Constrained nucleic acids (CNA). Part 2. Synthesis of conformationally restricted dinucleotide units featuring noncanonical α/β/γ or δ/ε/ζ torsion angle combinations

Article abstract of DOI:10.1021/jo048136t

(Chemical Equation Presented) Four dinucleotide building units of nucleic acids in which three out of six backbone torsion angles are included in a dioxaphosphorinane ring structure (D-CNA family) have been synthesized: two diastereoisomeric α,β,γ-D-CNA {

Full text of DOI:10.1021/jo048136t

Constrained Nucleic Acids (CNA). Part 2. Synthesis of
Conformationally Restricted Dinucleotide Units Featuring
Noncanonical r/â/γ or δ/E/ú Torsion Angle Combinations
Ingrid Le Cle´zio, Heinz Gornitzka,† Jean-Marc Escudier,* and Alain Vigroux*
Laboratoire de Synthe`se et Physico-Chimie de Mole´cules d’Inte´reˆt Biologique (UMR 5068),
Universite´ Paul Sabatier, 118 Route de Narbonne, 31062 Toulouse Cedex 4, France
escudier@chimie.ups-tlse.fr; vigroux@chimie.ups-tlse.fr
Received October 21, 2004
Four dinucleotide building units of nucleic acids in which three out of six backbone torsion angles
are included in a dioxaphosphorinane ring structure (D-CNA family) have been synthesized: two
diastereoisomeric R,â,γ-D-CNA {cis and trans} and two diastereoisomeric δ,ꢀ,ú-D-CNA {(SC4′,RP)
and (SC4′,SP)}. The structural analysis of these conformationally restricted dinucleotides, using NMR
spectroscopy and X-ray crystallography, shows that these D-CNA structural elements allow the
stabilization of torsion angle combinations, R/â/γ and δ/ꢀ/ú, that are significantly different from
those typically observed in canonical A- or B-form duplexes.
Introduction
The development of conformationally restricted nucleo-
sides has attracted a lot of attention mainly due to
important potential applications of antisense oligonucle-
otides.1 However, much less attention has been devoted
to the design of conformationally restricted nucleotides
for the special purpose of mimicking biologically relevant
helical distortions of B-DNA or important nonhelical
secondary structures of functional RNA.2
In addition to the double-stranded helical conforma-
tion, nucleic acids may adopt many other alternative
structures such as bulges, hairpins, U-turns, or branched
junctions.3 These secondary structures always contain
unpaired nucleotides or non-Watson-Crick pairs and are
FIGURE 1. The six backbone torsion angles (labeled R to ú)
of nucleic acids.
characterized by a variety of backbone conformations that
markedly differ from the regular conformational states
of double-stranded helices (Figure 1 and Table 1). It is
now well established that these disparate structures,
which are prone to promote a significant local conforma-
tional heterogeneity in the sugar-phosphate backbone,
play a crucial role in fundamental biological processes
where protein-nucleic acid interactions, RNA folding, or
RNA catalytic activity are involved.4
† Current address: Laboratoire He´te´rochimie Fondamentale et
Applique´e, Universite´ Paul Sabatier.
(1) For reviews, see: (a) Leumann, C. J. Bioorg. Med. Chem. 2002,
10, 841. (b) Wengel, J. A. Acc. Chem. Res. 1999, 32, 301.
(2) See for example: (a) Seio, K.; Wada, T.; Sakamoto, K.; Yokoyama,
S.; Sekine, M. J. Org. Chem. 1998, 63, 1429. (b) Sekine, M.; Kurasawa,
O.; Shohda, K.; Seio, K.; Wada, T. J. Org. Chem. 2000, 65, 6515. (c)
Kirchhoff, C.; Nielsen, P. Tetrahedron Lett. 2003, 44, 6475. (d) Børsting,
P.; Sørensen, A. M.; Nielsen, P. Synthesis 2002, 797. (e) Sørensen, A.
M.; Nielsen, K. E.; Vogg, B.; Jacobsen, J. P.; Nielsen, P. Tetrahedron
2001, 57, 10191.
The determination of the precise biological role played
by nonstandard helical conformations during biochemi-
cally important processes (e.g., protein-DNA complex-
(3) Belmont, P.; Constant, J.-F.; Demeunynck, M. Chem. Soc. Rev.
2001, 30, 70 and references therein.
10.1021/jo048136t CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/04/2005
1620
J. Org. Chem. 2005, 70, 1620-1629

R,â,γ- and δ,ꢀ,ú-D-CNA
TABLE 1. Summary of the Backbone Torsion Angles
Derived from the Canonical B- and A-DNA Duplex
Structuresa
torsion angle
duplex
conformation
R
â
γ
δ
ꢀ
ú
BI
g-
t
g+
a+
t
g-/a-
B
BII
g-
g-
t
t
g+
g+
a+/t
g-/a-
a-/t
t
A
g+/a+
g-
a The following 6-fold staggered pattern of the torsional angles
is used: cis ) 0 ( 30° (c), gauche(+) ) 60 ( 30° (g+), anticlinal(+)
) 120 ( 30° (a+), trans ) 180 ( 30° (t), anticlinal(-) ) 240 ( 30°
(a-), gauche(-) ) 300 ( 30° (g-). The notation a+/t is used to
designate a torsion angle on the border of anticlinal(+) and trans.
FIGURE 2. R,â,γ-D-CNA and δ,ꢀ,ú-D-CNA are constrained
nucleic acids in which the backbone torsion angles (R, â, γ)
and (δ, ꢀ, ú), respectively, are stereocontrolled by a dioxaphos-
phorinane ring structure.
ation, DNA processing, and DNA packaging) is also an
area of intense study.5 Recently, an important study
based on an analysis of available high-resolution crystal-
lographic data and molecular simulation techniques has
shown that, in contrast to free B-DNA structures, protein-
bound B-DNA oligomers regularly involve noncanonical
backbone geometries.6 It was found that in the crystal
structures of protein-DNA complexes, the R and γ
torsion angles can adopt noncanonical combinations
where R populates the gauche(+) or trans conformation
while γ populates the gauche(-) or trans conformation.
These unusual backbone states are believed to contribute
to the specific recognition of DNA by proteins in assisting,
at some stage, the fine structural adjustments that are
required between DNA and proteins to form stable
complexes. 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
backbone states. Stable structural analogues of these
distorted backbone geometries would be very useful in
the elucidation of the role that helical deformations play
in nucleic acid interactions.
With this in mind, we became interested in the
development of covalently constrained dinucleotide build-
ing units in which the backbone torsional angles of
nucleic acids can have predefined values that are sig-
nificantly different from the typical values observed in
A- or B-type duplexes (Table 1). Work in our laboratory
on the development of constrained nucleic acids (CNA)
has led to an interest in the 1,3,2-dioxaphosphorinane
ring structure. Specifically, we are interested in the
structural properties that this system may impart to the
DNA double helix depending on its position along the
sugar-phosphate backbone.
D-CNA members, R,â,γ-D-CNA and δ,ꢀ,ú-D-CNA, in
which the torsion angles R, â, γ and δ, ꢀ, ú, respectively,
are constrained to noncanonical values that depend on
the spatial arrangement of the dioxaphosphorinane ring
structure (Figure 2).
Results and Discussion
Synthesis of Cis and Trans r,â,γ-D-CNA. The
dioxaphosphorinane ring structure was introduced as
previously described,7 i.e., from the cyclization reaction
of a dinucleotide precursor in which the phosphate
oxyanions can attack an electrophilic tosyloxy-substituted
carbon atom. The corresponding acyclic precursor in-
volved in the present work is the diastereoisomeric 4′-
substituted dithymidine 4 prepared by coupling 5′-O-
tosyl-4′-C-hydroxymethylthymidine 3 with the com-
mercially available thymidine phosphoramidite by using
standard phosphoramidite technology8 (Scheme 1). Modi-
fied thymidine 3 has been obtained by treatment of 2
with tosyl chloride in the presence of pyridine followed
by the removal (with trifluoroacetic acid) of the dimethoxy-
trityl group selectively introduced on the 5′′-hydroxyl
function of starting 4′-C-hydroxymethylthymidine 1.9
By treatment with K2CO3 in dry dimethylformamide
at 90 °C for 2 h, 4 quantitatively cyclized into the cis and
trans isomers of phosphotriester 5 in a 2:1 ratio as
observed by 31P NMR (δP -9.6 and -7.8, respectively).
Removal of the silyl protective group with fluoride ion
provided the corresponding mixture of cis-6 and trans-6
isomers, which were separated at this stage (these two
compounds will be subsequently converted into their
corresponding phosphoramidites for incorporation into
oligonucleotides). Finally, R,â,γ-D-CNA cis-7 and trans-7
were obtained upon removal of the remaining dimethoxy-
trityl protective group in acidic conditions.
We have already reported on the diastereoselective
synthesis of a dioxaphosphorinane-CNA (D-CNA) di-
nucleotide building unit {referred to as (SC5′,RP)-R,â-D-
CNA} in which the R and â torsion angles are locked in
a (g+, t) conformation that frequently occurs in protein-
DNA complexes and in bulged regions of nucleic acids.7
In this paper, we report the synthesis of two additional
Structural Assigment of Cis and Trans r,â,γ-D-
CNA. The dinucleotide analogues, cis-7 and trans-7, were
fully characterized by mass spectroscopy and by 1H, 13C,
and 31P NMR. The chair conformation of the dioxaphos-
phorinane structure of cis-7 is clearly established from
1
the H NMR spectra, with the observation of small and
3
large JH/P coupling constants between the 5′- and 5′′-H
protons and phosphorus, which is characteristic of an
(4) See for example: Antson, A. A. Curr. Opin. Struct. Biol. 2000,
10, 87 and references therein.
axial position (3JHax/P ≈ 2 Hz) and an equatorial position
(5) (a) Lu, X.-J.; Shakked, Z.; Olson, W. K. J. Mol. Biol. 2000, 300,
819-840. (b) Olson, W. K.; Gorin, A. A.; Lu, X.-J.; Hock, L. M.; Zhurkin,
V. B. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 11163-11168.
(6) Va´rnai, P.; Djuranovic, D.; Lavery, R.; Hartmann, B. Nucleic
Acids Res. 2002, 30, 5398.
