Synthesis and structure of dinucleotides with S-type sugar puckering and noncanonical ε and ζ torsion angle combination (ν2, ε,ζ-D-CNA)

Article abstract of DOI:10.1002/ejoc.200701022

The synthesis of the diastereoisomers of ν₂,ε,ζ-D- CNA dinucleotide building units of nucleic acids and their (2′,5′)- ν₂,ε′,ζ′-D-CNA analogues, in which the ε and ζ torsional angles are stereocontrolled by a dioxaphosphorinane ring

Full text of DOI:10.1002/ejoc.200701022

FULL PAPER
DOI: 10.1002/ejoc.200701022
Synthesis and Structure of Dinucleotides with S-Type Sugar Puckering and
Noncanonical ε and ζ Torsion Angle Combination (ν2,ε,ζ-D-CNA)
Christelle Dupouy,[a] Pierre Lavedan,[b] and Jean-Marc Escudier*[a]
Keywords: Strained molecules / Nucleotides / DNA structure / Phosphorus heterocycles / Conformation analysis
The synthesis of the diastereoisomers of ν2,ε,ζ-D-CNA dinu-
cleotide building units of nucleic acids and their (2Ј,5Ј)-
ν2,εЈ,ζЈ-D-CNA analogues, in which the ε and ζ torsional
angles are stereocontrolled by a dioxaphosphorinane ring
structure (D-CNA family) is described from a common 3-de-
ν2,ε,ζ-D-CNA shows that the sugar puckering is fixed in the
C-2Ј-endo conformation and that these D-CNA structural
elements allow stabilization of the ε/ζ torsion angle combina-
tions, which are significantly different from the typical com-
binations observed in canonical A- or B-form duplexes.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2008)
oxy-3-(hydroxymethyl)-D-allofuranose intermediate. NMR
spectroscopic and circular dichroism structure analysis of
Introduction
cases, the α and γ torsion angles (Figure 1) can exhibit non-
canonical conformations with gauche(+) or trans and
gauche(–) or trans values, respectively, in protein-bound B-
DNA oligomers.[13] In contrast, it was shown that ε and ζ
conformations need to be shifted from the canonical BI
state to the BII state to achieve the helical changes that
are necessary so that DNA can properly interact with the
proteins.[14] Interestingly, many biologically relevant nucleic
acid structures exhibit more unusual conformations of the
α to ζ torsional angles that participate in structural distor-
tions specifically involved in particular macromolecular
processes.[15] Unfortunately, experimental studies aimed at
determining the structural and functional implications of
such helical deformations are somewhat complicated by the
intrinsically transient nature of the corresponding back-
bone states. Stable structural analogues of these distorted
backbone geometries would be very useful in the elucida-
tion of the role that helical deformations play in nucleic
acid interactions.
We are interested in the development of conformation-
ally constrained dinucleotide building units, in which the
backbone α to ζ torsion angles of nucleic acids can have
predefined values that closely match, or on the contrary
significantly deviate from, the typical values observed in
DNA and RNA duplexes. Our general strategy used to lock
the phosphate backbone torsional angles is based on the
introduction of the 1,3,2-dioxaphosphorinane structure at
key positions along the phosphate backbone (D-CNA fam-
ily).
The synthesis and evaluation of oligonucleotide ana-
logues for the construction of tools in DNA diagnostics, in
therapy, and in bio- or nanotechnology have recently been
the topics of intense research interest.[1–3] As part of these
analogues, conformationally constrained nucleosides such
as LNA or di- and tricyclo-DNA are a useful class of ana-
logues.[4–8] They were especially designed to mimic the con-
formation adopted by the sugar moiety of nucleotides in
duplexes in order to enhance, once incorporated in a single-
stranded DNA or RNA, the duplex formation ability.[9] In
contrast, mimicking important biologically relevant nonhe-
lical secondary structures of functional DNA or RNA by
designing conformationally restricted nucleotides has re-
ceived less attention.[10,11]
Together with the double-helix structure, nucleic acids
can adopt alternative conformations such as bulges, hair-
pins, branched junctions, or U-turns.[12] These secondary
structures are often characterized by non-Watson–Crick
pairs or unpaired nucleotides and remarkable sugar–phos-
phate backbone distortions with torsion-angle conforma-
tions that are significantly different from those observed in
regular double-stranded helices.
Sequence-specific protein–DNA recognition processes
very often require fine adjustment of the sugar–phosphate
backbone conformation to form stable duplexes. In some
[a] Laboratoire de Synthèse et Physico-Chimie de Molécules
d’Intérêt Biologique, UMR 5068 CNRS, Université Paul Sabatier,
31062 Toulouse Cedex 9, France
E-mail: escudier@chimie.ups-tlse.fr
[b] Service Commun de RMN, Université Paul Sabatier,
31062 Toulouse Cedex 9, France
We previously reported the synthesis of α,β-D-CNA
(dioxaphosphorinane-constrained nucleic acid) featuring
canonical or noncanonical values of the α and β torsional
angles,[16,17] and subsequently, on their properties within
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Figure 1. Left: the six backbone torsion angles (labeled αa to ζa for the upper nucleotide and αb to ζb for the lower nucleotide) of
nucleic acids; middle: ν2,ε,ζ-D-CNA dinucleotide, in which ν2a, εa, and ζa are stereocontrolled to canonical or noncanonical values by a
dioxaphosphorinane ring structure exhibiting two new asymmetric centers; right: (2Ј,5Ј)-ν2,εЈ,ζЈ-D-CNA dinucleotide, in which ν2a, εЈ,
and ζЈ are stereocontrolled by a dioxaphosphorinane ring structure exhibiting an asymmetric phosphorus center.
oligonucleotides.[18,19] The methodology was then and the (RC2Ј,RP) and (RC2Ј,SP) isomers of ν2,ε,ζ-D-CNA,
extended to enlarge the α,β-D-CNA family[20,21] and to which are two novel classes of constrained dinucleotide
prepare α,β,γ-D-CNA, δ,ε,ζ-D-CNA, or xylo-ε,ζ-D- building units.
CNA.[22,23] The ε/ζ combinations reached to date were in
most cases noncanonical forms of the B-type duplex, which
were excepted with one isomer of δ,ε,ζ-D-CNA featuring a
Results and Discussion
BII-like conformation with ε and ζ in a {gauche(–)/
Our retrosynthetic analysis for the preparation of the two
anticlinal(+),trans} conformation. In most of these con- diastereoisomers of the ν2,ε,ζ-D-CNA unit is described in
strained dinucleotides, the sugar puckering was not altered Figure 2 (Path A). The dioxaphosphorinane structure will
and the 2Ј-deoxyribose units were in a C-2Ј-endo conforma- be formed by the cyclization process already reported for
tion.
the synthesis of other D-CNA, that is, ring closure reaction
To complete the ε,ζ combination set and therefore to dis- of a 2Ј-C-substituted dinucleotide precursor in which the
poseofnewmembersoftheD-CNAfamily,wefusedthedioxa- pro-(R) and pro-(S)-phosphate oxyanions can attack an
phosphorinane ring to the sugar ring by connecting an oxy- electrophilic carbon atom.[16] The acyclic dinucleotide pre-
gen atom of the phosphate group to the 3Ј- or 2Ј-C with a cursor can be prepared from protected 2-deoxy-2-hy-
methylene bridge. In these new D-CNAs, termed ν2,ε,ζ-D- droxymethyl--ribofuranosyl nucleoside II by standard
CNA or (2Ј,5Ј)-ν2,εЈ,ζЈ-D-CNA, the inner sugar ν2 tor- phosphoramidite procedures.[24] The base will be intro-
sional angle of the sugar is constrained together with ε and duced by Vorbrüggen’s reaction[25] on protected 2-deoxy-2-
ζ or newly defined εЈ and ζЈ, respectively. In the present hydroxymethyl--ribofuranose I.[26] By following the pro-
work, we report the synthesis, from 3-deoxy-3-(hydroxy- cedure described by Marquez and Driscoll, this substituted
methyl)--allofuranose, and conformational behavior of the ribofuranose will be synthesized by oxidative cleavage of the
(RC3Ј,SP) and (RC3Ј,RP) isomers of (2Ј,5Ј)-ν2,εЈ,ζЈ-D-CNA C-1/C-2 bond of 3-deoxy-3-(hydroxymethyl)--allofuranose
Figure 2. Retrosynthetic pathway for the synthesis of ν2,ε,ζ-D-CNA dimers and their (2Ј-5Ј)-ν2,εЈ,ζЈ-D-CNA analogues from common
intermediate 3-deoxy-3-(hydroxymethyl)--allofuranose.
