after isolation and purification, diastereomerically
pure key intermediate 17. For the synthesis of 2Ј-spiro arabino-
nucleosides containing an additional O2Ј-C2Ј-linked five-
membered ring, regioselective hydroboration of 17 was carried
out using five equivalents of 9-BBN followed by oxidation with
alkaline hydrogen peroxide (15 equivalents). Mesylation of
the resulting 3Ј-C-(3-hydroxypropyl) arabinonucleoside 18
occurred selectively at the primary hydroxy group, and
subsequent base-induced cyclization proceeded in 56% overall
yield to give nucleoside 20 that was desilylated using two
equivalents of Et3Nؒ3HF to give in 91% yield the 2Ј-spiro
arabinonucleoside 21 ready for biological evaluation. O5Ј-
Dimethoxytritylation (to give 22) followed by O3Ј-phosphityl-
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 5 1 4 – 3 5 2 6
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discussed above; 8a (4.7%/5.9%); 13a (5.3%/5.7%)) strongly
support that both the monocyclic and the spirocyclic ribo-
nucleosides adopt the expected N-type (C3Ј-endo) furanose
conformation (see below).
Nucleoside conformations
Pseudorotation energy profiles were calculated by ab initio
calculations at the MP2/cc-pVTZ level of theory for the
X4-Ribo and X4-Ara nucleosides (compounds 14b and 27,
respectively). For the nucleosides there are several possible
conformations of the β, γ, and ε torsion angles.27 In the calcu-
lations, these values were kept at values close to those expected
upon incorporation into oligonucleotides and subsequent
Watson–Crick hybridization.
X4-Ribo. Two local energy minima were found in the pseudo-
rotation profile for this nucleoside (14b), these being P = 13Њ and
159Њ, with the energy difference between these conformations
depending on the value of the ε torsion angle (see Fig. 2). Using
a generalized Karplus equation,28–30 we found J3Ј4Ј = 8.5 Hz and
0.8 Hz for these N-type and S-type sugar puckers, respectively.
Experimentally J3Ј4Ј = 9.7 Hz was found which shows that the
X4-Ribo nucleoside is adopting an N-type furanose conform-
ation in solution. Likewise, a large J3Ј4Ј was found experi-
mentally for the X5-Ribo nucleoside (8.1 Hz). Although we have
not carried out high-level calculations for this nucleoside,
simple considerations show that this nucleoside adopts an
N-type furanose conformation as well. Thus both the X4-Ribo
and X5-Ribo nucleosides adopt N-type (RNA-like) furanose
conformations.
Scheme 2 Reagents, conditions (and yields): i) allylMgBr, CeCl3, THF,
Ϫ78 ЊC (75%); ii) 9-BBN, THF, aq. NaOH (2 M), H2O2 (64%); iii)
MsCl, DMAP, Et3N, CH2Cl2 (19: 74%; 25: 86%); iv) NaH, THF (20:
76%; 26: 54%); v) Et3Nؒ3HF, THF (21: 91%; 27: 82%); vi) DMTCl,
pyridine (22: 100%; 28: 89%); vii) NC(CH2)2OP(Cl)N(i-Pr)2, EtN(i-Pr)2,
CH2Cl2 (23: 61%; 29: 60%); viii) a) OsO4 (2.5 M in n-butanol), NaIO4,
THF, H2O, b) NaBH4, THF, H2O (47%). DMT = 4,4Ј-dimethoxytrityl.
U = uracil-1-yl.
ation using standard procedures afforded the phosphoramidite
building block 23 (Scheme 2).
By oxidative cleavage followed by reduction, the allyl group
of nucleoside 17 was converted into a 2-hydroxyethyl group
furnishing the arabinonucleoside 24 in moderate yield. Sub-
sequent selective mesylation, base induced intramolecular
cyclization and desilylation (via 25 and 26) afforded the 2Ј-spiro
arabinonucleoside 27 containing an O2Ј–C2Ј-linked four-
membered ring. Also this 2Ј-spironucleoside was prepared for
incorporation into ODNs by standard dimethoxytritylation
of the 5Ј-hydroxy group followed by phosphitylation of the
3Ј-hydroxy group to give the phosphoramidite building block
29 (via 28) (Scheme 2).
Nucleoside configurations
The configurations of the 2Ј-spironucleosides were determined
by NOE difference spectroscopy. Irradiation of H1Ј of nucleo-
side 20 induced a 7% enhancement of H1Љ (the protons of the
2Ј-C-methylene group) and vice versa, which suggests that these
protons are positioned at the same side of the furanose ring.
The arabino-configuration was further confirmed by the
observation of strong NOE effects of H6 (6.5%) and H3Љ(8.5%)
upon irradiation of H3Ј. Similarly in nucleoside 26, mutual
significant NOE contacts were observed between H1Ј and H1Љ.
Unfortunately, no mutual NOE contacts could be observed
between the additional ring protons and the parent nucleoside
in case of the 2Ј-spiro ribonucleoside series. The only key
enhancement observed for these compounds was of H6 when
irradiating H3Ј (supporting ribo-configuration). Since these
compounds were obtained following coupling between
persilylated thymine and O2Ј-acylated ribofuranose 2, the trans
rule invoking anchimeric assistance from an O2Ј-acyl substi-
tuent, in this case supported by the exclusive isolation of one
stereoisomer of nucleoside 3, implies β--ribo configuration
(i.e. R-configuration at C2 in compound 2). The assigned con-
figuration was furthermore confirmed by NOE experiments on
the common intermediate 5b. Significant NOE contacts were
observed between H6, H3Ј and H1Љa (4.6%/4.9% between H6
and H3Ј, 1.1%/1.8% between H6 and H1Љa, 1.8%/2.9% between
H-3Ј and H1Љa) suggesting that these protons are positioned at
the same side of the furanose ring. Furthermore, the NOE
effects observed between the H6 and the H3Ј protons (for 5b as
Fig. 2 MP2/cc-VTZ (full line) and force field (dotted line) potential
energies (kcal molϪ1) as a function of pseudorotation angle P for X4-
Ribo. All energies are given relative to the C3Ј-endo conformation. a
With the ε torsion angle constrained to Ϫ124Њ. b With the ε torsion
angle constrained to Ϫ165Њ.
X4-Ara. As for the corresponding ribo-configured com-
pound, two energy minima for pseudorotation were found for
this nucleoside (27) (P = 16Њ and 141Њ) (Fig. 2) with corre-
sponding J3Ј4Ј coupling constants of 8.5 Hz and 1.8 Hz, respect-
ively. The experimental value measured in solution was 6.1 Hz
which shows that this nucleoside is not adopting a pure N- or
S-type furanose conformation in solution but rather exists in an
equilibrium between two (or more) conformations. Assuming
an equilibrium between the two low energy conformations
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 5 1 4 – 3 5 2 6
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Table 1 Thermal denaturation experiments (Tm values shown)a
DNA complement
RNA complement
Ribo series
Sequence (5Ј-3Ј)
Ribo series
Ara series
Ara series
ON1: T7(X5)T6
ON2: T7(X4)T6
ON3: T5(X5)4T5
ON4: T5(X4)4T5
ON5: T14
19
20
9
8
32
17
18
<5
<5
32
19
19
8
8
29
18
19
<5
<5
29
ON6: (X5)9T
ON7: (X4)9T
ON8: T10
<5
<5
20
<5
<5
20
<5
<5
18
<5
<5
18
ON 9: d[G(X5)GA(X5)A(X5)GC]
ON10: d[G(X4)GA(X4)A(X4)GC]
ON11: d(GTGATATGC)
12
12
29
<5
<5
29
n.m.
n.m.
n.m.
<5
<5
29
a Duplex melting temperatures (Tm values/ЊC) measured as the maximum of the first derivative of the melting curve (A260 vs. temperature) recorded in
medium salt buffer (10 mM sodium phosphate, 100 mM sodium chloride, 0.1 mM EDTA, pH 7.0) using 1.5 µM concentrations of the two strands; A
= adenin-9-yl monomer; C = cytosin-1-yl monomer; G = guanin-9-yl monomer; T = thymin-1-yl monomer; X5 = 2Ј-C,2Ј-O-propano-linked 2Ј-spiro
ribonucleotide monomer (X5-Ribo; indicated by “Ribo series” in the headline) or X5 = 2Ј-C,2Ј-O-propano-linked 2Ј-spiro arabinonucleotide
monomer (X5-Ara; indicated by “Ara series” in the headline); X4 = 2Ј-C,2Ј-O-ethano-linked 2Ј-spiro ribonucleotide monomer (X4-Ribo; indicated by
“Ribo series” in the headline) or X4 = 2Ј-C,2Ј-O-ethano-linked 2Ј-spiro arabinonucleotide monomer (X4-Ara; indicated by “Ara series” in the
headline). “n.m.” = this experimental series not measured.
determined by calculations, the nucleoside possesses approx-
imately 65% N-type conformation as gauged from the J3Ј4Ј
coupling constant. This is in agreement with the quantum
mechanical calculations, which predict the N-type pucker to be
lower in energy than the S-type pucker, although a 65 : 35
Ara), significantly reduced thermal stabilities (duplex melting
temperatures, Tm values) were obtained compared with the Tm
values obtained for the unmodified reference duplexes (contain-
ing ON5, ON8 and ON11). For the singly modified oligo-
thymidylates ON1 and ON2, the Tm values obtained were at
least 10 ЊC lower than for the reference ON5 with similar results
obtained in the Ara and Ribo series. For the partly modified
oligothymidylates ON3 and ON4, and the partly modified
mixed-base 9-mers ON9 and ON10, Tm values corresponding
to decreases of approximately 5 ЊC per 2Ј-spironucleotide
monomer were obtained towards both the DNA and the RNA
complements in the Ribo series. No melting transitions were
observed for the corresponding Ara series of partly modified
ONs which shows the inability of ONs containing a larger
number of monomers X5-Ara and X4-Ara to form a duplex
with natural ONs. The general and clear difference in melting
behaviour between the partly modified ONs in the Ara and
Ribo series cannot be explained by the presence of the thymine
base in the Ribo series and the uracil base in the Ara series, but
is more likely due to different conformational and steric effects
(see next section). No melting transition was detected towards
the DNA or RNA complements for the almost fully modified
oligothymidylates ON6 and ON7. In all melting experiments,
similar results were obtained for the 2Ј-C,2Ј-O-ethano- and
2Ј-C,2Ј-O-propano-linked derivatives within an Ara or a Ribo
series.
distribution would give an energy difference of ∼0.4 kcal molϪ1
,
which is far lower than the ∼3.1 kcal molϪ1 as determined by
calculations. Assuming a single conformation of X4-Ara in
solution, the measured J3Ј4Ј coupling constant would yield an
∼
O4Ј-endo (P 90Њ) furanose conformation. This conformation
−
lies at the top of the pseudorotational energy barrier to inter-
conversion and is, as such, unlikely. A rather similar J3Ј4Ј = 5.4
Hz was measured for X5-Ara in solution. This shows that the
X5-Ara nucleoside adopts an equilibrium between two or more
furanose conformations, possibly an N S equilibrium as does
X4-Ara.
Biological evaluation of the 2Ј-spironucleosides
The 2Ј-spironucleosides 9b, 14b, 21 and 27 and the 2Ј-alkyl-
ribonucleosides 3 and 16 were evaluated for antiviral activity
against HIV-1 in MT-4 cells (Medical Research Council,
Centralised Facility for AIDS Reagents, UK) as described
earlier.31 All compounds were inactive against HIV-1 at 300 µM
and displayed no cytotoxicity at 300 µM.
Syntheses of ONs and thermal denaturation studies
All oligomers ON1–ON11 (Table 1) were prepared on a 0.2
µmol scale using the phosphoramidite approach. It should be
noted that two variants of all modified ONs were prepared, i.e.,
one in the “Ara series” and one in the “Ribo series” (see caption
below Table 1). The stepwise coupling efficiencies of the phos-
phoramidites 10, 15, 23 and 29 (10 min coupling time) and of
unmodified deoxynucleoside phosphoramidites were > 98%
using 1H-tetrazole as activator (see experimental section for
further details). After standard deprotection and ethanol
precipitation, the composition of oligomers ON1–ON4, ON6,
ON7, ON9 and ON10 was verified by MALDI-MS analysis
and their purity (> 90%) by capillary gel electrophoresis.
