Synthesis and structural study of ribo-dioxaphosphorinane-constrained nucleic acid dinucleotides (ribo-α,β-D-CNA)

Article abstract of DOI:10.1002/ejoc.201101353

With increasing interest in the control of the conformations of nucleic acids to define active riboswitch or siRNA, we present herein our efforts towards the stereocontrolled synthesis and conformational analysis of ribodinucleotides in which α and β torsional angles are constrained within a dioxaphosphorinane structure (ribo-α,β-D-CNA). The crystal structure analysis of one α,β-D-CNA TU dimer demonstrates the absolute stereochemistry of the newly created asymmetric centres. NMR and CD analyses allowed complete assignment and the determination of the torsional angles in the α,β-D-CNA UU diastereoisomers. Diastereoisomers of α,β-D-CNA ribodinucleotide building blocks of nucleicacids in which the α and β torsional angles are stereocontrolled by a dioxaphosphorinane ring structure (D-CNA family) have been synthesised. The structural analyses showed that these dinucleotides exhibit a South/North junction associated with either canonical or non-canonical states of the sugar phosphate backbone. Copyright

Full text of DOI:10.1002/ejoc.201101353

FULL PAPER
DOI: 10.1002/ejoc.201101353
Synthesis and Structural Study of ribo-Dioxaphosphorinane-Constrained
Nucleic Acid Dinucleotides (ribo-α,β-D-CNA)
Marie Maturano,[a] Dan-Andrei Catana,[a] Pierre Lavedan,[b] Nathalie Tarrat,[c,d,e]
Nathalie Saffon,[f] Corinne Payrastre,[a] and Jean-Marc Escudier*[a]
Keywords: Nucleotides / DNA structures / Strained molecules / Phosphorus heterocycles / Conformation analysis
With increasing interest in the control of the conformations
of nucleic acids to define active riboswitch or siRNA, we
present herein our efforts towards the stereocontrolled syn-
thesis and conformational analysis of ribodinucleotides in
which α and β torsional angles are constrained within a di-
oxaphosphorinane structure (ribo-α,β-D-CNA). The crystal
structure analysis of one α,β-D-CNA TU dimer demonstrates
the absolute stereochemistry of the newly created asymmet-
ric centres. NMR and CD analyses allowed complete assign-
ment and the determination of the torsional angles in the α,β-
D-CNA UU diastereoisomers.
switch and siRNA has strengthened the interest in chemi-
cally modified oligonucleotides.[5] However, the main effort
has been directed towards defining analogues with a high
affinity for nucleic acid targets, biostability, and efficient
cellular uptake. On the other hand, little attention has been
devoted to the design and elaboration of constrained nu-
cleotides with a view to controlling the shape of the sugar/
phosphate backbone of nucleic acids.[6] Therefore, in this
context, it is of interest to elaborate modified nucleotides
that could help in folding control or that could mimic biolo-
gically important local distortion of nucleic acids.
Introduction
Many biologically important nucleic acids exhibit un-
usual conformations of the sugar/phosphate backbone tor-
sional angles α–ζ that lead to structural distortions either
of the double helix or of the single strand (Figure 1). These
structures are very often specifically involved in particular
macromolecular processes.[1] X-ray data analyses of biolo-
gically important RNAs have shown that many torsional
angles deviate from the regular values observed in A- or B-
type duplexes.[2,3] Unfortunately, experimental studies
aimed at determining the structural and functional implica-
tions of such helical deformations are somewhat compli-
cated by the intrinsically transient nature of the correspond-
ing 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 nu-
cleic acid reactivity and interactions. Much effort has been
devoted to the elaboration of modified nucleotide ana-
logues that are conformationally constrained for thera-
peutic purposes.[4] More recently, the discovery of the ribo-
[a] Laboratoire de Synthèse et Physico-Chimie de Molécules
d’Intérêt Biologique, UMR 5068 CNRS, Université
Paul Sabatier,
Figure 1. Left: the six backbone torsion angles (labelled α to ζ) of
nucleic acids. Right: the α,β-D-CNA unit is a dinucleotide in which
α and β are controlled to canonical or noncanonical values by a
dioxaphosphorinane ring structure exhibiting two new asymmetric
centres.
118 Route de Narbonne, 31062 Toulouse Cedex 9, France
Fax: +33-5-61556011
E-mail: escudier@chimie.ups-tlse.fr
[b] Service Commun de RMN, Université Paul Sabatier, ICT,
118 Route de Narbonne, 31062 Toulouse Cedex 9, France
[c] Université de Toulouse; INSA, UPS, INP, LISBP,
135 Avenue de Rangueil, 31077 Toulouse, France
[d] INRA, UMR792, Ingénierie des Systèmes Biologiques et des
Procédés,
With this in mind, we have developed and reported on
the construction of covalently constrained nucleic acid
(CNA) building blocks in which the backbone torsion
angles are part of a dioxaphosphorinane ring structure in
the DNA series (2Ј-deoxyribose).[7] Herein we disclose the
detailed synthesis and structural study of ribose α,β-D-
CNA dinucleotides (Figure 1) in which the α and β tor-
sional angles can adopt either the canonical [gauche(–),
31400 Toulouse, France
[e] CNRS, UMR5504,
31400 Toulouse, France
[f] Service Commun Rayons X, ICT-FR2599, Université
Paul Sabatier,
118 Route de Narbonne, 31062 Toulouse Cedex 9, France
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J.-M. Escudier et al.
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trans] conformation found in A- or B-type duplexes[3] or the chloride in the presence of pyridine and chloroform in mod-
non-canonical [gauche(+), trans] combination frequently erate-to-good yields.[12] The diastereomerically pure diols
found in bulged regions of nucleic acids.[8,9]
were prepared in yields of 80–95% by reduction of the ester
function of 5Ј-C-methoxycarbonylmethyl-uridines 8 and 9
with NaBH4. A diastereoselective Mukaiyama reaction of
the corresponding 5Ј-C-aldehyde uridines 5 and 6 with the
Results and Discussion
[13]
silyl ketene of methyl acetate catalysed by BiCl3/ZnI2
The retrosynthetic analysis for the preparation of both
stereoisomers of ribo-α,β-D-CNA dinucleotides is summa-
rised in Scheme 1. The formation of the dioxaphosphorin-
ane ring is based on a very simple strategy that involves
the attack of the phosphate oxyanion on an electrophilic
tosyloxy-substituted carbon atom. The linear dinucleotide
precursors with 5Ј-C-(tosyloxyethyl) substitution can be
easily obtained according to standard phosphoramidite
technology by coupling commercially available phos-
phoramidites with 5Ј-C-(tosyloxyethyl) nucleosides. The lat-
ter can be prepared from 5Ј-C-methoxycarbonylmethyl nu-
cleosides, which can be synthesised by a Mukaiyama-type
aldolisation reaction of the corresponding 5Ј-C-aldehyde
nucleosides.
gave access to 8 with a 9:1 ratio in favour of the 5ЈS isomer
whereas 9 was obtained as the pure 5ЈS diastereoisomer.
The bulky 3Ј-O-tert-butyldiphenylsilyl protecting group on
the aldehyde seems to be responsible for the high selectivity
achieved in the addition to 4 and 6 in comparison with 5
in which it is replaced by the smaller tert-butyldimethylsilyl
group.
Scheme 1. Retrosynthetic analysis for the preparation of ribo-α,β-
D-CNA.
Scheme 2. Synthesis of 5Ј-C-tosyloxy nucleosides. Reagents:
a) i. TBDMSCl, Py, 95%; ii. TBDPSCl or TBDMSCl, DMF, Imid,
90%; iii. PTSA, MeOH, 95%; b) DCC, DMSO, DCAA, then ox-
alic acid, 60–90%; c) methyl acetate silyl ketene, BiCl3/ZnI2, DCM,
65–90%; d) NaBH4, EtOH, 80–95%; e) TsCl, Py, CHCl3, 55–86%.
This methodology has been successfully applied to the
synthesis of α,β-D-CNA starting from 2Ј-deoxy-ribo-
thymidine.[10] However, in the ribose nucleoside series a few
points remain to be addressed: 1) the behaviour of the di-
oxaphosphorinane structure in the presence of a 2Ј-hydroxy
The absolute configurations at the chiral C-5Ј centre
functionality on the upstream nucleoside, 2) the stereocon- were not determined at this stage but in a structural analysis
trol of the newly created asymmetric centres (5Ј-carbon and of the corresponding synthesised d-CNA. Note that the
phosphorus) and 3) the influence of the neutral dioxaphos- stereochemical outcome of this nucleophilic addition is op-
phorinane internucleotidic linkage.
posite to the stereochemistry depicted in the synthesis of
To provide an insight into these questions, we investi- Tunicamycin involving the addition of allyltrimethylsilane
gated the synthesis of the three following dimer combina- to a similar aldehyde in the presence of BF3 etherate.[14]
tions: ribo/2Ј-deoxy-ribo-, 2Ј-deoxy-ribo/ribo- and ribo/ribo-
α,β-D-CNA on the ribose puckering.