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Dinucleotides with S-Type Sugar Puckering and ε and ζ Torsion Angles
prepared from commercially available 1,2:5,6-diisopropyl-
idene--glucose. We chose to use 3-deoxy-3-(hydroxy-
methyl)--allofuranose as the precursor instead of the more
obvious 3-deoxy-3-(hydroxymethyl)--ribofuranose[27] or
2Ј-C-functionalized nucleosides[28,29] because it can undergo
additional oxidative cleavage of the C-5/C-6 bond (Figure 2,
Path B) to provide protected 3-deoxy-3-hydroxymethyl--
ribofuranose III. Compound III is the starting material for
the synthesis of the (2Ј,5Ј)-ν2,εЈ,ζЈ-D-CNA analogues of
ν2,ε,ζ-D-CNA. Protected 3-deoxy-3-hydroxymethyl--ri-
bofuranosyl nucleoside IV will be coupled to a 5Ј-O-phos-
phoramidite nucleotide to provide the acyclic precursor that
will be cyclized to form the dioxaphosphorinane structure.
In (2Ј,5Ј)-ν2,εЈ,ζЈ-D-CNA, we introduced the new torsional
angles εЈ and ζЈ defined as follows: εЈ (P–O2Ј–C2Ј–C3Ј) and
ζЈ (O5Ј–P–O2Ј–C2Ј).
Scheme 1. Reagents and conditions: (a) TMSOTf, bis(trimethylsi-
lyl)thymine, CH3CN, reflux, 2 h; (b) concentrated NH4OH, room
temp., 2 h; (c) 3Ј-O-tert-butyldiphenylsilylthymidine-5Ј-O- phos-
phoramidite (2 equiv.), tetrazole (10 equiv.), CH3CN then collidine,
I2/H2O, room temp., 3 h.
Synthesis of ν2,ε,ζ-D-CNA and (2Ј,5Ј)-ν2,εЈ,ζЈ-D-CNA
Dinucleotides
Starting 2Ј- or 3Ј-C-hydroxymethyl-substituted ribo-
furanose 1 and 2 were obtained after oxidative cleavage of
the suitably protected 3-deoxy-3-(hydroxymethyl)--allofu-
ranose as described by Marquez and Driscoll.[26] Introduc-
tion of the thymine base by the Vorbrüggen procedure pro-
ceeded in high yield with a high selectivity in favor of the
β-anomer, as expected for 2-O-acetylribose 1 (Scheme 1). In
contrast, a lower yield (56%) and a lower selectivity (β/α =
1
9:1 as determined by H NMR spectroscopy) was obtained
from 3-O-formylribose 2. This could be explained by the
fact that the 2Ј-benzoyloxymethyl group is less efficient in
the stabilization of the intermediate carbocation, which
therefore allows a small level of undesirable α-face attack.
The 2Ј- or 3Ј-hydroxy functionalities were released by treat-
ment of nucleosides 3 or 4 with ammonia. The resulting
nucleosides were then coupled with the readily available 3Ј-
O-tert-butyldiphenylsilyl-5Ј-O-phosphoramidite thymidine
according to a standard phosphoramidite procedure[24] to
give dinucleotides 5 and 6 in each case as a diastereoiso-
meric mixture (1:1 ratio as determined by 31P NMR spec-
troscopy: δP = –2.1 and –2.3 ppm for 5; δP = –1.8 and
–2.1 ppm for 6).
Scheme 2. Reagents and conditions: (a) K2CO3, MeOH/H2O (4:1),
room temp., 6 h; (b) 1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole
(MSNT), pyridine, room temp. for 1 h, then 80 °C for 30 min, and
90 °C for 30 min.
We applied the well-known phosphotriester methodology
in the presence of 1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-tri-
azole (MSNT) as an activating agent[30] to prepare the
(RC3Ј, SP) and (RC3Ј, RP) (2Ј,5Ј)-ν2,εЈ,ζЈ-D-CNA diastereo-
Surprisingly, when applied to the synthesis of ν2,ε,ζ-D-
isomers 8 and 9, respectively, in good yield (80%). They CNA, the removal of the benzoyl and cyanoethyl protecting
were obtained as a 1.8:1 mixture of diastereoisomers as de- groups only proceeded in moderate yield (45%), and we
termined by 31P NMR spectroscopy: δP = –9.7 and noticed concomitant cleavage of the tert-butyldiphenylsilyl
–6.0 ppm. In this particular case, the diastereoselectivity protecting group that provided acyclic dinucleotides 10 and
observed was certainly due to a more favorable axial posi- 11 in a 1:1 ratio (Scheme 3). Nevertheless, when treated
tion of the lower nucleoside in the transition state that fa- with 1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole, 10
vored the (RC3Ј,SP)-configured dioxaphosphorinane dia- underwent diastereoselective cyclization to provide 3Ј-O-
stereoisomer. Acyclic phosphodiester 7 was generated in protected ν2,ε,ζ-D-CNA 12 and 13 in a 1:1.8 ratio (31P
high yield from 5 by removal of the benzoyl and cyanoethyl NMR: δP = –6.0 and –9.4 ppm, respectively) in 75% yield.
groups with K2CO3 (90%, Scheme 2).
This result is highly comparable with that obtained for the
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C. Dupouy, P. Lavedan, J.-M. Escudier
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Scheme 3. Reagents and conditions: (a) K2CO3, MeOH/H2O (4:1), room temp., 6 h; (b) 1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole
(MSNT), pyridine, room temp. for 1 h, then 80 °C for 30 min, and 90 °C for 30 min.
formation of (2Ј,5Ј)-ν2,εЈ,ζЈ-D-CNA. In contrast, the diox-
The chair conformation of the dioxaphosphorinane
aphosphorinane rings were formed with a lower diastereo- structure of 8 is clearly established from the 1H NMR spec-
selectivity from fully deprotected dinucleotide 11 to give tra, and small (3JH6Ј,P Ͻ1 Hz) and large (3JH6Ј,P ≈ 22 Hz)
ν2,ε,ζ-D-CNA 14 and 15 in a 1:1.4 ratio (31P NMR: δP = 3JH,P coupling constants between the 6Јa-H and 6"a-H pro-
–6.6 and –7.8 ppm, respectively) in 60% yield.
tons and phosphorus are observed, which is characteristic
of an axial position (0 Յ 3JHax,P Յ 3 Hz) and an equatorial
3
position (20 Յ JHeq,P Յ 30 Hz) of these protons, respec-
Structural Assignment of the (2Ј,5Ј)-ν2,εЈ,ζЈ-D-CNA TT
Dimers
tively (Table 2).[33] The undetectable coupling constant
between the 2Јa-H proton and the phosphorus atom also
provided evidence for the axial position of this proton and
for the chair conformation of the dioxaphosphorinane
ring.
To obtain insight into the structural behavior of each
1
1
1
constrained dinucleotide, H, H–{31P}, 2D COSY H–1H,
1
2D COSY H–31P, and 2D NOESY NMR spectra were re-
corded at 300 and 500 MHz in deuterated chloroform for
(RC3Ј,SP) (2Ј,5Ј)-ν2,εЈ,ζЈ-D-CNA 8 and (RC3Ј,RP) (2Ј,5Ј)-
ν2,εЈ,ζЈ-D-CNA 9.
Table 2. H,P coupling constants in the 1H NMR spectra (500 MHz,
CDCl3) of (RC3Ј,SP) and (RC3Ј,RP) (2Ј,5Ј)-ν2,εЈ,ζЈ-D-CNA dia-
stereoisomers.
The puckering of the 2Ј- and 3Ј-deoxyribose moieties of
8 and 9 was assigned by examination of the sugar ring H,H
coupling constants (Table 1). The C3Ј-endo conformation
of the upper nucleosides was clearly established with an un-
detectable coupling constant between 1Ј-H and 2Ј-H and a
large coupling constant between 3Ј-H and 4Ј-H. For the
lower sugar, the relatively small JH3Ј,H4Ј measured for the
lower nucleosides and the values of JH2Ј,H3Ј and JH1Ј,H2Ј are
close in each case to those found in the standard C2Ј-endo
conformation of the natural 2Ј-deoxyribose.[31,32] Therefore,
as a consequence of the introduction of the dioxaphosphor-
inane ring, these dinucleotides display a constrained North/
South junction.
Dinucleotide
Coupling constant J [Hz]
3J2Јa,P
4J4Јb,P
3J5Јb,P
3J6Јa,P
8
9
0
4.3
Ͻ1
0
8.0; 3.0
6.7; 2.5
22.0
20.3
Ͻ1
5.6
In contrast, average values of 4.3, 5.6, and 20.3 Hz were
observed for the 3JH,P coupling constants involving the 2Јa-
H, 6Јa-H, and 6ЈЈa-H protons, respectively of (RC3Ј,RP)
(2Ј,5Ј)-ν2,εЈ,ζЈ-D-CNA 9, which thus suggests that the di-
oxaphosphorinane structure of this isomer is in a twist-
chair conformation. In both diastereoisomers, there is no
observable coupling constant between 4Јb-H and phospho-
rus; this is indicative of the fact that these compounds do
not adopt the canonical gauche(+) γ conformation fre-
quently observed in nucleotides.[31,32] Thus, on the basis of
Table 1. H,H coupling constants in the 1H NMR spectra
(500 MHz, CDCl3) of (RC3Ј,SP) and (RC3Ј,RP) (2Ј,5Ј)-ν2,εЈ,ζЈ-D-
CNA diastereoisomers (n.d. = not determined).