Thermal denaturation studies towards complementary DNA
and RNA target strands were performed in a medium salt
buffer with 10-mer and 14-mer oligothymidylate sequences and
with a mixed-base 9-mer sequence (Table 1). Regardless of
sequence context and the number and structure of 2Ј-spiro-
nucleotides incorporated (X5-Ribo, X4-Ribo, X5-Ara and X4-
Modelling of X4-Ribo/DNA:DNA and X4-Ara/DNA:DNA
duplexes
To assess the duplex geometry of X4-Ribo and X4-Ara modified
dsDNA duplexes, we have carried out molecular dynamics
(MD) simulations of the 5Ј-d(GXGAXAXGC)-3Ј:5Ј-
d(GCATATCAC)-3Ј duplex, with X being either X4-Ribo-T or
X4-Ara-U modifications. The simulations were carried out
using explicit solvent, periodic boundary conditions and Ewald
treatment of electrostatic interactions. For each of the two
duplexes, both standard A- and B-type duplex geometries were
used as starting coordinates. This resulted in four simulations
of at least 1 ns duration each. For comparison, two simulations
(A- and B-form starting geometry; 1 ns) were carried out of
the unmodified reference duplex. The calculations of the
unmodified duplexes converged to identical structures (RMSD
between the average structures from the final 500 ps of each
simulation was 1.0 Å), and usual duplex features prevailed
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 5 1 4 – 3 5 2 6
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throughout the simulation, e.g. normal Watson–Crick base
pairing and a regular duplex geometry.
the range 70 to 100%, except for C11 and C16, which have ∼40%
S-type conformation. The average minor groove width in the
central part of the duplex is 9.5 Å, which is intermediate
between A- and B-type duplex geometries as would be expected
with the large proportion of N-type sugar conformations in the
modified strand. The backbone angles of the duplex generally
fall in the standard genus for right-handed nucleic acids, that is
α to ζ: scϪ, ap, scϩ, scϩ or acϩ, ap, scϪ, except the ε torsion angle
in the modified nucleotides, which have values of ∼Ϫ124Њ. The
global structure of the duplex is intermediate between A- and
B-type geometries and highly irregular.
Two of the modified X4-Ribo nucleotides partake in non-
Watson–Crick hydrogen bonding at times in the simulation.
After 1440 ps of simulation, the T7:A12 base pair is partly
broken and T7 forms a bifurcated base pair with T13 and A12.
Two hydrogen bonds are formed between T7 and T13 (T7:H3–
T13:O4 and T7:O2–T13:H3) and the bond between A12:NH6
and T7:O4 remains intact (Fig. 4). For most of the duration of
the bifurcated base pairing, A12 assumes an N-type sugar
pucker. The bifurcated base pair persists for ∼350 ps thereafter
normal Watson–Crick base pairing resumes. While T7 and T13
base pair, A6 stacks with T7. In the T2:A17 base pair normal
Watson–Crick base pairing is broken after 1065 ps and a non-
Watson–Crick base pair is formed with hydrogen bonding
between T2:O2 and A17:NH6. This hydrogen bond scheme
remains for the duration of the simulation. In the remaining
base pairs, usual Watson–Crick base pairing is observed.
X4-Ribo/DNA:DNA. Two MD simulations were performed,
starting from either A- or B-type duplex geometries. Both
calculations produced stable trajectories as indicated by average
RMSDs along the trajectories of 2.1 Å and 2.6 Å, respectively.
The two calculations did not converge to identical structures
for the X4-Ribo modified dsDNA duplex. In the B-structure,
all sugars remain in S-type conformation, whereas in the
A-structure, the three X4-Ribo furanoses remain in N-type con-
formations as does A4, while the deoxyriboses (except A4)
repucker to S-type puckers (within ∼400 ps). The difference
in furanose conformation of the modified nucleotides is
accompanied by differences in their ε torsion angles: in the
A-type structure, we observe ε = Ϫ124Њ whilst in the B-structure,
the ε angles take values of ∼Ϫ85Њ and ∼Ϫ165Њ. To investigate
this phenomenon, we calculated pseudorotation energy profiles
of the X4-Ribo nucleoside with two different values of the
ε torsion angle (Ϫ124Њ and Ϫ165Њ) at both force field and
MP2/cc-pVTZ levels of theory (see details below and Fig. 2).
In both pseudorotation profiles, it is seen that the global
energy minimum is N-type and that there is a local S-type min-
imum. Furthermore, the very close similarity between the
energy profiles calculated at the two, very different, levels of
theory (force field and ab initio) is striking, albeit fortuitous.
The propensity of the modified sugars to remain in an S-type
conformation in the B-structure is obvious from the pseudo-
rotation profile calculated with ε = Ϫ165Њ as there is a very deep
local S-type minimum with an energy barrier to interconversion
to N-type too large to be surmounted in the simulation time
used (1 ns). A similar problem of incomplete conformational
sampling is observed with stabilization of B-type RNA in MD
simulations.32 Therefore, we consider that the structure derived
in the A-simulation is the structure most likely to be present in
solution and thus we discuss the features of this structure. A
view of the average structure from the final 1500 ps of the
simulation is shown in Fig. 3. As can be observed, the presence
of the C2Ј-spiro rings introduces pronounced kinks in the
duplex owing to their steric clashes with primarily the O4Ј, O5Ј
and H5Ј atoms of the 3Ј-flanking residue, with the bending at
these base pair steps being approximately 30Њ towards the major
groove. Consequently, the nucleobases of the modified nucleo-
tides destack and tilt into the major groove and an irregular
duplex structure ensues. Owing to the duplex bending into the
major groove, the C2Ј-spiro rings are located at the brim of the
minor groove, being exposed to the solvent there.
Fig. 4 MD simulation of X4-Ribo/DNA:DNA duplex. Stereo view of
snapshot at 1600 ps showing the bifurcated base pair between T7 and
A12/T13. Hydrogen bonds are shown as dotted lines. The colouring
scheme used is: carbon, green; nitrogen, blue; oxygen, red;
phosphorous, magenta; hydrogen, off-white.
As the control simulation of the unmodified dsDNA duplex
showed no irregularities, i.e. pronounced bending of the duplex
or non-Watson–Crick base pairing, these structural features are
a consequence of the spiro modified nucleotides. Our MD simu-
lation of the X4-Ribo modified dsDNA reveals convincingly
that this modification fits poorly into the Watson–Crick frame-
work of a nucleic acid duplex thereby also offering explanations
for the negative influence of monomers X4-Ribo and X5-Ribo
on duplex thermal stabilities.
During the simulation, all deoxyriboses, with the exception
of A4, repucker with populations of S-type conformation in
X4-Ara/DNA:DNA. As the X4-Ara sugar is able to repucker,
one must require the A- and B-simulations to converge in order
to get a reliable picture of the geometry of the X4-Ara modified
duplex. Unfortunately, our simulations did not converge, with
the RMSD between the two average structures being 1.6 Å.
This is probably a consequence of incomplete conformational
sampling and longer trajectories would be required to alleviate
this problem. Both simulations produce stable trajectories
(RMSDs 2.2 Å and 2.6 Å for the A- and B-simulations, respect-
ively). In the A-simulation, the deoxyriboses repucker to S-type
conformations, as does one of the X4-Ara modified sugars,
whilst in the B-simulation, all sugars, including the modified
ones, remain in the S-type range. This suggests that in a duplex
context, the S-type conformation is preferred by the modified
sugars, although longer simulations would be required to
establish this beyond doubt. In both simulations, normal
Watson–Crick base pairing is observed throughout. As in the
Fig. 3 MD simulation of X4-Ribo/DNA:DNA duplex. Stereo view of
the average structure from the final 1500 ps of simulation. The
colouring scheme used is: sugar–phosphate backbone, red; nucleobases,
yellow; 2Ј-spiro-rings, blue.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 5 1 4 – 3 5 2 6
3518

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X4-Ribo/DNA:DNA simulation, the spiro rings introduce some
bending of the duplex at the base pair steps between the
modified nucleotides and the 3Ј-flanking nucleotides, albeit the
bending is less pronounced than in the X4-Ribo modified
duplex. Altogether, it appears that the X4-Ara modifications
introduce less distortion of the Watson–Crick duplex as
compared with the X4-Ribo modification. However, to assess
the structure in detail and its influence on thermal stabilities of
duplexes, simulations longer than the present ones are required.
over 15 min whereupon stirring was continued for 2 h at Ϫ78
ЊC. The ice–acetone cooling bath was removed and saturated
NH4Cl (100 cm3) was added with stirring. After warming to rt,
the mixture was filtered through Celite and washing was per-
formed, successively, with diethyl ether (200 cm3) and saturated
aq. NH4Cl (200 cm3). The organic phase was separated and
washed with saturated NH4Cl (50 cm3). The combined aq.
phase was extracted with diethyl ether (100 cm3), and the
combined organic phase was dried (Na2SO4), filtered and evap-
orated to dryness to give a yellow oil. This oil was coevaporated
with pyridine (2 × 5 cm3) and the residue was dissolved in
anhydrous CH2Cl2 (80 cm3) whereupon DMAP (2.00 g, 16.4
mmol), benzoyl chloride (3.50 cm3, 30.1 mmol) and Et3N (100
cm3) were added. After stirring for 12 h at rt, the reaction
mixture was diluted with CH2Cl2 (100 cm3) and then washed,
successively, with 1 M aq. HCl (3 × 100 cm3) and saturated aq.
NaHCO3 (50 cm3). The combined aq. phase was extracted with
diethyl ether (100 cm3) and the combined organic phase was
dried (Na2SO4), filtered and evaporated to dryness under
reduced pressure to give a yellow oil which was purified by
column chromatography [90–95% (v/v) CH2Cl2 in petroleum
ether] to yield furanose 2 (3.35 g, 53%, mixture of anomers) as a
white foam; Rf 0.40, 0.35 (CH2Cl2); δH (CDCl3, major anomer)
8.15–8.12 (2H, m), 8.07 (4H, d, J 7.8), 7.91–7.88 (2H, m), 7.66–
7.59 (3H, m), 7.53–7.39 (7H, m), 7.19–7.14 (2H, m), 7.06 (1H,
s), 6.12 (1H, d, J 8.0), 5.73 (1H, m), 5.09 (1H, dd, J 1.4 and
17.0), 4.90 (1H, dd, J 1.4 and 10.2), 4.79–4.68 (2H, m), 4.52
(1H, dd, J 4.5 and 12.0), 3.43 (1H, dd, J 7.3 and 14.9), 3.20 (1H,
dd, J 7.6 and 14.9); δC (CDCl3, major anomer) 166.2, 165.6,
164.8, 164.6, 133.8, 133.6, 133.0, 130.3, 130.2, 130.1, 130.0,
129.9, 129.7, 129.6, 129.5, 129.2, 129.1, 128.8, 128.7, 128.3,
120.0, 98.2, 88.5, 79.3, 75.7, 64.0, 35.3; MALDI-HRMS m/z
629.1755 ([M ϩ Na]ϩ, C36H30O9Naϩ calc. 629.1782).
Conclusion
2Ј-Spiro nucleosides have been synthesized as a novel class of
conformationally restricted nucleosides. The furanose ring
of the ribo-configured 2Ј-spiro nucleosides adopts N-type
conformations, whereas the furanose ring of the arabino-
configured 2Ј-spiro nucleosides exists as an N S equilibrium.
These 2Ј-spiro nucleosides and selected 2Ј-C-branched pre-
cursors did not show anti-HIV-1 activity or cytotoxicity.
Irrespective of monomeric configuration, hybridization studies
revealed that incorporation of these 2Ј-spironucleotide
monomers induces decreased duplex thermostabilities com-
pared to the corresponding unmodified reference duplexes.
Molecular modelling clearly revealed unfavourable steric
interactions as a plausible explanation for the lowered binding
affinities.
Experimental
General
Reactions were conducted under an atmosphere of nitrogen
when anhydrous solvents were used. All reactions were moni-
tored by thin-layer chromatography (TLC) using silica plates
with fluorescence indicator (SiO2-60, F-254) visualizing under
UV light and by spraying with 5% conc. sulfuric acid in ethanol
(v/v) followed by heating. Silica gel 60 (particle size 0.040–0.063
mm, Merck) was used for flash column chromatography.
Petroleum ether of the distillation range 60–80 ЊC was used.
After column chromatography fractions containing product
were pooled, evaporated to dryness under reduced pressure and
dried for 12 h under vacuum to give the product unless other-
1-(2-C-Allyl-ꢀ-D-ribofuranosyl)thymine (3)
N,O-Bis(trimethylsilyl)acetamide (25.0 cm3, 101 mmol) was
added to a suspension of furanose 2 (14.0 g, 23.1 mmol) and
thymine (5.72 g, 45.4 mmol) in anhydrous acetonitrile (200
cm3). The reaction mixture was refluxed for 1 h whereupon the
clear solution was allowed to cool to rt. SnCl4 (10 cm3, 85.6
mmol) was added dropwise and the mixture was refluxed
for 3 h. The mixture was then cooled to rt, EtOAc (250 cm3)
followed by saturated aq. NaHCO3 (250 cm3) were added and
the mixture was stirred for 15 min. The mixture was filtered
through Celite and washing was performed successively with
EtOAc (200 cm3) and saturated aq. NaHCO3 (200 cm3). The
organic phase was separated and washed with saturated aq.