The 5Ј-C-aldehyde nucleosides 5[15] and 6 were prepared
by a Pfitzner–Moffatt oxidation procedure[16] of the 5Ј-hy-
In the first step we had to prepare two 5Ј-C-(tosyloxy- droxy function of 2Ј,3Ј-O-protected uridines 2 and 3 in
ethyl)uracils 14 and 15 differentiated by the 2Ј- and 3Ј-O- moderate-to-good yields and used in the subsequent step
protecting groups, as shown in Scheme 2 (together with the without further purification. Starting from 2Ј-O-methyl-
previously described (S)-5Ј-C-(tosyloxyethyl)thymidine uridine or uridine, the protected derivatives 2 and 3 were
13[11]). They were obtained from the corresponding dia- synthesised in three steps by selective silylation protection/
stereopure diols 11 and 12 by selective tosylation with tosyl deprotection in high yields.[17]
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Dioxaphosphorinane-Constrained Nucleic Acid Dinucleotides
With the three 5Ј-C-(tosyloxyethyl) nucleosides 13–15 in the target compound. This particular in-line geometry is
hand we synthesised ribo-α,β-D-CNA according to the typically observed within the hammerhead ribozyme at the
methodology developed for 2Ј-deoxy-ribo-α,β-D-CNA active site during intramolecular phosphodiester cleav-
(Scheme 3).[11]
age.[19]
Figure 2. Results of the DFT calculation evidencing the in-line con-
formation of α,β-D-CNA UT 18.
2Ј-Deoxy-ribo/ribo-α,β-D-CNA TU 21 was readily syn-
thesised in three steps in 47% overall yield from 5Ј-C-(tosy-
loxyethyl)uridine 14 by phosphoramidite coupling with
standard thymidine phosphoramidite followed by K2CO3-
mediated dioxaphosphorinane ring formation and removal
of the 5Ј-O-protecting group in TFA (Scheme 4). However,
the cyclisation process did not occur with high diastereo-
selectivity (5.3:1 ratio) with 16% of the minor SP isomer
being formed, as depicted by 31P NMR (δ = –3.8 ppm).
The two diastereoisomers were easily separable by silica gel
chromatography. At this level the determination of the ab-
solute configurations at the newly created C-5Ј and P asym-
metric centres of 14 and α,β-D-CNA TU 21 was greatly
simplified by the X-ray structure solved for the latter (Fig-
ure 3).
Scheme 3. Synthesis of ribo/2Ј-deoxy-ribo-α,β-D-CNA UT. Rea-
gents: a) 2Ј-OTBDMS uridine phosphoramidite, tetrazole, CH3CN
then I2/H2O/Coll, 71%; b) K2CO3, DMF, 92%; c) i. NEt3, 3 HF,
THF, 85%; ii. 3% TFA/CH2Cl2, 80%.
As expected, the acyclic dinucleotide 16 precursor of
ribo/2Ј-deoxy-ribo-α,β-D-CNA was assembled by standard
phosphoramidite technology[18] by coupling the commer-
cially available 2Ј-O-(tert-butyldimethylsilyl)uridine phos-
phoramidite to 5Ј-C-(tosyloxyethyl)thymidine 13 in 71%
yield. Dioxaphosphorinane ring formation mediated by
K2CO3 in DMF occurred in good yield (92%) with very
high diastereoselectivity and only compound 17 was de-
tected by 31P NMR (δ = –8.5 ppm). Attempts to remove the
silyl protecting groups with TBAF failed and only furnished
mixtures with no detectable dioxaphosphorinane ring by
31P NMR spectroscopy. Treatment with the mild reagent
NEt3·3HF in THF at room temperature for 24 h provided
a compound with a single peak at δP = –7.9 ppm that
proved to be unstable over time. After removal of the 5Ј-O-
DMTr protecting group in an acidic medium, a mixture of
two compounds was isolated and characterised by 31P
NMR with the signals at –7.8 and –7.3 ppm (2:1 ratio) cor-
responding to the dioxaphosphorinane structures. However,
efforts to separate them by reversed-phase HPLC (retention
times of 6.5 and 9 min) showed that each isolated fraction
provided the same chromatogram (two peaks with the same
ratio and retention times), which is indicative of an equilib-
rium between the proposed compounds 18 and 19.
Scheme 4. Synthesis of 2Ј-deoxy-ribo/ribo-α,β-D-CNA TU. Rea-
gents: a) thymidine phosphoramidite, tetrazole, CH3CN then I2/
H2O/coll., 70%; b) i. K2CO3, DMF, 95%; ii. 3% TFA/CH2Cl2to
give 21, 85% and iii. TBAF, THF to give 22, 79%.
This crystal structure gives further evidence that the C-
Therefore we concluded that the presence of the free 2Ј- 5Ј atom of the modified uridine is S-configured allowing
hydroxy function on the upper nucleoside induced instabil- the assignment of the absolute configuration of 14 and thus
ity in this UT d-CNA series. DFT calculations clearly evi- indicating the stereochemical outcome of the Mukaiyama
denced that the expected α,β-D-CNA UT can adopt a ge- reaction of 5Ј-C-aldehyde uridine 5 (Scheme 2). The X-ray
ometry in which the 2Ј-hydroxy function of the upper urid- structure also revealed that the six-membered cyclic phos-
ine, the phosphorus atom and the 5’-oxygen of the lower photriester adopts the chair conformation with non-canoni-
thymidine are in line (Figure 2). This particular conforma- cal dihedral values of α = +68.9° and β = +176.1°. Surpris-
tion, induced by the dioxaphosphorinane ring, can strongly ingly, in the solid state, the thymidine sugar moiety features
favour a transesterification process, which could be respon- C-2Ј exo puckering, which is unexpected due to the pres-
sible for the observed instability and further degradation of ence of the neutral internucleotidic linkage.
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Figure 3. X-ray molecular structure of the (SC5Ј,RP)-configured
α,β-D-CNA TU dimer 21 displaying the chair and C-2Ј exo confor-
mations of the dioxaphosphorinane and sugars subunits. For clar-
ity, hydrogen atoms and co-crystallised solvent molecules have been
omitted. Thermal ellipsoids are shown at the 40% probability level.
Figure 4. CD spectra of TpU (top) and (SC5Ј,RP)-α,β-D-CNA TU
22 (bottom) in 10 mm sodium phosphate (pH 7.0) at 80, 60, 40 and
20 °C.
Table 1. H–H and H–P coupling constants in the 1H NMR spectra
(500 MHz) of ribo-α,β-D-CNA.
To gain information on the conformational behaviour of
this 2Ј-deoxy-ribo/ribo-α,β-D-CNA TU in solution, the 2Ј-
O- and 3Ј-O-tert-butyldimethylsilyl protecting groups were
removed with fluoride ion to give 22, which was analysed
1
by circular dichroism and H and 31P NMR (Figure 4 and
Table 1).
To evaluate the relative positions of the nucleobases in
this constrained dinucleotide, CD spectra were recorded in
phosphate buffer (pH 7.0) at 20, 40, 60 and 80 °C and com-
pared with those of the corresponding unmodified thymid-
ine/uridine dinucleotide (denoted as TpU in Figure 4). As
previously observed for the α,β-D-CNA TT analogue,[20]
the striking features of the CD spectra of α,β-D-CNA TU
are the temperature independence and the low intensity of
the positive Cotton effect band at around 270 nm, whereas
TpU shows variations due to base-stacking disruption with
increasing temperature. With the α torsional angle locked
into the non-canonical gauche(+) conformation, the rigidity
of the dioxaphosphorinane structure does not allow any
base stacking in α,β-D-CNA TU 22.