3
3
3
careful examination of the JH,H and JH,P coupling con-
stants for the P-coupled CH and CH2 signals of 8 and 9,
we were able to establish conclusively the conformation of
the backbone torsional angles εЈ (P–O2Ј–C2Ј–C3Ј) and ζЈ
(O5Ј–P–O2Ј–C2Ј)as(c+,g+/a+)and(c–,a–)forthe(RC3Ј,SP)and
(RC3Ј, RP) diastereoisomers, respectively.
Nucleoside
Coupling constant J [Hz]
J1Ј,2Ј
J2Ј,3Ј
J3Ј,4Ј
8
9
upper
lower
upper
lower
0
7.0
0
–
7.0
–
4.5
5.5
6.0
2.5
–
4.0
–
11.0
n.d.
9.7
8.0
5.5
n.d.
2.5
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Dinucleotides with S-Type Sugar Puckering and ε and ζ Torsion Angles
Structural Assignment of the ν2,ε,ζ-D-CNA TT Dimers
Table 3. H,H coupling constants in the 1H NMR spectra
(500 MHz) of (RC2Ј,RP) and (RC2Ј,SP) ν2,ε,ζ-D-CNA diastereoiso-
mers (12 and 13 in CDCl3, 14 and 15 in CD3OD, n.d. = not deter-
mined).
CD spectra were recorded in phosphate buffer (pH 7.0)
at 20, 40, 60, and 80 °C and compared with those of an
3
Nucleoside
Coupling constant J [Hz]
unmodified TPT dinucleotide (Figure 3) in order to have
information about the behavior of the two thymine moieties
in ν2,ε,ζ-D-CNA 14 and 15.
J1Ј,2Ј
J2Ј,3Ј
J3Ј,4Ј
12
13
14
15
upper
lower
upper
lower
upper
lower
upper
lower
9.0
8.0
9.5
6.5
9.0
7.0
9.5
7.0
–
6.0
–
6.5
–
7.0
–
7.0
4.5
6.0
4.5
6.5
5.0
6.5
4.5
6.5
–
2.0
–
7.0
–
6.5
–
6.5
0
Ͻ1
Ͻ1
n.d.
Ͻ1
3.6
Ͻ1
3.3
The most striking features are the low intensity and the
very little temperature dependence of the positive Cotton
effect observed around 270 nm for ν2,ε,ζ-D-CNA 14 and 15
relative to that of the unstrained TPT. These results can be
undoubtedly explained in terms of the relative conforma-
tional rigidity of the dinucleotide structure, which does not
allow base stacking in this particular conformation of the
ε and ζ torsion angles. The CD spectra of these di-
nucleotides are therefore highly comparable to the CD
spectrum of the corresponding monomer.[34] Surprisingly,
the minimum intensity of the positive Cotton effect
(∆ε = 0.66 –1 cm–1) was observed at 20 °C for (RC2Ј,RP)
ν2,ε,ζ-D-CNA 14, whereas ∆ε increased when the tempera-
ture reached 80 °C (∆ε = 0.94 –1 cm–1); this was ac-
companied by a shift in the absorption maximum (∆λ =
7 nm). This result suggests that the dioxaphosphorinane
structure could be affected by increasing temperature and,
therefore, should not be in a rigid chair conformation.
To determine the relative spatial arrangement of the two
The chair conformation of the dioxaphosphorinane
structure of 3Ј-O-protected (RC2Ј,SP) ν2,ε,ζ-D-CNA 13 and
1
(RC2Ј,SP) ν2,ε,ζ-D-CNA 15 is clearly established from H
and H–{31P} NMR spectra (Table 4), and no detectable
1
coupling constant between the 3Јa-H involved in the di-
oxaphosphorinane system and phosphorus (3JH3Јa,P ≈ 0)
was observed, which is characteristic of an axial position of
this proton.[33] The observation of small (Ͻ1 Hz) and large
3
(Ͼ24 Hz) JH,P coupling constants between the dioxaphos-
phorinane 6Јa-H protons and the phosphorus atom are also
indicative of an axial and an equatorial position of these
protons, respectively.
1
1
1
nucleotides, H, H–{31P}, 2D COSY H–1H, 2D COSY
1H–31P, and 2D NOESY NMR spectra were recorded at
300 and 500 MHz in deuterated chloroform for 3Ј-O-pro-
tected ν2,ε,ζ-D-CNA 12 and 13 and in deuterated methanol
for ν2,ε,ζ-D-CNA 14 and 15.
Table 4. H,P coupling constants in the 1H NMR spectra (500 MHz)
of (RC2Ј,RP) and (RC2Ј,SP) ν2,ε,ζ-D-CNA diastereoisomers (12 and
13 in CDCl3, 14 and 15 in CD3OD, n.d. = not determined).
Dinucleotide
Coupling constant J [Hz]
3J3Јa,P
4J4Јb,P
3J5Јb,P
3J6Јa,P
The puckering of the 2Ј-deoxyribose moieties in 12, 13,
14, and 15 was assigned by examination of the sugar ring
H,H coupling constants (Table 3). In all cases, according to
the approximation of Altona and Sundaralingam: South
12
13
14
15
Ͻ1
Ͻ1
2.5
0
Ͻ1
Ͻ1
2.2
0
5.5; 3.5
6.0; Ͻ1
n.d.
20.0
26.0
20.1
24.0
2.4
Ͻ1
6.9
1.5
3
(%) = [3JH1Ј,H2Ј/(3JH1Ј,H2Ј + JH3Ј,H4Ј)]ϫ100.[35] The sugar
7.7; 3.3
puckering of the upper nucleoside of ν2,ε,ζ-D-CNA was de-
termined to be in a constrained South (C2Ј-endo) confor-
The analysis of minor isomer (RC2Ј,RP) ν2,ε,ζ-D-CNA 14
3
3
mation (100%). With smaller JH1Ј,H2Ј and larger JH3Ј,H4Ј was complicated by the overlapping of the 6Јa-H, 3Јb-H,
3
and JH2Ј,H3Ј values, the lower sugar moieties of ν2,ε,ζ-D- and 5Јb-H signals (Figure 4). Whereas the 3JH6"a,P coupling
1
CNA exhibit coupling constants close to those found for constants were readily accessible (6.9 Hz) from the H and
the unmodified TT dimer (63% of C2Ј-endo conformation 1H–{31P} NMR spectra, the JH6Јa,P was not. We recorded
3
for the lower thymidine).[31]
a 1H COSY spectrum performed with a selective irradiation
Figure 3. CD spectra in 10 m sodium phosphate (pH 7.0) at 80, 60, 40, and 20 °C of: (a) TpT; (b) (RC2Ј,RP) ν2,ε,ζ-D-CNA TT 14; and
(c) (RC2Ј,SP) ν2,ε,ζ-D-CNA TT 15.
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C. Dupouy, P. Lavedan, J.-M. Escudier
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at δ = 2.89 ppm corresponding to saturation of the 2Јa-H ther investigated by examination of the 2D NOESY NMR
signal. The resulting pattern obtained around 4.46 ppm spectra and their comparison with an unmodified dinucleo-
only exhibited a signal from the 6Јa-H proton, which has tide (Figure 5). Although many cross peaks are present for
an effective scalar coupling with 2Јa-H. We were, therefore, 14 and 15, all of them are derived from intraresidual H,H
3
able to determine the value of JH6Јa,P (20.1 Hz), together interactions that are indicative of the sugar puckering
3
3
with values of JH6Јa,H2Јa and JH6Јa,H6"a (2.1 and 12.2 Hz, (South-type) and the relative position of the thymine bases
respectively).
towards theirs corresponding 2Ј-deoxyribose rings.
All together, the data collected from NMR spectroscopy
helped us to propose the conformational ranges of the δ–
ζ torsional angles for each ν2,ε,ζ-D-CNA diastereoisomer
(Table 5). Those of a dinucleotide involved in a natural A-,
BI-, or BII-type duplex are given as reference.[37]
Table 5. Summary of the backbone δ, ε, and ζ torsion angles de-
rived from the canonical A-DNA duplex structure and that of ν2-
ε,ζ-D-CNA dimeric units 14 and 15.[a]
Structure
δ
ε
ζ
ε–ζ
A-type
BI-type
BII-type
(RC2Ј,RP)-14
(RC2Ј,SP)-15
g+/a+
a+
–/t
t
g–
g–/a–
t
Ͻ0
0
Ͼ0
Ͻ0
0
a+/t
a+/t
a+/t
g–/a–
a+/t
a+/t
a–
g+
[a] The following sixfold staggered pattern of the torsional angles
is used: cis = 0Ϯ30° (c), gauche(+) = 60Ϯ30° (g+), anticlinal(+) =
120Ϯ30° (a+), trans = 180Ϯ30° (t), anticlinal(–) = 240Ϯ30° (a–),
gauche(–) = 300Ϯ30° (g–). The notation a–/t is used to designate a
torsion angle on the border of anticlinal(–) and trans.