NaHCO3 (250 cm3). The combined aq. phase was extracted
with EtOAc (250 cm3) and the combined organic phase was
dried (Na2SO4), filtered and evaporated to dryness under
reduced pressure. The resulting yellow oil was purified by
column chromatography [60–75% (v/v) EtOAc in petroleum
ether] affording a white solid material. Saturated methanolic
ammonia (150 cm3) was added and stirring at rt was continued
for 48 h. The mixture was evaporated under reduced pressure
to give a residue that was coevaporated with acetonitrile (2 × 50
cm3) and then purified by column chromatography [8–10% (v/v)
MeOH in CH2Cl2] to give nucleoside 3 (3.71 g, 54%) as a white
solid material; Rf 0.3 (CH2Cl2–MeOH 90 : 10, v/v); δH (CD3OD)
7.85 (1H, s, H-6), 6.00 (1H, s, H-1Ј), 5.87 (1H, m, H-2Љ), 4.97
(1H, d, J 13.7, H-3Љa), 4.96 (1H, d, J 13.1, H-3Љb), 4.07 (1H, d,
J 9.2, H-3Ј), 3.98 (1H, dd, J 1.9 and 12.7, H-5Јa), 3.88 (1H, ddd,
J 2.4, 2.4 and 9.2, H-4Ј), 3.79 (1H, dd, J 2.7 and 12.5, H-5Јb),
2.39 (1H, dd, J 6.7 and 14.8, H-1Љa), 2.22 (1H, dd, J 7.4 and
14.6, H-1Љb), 1.87 (3H, s, 5-CH3); δC (CD3OD) 166.2, 152.6,
138.8 (C-6), 133.6 (C-2Љ), 117.9 (C-3Љ), 111.2, 92.4 (C-1Ј), 83.5
(C-4Ј), 81.4 (C-2Ј), 72.6 (C-3Ј), 60.5 (C-5Ј), 40.3 (C-1Љ), 12.4
(5-CH3); MALDI-HRMS m/z 321.1049 ([M ϩ Na]ϩ, C13H18-
N2O6Naϩ calc. 321.1057).
wise specified. 1H NMR spectra were recorded at 300 MHz, 13
C
NMR spectra at 75.5 MHz, and 31P NMR spectra at 121.5
MHz. Chemical shifts are reported in ppm relative to either
tetramethylsilane or the deuterated solvent as internal standard
1
for H NMR and 13C NMR, and relative to 85% H3PO4 as
external standard for 31P NMR. Assignments of NMR spectra,
when given, are based on 2D spectra and follow the standard
carbohydrate/nucleoside nomenclature. Coupling constants
(J-values) are given in Hertz. Fast atom bombardment mass
spectra (FAB-MS) were recorded in positive ion mode on a
Kratos MS50TC spectrometer and MALDI-HRMS were
recorded in positive ion mode on an IonSpec Fourier Trans-
form mass spectrometer. The composition of the ONs was
verified by MALDI-MS (negative ion mode) on a Micromass
Tof Spec E mass spectrometer using a matrix of diammonium
citrate and 2,6-dihydroxyacetophenone.
1,2,3,5-Tetra-O-benzoyl-2-C-allyl-D-ribofuranose (2)
CeCl3ؒ7H2O (98.5%, 18.0 g, 48.3 mmol) was in a three-necked
flask heated at 170 ЊC under vacuum for 4 h. The flask was
purged with N2 and after cooling on an ice bath, THF (140 cm3,
freshly distilled from sodium–benzophenone) was slowly added
and stirring was continued for 12 h at rt. The suspension was
cooled to Ϫ78 ЊC and allyl magnesium bromide (46.0 cm3 of a
1.0 M solution in diethyl ether, 46 mmol) was added dropwise
over 20 min and the resulting mixture stirred for another 2 h at
Ϫ78 ЊC. A solution of 1,3,5-tri-O-benzoyl-2-keto-α--ribo-
furanose (1)22 (4.80 g, 10.4 mmol) in THF (50 cm3) was added
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 5 1 4 – 3 5 2 6
3519

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1-[2-C-Allyl-5-O-(4,4Ј-dimethoxytrityl)-ꢀ-D-ribofuranosyl]-
thymine (4)
5-CH3), 0.95 (9H, s, –C(CH3)3), 0.93 (9H, s, –C(CH3)3), 0.17
(3H, s, –SiCH3), 0.16 (3H, s, –SiCH3), 0.13 (6H, s, 2 × –SiCH3);
δC (CDCl3) 163.7, 150.5, 136.3 (C-6), 132.2 (C-2Љ), 118.0 (C-3Љ),
110.4, 90.8 (C-1Ј), 82.3 (C-4Ј), 79.6 (C-2Ј), 73.0 (C-3Ј), 60.9
(C-5Ј), 39.6 (C-1Љ), 26.1 and 25.8 (2 × –C(CH3)3), 18.6 and 18.0
(2 × –C(CH3)3), 12.7 (5-CH3), Ϫ4.1, Ϫ4.2, Ϫ5.1 and Ϫ5.2 (4 ×
–SiCH3); MALDI-HRMS m/z 549.2791 ([M ϩ Na]ϩ, C25H46-
N2O6Si2.Naϩ calc. 549.2787).
4,4Ј-Dimethoxytrityl chloride (3.42 g, 10.1 mmol) was added in
one portion to a stirred solution of nucleoside 3 (2.5 g, 8.4
mmol) in anhydrous pyridine (20 cm3). After stirring for 12 h at
rt, methanol (0.2 cm3) was added and the resulting mixture was
evaporated to dryness under reduced pressure. The residue was
dissolved in CH2Cl2 (100 cm3) and washed with saturated aq.
NaHCO3 (2 × 50 cm3). The combined aq. phase was extracted
with CH2Cl2 (50 cm3). The combined organic phase was dried
(Na2SO4), filtered and evaporated to dryness under reduced
pressure. The residue was purified by column chromatography
[4–5% MeOH in CH2Cl2, containing 0.5% Et3N (v/v/v)] yielding
nucleoside 4 as a white solid material (4.33 g, 86%); Rf 0.3
(CH2Cl2–MeOH 95 : 5, v/v); δH (CDCl3) 10.35 (1H, br s), 7.67
(1H, s), 7.43 (2H, d, J 7.7), 7.33–7.20 (7H, m), 6.83 (4H, d,
J 8.8), 6.11 (1H, s), 5.91 (1H, m), 5.20 (1H, br s), 5.11 (1H, d,
J 10.0), 5.08 (1H, d, J 18.3), 4.24 (1H, d, J 7.2), 4.06 (1H, d,
J 8.1), 3.78 (6H, s), 3.59 (1H, d, J 10.5), 3.45 (1H, d, J 10.7),
3.10 (1H, d, J 7.3), 2.47 (1H, dd, J 6.8 and 14.4), 2.32 (1H, dd, J
7.3 and 14.1), 1.41(3H, s); δC (CDCl3) 164.3, 158.8, 151.3, 144.5,
136.0, 135.7, 135.5, 131.7, 130.3, 128.4, 128.0, 127.2, 119.2,
113.3, 111.6, 92.5, 86.7, 82.2, 79.5, 72.8, 61.6, 55.3, 39.5, 11.8;
MALDI-HRMS m/z 623.2347 ([M ϩ Na]ϩ, C34H36N2O8Naϩ
calc. 623.2364).
1-[3-O-(tert-Butyldimethylsilyl)-5-O-(4,4Ј-dimethoxytrity)-2-C-
(3-hydroxypropyl)]-ꢀ-D-ribofuranosyl]thymine (6a)
To a stirred solution of nucleoside 5a (3.57 g, 5.0 mmol) in
anhydrous THF (25 cm3) was added dropwise BH3 (1.0 cm3 of a
7.8 M solution in 1,4-oxathiane, 7.8 mmol). The mixture was
stirred for 12 h at rt whereupon it was cooled to 0 ЊC using an
ice bath. A mixture of 2 M aq. NaOH (4.0 cm3, 8.0 mmol) and
aq. H2O2 (35%, 1.5 cm3, 17.4 mmol) was added dropwise and
the mixture was stirred for 2 h at rt. EtOAc (200 cm3) was added
and washing was performed, successively, with H2O (2 × 50
cm3) and saturated aq. NaHCO3 (2 × 50 cm3). The combined
aq. phase was extracted with EtOAc (50 cm3) and the combined
organic phase was dried (Na2SO4), filtered and evaporated to
dryness under reduced pressure. The residue obtained was
purified by column chromatography [4% (v/v) MeOH in
CH2Cl2] affording nucleoside 6a as a white solid material (2.02
g, 55%); Rf 0.28 (CH2Cl2–MeOH 95 : 5, v/v); δH (CDCl3) 9.46
(1H, s), 7.54 (1H, s), 7.38 (2H, d, J 7.7), 7.33–7.23 (7H, m), 6.83
(4H, d, J 8.9), 6.19 (1H, s), 4.27 (1H, d, J 8.2), 3.99 (1H, m),
3.79 (6H, s), 3.75 (1H, m), 3.63 (1H, m), 3.53 (1H, m), 3.27 (1H,
m), 3.24 (1H, s), 2.47 (1H, br s), 1.69–1.54 (4H, m), 1.33 (3H, s),
0.79 (9H, s), 0.10 (3H, s), Ϫ0.25 (3H, s); δC (CDCl3) 164.1,
158.9, 150.9, 144.1, 136.4, 135.3, 130.5, 130.4, 128.6, 128.0,
127.4, 113.3, 113.2, 111.5, 91.0, 86.8, 81.4, 80.8, 74.3, 62.7, 61.5,
55.4, 31.4, 26.6, 25.8, 18.0, 11.6, Ϫ4.1, Ϫ4.2; MALDI-HRMS
m/z 755.3349 ([M ϩ Na]ϩ, C40H52N2O9SiNaϩ calc. 755.3334).
1-[2-C-Allyl-3-O-(tert-butyldimethylsilyl)-5-O-(4,4Ј-dimethoxy-
trityl)-ꢀ-D-ribofuranosyl]thymine (5a)
To a solution of nucleoside 4 (5.00 g, 8.30 mmol) in anhydrous
DMF (25 cm3) were added imidazole (2.80 g, 41.1 mmol) and
tert-butyldimethylsilyl chloride (3.01 g, 20.0 mmol) and stirring
was continued for 12 h at 36 ЊC. The reaction mixture was
evaporated to dryness under reduced pressure and the residue
obtained was dissolved in CH2Cl2 (100 cm3) and washed with
saturated aq. NaHCO3 (2 × 50 cm3). The combined aq. phase
was extracted with CH2Cl2 (50 cm3) and the combined organic
phase was dried (Na2SO4), filtered and evaporated to dryness
under reduced pressure. The residue obtained was purified by
column chromatography [3–4% MeOH in CH2Cl2, containing
0.5% Et3N (v/v/v)] affording nucleoside 5a as a white solid
material (5.01 g, 84%); Rf 0.39 (CH2Cl2–MeOH 95 : 5, v/v); δH
(CDCl3) 8.98 (1H, br s), 7.51 (1H, s), 7.40 (2H, d, J 7.9), 7.32–
7.22 (7H, m), 6.83 (4H, d, J 8.6), 6.11 (1H, s), 5.85 (1H, m), 5.06
(1H, d, J 10.4), 4.99 (1H, d, J 18.1), 4.31 (1H, d, J 7.7), 3.98
(1H, m), 3.79 (6H, s), 3.74 (1H, m), 3.28 (1H, dd, J 3.2 and
10.9), 3.23 (1H, s), 2.39 (1H, dd, J 6.5 and 14.8), 2.21 (1H, dd,
J 7.1 and 14.8), 1.40 (3H, s), 0.79 (9H, s), 0.07 (3H, s), Ϫ0.27
(3H, s); δC (CDCl3) 163.8, 158.9, 150.5, 144.1, 136.6, 135.3,
132.2, 130.4, 130.3, 128.6, 128.0, 127.3, 118.4, 113.3, 113.2,
113.1, 110.8, 91.7, 86.7, 81.7, 80.0, 73.6, 61.5, 55.4, 40.0, 25.8,
18.0, 11.6, Ϫ4.2; MALDI-HRMS m/z 737.3252 ([M ϩ Na]ϩ,
C40H50N2O8SiNaϩ calc. 737.3229).
1-[3,5-Di-O-(tert-butyldimethylsilyl)-2-C-(3-hydroxypropyl)-ꢀ-
D-ribofuranosyl]thymine (6b)
By the procedure described above for synthesis of nucleoside
6a, reaction of nucleoside 5b (600 mg, 1.14 mmol) with BH3
(0.2 cm3 of a 7.8 M solution in 1,4-oxathiane, 1.56 mmol) in
anhydrous THF (8 cm3) followed by oxidation with a mixture
of 2 M aq. NaOH (0.8 cm3, 1.6 mmol) and aq. H2O2 (35%, 0.3
cm3, 3.5 mmol) afforded a residue that was purified by column
chromatography [50–60% (v/v) EtOAc in petroleum ether] to
yield nucleoside 6b as a white solid material (242 mg, 39%); Rf
0.20 (CH2Cl2–MeOH 95 : 5, v/v);δH (CDCl3) 9.22 (1H, br s),
7.23 (1H, d, J 1.1), 6.10 (1H, s), 4.07 (1H, d, J 7.3),.4.02 (1H,
dd, J 1.5 and 11.7), 3.97–3.78 (2H, m), 3.61 (1H, m), 3.50 (1H,
m), 3.27 (1H, s), 2.33 (1H, br s), 1.92 (3H, d, J 0.9), 1.68 (1H,
m), 1.59–1.42 (3H, m), 0.94 (9H, s), 0.93 (9H, s), 0.17 (6H, s),
0.13 (6H, s); δC (CDCl3) 163.9, 150.7, 136.3, 110.9, 90.4, 82.1,
80.4, 73.4, 62.6, 60.9, 30.1, 26.3, 26.2, 25.8, 18.7, 18.1, 12.8,
Ϫ4.0, Ϫ4.1, Ϫ5.0, Ϫ5.2; MALDI-HRMS m/z 567.2879
([MϩNa]ϩ, C25H48N2O7Si2Naϩ calc. 567.2892).