[a] From the approximation of Altona and Sundaralingam, South
(%) = [J1ЈH,2ЈH/(J1ЈH,2ЈH + J3ЈH,4ЈH)] ϫ100.[22]
To complement this information, the solution-state struc-
ture determination in terms of sugar puckering and dioxa-
phosphorinane conformation was established after careful conformation) observed by X-ray analysis for the 2Ј-deoxy-
2
analysis of the coupling constants depicted in the 1H NMR ribose moiety of the thymidine of 21, the small J3ЈH-4ЈH
2
spectrum of 22 recorded at 500 MHz in deuteriated meth- coupling constants measured and the values of J2ЈH-3ЈH
2
anol (Table 1). In contrast to the C-2Ј exo puckering (North and J1ЈH-2ЈH for 22 are consistent with the standard C-2Ј
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Dioxaphosphorinane-Constrained Nucleic Acid Dinucleotides
endo conformation (South conformation) previously ob- ketone (with NaBH4) to give 23 as a 1:1 mixture of dia-
served in natural 2Ј-deoxyribose units.[21] Likewise, the stereoisomers in 65% overall yield. Removal of the silyl pro-
lower uridine ribose sugar adopts the expected C-2Ј exo tecting group under mild acidic conditions followed by
conformation (North conformation) according to the Al- treatment with tosyl chloride in the presence of pyridine
tona–Sundaralingam approximation.[22] On the other hand, and chloroform provided a 1:1 mixture of 15 and 24 in a
the chair conformation of the dioxaphosphorinane ring dis- moderate 60% yield. Phosphoramidite coupling of sepa-
closed in the solid state for 21 is corroborated for 22 by the rated 15 and 24 with commercially available 2Ј-O-methylur-
very small coupling constant exhibited by 5Ј-H of the lower idine phosphoramidite produced the acyclic dinucleotides
nucleoside with phosphorus, which is characteristic of an 25 and 26 as an equimolar mixture of each diastereoisomer
axial position of the proton in this six-membered ring.[23]
(31P NMR: δ = –2.2, –2.3, –2.4 and –2.6 ppm, respectively)
Finally, with all these observations in hand, we proposed in 90% yield after the oxidation step.
to synthesise ribo/ribo-α,β-D-CNA in the 2Ј-OMe form to
2Ј-OMe-ribo-α,β-D-CNA 27 and 28 were isolated from
avoid any dioxaphosphorinane ring-opening and to enforce 25 and 26, respectively, in overall yields of 56% after re-
the North conformations of the puckered sugar units.
moval of the protecting groups and dioxaphosphorinane
Because we wished to obtain the two main isomers of ring formation. In each case the cyclisation process oc-
2Ј-O-methyl-ribo-α,β-D-CNA (featuring either canonical or curred with a modest diastereoselectivity of 4:1. As ob-
non-canonical values of α and β) we prepared a mixture of served by 31P NMR, minor isomers were detected at –4.8
(S)-5Ј-C- and (R)-5Ј-C-tosyloxyethyl-2Ј-O-methyluridine 15 and –5.2 ppm, respectively. The major isomers were sepa-
and 24 (Scheme 5). Starting from diastereopure diol 12, se- rated from the minor isomers by silica gel chromatography
lective protection of the primary hydroxy function with a before the deprotection steps.
tert-butyldimethylsilyl group allowed the oxidation of the
The CD spectra of ribo-α,β-D-CNA 27 and 28 were re-
5Ј-hydroxy function with Dess–Martin periodinane[24] im- corded in phosphate buffer (pH 7.0) at 20, 40, 60 and 80 °C
mediately followed by the reduction of the corresponding (Figure 5). As expected, the two diastereoisomers exhibit
striking different features. Although ribo-α,β-D-CNA 28
shows a temperature dependence and a high intensity of the
positive Cotton effect at around 270 nm, its diastereoisomer
27 exhibits neither temperature dependence nor a high in-
tensity of the same band. This difference in behaviour is
related to the relative positions of the two nucleobase moie-
ties. Therefore it is clear that in the ribo-α,β-D-CNA 28 the
Scheme 5. Synthesis of 2Ј-OMe-ribo-α,β-D-CNA UU. Reagents:
a) i. TBDMSCl, Py, 90%; ii. Dess–Martin periodinane, Py, DCM,
then NaBH4, 72%; b) i. PTSA, MeOH, 85%; ii. TsCl, Py, CHCl3,
60%; c) 2Ј-OMe-uridine phosphoramidite, tetrazole, CH3CN then Figure 5. CD spectra of (RC5Ј,RP)-α,β-D-CNA 28 (top) and
I2/H2O/coll, 70%; d) i. K2CO3, DMF, 80%; ii. TBAF, THF, then
3% TFA/CH2Cl2, 70%.
(SC5Ј,SP)-α,β-D-CNA 27 (bottom) in 10 mm sodium phosphate
(pH 7.0) at 80, 60, 40 and 20 °C.
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torsional constraints allow for base stacking whereas in 27 CNA, 3) due to the neutral internucleotidic linkage, the 5Ј-
it is avoided. Thus, circular dichroism is efficient for easily top nucleotides adopt a C-2Ј endo (South) conformation in
distinguishing and assigning the relative structures of the solution but a C-2Ј exo pucker can be observed in the solid
two diastereoisomers.
state for a 2Ј-deoxyribose and 4) 2Ј-OMe-ribo-α,β-D-CNA
The furanose and dioxaphosphorinane solution-state can be obtained either with α and β locked in the gauche-
1
structures of 27 and 28 were determined by means of H (–)/trans conformation depicted in A- and B-type duplexes
and 31P NMR spectroscopy in CD3OD (Table 1). The chair or in the atypical gauche(+)/trans conformation frequently
conformation of the dioxaphosphorinane structure of 27 observed in bulged or looped regions of RNA.
and 28 was clearly established on the basis of the 1H NMR
A general comparison of the behaviour within oligonu-
3
spectra, with the observation of small and large JH,P cou- cleotides of all α,β-CNAs synthesised by our group[7b,7d,27]
pling constants between the 7Ј-H proton and phosphorus, is underway and will be presented in a forthcoming paper.
which are characteristic of the axial (3JHax-P ≈ 2 Hz) and
equatorial positions (3JHeq-P ≈ 22 Hz) of these protons.[23]
3
Experimental Section
The small JH-P coupling constant between the 5Ј-H pro-
tons and phosphorus (Ͻ1 Hz) in both compounds is also
consistent with an axial position of these protons in the
dioxaphosphorinane ring. Therefore, together with the CD
results, it can be concluded that 27 is the (RC5Ј,RP)-ribo-
α,β-D-CNA featuring canonical values gauche(–)/trans for
α and β, and 28 is the (SC5Ј,SP)-ribo-α,β-D-CNA featuring
non-canonical values gauche(+)/trans for α and β.
General: Products were purified by medium-pressure liquid
chromatography with a Jobin and 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 spec-
1
trometer (250, 300 or 500 MHz for H and 63, 75 or 125 MHz for
13C). Chemical shifts were referenced to the tetramethylsilane. Mass
spectra were recorded with a Nermag R10-10 or Perkin–Elmer API
365 spectrometer. All solvents were distilled and dried before use.
Examination of the sugar ring H–H coupling constants
allowed the assignment of the puckering of the 2Ј-OMe ri-
bose moieties of 27 and 28 (Table 1). In each case the upper
ribose residue features small J3ЈH-4ЈH (ca. 3 Hz) and rather
large J1ЈH-2ЈH (ca. 6 Hz) values close to those found in the
standard sugar C-2Ј endo conformation[21] and similar to
those of the 2Ј-deoxyribose of 22. On the other hand, the
lower units behave as regular ribose units with a preferred
C-2Ј exo conformation that is even strengthened for the di-
nucleotide 28 exhibiting canonical constraints. Therefore it
can be concluded that these ribose dinucleotides mainly
present a South/North junction in solution. The neutral in-
ternucleotidic linkage induces a more favourable South con-
formation even for 2Ј-OMe ribose.[25] Similar observations
have been reported in the case of macrocyclic phosphotri-
ester-linked diuridines.[26]
The syntheses of compounds 1, 4, 7, 10 and 13 have been described
previously,[11] as have compounds 2 and 5.[15,17]
3Ј-O-(tert-Butyldiphenylsilyl)-2Ј-O-methyluridine (3): tert-Butyldi-
methylsilyl chloride (9.63 g, 63.8 mmol) was added to a solution of
2Ј-O-methyluridine (15 g, 58 mmol) in anhydrous pyridine
(200 mL) at room temperature. After stirring for 12 h, the reaction
mixture was diluted with ethyl acetate (500 mL) and the organic
layer washed with a saturated aqueous solution of NH4Cl, water
and brine. After removal of the solvent under reduced pressure,
the crude product was dissolved in anhydrous DMF and imidazole
(23.72 g, 0.35 mol) and tert-butyldimethylsilyl chloride (17.50 g,
63.8 mmol) were added at room temperature. After stirring for 12 h
the reaction mixture was diluted with diethyl ether (500 mL) and
the organic layer washed three times with water and brine. After
removal of the solvent under reduced pressure, the crude product
was dissolved in methanol (300 mL) and p-toluenesulfonic acid
(0.95 g) was added. A saturated aqueous solution of NaHCO3
(100 mL) was added dropwise after 3 h at room temperature. The
methanol was evaporated and the residual mixture diluted with
ethyl acetate and the organic layer washed with water and brine.