Figure 4. NMR spectroscopic data for 6Јb-H of (RC2Ј,RP)
ν2,ε,ζ-D-CNA TT 14: (a) 1H NMR and (b) 1H COSY with selective
irradiation at δ = 2.89 ppm.
The average values of 2.4, 6.9, 20.0, and 20.1 Hz ob-
3
served for the JH,P coupling constants involving the 6Јa-H
and 6"a-H protons of 12 and 14, respectively, suggest that
When compared with the backbone conformations sum-
the dioxaphosphorinane structure of these (RC2Ј,RP)-con- marized in Table 5, we can expect that DNA oligonucleo-
figured isomers are in a twist-chair conformation. This “in- tides incorporating the (RC2Ј, RP) dinucleotide element
between” conformation probably results from the involve- would help to better understand the relative contribution of
ment of conflicting steric and anomeric effects in the chair a fixed C2Ј-endo sugar puckering with respect to a slightly
conformations of 12 and 14 as a result of the preferred C2Ј- distorted BI-like substrate (ε–ζ Ͻ 0°, centered around –90°).
endo puckering of the sugar of the upper nucleoside and In contrast, stereoisomer (RC2Ј,SP)-15 exhibits a more un-
the equatorial positioning of the dioxaphosphorinane ring usual δ/ε/ζ combination [anticlinal(+)/trans, anticlinal(+)/
in the lower nucleoside substituent. Interestingly, only iso- trans, gauche(+)], which could represent an important struc-
4
mer 14 exhibits a JH4Јb,P (2.2 Hz) frequently observed in tural element that can be used to stabilize unpaired struc-
nucleotides as a consequence of the canonical gauche(+) γ tures in DNA or RNA, such as bulges or hairpins, that
conformation.[32]
could require a distorted conformation of the backbone
The overall conformations of dinucleotides (RC2Ј,RP) (ε–ζ Ͼ 0°, centered around +90°) while maintaining a C2Ј-
ν2,ε,ζ-D-CNA 14 and (RC2Ј,SP) ν2,ε,ζ-D-CNA 15 were fur- endo conformation of the sugar moiety.
Figure 5. Tentative model of the conformation of the (RC2Ј,SP) and (RC2Ј,RP) ν2,ε,ζ-D-CNA diastereoisomers derived from the NMR
spectroscopic data and that of TpT.[36]
1290
www.eurjoc.org
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 1285–1294

Dinucleotides with S-Type Sugar Puckering and ε and ζ Torsion Angles
1 H, NH), 8.05–7.96 (m, 4 H, H, ph), 7.62–7.39 (m, 6 H, ph), 7.15
(d, J = 1.2 Hz, 1 H, 6-H), 5.85 (d, J = 3.0 Hz, 1 H, 1Ј-H), 5.65 (dd,
J = 2.7 and 6.9 Hz, 1 H, 2Ј-H), 4.83 (m, 1 H, 6Ј-H), 4.60 (A part
of an ABX syst., J = 6.3 and 11.7 Hz, 1 H, 5Ј-H), 4.57–4.50 (m, 2
H, 4Ј-H and 6Ј-H), 4.45 (B part of an ABX syst., J = 6.6 and
11.7 Hz, 1 H, 5Ј-H), 3.21–3.11 (m, 1 H, 3Ј-H), 2.13 (s, 3 H, CH3),
1.67 (d, J = 1.2 Hz, 3 H, CH3) ppm. 13C NMR (75 MHz, CDCl3):
δ = 170.1, 166.1, 164.8, 150.3, 137.7, 137.0, 136.7, 133.5, 133.1,
129.6–128.2, 110.4, 90.9, 80.1, 76.8, 66.5, 64.1, 64.4, 60.7, 40.7,
32.9, 27.4, 23.0, 19.2, 10.9 ppm. MS (electrospray): m/z = 545.5.0
[M + Na]+, 523.2 [M + H]+. C27H26N2O9 (522.51): calcd. C 62.07,
H 5.02, N 5.36; found C 61.89, H 5.12, N 5.32.
Conclusions
From a common 3-deoxy-3-(hydroxymethyl)--allofura-
nose intermediate, we synthesized two new members of the
D-CNA family, in which the backbone torsional angles (ε,
ζ or εЈ, ζЈ) of nucleic acids are simultaneously locked in a
dioxaphosphorinane ring structure. Structural analysis indi-
cated that the sugar rings of ν2-ε,ζ-D-CNA dinucleotides
are locked in a C2Ј-endo (South) conformation, whereas
their (2Ј,5Ј)-ν2,εЈ,ζЈ-D-CNA analogues exhibit a C3Ј-endo
(North) conformation for the upper nucleoside sugar moi-
ety. The dioxaphosphorinane ring structure introduced as
an internucleotidic linkage in the (RC2Ј,SP) and (RC2Ј,RP)
ν2,ε,ζ-D-CNA diastereoisomers provide a (δ, ε, ζ) torsional
angle set locked in the (a+/t, a+/t, g+) and (a+/t, a+/t, a–)
conformations, respectively. In contrast, the two (RC3Ј, RP)
and (RC3Ј, SP) diastereoisomers of (2Ј,5Ј)-ν2,εЈ,ζЈ-D-CNA
exhibit very unusual backbone conformations due to the
transposition of the O3Ј–O5Ј internucleotidic linkage to
O2Ј–O5Ј. As can be seen, all of these structures are associ-
ated with very different backbone conformations and could
provide interesting tools to discriminate between the impact
of sugar puckering and backbone constrains on duplex for-
mation ability. The incorporation of D-CNA building
blocks at preselected positions in an oligonucleotide is ex-
pected to create a remarkable variety of different shapes
including helical distortions of B-DNA or nonhelical sec-
ondary structures of functional RNA.
5Ј-O-Benzoyl-2Ј-deoxy-2Ј-[(benzoyloxy)methyl]-3Ј-O-formyl-5-meth-
yluridine (4): Compound 4 was prepared following the same pro-
cedure as that described for 3. Starting from 5-O-benzoyl-3-O-for-
myl-2-deoxy-2-[(benzoyloxy)methyl]-1-O-acetylribose (2; 380 mg,
0.86 mmol), 4 (240 mg, 55%) was recovered as an oil after silica
gel chromatography (ethyl acetate/dichloromethane, 1:4). 1H NMR
(300 MHz, CDCl3): δ = 8.30 (br. s, 1 H, NH), 8.17 (s, 1 H, HCOO),
8.06–8.03 (m, 2 H, ph), 7.90–7.87 (m, 2 H, ph), 7.66–7.38 (m, 6 H,
ph), 7.10 (d, J = 1.5 Hz, 1 H, 6-H), 6.40 (d, J = 9.0 Hz, 1 H, 1Ј-
H), 5.69 (br. d, J = 6.0 Hz, 1 H, 3Ј-H), 4.75–4.66 (m, 2 H, 5Ј-H
and 6Ј-H), 4.61 (B part of an ABX syst., J = 3.0 and 12.0 Hz, 1
H, 5Ј-H), 4.51 (dd, J = 9.0 and 12.0 Hz, 1 H, 6Ј-H), 4.42 (m, J =
3.0 Hz, 1 H, 4Ј-H), 3.13–3.04 (m, 1 H, 2Ј-H), 2.04 (s, 3 H, CH3),
1.29 (d, J = 1.5 Hz, 3 H, CH3) ppm. 13C NMR (75 MHz, CDCl3):
δ = 166.0, 165.9, 163.3, 159.9, 150.6, 134.1–128.5, 112.2, 86.4, 82.0,
74.1, 64.1, 59.9, 45.6, 12.0 ppm. MS (DCI): m/z = 526.3 [M +
NH4]+. C26H24N2O9 (508.48): calcd. C 61.42, H 4.76, N 5.51; found
C 61.58, H 4.79, N 5.36.
(3Ј-O-tert-Butyldiphenylsilyl-5Ј-thymidinyl)-(2-cyanoethyl)-5Ј-O-ben-
zoyl-3Ј-deoxy-3Ј-[(benzoyloxy)methyl]-5-methyluridine-2Ј-phosphate
(5): Compound
3 (0.1 g, 0.19 mmol) dissolved in methanol
Experimental Section
(0.5 mL) was treated with a concentrated ammonia solution (28%
in water, 2.5 mL), at room temperature for 120 min. After complete
removal of the solvent, the crude material was dried under high
vacuum and used without further purification. To this compound
was added 3Ј-O-tert-butyldiphenylsilylthymidine-5Ј-O- phos-
phoramidite (388 mg, 0.57 mmol) and freshly sublimed tetrazole
(93 mg, 1.33 mmol), and the mixture was diluted with anhydrous
acetonitrile (7.8 mL) and stirred for 3 h at room temperature under
an inert atmosphere of argon. After the addition of collidine
(150 µL), the phosphite was oxidized with a solution of iodine
[0.1  in THF/H2O, 2:1] until the dark-brown color persisted. After
extraction with ethyl acetate, the organic layer was washed with an
aqueous solution of sodium thiosulfate (15%), water, and brine and
dried with MgSO4 before removal of the solvent. Compound 5
(140 mg, 68%) was isolated (two diastereoisomers) as a white foam
after chromatography on silica gel (CH2Cl2/AcOEt, 1:1, then Ac-
OEt). Rf (CH2Cl2/AcOEt, 1:1) = 0.19. 31P NMR (81 MHz, CDCl3):
δ = –2.1, –2.3 ppm. MS (ESI): m/z = 1098.54 [M + Na]+.