1-[2-C-Allyl-3,5-di-O-(tert-butyldimethylsilyl)-ꢀ-D-
ribofuranosyl]thymine (5b)
To a stirred solution of nucleoside 3 (800 mg, 2.68 mmol) in
anhydrous DMF (8 cm3) was added tert-butyldimethylsilyl
chloride (1.62 g, 10.8 mmol) and imidazole (1.46 g, 21.4 mmol)
and stirring was continued overnight at 36 ЊC. After the usual
work-up procedure, the residue obtained was purified by
column chromatography [40% (v/v) EtOAc in petroleum ether]
affording nucleoside 5b as a white solid material (1.19 g, 84%);
Rf 0.28 (CH2Cl2–MeOH 95 : 5, v/v); δH (CDCl3) 9.02 (1H, br s,
–NH), 7.19 (1H, s, H-6), 6.05 (1H, s, H-1Љ), 5.85 (1H, m, H-2Љ),
5.03 (1H, d, J 10.4, H-3Љa), 4.96 (1H, d, J 17.4, H-3Љb), 4.10
(1H, d, J 7.4, H-3Ј), 4.01 (1H, d, J 10.9, H-5Јa), 3.84–3.80 (2H,
m, H-4Ј and H-5Љb), 3.23 (1H, s, –OH), 2.29 (1H, dd, J 6.2 and
14.7, H-1Љa), 2.11 (1H, dd, J 7.3 and 14.8, H-1Љb), 1.93 (3H, s,
1-[3-O-(tert-Butyldimethylsilyl)-5-O-(4,4Ј-dimethoxytrityl)-2-C-
(3-methanesulfonyloxypropyl)-ꢀ-D-ribofuranosyl]thymine (7a)
A solution of methanesulfonyl chloride (115 mg, 1.0 mmol) in
anhydrous CH2Cl2 (1.0 cm3) was added dropwise at rt to a
solution of nucleoside 6a (600 mg, 0.82 mmol) in anhydrous
pyridine (2.0 cm3). After stirring for 12 h at rt, methanol
(0.1 cm3) was added and the resulting mixture was evaporated
to dryness under reduced pressure and coevaporated with
anhydrous toluene (2 × 5 cm3). The residue was dissolved in
CH2Cl2 (50 cm3) and washed with saturated aq. NaHCO3 (2 ×
25 cm3). The combined aq. phase was extracted with CH2Cl2
(50 cm3). The combined organic phase was dried (Na2SO4),
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 5 1 4 – 3 5 2 6
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filtered and evaporated to dryness under reduced pressure. The
residue obtained was purified by column chromatography [2.0%
(v/v) MeOH in CH2Cl2] affording nucleoside 7a as a white solid
material (385 mg, 58%); Rf 0.33 (CH2Cl2–MeOH 95 : 5, v/v); δH
(CDCl3) 9.22 (1H, s), 7.54 (1H, s), 7.38 (2H, d, J 8.2), 7.34–7.24
(7H, m), 6.84 (4H, d, J 9.0), 6.18 (1H, s), 4.27 (1H, d, J 8.0),
4.20 (2H, t, J 5.6), 3.97 (1H, d, J 7.8), 3.79 (6H, s), 3.76 (1H, m),
3.26 (1H, dd, J 3.1 and 11.0), 3.09 (1H, s), 2.96 (3H, s), 1.96
(1H, m), 1.84 (1H, m), 1.68 (1H, m), 1.50 (1H, m), 1.31 (3H, s),
0.80 (9H, s), 0.11 (3H, s), Ϫ0.25 (3H, s); δC (CDCl3) 163.8,
159.0, 150.7, 143.9, 136.1, 135.2, 135.1, 130.6, 130.5, 130.4,
128.7, 128.0, 127.4, 113.3, 113.2, 111.9, 90.7, 86.9, 81.3, 80.6,
74.2, 70.1, 61.3, 55.4, 37.4, 30.9, 25.7, 23.3, 17.9, 11.4, Ϫ4.2;
FAB-MS: m/z 810 [Mϩ].
s), 0.92 (9H, s), 0.15 (3H, s), 0.14 (3H, s), 0.13 (3H, s), 0.12 (3H,
s); δC (CDCl3) 163.9, 150.6, 135.4, 110.9, 90.4, 90.1, 81.3, 72.2,
69.5, 60.4, 29.4, 26.3, 26.2, 25.8, 18.9, 18.1, 12.8, Ϫ4.0, Ϫ4.1,
Ϫ4.7, Ϫ5.2; MALDI-HRMS m/z 549.2776 ([M ϩ Na]ϩ,
C25H46N2O6Si2Naϩ calc. 549.2786).
1-[5-O-(4,4Ј-Dimethoxytrityl)-2-O,2-C-propano-ꢀ-D-ribo-
furanosyl]thymine (9a)
Tetrabutylammonium fluoride (0.60 cm3 of a 1.1 M solution in
THF, 0.66 mmol) was added to a stirred solution of nucleoside
8a (300 mg, 0.42 mmol) in anhydrous THF (2.0 cm3). After
stirring for 2 h at rt, anhydrous toluene (2.0 cm3) was added and
the mixture was evaporated under reduced pressure to one-fifth
of its original volume. The residue obtained was purified by
column chromatography [3.0% (v/v) MeOH in CH2Cl2] afford-
ing nucleoside 9a as a white solid material (222 mg, 88%); Rf
0.27 (CH2Cl2–MeOH 95 : 5, v/v); δH (CDCl3) 9.16 (1H, s), 7.59
(1H, d, J 0.9), 7.43 (2H, d, J 8.5), 7.34–7.22 (7H, m), 6.83 (4H,
d, J 9.2), 6.00 (1H, s), 4.24 (1H, dd, J 9.8 and 10.6), 4.08–4.01
(2H, m), 3.86 (1H, d, J 9.3), 3.78 (6H, s), 3.58 (1H, dd, J 1.5 and
10.7), 3.45 (1H, dd, J 3.3 and 11.1), 2.45 (1H, d, J 11.3), 2.04–
1.94 (3H, m), 1.80 (1H, m), 1.36 (3H, s); δC (CDCl3) 163.9,
158.8, 150.7, 144.5, 135.6, 135.5, 135.3, 130.3, 130.2, 128.4,
128.2, 128.0, 127.2, 113.3, 111.5, 90.2, 90.0, 86.8, 81.9, 72.3,
69.9, 61.3, 55.3, 29.6, 26.4, 11.7; MALDI-HRMS m/z 623.2348
([M ϩ Na]ϩ, C34H36N2O8Naϩ calc. 623.2364).
1-[3-O-(tert-Butyldimethylsilyl)-5-O-(4,4Ј-dimethoxytrityl)-2-
O,2-C-propano-ꢀ-D-ribofuranosyl]thymine (8a)
A solution of nucleoside 7a (120 mg, 0.15 mmol) in THF (1.0
cm3) was added dropwise to a stirred suspension of NaH (60%
in mineral oil, 20 mg, 0.5 mmol) in anhydrous THF (2.0 cm3),
at 0 ЊC. After stirring for 2 h at rt, ice-cold H2O (10 cm3) was
slowly added followed by addition of CH2Cl2 (25 cm3). The
separated organic phase was washed with saturated aq.
NaHCO3 (2 × 20 cm3). The combined aq. phase was extracted
with CH2Cl2 (25 cm3) and the combined organic phase was
dried (Na2SO4), filtered and evaporated to dryness under
reduced pressure. The residue obtained was purified by column
chromatography [2.0–2.5% (v/v) MeOH in CH2Cl2] affording
nucleoside 8a as a white solid material (92 mg, 87%); Rf 0.37
(CH2Cl2–MeOH 95 : 5, v/v); δH (CDCl3) 9.10 (1H, s), 7.78 (1H,
s), 7.38 (2H, d, J 6.8), 7.32–7.23 (7H, m), 6.83 (4H, d, J 8.9),
5.98 (1H, s), 4.21 (1H, d, J 9.3), 4.12–4.04 (2H, m), 3.91 (1H,
dd, J 7.5 and 14.5), 3.82 (1H, m), 3.79 (6H, s), 3.28 (1H, dd,
J 2.3 and 11.1), 2.10–1.76 (4H, m), 1.19 (3H, s), 0.75 (9H, s),
0.07 (3H, s), Ϫ0.27 (3H, s); δC (CDCl3) 164.0, 158.9, 150.8,
144.0, 135.5, 135.3, 130.6, 130.5, 128.8, 128.0, 127.4, 113.2,
113.1, 113.0, 111.2, 91.2, 90.3, 86.9, 81.0, 73.1, 69.8, 61.2, 55.4,
29.4, 26.3, 25.7, 18.0, 11.4, Ϫ4.0, Ϫ4.2; MALDI-HRMS m/z
737.3213 ([M ϩ Na]ϩ, C40H50N2O8SiNaϩ calc. 737.3229).
1-(2-O,2-C-Propano-ꢀ-D-ribofuranosyl)thymine (9b)
By the procedure described above for synthesis of nucleoside
9a, desilylation of nucleoside 8b (135 mg, 0.26 mmol) using
tetrabutylammonium fluoride (0.77 cm3 of a 1.0 M solution
in THF, 0.77 mmol) in anhydrous THF (1.0 cm3) furnished a
residue that was purified by column chromatography [3.0%
(v/v) MeOH in CH2Cl2] to yield nucleoside 9b as a white solid
material (60 mg, 78%); Rf 0.21 (CH2Cl2–MeOH 90 : 10, v/v); δH
((CD3)2SO) 11.36 (1H, s, –NH–), 7.93 (1H, s, H-6), 5.75 (1H, s,
H-1Ј), 5.25 (1H, t, J 4.8, 5Ј-OH), 5.01 (1H, d, J 8.1, H-3Ј),
3.88–3.69 (4H, m, H-4Ј, H-5Јa, H-3Љ), 3.62 (1H, m, H-5Јb),
1.87–1.79 (3H, m, H-1Љa, H-2Љ), 1.75 (3H, s, 5-CH3), 1.56 (1H,
m, H-1Љb); δC ((CD3)2SO) 163.6 (C-4), 150.7 (C-2), 135.9 (C-6),
109.1 (C-5), 89.9 (C-1Ј), 89.7 (C-2Ј), 81.9 (C-4Ј), 70.9 (C-3Ј),
68.9 (C-3Љ), 58.5 (C-5Ј), 28.7 (C-2Љ), 25.7 (C-1Љ), 12.3 (5-CH3);
MALDI-HRMS m/z 321.1063 ([M ϩ Na]ϩ, C13H18N2O6Naϩ
calc. 321.1057).
1-[3,5-Di-O-(tert-butyldimethylsilyl)-2-O,2-C-propano-ꢀ-D-ribo-
furanosyl]thymine (8b)
A solution of nucleoside 6b (225 mg, 0.410 mmol) and DMAP
(77 mg, 0.63 mmol) in Et3N (1.0 cm3) was stirred at rt for 15 min
whereupon methanesulfonyl chloride (57 mg, 0.50 mmol) dis-
solved in CH2Cl2 (0.5 cm3) was added dropwise. After stirring
for 12 h at rt, methanol (0.1 cm3) was added dropwise and the
resulting mixture was evaporated to dryness under reduced
pressure, and coevaporated with anhydrous toluene (2 × 2 cm3).
The residue was dissolved in CH2Cl2 (25 cm3) and washed with
saturated aq. NaHCO3 (2 × 25 cm3). The combined aq. phase
was extracted with CH2Cl2 (25 cm3) and the combined organic
phase was dried (Na2SO4), filtered and evaporated to dryness
under reduced pressure. The residue was dissolved in THF
(1.0 cm3) and this solution was added dropwise to a stirred
suspension of NaH (60% in mineral oil, 20 mg, 0.50 mmol) in
anhydrous THF (2.0 cm3) at 0 ЊC. After stirring for 2 h at rt, ice-
cold H2O (10 cm3) was slowly added followed by addition of
CH2Cl2 (25 cm3). The separated organic phase was washed with
saturated aq. NaHCO3 (2 × 20 cm3) and the combined aq.
phase was extracted with CH2Cl2 (25 cm3). The combined
organic phase was dried (Na2SO4), filtered and evaporated to
dryness under reduced pressure. The residue obtained was
purified by column chromatography [40% (v/v) EtOAc in
petroleum ether] affording nucleoside 8b as a white solid
material (161 mg, 74%); Rf 0.33 (CH2Cl2–MeOH 90 : 10, v/v);
δH (CDCl3) 9.33 (1H, br s), 7.43 (1H, d, J 1.0), 6.00 (1H, s),
4.11–3.99 (3H, m), 3.96–3.88 (2H, m), 3.82 (1H, dd, J 1.8 and
11.9), 1.95 (3H, s), 1.92–1.75 (3H, m), 1.63 (1H, m), 0.95 (9H,
1-[3-O-(2-Cyanoethoxy)(diisopropylamino)phosphino-5-O-(4,4Ј-
dimethoxytrityl)-2-O,2-C-propano-ꢀ-D-ribofuranosyl]thymine
(10)
2-Cyanoethyl N,NЈ-diisopropylphosphoramidochloridite (119
mg, 0.50 mmol) was added dropwise to a stirred solution of the
nucleoside 9a (250 mg, 0.42 mmol) and N,NЈ-diisopropylethyl-
amine (0.4 cm3) in anhydrous CH2Cl2 (2.5 cm3). After stirring
for 12 h at rt, methanol (0.2 cm3) was added and the mixture
diluted with EtOAc (20 cm3). Washing was performed with
saturated aq. NaHCO3 (2 × 10 cm3) and the combined aq.
phase was extracted with EtOAc (25 cm3). The combined
organic phase was dried (Na2SO4), filtered and evaporated to
dryness under reduced pressure. The residue obtained was
purified by column chromatography [60–75% EtOAc in n-hexa-
ne, containing 0.5% Et3N (v/v/v)] yielding the phosphoramidite
10 as a white solid material (255 mg, 76%); Rf 0.29 (CH2Cl2–
MeOH 95 : 5, v/v); δP (CDCl3) 152.6, 149.7.