(8) Caruthers, M. H. Acc. Chem. Res. 1991, 24, 278.
(9) (a) Youssefyeh, R. D.; Verheyden, J. P. H.; Moffatt, J. G. J. Org.
Chem. 1979, 44, 1301. (b) Jones, G. H.; Taniguchi, M.; Tegg, D.; Moffatt,
J. G. J. Org. Chem. 1979, 44, 1309.
(7) Le Cle´zio, I.; Escudier, J.-M.; Vigroux, A. Org. Lett. 2003, 5, 161.
J. Org. Chem, Vol. 70, No. 5, 2005 1621

Le Cle´zio et al.
SCHEME 1. Synthesis of Cis and Trans
r,â,γ-D-CNA Dinucleotides
FIGURE 3. Schematic drawings showing electronic interac-
tions favoring the flipping of trans-7 from the chair conforma-
tion to the twist-chair conformation.
FIGURE 4. Schematic drawings of cis-7 and trans-7 showing
the main NOE effects observed between the sugar, the base,
and the dioxaphosphorinane subunits of R,â,γ-D-CNA.
TABLE 2. H/H Coupling Constants (Hz) in the 1H NMR
Spectra (400 MHz) of cis-7 and trans-7 r,â,γ-D-CNA
coupling constant J (Hz)
J(1′,2′)
J(2′,3′)
J(3′,4′)
cis-7
upper nucleoside
lower nucleoside
upper nucleoside
lower nucleoside
5.8
8.4
6.9
5.8
5.9
6.0
2.1
3.8
1.8
2.1
2.0
6.9
5.8
5.9
6.8
5.8
5.5
trans-7
1.9
NOESY NMR study. NMR spectra of cis-7 and trans-7
were acquired at 400 MHz. Schematic views of both
diastereoisomers showing the observed proton proximity
relationships are given in Figure 4. The chair conforma-
tion of the dioxaphosphorinane ring of cis-7 is cor-
roborated by a single strong NOESY cross-peak observed
between the H3′ of the lower nucleoside and the H5′
equatorial proton of the six-membered ring. The twist-
chair conformation of trans-7 is corroborated by the cross-
peak exhibited by H1′ (of the lower nucleoside) with both
of the H5′′ protons. The strong cross-peaks displayed by
each thymine H-6 proton with their corresponding sugar
H1′ and H2′ protons are indicative of the relative position
of the thymine bases toward the sugar rings. The
empirical equation established by Lankhorst et al.11
allowed us to determine, from the coupling constant JH3′/P
(6.2 Hz for cis-7 and 6.8 Hz for trans-7), the value of the
torsional angle ꢀ (-158° for cis-7 and -155° for trans-7)
and therefore the relative position of the upper nucleo-
sides toward the dioxaphosphorinane ring. Although
many cross-peaks are present for cis-7 and trans-7, all
of them are derived from intraresidual H/H interactions.
This suggests that these dinucleotides have conforma-
(3JHeq/P ) 22 Hz) of these protons. The observation of a
long-range coupling constant between the 5′- and 5′′-H
protons (4JH/H ) 2.9 Hz) is also consistent with the typical
W-shaped Heq-C-C-C-Heq junction involved in chair
conformations. In contrast, average values of 11.5 and
3
13.2 Hz were observed for the JH/P coupling constants
involving the 5′- and 5′′-H protons of trans-7, thus
suggesting that the dioxaphosphorinane structure of this
isomer is in a twist-chair conformation. By flipping from
the chair conformation to the twist-chair conformation,
the trans isomer should minimize the repulsive electro-
static interaction between the lone pairs of the endocyclic
phosphate oxygens and the O4′ sugar atom (Figure 3).
The puckering of the 2′-deoxyribose moieties of cis-7
and trans-7 was assigned by examination of the sugar
ring H/H coupling constants (Table 2). Whereas the H3′/
H4′ coupling constant cannot be used to clearly establish
the puckering of the lower nucleosides, the small JH3′/H4′
(∼2 Hz) measured for the upper nucleosides and the
values of JH2′/H3′ and JH1′/H2′ are close in each case to those
found in the standard C2′-endo conformation of the
natural 2′-deoxyribose.10
(10) Wood, D. J.; Ogilvie, K. K.; Hruska, F. E. Can. J. Chem. 1975,
53, 2781.
(11) Lankhorst, P. P.; Haasnoot, C. A. G.; Erkelens, C.; Altona, C.
J. J. Biomol. Struct. Dynam. 1984, 1, 1387. The torsional angle ꢀ was
determined by using the relation ꢀ° ) -θ - 120° and θ was calculated
with JH3′/P ) 15.3 cos2(θ) - 6.1 cos(θ) + 1.6.
To further investigate the overall conformation of
dinucleotides cis-7 and trans-7, we carried out a 2D
1622 J. Org. Chem., Vol. 70, No. 5, 2005

R,â,γ- and δ,ꢀ,ú-D-CNA
FIGURE 5. (a) Tentative model of the conformation of cis-7 and trans-7 R,â,γ-D-CNA derived from NMR data. (b) Superimposition
of the structures of cis-7 (blue) and trans-7 (red) R,â,γ-D-CNA with an X-ray structure of unmodified pTpT12 (green).
SCHEME 2. Synthesis of (SC4′,RP) and (SC4′,SP)
δ,E,ú-D-CNA
tions in which the distance between the protons of the
upper and the lower nucleoside are greater than 4 Å.
Thus, the observed NOESY spectra corroborate the 3JH/P
evidence discussed above for the chair and twist-chair
conformations of cis-7 and trans-7. A hand-built model
of both compounds constructed to fit the observed prox-
imity relationships is shown in Figure 5a.
The chair and twist-chair conformations of cis and
trans R,â,γ-D-CNA allow the stabilization of unusual
conformational states {(g-, g-, g-) and (g+, c/g+, g-/a-)},
which greatly differ from the canonical (g-, t, g+) back-
bone conformation adopted by the B- and A-forms of the
DNA double helix (Table 1 and Figure 5b). Although a
thorough analysis is required to precisely determine what
structural consequences can be expected from these
alternative backbone conformations, it is likely that once
incorporated into DNA or RNA oligomers,13 cis and trans
R,â,γ-D-CNA dinucleotides will either favor the formation
of unpaired secondary motifs or induce a significant
conformational distortion from the ideal B- or A-form
helical geometry.
Synthesis of (SC4′,RP) and (SC4′,SP) δ,E,ú-D-CNA. The
1/1 diastereoisomeric mixture of 11a and 11b was
quantitatively obtained from the expected base-induced
cyclization of the O-5′′-tosylated acyclic precursor 10
(Scheme 2). The 1/1 mixture of P epimers of 10 was
generated (following the standard procedure) from the
coupling reaction of the diastereopure (4′S)-C-tosyloxy-
methylthymidine 9 with 5′-thymidine phosphoramidite.
Diastereomerically pure nucleoside 9 was prepared
through the selective tosylation of the 5′′-OH function of
1, followed by removal of the tert-butyldiphenylsilyl group
(producing 8 in 25% combined yield) and dimethoxytri-
tylation of the remaining primary 5′′-hydroxyl function.
Although the synthetic pathway of Scheme 2 gives
access to the easily separable, fully protected, diastere-
oisomers 11a and 11b, the overall yield from 1 is low
(18%). This is due to the selective tosylation of 1 which,
under standard conditions, proceeds only with limited
success. In an attempt to increase the overall yield of 11,
we decided to take advantage of the greater reactivity of
the 5′′-hydroxyl function later in the synthetic pathway,
i.e., right at the cyclization step producing the dioxaphos-
phorinane structure (dinucleotide 14 in Scheme 3). Thus,
we applied the well-known phosphotriester methodology14
in the presence of the 1-(mesitylene-2-sulfonyl)-3-nitro-
1,2,4-triazole (MSNT) as an activating agent15 to quan-
titatively prepare the (SC4′,RP) and (SC4′,SP) diastereo-
isomers 15a and 15b, respectively. The acyclic phospho-
diester 14 was obtained in a good overall yield (80%) from
starting nucleoside 1 in a five-step sequence (Scheme 3).
Dimethoxytritylation of both primary hydroxyl functions
of 1 followed by desilylation of the O3′ oxygen atom (with
tetrabutylamonium fluoride) gave nucleoside 12 in a high
combined yield.16 After coupling 12 with a thymidine
bearing a phosphoramidite function at the 5′ position,
both dimethoxytrityl groups were removed under acidic
conditions to give the expected 1/1 diastereoisomeric
(12) Camerman, N.; Fawcett, J. K.; Camerman, A. J. Mol. Biol. 1976,
107, 601.
(13) In progress. Note that in contrast to acyclic or macrocyclic
phosphotriesters, the dioxaphosphorinane structure is very stable in
concentrated aqueous ammonia and is therefore perfectly compatible
with automated synthetic procedures.
(14) Sonveaux, E. Bioorg. Chem. 1986, 14, 274.
(15) (a) Reese, C. B.; Titmas, R. C.; Yan, L. Tetrahedron Lett. 1978,
2727. (b) Jones, S. S.; Rayner, B.; Reese, C. B.; Ubasawa, A.; Ubasawa,
M. Tetrahedron 1980, 36, 3075.
(16) Thrane, H.; Fensholdt, J.; Regner, M.; Wengel, J. Tetrahedron
1995, 51, 10389.
J. Org. Chem, Vol. 70, No. 5, 2005 1623

Le Cle´zio et al.