C54H58N5O15PSi (1076.13): calcd. C 60.27, H 5.43, N 6.51; found
C 60.07, H 5.23, N 6.65.
Products were purified by medium-pressure liquid chromatography
with a Jobin et Yvon Modoluprep apparatus by using Amicon 6–
35 µm or Merck 15 µm silica. NMR spectra were recorded with a
Bruker AC-250, Avance-300, or Avance 500 spectrometer (250, 300,
1
or 500 MHz, respectively, for H and 63, 75, or 125 MHz, respec-
tively, for 13C). Chemical shifts were referenced to tetramethylsil-
ane. Mass spectra were recorded with a Nermag R10–10 or a Per-
kin–Elmer API 365. All solvents were distilled and dried before
use.
5Ј-O-Benzoyl-3Ј-deoxy-3Ј-[(benzoyloxy)methyl]-2Ј-O-acetyl-5-meth-
yluridine (3): A mixture of thymine (1 g, 7.9 mmol), hexamethyldi-
silazane (15 mL), and trimethylsilyl chloride (0.3 mL) were heated
at reflux in anhydrous pyridine (10 mL) until the solution became
clear. After removal of the excess amounts of reactants under vac-
uum, the bis(silylated) thymine was distilled under reduced pressure
and stored under an inert atmosphere of argon. At room tempera-
ture, under an inert atmosphere of argon, to 5-O-benzoyl-3-deoxy-
3-[(benzoyloxy)methyl]-1,2-di-O-acetylribose (1; 210 mg, 0.46
mmol) dissolved in anhydrous acetonitrile (1.8 mL) was added si-
lylated thymine (249 mg, 0.92 mmol) and a solution of trimethyl-
silyltrifluoromethanesulfonate in anhydrous toluene (1 mL, 0.55 ,
0.55 mmol). The mixture was heated at 85 °C for 2 h. The reaction
mixture was diluted with ethyl acetate and washed with a saturated
aqueous solution of NaHCO3. The organic layer was collected and
washed with water and brine and dried with MgSO4. After removal
of the solvent under reduced pressure, the crude product was de-
posited on a silica-gel column and eluted with ethyl acetate/dichlo-
romethane, 1:4. Compound 3 was recovered as a white foam in
(3Ј-O-tert-Butyldiphenylsilyl-5Ј-thymidinyl)-(2-cyanoethyl)-5Ј-O-ben-
zoyl-2Ј-deoxy-2Ј-[(benzoyloxy)methyl]-5-methyluridine-3Ј-phosphate
(6): Compound 6 was prepared following the same procedure as
that described for 5. Starting from 4 (190 mg, 0.37 mmol), com-
pound 6 (two diastereoisomers) was recovered as a white foam
(290 mg, 75%) after silica-gel chromatography (ethyl acetate/
dichloromethane, 1:1, then ethyl acetate. 31P NMR (121 MHz,
CD3OD): δ = –1.8 and –2.1 ppm. MS (ESI): m/z = 1098.50 [M +
Na]+. C54H58N5O15PSi (1076.13): calcd. C 60.27, H 5.43, N 6.51;
found C 59.85, H 5.61, N 6.42.
1
93% yield (224 mg). H NMR (300 MHz, CDCl3): δ = 8.48 (br. s,
Eur. J. Org. Chem. 2008, 1285–1294
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1291

C. Dupouy, P. Lavedan, J.-M. Escudier
FULL PAPER
(3Ј-O-tert-Butyldiphenylsilyl-5Ј-thymidinyl)-3Ј-deoxy-3Ј-[(hydroxy)-
methyl]-5-methyluridine-2Ј-phosphate (7): To compound 5 (140 mg,
0.13 mmol) in a solution of methanol/water (4:1, 2.5 mL) was
added potassium carbonate (36 mg, 0.26 mmol), and the mixture
was stirred for 6 h. After removal of the solvents, 7 was isolated
(95 mg, 90%) by HPLC (Kromasil C18, 7 µm, 250ϫ4.6 mm; water/
1.89 (s, 3 H, Me), 1.11 (s, 9 H, tBu) ppm. 13C NMR (125 MHz,
CDCl3): δ = 163.4, 163.1, 150.1, 149.8, 136.9, 135.7, 135.5, 132.9,
132.7, 130.3, 130.2, 128.1, 111.3, 85.3, 85.2, 85.1, 85.0, 83.9, 83.8,
80.6, 72.9, 68.2, 68.1, 64.3, 60.8, 40.4, 30.1, 29.7, 26.8, 19.0, 12.5,
12.4 ppm. 31P NMR (202 MHz, CDCl3): δ = –6.0 ppm. MS (ESI):
m/z = 819.5 [M + Na]+, 836.0 [M + K]+. C37H45N4O12PSi (796.84):
acetonitrile, gradient 95:5Ǟ50:50 in 30 min), tR = 16.80 min. 1H calcd. C 55.77, H 5.69, N 7.03; found C 55.53, H 5.78, N 7.10.
NMR (300 MHz, CD3OD): δ = 7.72–7.58 (m, 6 H, ph), 7.48–7.42
(3Ј-O-tert-Butyldiphenylsilyl-5Ј-thymidinyl)-2Ј-deoxy-2Ј-[(hydroxy)-
(m, 6 H, ph, 6a-H and 6b-H), 6.49 (dd, J = 6.3 and 8.4 Hz, 1 H,
methyl]-5-methyluridine-2Ј-phosphate (10) and (5Ј-Thymidinyl)-2Ј-
1Јb-H), 5.94 (d, J = 1.2 Hz, 1 H, 1Јa-H), 4.70 (dd, J = 6.0 and
deoxy-2Ј-[(hydroxy)methyl]-5-methyluridine-2Ј-phosphate (11): To
9.3 Hz, 1 H), 4.58 (m, 1 H), 4.11 (s, 1 H), 4.01–3.87 (m, 4 H), 3.78
compound 6 (290 mg, 0.27 mmol) in a solution of methanol/water
(4:1, 5 mL) was added potassium carbonate (224 mg, 1.62 mmol),
and the mixture was stirred for 6 h. After removal of the solvents,
10 and 11 were isolated (40 mg each, 45%) by HPLC [Kromasil
(dt, J = 3.6 and 11.0 Hz, 1 H), 3.67 (A part of an ABX syst., J =
3.0 and 12.7 Hz, 1 H), 3.59 (B part of an ABX syst., J = 5.1 and
12.7 Hz, 1 H), 2.67–2.57 (m, 1 H, 2Јa-H), 2.14–2.10 (m, 2 H, 2Јb-
H), 1.89 (s, 3 H, Me), 1.81 (s, 3 H, Me), 1.12 (s, 9 H, tBu) ppm.
C18, 7 µm, 250ϫ4.6 mm; water/acetonitrile, gradient 95:5 (t = 0 to
13C NMR (125 MHz, CD3OD): δ = 165.3, 165.1, 151.3, 150.7,
12 min) to 50:50 in 42 min], tR = 25.07 (for 10), 3.75 (for 11) min.
136.9, 136.4, 135.5, 133.0, 132.9, 129.8,129.7, 127.7, 127.6, 110.6,
Data for 10: 1H NMR (300 MHz, CD3OD): δ = 7.82 (d, J = 1.2 Hz,
1 H, 6a-H), 7.67 (d, J = 1.2 Hz, 1 H, 6b-H), 7.63–7.60 (m, 4 H,
109.8, 90.8, 86.4, 84.5, 82.1, 79.9, 74.7, 64.7, 60.8, 57.0, 44.9, 39.9,
26.0, 18.4, 11.4, 11.1 ppm. 31P NMR (121 MHz, CD3OD): δ =
ph), 7.42–7.39 (m, 6 H, ph), 6.43 (dd, J = 5.4 and 9.0 Hz, 1 H, 1Јb-
–0.5 ppm. MS (ESI): m/z = 813.24 [M]+. C37H46KN4O13PSi
H), 5.97 (d, J = 9.0 Hz, 1 H, 1Јa-H), 4.76–4.70 (m, 2 H), 4.47 (d,
J = 4.8 Hz, 1 H, 3Јa-H), 4.02 (m, 1 H), 3.98 (m, 1 H), 3.87–3.81
(m, 2 H), 3.57 (d, J = 2.7 Hz, 1 H), 3.50–3.44 (m, 2 H), 2.53 (m, 1
(852.94): calcd. C 52.10, H 5.44, N 6.57; found C 51.87, H 5.55, N
6.32.