1-[3-O-(tert-Butyldimethylsilyl)-5-O-(4,4Ј-dimethoxytrityl)-2-C-
(2-hydroxyethyl)-ꢀ-D-ribofuranosyl]thymine (11a)
To a stirred solution of nucleoside 5a (1.43 g, 2.00 mmol) in a
mixture of THF (10 cm3) and H2O (5 cm3) was added sodium
periodate (1.34 g, 6.26 mmol) followed by osmium tetraoxide
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 5 1 4 – 3 5 2 6
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(0.27 cm3 of a 2.5% solution in tert-butyl alcohol, 21.5 µmol)
and stirring was continued for 2 h at rt. H2O (25 cm3) was added
and extraction was performed with EtOAc (2 × 50 cm3). The
organic phase was washed with saturated aq. NaHCO3 (2 × 50
cm3). The combined aq. phase was extracted with EtOAc
(50 cm3) and the combined organic phase was dried (Na2SO4),
filtered and evaporated to dryness under reduced pressure. The
residue obtained was dissolved in a mixture of THF (10 cm3)
and H2O (2 cm3) and sodium borohydride (120 mg, 3.1 mmol)
was added. The reaction mixture was stirred for 12 h at rt
whereupon H2O (25 cm3) was added and extraction was
performed with EtOAc (2 × 50 cm3). The combined organic
phase was washed with saturated aq. NaHCO3 (2 × 50 cm3), the
combined aq. phase was extracted with EtOAc (50 cm3), and
the combined organic phase was dried (Na2SO4), filtered and
evaporated to dryness under reduced pressure. The residue
obtained was purified by column chromatography [4–5% (v/v)
MeOH in CH2Cl2] affording nucleoside 11a as a white solid
material (608 mg, 42%); Rf 0.28 (CH2Cl2–MeOH 95 : 5, v/v);
δH (CDCl3) 9.49 (1H, br s), 7.49 (1H, s), 7.38 (2H, d, J 6.6),
7.31–7.24 (7H, m), 6.83 (4H, d, J 8.6), 6.17 (1H, s), 4.30 (1H, d,
J 8.0), 3.99 (1H, d, J 7.1), 3.79 (6H, s), 3.78–3.69 (3H, m), 3.46
(1H, s), 3.28 (1H, dd, J 3.1 and 11.0), 2.95 (1H, br s), 1.88 (1H,
m), 1.73 (1H, m), 1.37 (3H, s), 0.80 (9H, s), 0.11 (3H, s), Ϫ0.23
(3H, s); δC (CDCl3) 163.9, 158.9, 150.9, 144.1, 136.4, 135.3,
130.4, 130.3, 130.2, 128.6, 128.0, 127.3, 113.3, 113.2, 111.6,
91.3, 86.8, 81.4, 81.0, 74.3, 61.6, 58.3, 55.4, 36.2, 25.8, 18.0,
11.6, Ϫ4.2; MALDI-HRMS m/z 741.3160 ([M ϩ Na]ϩ,
C39H50N2O9SiNaϩ calc. 741.3178).
1-[3,5-Di-O-(tert-butyldimethylsilyl)-2-C-(2-methanesulfonyl-
oxyethyl)-ꢀ-D-ribofuranosyl]thymine (12b)
By the procedure described above during synthesis of com-
pound 8b, mesylation of nucleoside 11b (185 mg, 0.35 mmol)
using a solution of methanesulfonyl chloride (57 mg, 0.50
mmol) in CH2Cl2 (0.5 cm3) in the presence of DMAP (61 mg,
0.50 mmol) and Et3N (1.0 cm3) afforded a residue that was
purified by column chromatography [40–50% (v/v) EtOAc in
petroleum ether] to give nucleoside 12b as a white solid material
(135 mg, 64%); Rf 0.49 (CH2Cl2–MeOH 90 : 10, v/v); δH
(CDCl3) 9.25 (1H, s), 7.21 (1H, d, J 1.2), 6.06 (1H, s), 4.35 (2H,
t, J 7.4), 4.12 (1H, d, J 7.2), 4.04 (1H, d, J 11.4), 3.86–3.79 (2H,
m), 3.17 (1H, s), 2.99 (3H, s), 2.03 (1H, m), 1.94 (3H, s), 1.83
(1H, m), 0.94 (9H, s), 0.93 (9H, s), 0.19 (6H, s), 0.13 (6H, s);
δC (CDCl3) 163.6, 150.8, 135.9, 111.7, 90.3, 81.9, 79.4, 73.1,
66.1, 60.8, 37.0, 33.5, 26.2, 26.1, 25.8, 18.8, 18.0, 12.9, Ϫ4.1,
Ϫ4.2, Ϫ4.9, Ϫ5.2; MALDI-HRMS m/z 631.2484 ([M ϩ Na]ϩ,
C25H48N2O9SSi2Naϩ calc. 631.2511).
1-[3-O-(tert-Butyldimethylsilyl)-5-O-(4,4Ј-dimethoxytrityl)-2-
O,2-C-ethano-ꢀ-D-ribofuranosyl]thymine (13a)
By the procedure described above for synthesis of compound
8a, cyclization of nucleoside 12a (680 mg, 0.85 mmol) using
NaH (60% suspension in mineral oil, 100 mg, 2.5 mmol) in
anhydrous THF (2.0 cm3) afforded a residue that was purified
by column chromatography [2.0–3.0% (v/v) MeOH in CH2Cl2]
to yield nucleoside 13a as a white solid material (556 mg, 93%);
Rf 0.35 (CH2Cl2–MeOH 95 : 5, v/v); δH (CDCl3) 9.09 (1H, s,
–NH–), 7.69 (1H, d, J 1.1, H-6), 7.39–7.23 (9H, m), 6.83 (4H, d,
J 9.0), 6.29 (1H, s, H-1Ј), 4.66 (1H, dd, J 1.2 and 5.6, H-2Љa),
4.46 (1H, dd, J 2.5 and 5.8, H-2Љb), 4.22 (1H, d, J 9.2, H-3Ј),
3.91 (1H, d, J 9.0, H-4Ј), 3.79 (6H, s, 2 × –OCH3), 3.77 (1H, m,
H-5Јa), 3.25 (1H, dd, J 2.6 and 11.0, H-5Јb), 2.62–2.54 (2H, m,
H-1Љ), 1.26 (3H, s, 5-CH3), 0.82 (9H, s, –C(CH3)3), 0.17 (3H, s,
–Si–CH3), Ϫ0.20 (3H, s, –Si–CH3); δC (CDCl3) 163.9, 158.9,
150.8, 144.0, 135.7, 135.3, 135.2, 130.5, 130.4, 128.7, 128.0,
127.4, 113.3, 113.2, 111.3, 91.7 (C-1Ј), 91.4 (C-2Ј), 86.9 (Ar3C),
79.7 (C-4Ј), 72.9 (C-3Ј), 66.9 (C-2Љ), 61.2 (C-5Ј), 55.3 (2 ×
–OCH3), 25.7 (–C(CH3)3), 24.8 (C-3Љ), 18.1 (–C(CH3)3), 11.5
(5-CH3), Ϫ4.0 (–Si–CH3), Ϫ4.3 (–Si–CH3); MALDI-HRMS
m/z 723.3059 ([M ϩ Na]ϩ, C39H48N2O8SiNaϩ calc. 723.3072).
1-[3,5-Di-O-(tert-butyldimethylsilyl)-2-C-(2-hydroxyethyl)-ꢀ-D-
ribofuranosyl]thymine (11b)
By the procedure described above for synthesis of nucleoside
11a, oxidative cleavage of the terminal double bond in nucleo-
side 5b (400 mg, 0.76 mmol) using osmium tetraoxide (0.11 cm3
of a 2.5% solution in tert-butyl alcohol, 8.8 µmol) and sodium
periodate (540 mg, 2.52 mmol) in a mixture of THF (4 cm3) and
H2O (2 cm3), followed by reduction with sodium borohydride
(50 mg, 1.32 mmol), work-up and column chromatography
[50–60% (v/v) EtOAc in petroleum ether] afforded nucleoside
11b as a white solid material (190 mg, 47%); Rf 0.34 (CH2Cl2–
MeOH 90 : 10, v/v); δH (CDCl3) 9.44 (1H, br s), 7.19 (1H, d,
J 1.0), 6.09 (1H, s), 4.12 (1H, d, J 6.8), 4.00 (1H, dd, J 1.2 and
11.4), 3.88–3.83 (2H, m), 3.79–3.77 (2H, m), 3.50 (1H, s), 3.01
(1H, br s), 1.91 (3H, s), 1.82 (1H, m), 1.65 (1H, m), 0.93 (18H,
s), 0.18 (3H, s), 0.17 (3H, s), 0.12 (6H, s); δC (CDCl3) 163.8,
150.8, 136.3, 111.0, 90.5, 82.5, 80.7, 73.4, 61.1, 58.3, 35.3, 26.1,
25.8, 18.7, 18.1, 12.8, Ϫ4.1, Ϫ4.2, Ϫ5.1, Ϫ5.3; MALDI-HRMS
m/z 553.2731 ([M ϩ Na]ϩ, C24H46N2O7Si2Naϩ calc. 553.2736).
1-[3,5-Di-O-(tert-butyldimethylsilyl)-2-O,2-C-ethano-ꢀ-D-
ribofuranosyl]thymine (13b)
By the procedure described above for synthesis of compound
8a, cyclization of nucleoside 12b (115 mg, 0.19 mmol) using
NaH (60% suspension in mineral oil, 40 mg, 1.0 mmol) in
anhydrous THF (1.5 cm3) afforded a residue that was purified
by column chromatography [40% (v/v) EtOAc in petroleum
ether] to yield nucleoside 13b as a white solid material (74 mg,
77%); Rf 0.35 (CH2Cl2–MeOH 90 : 10, v/v); δH (CDCl3) 9.24
(1H, s), 7.28 (1H, d, J 1.0), 6.24 (1H, s), 4.58 (1H, m), 4.47 (1H,
m), 4.08–4.02 (2H, m), 3.82–3.73 (2H, m), 2.55 (1H, m), 2.45
(1H, m), 1.93 (3H, d, J 1.2), 0.98 (9H, s), 0.94 (9H, s), 0.23 (3H,
s), 0.19 (3H, s), 0.12 (6H, s); δC (CDCl3) 163.9, 150.6, 135.7,
111.0, 91.4, 90.8, 80.1, 72.4, 66.7, 60.5, 29.8, 26.2, 25.8, 25.2,
18.8, 18.2, 12.7, Ϫ4.1, Ϫ4.3, Ϫ4.9, Ϫ5.2; MALDI-HRMS m/z
535.2602 ([M ϩ Na]ϩ, C24H44N2O6Si2Naϩ calc. 535.2630).
1-[3-O-(tert-Butyldimethylsilyl)-5-O-(4,4Ј-dimethoxytrityl)-2-C-
(2-methanesulfonyloxyethyl)-ꢀ-D-ribofuranosyl]thymine (12a)
By the procedure described above during synthesis of
compound 8b, mesylation of nucleoside 11a (530 mg, 0.74
mmol) using a solution of methanesulfonyl chloride (95 mg,
0.83 mmol) in CH2Cl2 (0.5 cm3) in the presence of DMAP (98
mg, 0.80 mmol) and Et3N (2.0 cm3) afforded a residue that was
purified by column chromatography [3.0% (v/v) MeOH in
CH2Cl2] to yield nucleoside 12a as a white solid material (491
mg, 83%); Rf 0.36 (CH2Cl2–MeOH 95 : 5, v/v); δH (CDCl3) 9.33
(1H, br s), 7.56 (1H, s), 7.38–7.23 (9H, m), 6.84 (4H, d, J 8.8),
6.12 (1H, s), 4.45–4.38 (2H, m), 4.33 (1H, d, J 8.2), 3.98 (1H, d,
J 7.9), 3.79 (6H, s), 3.75 (1H, m), 3.29 (1H, dd, J 3.0 and 11.1),
3.15 (1H, s), 3.00 (3H, s), 2.10 (1H, m), 1.87 (1H, m), 1.28 (3H,
s), 0.80 (9H, s), 0.12 (3H, s), Ϫ0.27 (3H, s); δC (CDCl3) 163.8,
159.0, 150.9, 143.8, 135.1, 130.5, 130.4, 128.7, 128.0, 127.5,
113.3, 113.2, 112.2, 90.9, 87.0, 81.1, 79.8, 73.9, 66.5, 61.2, 55.4,
37.0, 34.1, 25.7, 17.9, 11.4, Ϫ4.3.