SCHEME 3. Improved Synthesis of (SC4′,RP) and
(SC4′,SP) δ,E,ú-D-CNA
FIGURE 6. X-ray molecular structure of (SC4′,SP) δ,ꢀ,ú-D-CNA
15b with the adopted numbering scheme, displaying the twist-
chair and C2′-endo conformations of the dioxaphosphorinane
and sugar subunits, respectively. For clarity, hydrogen atoms
and cocrystallized solvent molecules have been omitted and
thermal ellipsoids are shown at 40% probability.
TABLE 3. H/H Coupling Constants (Hz) in the 1H NMR
Spectra (400 MHz) of 15a and 15b
coupling constant J (Hz)
J(1′,2′)
J(2′,3′)
J(3′,4′)
15a
15b
upper nucleoside
lower nucleoside
upper nucleoside
lower nucleoside
5.6
8.9
7.5
9.2
8.0
5.4
0
6.2
5.5
6.1
5.4
6.2
6.5
2
<1
0
2.7
2.5
mixture of 13. The negatively charged phosphodiester
was released by treatment of 13 with triethylamine,
producing the acyclic precursor 14 which, in the presence
of MSNT in hot pyridine, quantitatively cyclized into the
1/1 diastereoisomeric mixture of 15a and 15b as observed
by 31P NMR (δp -2.3 and -3.8).
That the dioxaphosphorinane ring was actually formed
by reaction of the 5′′-hydroxyl group was shown by
recovering the 1/1 diastereoisomeric mixture of 11a and
11b upon dimethoxytritylation of 15. Finally, removal of
the tert-butyldiphenylsilyl protective group with floride
ion provided the (SC4′,RP) and (SC4′,SP) diastereoisomers
16a and 16b, respectively, which are ready to be con-
verted into their corresponding phosphoramidite building
blocks for oligonucleotide synthesis purposes. This syn-
thetic pathway allows for the preparation of 16a and 16b
in 55% overall yield from 1 compared with 18% for the
first approach described in Scheme 2.
Structural Assignment of δ,E,ú-D-CNA. The deter-
mination of the absolute configuration at the newly
created asymmetric centers 4′-C and P of δ,ꢀ,ú-D-CNA
11, 15, and 16 was greatly simplified by the X-ray
structure solved for 15b identified as the (SC4′,SP) di-
astereoisomer of 15 (Figure 6). This crystal structure
gives further evidence that the dioxaphosphorinane ring
system was formed via the reaction of the 5′′-hydroxyl
function of 14. The major information revealed by the
X-ray structure of 15b is the twist-chair conformation of
the six-membered cyclic phosphotriester with dihedral
angle values δ ) 140.7°, ꢀ ) -62.4°, and ú ) 152.5°. Both
sugar rings are in the C2′-endo conformation, which is
typical of B-DNA, with δ ) 140.7° for the fused dioxa-
phosphorinane/sugar system and δ ) 149.9° for the
unmodified sugar moiety. The C2′-endo conformation is
also observed for the 2′-deoxyribose units of 15a as
outlined by the analysis of the coupling constants of the
sugar ring protons of 15a and 15b (Table 3).
The solid-state structure of 15b agrees with the solu-
tion-state structure of 11 and 15 obtained by NMR
spectroscopy.17 The 3JH/P coupling constants measured for
the H3′ and H5′′ protons of the dioxaphosphorinane unit
in all compounds have intermediate values ranging
between 6.3 and 10.7 Hz (H3′) and between 11.6 and 16.8
Hz (H5′′), indicating that neither the (SC4′,SP) nor the
(SC4′,RP) stereoisomers of 11 and 15 are in a true chair
conformation. From the inspection of hand-built models,
it appears that the (SC4′,RP) stereoisomers of 11, 15, and
16 (i.e., 11a, 15a, and 16a) might well adopt a chair
conformation with the favorable 1,2-trans diequatorial
orientation of the C2′ and C5′ sugar atoms, and with both
sugar rings in a C2′-endo conformation. However, as
already mentioned in the case of trans-7, this arrange-
ment probably leads to important repulsive electrostatic
interactions between the lone pairs of the axially oriented
(17) Sartillo-Piscil, F.; Cruz, S.; Sa´nchez, M.; Ho¨pfl, H.; Anaya de
Parrodi, C.; Quintero, L. Tetrahedron 2003, 59, 4077.
1624 J. Org. Chem., Vol. 70, No. 5, 2005

R,â,γ- and δ,ꢀ,ú-D-CNA
FIGURE 7. Conformation within the dioxaphosphorinane ring of (SC4′,RP) δ,ꢀ,ú-D-CNA (a) and (SC4′,SP) δ,ꢀ,ú-D-CNA (b).
O4′ sugar atom and the two endocyclic phosphate oxygens
(Figure 7a). By flipping from the chair to the twist-chair
conformation, the (SC4′,RP) stereoisomers 11a, 15a, and
16a should minimize these interactions. On the other
hand, the (SC4′,SP) stereoisomers 11b, 15b, and 16b are
unlikely to adopt a chair conformation based on the
unfavorable 1,2-trans diaxial orientation of the C2′ and
C5′ sugar atoms and the required 2′-exo conformation of
the fused sugar unit (Figure 7b).
Experimental Section
3′-O-tert-Butyldiphenylsilyl-4′-C-(hydroxymethyl)thy-
midine (1) was prepared (45% yield) according to the proce-
dure described by Jones and co-workers9b (see compound 28a)
and purified by silica gel chromatography with use of a 1/1
mixture of ethyl acetate/ dichloromethane as eluent. TLC, Rf
(CH2Cl2/AcOEt 1/1) ) 0.3. 1H NMR (250 MHz, CDCl3) δppm 9.67
(s, 1H, NH); 7.66-7.63 (m, 4H, ph); 7.46-7.35 (m, 6H, ph);
6.86 (s, 1H, H6); 6.39 (dd, 1H, J ) 7.6, 4.3 Hz, H1′); 4.71 (t, 1H,
J ) 7.5 Hz, H3′); 3.94, 3.92, 3.62 and 3.42 (A and B part of AB
systems, 4H, J ) 12.5 Hz, H5′ and H5′′); 2.33 and 1.77 (m, 2H,
H2′); 1.66 (s, 3H, Me7); 1.08 (s, 9H, tBu). 13C NMR (63 MHz,
CDCl3) δppm 164.2; 150.9; 135.8; 133.0; 132.4; 130.3; 127.9;
111.1; 88.8; 83.4; 72.7; 62.6; 62.2; 39.7; 26.9; 19.1; 12.3. Anal.
Calcd (found): C 63.51 (63.71), H 6.71 (6.74), N 5.49 (5.22).
4′-C(S)-(4,4′-Dimethoxytrityloxymethyl)-3′-O-tert-bu-
tyldiphenylsilylthymidine (2). To a solution of 3′-O-tert-
butyldiphenylsilyl-4′-C-(hydroxymethyl)thymidine (1) (1.0 g,
1.96 mmol) in anhydrous pyridine (10 mL) is added dimethoxy-
trityl chloride (0.9 g, 2.65 mmol). After 12 h of stirring, the
reaction is stopped by addition of a saturated aqueous solution
of NH4Cl (20 mL) and extracted with ethyl acetate. The organic
layer is washed with water and brine and dried over MgSO4.
After removal of the solvent under reduced pressure, the crude
product was chromatographed on silica gel with ethyl acetate
as solvent. Compound 2 is obtained as a white foam (m ) 1.11
g, 70% yield). Its isomer (m ) 223 mg, 20%) bearing the
dimethoxytrityl group on the 5′-O position was also collected
(Rf ) 0.16). TLC, Rf (CH2Cl2/AcOEt 4/1) ) 0.23. 1H NMR (250
MHz, CDCl3) δppm 8.60 (s, 1H, NH); 7.47-7.21 (m, 20H, ph
and H6); 6.84-6.79 (m, 4H, ph); 6.32 (t, 1H, J ) 6.3 Hz, H1′);
4.49 (t, 1H, J ) 6.6 Hz, H3′); 3.84 (A part of an ABX system,
1H, J ) 4.6, 11.9 Hz, H5′); 3.77 (s, 6H, OMe); 3.59 (A part of
an AB system, 1H, J ) 10.4 Hz, H5′′); 3.26 (B part of an ABX
system, 1H, J ) 7.3, 11.9 Hz, H5′); 3.09 (B part of an AB
system, 1H, J ) 10.4 Hz, H5′′); 2.39 (m, 1H, H2′); 1.98 (m, 1H,
H2′); 1.84 (s, 3H, Me7); 0.91 (s, 9H, tBu). 13C NMR (63 MHz,
Thus, based on the X-ray structure of 15b and the
3
careful examination of the JH/P coupling constants for
the P-coupled CH and CH2 signals of 11, 15, and 16, we
were able to establish conclusively the conformation of
the backbone torsional angles δ (C5′-C4′-C3′-O3′), ꢀ
(C4′-C3′-O3′-P), ú (C3′-O3′-P-O5′) as (a+/t, g-, a+/t)
and (a+/t, g-, g-) for the (SC4′,SP) and (SC4′,RP) diastereo-
isomers, respectively. When compared with the backbone
conformations summarized in Table 1, we can expect that
DNA oligonucleotides incorporating the (SC4′,SP) dinucleo-
tide element would form B-type duplexes with unique BII-
like substates conformationally fixed. If this structural
feature is confirmed this compound might find important
applications in determining the role played by the
unusual BII substate in DNA curvature as well as in
structures of protein-bound DNA.18 On the other hand,
the (SC4′,RP) stereoisomer exhibits an even more unusual
ꢀ/ú combination (ꢀ - ú ) 0°), which could represent an
important transient substate between the BI (ꢀ - ú < 0°,
centered around -90°) and BII (ꢀ - ú > 0°, centered
around +90°) conformations of the DNA backbone.