H, 2Јa-H), 2.16 (A part of an ABXY syst., J = 1.5, 5.8, and
3Ј-O-(tert-Butyldiphenylsilyl)-(2Ј,5Ј)-ν2,εЈ,ζЈ-D-CNA (RC3Ј,SP) (8)
13.3 Hz, 1 H, 2Јb-H), 2.06 (m, J = 5.1, 9.0 and 13.8 Hz, 1 H, 2Јb-
and (RC3Ј,RP) (9): To compound 7 (30 mg, 0.038mmol) in anhy-
H), 1.85 (d, J = 1.2 Hz, 3 H, Me), 1.82 (d, J = 1.2 Hz, 3 H, Me),
drous pyridine (1 mL) was added, under an inert atmosphere
of argon, 1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole (22 mg,
1.04 (s, 9 H, tBu) ppm. 13C NMR (125 MHz, CD3OD): δ = 165.1,
165.0, 151.3, 151.2, 136.8, 136.6, 135.5, 133.0, 132.9, 130.0, 129.9,
0.075 mmol), and the mixture was stirred for 1 h at room tempera-
127.8, 127.7, 110.9, 110.5, 86.7, 86.6, 86.3, 86.2, 85.0, 76.5, 76.4,
ture, 1 h at 80 °C, and then 30 min at 90 °C. After addition of ethyl
74.8, 65.4, 65.3, 61.8, 56.9, 51.2, 51.2, 40.0, 26.0, 18.4, 11.2,
acetate/methanol (10%, 5 mL), the solvent were removed under
11.1 ppm. 31P NMR (121 MHz, CD3OD): δ = –0.2 ppm. MS (ESI):
high vacuum and compounds 8 (16 mg) and 9 (9 mg) were isolated
m/z = 813.5 [M]–. C37H46KN4O13PSi (852.94): calcd. C 52.10, H
(80%) by HPLC (Kromasil C18, 7 µm, 250ϫ4.6 mm; water/aceto-
5.44, N 6.57; found C 51.92, H 5.66, N 6.61. Data for 11: 1H NMR
nitrile, 1:1), tR = 13.33 (for 8), 15.03 (for 9) min. Data for 8: 1H
(300 MHz, D2O): δ = 7.54 (d, J = 1.2 Hz, 1 H, 6a-H), 7.53 (d, J =
NMR (500 MHz, CDCl3): δ = 7.68–7.65 (m, 4 H, ph), 7.55 (s, 1
1.2 Hz, 1 H, 6b-H), 6.17 (t, J = 6.9 Hz, 1 H, 1Јb-H), 5.95 (d, J =
H, 6-H), 7.49–7.41 (m, 6 H, ph), 7.07 (s, 1 H, 6-H), 6.23 (t, J =
8.7 Hz, 1 H, 1Јa-H), 4.68 (m, 1 H, 3Јa-H), 4.42 (m, 1 H, 3Јb-H),
7.0 Hz, 1 H, 1Јb-H), 5.61 (s, 1 H, 1Јa-H), 5.09 (d, J = 4.5 Hz, 1 H,
4.15 (m, 1 H, 4Јa-H), 4.01 (m, 2 H, 4Јb-H and 5Јb-H), 3.96 (m, 1
2Јa-H), 4.48 (br. d, J = 11.0 Hz, 1 H, 4Јa-H), 4.43–4.37 (m, 2 H,
H, 5Јb-H), 3.80 (A part of an ABX syst., J = 6.6 and 11.4 Hz, 1
6Јa-H and 3Јb-H), 4.21–4.14 (m, J = 12.0, JH/P = 22.0 Hz, 1 H,
H, 6Јa-H), 3.65 (m, 2 H, 5Јa-H), 3.57 (B part of an ABX syst., J
6Јa-H), 4.18 (A part of an AB syst., J = 12.0 Hz, 1 H, 5Јa-H), 4.11
= 6.6 and 11.4 Hz, 1 H, 6Јa-H), 2.57 (m, 1 H, 2Јa-H), 2.25 (dd, J
(br. s, 1 H, 4Јb-H), 4.10–4.07 (m, J = 11.5 and 3.0, JH,P = 2.0 Hz,
= 5.4 and 6.9 Hz, 1 H, 2Јb-H), 1.77 (d, J = 1.2 Hz, 3 H, Me), 1.75
1 H, 5Јb-H), 3.82 (B part of an AB syst., J = 12.0 Hz, 1 H, 5Јa-
(d, J = 1.2 Hz, 3 H, Me) ppm. 13C NMR (125 MHz, D2O): δ =
H), 3.82–3.77 (m, J = 11.5 and 6.0, JH,P = 8.0 Hz, 1 H, 5Јb-H),
166.4, 166.3, 151.7, 151.6, 137.3, 137.2, 111.8, 111.5, 86.8, 85.3,
2.82 (m, J = 5.5 Hz, 1 H, 3Јa-H), 2.40 (A part of an ABXY syst.,
J = 4.0, 6.8, and 13.4 Hz, 2Јb-H), 2.06 (B part of an ABXY syst.,
85.1, 75.9, 70.7, 65.3, 61.2, 57.0, 48.9, 38.5, 11.6, 11.5 ppm. 31P
NMR (121 MHz, D2O): δ = –0.5 ppm. MS (ESI): m/z = 575.3
J = 5.5, 7.0, and 13.4 Hz, 2Јb-H), 1.91 (s, 3 H, Me), 1.88 (s, 3 H,
[M]–. C21H28KN4O13P (614.54.25): calcd. C 41.04, H 4.59, N 6.36;
Me), 1.10 (s, 9 H, tBu) ppm. 13C NMR (125 MHz, CDCl3): δ =
164.0, 150.3, 150.2, 137.3, 136.3, 135.8, 135.7, 132.8, 132.7, 130.3,
found C 39.78, H 4.73, N6.63.
130.2, 128.1, 128.0, 111.3, 110.5, 86.0, 85.1, 84.4, 80.4, 71.9, 66.1,
64.7, 59.6, 39.7, 26.8, 21.1, 19.0, 12.4 ppm. 31P NMR (202 MHz,
CDCl3): δ = –9.7 ppm. MS (ESI): m/z = 797.8 [M + H]+, 819.5 [M
+ Na]+, 835.8 [M + K]+. C37H45N4O12PSi (796.84): calcd. C 55.77,
H 5.69, N 7.03; found C 56.01, H 5.57, N 6.99. Data for 9: 1H
NMR (500 MHz, CDCl3): δ = 7.68–7.65 (m, 4 H, Ph), 7.51–7.43
3Ј-O-(tert-Butyldiphenylsilyl)-ν2,ε,ζ-D-CNA (RC3Ј,RP) (12) and
(RC3Ј,SP) (13): Compounds 12 and 13 were prepared following the
same procedure as that described for 8 and 9. Starting from 10
(40 mg, 0.049 mmol), compounds 12 (10 mg) and 13 (19 mg) were
isolated (75%) by HPLC (Kromasil C18, 7 µm, 250ϫ4.6 mm;
water/acetonitrile, 1:1), tR = 14.82 (for 12), 17.00 (for 13) min. Data
1
(m, 6 H, Ph), 7.40 (s, 1 H, 6b-H), 7.26 (s, 1 H, 6a-H), 6.44 (dd, J for 12: H NMR (500 MHz, CDCl3): δ = 7.67–7.61 (m, 5 H, ph),
= 5.5 and 8.0 Hz, 1 H, 1Јb-H), 5.67 (s, 1 H, 1Јa-H), 5.29 (t, J = 4.3
and 6.0 Hz, 1 H, 2Јa-H), 4.73 (m, J = 3.5, 5.6 and 12.5 Hz, 1 H,
6Јa-H), 4.42 (m, J = 2.5 and 6.1 Hz, 1 H, 3Јb-H), 4.29 (dt, J = 2.5
and 9.7 Hz, 1 H, 4Јa-H), 4.20 (ddd, J = 2.0, 12.5, and 20.3 Hz, 1
H, 6Јa-H), 4.11 (A part of an ABX syst., J = 2.5 and 12.4 Hz, 1
H, 5Јa-H), 4.08 (br. s, 1 H, 4Јb-H), 4.09–4.05 (m, J = 2.5, 6.7 and
7.50–7.41 (m, 6 H, ph), 7.28 (s, 1 H, 6a-H), 6.41 (dd, J = 6.0 and
8.0 Hz, 1 H, 1Јb-H), 6.22 (d, J = 9.0 Hz, 1 H, 1Јa-H), 5.26 (d, J =
4.5 Hz, 1 H, 3Јa-H), 4.69 (m, J = 3.0 and 12.0, JH,P = 2.4 Hz, 1 H,
6Јa-H), 4.40 (m, J = 2.5 and 5.5 Hz, 1 H, 3Јb-H), 4.29 (dd, J =
11.5, JH,P = 20.0 Hz, 1 H, 6Јa-H), 4.10–4.07 (m, 3 H, 4Јa-H, 4Јb-
H and 5Јb-H), 3.85 (AB syst., J = 10.8 Hz, 2 H, 5Јa-H), 3.66 (m,
16.0 Hz, 1 H, 5Јb-H), 3.81 (B part of an ABX syst., J = 2.5 and J = 3.0 and 11.0, JH,P = 3.5 Hz, 1 H, 5Јb-H), 2.82 (m, 1 H, 2Јa-
12.4 Hz, 1 H, 5Јa-H), 3.73–3.69 (m, J = 2.5 Hz, 1 H, 5Јb-H), 3.00 H), 2.39 (A part of an ABXY syst., J = 2.0, 5.5, and 13.5 Hz, 1 H,
(m, 1 H, 3Јa-H), 2.39 (A part of an ABXY syst., J = 2.5, 5.5, and
13.5 Hz, 1 H, 2Јb-H), 1.95–1.90 (m, 1 H, 2Јb-H), 1.92 (s, 3 H, Me),
2Јb-H), 1.98 (m, J = 6.0, 7.5, and 14.0 Hz, 1 H, 2Јb-H), 1.88 (s, 3
H, Me), 1.86 (s, 3 H, Me), 1.10 (s, 9 H, tBu) ppm. 13C NMR
1292
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Eur. J. Org. Chem. 2008, 1285–1294

Dinucleotides with S-Type Sugar Puckering and ε and ζ Torsion Angles
(125 MHz, CDCl3): δ = 164.2, 163.8, 150.9, 150.4, 136.0, 135.9, m/z = 559.3 [M + H]+, 581.2 [M + Na]+, 597.5 [M + K]+.
