Synthesis of phostone-constrained nucleic acid (P-CNA) dinucleotides through intramolecular Arbuzov's reaction

Article abstract of DOI:10.1002/ejoc.201101061

P-CNAs are dinucleotide building blocks in which the torsional angles α and β of the sugar/phosphate backbone are constrained to non-canonical values within a cyclic phosphonate structure (phostone) synthesised by diastereoselective intramolecular Arbuzov reaction. The reaction has been improved through the use of microwave activation and addition of lithium bromide.

Full text of DOI:10.1002/ejoc.201101061

FULL PAPER
DOI: 10.1002/ejoc.201101061
Synthesis of Phostone-Constrained Nucleic Acid (P-CNA) Dinucleotides
Through Intramolecular Arbuzov’s Reaction
Dan-Andrei Catana,[a] Marie Maturano,[a] Corinne Payrastre,[a] Pierre Lavedan,[b]
Nathalie Tarrat,[c,d,e] and Jean-Marc Escudier*[a]
Keywords: Nucleotides / DNA structures / Strained molecules / Phosphorus heterocycles / Conformation analysis
P-CNAs are dinucleotide building blocks in which the tor- tive intramolecular Arbuzov reaction. The reaction has been
sional angles α and β of the sugar/phosphate backbone are
constrained to non-canonical values within a cyclic phos-
phonate structure (phostone) synthesised by diastereoselec-
improved through the use of microwave activation and ad-
dition of lithium bromide.
what complicated by the fragile and flexible nature of sin-
gle-stranded fragments. With stable structural analogues of
these secondary structures, we could expect to learn more
about their role in vivo, the information they carry, and the
key elements of their recognition by other macromolecules.
With this in mind, we became interested in the develop-
ment of covalently constrained dinucleotide building units
in which the backbone torsion angles of nucleic acids
(Scheme 1) can have predefined values that are significantly
different from the typical values observed in DNA and
RNA duplexes.[5] Our general strategy is based on the intro-
duction of the 1,3,2-dioxaphosphorinane ring structure at
key positions along the phosphate backbone. We have al-
ready reported on the diastereoselective synthesis of a di-
oxaphosphorinane-CNA (D-CNA) dinucleotide building
unit in which the α and β torsion angles are locked in a
(g+,t) conformation (Scheme 1).[6]
Introduction
The development of conformationally restricted nucleo-
sides has attracted a lot of attention, mainly due to the im-
portant potential applications of antisense oligonucleo-
tides.[1] However, much less attention has been devoted to
the design of conformationally restricted nucleosides for the
special purpose of mimicking biologically important non-
helical secondary structures of DNA and RNA.[2]
In addition to the double-stranded helical conformation,
nucleic acids may adopt many other alternative structures
such as bulges, hairpins, U-turns, or branched junctions.[3]
These secondary structures always contain unpaired nucleo-
tides or non-Watson–Crick pairs and are characterized by
a variety of backbone conformations that differ markedly
from the regular conformational states of double-stranded
helices.[4] It is now well-established that these disparate
structures play a crucial role in fundamental biological pro-
cesses where protein–nucleic acid interactions, DNA/RNA
folding, or RNA catalytic activity are involved. The deter-
mination of the precise biological role of a particular back-
bone conformation is currently an area of intense study.
Unfortunately, structural and functional studies are some-
[a] Laboratoire de Synthèse et Physico-Chimie de Molécules
d’Intérêt Biologique, UMR 5068 CNRS, Université Paul
Sabatier,
118 Route de Narbonne, 31062 Toulouse Cedex 9, France
Fax: +33-5-61556011
Scheme 1. Left: the six backbone torsion angles (labeled α to ζ) of
nucleic acids. Right: D- or P-CNA dinucleotides in which α and β
are stereocontrolled by a dioxaphosphorinane or a phostone ring
structure, respectively.
E-mail: escudier@chimie.ups-tlse.fr
[b] Service Commun de RMN, Université Paul Sabatier,
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,
However, the high level of diastereoselectivity observed
for its formation led us to investigate the synthesis of
phostone-constrained nucleic acids (P-CNA) in which the
dioxaphosphorinane structure was replaced by a cyclic
phosphonate to provide access to all possible diastereoiso-
mers of the cyclic structure (Scheme 1).
31400 Toulouse, France
[e] CNRS, UMR5504,
31400 Toulouse, France
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201101061 or from
the author.
Eur. J. Org. Chem. 2011, 6857–6863
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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J.-M. Escudier et al.
FULL PAPER
Similar compounds, featuring a seven-membered cyclic
phosphonate ring have been obtained through a ring clos-
ing metathesis (RCM) reaction between a vinyl phos-
phonate and the 2Ј-C-allyl moiety of a dinucleotide.[7]
Seven-membered phostone rings can also be prepared by
cyclic acetal ring enlargement,[8] and smaller ring sizes can
be achieved by esterification or cyclization of the formed
phosphonate.[9] Because, in our case, the 5Ј-oxygen of the
lower nucleoside must be involved in a six-membered cyclic
phosphonate structure as an internucleotidic linkage, we
speculated that an intramolecular Arbuzov reaction could
be suitable for this task.[10]
Results and Discussion
Scheme 2. Retrosynthesis of phostone-constrained nucleic acid (P-
CNA).
In an initial analysis, the key intermediate in the retro-
synthetic analysis would be a dinucleotide composed of an
ethoxy phosphite and a 5Ј-C-bromopropyl-substituted
lower nucleoside (Scheme 2). The latter would be prepared
5Ј-O-Protection by trimethylsilane of diastereomerically
from 5Ј-C-allyl nucleoside, which can be synthesized from pure 5Ј-C-allylthymidine (1) and subsequent hydroboration/
natural nucleoside through a Sakuraï addition on the corre- oxidation afforded 3, which was converted into 4 by treat-
sponding 5Ј-aldehyde.[11] The (5ЈS)-C-tosyloxy- or -(bro- ment with tosyl chloride in pyridine (Scheme 3).
mopropyl)thymidine (7 and 6, respectively Scheme 3) were
Dinucleoside phosphites 8–10 were formed as a 1:1 dia-
prepared from protected (5ЈS)-C-tosyloxypropylthymidine stereoisomeric mixture (31P NMR analysis) by coupling 5Ј-
(4); the former by direct removal of the silyl group under C-functionalized nucleosides 6 or 7 with commercially
acidic conditions to prevent the formation of the furan ring, available thymidine ethyl or cyanoethyl phosphoramidites
and the latter through displacement of the tosyl group with using standard phosphoramidite technology, without an
lithium bromide followed by a desilylation step.
oxidation step.[12]
Scheme 3. Synthesis of α,β-P-CNA. Reagents and conditions: (a) Me3SiCl, pyr, room temp., 3 h, 95%. (b) BH3/Me2S, THF, room temp.,
2 h then NaOH/H2O2, room temp., 0.5 h, 90%. (c) TsCl, CHCl3, pyr, room temp., 16 h, 85%. (d) LiBr, Me2CO/DMF (4:1), 60 °C, 0.75 h,
quant. (e) PTSA, MeOH, room temp., 1 h, 70–90%. (f) thymidine phosphoramidite, tetrazole, room temp., 60–85%. (g) (i) see Table 1
for Arbuzov reaction conditions. (ii) TFA, CH2Cl2, quant. (h) TBAF, THF, room temp., 1 h, 90%.
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Eur. J. Org. Chem. 2011, 6857–6863