Conclusion
CDCl3) δ
164.4; 158.5; 150.5; 144.9; 136.6; 135.8; 135.7;
ppm
We have synthesized two new members of the D-CNA
family in which the backbone torsional angles (R, â, γ)
or (δ, ꢀ, ú) of nucleic acids are simultaneously locked in
a dioxaphosphorinane ring structure. Structural analysis
indicates that cis and trans R,â,γ-D-CNA have R, â, and
γ torsions locked in the (g-, g-, g-) and (g+, c/g+, g-/a-)
conformations, respectively, while the two diastereoiso-
mers (SC4′,RP) and (SC4′,SP) of δ,ꢀ,ú-D-CNA have δ, ꢀ, and
ú torsions locked in the (a+/t, g-, g-) and (a+/t, g-, a+/t)
conformations. As can be seen, these structures are
associated with very different backbone conformations.
The incorporation of D-CNA building blocks at prese-
lected positions in an oligonucleotide is expected to create
a remarkable variety of different shapes including helical
distortions of B-DNA or nonhelical secondary structures
of functional RNA and we are currently examining this
possibility with R,â,γ- and δ,ꢀ,ú-D-CNA.
132.9; 132.8; 130.1; 128.2; 128.0; 127.8; 126.8;113.3; 111.8;
88.7; 86.6; 84.2; 72.7; 64.7; 63.6; 55.2; 40.3; 26.9; 19.1; 12.5.
Anal. Calcd (found): C 70.91 (71.26), H 6.45 (6.74), N 3.45
(3.14).
5′-O-Tosyl-4′-C(R)-hydroxymethyl-3′-O-tert-butyldiphen-
ylsilylthymidine (3). To 4′-C(S)-(4,4′-dimethoxytrityloxy-
methyl)-3′-O-tert-butyldiphenylsilylthymidine (2) (560 mg, 0.69
mmol) dissolved in anhydrous pyridine is added at 0 °C the
tosyl chloride (264 mg, 1.38 mmol). Stirring was maintained
for 12 h and the reaction mixture was diluted with ethyl
acetate and washed with a saturated aqueous solution of NH4-
Cl. The organic layer is collected and washed with water and
brine and dried over MgSO4. After removal of the solvent and
pyridine under reduced pressure the crude product was diluted
with a solution of trifluoroacetic acid (3%) in dichloromethane
(50 mL) and stirred for 6 h. After evaporation of the solvent
and trifluoroacetic acid under high vacuum, the crude material
is diluted with ethyl acetate (150 mL) and washed twice with
a saturated aqueous solution of NaHCO3 (50 mL), and with
water and brine. After silica gel chromatography eluted with
ethyl acetate/dichloromethane 1/1, compound 3 was recovered
(18) Djuranovic, D.; Hartmann, B. Biopolymers 2004, 73, 356.
J. Org. Chem, Vol. 70, No. 5, 2005 1625

Le Cle´zio et al.
as a white foam (458 mg, 90% yield). TLC, Rf (CH2Cl2/AcOEt
1/1) ) 0.44. 1H NMR (250 MHz, CDCl3) δppm 9.35 (s, 1H; NH);
7.65 (A part of an AB system, 2H, J ) 8.6 Hz, ph Ts); 7.61-
7.57 (m, 4H, ph); 7.46-7.35 (m, 6H; ph); 7.27 (B part of an AB
system, 2H, J ) 8.6 Hz, ph Ts); 7.13 (s, 1H, H6); 6.36 (t, 1H,
J ) 6.7; H1′); 4.59 (dd, 1H; J ) 4.3; 7.0; H3′); 4.14 (A part of an
AB system, 1H, J ) 12.4 Hz, H5′); 3.89 (A part of an AB system,
1H, J ) 12.5 Hz, H5′′); 3.87 (B part of an AB system, 1H, J )
12.4 Hz, H5′); 3.58 (B part of an AB system, 1H, J ) 12.5 Hz,
H5′′); 2.42 (s, 3H, Me Ts); 2.28 (A part of an ABX system, 1H,
J ) 4.3, 6.7, 13.3 Hz; H2′); 1.89 (m, 1H, H2′); 1.85 (s, 3H; Me);
1.06 (s, 9H; tBu). 13C NMR (63 MHz, CDCl3) δppm 163.8; 150.4;
145.3; 135.7; 135.2; 132.4; 131.9; 130.5; 130.0; 128.1; 127.8;
111.8; 86.6; 83.3; 73.4; 70.1; 62.7; 40.5; 30.9; 26.9; 21.7; 19.1;
12.3. Anal. Calcd (found): C 61.42 (61.22), H 6.06 (6.01), N
4.21 (4.24).
Cyanoethyl 3′-O-(5′-O-Dimethoxytrityl)thymidinyl-4′-
C(R)-oxymethyl(3′-tert-butyldiphenylsilyl)-5′-O-tosylthy-
midinyl Phosphoric Ester (mixture of diastereoisomers)
(4). 5′-O-tosyl-4′-C(R)-hydroxymethyl-3′-O-tert-butyldiphenyl-
silylthymidine (3) (500 mg, 0.75 mmol), 3′-O-thymidine phos-
phoramidite (1.12 g, 1.5 mmol), and freshly sublimed tetrazole
(525 mg, 7.5 mmol) were dissolved with anhydrous acetonitrile
(5 mL) and stirred for 20 min at room temperature. After
addition of collidine (454 µL, 3.0 mmol), the phosphite was
oxidized with iodine [0.1 M solution in THF(2)/H2O(1)] until
the dark brown color persists. The reaction mixture was
diluted with ethyl acetate and washed with an aqueous
solution of sodium thiosulfate (15%) to remove an excess of
iodine. The organic layer was washed with water and brine
and the solvent was removed in vacuo. The crude material was
chromatographed on silica gel with ethyl acetate as solvent.
After evaporation of the solvent, compound 4 (mixture of
diastereoisomers) was recovered as a white foam (0.79 g, 80%
yield). TLC, Rf (AcOEt/CH2Cl2 1/1) ) 0.25. 1H NMR (250 MHz,
CDCl3) δppm 9.46, 9.41, 9.35, 9.31 (4s, 4H, NH); 7.64-6.81 (m,
58H, ph and H6); 6.44-6.29 (m, 4H, H1′); 5.18 (m, 2H, H3′a);
4.60-4.55 (m, 2H, H3′b); 4.54-4.37 and 4.27-4.00 (m, 8H, H5′b
and H5′′b); 4.26 (br s, 2H, H4′); 3.86 (m, 4H, CH2OP); 3.77 (s,
12H, OCH3); 3.48-3.33 (m, 4H, H5′a); 2.75 and 2.58 (2t, 4H,
NCCH2); 2.76-2.30 (m, 4H, H2′); 2.42 (br s, 6H, CH3 Ts); 2.04-
1.86 (m, 2H, H2′); 1.80 and 1.37 (s, 12H, Me7); 1.29-1.19 (m,
2H, H2′); 1.08, 1.06 (2s, 18H, tBu). 13C NMR (63 MHz, CDCl3)
δppm 163.7; 158.8; 150.5; 149.9; 145.3; 144.0; 136.6; 135.8; 134.9;
132.6; 130.4; 131.9; 130.4; 130.2; 130.0; 128.1; 127.3; 116.3;
113.3; 111.8; 111.2; 87.3; 86.9; 85.8; 84.3; 76.6; 74.3; 68.6; 62.3;
55.3; 39.3; 30.9; 26.8; 21.7; 19.5; 19.1; 14.2; 12.3; 11.6. 31P NMR
(81 MHz, CDCl3) δppm -2.06; -3.05; MS (FAB) 1362 (MK+,
5%); 1346 (MNa+, 84%); 1323 (M+, 13%).
127.3; 113.6; 113.4; 112.0; 111.8. 111.4; 111.1. 88.0; 87.6; 87.5;
86.0. 85.5; 84.9; 84.5, 81.3; 81.1. 78.3; 74.2; 73.9; 72.1; 63.6;
62.1; 55.5; 40.2; 39.9; 37.9; 27.1; 19.4; 19.3; 15.5; 12.7; 12.6;
12.5; 11.9. MS (electrospray) 1121 (100%, [M + Na]+), 1137
(10%, [M + K]+).
5′-O-Dimethoxytrityl-r,â,γ-CNA cis-(6) and trans-(6).
To compounds 5a and 5b (110 mg; 0.1 mmol) dissolved in
anhydrous THF (2 mL) is added at room temperature the
tetrabutylammonium fluoride (150 µL, 0.15 mmol). Stirring
was maintained for 1 h. After removal of the solvent under
reduce pressure the crude product was deposed on a silica gel
column and eluted with ethyl acetate 3% methanol. Com-
pounds R,â,γ-CNA cis-6 and trans-6 were recovered as a white
foam (73 mg, 85% yield). cis-6 was separated from trans-6 by
means of reverse-phase HPLC (Kromasil C18, φ 20 mm, 250
mm) with CH3CN/H2O 1/1 as solvent. Data for cis-6: HPLC:
Tr ) 4.22 min. Probably due to the formation of aggregates in
solution, the NMR spectra were obtained with a very poor
resolution for 1H and without detection of the aliphatic carbon
for 13C. This phenomenon has already been observed on the
corresponding derivative of R,â-CNA (see Supporting Informa-
tion of ref 7). 1H NMR (250 MHz, CDCl3) δppm 7.56 (m, 1H,
H6); 7.31-7.22 (m, 10H, ph and H6); 6.83-6.79 (m, 4H, ph);
6.44 (m, 1H, H1′); 6.13 (m, 1H, H1′); 5.19 (m, 1H, H3′); 4.70-
4.20 (m, 6H); 3.76 (s, 6H, OMe); 3.46 (m, 1H, H5′a); 2.63-2.25
(m, 4H, H2′); 1.86 and 1.37 (2s, 6H, H7). 13C NMR (63 MHz,
CDCl3) δppm 164.1; 163.2; 150.9; 150.7; 144.1; 135.0; 129.9;
128.3; 113.3; 111.9; 111.5; 88.1; 80.4; 55.3. 31P NMR (81 MHz,
CDCl3) δppm -9.4. MS (electrospray) 883.1 ([M + Na]+); 899.1
([M + K]+). Data for trans-6: HPLC: Tr ) 3.17 min. Same
remark as for cis-6 (above) concerning the lack of resolution
1
1
of the H and 13C NMR spectra. H NMR (250 MHz, CDCl3)
δppm 7.58 (m, 1H, H6); 7.32-7.21 (m, 9H, ph); 7.00 (m, 1H, H6);
6.84-6.80 (m, 4H, ph); 6.42 (m, 1H, H1′); 6.06 (m, 1H, H1′);
5.20 (m, 1H, H3′); 4.69-4.20 (m, 6H); 3.76 (s, 6H, OMe); 3.43
(m, 1H, H5′a); 2.65 and 2.44 (m, 4H, H2′); 1.81 and 1.38 (2s,
6H, H7). 13C NMR (63 MHz, CDCl3) δppm 164.2; 163.2; 151.1;
150.8; 144.1; 135.1; 130.1; 128.1; 113.4; 111.9; 111.4; 87.3; 80.7;
55.3. 31P NMR (81 MHz, CDCl3) δppm -7.4. MS (electrospray)
883.1 ([M + Na]+); 899.2 ([M + K + Na]+).