135.7, 132.9, 132.7, 130.4, 130.2, 128.1, 128.0, 111.9, 111.1, 86.1,
85.9, 85.8, 85.4, 85.3, 85.2, 81.8, 73.2, 68.5, 68.4, 63.5, 62.3, 60.4,
43.8, 40.4, 26.9, 12.6, 12.4 ppm. 31P NMR (202 MHz, CDCl3): δ =
–6.0 ppm. MS (ESI): m/z = 819.5 [M + Na]+, 836.0 [M + K]+.
C37H45N4O12PSi (796.84): calcd. C 55.77, H 5.69, N 7.03; found C
C21H27N4O12P (558.43): calcd. C 45.17, H 4.87, N 10.03; found C
44.97, H 4.92, N 10.14.
Circular Dichroism Studies: These experiments were carried out
with a Jasco J-815 CD spectrometer equipped with a Peltier con-
troller Jasco PTC-4235/15 at a dinucleotide concentration range of
0.1 m in a 10 m Na2HPO4, 100 m NaCl, 0.1 m Na2EDTA,
buffer, pH 7.00Ϯ0.02. Molar extinction coefficients were calcu-
lated from those of dinucleotides by using the nearest-neighbor
approximation method by assuming that ν2,ε,ζ-D-CNA TT has the
same molar extinction coefficient as DNA. Dinucleotide concentra-
tion was determined from UV absorbance at high temperature
(90 °C). All CD spectra were recorded after stabilization of the
temperature for 10 min and were normalized by subtraction of the
background scan with buffer. By taking the known dinucleotide
concentration into account, the normalized spectra were converted
into a variation of the molar extinction coefficient (∆ε).
1
55.89, H 5.78, N 7.14. Data for 13: H NMR (500 MHz, CDCl3):
δ = 7.68–7.65 (m, 4 H, ph), 7.52–7.42 (m, 6 H, ph), 7.40 (s, 1 H,
6a-H), 7.10 (s, 1 H, 6b-H), 6.16 (t, J = 6.5 Hz, 1 H, 1Јb-H), 6.02
(d, J = 9.5 Hz, 1 H, 1Јa-H), 5.10 (d, J = 4.5 Hz, 1 H, 3Јa-H), 4.43–
4.39 (m, 2 H, 3Јb-H and 6Јa-H), 4.26 (m, J = 8.5, JH,P = 26.0 Hz,
1 H, 6Јa-H), 4.25 (s, 1 H, 4Јa-H), 4.11 (m, J = 3.0, and 6.0 Hz, 1 H,
4Јb-H), 3.98 (A part of an ABXY syst., J = 3.0, 6.0, and 11.2 Hz, 1
H, 5Јb-H), 3.89 (A part of an AB syst., J = 11.0 Hz, 1 H, 5Јa-H),
3.84 (B part of an ABX syst., J = 11.0, JH,P = 6.0 Hz, 1 H, 5Јb-
H), 3.72 (B part of an AB syst., J = 11.5 Hz, 1 H, 5Јa-H), 3.03 (m,
1 H, 2Јa-H), 2.37 (A part of an ABXY syst., J = 3.5, 6.5, and
13.6 Hz, 1 H, 2Јb-H), 2.14 (B part of an ABXY syst., J = 7.0 and
13.5 Hz, 1 H, 2Јb-H), 1.90 (s, 3 H, Me), 1.88 (s, 3 H, Me), 1.10 (s,
9 H, tBu) ppm. 13C NMR (125 MHz, CDCl3): δ = 164.4, 164.2,
135.7, 135.6, 132.8, 132.7, 130.4, 130.3, 128.1, 128.0, 111.6, 111.1,
86.7, 86.2, 86.1, 86.0, 84.9, 84.8, 83.1, 83.0, 72.3, 66.2, 64.4, 62.3,
42.2, 39.5, 26.9, 19.0, 12.4, 12.3 ppm. 31P NMR (202 MHz,
CDCl3): δ = –9.4 ppm. MS (ESI): m/z = 819.5 [M + Na]+, 836.0
[M + K]+. C37H45N4O12PSi (796.84): calcd. C 55.77, H 5.69, N
7.03; found C 55.81, H 5.55, N 7.16.
[1] C. J. Leumann, Bioorg. Med. Chem. 2002, 10, 841–854.
[2] J. Wengel, Acc. Chem. Res. 1999, 32, 301–310.
[3] P. Herdewijn, Biochim. Biophys. Acta 1999, 1489, 167–179.
[4] S. Obika, D. Nanbu, Y. Hari, K.-I. Morio, Y. In, T. Ishida, T.
Imanishi, Tetrahedron Lett. 1997, 38, 8735–8738.
[5] S. Obika, D. Nanbu, Y. Hari, J.-I. Andoh, K.-I. Morio, T. Doi,
T. Imanishi, Tetrahedron Lett. 1998, 39, 5401–5404.
[6] S. K. Singh, P. Nielsen, A. A. Koshkin, J. Wengel, Chem. Com-
mun. 1998, 455–456.
ν2,ε,ζ-D-CNA (RC3Ј,RP) (14) and (RC3Ј,SP) (15): Compounds 14
and 15 were prepared following the same procedure as that de-
scribed for 8 and 9. Starting from 11 (40 mg, 0.049 mmol), com-
pounds 14 (10 mg) and 15 (13 mg) were isolated (60%) by HPLC
(Kromasil C18, 7 µm, 250ϫ4.6 mm; water/acetonitrile, gradient
95:5Ǟ60:40 in 30 min), tR = 27.91 (for 14), 27.12 (for 15) min.
Data for 14: 1H NMR (500 MHz, CD3OD): δ = 7.89 (d, J = 1.0 Hz,
1 H, 6a-H), 7.53 (d, J = 1.0 Hz, 1 H, 6b-H), 6.47 (d, J = 9.0 Hz, 1
H, 1Јa-H), 6.31 (t, J = 7.0 Hz, 1 H, 1Јb-H), 5.31 (m, J = 5.0, JH,P
= 2.5 Hz, 1 H, 3Јa-H), 4.76 (A part of an ABXY syst., J = 3.7 and
12.5 Hz, JH,P = 6.9 Hz, 1 H, 6Јa-H), 4.49–4.39 (m, 4 H, 6Јa-H, 5Јb-
[7] M. Maderia, S. Shenoy, Q. N. Van, V. E. Marquez, J. J. Bar-
chi Jr, Nucleic Acids Res. 2007, 35, 1978–1991.
[8] D. Renneberg, C. J. Leumann, J. Am. Chem. Soc. 2002, 124,
5993–6002.
[9] E. T. Kool, Chem. Rev. 1997, 97, 1473–1487.
[10] a) K. Seio, T. Wada, K. Sakamoto, S. Yokoyama, M. Sekine,
J. Org. Chem. 1998, 63, 1429–1443; b) K. Seio, T. Wada, M.
Sekine, Helv. Chim. Acta 2000, 83, 162–180; c) M. Sekine, O.
Kurasawa, K. Shohda, K. Seio, T. Wada, J. Org. Chem. 2000,
65, 3571–3578; d) M. Sekine, O. Kurasawa, K. Shohda, K.
Seio, T. Wada, J. Org. Chem. 2000, 65, 6515–6524; e) M. Sek-
ine, O. Kurasawa, K. Shohda, K. Seio, T. Wada, Eur. J. Org.
Chem. 2001, 1989–1999.