1-[5-O-(4,4Ј-Dimethoxytrityl)-2-O,2-C-ethano-ꢀ-D-
ribofuranosyl]thymine (14a)
By the procedure described above for synthesis of compound
9a, desilylation of nucleoside 13a (550 mg, 0.78 mmol) with
tetrabutylammonium fluoride (1.0 cm3 of a 1.0 M solution in
THF, 1.0 mmol) in THF (2.0 cm3) afforded a residue that was
purified by column chromatography [3.0–3.5% (v/v) MeOH in
CH2Cl2] to yield nucleoside 14a as a white solid material (424
mg, 92%); Rf 0.25 (CH2Cl2–MeOH 95 : 5, v/v); δH (CDCl3) 9.35
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 5 1 4 – 3 5 2 6
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(1H, s, –NH–), 7.51 (1H, s, H-6), 7.42 (2H, d, J 7.6), 7.32–7.16
(7H, m), 6.83 (4H, d, J 9.0), 6.29 (1H, s, H-1Ј), 4.76 (1H, dd,
J 7.9 and 13.7, H-2Љa), 4.50 (1H, dd, J 5.7 and 14.4, H-2Љb), 4.25
(1H, dd, J 9.9 and 10.6, H-3Ј), 3.78 (6H, s, 2 × –OCH3), 3.74
(1H, m, H-4Ј), 3.58 (1H, dd, J 1.6 and 10.9, H-5Јa), 3.44 (1H,
dd, J 2.8 and 11.0, H-5Јb), 2.95 (1H, d, J 11.1, 3Ј-OH), 2.82
(1H, m, H-3Љa), 2.51 (1H, m, H-3Љb), 1.41 (3H, s, 5-CH3); δC
(CDCl3) 163.9, 158.8, 150.7, 144.5, 135.6, 135.5, 130.3, 130.2,
128.3, 128.0, 127.2, 113.3, 111.5, 92.1 (C-2Ј), 90.5 (C-1Ј), 86.8
(Ar3C–), 80.5 (C-4Ј), 72.3 (C-3Ј), 67.9 (C-2Љ), 61.3 (C-5Ј), 55.3
(2 × –OCH3), 24.9 (C-3Љ), 11.8 (5-CH3); MALDI-HRMS m/z
609.2198 ([MϩNa]ϩ, C33H34N2O8Naϩ calc. 609.2207).
(20 cm3) was added dropwise at Ϫ78 ЊC and the mixture was
stirred for another 2 h. The cooling bath was removed and
glacial acetic acid (10 cm3) was poured into the reaction mix-
ture. After being allowed to warm to rt, ethyl acetate (400 cm3)
was added and washing was performed, successively, with H2O
(2 × 200 cm3) and brine (2 × 200 cm3). The organic phase was
dried (Na2SO4), filtered and evaporated to dryness under
reduced pressure to give a residue that was purified by column
chromatography [0–4% (v/v) CH3OH in CH2Cl2) yielding
nucleoside 17 as a white solid material (4.84 g, 75%); Rf 0.55
(CH2Cl2–MeOH 92 : 8, v/v); δH (CDCl3) 9.47 (1H, br s, –NH–),
7.82 (1H, d, J 8.2, H-6), 6.08 (1H, m, H-2Љ), 5.91 (1H, s, H-1Ј),
5.69 (1H, dd, J 1.3 and 8.0, H-5), 5.36.5–27 (2H, m, H-3Љ), 4.24
(1H, d, J 9.3, H-3Ј), 4.14 (1H, dd, J 1.6 and 13.2, H-5Јa), 3.99
(1H, dd, J 2.9 and 13.5, H-5Јb), 3.79 (1H, ddd, J 1.9, 2.6 and
9.3, H-4Ј), 2.72 (1H, m, H-1Љa), 2.51 (1H, m, H-1Љb), 1.13–0.97
(28H, m, –SiCH(CH3)2); δC 163.5 (C-4), 151.1 (C-2), 140.4
(C-6), 131.1 (C-2Љ), 121.8 (C-3Љ), 101.4 (C-5), 87.4 (C-1Ј), 80.8
(C-4Ј), 78.8 (C-2Ј), 73.0 (C-3Ј), 60.2 (C-5Ј), 37.8 (C-1Љ), 17.4,
17.3, 17.2, 17.1, 17.0 and 16.7 (SiCH(CH3)2), 13.4, 12.9, 12.8,
and 12.3 (SiCH(CH3)2); FAB-MS m/z 527 [M ϩ H]ϩ.
1-(2-O,2-C-Ethano-ꢀ-D-ribofuranosyl)thymine (14b)
By the procedure described above for synthesis of compound
9a, desilylation of nucleoside 13b (60 mg, 0.12 mmol) with
tetrabutylammonium fluoride (0.15 cm3 of a 1.0 M solution in
THF, 0.15 mmol) in THF (1.0 cm3) afforded a residue that was
purified by column chromatography (EtOAc) to yield nucleo-
side 14b as a white solid material (26 mg, 79%); Rf 0.25
(CH2Cl2–MeOH 90 : 10, v/v); δH (CD3OD ϩ CDCl3, 3 : 1) 7.80
(1H, d, J 0.9), 6.22 (1H, s), 4.67–4.51 (2H, m), 4.06 (1H, d,
J 9.7), 4.01 (1H, dd, J 2.1 and 12.6), 3.81 (1H, dd, J 2.5 and
12.6), 3.72 (1H, ddd, J 2.3, 2.3 and 9.5), 2.72 (1H, ddd, J 6.9, 8.6
and 11.8), 2.51 (1H, ddd, J 6.3, 8.7 and 11.7), 1.89 (3H, d,
J 1.0); δC (CD3OD ϩ CDCl3, 3 : 1) 165.4, 151.7, 137.3, 111.2,
92.6, 91.0, 81.3, 71.7, 67.6, 59.7, 25.0, 12.4; MALDI-HRMS
m/z 307.0897 ([M ϩ Na]ϩ, C12H16N2O6Naϩ calc. 307.0901).
1-[2-C-(3-Hydroxypropyl)-3,5-di-O-(1,1,3,3-tetraisopropyldisil-
oxane-1,3-diyl)-ꢀ-D-arabinofuranosyl]uracil (18)
To a stirred solution of nucleoside 17 (2.21 g, 4.20 mmol) in
anhydrous THF (15 cm3) was added 9-borabicyclo[3.3.1]-
nonane (42.0 cm3 of a 0.5 M solution in THF, 21.0 mmol). The
mixture was stirred for 3 h at rt, whereupon it was cooled to
0 ЊC and 2 M aq. NaOH (10.5 cm3, 21.0 mmol) and 35% aq.
H2O2 (6.30 cm3, 63.0 mmol) were added slowly. The mixture
was allowed to warm to rt, stirred for 2 h and then poured into
a mixture of diethyl ether (150 cm3) and H2O (150 cm3). The aq.
phase was extracted with diethyl ether (50 cm3), the organic
extracts were combined with the separated organic phase and
washed, successively, with saturated aq. NaHCO3 (2 × 40 cm3)
and H2O (2 × 40 cm3), dried (MgSO4), filtered and evaporated
to dryness under reduced pressure. The residue obtained was
purified by dry column chromatography33 [0–3% (v/v) MeOH
in CH2Cl2] yielding nucleoside 18 as a white solid material
which was used in the next step without further purification
(1.45 g, 64%); Rf 0.38 (CH2Cl2–MeOH 92 : 8, v/v); δH (CDCl3)
10.76 (1H, br s, NH), 7.99 (1H, d, J 8.2, H-6), 5.85 (1H, s, H-1Ј),
5.70 (1H, dd, J 1.8 and 8.1, H-5), 4.16–4.12 (2H, m, H-3Ј and
H-5Јa), 3.99–3.94 (2H, m, H-5Јb and H-3Љb), 3.71 (1H, dd, J 1.8
and 9.7, H-4Ј), 3.50 (1H, m, H-3Љb), 2.21–2.08 (2H, m, H-1Љa
and H-2Љa), 1.99 (1H, m, H-2Љb), 1.87 (1H, m, H-1Љb), 1.11–
0.94 (28H, m, –SiCH(CH3)2); δC (CDCl3) 164.7 (C-4), 152.3
(C-2), 140.8 (C-6), 101.6 (C-5), 88.1 (C-1Ј), 80.9 (C-4Ј), 78.4
(C-2Ј), 73.1 (C-3Ј), 62.9 (C-3Љ), 60.2 (C-5Ј), 31.4 (C-2Љ), 25.9 (C-
1Љ), 17.5, 17.4, 17.2, 17.1, 17.0, 16.8 and 16.7 (SiCH(CH3)2),
13.5, 13.1, 12.9 and 12.4 (SiCH(CH3)2); FAB-MS m/z 545 [M ϩ
H]ϩ.
1-[3-O-(2-Cyanoethoxy)(diisopropylamino)phosphino-5-O-(4,4Ј-
Dimethoxytrityl)-2-O,2-C-ethano-ꢀ-D-ribofuranosyl]thymine
(15)
By the procedure described above for synthesis of compound
10, treatment of compound 14a (386 mg, 0.66 mmol) with 2-
cyanoethyl N,NЈ-diisopropylphosphoramidochloridite (166 mg,
0.70 mmol) in the presence of N,NЈ-diisopropylethylamine
(0.4 cm3) and anhydrous CH2Cl2 (3.0 cm3) afforded a residue
that was purified by column chromatography [60–75% EtOAc
in n-hexane, containing 0.5% Et3N (v/v/v)] to yield phosphor-
amidite 15 as a white solid material (412 mg, 80%). Rf 0.32
(CH2Cl2–MeOH 95 : 5, v/v); δP (CDCl3) 152.4, 149.6.
1-[2-C-(3-Hydroxypropyl)-ꢀ-D-ribofuranosyl]thymine (16)
By the procedure described above for synthesis of compound
9a, desilylation of nucleoside 6b (55 mg, 0.1 mmol) with tetra-
butylammonium fluoride (0.20 cm3 of a 1.0 M solution in THF,
0.20 mmol) in THF (1.0 cm3) afforded a residue that was puri-
fied by column chromatography [15% (v/v) MeOH in CH2Cl2]
to yield nucleoside 16 as a white solid material (25 mg, 78%); Rf
0.23 (CH2Cl2/MeOH 85:15, v/v); δH (CD3OD) 7.92 (1H, s), 6.04
(1H, s), 4.05 (1H, d, J 9.0), 4.00 (1H, d, J 12.2), 3.90 (1H, d,
J 8.8), 3.81 (1H, d,J 12.3), 3.51–3.49 (2H, m), 3.37 (1H, s), 3.34
(1H, br s), 1.89 (3H, s), 1.83–1.52 (4H, m); δC (CD3OD) 166.2,
152.6, 138.8, 111.4, 93.0, 84.0, 81.4, 73.4, 63.2, 60.5, 32.4, 26.9,
12.5.
1-[2-C-(3-Methanesulfonyloxypropyl)-3,5-di-O-(1,1,3,3-tetra-
isopropyldisiloxane-1,3-diyl)-ꢀ-D-arabinofuranosyl]uracil (19)
By the procedure described above for synthesis of compound
7a, mesylation of nucleoside 18 (1.43 g, 2.63 mmol) using
methanesulfonyl chloride (0.31 cm3, 3.94 mmol) in pyridine
(10 cm3) afforded a residue that was purified by dry column
chromatography33 [1–3% (v/v) MeOH in CH2Cl2] to yield
nucleoside 19 as a white solid material (1.22g, 74%); Rf 0.64
(CH2Cl2–MeOH 92 : 8, v/v); δH (CDCl3) 7.86 (1H, d, J 8.1,
H-6), 5.79 (1H, s, H-1Ј), 5.68 (1H, d, J 8.1, H-5), 4.34–4.31 (2H,
m, H-3Љ), 4.21 (1H, d, J 9.2, H-3Ј), 4.14 (1H, dd, J 1.5 and 13.5
Hz, H-5Јa), 3.99 (1H, dd, J 2.8 and 13.4, H-5Јb), 3.79 (1H, d,
J 9.2, H-4Ј), 3.03 (3H, s, –SO2CH3), 2.16–2.13 (3H, m, H-2Љ and
H-1Љa), 1.82 (1H, m, H-1Љb), 1.11–0.97 (28H, m, –SiCH(CH3)2);
δC(CDCl3) 163.7 (C-4), 151.5 (C-2), 140.6 (C-6), 101.6 (C-5),
88.4 (C-1Ј), 81.2 (C-4Ј), 79.9 (C-2Ј), 74.0 (C-3Ј), 70.5 (C-3Љ),
1-[2-C-Allyl-3,5-di-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-
ꢀ-D-arabinofuranosyl]uracil (17)
CeCl3ؒ7H2O (20.0 g, 53.7 mmol) was added to a three-necked
flask and heated at 140 ЊC under vacuum for 12 h. The flask was
purged with N2 and after cooling on an ice bath, THF (150 cm3,
freshly distilled from sodium–benzophenone) was slowly added
and stirring was continued for 2 h at rt. The suspension was
cooled to Ϫ78 ЊC and allyl magnesium bromide (53.7 cm3 of a
1.0 M solution in diethyl ether, 53.7 mmol) was added dropwise
over 20 min and the mixture was stirred for an additional 2 h. A
solution of 3Ј,5Ј-di-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-
diyl)-2Ј-ketouridine26 (5.95 g, 12.3 mmol) in anhydrous THF
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 5 1 4 – 3 5 2 6
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60.2 (C-5Ј), 37.4 (–SO2CH3), 29.6 (C-1Љ), 22.8 (C-2Љ), 17.5, 17.4,
17.2, 17.1, 17.0 and 16.9 (–SiCH(CH3)2), 13.5, 13.1, 12.9 and
12.5 (–SiCH(CH3)2); FAB-MS m/z 623 [M ϩ H]ϩ.
