Diastereoselective synthesis of a conformationally restricted dinucleotide with predefined alpha and beta torsional angles for the construction of alpha,beta-constrained nucleic acids (alpha,beta-CNA).

Article abstract of DOI:10.1021/ol027222h

[reaction: see text] The synthesis of a diastereopure 1,3,2-dioxaphosphorinane linkage in which two, alpha and beta, out of six torsion angles of the natural phosphodiester backbone are constrained with predefined values of ca. +60 degrees (g(+)) and 180

Full text of DOI:10.1021/ol027222h

ORGANIC
LETTERS
2003
Vol. 5, No. 2
161-164
Diastereoselective Synthesis of a
Conformationally Restricted Dinucleotide
with Predefined r and â Torsional
Angles for the Construction of
r,â-Constrained Nucleic Acids
(r,â-CNA)
Ingrid Le Cle´zio, 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
Received November 4, 2002
ABSTRACT
The synthesis of a diastereopure 1,3,2-dioxaphosphorinane linkage in which two, r and â, out of six torsion angles of the natural phosphodiester
backbone are constrained with predefined values of ca. +60° (g+) and 180° (t), respectively, is described. The stable and unstrained six-
membered cyclic phosphotriester structure represents the smallest possible ring allowing the conformational locking of the r torsion angle
at a significant positive value that is typical of many bulged regions of nucleic acids.
While the backbone organization of double-stranded DNA
and RNA is normally quite regular, there are many other
secondary and tertiary structures that DNA and RNA
molecules can adapt in vivo.1 It is now well established that
the diverse biological functions of DNA and RNA are linked
to their capacity to support a significant local conformational
heterogeneity in the sugar-phosphate backbone. This con-
formational property allows RNA to adopt biologically
important disparate structures such as bulges, hairpin loops,
U-turns, adenosine platforms or branched junctions. An
increasing number of studies indicate that these structural
motifs are indeed characterized by a variety of backbone
conformations that markedly differ from the regular confor-
mational states of double-stranded DNA and RNA mol-
ecules.2 The actual role played by the phosphate diester
backbone in defining these structures is still not well
understood. The flexibility of the phosphodiester linkage is
expected to be an important component of this “second
genetic code”.
(2) See for example: (a) Ennifar, E.; Yusupov, M.; Walter, P.; Marquet,
R.; Ehresmann, B.; Ehresmann, C.; Dumas, P. Structure 1999, 7, 1439. (b)
Tereshko, V.; Wallace, S. T.; Usman, N.; Wincott, F. E.; Egli, M. RNA
2001, 7, 405. (c) Hung, L.-W.; Holbrook, E. L.; Holbrook, S. R. Proc.
Nat. Acad. Sci. U.S.A. 2000, 97, 5107. (d) Scott, W. G.; Finch, J. T.; Klug,
A. Cell 1995, 81, 991. (e) Ippolito, J. A.; Steitz, T. A. Proc. Nat. Acad.
Sci. U.S.A. 1998, 95, 9819. (f) Cate, J. H.; Gooding, A. R.; Podell, E.;
Zhou, K.; Golden, B. L.; Szewczak, A. A.; Kundrot, C. E.; Cech, T. R.;
Doudna, J. A. Science 1996, 273, 1696.
* Corresponding authors. E-mail address for A.V.: vigroux@
chimie.ups-tlse.fr.
(1) Belmont, P.; Constant, J-F.; Demeunynck, M. Chem. Soc. ReV. 2001,
30, 70 and references therein.
10.1021/ol027222h CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/17/2002