H, and 3Јb-H), 4.30 (br. s, 1 H, 4Јa-H), 4.09 (m, J = 3.6, JH,P
=
[11] a) A. M. Sørensen, P. Nielsen, Org. Lett. 2000, 2, 4217–4219;
b) A. M. Sørensen, K. E. Nielsen, B. Vogg, J. P. Jacobsen, P.
Nielsen, Tetrahedron 2001, 57, 10191–10201; c) P. Børsting, P.
Nielsen, Chem. Commun. 2002, 2140–2141; d) C. Kirchhoff, P.
Nielsen, Tetrahedron Lett. 2003, 44, 6475–6478; e) P. Børsting,
M. Freitag, P. Nielsen, Tetrahedron 2004, 60, 10955–10966; f)
P. B ørsting, K. E. Nielsen, P. Nielsen, Org. Biomol. Chem. 2005,
3, 2183–2190; g) P. K. Sharma, B. H. Mikkelsen, M. S. Chris-
tensen, K. E. Nielsen, C. Kirchhoff, S. L. Pedersen, A. M.
Sørensen, K. Østergaard, M. Petersen, P. Nielsen, Org. Biomol.
Chem. 2006, 4, 2433–2445.
2.2 Hz, 1 H, 4Јb-H), 3.84 (d, J = 3.0 Hz, 2 H, 5Јa-H), 2.89 (m, 1
H, 2Јa-H), 2.33–2.24 (m, J = 6.5, 6.5, and 7.0 Hz, 2 H, 2Јb-H), 1.90
(d, J = 1.0 Hz, 3 H, Me), 1.89 (d, J = 1.0 Hz, 3 H, Me) ppm. 13C
NMR (125 MHz, CD3OD): δ = 164.9, 164.8, 151.5, 150.9, 136.1,
135.9, 111.0,110.5, 86.0, 85.9, 84.9, 84.7, 84.6, 84.4, 82.7, 70.5, 68.9,
68.8, 63.9, 63.8, 61.4, 61.3, 43.8, 43.7, 39.0, 11.1 ppm. 31P NMR
(202 MHz, CD3OD): δ = –6.6 ppm. MS (ESI): m/z = 559.3 [M +
H]+, 581.2 [M + Na]+, 597.5 [M + K]+. C21H27N4O12P (558.43):
calcd. C 45.17, H 4.87, N 10.03; found C 45.23, H 4.78, N 9.92.
Data for 15: 1H NMR (500 MHz, CD3OD): δ = 7.92 (d, J = 1.0 Hz,
1 H, 6a-H), 7.55 (d, J = 1.0 Hz, 1 H, 6b-H), 6.51 (d, J = 9.5 Hz, 1
H, 1Јa-H), 6.28 (t, J = 7.0 Hz, 1 H, 1Јb-H), 5.27 (d, J = 4.5 Hz, 1
H, 3Јa-H), 4.69 (A part of an ABXY syst., J = 3.2 and 12.5 Hz,
JH,P = 1.5 Hz, 1 H, 6Јa-H), 4.46 (m, 1 H, 3Јb-H), 4.42 (B part of
an ABXY syst., J = 12.5 Hz, JH,P = 24.0 Hz, 1 H, 6Јa-H), 4.40 (A
part of an ABXY syst., J = 11.3 Hz, JH,P = 3.3 Hz, 1 H, 5Јb-H),
4.36 (B part of an ABXY syst., J = 5.8 and 11.3 Hz, JH,P = 7.7 Hz,
1 H, 5Јb-H), 4.27 (br. s, 1 H, 4Јa-H), 4.11 (m, J = 3.3, 3.4, and
5.8 Hz, 1 H, 4Јb-H), 3.81 (ABX syst., J = 2.5, 3.0, and 12.0 Hz, 2
H, 5Јa-H), 2.84 (m, 1 H, 2Јa-H), 2.32 (m, J = 6.5 and 7.0 Hz, 2 H,
2Јb-H), 1.92 (d, J = 1.0 Hz, 3 H, Me), 1.90 (d, J = 1.0 Hz, 3 H,
Me) ppm. 13C NMR (125 MHz, CD3OD): δ = 164.9, 164.8, 151.5,
150.8, 136.6, 136.1, 110.9, 110.5, 86.2, 86.1, 85.6, 84.7, 84.6, 84.1,
83.8, 83.7, 70.3, 67.1, 67.0, 64.7, 64.6, 61.5, 43.1, 43.0, 38.9, 11.1,
11.0 ppm. 31P NMR (202 MHz, CD3OD): δ = –7.8 ppm. MS (ESI):
[12] P. Belmont, J.-F. Constant, M. Demeunynck, Chem. Soc. Rev.
2001, 30, 70–81.
[13] P. Várnai, D. Djuranovic, R. Lavery, B. Hartmann, Nucleic Ac-
ids Res. 2002, 30, 5398–5406.
[14] a) D. Djuranovic, B. Hartmann, Biopolymers 2004, 73, 356–
368; b) B. Heddi, N. Foloppe, N. Bouchemal, E. Hantz, B.
Hartmann, J. Am. Chem. Soc. 2006, 128, 9170–9177.
[15] C. M. Duarte, A. M. Pyle, J. Mol. Biol. 1998, 284, 1465–1478.
[16] I. Le Clezio, J.-M. Escudier, A. Vigroux, Org. Lett. 2003, 5,
161–164.
[17] C. Dupouy, I. Le Clezio, P. Lavedan, H. Gornitzka, J.-M. Es-
cudier, A. Vigroux, Eur. J. Org. Chem. 2006, 5515–5525.
[18] C. Dupouy, N. Iché-Tarrat, M.-P. Durrieu, F. Rodriguez, J.-
M. Escudier, A. Vigroux, Angew. Chem. 2006, 118, 3705–3709;
Angew. Chem. Int. Ed. 2006, 45, 3623–3627.
[19] I. Le Clezio, C. Dupouy, P. Lavedan, J.-M. Escudier, Eur. J.
Org. Chem. 2007, 3894–3900.
Eur. J. Org. Chem. 2008, 1285–1294
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1293

C. Dupouy, P. Lavedan, J.-M. Escudier
FULL PAPER
[20]
[21]
[22]
[23]
[24]
[30] a) S. S. Jones, B. Rayner, C. B. Reese, A. Ubazawa, M. Ubas-
awa, Tetrahedron 1980, 36, 3075–3085; b) C. B. Reese, R. C.
Titmas, L. Yau, Tetrahedron Lett. 1978, 19, 2727–2730.
[31] D. J. Wood, F. E. Hruska, K. K. Ogilvie, Can. J. Chem. 1974,
52, 3353–3366.
I. Le Clezio, A. Vigroux, J.-M. Escudier, Eur. J. Org. Chem.
2007, 1935–1941.
C. Dupouy, P. Lavedan, J.-M. Escudier, Eur. J. Org. Chem.
2007, 5256–5264.
I. Le Clezio, H. Gornitzka, J.-M. Escudier, A. Vigroux, J. Org.
Chem. 2005, 70, 1620–1629.
C. Dupouy, P. Lavedan, J.-M. Escudier, Tetrahedron 2007, 63,
11235–11243.
[32] D. J. Wood, K. K. Ogilvie, F. E. Hruska, Can. J. Chem. 1975,
53, 2781–2790.
[33] D. G. Gorenstein, R. Rowell, J. Findlay, J. Am. Chem. Soc.
1980, 102, 5077–5081.
[34] W. C. Johnson in Circular Dichroism: Principles and Applica-
tions (Eds.: N. Berova, K. Nakanishi, R. W. Woody), John
Wiley & Sons, New York, 2000, pp. 703–718.
[35] C. Altona, M. Sundaralingam, J. Am. Chem. Soc. 1973, 95,
2333–2344.
M. H. Caruthers, Acc. Chem. Res. 1991, 24, 278–284.
[25] H. Vorbrüggen, K. Krolikiewiecz, B. Bennua, Chem. Ber. 1981,
114, 1234–1255.
[26] C. K. H. Tseng, V. E. Marquez, G. W. A. Milne, R. J. Wysocki,
H. Mitsuya, T. Shirasaki, J. S. Driscoll, J. Med. Chem. 1991,
34, 343–349.
[27] N.-S. Li, J. A. Piccirilli, J. Org. Chem. 2004, 69, 4751–4759.
[28] A. Matsuda, K. Takenuki, M. Tanaka, T. Sasaki, T. Ueda, J.
Med. Chem. 1991, 34, 812–819.
[29] J. B. J. Pavey, A. J. Lawrence, A. J. Potter, R. Cosstick, I. A.
O’Neil, Tetrahedron Lett. 1998, 39, 6967–6970.
[36] N. Camerman, J. K. Fawcett, A. Camerman, J. Mol. Biol. 1976,
107, 601–621.
[37] B. Schneider, S. Neidle, H. M. Berman, Biopoly. 1997, 42, 113–
124.
Received: October 29, 2007
Published Online: January 3, 2008
1294
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Eur. J. Org. Chem. 2008, 1285–1294