r,â,γ-CNA cis-7 and r,â,γ-CNA trans-7. Compound cis-6
(45 mg, 0.052 mmol) (or trans-6, 32 mg, 0.037 mmol) is
dissolved in a solution of trifluoroacetic acid (3%) in dichlo-
romethane (2 mL) at room temperature. After 15 min the red
solution is evaporated to dryness. The crude material is
dissolved with THF and submitted to silica gel chromatogra-
phy. It was first eluted with ethyl acetate to remove the
dimethoxytrityl residue and then with ethyl acetate/methanol
(20%) to collect the R,â,γ-CNA cis-7 (29 mg, yield: 99%) or
R,â,γ-CNA trans-7 (20 mg, 98% yield) obtained as a white foam
after evaporation of the solvent. Data for cis-7: TLC, Rf
(AcOEt, 20% MeOH) ) 0.25. 31P NMR (200 MHz, CD3OD) δppm
5′-O-Dimethoxytrityl-3′-O-tert-butyldiphenylsilyl-r,â,γ-
CNA cis-(5) and trans-(5). To a solution of 4 (150 mg, 0.11
mmol) in anhydrous DMF (4 mL) was added K2CO3 (62 mg,
0.45 mmol). After 2 h of heating at 90 °C the reaction mixture
is cooled and diluted with ethyl acetate (50 mL) and washed
three times with water (3 × 10 mL) and once with brine (10
mL). The organic layer was dried over MgSO4 and the solvent
was removed in vacuo. The cyclic phosphotriesters 5 and 6
(2/1 ratio) were recovered as a white foam (123 mg, 100%
yield). TLC, Rf (AcOEt/CH2Cl2 1/1) ) 0.2. 31P NMR (81 MHz,
CDCl3) δppm -7.8; -9.6. 1H NMR (250 MHz, CDCl3) δppm 9.19,
9.16, 9.07 and 9.06 (4s, 4H, NH); 7.74-7.26 (m, 40H, ph and
H6); 7.00 (d, 1H, H6); 6.87-6.84 (m, 8H, ph); 6.82 (d, 1H, H6);
6.51-6.46 (m, 2H, H1′); 6.11-6.07 (m, 2H, H1′); 5.29 (m, 1H,
1
-6.05. H NMR (400 MHz, CD3OD) δppm 7.83 (d, 1H, J ) 1.2
Hz, H6a); 7.51 (d, 1H, J ) 1.2 Hz, H6b); 6.36 (dd, 1H, J ) 5.8;
8.4 Hz, H1′a); 6.31 (t, 1H, J ) 6.9 Hz, H1′b); 5.13 (tt, 1H, J )
2.0; 6.0; Hz, J3′a/P ) 6.2 Hz, H3′a); 4.68 (A part of an ABX, 1H,
J ) 11.9 Hz, J5′bax/P ) 2.1 Hz, H5′bax); 4.66 (A part of an ABX,
1H, J ) 12.3 Hz, J5′′bax/P ) 1.9 Hz H5′′bax); 4.50 (B part of an
ABX(Y), 1H, J ) 12.3; 2.9 Hz, J5′′beq/P ) 22.0 Hz, H5′′beq); 4.46
(B part of an ABX(Y), 1H, J ) 11.9; 2.9 Hz, J5′beq/P ) 22.0 Hz,
H
5′beq); 4.27 (∼q, 1H, J ) 2.0; 3.1 Hz, H4′a); 4.12 (X part of an
ABX, 1H, J ) 3.8; 6.6 Hz H3′b); 3.84 (d, 1H, J ) 3.2 Hz, H5′a);
2.59 (A part of an ABX(Y), 1H, J ) 14.1; 5.8; 2.1 Hz, H2′a);
2.51 (B part of an ABX(Y), 1H, J ) 14.0, 6.9; 6.8 Hz, H2′b);
H
3′a); 5.20 (m, 1H, H3′a); 4.85 (m, 1H, H3′b); 4.66 (m, 1H, H4′a);
4.59-4.41 (m, 4H, H5′b and H5′′b); 4.38 (q, 1H, J ) 4.8; 7.2 Hz,
3′b); 4.33-4.18 (m, 2H, H5′b or H5′′b); 3.98-3.84 (m, 2H, H5′b
2.44 (B part of an ABX(YZ), 1H, J ) 14.2; 8.4; 6.1 Hz, J2′a/P
)
H
1.1 Hz, H2′a); 2.39 (A part of an ABX(Y), 1H, J2′b1/2′b2 ) 14.0;
6.7; 3.8 Hz, H2′b); 1.92 (d, 3H, J ) 1.1 Hz, H7b); 1.90 (d, 3H, J
) 1.2 Hz, H7a). 13C NMR (100 MHz, CD3OD) δppm 165.3 (C4a,
C4b); 151.4 (C2a); 151.1 (C2b); 137.5 (C6b); 136.9 (C6a); 110.9 (C5a,
C5b); 86.8 (C1′b); 86.1 (C4′a); 85.0 (C1′a); 81.3 (C4′b); 79.0 (C3′a);
74.0 (C5′b); 72.9 (C5′′b); 71.9 (C3′b); 61.6 (C5′a); 39.1 (C2′b); 38.5
(C2′a); 11.4 (C7a, C7b). Anal. Calcd (found): N 10.03 (9.98); C
or H5′′b); 3.81 and 3.80 (2s, 12H, OMe); 3.55-3.41 (m, 4H, H5′a);
2.73-2.41 (m, 6H, H2′); 2.31-2.15 (m, 2H, H2′); 1.88, 1.83, 1.43
and 1.40 (4d, 12H, H7); 1.12 and 1.11 (2s, 18H, tBu). 13C NMR
(63 MHz, CDCl3) δppm 164.2; 164.0; 159.0; 158.8; 150.8; 150.2;
150.0; 144.2; 139.7; 136.1; 136.0; 135.9; 135.2; 133.1. 133.0;
132.3; 132.1; 131.0; 130.8; 130.6; 130.3. 128.5; 128.0; 127.5;
1626 J. Org. Chem., Vol. 70, No. 5, 2005

R,â,γ- and δ,ꢀ,ú-D-CNA
45.17 (45.01); H 4.87 (4.92). Data for trans-7: TLC, Rf (AcOEt,
20% MeOH) ) 0.25. 31P NMR (200 MHz, CD3OD) δ ppm -4.77.
1H NMR (400 MHz, CD3OD) δppm 7.80 (d, 1H, J ) 1.2 Hz, H6a);
7.44 (d, 1H, J ) 1.2 Hz, H6b); 6.34 (t, 1H, J ) 5.9 Hz, H1′b);
6.32 (t, 1H, J ) 5.8 Hz, H1′a); 5.14 (tt, 1H, J ) 1.8; 5.8 Hz,
J3′a/P ) 6.8 Hz, H3′a); 4.74 (B part of an ABXY, 1H, J ) 11.5;
13.2; 1.0 Hz, H5′′beq); 4.58 (B part of an ABXY, 1H, J ) 11.7
Hz, J5′beq/P ) 13.1 Hz, J5′beq/5′′beq ) 1.0 Hz, H5′beq); 4.55 (X part
of an ABX, 1H, J ) 2.1; 5.5 Hz, H3′b); 4.47 (A part of an ABX,
1H, J ) 11.7; 11.5 Hz, H5′bax); 4.42 (A part of an ABX, 1H, J )
11.5 Hz, J5′′bax/P ) 11.2 Hz, H5′′bax); 4.24 (∼q, 1H, J ) 1.9; 3.1
Hz, H4′a); 3.78 (A part of an ABX, 1H, J ) 12.1; 3.0 Hz, H5′a);
3.75 (B part of an ABX, 1H, J ) 12.1; 3.1 Hz, H5′a); 2.59 (A
part of an ABX(Y), 1H, J ) 13.8; 5.7; 5.5 Hz, H2′b); 2.56 (A
part of an ABX(Y), 1H, J ) 14.2; 5.7; 1.7 Hz H2′a); 2.39 (B part
of an ABX(YZ), 1H, J ) 14.2; 5.9 Hz, J2′a/P ) 1.4 Hz, H2′a);
2.33 (B part of an ABX(Y), 1H, J ) 13.8; 5.7; 2.1 Hz, H2′b);
ppm 164.2; 158.9; 157.4; 150.6; 148.0; 145.2; 144.3; 135.9; 135.3;
132.7; 130.3; 130.0; 129.4; 128.4; 128.2; 128.0; 127.4; 121.6;
113.5; 113.4; 113.2; 111.4; 87.4; 86.9; 73.0; 70.0; 64.9; 55.5; 40.9;
24.2; 15.6; 12.1. Anal. Calcd (found): N 3.84 (3.92); C 64.27
(64.56); H 5.53 (5.61).