The crude oil obtained after removal of the solvent was precipitated
in cold petroleum ether to provide 3 as a white powder (27 g, 93%
yield). 1H NMR (300 MHz, CDCl3): δ = 9.50 (m, 1 H, NH), 7.73–
760 (m, 5 H, Ph, 6-H), 7.45–7.40 (m, 6 H, Ph), 5.79 (d, J = 3.6 Hz,
1 H, 1Ј-H), 5.63 (d, J = 8.1 Hz, 1 H, 5-H), 4.24 (t, J = 5.1 Hz, 1
H, 3Ј-H), 4.12 (m, 1 H, 4Ј-H), 3.80 (A part of an ABX syst., J =
12.3, 1.5 Hz, 1 H, 5Ј-H), 3.61 (t, J = 4.2 Hz, 1 H, 2Ј-H), 3.44 [B
part of an AB(X) syst., J = 12.3 Hz, 1 H, 5Ј-H], 3.37 (s, 3 H, OMe),
1.12 (s, 9 H, tBu) ppm. 13C NMR (75 MHz, CDCl3): δ = 163.7,
150.2, 141.5, 135.8, 133.1, 132.9, 130.2, 129.6, 127.9, 127.8, 127.7,
102.1, 89.7, 84.8, 82.7, 69.8, 60.7, 58.3, 26.9, 19.3 ppm.
C26H32N2O6Si (496.63): calcd. C 62.87, H 6.49, N 5.64; found C
63.12, H 6.40, N 5.60.
Conclusions
We have synthesised constrained ribo dinucleotides with
a dioxaphosphorinane structure as the internucleotidic link-
age that restrains the α and β torsional angles (α,β-D-CNA)
by connection between the C-5Ј of the downstream nucleo-
tide to an oxygen of the phosphate through an ethylene
chain.
We showed that in the case of a ribose residue at the
5Ј-top (ribo/2Ј-deoxy-ribo-α,β-D-CNA UT), the 2Ј-hydroxy
function must be protected to avoid a transesterification
process due to a highly favourable in-line attack conforma-
tion imposed by the cyclic phosphotriester moiety.
Structural analysis, including X-ray crystallography and
NMR spectroscopy, allowed us to determine that 1) the
Mukaiyama aldol condensation on uridine aldehyde pro-
vides an S-configured C-5Ј, 2) the dioxaphosphorinane
linkage adopts a true chair conformation in major dia-
3Ј-O-(tert-Butyldiphenylsilyl)-2Ј-O-methyl-5Ј-oxouridine (6): Di-
chloroacetic acid (1.15 mL in 28 mL DMSO) was added dropwise
to 3 (14.4 g, 29 mmol) and dicyclohexylcarbodiimide (23.8 g,
116 mmol) dissolved in anhydrous DMSO (87 mL) at room tem-
perature. After stirring for 16 h, oxalic acid (10.92 g, 87 mmol) was
stereoisomers of 2Ј-deoxy-ribo/ribo- and ribo/ribo-α,β-D- added portionwise. The reaction mixture was diluted with ethyl
726
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Dioxaphosphorinane-Constrained Nucleic Acid Dinucleotides
acetate (250 mL) and cooled to 0 °C. After 1 h, the dicyclohex-
ylurea was filtered off and the organic layer was washed with a
saturated aqueous solution of NaHCO3 and three times with water
and brine. After removal of the solvent the residual oil was precipi-
tated in cold petroleum ether to provide 6 as a white powder (13 g,
2.3 Hz, 1 H, 5-H), 5.56 (d, J = 5.4 Hz, 1 H, 1Ј-H), 4.48 (t, J =
4.8 Hz, 1 H, 3Ј-H), 4.17–4.11 (m, 2 H, 2Ј-H, 4Ј-H), 3.98–3.90 (2 H,
5Ј-H, 7Ј-H), 1.68 (m, 2 H, 6Ј-H), 0.91, 0.88 (2 s, 18 H, tBu), 009,
008, 006, 003 (4 s, 12 H, SiMe2) ppm. 13C NMR (63 MHz, CDCl3):
δ = 163.9, 150.7, 142.9, 102.1, 92.4, 88.3, 74.0, 72.5, 69.9, 61.2,
35.6, 25.9, 18.1, –4.5 ppm. C23H44N2O7Si2 (516.78): calcd. C 53.46,
H 8.58, N 5.42; found C 53.37, H 8.42, N 5.47.
1
90% yield). H NMR (300 MHz, CDCl3): δ = 9.39 (d, J = 2.1 Hz,
1 H, 5Ј-H), 9.28 (s, 1 H, NH), 7.74–7.65 (m, 5 H, Ph, 6-H), 7.47–
7.40 (m, 6 H, Ph), 6.02 (d, J = 5.1 Hz, 1 H, 1Ј-H), 5.75 (d, J =
8.1 Hz, 1 H, 5-H), 4.62 (d, J = 4.2 Hz, 1 H, 3Ј-H), 4.33 (m, 1 H,
4Ј-H), 3.53 (t, J = 4.6 Hz, 1 H, 2Ј-H), 3.29 (s, 3 H, OMe), 1.14 (s,
9 H, tBu) ppm. 13C NMR (75 MHz, CDCl3): δ = 198.2, 163.3,
150.1, 140.6, 135.8, 132.4, 130.4, 128.1, 127.9, 102.8, 894, 87.7,
81.9, 71.3, 58.4, 24.9, 19.3 ppm.
(5ЈS)-3Ј-O-(tert-Butyldiphenylsilyl)-5Ј-C-(hydroxyethyl)-2Ј-O-
methyluridine (12): The same procedure as followed for 11, starting
from 9 (1.0 g, 1.76 mmol); 12 (0.9 g, 95% yield) was isolated as a
1
white foam. H NMR (300 MHz, CDCl3): δ = 8.91 (s, 1 H, NH),
7.82 (d, J = 8.1 Hz, 1 H, 6-H), 7.74–7.40 (m, 10 H, Ph), 5.82 (d, J
= 3.9 Hz, 1 H, 1Ј-H), 5.64 (dd, J = 8.1, 2.1 Hz, 1 H, 5-H), 4.28 (t,
J = 5.0 Hz, 1 H, 3Ј-H), 3.96 (dd, J = 5.1, 1.5 Hz, 1 H, 4Ј-H), 3.83–
3.60 (m, 5 H, OH, 7Ј-H, 5Ј-H, 2Ј-H), 3.33 (s, 3 H, OMe), 1.83 (m,
1 H, 6Ј-H), 1.55 (m, 1 H, 6Ј-H), 1.12 (s, 9 H, tBu) ppm. 13C NMR
(75 MHz, CDCl3): δ = 163.2, 150.1, 141.7, 135.8, 133.2, 132.9,
130.2, 130.1, 127.9, 127.8, 102.1, 89.4, 87.3, 70.9, 69.5, 61.4, 58.2,
35.4, 26.9, 19.3 ppm. C28H36N2O7Si (540.68): calcd. C 62.20, H
6.71, N 5.18; found C 62.10, H 6.77, N 5.02.
(5ЈS)-2Ј,3Ј-O-(tert-Butyldimethylsilyl)-5Ј-C-(methoxycarbonyl-
methyl)uridine (8): BiCl3 (82 mg) and ZnI2 (124 mg) were added to
a solution of compound 5 (1.8 g, 3.9 mmol) in anhydrous dichloro-
methane (11 mL) at room temperature. After stirring for 30 min,
silyl ketene (1.92 g, 10.2 mmol) diluted with dichloromethane
(3 mL) was added. The black solution turned to a bright-orange
colour. After 2 h, the reaction mixture was diluted with ethyl acet-
ate and washed with a saturated aqueous solution of NH4Cl. 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 deposited on a silica gel column
and eluted with diethyl ether/petroleum ether (7:3): 1.7 g of 8 was
recovered as a white foam together with 0.15 g of its C-5Ј epimer
(5ЈS)-2Ј,3Ј-O-(tert-Butyldimethylsilyl)-5Ј-C-(tosyloxyethyl)uridine
(14): Tosyl chloride (442 mg, 2.3 mmol) was added to compound
11 (0.8 g, 1.55 mmol) dissolved in anhydrous pyridine (1.4 mL) and
chloroform (5 mL) at 0 °C. Stirring was maintained for 12 h and
the reaction mixture was diluted with ethyl acetate and washed with
a saturated aqueous solution of NH4Cl. The organic layer was col-
lected and washed with water and brine and dried with MgSO4.