Considering the importance of the relationship between
the local conformational and functional properties of nucleic
acids, conformationally restricted oligonucleotides with
modifications in the phosphodiester backbone unit, in the
sugar unit and, to a limited extent, in the base units have
been designed to efficiently sequester specific mRNA
sequences with the background of antisense applications.3
Since these modifications are introduced for the purpose of
forming the strongest duplexes with the target complementary
RNA, they do not lead to dramatic changes of the backbone
torsion angles R-ú (Figure 1) compared to the values
Our general strategy for the conformational locking of the
phosphate backbone torsional angles is based on the intro-
duction of the 1,3,2-dioxaphosphorinane ring structure at key
positions along the phosphate backbone. To lock torsional
angles R and â simultaneously, we selected the cyclic
structure in which the P-O5′ and O5′-C5′ bonds are part
of the dioxaphosphorinane system (Figure 1). In such a
structure, both the phosphorus atom and the 5′-carbon atom
are asymmetric centers. Thus, in the absence of a diastereo-
selective synthetic method, the R,â-constrained dinucleotide
product (R,â-CNA) should consist of a mixture of four
diastereoisomers.
It is well documented that six-membered phosphorus
compounds containing endocyclic oxygen atoms adjacent to
phosphorus have chair conformations that are strongly
influenced by the ground-state anomeric (stereoelectronic)
effect: exocyclic electronegative phosphorus substituents
prefer an axial position (equatorial PdO), whereas electro-
positive and carbon substituents prefer an equatorial position
(axial PdO).8 Accordingly, it is expected that among the
four possible diastereoisomers of R,â-CNA, those with the
alkoxy group ON1 axial (equatorial PdO) and the carbon
group N2 equatorial should have an energetically more
favorable chair conformation due to both the sterically and
anomerically favorable trans relationship between ON1 and
N2 (Figure 2).9
Figure 1. Six torsion angles R-ú describing the sugar-backbone
geometry of DNA. R,â-CNA (see ref 6) are nucleic acids in which
the R and â torsion angles are conformationally constrained at
predefined values.
observed in a natural nucleotide unit in an A-form duplex.4
To our knowledge, much less attention has been paid to
the design of conformationally restricted nucleosides with
the aim of mimicking nucleic acid secondary structures
containing non-Watson-Crick pairs or unpaired nucleotides.5
We are interested in the development of conformationally
constrained dinucleotide building units in which the backbone
torsional angles R-ú can have predefined values that are
significantly different from the typical values observed in
DNA and RNA duplexes. Herein, we report the diastereo-
selective synthesis of a conformationally fixed dinucleotide
building unit {(SC,RP)-R,â-CNA,6 Scheme 2} in which the
R and â torsion angles are locked in a (g+, t) conformation
that frequently occurs in bulged regions of nucleic acids.2a-c
(3) For a recent review, see: Leumann, C. J. Bioorg. Med. Chem. 2002,
10, 841.
Figure 2. Retrosynthetic pathway for the diastereoselective
synthesis of the (SC,RP)-stereoisomer of the R,â-CNA dinucleotide
building unit with the (g+, t) backbone conformation.
(4) (a) Wengel, J. A. Acc. Chem. Res. 1999, 32, 301. (b) Petersen, M.;
Bondensgaard, K.; Wengel, J.; Jacobsen, J. P. J. Am. Chem. Soc. 2002,
124, 5974. (c) Sørensen, A. M.; Nielsen, P. Org. Lett. 2000, 2, 4217. (d)
Tarko¨y, M.; Leumann, C. Angew. Chem., Int. Ed. Engl. 1993, 32, 1432.
(e) Marquez, V. E.; Siddiqui, M. A.; Ezzitouni, A.; Russ, P.; Wang, J.;
Wagner, R. W.; Matteucci, M. D. J. Med. Chem. 1996, 39, 3739. (f) Singh,
S. K.; Nielsen, P.; Koshkin, A. A.; Wengel, J. Chem. Commun. 1998, 455.
(g) Steffens, R.; Leumann, C. J. J. Am. Chem. Soc. 1999, 121, 3249. (h)
Rajwanshi, V. K.; Håkansson, A. E.; Sørensen, M. D.; Pitsch, S.; Singh, S.
K.; Kumar, R.; Nielsen, P.; Wengel, J. Angew. Chem., Int. Ed. 2000, 39,
1656.
As shown in Figure 2, the trans (SC,RP)-stereoisomer has
torsional angles R and â constrained to values ca. g+ (+60°)
and t (180°), whereas in the (RC,SP)-component, R and â
are constrained to the (g-, t) conformation, which is typical
(5) (a) Seio, K.; Wada, T.; Sakamoto, K.; Yokoyama, S.; Sekine, M. J.
Org. Chem. 1998, 63, 1429. (b) Seio, K.; Wada, T.; S.; Sekine, M. HelV.
Chim. Acta 2000, 83, 162. (c) Sekine, M.; Kurasawa, O.; Shohda, K.; Seio,
K.; Wada, T. J. Org. Chem. 2000, 65, 3572. (d) Sekine, M.; Kurasawa, O.;
Shohda, K.; Seio, K.; Wada, T. J. Org. Chem. 2000, 65, 6515. An alternative
synthetic method for a structure very similar to that previously described
in ref 5d has been recently proposed. See: Børsting, P.; Nielsen, P. Chem.
Commun., 2002, 2140.
(6) We first wished to use the term R,â-LNA (R,â-locked nucleic acids)
in the interest of semantic economy. The general term LNA (locked nucleic
acid) was first introduced in reference to modifications in which the
ribofuranose subunit of nucleic acids is locked (via covalent bridges between
the 2′-oxygen and the 4′-carbon atoms) into the 3′-endo conformation with
the aim of increasing the thermal stabilities of the corresponding duplexes
formed with DNA and RNA. Therefore, the so-called LNA family is
162
Org. Lett., Vol. 5, No. 2, 2003