Cyanoethyl 3′-O-(5′-O-dimethoxytrityl-4′-C-tosyloxym-
ethyl)thymidinyl-5′-O-(3′-O-tert-butyldiphenylsilyl)thy-
midinyl Phosphoric Ester (mixture of diastereoisomers)
(10). Compound 9 (83 mg, 0.114 mmol), 5′-phosphoramidite-
3′-tert-butyldiphenylsilylthymidine (155 mg, 0.228 mmol), and
freshly sublimed tetrazole (80 mg, 1.14 mmol) were diluted
into anhydrous acetonitrile (1.5 mL) and stirred for 45 min at
room temperature. After addition of collidine (70 µL), the
phosphite was oxydized with a solution of iodine [0.1 M in
THF(2)/H2O(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 over MgSO4 before removal of the solvent.
Compound 10 (140 mg, 93% yield) was isolated (mixture of
diastereoisomers) as a white foam after chromatography on
silica gel eluted first with CH2Cl2/AcOEt 1/1 and with AcOEt.
TLC, Rf (CH2Cl2/AcOEt 1/1) ) 0.14. 31P NMR (81 MHz, CDCl3)
δppm -2.8 and -3.1 (dias). 1H NMR (250 MHz, CDCl3) δppm
8.89; 8.82 and 8.79 (3s, 4H; NH); 7.68-7.13 (m, 58H; ph and
H6); 6.38-6.32 (m, 2H, H1′); 6.18-6.13 (m, 1H, H1′); 6.10-6.04
(m, 1H, H1′); 5.09 (m, 2H, H3′a), 4.34 (m, 2H); 4.16-3.79 (m,
10H); 3.77 (s, 12H, Me DMTr); 3.38-3.12 (m, 4H, CH2); 2.47
and 2.46 (2s, 6H, Me Ts); 2.63-2.29 (m, 8H, CH2), 1.82; 1.79;
1.50 and 1.49 (4s, 12H, Me7); 1.92; 1.90; 1.32 and 1.27 (4m,
8H, H2′); 1.08 and 1.07 (s, 18H, tBu). 13C NMR (63 MHz, CDCl3)
δppm 164.2; 164.0; 159.0; 158.9; 150.8; 150.7; 150.4; 145.5; 145.4;
144.0; 136.2; 135.9; 134.9; 134.8; 133.1; 133.0; 132.9; 130.5;
130.4; 130.3; 130.2; 130.1; 128.3; 128.2; 128.0; 127.5; 127.3;
121.6; 116.7; 116.5; 113.5; 111.8; 111.7; 111.6; 87.7; 87.6; 86.2;
86.0; 85.8; 85.5; 85.4; 85.3; 85.1; 85.0; 78.9; 78.7; 73.3; 73.0;
69.0; 68.9; 67.9; 64.8; 64.7; 62.7; 62.6; 55.5; 40.2; 40.0; 38.9;
38.7; 27.0; 21.9; 19.7; 19.2; 12.5; 12.1. Anal. Calcd (found): N
5.29 (5.42); C 61.67 (62.02); H 5.63 (5.57).
5′-O-Dimethoxytrityl-3′-O-tert-butyldiphenylsilyl-
(SC,RP)-δ,E,ú-CNA (11a) and 5′-O-Dimethoxytrityl-3′-O-
tert-butyldiphenylsilyl-(SC,SP)-δ,E,ú-CNA (11b). Starting
from 10 (140 mg, 0.1 mmol), the fully protected δ,ꢀ,ú-CNA 11a
and 11b (116 mg, 100% yield) were obtained following the
procedure described for the R,â,γ-CNA 5. Starting from a
mixture of 15a and 15b (200 mg, 0.25 mmol), 11a and 11b
are prepared by adding dimetoxytrityl chloride (0.63 mmol,
214 mg), silver nitrate (0.63 mmol, 107 mg), and collidine (2.5
mmol, 0.33 mL) in anhydrous DMF (6 mL). The reaction
mixture was stirred for 5 h at 90 °C. The reaction was stopped
by addition of a saturated aqueous solution of NaHCO3 (10
mL) at 0 °C and extracted with ethyl acetate. The organic layer
was washed with water (twice) and brine and dried over
MgSO4. After removal of the solvent 11a and 11b were
separated by chromatography on silica gel with ethyl acetate/
dichloromethane 1/1 as eluent. Data for 11a: TLC, Rf (CH2-
Cl2/AcOEt 1/1) ) 0.2. 31P NMR (81 MHz, CDCl3) δppm -1.4. 1H
NMR (250 MHz, CDCl3) δppm 9.59 and 9.35 (2s, 2H; NH); 7.67-
6.83 (m, 25H; ph, H6a and H6b); 6.57 (dd, 1H, J ) 8.8, 5.7 Hz,
1.91 (d, 1H, J ) 1.1 Hz, H7b); 1.89 (d, 1H, J ) 1.2 Hz, H7a). 13
C
NMR (100 MHz, CD3OD) δppm 165.3 (C4a, C4b); 151.3 (C2a, C2b);
137.1 (C6b); 136.9 (C6a); 111.2 (C5b); 110.9 (C5a); 86.4 (C1′b); 86.1
(C4′a); 85.1 (C1′a); 80.6 (C4′b); 80.1 (C3′a); 70.6 (C5′b); 72.6 (C5′′b);
71.5 (C3′b); 61.6 (C5′a); 38.2 (C2′b); 38.1 (C2′a); 11.5 (C7b); 11.4
(C7a). Anal. Calcd (found): N 10.03 (10.21); C 45.17 (45.42); H
4.87 (4.65).
4′-C(S)-Tosyloxymethylthymidine (8). Compound 1 (500
mg, 0.98 mmol) was diluted in anhydrous pyridine (1.7 mL)
and chloroform (3 mL), then tosyl chloride was added at 0 °C
(187 mg, 0.98 mmol). After 12 h of stirring the reaction mixture
was diluted with ethyl acetate and washed with a saturated
aqueous solution of NH4Cl. The organic layer is collected and
washed with water and brine and dried over MgSO4. After
removal of the solvent and pyridine under reduced pressure
the crude material is submitted to NEt3‚2HF (386 µL, 2.48
mmol) in THF (7 mL) for 15 h at 60 °C. The reaction medium
was cooled to 0 °C and the reaction was stopped by addition
of a saturated aqueous solution of NaHCO3 (20 mL) and
extracted with ethyl acetate. The organic layer was washed
with water and brine and dried over MgSO4. After evaporation
of the solvent the crude material was chromatographed on
silica gel with a first elution with ethyl acetate/dichlo-
romethane (1/1) as solvent and then with ethyl acetate to
collect compound 8 (104 mg, 25% yield) as a white foam after
1
evaporation of the solvent. TLC, Rf (AcOEt) ) 0.20. H NMR
(250 MHz, CDCl3) δppm 9.05 (s, 1H; NH); 7.75 (A part of an AB
system, 2H, J ) 8.3 Hz; Ts); 7.58 (s, 1H, H6); 7.33 (B part of
an AB, 2H, J ) 8.3 Hz; Ts); 6.11 (t, 1H, J ) 6.8 Hz; H1′); 4.46
(t, 1H; J ) 4.9 Hz; H3′); 4.15 (s, 2H, H5′′ or H5′); 3.64 (s, 2H,
H5′′ or H5′); 2.40 (s, 3H, Me Ts); 2.26 (dd, 2H, J ) 5.3; 6.7 Hz;
H2′); 1.84 (s, 3H; Me7). 13C NMR (63 MHz, CDCl3) δppm 166.4;
152.2; 146.6; 138.2; 133.7; 131.3; 129.4; 112.2; 88.5; 86.4; 73.1;
71.4; 64.1; 62.0; 41.8; 22.7; 15.2; 13.4. Anal. Calcd (found): N
6.57 (6.80); C 50.70 (50.32); H 5.20 (5.25).
5′-O-Dimethoxytrityl-4′-C(S)-tosyloxymethylthymi-
dine (9). To a solution of compound 8 (100 mg, 0.234 mmol)
in anhydrous dichloromethane (5 mL) was added silver nitrate
(40 mg, 0.234 mmol), dimethoxytrityl chloride (85 mg, 0.257
mmol), and collidine (39 µL, 0.292 mmol). After 12 h of stirring
at room temperature, the reaction was stopped by addition of
a saturated aqueous solution of NaHCO3 (10 mL) at 0 °C and
extracted with ethyl acetate. The organic layer was washed
with water and brine and dried over MgSO4. After removal of
the solvent compound 9 (153 mg, 90% yield) was isolated as a
white foam after chromatography on silica gel eluted first with
CH2Cl2/AcOEt 4/1 and with 1/1. TLC, Rf (CH2Cl2/AcOEt 1/1)
) 0.4. 1H NMR (250 MHz, CDCl3) δppm 8.69 (s, 1H; NH); 7.72
(A part of an AB, 2H, J ) 8.2 Hz; ph Ts); 7.31-6.78 (m, 16H,
ph and H6); 6.25 (t, 1H, J ) 7.0 Hz; H1′); 4.52 (t, 1H; J ) 6.1
Hz; H3′); 4.28 (A part of an AB system, 1H, J ) 10.4 Hz; H5′);
4.12 (B part of an AB, 1H, J ) 10.4 Hz; H5′); 3.79 (s, 6H, Me
DMTr); 3.38 (A part of an AB, 1H, J ) 10.0 Hz; H5′′); 3.19 (B
part of an AB, 1H, J ) 10.0 Hz; H5′′); 2.40 (s, 3H, Me Ts); 2.30
(m, 2H, H2′); 2.25 (s, 3H; Me7). 13C NMR (63 MHz, CDCl3) δ
H
1′a); 6.45 (dd, 1H, J ) 8.4, 6.0 Hz, H1′b); 5.01 (br t, 1H, J )
6.0 Hz, J3′a/P) 6.4 Hz, H3′a); 4.30 (m, 1H, H3′b); 4.25 (A part of
an ABX, 1H, J ) 12.0 Hz, J5′′a/P ) 11.6 Hz, H5′′a); 4.07 (m, 1H,
H5′b); 4.01 (B part of an ABX, 1H, J ) 12.0 Hz, J5′′a/P ) 16.8
Hz, H5′′a); 3.83 (m, 1H, H5′b); 3.79 (s, 6H, Me DMTr); 3.71 (m,
1H, J ) 6.1, 11.2 Hz, J4′b/P ) 8.2 Hz, H4′b); 3.46 (A part of an
AB, 1H, J ) 10.4 Hz, H5′a); 3.31 (B part of an AB, 1H, J )
10.0 Hz, H5′a); 2.75 (dd, 1H, J ) 14.0, 5.6 Hz, H2′a); 2.58 (m,
1H, J2′a/P < 1 Hz, H2′a); 2.34 (m, 1H, J ) 12.0, 5.6, <1 Hz, H2′b);
1.83 (m, 1H, H2′b); 1.80 and 1.51 (2s, 6H, Me7); 1.11 (s, 9H,
tBu). 13C NMR (63 MHz, CDCl3) δppm 164.5; 164.4; 151.0; 150.8;
144.4; 136.4-128.1; 114.1; 112.5; 112.0; 111.8; 88.2; 86.3; 85.8;
85.0; 84.9; 82.5; 73.6; 70.0; 68.5; 65.8; 56.0; 41.0; 40.4; 39.8;
30.4; 27.5; 19.7; 13.0; 12.5. Anal. Calcd (found): N 5.10 (5.12);
J. Org. Chem, Vol. 70, No. 5, 2005 1627

Le Cle´zio et al.