After removal of the solvent under reduce pressure, the crude prod-
uct was deposited on a silica gel column and eluted with ethyl acet-
ate/dichloromethane (1:3). Compound 14 was recovered as a white
1
in 85% yield. H NMR (250 MHz, CDCl3): δ = 8.86 (s, 1 H, NH),
7.93 (d, J = 8.1 Hz, 1 H, 6-H), 5.73 (d, J = 8.2 Hz, 1 H, 5-H), 5.67
(d, J = 6.7 Hz, 1 H, 1Ј-H), 4.53 (t, J = 4.6 Hz, 1 H, 2Ј-H), 4.19–
4.13 (m, 2 H, 3Ј-H, 5Ј-H), 3.92–3.90 (m, 2 H, 4Ј-H, OH), 3.73 (s,
3 H, MeO), 2.83–2.50 [AB part of AB(X) syst., J = 17.0, 9.7,
3.3 Hz, 2 H, 6Ј-H], 0.89, 0.87 (2 s, 18 H, tBu), 0.11, 0.10, 0.09, 0.05
(4 s, 12 H, SiMe2) ppm. 13C NMR (63 MHz, CDCl3): δ = 173.5,
163.6, 150.5, 141.7, 102.1, 96.1, 91.2, 74.5, 72.0, 66.6, 52.1, 36.0,
25.8, 18.0, –4.6 ppm. C24H44N2O8Si2 (544.79): calcd. C 52.91, H
8.14, N 5.14; found C 52.63, H 8.17, N 5.01.
1
foam (0.89 g, 86% yield). H NMR (250 MHz, CDCL3): δ = 8.35
(s, 1 H, NH), 7.80 (d, J = 8.0 Hz, 2 H, Ph), 7.60 (d, J = 8.1 Hz, 1
H, 6-H), 7.35 (d, J = 8.0 Hz, 2 H, Ph), 5.73 (dd, J = 8.1, 2.3 Hz, 1
H, 5-H), 5.43 (d, J = 5.8 Hz, 1 H, 1Ј-H), 4.55 (dd, J = 5.8, 4.6 Hz,
1 H, 2Ј-H), 4.16–4.12 (m, 2 H, 3Ј-H, 4Ј-H), 3.90–3.85 (m, 2 H, 5Ј-
H, 7Ј-H), 2.45 (s, 3 H, CH3 Ts), 1.90 (br. s, 1 H, OH), 1.62 (br. s,
2 H, 6Ј-H), 0.91, 0.87 (2 s, 18 H, tBu), 0.09, 0.08, 0.02, 0.01 (4 s,
12 H, SiMe2) ppm. 13C NMR (63 MHz, CDCL3): δ = 163.7, 150.5,
145.0, 143.3, 132.3, 130.0, 128.8, 127.9, 126.0, 102.2, 93.4, 88.2,
73.4, 72.8, 67.7, 66.4, 33.5, 25.7, 21.7, 18.1, 1.0, –4.6 ppm.
C30H50N2O9SSi2 (670.96): calcd. C 53.70, H 7.51, N 4.18; found C
53.95, H 7.49, N 4.11.
(5ЈS)-3Ј-O-(tert-Butyldiphenylsilyl)-5Ј-C-(methoxycarbonylmethyl)-
2Ј-O-methyluridine (9): The same procedure as for 8 was followed
starting from 6 (6.0 g, 12.9 mmol); 9 (6.2 g, 90% yield) was isolated
after precipitation in petroleum ether. 1H NMR (300 MHz,
CDCl3): δ = 9.58 (s, 1 H, NH), 7.87 (d, J = 8.1 Hz, 1 H, 6-H),7.73–
7.40 (m, 10 H, Ph), 5.91 (d, J = 3.3 Hz, 1 H, 1Ј-H), 5.63 (dd, J =
8.1, 2.1 Hz, 1 H, 5-H), 4.28 (t, J = 5.2 Hz, 1 H, 4Ј-H), 3.97 (d, J =
6. 0 Hz, 1 H, 3Ј-H), 3.92 (m, 1 H, 5Ј-H), 3.71 (s, 3 H, OMe), 3.44
(dd, J = 4.8, 3.6 Hz, 1 H, 2Ј-H), 3.34 (s, 3 H, OMe), 2.71, 2.43 [AB
part of AB(X) syst., J = 16.8, 9.9, 3.0 Hz, 2 H, 6Ј-H], 1.11 (s, 9 H,
tBu) ppm. 13C NMR (75 MHz, CDCl3): δ = 173.3, 163.7, 150.3,
140.8, 135.9, 135.8, 133.0, 130.2, 130.1, 127.9, 127.8, 102.0, 88.0,
85.6, 83.0, 70.5, 65.9, 58.1, 52.0, 38.0, 26.9, 19.3 ppm.
C29H36N2O8Si (568.69): calcd. C 61.25, H 6.38, N 4.93; found C
59.88, H 6.42, N 4.77.
(5ЈS)-3Ј-O-(tert-Butyldiphenylsilyl)-2Ј-O-methyl-5Ј-C-(tosyloxy-
ethyl)uridine (15): The same procedure was followed as for 14, start-
ing from 12 (800 mg, 1.48 mmol); 15 (600.0 mg, 58% yield) was
isolated as a white foam after column chromatography with ethyl
acetate/dichloromethane (1:4) as eluent. 1H NMR (300 MHz,
CDCl3): δ = 8.60 (s, 1 H, NH), 7.81–7.38 (m, 15 H, Ph, 6-H), 5.74
(d, J = 3.9 Hz, 1 H, 1Ј-H), 5.62 (dd, J = 8.1, 1.8 Hz, 1 H, 5-H),
4.25–3.99 (m, 3 H, 3Ј-H, 7Ј-H), 3.90 (dd, J = 5.4, 1.2 Hz, 1 H, 4Ј-
H), 3.64 (dd, J = 4.8, 3.9 Hz, 1 H, 2Ј-H), 3.56 (m, 1 H, 5Ј-H), 3.42
(s, 3 H, OMe), 2.48 (s, 3 H, MeTs), (m, 1 H, 6Ј-H), 1.13 (s, 9 H,
tBu) ppm. 13C NMR (75 MHz, CDCl3): δ = 162.9, 149.9, 145.0,
141.5, 135.8, 132.9, 130.5, 130.2, 130.0, 102.0, 89.7, 86.6, 82.4, 70.7,
65.4, 58.4, 33.2, 26.9, 21.7, 19.2 ppm. C35H42N2O9SSi (694.87):
calcd. C 60.50, H 6.09, N 5.42; found C 60.58, H 6.06, N 5.39.
(5ЈS)-2Ј,3Ј-O-(tert-Butyldimethylsilyl)-5Ј-C-(hydroxyethyl)uridine
(11): Sodium borohydride (416 mg, 11.0 mmol) was added to com-
pound 8 (1.0 g; 1.83 mmol) dissolved in ethanol (10 mL) at 0 °C.
The reaction mixture was allowed to reach room temperature and
stirred for 12 h. After addition of a saturated aqueous solution of
NH4Cl (30 mL), ethanol was removed under reduced pressure. The
residue was extracted with ethyl acetate and the organic layer
washed with water and brine and dried with MgSO4. The solvent
[3Ј-O-(tert-Butyldiphenylsilyl)-(5ЈS)-5Ј-C-(tosyloxyethyl)-5Ј-thymid-
inyl]-2-cyanoethyl-2Ј-O-(tert-butyldimethylsilyl)-5Ј-O-dimethoxy-
trityluridine-2Ј-phosphate (16): Compound 13 (200 mg, 0.29 mmol),
uridine phosphoramidite (0.5 g, 0.58 mmol) and freshly sublimed
was removed in vacuo and compound 11 was obtained as a white
1
foam (900 mg, 95% yield). H NMR (250 MHz, CDCl3): δ = 8.41 tetrazole (203 mg, 2.9 mmol) were dissolved in anhydrous acetoni-
(s, 1 H, NH), 7.82 (d, J = 8.1 Hz, 1 H, 6-H), 5.73 (dd, J = 8.1,
trile (5 mL) and stirred for 120 min at room temperature. After
Eur. J. Org. Chem. 2012, 721–730
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727

J.-M. Escudier et al.