1-[3-O-(2-Cyanoethoxy)(diisopropylamino)phosphino-5-O-(4,4Ј-
dimethoxytrityl)-2-O,2-C-propano-ꢀ-D-arabinofuranosyl]uracil
(23)
By the procedure described above for synthesis of compound
10, treatment of compound 22 (340 mg, 0.59 mmol) with
2-cyanoethyl N,NЈ-diisopropylphosphoramidochloridite (166
mg, 0.70 mmol) in the presence of N,NЈ-diisopropylethylamine
(0.4 cm3) and anhydrous CH2Cl2 (3.0 cm3) afforded a residue
that was purified by column chromatography [EtOAc–n-
hexane–Et3N (49.5 : 50 : 0.5 (v/v/v)] to yield phosphoramidite
23 (277 mg, 61%) as a white solid material.. Rf 0.61 (CH2Cl2–
MeOH 95 : 5, v/v); δP (CDCl3) 152.2 and 151.3; FAB-MS m/z
787 [M ϩ H]ϩ.
1-[2-O,2-C-Propano-3,5-di-O-(1,1,3,3-tetraisopropyldisiloxane-
1,3-diyl)-ꢀ-D-arabinofuranosyl]uracil (20)
By the procedure described above for synthesis of compound
8a, cyclization of nucleoside 19 (1.20 g, 1.93 mmol) using
NaH (60% suspension in mineral oil, 154 mg, 3.85 mmol) in
anhydrous THF (15.0 cm3) afforded a residue that was purified
by dry column chromatography33 [0–2% (v/v) MeOH in
CH2Cl2] to furnish nucleoside 20 as a white solid material (772
mg, 76%); Rf 0.52 (CH2Cl2–MeOH 95 : 5, v/v); δH (CDCl3) 9.18
(1H, br s, –NH), 7.80 (1H, d, J 8.2, H-6), 5.76 (1H, s, H-1Ј), 5.67
(1H, dd, J 1.8 and 8.1, H-5), 4.22 (1H, d, J 9.2, H-3Ј), 4.12 (1H,
dd, J 1.6 and 13.2, H-5Јa), 3.98 (1H, dd, J 2.8 and 13.2, H-5Јb),
3.77–3.74 (2H, m, H-3Љ), 3.65 (1H, ddd, J 1.7, 2.6 and 9.3,
H-4Ј), 2.41 (1H, m, H-1Љa), 2.14 (1H, m, H-2Љa), 1.99–1.86 (2H,
m, H-1Љb and H-2Љb), 1.11–0.94 (28H, m, –SiCH(CH3)2);
δC (CDCl3) 163.4 (C-4), 150.4 (C-2), 140.1 (C-6), 101.2 (C-5),
88.8 and 88.5 (C-1Ј and C-2Ј), 80.9 (C-4Ј), 71.0 (C-3Ј), 69.7
(C-3Љ), 60.1 (C-5Ј), 29.3 (C-1Љ), 25.7 (C-2Љ), 17.4, 17.3, 17.2,
17.1, 17.0 and 16.9 (–SiCH(CH3)2), 13.4, 13.0, 12.8 and 12.4
(–SiCH(CH3)2); FAB-MS m/z 527 [M ϩ H]ϩ; Anal. Calcd for
C24H42N2O7Si2: C, 54.7; H, 8.0; N, 5.3. Found: C, 54.6; H, 8.1;
N, 5.0%.
1-[2-C-(2-Hydroxyethyl)-3,5-di-O-(1,1,3,3-tetraisopropyldisil-
oxane-1,3-diyl)-ꢀ-D-arabinofuranosyl]uracil (24)
By the procedure described above for synthesis of compound
11a, oxidative cleavage of the terminal double bond of nucleo-
side 17 (4.83 g, 9.18 mmol) using osmium tetraoxide (1.0 cm3 of
a 2.5% solution in tert-butyl alcohol, 98 µmol) and sodium
periodate (7.52 g, 35.2 mmol) in a mixture of THF (25.0 cm3)
and H2O (25.0 cm3), followed by reduction with sodium boro-
hydride (1.04 g, 27.5 mmol), afforded a residue that was purified
by column chromatography [3% (v/v) MeOH in CH2Cl2] to
yield nucleoside 24 as a white solid material (2.27 g, 47%); Rf
0.47 (CH2Cl2–MeOH 92 : 8, v/v); δH (CDCl3) 11.23 (1H, br s, –
NH), 7.91 (1H, d, J 8.1, H-6), 6.01 (1H, s, H-1Ј), 5.71 (1H, dd,
J 2.2 and 8.2, H-5), 4.61 (1H, m, H-2Љa), 4.17–3.94 (4H, m,
H-2Љb, H-3Ј, H-5Ј), 3.66 (1H, dd, J 1.8 and 9.9, H-4Ј), 2.50 (1H,
m, H-1Љa), 1.60 (1H, m, H-1Љb), 1.11–0.94 (28H, m, –SiCH-
(CH3)2), δC (CDCl3) 164.5 (C-4), 152.5 (C-2), 140.2 (C-6), 101.8
(C-5), 87.4 (C-1Ј), 80.9 (C-2Ј), 80.1 (C-4Ј), 73.1 (C-3Ј), 59.9
(C-2Љ), 58.5 (C-5Ј), 30.3 (C-1Љ), 17.5, 17.4, 17.3, 17.2, 17.1, 17.0,
16.8 and 16.7 (–SiCH(CH3)2), 13.4, 13.0, 12.8 and 12.3
(–SiCH(CH3)2); FAB-MS m/z 531 [M ϩ H]ϩ.
1-(2-O,2-C-Propano-ꢀ-D-arabinofuranosyl)uracil (21)
Compound 20 (734 mg, 1.4 mmol) was coevaporated with
anhydrous acetonitrile (2 × 3 cm3), dissolved in anhydrous THF
(10 cm3) and Et3N,3HF (0.45 cm3, 2.79 mmol) was added, and
the mixture was stirred for 2 h at rt. The mixture was then
evaporated to dryness under reduced pressure, and the residue
was purified by column chromatography [3–6% (v/v) MeOH in
CH2Cl2] affording nucleoside 21 as a white solid material (361
mg, 91%); Rf 0.19 (CH2Cl2–MeOH 92 : 8, v/v); δH (pyridine-d5)
13.33 (1H, br s, –NH), 8.45 (1H, d, J 8.1, H-6), 6.57 (1H, s,
H-1Ј), 5.84 (1H, d, J 7.9, H-5), 4.78 (1H, d, J 5.4, H-3Ј),
4.37–4.26 (3H, m, H-4Ј and H-5Ј), 3.81–3.72 (2H, m, H-3Љ),
2.78 (1H, m, H-1Љa), 2.16 (1H, m, H-1Љb), 1.97 (1H, m, H-2Љa),
1.86 (1H, m, H-2Љb); δC (pyridine-d5) 164.6 (C-4), 152.6 (C-2),
142.7 (C-6), 101.7 (C-5), 91.6 (C-2Ј), 89.0 (C-1Ј), 85.3 (C-4Ј),
74.3 (C-3Ј), 70.0 (C-3Љ), 61.5 (C-5Ј), 29.5 (C-1Љ), 26.4 (C-2Љ);
FAB-MS m/z 285 [M ϩ H]ϩ; Anal. Calcd for C12H16N2O6: C,
50.7; H, 5.7; N, 9.9. Found: C, 50.3; H, 5.8; N, 9.6%.
1-[2-C-(2-Methanesulfonyloxyethyl)-3,5-di-O-(1,1,3,3-tetraiso-
propyldisiloxane-1,3-diyl)-ꢀ-D-arabinofuranosyl]uracil (25)
By the procedure described above for synthesis of compound
7a, mesylation of nucleoside 24 (1.68 g, 3.17 mmol) using
methanesulfonyl chloride (0.74 cm3, 9.5 mmol) in the presence
of pyridine (20 cm3) afforded nucleoside 25 as a white solid
material which was used without further purification in the next
step; Rf 0.66 (CH2Cl2–MeOH 92 : 8, v/v); δH (CDCl3) 9.82 (1H,
br s, –NH), 7.80 (1H, d, J 8.1, H-6), 5.86 (1H, s, H-1Ј), 5.66 (1H,
d, J 8.2, H-5), 4.75 (1H, m, H-2Љa), 4.62 (1H, m, H-2Љb), 4.22
(1H, d, J 9.0, H-3Ј), 4.14 (1H, dd, J 1.8 and 13.4, H-5Јa), 4.00
(1H, dd, J 2.7 and 13.4, H-5Јb), 3.81 (1H, m, H-4Ј), 3.05 (3H, s,
–SO2CH3), 2.53 (1H, m, H-1Љa), 2.25 (1H, m, H-1Љb), 1.11–0.97
(28H, m, –SiCH(CH3)2); δC (CDCl3) 163.6 (C-4), 151.4 (C-2),
140.4 (C-6), 101.6 (C-5), 88.1 (C-1Ј), 81.0 (C-4Ј), 79.5 (C-2Ј),
74.3 (C-3Ј), 66.3 (C-2Љ), 60.1 (C-5Ј), 37.2 (–SO2CH3), 33.4
(C-3Љ), 17.4, 17.3, 17.2, 17.1, 17.0, 16.9, 16.8 and 16.7 (–SiCH-
(CH3)2), 13.4, 12.9, 12.8 and 12.4 (–SiCH(CH3)2); FAB-MS m/z
509 [M ϩ H]ϩ.
1-[5-O-(4,4Ј-Dimethoxytrityl)-2-O,2-C-propano-ꢀ-D-arabino-
furanosyl]uracil (22)
By the procedure described above for synthesis of compound 4,
compound 21 was dimethoxytritylated (260 mg, 0.91 mmol)
using 4,4Ј-dimethoxytrityl chloride (465 mg, 1.37 mmol) in
anhydrous pyridine (5 cm3) affording a yellow oil that was
purified by dry column chromatography33 [0–2% MeOH in
CH2Cl2, containing 0.5% of pyridine (v/v/v)] to yield nucleoside
22 as a white solid material (536 mg, ∼100%) Rf 0.45 (CH2Cl2–
MeOH 92 : 8, v/v); δH (CDCl3) 9.03 (1H, br s, –NH), 7.82 (1H,
d, J 8.2, H-6), 7.35–7.08 (9H, m, ArH), 6.77–6.74 (4H, m,
ArH), 5.87 (1H, s, H-1Ј), 5.35 (1H, dd, J 2.0 and 8.1, H-5), 4.14
(1H, d, J 7.0, H-3Ј), 3.76 (1H, m, H-3Љa), 3.71–3.68 (7H, m,
2 × –OCH3 and H-4Ј), 3.60 (1H, m, H-3Љb), 3.44–3.41 (2H, m,
H-5Ј), 2.24 (1H, m, H-1Љa), 1.90 (1H, m, H-2Љa), 1.84–1.80 (2H,
m, H-1Љb and H-2Љb); δC (CDCl3) 163.3 (C-4), 158.5, 150.5
(C-2), 144.4, 141.5 (C-6), 136.0, 135.4, 130.0, 129.9, 128.1,
128.0, 127.8, 126.9, 125.1, 113.1, 101.2 (C-5), 89.7 and 88.0
(C-1Ј and C-2Ј), 86.7 (–CPh3), 81.4 (C-4Ј), 73.2 (C-3Ј), 69.5 (C-
3Љ), 61.7 (C-5Ј), 55.1 (2 × –OCH3), 28.4 (C-1Љ), 25.5 (C-2Љ);
FAB-MS m/z 587 [M ϩ H]ϩ and 586 [M]ϩ.