of DNA and RNA duplexes. Therefore, the (SC,RP)-
compound appears as the most interesting stereoisomer for
the purpose of mimicking nucleic acid bulged structures.
We anticipated that the ground-state anomeric effect of
the 1,3,2-dioxaphosphorinane system could be used as an
essential driving force for the stereocontrolled chemical
synthesis of this compound. According to our analysis, the
pro-(R)-phosphate oxyanion of the (5′S)-isomer of the acyclic
phosphate precursor shown in Figure 2 should attack much
more readily the electrophilic 7′-carbon atom than the pro-
(S)-oxyanion. If we are correct, then the cyclization reaction
from the (5′S)-acyclic stereoisomer (referred to as compound
6 in Scheme 2) should occur stereospecifically to give the
(SC,RP)-R,â-CNA as the dominant product. Finally, the key
starting material that results from the retrosynthetic pathway
shown in Figure 2 is a diastereopure (5′S)-C-tosyloxyethyl-
substituted nucleoside.
(5′S)-C-Tosyloxyethylthymidine 4 was coupled with the
commercially available thymidine phosphoramidite (Scheme
2) using standard phosphoramidite technology12 to give two
Scheme 2. Diastereoselective Synthesis of the
(SC,RP)-R,â-CNA Dinucleotidea
The corresponding (5′S)-C-tosyloxyethylthymidine 4 was
prepared from diastereopure diol 3 with tosyl chloride in the
presence of pyridine (Scheme 1). Diastereomerically pure
Scheme 1. Diastereoselective Synthesis of the Key Starting
Material 5′-C-Tosyloxyethylthymidine 4a
a Reaction conditions: (a) 1H-tetrazole, CH3CN, 20 min, then
collidine, I2/H2O-THF, 89%; (b) Et3N, DMF, 95%; (c) Bu4NF,
THF, 90%; (d) 2% TFA in CH2Cl2, 95%. T ) thymin-1-yl.
n
diastereoisomeric dinucleotides 5 in an equimolar ratio
(characterized by two 31P NMR signals at δP -2.6 and -2.5).
The expected ring closure reaction occurred by treatment of
5 with triethylamine in dry dimethylformamide at 90 °C for
2 h. Cyclic phosphotriester 7 was obtained in 95% yield as
a single diastereoisomer as observed by 31P (δP -9.0), H,
1
a Reaction conditions: (a) Methyl acetate silylketene, BiCl3/ZnI2
cat, CH2Cl2, 90%; (b) NaBH4, EtOH, 85%; (c) TsCl, pyridine, 90%.
T ) thymin-1-yl.
and 13C NMR. The subsequent removal of the protective
groups (using fluoride ion and trifluoroacetic acid) provided
the final compound (SC,RP)-R,â-CNA. Finally, it should be
noted that the cyclic structure described here is perfectly
compatible with the chemistry of nucleotide structures.13
The (SC,RP)-R,â-CNA dinucleotide product was fully
diol 3 has been obtained starting from 5′-C-aldehyde
thymidine 1, which, via a Muka¨ıyama’s reaction with the
silylketene of methyl acetate catalyzed by BiCl3/ZnI2,10
provided compound 2 with a (S)-configuration at the newly
created asymmetric center 5′-C in 90% yield (Scheme 1).11
Reduction of the ester function by NaBH4 produced 3 in
85% yield.
characterized by elemental analysis and by 1H, 13C, and 31
P
NMR spectroscopy.14 The chair conformation of this com-
1
pound is evident from the H NMR spectra,15 with no
detectable coupling constant between the 5′-H involved in
(11) (a) Banuls, V.; Escudier, J.-M. Tetrahedron 1999, 55, 5831. (b)
Escudier, J.-M.; Tworkowski, I.; Bouziani, I.; Gorrichon, L. Tetrahedron
Lett. 1996, 50, 4689.
characterized by the extra conformational locking of only one out of six
torsion angles of the sugar-phosphate backbone, the δ torsion angle, and
could thus have been referred to as δ-LNA according to this nomenclature.
However, to avoid any confusion with R-L-ribo-configured locked nucleic
acids (termed R-L-LNA7) and the natural â-D-ribo configuration of LNA,
we used the term “constrained” instead of “locked” to assign the meaning
of torsional angle to R and â when they precede the acronym CNA
(constrained nucleic acids).
(12) Caruthers, M. H. Acc. Chem. Res. 1991, 24, 278.
(13) The uncharged (SC,RP)-R,â-CNA dimer has been successfully
incorporated at preselected positions in otherwise unmodified oligonucleo-
tides using the conventional automatic solid-phase technology based on
phosphoramidite chemistry.12 Unpublished results.
(14) Selected data for (SC,RP)-R,â-CNA. Elemental analysis: found
(calcd) C, 46.35 (46.26); H, 5.08 (5.11); N, 9.65 (9.79). 31P NMR δP (81
MHz, CD3OD): -6.50. 1H NMR δH (400 MHz, CD3OD): H5′b 4.85; J5′b/6′bax
) 11.9 Hz, J5′b/6′beq ) 1.9 Hz, J5′b/4′b ) 2.0 Hz, J5′b/P ≈ 0 Hz. For full
characterization, see Supporting Information.
(7) See: Sørensen, M. D.; Kværnø, L.; Bryld, T.; Håkansson, A. E.;
Verbeure, B.; Gaubert, G.; Herdewijn, P.; Wengel, J. J. Am. Chem. Soc.
2002, 124, 2164 and references therein.
(8) Gorenstein, D. G. Chem. ReV. 1987, 87, 1047 and references therein.
(9) The two remaining stereoisomers, (RC,RP) and (SC,SP), with a cis
relationship between ON1 and N2 have either a sterically or anomerically
favorable chair conformation.
(10) Le Roux, C.; Gaspard Iloughmane, H.; Dubac, J.; Jaud, J.; Vignaux,
P. J. Org. Chem. 1993, 58, 1835.
(15) Independent preparation of the (RC,SP)-stereoisomer of R,â-CNA
(de 70%) was carried out directly from the cyclization reaction of the (5′R)-
isomer of the acyclic phosphate precursor shown in Figure 2. The structure
and geometry of (RC,SP)-R,â-CNA was confirmed by 1H, 13C, and 31P NMR
spectroscopy and ultimately by X-ray crystallography, establishing the chair
conformation and the trans relationship between the ON1 (axial) and N2
Org. Lett., Vol. 5, No. 2, 2003
163