FULL PAPER
addition of collidine (157 μL, 1.16 mmol), the phosphite was oxi-
dised with iodine (0.1 m solution in THF/H2O, 2:1) until the dark-
brown colour persisted. The reaction mixture was diluted with ethyl
acetate and washed with an aqueous solution of sodium thiosulfate
(15%) to remove excess iodine. The organic layer was washed with
water and brine and the solvent was removed in vacuo. The crude
material was purified by chromatography on silica gel with ethyl
acetate as eluent. After evaporation of the solvent compound 16
(mixture of diastereoisomers) was recovered as a white foam (0.3 g,
CH2Cl2, 1:1) the cyclic phosphotriester 21 was recovered as a white
foam (0.193 g, 80% yield) along with the slower-eluted phosphorus
epimer (δP = –3.2 ppm, 37 mg, 15%). The major isomer was treated
with TFA (3% in CH2Cl2, 10 mL). After 15 min the red solution
was evaporated to dryness. The crude material was dissolved with
THF and submitted to silica gel chromatography. It was first eluted
with ethyl acetate to remove the dimethoxytrityl residue and then
with 20% methanol in ethyl acetate to give 21 as a white foam
after evaporation of the solvent (120 mg, 85% yield). 31P NMR
1
71% yield). 31P NMR (81 MHz, CDCl3): δ = –2.9, –1.2 ppm. 1H (100 MHz, CDCl3): δ = –8.8 ppm. H NMR (500 MHz, CDCl3): δ
NMR (250 MHz, CDCl3): δ = 7.80–6.80 (m, 29 H, Ph, 6-H), 6.45,
= 9.95, 9.57 (s, 2 H, NH), 7.59 (m, 1 H, 6-HT), 7.35 (d, J = 8.2 Hz,
5.95 (2 m, 2 H, 1Ј-HU, 1Ј-HT), 5.28 (m, 1 H, 7-HU), 4.81 (m, 1 H, 1 H, 6-HU), 6.38 (dd, J = 8.0, 6.0 Hz, 1 H, 1Ј-HT), 5.82 (dd, J =
5Ј-H), 4.50 (m, 9 H, 3Ј-HT, 4Ј-H, 5Ј-H, 2Ј-HU, 2 CH2), 3.75, 3.73
(s, 12 H, Me DMTr), 2.71 (m, 2 H, CH2), 2.44 (s, 3 H, Me Ts),
2.30 (m, 2 H, CH2), 1.82 (s, 3 H, 7-MeT), 1.59, 1.24 (2 m, 4 H, 2
CH2), 1.04, 0.89 (2 s, 18 H, tBu), 0.14, 0.05 (2 s, 6 H, Me) ppm.
C74H88N5O18PSSi2 (1454.74): calcd. C 61.10, H 6.10, N 4.81; found
C 61.58, H 6.02, N 4.85.
8.0, 2.0 Hz, 1 H, 5-HU), 5.49 (d, J = 6.4 Hz, 1 H, 1’-HU), 5.21 (tdd,
J = 6.2, 2.3, JH-P = 6.0 Hz, 1 H, 3Ј-HT), 4.79 (ddd, J = 11.3, 6.0,
2.3, JH-P Ͻ1.0 Hz, 1 H, 5Ј-HU), 4.75 (dd, J = 6.2, 4.5 Hz, 1 H, 2Ј-
HU), 4.54–4.43 (m, 2 H, 7Ј-HU), 4.35 (br. d, J = 8.0, 6.0 Hz, 1 H,
4Ј-HT), 4.28 (dd, J = 4.3, 2.0 Hz, 1 H, 3Ј-HU), 3.99–3.91 (m, 3 H,
4Ј-HU, 5Ј-HT), 2.60, 2.44 [AB part of an ABX(Y) syst., J = 14.0,
5.8, 2.0, 8.0, 6.0 Hz, 2 H, 2Ј-HT], 2.11 (m, 1 H, 6Ј-HU), 2.00 (m, 1
H, 6Ј-HU), 0.94 (s, 9 H, tBu), 0.87 (s, 9 H, tBu), 0.16, 0.14, 0.07,
0.01 (s, 12 H, Me) ppm. 13C NMR (125 MHz, CDCl3): δ = 164.0,
163.5, 150.6, 142.9, 136.3, 111.5, 102.7, 93.6, 86.5, 86.4, 85.6, 79.5,
79.4, 78.0, 72.1, 71.7, 68.5, 62.1, 38.5, 29.7, 28.0, 25.8, 18.0, 12.6,
–4.4, –4.5, –4.6 ppm. C33H55N4O13PSi2 (802.96): calcd. C 49.36, H
6.90, N 6.98; found C 48.92, H 6.75, N 6.70.
2Ј-O-(tert-Butyldimethylsilyl)-3Ј-O-(tert-butyldiphenylsilyl)-5Ј-O-di-
methoxytrityl-α,β-D-CNA UT (17): K2CO3 (180 mg, 1.24 mmol)
was added to a solution of 16 (0.45 g, 0.31 mmol) in anhydrous
DMF (7 mL). After 4 h at room temperature the reaction mixture
was diluted with ethyl acetate (100 mL) and washed three times
with water (3ϫ20 mL) and once with brine. The organic layer was
dried with MgSO4 and the solvent was removed in vacuo. The cy-
clic phosphotriester 17 was recovered as a white foam (0.35 g, 92%
yield). 31P NMR (81 MHz, CDCl3): δ = –8.5 ppm. 1H NMR
(250 MHz, CDCl3): : δ = 9.10, 8.77 (s, 2 H, NH), 7.71 (d, J =
8.1 Hz, 1 H, 6-HU), 7.60–7.14 (m, 20 H, Ph, 6-HT), 6.59 (dd, J =
9.4, 5.4 Hz, 1 H, 1Ј-HT), 6.14 (d, J = 7.8 Hz, 1 H, 1Ј-HU), 5.25 (d,
J = 8.1 Hz, 1 H, 7-HU), 4.95 (dd, J = 4.8, JH-P = 8.0 Hz, 1 H, 3Ј-
HU), 4.50 (dd, J = 7.8, 4.8 Hz, 1 H, 2Ј-HU), 4.25 (m, 1 H, 3Ј-HT),
4.19 (m, JH-P = 20.0, 2.5 Hz, 2 H, 7Ј-HT), 3.89 (m, 1 H, 4Ј-HU),
3.69 (m, JH-P = 6.0 Hz, 1 H, 4Ј-HT), 3.34 (m, 3 H, 5Ј-HT, 5Ј-HU),
2.24 (m, J = 13.3, 5.8, 5.6 Hz, 1 H, 2Ј-HT), 2.15 (m, 1 H, 6Ј-HT),
1.90 (s, 3 H, 7-HT), 1.89 (m, 1 H, 2Ј-HT), 1.30 (m, 1 H, 6Ј-HT),
0.84, 0.85 (s, 18 H, tBu), 0.08, 0.03 (s, 6 H, Me) ppm. 13C NMR
(63 MHz, CDCl3): δ = 163.8, 162.9, 159.1, 159.0, 150.9, 150.7,
144.0, 135.9–127.6, 113.6, 113.3, 112.4, 102.1, 88.5, 88.0, 86.2, 85.4,
84.8, 83.8, 79.5, 74.8, 67.5, 63.5, 55.5, 40.1, 27.0, 25.7, 19.1, 18.2,
12.6, 0.4, 0.1 ppm. C64H77N4O15PSi2 (1229.47): calcd. C 62.52, H
6.31, N 4.56; found C 61.99, H 6.25, N 4.52.
α,β-D-CNA TU (22): TBAF (1 m in THF, 136 μL) was added to 21
(50 mg, 0.06 mmol) in anhydrous THF (1 mL) at 0 °C. After stir-
ring for 2 h at room temperature, THF was removed under reduced
pressure and the crude was submitted to silica gel chromatography
first eluting with ethyl acetate and then with 10% methanol in ethyl
acetate to give 22 (28 mg, 79 % yield). 31P NMR (81 MHz,
1
CD3OD): δ = –8.6 ppm. H NMR (500 MHz, CD3OD): δ = 7.79
(m, 1 H, 6-HT), 7.72 (d, J = 8.1 Hz, 1 H, 6-HU), 6.34 (dd, J = 8.4,
5.8 Hz, 1 H, 1Ј-HT), 5.94 (d, J = 4.4 Hz, 1 H, 1Ј-HU), 5.76 (d, J =
8.1 Hz, 1 H, 7-HU), 5.11 (br. q, J = 6.0, 2.1 Hz, 1 H, 3Ј-HT), 4.84
(m, J = 11.7, 2.3, JH-P Ͻ1.0 Hz, 1 H, 5Ј-HU), 4.55–4.50 (m, 2 H,
7Ј-HU), 4.26 (t, J = 2.8 Hz, 1 H, 4Ј-HT), 4.23 (d, J = 5.3 Hz, 1 H,
3Ј-HU), 4.16 (d, J = 4.8 Hz, 1 H, 2Ј-HU), 4.05 (m, J = 5.3, 2.4, JH-
= 4.6 Hz, 1 H, 4Ј-HU), 3.81 (d, J = 3.0 Hz, 1 H, 5Ј-HT), 2.57 (m,
P
J = 14.3, 8.4, JH-P = 1.2 Hz, 1 H, 2Ј-HT), 2.43 (m, J = 14.3, 5.8 Hz,
1 H, 2Ј-HT), 2.38 (m, 1 H, 6Ј-HU), 1.88 (s, 3 H, Me) ppm. 13C
NMR (100 MHz, CD3OD): δ = 152.4, 141.9, 137.8, 111.9, 111.4,
108.0, 103.1, 90.8, 87.0, 86.0, 85.4, 79.8, 75.0, 71.1, 62.5, 39.4, 28.8,
12.4, 9.6 ppm. C21H27N4O13P (574.43): calcd. C 43.91, H 4.74, N
9.75; found C 43.88, H 4.78, N 10.02.