C 63.38 (63.23); H 5.78 (5.82). Data for 11b: TLC Rf (CH2Cl2/
AcOEt 1/1) ) 0.3. 31P NMR (81 MHz, CDCl3) δppm -2.8. 1H
NMR (250 MHz, CDCl3) δppm 9.49 and 9.40 (2s, 2H; NH); 7.68-
6.85 (m, 25H; ph, H6a and H6b); 6.57 (dd, 1H, J ) 9.0, 5.7 Hz,
acetate and washed with a saturated aqueous solution of
NaHCO3 (10 mL). The organic layer was washed with water
and brine and dried over MgSO4. After removal of the solvent
the two δ,ꢀ,ú-CNA 15a (210 mg) and 15b (208 mg) (1/1 ratio,
98% yield) were separated by silica gel chromatography with
CH2Cl2/5% MeOH as eluent. Data for 15a: TLC, Rf (CH2Cl2/
H1′a); 6.38 (dd, 1H, J ) 8.0, 5.7 Hz, H1′b); 5.19 (t, 1H, J ) 5.3
Hz, J3′a/P ) 6.3 Hz, H3′a); 4.38 (m, 1H, H3′b); 4.09 (m, 2H; H4′b
and H5′b); 3.96 (m, 2H, J5′′a/P ) 13.2 Hz, H5′′a); 3.81 (s, 6H, Me
DMTr); 3.65 (m, 1H, Hz, H5′b); 3.54 (A part of an AB, 1H, J )
10.1 Hz, H5′a); 3.37 (B part of an AB, 1H, J ) 10.2 Hz, H5′a);
2.55 (m, 2H, H2′a); 2.43 (m, 1H, J ) 13.4, 5.7, 2.2 Hz; H2′b);
1.91 (m, 1H, J ) 14.0, 8.0, 2.0 Hz, H2′b); 1.85 and 1.36 (2s, 6H,
Me7); 1.09 (s, 9H, tBu). 13C NMR (63 MHz, CDCl3) δ ppm 164.5;
164.1; 159.6; 150.9; 144.2; 136.4-128.2; 114.1; 112.9; 111.7;
88.5; 86.1; 85.8; 85.5; 84.9; 81.7; 73.4; 70.0; 68.6; 65.9; 56.0;
41.3; 39.6; 30.4; 27.5; 19.6; 13.0; 12.3. Anal. Calcd (found): N
5.10 (5.22); C 63.38 (63.05); H 5.78 (5.67).
1
5% MeOH) ) 0.10. 31P NMR (162 MHz, CDCl3) δppm -2.3. H
NMR (400 MHz, CDCl3) δppm 10.11 and 9.97 (s, 1H, NH); 7.67-
7.64 (m, 4H, ph); 7.49-7.40 (m, 7H, ph and H6); 7.22 (s, 1H,
H6); 6.29 (dd, 1H, J ) 6.2, 7.5 Hz, H1′b); 6.18 (dd, 1H, J ) 5.6,
8.9 Hz, H1′a); 5.01 (dd, 1H, J ) 5.4 Hz, J3′a/P ) 7.8 Hz, H3′a);
4.41 (m, 1H, OH); 4.38 (m, 1H, H3′b); 4.20 and 4.11 (AB part of
an ABX, 2H, J ) 12.1 Hz, J5′′a/P ) 15.1 Hz, H5′′a); 4.02 (A part
of an ABX(Y), 1H, J ) 10.0; 2.2 Hz, J5′b/P ) 5.0 Hz, H5′b); 4.09
(br s, 1H, H4′b); 3.76 (B part of an ABX(Y), 1H, J ) 10.0; 4.1
Hz, J5′b/P ) 5.5 Hz, H5′b); 3.68 (br s, 2H, H5′a); 2.86 (m, 1H, J )
14.2, 8.9, 5.4 Hz, H2′a); 2.56 (m, 1H, J ) 14.2, 5.6 Hz, H2′a);
2.36 (m, 1H, J ) 13.4, 6.0, 2.0 Hz, H2′b); 2.00 (m, 1H, J ) 13.4,
7.4, 5.4 Hz, H2′b); 1.85 (s, 6H, Me7); 1.09 (s, 9H, tBu). 13C NMR
(63 MHz, CDCl3) δppm 165.1; 165.0; 151.3; 151.1; 136.2; 133.4;
133.3; 130.8; 128.6; 111.8; 111.6; 88.9; 86.4; 85.7; 85.4; 85.3;
83.3; 73.4; 64.2; 63.2; 61.7; 46.4; 40.7; 39.6; 27.4; 26.4; 19.6;
12.9; 9.1. MS (electrospray) 819.2 (M + Na+). Anal. Calcd
(found): N 7.03 (7.12); C 55.77 (55.85); H 5.69 (5.73). Data for
15b: TLC, Rf (CH2Cl2/5% MeOH) ) 0.05. 31P NMR (202 MHz,
CD3OD) δppm -3.8. 1H NMR (500 MHz, CD3OD) δppm 7.74-
7.70 (m, 5H, ph and H6); 7.50-7.43 (m, 7H, ph and H6); 6.44
(dd, 1H, J ) 5.5; 9.2 Hz, H1′a); 6.38 (dd, 1H, J ) 6.1, 8.0 Hz,
Cyanoethyl 3′-O-(4′-C-Hydroxymethyl)thymidinyl-5′-
O-(3′-O-tert-butyldiphenylsilyl)thymidinyl Phosphoric
Ester (13). Compound 12 (1.86 g; 2.12 mmol) was coupled with
5′-O-(cyanoethyldiisopropylphosphoramidite)-3′-O-tert-butyl-
diphenylsilylthymidine (2.89 g, 4.24 mmol) in the presence of
tetrazole (1.48 g, 21.2 mmol) according to protocol described
for 10. The crude material obtained was dissolved in 20 mL of
a solution of dichloromethane with 3% of TFA, at room
temperature. After 15 min, the red solution was evaporated
to dryness. The dinucleotide 13 (1.56 g, 85% yield) was
obtained as a white foam by purification on silica gel with CH2-
Cl2/AcOEt 1/1 then AcOEt and finally AcOEt/5% MeOH as
eluent. TLC, Rf (AcOEt/5%MeOH) ) 0.22. 31P NMR (81 MHz,
H1′b); 5.14 (dd, 1H, J ) 6.2 Hz, J3′a/P ) 10.7 Hz, H3′a); 4.51 (m,
1
1H, J ) 2.5 Hz, H3′b); 4.34 (A part of an ABX, 1H, J ) 12.0
Hz, J5′′a/P ) 14.1 Hz, H5′′a); 4.17-4.09 (m, 3H, H4′b, H5′′a, H5′b);
3.86 (B part of an ABX(Y), 1H, J ) 11.2; 4.1 Hz, J5′b/P ) 6.8
Hz, H5′b); 3.77 and 3.68 (AB, 2H, J ) 11.7 Hz, H5′a); 2.51 (A
part of an ABX(YZ), 1H, J ) 14.5; 6.2; 9.2 Hz, J2′a/P ) 1.5 Hz,
CDCl3) δppm -2.9 and -2.7 (diastereoisomers). H NMR (250
MHz, CDCl3) δppm 7.61-7.13 (m, 24H, ph and H6); 6.32-6.08
(m, 4H, H1′); 5.07 (m, 2H, H3′a); 4.29 (m, 2H); 4.11 (m, 8H);
3.83 (m, 4H); 3.68 (m, 6H); 2.94 and 2.66 (2m, 8H); 2.43 and
2.25 (2m, 8H, H2′); 1.80 and 1.79 (2s, 12H, Me7); 1,03 (s. 18H,
tBu). 13C NMR (63 MHz, CDCl3) δ
164.5; 150.7; 144.8;
H2′a); 2.41 (B part of an ABX(Y), 1H, J ) 14.5; 5.5 Hz, H2′a);
ppm
135.6; 132.7; 132.5; 130.2; 128.7; 128.1; 128.0; 127.9; 125.6;
116.7; 111.3; 110.9; 88.7; 84.9; 79.0; 72.7; 62.8; 62.6; 61.5; 39.7;
39.0; 26.6; 21.1; 19.4; 19.3; 19.2; 18.8; 12.2; 12.0. MS (FAB)
890 (M + Na+). Anal. Calcd (found): N 5.81 (5.67); C 55.36
(54.92); H 5.81 (5.67).