1-[2-O,2-C-Ethano-3,5-di-O-(1,1,3,3-tetraisopropyldisiloxane-
1,3-diyl)-ꢀ-D-arabinofuranosyl]uracil (26)
By the procedure described above for synthesis of compound
8a, cyclization of nucleoside 25 (1.54 g, 2.53 mmol) using NaH
(60% suspension in mineral oil, 404 mg, 10.1 mmol) in
anhydrous THF (20.0 cm3) afforded a residue that was purified
by anhydrous column chromatography [1–3% (v/v) MeOH in
CH2Cl2] to yield nucleoside 26 as a white solid material (704
mg, 54%); Rf 0.52 (CH2Cl2–MeOH 95 : 5, v/v); δH (CDCl3) 9.05
(1H, br s, NH), 7.61 (1H, d, J 8.1, H-6), 6.20 (1H, s, H-1Ј), 5.66
(1H, d, J 2.1 and 8.1, H-5), 4.59 (1H, ddd, J 5.3, 7.3 and 8.2,
H-2Љa), 4.42 (1H, m, H-2Љb), 4.23–4.09 (2H, m, H-3Ј and
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 5 1 4 – 3 5 2 6
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Table 2 MALDI-MS m/z [M Ϫ H]Ϫ
Ribo series
Found
Ara series
Found
Calcd
Calcd
ON1: T7(X5)T6
4250
4240
4419
4367
3484
3361
2922
2881
4252
4238
4420
4364
3484
3358
2921
2879
4234
4224
4359
4303
3356
3229
2876
2846
4238
4224
4364
4308
3358
3232
2879
2837
ON2: T7(X4)T6
ON3: T5(X5)4T5
ON4: T5(X4)4T5
ON6: (X5)9T
ON7: (X4)9T
ON9: G(X5)GA(X5)A(X5)GC
ON10: G(X4)GA(X4)A(X4)GC
H-5Јa), 3.97 (1H, dd, J 2.7 and 13.2, H-5Јb), 3.55 (1H, ddd,
J 1.7, 2.6 and 9.4, H-4Ј), 3.23 (1H, m, H-1Љa), 2.57 (1H, m,
H-1Љb), 1.11–0.96 (28H, m, –SiCH(CH3)2); δC (CDCl3) 163.3
(C-4), 150.8 (C-2), 139.7 (C-6), 101.7 (C-5), 90.1 (C-2Ј), 87.9
(C-1Ј), 79.1 (C-4Ј), 72.2 (C-3Ј), 67.9 (C-2Љ), 60.0 (C-5Ј), 24.4
(C-1Љ), 17.5, 17.4, 17.2, 17.1, 17.0 and 16.9 (SiCH(CH3)2), 13.6,
12.9, 12.8 and 12.5 (SiCH(CH3)2); FAB-MS m/z 513 [M ϩ H]ϩ;
Anal. Calcd for C23H40N2O7Si2: C, 53.9; H, 7.9; N, 5.5. Found:
C, 53.5; H, 7.9; N, 5.3%.
cm3, 1.62 mmol) in the presence of N,NЈ-diisopropylethylamine
(1.0 cm3) and anhydrous CH2Cl2 (5.0 cm3) afforded a residue
that was purified by column chromatography [EtOAc–n-
hexane–Et3N (49.5 : 50 : 0.5 (v/v/v)] to yield the phos-
phoramidite 29 (251 mg, 60%) as a white solid material; Rf 0.60
(CH2Cl2–MeOH 95 : 5, v/v); δP (CDCl3) 152.6 and 152.0; FAB-
MS m/z 773 [M ϩ H]ϩ.
Synthesis, deprotection and purification of oligonucleotides
All oligomers ON1–ON11 were prepared using the phosphor-
amidite approach on a Biosearch 8750 DNA synthesizer at 0.2
µmol scale on CPG solid supports (BioGenex). The stepwise
coupling efficiencies of the phosphoramidites 10, 15, 23 and 29
(10 min coupling time) and of unmodified deoxynucleoside
phosphoramidites were > 98% using 1H-tetrazole as activator.
Standard conditions were used on the synthesizer except that
“hand-coupling conditions”34 were used when coupling the
phosphoramidites 10, 15, 23 and 29. After standard depro-
tection and cleavage from the solid support using 32% aqueous
ammonia (12 h, 55 ЊC), the oligomers were purified by precipit-
ation from ethanol. The composition of the oligomers was
verified by MALDI-MS analysis and the purity (> 80%) by
capillary gel electrophoresis. Table 2 shows the MALDI-MS
data ([M Ϫ H]Ϫ; found/calcd).
1-(2-O,2-C-Ethano-ꢀ-D-arabinofuranosyl]uracil (27)
By the procedure described above for synthesis of compound
21, desilylation of nucleoside 26 (103 mg, 0.195 mmol) using
Et3Nؒ3HF (0.1 cm3, 0.61 mmol) in THF (3.0 cm3) afforded
a residue that was purified by dry column chromatography33
[3–8% (v/v) MeOH in CH2Cl2] to yield nucleoside 27 as a white
solid material which was used in the next step without further
purification (45 mg, 82%); Rf 0.20 (CH2Cl2–MeOH 98 : 2, v/v);
δH (pyridine-d5) 13.43 (1H, br s, NH), 8.32 (1H, d, J 8.1, H-6),
6.86 (1H, s, H-1Ј), 5.83 (1H, d, J 8.1, H-5), 4.88 (1H, d, J 6.1,
H-3Ј), 4.55 (1H, ddd, J 5.5, 7.2 and 8.5, H-2Љa), 4.44 (1H, m,
H-2Љb), 4.33–4.22 (3H, m, H-4Ј, H-5Ј), 3.45 (1H, ddd, J 7.2, 8.9
and 11.3, H-1Љa), 2.79 (1H, ddd, J 6.0, 8.5 and 11.4, H-1Љb); δC
(pyridine-d5) 164.8 (C-4), 152.9 (C-2), 142.5 (C-6), 101.9 (C-5),
92.7 (C-2Ј), 88.6 (C-1Ј), 83.9 (C-4Ј), 74.6 (C-3Ј), 68.0 (C-2Љ),
61.3 (C-5Ј), 25.0 (C-1Љ); FAB-MS m/z 271 [M ϩ H]ϩ.
Thermal denaturation studies
The thermal denaturation experiments were performed on a
Perkin-Elmer UV/VIS spectrometer fitted with a PTP-6 Peltier
temperature-programming element using a medium salt buffer
solution (10 mM sodium phosphate, 100 mM sodium chloride,
0.1 mM EDTA, pH 7.0). Concentrations of 1.5 µM of the two
complementary strands were used assuming identical extinction
coefficients for modified and unmodified oligonucleotides. The
absorbance was monitored at 260 nm while raising the temper-
ature at a rate of 1 ЊC per min. The melting temperatures
(Tm values) of the duplexes were determined as the maximum
of the first derivatives of the melting curves obtained.
1-[5-O-(4,4Ј-Dimethoxytrityl)-2-O,2-C-ethano-ꢀ-D-arabino-
furanosyl]uracil (28)
By the procedure described above for synthesis of compound 4,
dimethoxytritylation of nucleoside 27 (101 mg, 0.37 mmol)
using 4,4Ј-dimethoxytrityl chloride (190 mg, 0.56 mmol) in
anhydrous pyridine (5 cm3) afforded a yellow oil that was puri-
fied by dry column chromatography33 [0–3% MeOH in CH2Cl2,
containing 0.5% of pyridine (v/v/v)] to yield nucleoside 28 as a
white solid material (190 mg, 89%); Rf 0.26 (CH2Cl2–MeOH
95 : 5, v/v); δH (CDCl3) 9.22 (1H, br s, NH), 7.71 (1H, d, J 8.1,
H-6), 7.33–7.07 (9H, m, ArH), 6.77–6.74 (4H, m, ArH), 6.19
(1H, s, H-1Ј), 5.37 (1H, d, J 8.2, H-5), 4.42–4.32 (2H, m, H-2Љ),
4.19 (1H, d, J 7.3, H-3Ј), 3.71–3.67 (7H, m, 2 × –OCH3 and
H-4Ј), 3.43 (1H, dd, J 3.3 and 10.8, H-5Јa), 3.35 (1H, dd, J 4.0
and 11.0 Hz, H-5Јb), 3.06 (1H, m, H-1Љa), 2.48 (1H, m, H-1Љb);
δC (CDCl3) 163.3 (C-4), 158.5, 150.8 (C-2), 144.3, 141.1 (C-6),
136.1, 135.3, 130.0, 129.0, 128.9, 128.1, 128.0, 126.9, 125.1 and
113.1, 101.4 (C-5), 90.9 (C-2Ј), 87.5 (C-1Ј), 86.7 (Ar3C), 80.1
(C-4Ј), 73.8 (C-3Ј), 67.7 (C-2Љ), 61.6 (C-5Ј), 55.1 (2 × –OCH3),
23.8 (C-1Љ); FAB-MS m/z 573 [M ϩ H]ϩ and 572 [M]ϩ.
Quantum mechanical calculation
All ab initio quantum mechanical calculations were carried out
using the Gaussian 98 program.35 To determine the potential
energy of pseudorotation, sugar torsion angles ν2 and ν4 were
calculated using the theory of Altona and Sundaralingam with
a maximal puckering amplitude of 36Њ.36 For each point along
the pseudorotation pathway, full geometry optimizations (HF/
6-31G) were carried out while maintaining the desired ν2 and ν4
angles and, in some cases, the ε torsion angle was constrained as
well. Single point energies were determined using second-order
Møller–Plesset theory (MP2) with the cc-pVTZ basis set.
1-[3-O-(2-Cyanoethoxy)(diisopropylamino)phosphino-5-O-(4,4Ј-
dimethoxytrityl)-2-O,2-C-ethano-ꢀ-D-arabinofuranosyl]uracil
(29)
Karplus equation
By the procedure described above for synthesis of compound
10, treatment of compound 28 (310 mg, 0.54 mmol) with
2-cyanoethyl N,NЈ-diisopropylphosphoramidochloridite (0.36
A Karplus relationship correlating JH3ЈH4Ј and the ν3 torsion
angle was constructed using a state-of-the-art Karplus equation
(eqn. 1) for nucleosides and nucleotides developed by Altona
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 5 1 4 – 3 5 2 6
3525

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and co-workers,28–30 where the electronegativity of the HCCH-
fragment substituents is accounted for in the coefficients Cm
and Sn:
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15 R. H. Griffey, B. P. Monia, L. L. Cummins, S. Freier, M. J. Greig,
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Force field calculations were carried out using the AMBER7
suite of programs.37 Atomic charges for the X4-Ribo and
X4-Ara nucleotides were calculated using the RESP pro-
cedure.38 Initial models of A- and B-type geometry were built
using the nucgen module and the relevant residues were modi-
fied. The duplexes were solvated with a truncated octahedral
box of TIP3P water molecules, so that no duplex atom was
closer to the boundary than 10 Å, which resulted in truncated
octahedral boxes with side lengths of ∼58 Å. To obtain electro-
static neutrality, 16 sodium ions were included in the calcu-
lations. The total system sizes varied between 12000 and 13000
atoms. All bonds with hydrogen atoms involved were con-
strained with SHAKE. The particle mesh Ewald (PME)
method was used in all calculations to evaluate electrostatic
interactions. The default parameter values in AMBER were
used in PME calculations. For equilibration, the systems were
initially energy minimised, followed by 70 ps of molecular
dynamics at 300 K and 1 atm pressure, in which the solute was
positionally restrained with incrementally decreasing force
18 P. Nielsen, K. Larsen and J. Wengel, Acta Chem. Scand., 1996, 50,
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21 M. S. Wolfe and R. E. Harry-O’kuru, Tetrahedron Lett., 1995, 36,
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22 G. P. Cook and M. M. Greenberg, J. Org. Chem., 1994, 59, 4704.
23 T. Imamoto, N. Takiyama, K. Nakamura, T. Hatajima and
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25 T. Iino, S. Shuto and A. Matsuda, Nucleosides Nucleotides, 1996, 15,
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constants (500 to 0.1 kcal molϪ1
Å
Ϫ2). In the production
26 M. J. Robins, J. S. Wilson and F. Hansske, J. Am. Chem. Soc., 1983,
105, 4059.
27 IUPAC-IUB Joint Commission on Biochemical Nomenclature, Eur.
J. Biochem., 1983, 131, 9.
28 L. A. Donders, F. A. A. M. de Leeuw and C. Altona, Magn. Reson.
Chem., 1989, 27, 556.
29 C. Altona, J. H. Ippel, A. J. A. Westra Hoekzema, C. Erkelens,
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31 K. Danel, E. B. Pedersen and C. Nielsen, J. Med. Chem., 1998, 41,
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calculations, temperature coupling was switched off and the
simulations were carried out in the microcanonical ensemble. A
time step of 2 fs was used. In the simulations starting from
A-type duplex geometry, the trajectories were extended to 2 ns,
while the B-simulations were run for 1 ns.
Acknowledgements
The Danish National Science Foundation, the Danish Natural
Science Research Council and the Danish Technical Research
Council are acknowledged for financial support. Ms Britta
M. Dahl is thanked for oligonucleotide synthesis and
Dr Michael Meldgaard, Exiqon A/S, for MALDI-MS analysis.
32 T. E. Cheatham III and P. A. Kollman, J. Am. Chem. Soc., 1997, 119,
4805.
33 D. S. Pedersen and C. Rosenbohm, Synthesis, 2001, 2431.
34 V. K. Rajwanshi, A. E. Håkansson, B. M. Dahl and J. Wengel,
Chem. Commun., 1999, 1395.
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