2Ј,3Ј-O-(tert-Butyldimethylsilyl)-(5ЈS)-5Ј-C-(tosyloxyethyl)-5Ј-
uridinyl-(2-cyanoethyl)-5Ј-O-dimethoxytritylthymidine-2Ј-phos-
phate (20): Following the same procedure as for 16, starting from
14 (450 mg, 6.7 mmol), 20 (613.0 mg, 70% yield) was isolated as a
3Ј-O-(tert-Butyldiphenylsilyl)-5Ј-C-(tert-butyldimethylsilyloxyethyl)-
2Ј-O-methyluridine (23): tert-Butyldimethylsilyl chloride (0.12 g,
0.81 mmol) was added to a solution of 12 (0.33 g, 0.61 mmol) in
anhydrous pyridine (5 mL) at room temperature. After stirring for
12 h, the reaction mixture was diluted with ethyl acetate (50 mL)
and the organic layer was washed with a saturated aqueous solu-
tion of NH4Cl, water and brine. After removal of the solvent under
reduced pressure, the crude product was dissolved in anhydrous
dichloromethane (5 mL) and pyridine (40 μL) and Dess–Martin
periodinane (0.517 mg, 1.22 mmol) was added. After 12 h at room
temperature ethanol (5 mL) and NaBH4 (140 mg, 3.66 mmol) were
1
1:1 mixture of diastereoisomers. H NMR (250 MHz, CDCl3): δ =
8.88 (2 br. s, 4 H, NH), 7.79–7.71 (m, 2 H, 6-H), 7.52–7.22 (m, 11
H, Ph), 6.85–6.80 (m, 6 H, Ph), 6.45 (m, 2 H, 1Ј-H), 5.77 (m, 2 H,
5Ј-H), 4.28–4.11 (m, 15 H, 2Ј-H, 3Ј-H, 4Ј-H, 5Ј-H, CH2), 3.78 (s, 6
H, Me DMTr), 2.43 (s, 3 H, Me OTs), 1.64–1.24 (m, 6 H), 0.88,
0.85 (2 s, 18 H, tBu), 0.08, 0.07, 0.05, 0.02 (4 s, 12 H, SiMe2) ppm.
3 1 P N M R ( 8 1 M H z , C D C l 3 ) : δ = – 2 . 6 , – 2 . 4 p p m .
C64H84N5O18PSSi2 (1330.59): calcd. C 57.77, H 6.36, N 5.26; found
C 57.52, H 6.26, N 5.44.
2Ј,3Ј-O-(tert-Butyldimethylsilyl)-α,β-D-CNA TU (21): K2CO3 added at 0 °C. After stirring for 20 h at room temperature the reac-
(130 mg, 0.9 mmol) was added to a solution of 20 (0.3 g,
0.22 mmol) in anhydrous DMF (5 mL). After 4 h at room tempera-
ture the reaction mixture was diluted with ethyl acetate (800 mL)
and washed three times with water (3 ϫ 20 mL) and once with
brine. The organic layer was dried with MgSO4 and the solvent
tion mixture was diluted with ethyl acetate (100 mL) and washed
with a saturated aqueous solution of NH4Cl, water and brine. After
silica gel chromatography with ethyl acetate/dichloromethane (1:4)
as eluent, 23 (0.3 g, 75% yield) was isolated as a white foam as a
1:1 mixture of C-5Ј epimers. Data for the 5ЈS epimer: 1H NMR
removed in vacuo. After silica gel chromatography (AcOEt/ (300 MHz, CDCl3): δ = 8.67 (s, 1 H, NH), 8.05 (d, J = 8.1 Hz, 1
728
www.eurjoc.org
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 721–730

Dioxaphosphorinane-Constrained Nucleic Acid Dinucleotides
H, 6-H), 7.75–7.39 (m, 10 H, Ph), 5.97 (d, J = 3.6 Hz, 1 H, 1Ј-H),
5.60 (dd, J = 8.1, 1.5 Hz, 1 H, 5-H), 4.31 (t, J = 5.0 Hz, 1 H, 3Ј-
101.8, 101.6, 87.6, 86.6, 83.8, 82.9, 79.6, 74.6, 68.7, 68.3, 60.3, 57.7,
57.6, 27.6 ppm. C22H29N4O14P (604.46): calcd. C 43.72, H 4.84, N
H), 3.93 (m, 2 H, 7Ј-H, 4Ј-H), 3.72 (m, 2 H, 7Ј-H, 5Ј-H), 3.36 (dd, 9.27; found C 43.91, H 4.75, N 9.56.
J = 4.8, 3.8 Hz, 1 H, 2Ј-H), 3.31 (s, 3 H, OMe), 1.92 (m, 1 H, 6Ј-
(RC5Ј,RP)-2Ј,2Ј-O-Methyl-α,β-D-CNA UU (28): 31P NMR
H), 1.53 (m, 1 H, 6Ј-H), 1.12 (s, 9 H, tBu), 0.93 (s, 9 H, tBu), 0.12,
0.10 (s, 6 H, Me) ppm. 13C NMR (75 MHz, CDCl3): δ = 162.9,
150.0, 140.8, 135.9, 135.8, 130.1, 130.0, 127.7, 101.8, 87.4, 86.9,
83.2, 70.7, 70.2, 62.8, 58.0, 35.0, 26.9, 25.8, 19.3, 18.01, –5.5 ppm.
MS (ESI): m/z = 667.8 [M + Na]+. C34H52N2O7Si2 (654.95): calcd.
C 62.35, H 7.69, N 4.28; found C 61.85, H 7.59, N 4.52.
(100 MHz, CD3OD): δ = –7.4 ppm. 1H NMR (500 MHz, CD3OD):
δ = 8.07 (d, J = 8.5 Hz, 1 H, 5a-H), 7.76 (d, J = 8.1 Hz, 1 H, 5b-
H), 6.11 (d, J = 6.1 Hz, 1 H, 1Јa-H), 5.97 (d, J = 4.8 Hz, 1 H, 1Јb-
H), 5.76 (d, J = 8.1 Hz, 2 H, 6b-H, 6a-H), 5.05 (m, J = 4.7, 3.1,
JH-P = 7.4 Hz, 1 H, 3Јa-H), 5.01 (m, J = 12.0, 3.8, 2.2, JH-P
Ͻ1.0 Hz, 1 H, 5Јb-H), 4.58 (m, J = 11.6, 2.2, JH-P = 22.9 Hz, 1 H,
7Јb-H), 4.55 (m, J = 11.6, 5.4, 1.5, JH-P = 1.5 Hz, 1 H, 7Јb-H), 4.46
(t, J = 5.1 Hz, 1 H, 3Јb-H), 4.32 (td, J = 2.9, 2.6 Hz, 1 H, 4Јa-H),
4.17 (dd, J = 6.0, 4.8 Hz, 1 H, 2Јa-H), 4.06 (m, J = 4.7, 4.0, JH-P
= 3.3 Hz, 1 H, 4Јb-H), 3.86, 3.82 [AB part of an ABX(Y), J = 12.5,
2.7, 2.4 Hz, 2 H, 5Јa-H], 3.52, 3.51 (s, 6 H, Me), 2.30 (m, J = 14.1,
12.2, 5.6, JH-P Ͻ1 Hz, 1 H, 6Јb-H), 2.02 (m, 1 H, 6Јb-H) ppm. 13C
NMR (125 MHz, CD3OD): δ = 164.6, 164.5, 150.9, 150.7, 140.7,
140.6, 101.9, 101.7, 87.5, 86.1, 84.7, 84.1, 82.2, 81.8, 79.9, 75.3,
69.0, 67.7, 60.4, 57.9, 57.4, 26.9 ppm. C22H29N4O14P (604.46):
calcd. C 43.72, H 4.84, N 9.27; found C 44.02, H 4.81, N 9.32.