the dioxaphosphorinane system and phosphorus, which is
characteristic of an axial position of the 5′-H proton.16
The present results clearly demonstrate that it is possible
to introduce the specific {R(g+), â(t)} conformation into the
phosphodiester linkage by simultaneously constraining tor-
sional angles R and â into a dioxaphosphorinane structure.
To our knowledge, we have reported the first example of a
(100%) diastereoselective synthesis of a cyclic phosphotri-
ester linkage. This very simple strategy that consists of using
both steric and anomeric effects to stereocontrol the ring-
closing reaction allowed us to independently prepare cyclic
phosphotriester linkages with either an {R(g-), â(t)} con-
formation,15 which is typical of RNA and DNA duplexes,
or an {R(g+), â(t)} conformation, which frequently occurs
in many bulged regions of nucleic acids (Figure 2).
system are expected to play an important role for the artificial
stabilization of biologically important nucleic acid secondary
structures.18 In this respect, the dioxaphosphorinane-CNA
family might provide new and promising tools for the
structural analysis of complex RNA edifices and might
ultimately contribute to the design of specific ligands to target
particular RNA structures in the hope of treating a variety
of chronic and degenerative diseases.
Further efforts in synthetic chemistry and detailed struc-
tural studies of potentially interesting dioxaphosphorinane-
CNA dinucleotides are in progress in our laboratory.
Acknowledgment. We thank Dr. Ste´phane Massou for
his assistance in NMR spectroscopy.
Supporting Information Available: Experimental pro-
cedures and characterization data. This material is available
free of charge via the Internet at http://pubs.acs.org.
Six-membered cyclic phosphotriesters are resistant com-
pounds relative to both hydrolysis and phosphohydrolase
enzymes.17 Thus, synthetically accessible dinucleotides that
are conformationally restricted by a dioxaphosphorinane
OL027222H
(equatorial) groups. From the 31P NMR spectra, the axial position of 5′-H
is revealed by the absence of a P-coupled 5′-CH signal. Chemical shifts of
δP -6.5 and -5.1 were conclusively assigned to the (SC,RP)- and (RC,SP)-
diastereoisomers of R,â-CNA, respectively. Unpublished results.
(17) (a) Westheimer, F. H. Acc. Chem. Res. 1968, 1, 70. (b) Thatcher,
G. R. J.; Kluger, R. AdV. Phys. Org. Chem. 1989, 25, 99. (c) Farquhar, D.;
Chen, R.; Khan, S. J. Med. Chem. 1995, 38, 488.
(18) The simple fact that the R,â-CNA dinucleotide unit has no charge
could in itself bring about a significant change in the conformation of the
nucleic acid. The resulting positive or negative effect of that aspect in the
possible induction of bulged regions will be discussed elsewhere (full paper
in preparation).
3
(16) Typical upper and lower limits observed for the JH/P coupling
constants for dioxaphosphorinane structures in chair conformation are 3JHax/P
3
< ca. 3 Hz and JHeq/P > ca. 20 Hz, respectively. See: Gorenstein, D. G.;
Rowell, R.; Findlay, J. J. Am. Chem. Soc. 1980, 102, 5077.
164
Org. Lett., Vol. 5, No. 2, 2003