3′-O-(4′-C-Hydroxymethyl)thymidinyl-5′-O-(3′-O-tert-
butyldiphenylsilyl)thymidinyl Phosphoric Acid Triethyl-
ammonium Salt (14). Compound 13 (1.6 g; 1.84 mmol) was
treated with triethylamine (530 µL, 3.68 mmol) in 15 mL of
acetonitrile at 60 °C for 3 h. After removal of the solvent and
excess of base under vacum, the dinucleotide 14 was obtained
as a white foam (1.7 g, 100% yield). TLC Rf (AcOEt/20%MeOH)
) 0.25. 31P NMR (81 MHz, CD3OD) δppm -0.25. 1H NMR (400
MHz, CD3OD) δppm 7.89 (d, 1H, J ) 1.5 Hz, H6); 7.69-7.67
(m, 5H, ph and H6); 7.46-7.44 (m, 6H, ph); 6.47 (dd, 1H, J )
5.5; 8.9 Hz, H1′b); 6.25 (t, 1H, J ) 7.0 Hz, H1′a); 4.91 (m, 1H,
2.37 (A part of an ABX(Y), 1H, J ) 13.5; 2.7; 6.0 Hz, H2′b);
2.13 (B part of an ABX(Y), 1H, J ) 13.5; 6.5; 8.0 Hz, H2′b);
1.91 and 1.85 (s, 3H, Me7); 1.11 (s, 9H, tBu). 13C NMR (126
MHz, CDCl3) δppm 164.8; 150.9; 150.7; 136.1; 136.0; 135.6;
132.9; 132.7; 130.0; 129.9; 127.8; 127.7; 110.7; 110.5; 85.1; 85.0;
84.9; 82.4; 729.4; 69.2; 67.6; 62.9; 39.7; 38.4; 38.3; 26.0; 18.4;
11.1. MS (electrospray) 819.2 (M + Na+). Anal. Calcd (found):
N 7.03 (7.02); C 55.77 (55.65); H 5.69 (5.66).
5′-O-Dimethoxytrityl-(SC,RP)-δ,E,ú-CNA (16a) and 5′-O-
Dimethoxytrityl-(SC,SP)-δ,E,ú-CNA (16b). Compound 11a (or
11b) was dissolved in anhydrous THF (5 mL/0.10 mmol) and
nBu4NF (110 µL, sol 1 M in THF) was added at 0 °C (120 µL,
0.11 mmol). After 1 h of stirring, the solvent was removed and
the crude material was chromatographed on silica gel with
first AcOEt as eluent and then AcOEt 10% MeOH to recover
16a (or 16b) (80 mg, 90% yield) as a white foam after removal
of the solvent. Data for 16a: 31P NMR (81 MHz, CD3OD) δppm
-3.5. 1H NMR (250 MHz, CDCl3) δppm 9.76 (m, 2H, NH); 7.35-
6.80 (m, 15H, ph and H6); 6.55 (t, 1H, H1′b); 6.26 (t, 1H, H1′a);
5.15 (m, 1H, H3′a); 4.35-4.06 (m, 6H); 3.74 (s, 6H, DMTr); 3.45
(m, 1H); 3.26 (m, 1H); 2.88 (m, 1H, H2′); 2.75 (m, 1H, H2′); 2.54
H
3′a); 4.56 (d, 1H, J ) 5.3; 1.5 Hz, H3′b); 4.12 (m, 1H, J ) 1.5
Hz; J4′b/P ) 2.3 Hz, H4′b); 3.92 (A part of an ABX, 1H, J ) 2.9;
11.2 Hz, J5′b/P ) 4.3 Hz, H5′b); 3.75 (m, 2H, J ) 6.0; 12.1 Hz,
H
5′a); 3.68 (B part of an ABX, 1H, J ) 11.4 Hz; J5′′b/P < 1 Hz,
5′′b); 3.62 (m, 2H, J ) 11.9 Hz, H5′′a); 3.18 (q, 6H, CH2 Et3N);
H
(m, 1H, H2′); 2.28 (m, 1H, H2′); 1.72 and 1.48 (2s, 6H, Me7). 13
C
2.41 (A part of an ABX(Y), 1H, J ) 3.6; 6.3; 13.7 Hz, H2′a);
2.30 (B part of an ABX(Y), 1H, J ) 6.6; 6.7; 13.7 Hz, H2′′a);
2.19 (A part of an ABX(Y), 1H, J ) 1.6; 5.6; 13.5 Hz, H2′b);
2.08 (B part of an ABX(Y), 1H, J ) 5.4; 9.0; 13.4 Hz, H2′b);
1.91 and 1.90 (2s, 6H, Me7); 1.28 (q, 9H, CH3 Et3N); 1.03 (s,
9H, tBu). 13C NMR (63 MHz, CD3OD) δppm 165.3; 165.2; 151.3;
151.7; 137.1; 136.7; 135.7; 133.2; 133.1; 130.0; 127.9; 111.3;
111.0; 89.1; 89.0; 86.6; 86.5; 85.0; 84.5; 75.7; 74.6; 65.4; 63.2;
61.7; 40.0; 39.6; 26.2; 18.6; 11.5; 11.4; 8.1. MS (electrospray)
813.3 (M+).
3′-O-tert-Butyldiphenylsilyl-(SC,RP)-δ,E,ú-CNA (15a) and
3′-O-tert-Butyldiphenylsilyl-(SC,SP)-δ,E,ú-CNA (15b). To
compound 14 (500 mg, 0.55 mmol) dissolved in 5 mL of
anhydrous pyridine was added 2,4,6-trimethylphenyl-3-nitro-
1,2,4-triazol-1-yl (MSNT) (380 mg, 1.25 mmol). After 2 h of
stirring at 90 °C, the reaction mixture was diluted with ethyl
NMR (75 MHz, CD3OD) δppm 164.1; 163.9; 158.9; 150.6; 150.5;
143.8; 135.8; 135.3;134.6; 130.0; 129.9; 128.2; 127.9; 127.4;
113.5; 111.9; 111.1; 87.5; 85.4; 85.2; 84.5; 84.2; 81.9; 70.3; 69.3;
67.8; 64.9; 60.4; 55.3; 51.9; 39.8; 39.2; 29.7; 29.5; 29.3; 25.7;
20.3; 12.4; 11.9. MS (electrospray) 883.1 ([M + Na+]+); 899.0
([M + K+]+). Anal. Calcd (found): N 6.51 (6.44); C 58.60 (58.47);
H 5.27 (5.21). Data for 16b: 31P NMR (81 MHz, CD3OD) δppm
1
-4.4. H NMR (250 MHz, CDCl3) δppm 10.50 and 9.82 (s, 2H,
NH); 7.48 and 7.39 (s, 2H, H6); 7.31-7.19 (m, 9H, ph); 6.86
(m, 4H, ph); 6.69 (dd, 1H, J ) 9.1, 5.5 Hz, H1′b); 6.27 (t, 1H, J
) 6.7 Hz, H1′a); 5.23 (dd, 1H, J ) 16.5, 6.1 Hz, H3′a); 4.55-
4.37 (m, 4H); 4.25-4.10 (m, 4H); 3.79 (s, 6H, DMTr); 3.37 (s,
2H, H5′a); 2.76 (m, 1H, H2′); 2.46 (m, 3H, H2′); 2.14 (m, 1H,
H2′); 1.88 and 1.34 (s, 6H, Me7). 13C NMR (63 MHz, CD3OD)
δppm 163.4; 159.7; 151.1; 150.3; 143.4; 134.3; 130.1; 130.0. 128.3;
1628 J. Org. Chem., Vol. 70, No. 5, 2005

R,â,γ- and δ,ꢀ,ú-D-CNA
128.0; 127.6; 113.6; 112.8; 111.1; 87.9; 85.5; 84.8; 84.6; 82.4;
70.6; 68.2; 55.3; 52.2; 40.1; 29.8; 20.4; 12.4; 11.6. MS (electro-
spray) 883.1 ([M + Na+]+); 899.0 ([M + K+]+). Anal. Calcd
(found): N 6.51 (6.39); C 58.60 (57.99); H 5.27 (5.34).
Crystal data for 15b: C39H47.25Cl5.75N4O12PSi, M ) 1026.96,
monoclinic, P21, a ) 10.670(1) Å, b ) 18.748(1) Å, c ) 23.941-
(2) Å, â ) 98.397(2)°, V ) 4737.6(6) Å3, Z ) 4, T ) 193(2) K.
21327 reflections (13121 independent, Rint ) 0.0507) were
collected at low temperatures, using an oil-coated shock-cooled
crystal on a Bruker-AXS CCD 1000 diffractometer with Mo
KR radiation (λ ) 0.71073 Å). The structure was solved by
direct methods (SHELXS-97)19 and all non-hydrogen atoms
were refined anisotropically using the least-squares method
on F2.20 Largest electron density residue: 0.483 e Å-3, R1 (for
I > 2σ(I)) ) 0.0614 and wR2 ) 0.1454 (all data) with R1
)
∑||Fo| - |Fc||/∑|Fo| and wR2 ) (∑w(Fo - Fc2)2/∑w(Fo )2)0.5
.
2
2
Acknowledgment. We thank Dr. Y. Coppel and Dr.
S. Massou for their assistance in NMR spectroscopy.
Supporting Information Available: General methods;
dinucleotide numbering; dioxaphosphorinane protons region
of 1H and 1H-{31P},1H-COSY, 2D-NOESY, and 1D-NOESY
NMR spectra of R,â,γ-CNA cis-7 and trans-7; dioxaphospho-
rinane protons region of 1H and 1H-{31P} NMR spectra of
(SC,RP) δ,ꢀ,ú-CNA. This material is available free of charge via
the Internet at http://pubs.acs.org.
(19) Sheldrick, G. M. Acta Crystallogr. 1990, A46, 467-473.
(20) SHELXL-97, Program for Crystal Structure Refinement; G. M.
Sheldrick, University of Go¨ttingen, 1997.
JO048136T
J. Org. Chem, Vol. 70, No. 5, 2005 1629