(5ЈR)-3Ј-O-(tert-Butyldiphenylsilyl)-2Ј-O-methyl-5Ј-C-(tosyloxy-
ethyl)uridine (24): p-Toluenesulfonic acid (50 mg) was added to a
mixture of 23 (0.3 g, 0.46 mmol) in MeOH (5 mL). A saturated
aqueous solution of NaHCO3 (10 mL) was added dropwise after
3 h at room temperature. The methanol was evaporated and the
residual mixture diluted with ethyl acetate and washed with water
and brine. After removal of the solvent, the residual oil was precipi-
tated into cold petroleum ether to provide a white powder (210 mg,
85 % yield). This powder was dissolved in anhydrous pyridine
(0.35 mL) and chloroform (1.5 mL) and tosyl chloride (111 mg,
0.58 mmol) was added at 0 °C. Stirring was maintained for 12 h
and the reaction mixture was diluted with ethyl acetate and washed
with a saturated aqueous solution of NH4Cl. The organic layer was
collected and washed with water, brine and dried with MgSO4. Af-
ter removal of the solvent under reduced pressure, the crude prod-
uct was deposited on a silica gel column and eluted with ethyl acet-
ate/dichloromethane (1:3). After two separation processes, com-
pounds 15 and 24 were isolated from each other and recovered as
Computational Details: All calculations were performed with the
Gaussian 98 set of programs using hybrid density functional meth-
ods.[28] The Lee, Yang and Parr correlation functional (B3LYP) was
used.[29] A double-ζ plus polarisation valence basis set was em-
ployed for each atom including hydrogen.mod3 The s and p diffuse
functions were added for oxygen atoms (ζs = 0.108151 and ζp
=
0.070214). Standard pseudo-potentials developed in Toulouse were
used to describe the atomic cores of all non-hydrogen atoms.[30]
The structural parameters were produced from full geometry op-
timisation procedures in the gas phase with no imposed constraints.
The harmonic frequencies of these B3LYP optimised structures
were calculated to verify that the localised stationary points coin-
cide with energy minima.
1
white foam (79 and 83 mg, 60% combined yield). Data for 24: H
NMR (300 MHz, CDCl3): δ = 8.40 (s, 1 H, NH), 7.78–7.35 (m, 15
H, Ph, 6-H), 5.77 (dd, J = 7.8, 2.1 Hz, 1 H, 5-H), 5.61 (d, J =
6.3 Hz, 1 H, 1Ј-H), 4.32 (dd, J = 4.8, 2.4 Hz, 1 H, 3Ј-H), 4.09–3.89
(m, 4 H, OH, 4Ј-H, 7Ј-H), 3.79 (qd, J = 10.8, 2.7 Hz, 1 H, 5Ј-H),
3.57 (br. d, J = 2.4 Hz, 1 H, 2Ј-H), 3.18 (s, 3 H, OMe), 2.47 (s, 3
H, MeTs), 1.62 (m, 1 H, 6Ј-H), 1.10 (s, 9 H, tBu) ppm. 13C NMR
(75 MHz, CDCl3): δ = 162.5, 150.1, 143.2, 136.0, 135.9, 132.9,
132.8, 129.9, 128.0, 102.7, 92.0, 89.2, 81.0, 70.0, 67.5, 67.2, 58.3,
31.3, 26.9, 21.7, 19.4 ppm. C35H42N2O9SSi (694.87): calcd. C 60.50,
H 6.09, N 5.42; found C 60.35, H 6.10, N 5.37.
Circular Dichroism Studies: These experiments were performed
with a Jasco J-815 CD spectrometer equipped with a Peltier con-
troller Jasco PTC-4235/15 at a dinucleotide concentration of
0.1 mm in 10 mm Na2HPO4, 100 mm NaCl, 0.1 mm Na2EDTA
buffer, pH 7.00Ϯ0.02. Molar extinction coefficients were calcu-
lated from those of dinucleotides using the nearest-neighbour
approximation method assuming that α,β-D-CNAs have the same
molar extinction coefficient as DNA. The dinucleotide concentra-
tion was determined from the UV absorbance at high temperature
(90 °C). All CD spectra were recorded after stabilisation of the tem-
perature for 10 min and were normalised by subtraction of the
background scan with buffer. Taking the known dinucleotide con-
centration into account, the normalised spectra were converted to
show the variation of the molar extinction coefficient (Δε) with
wavelength.
3Ј-O-(tert-Butyldimethylsilyl)-2Ј-O-methyl-C-(tosyloxyethyl)-5Ј-
uridinyl-2-cyanoethyl-2Ј-O-methyl-5Ј-O-dimethoxytrityluridine-2Ј-
phosphate (25 and 26): Following the same procedure as for 16,
starting from a 1:1 mixture of 15 and 24 (115 mg, 0.17 mmol), a
1:1 mixture of diastereoisomers 25 and 26 (158.0 mg, 70% yield)
was isolated after silica gel chromatography eluted with ethyl acet-
ate/dichloromethane (9:1). 31P NMR (100 MHz, C6D6): δ = –2.6,
–2.4, –2.3, –2.2 ppm. C66H72N4O19PSSi (1370.50): calcd. C 60.47,
H 5.59, N 5.11; found C 60.58, H 5.63, N 5.02.
(SC5Ј,SP)-2Ј,2Ј-O-Methyl-α,β-D-CNA UU (27): The same procedure
as for 21/22 was followed. 31P NMR (100 MHz, CD3OD): δ =
–6.3 ppm. H NMR (500 MHz, CD3OD): δ = 8.07 (d, J = 8.5 Hz,
Crystal Structure Determination for 21: To determine the structure
of compound 21 the selected crystal was mounted on a glass fibre
by using perfluoropolyether oil and cooled rapidly to 193 K in a
1
1 H, 5a-H), 7.78 (d, J = 8.0 Hz, 1 H, 5b-H), 6.08 (d, J = 6.0 Hz, 1 stream of cold N2. X-ray intensity data were collected with graph-
H, 1Јa-H), 6.00 (d, J = 3.0 Hz, 1 H, 1Јb-H), 5.77 (d, J = 8.0 Hz, 1 ite-monochromated Mo-Kα radiation (wavelength = 0.71073 Å)
H, 6b-H), 5.76 (d, J = 8.5 Hz, 1 H, 6a-H), 5.05 (m, J = 4.8, 3.7, with a Bruker-AXS kappa APEX II Quazar diffractometer by
JH-P = 7.4 Hz, 1 H, 3Јa-H), 4.90 (m, J = 11.0, 4.0, 2.2,
JH-P Ͻ1.0 Hz, 1 H, 5Јb-H), 4.66 (m, J = 11.0, 4.9, 1.5, JH-P
using φ and ω scans and a 30-W air-cooled microfocus source
(ImS) with focusing multilayer optics at a temperature of 193(2) K.
The data were integrated with SAINT[31] and an empirical absorp-
tion correction was applied with SADABS.[32] The structure was
=
23.5 Hz, 1 H, 7Јb-H), 4.55 (m, J = 13.0, 11.0, 1.8, JH-P = 1.5 Hz,
1 H, 7Јb-H), 4.32 (m, 2 H, 4Јa-H, 3Јb-H), 4.19 (br. t, J = 6.0,
5.0 Hz, 1 H, 2Јa-H), 4.05 (m, J = 6.8, 2.2, JH-P = 4.6 Hz, 1 H, 4Јb- solved by direct methods (SHELXS-97)[33] and refined against all
H), 3.84 (m, J = 12.5, 2.8, 2.5 Hz, 2 H, 5Јa-H), 3.52, 3.51 (s, 6 H,
Me), 2.42 (m, 1 H, 6Јb-H), 2.00 (m, 1 H, 6Јb-H) ppm. 13C NMR
(125 MHz, CD3OD): δ = 164.6, 164.5, 150.9, 150.6, 140.7, 140.1,
data by full-matrix least-squares methods on F2 (SHELXL-97).[34]
All non-hydrogen atoms were refined with anisotropic displace-
ment parameters. The hydrogen atoms were refined isotropically
Eur. J. Org. Chem. 2012, 721–730
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
729

J.-M. Escudier et al.
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Published Online: December 5, 2011
730
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© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 721–730