Phostone-Constrained Nucleic Acid Dinucleotides
Initially, we attempted to conduct the Arbuzov reaction
The two isomers 13 and 14 were first characterized by
with dinucleotide 5Ј-C-bromopropyl-ethoxyphosphite (8) their chemical shifts in their respective 31P NMR spectra
under standard conditions (i.e., high temperature in diox- (recorded in CD3OD), which were measured at δ = +26.9
ane or toluene; Table 1, entries 1 and 2), however, partial and +31.3 ppm, respectively. In the CNA dinucleotide
loss of the dimethoxytrityl protective group occurred. The series, this difference in chemical shift was always observed
crude material was submitted to acidic conditions (tri- between phosphorus epimers, and the isomer featuring
fluoroacetic acid, TFA) to provide phostones 11 and 12, as downfield chemical shift was always assigned as the isomer
revealed by 31P NMR analysis (δP = 28.2 and 32.1 ppm, with all the substituents in the most stabilizing position on
respectively). As described for the intermolecular Arbuzov- the internucleotidic six-membered ring.[5,6]
1
type reaction involving nucleoside phosphites,[13] the reac-
The phostone chair conformation is evident from the H
tion was difficult to reproduce, and yields of phostones NMR spectra (Table 2), with a very small coupling constant
ranged from 15 to 70% in a heterogeneous mixture of com- between the 5Ј-H involved in the oxaphosphorinane system
pounds that consisted of mainly alkylphosphonates or and the phosphorus atom, which is characteristic of an ax-
phosphates (31P NMR analysis). Changing the solvent to ial position of the 5Ј-H proton.[16] The observation of a
dioxane or pyridine also failed to provide any of the desired long-range coupling between the 4Ј-H of the lower sugar
phostone. We then investigate the use of microwave-assisted unit and the phosphorus atom is indicative of a typical W-
reaction conditions in various solvents and found that, as shaped P–O5Ј–C5Ј–C4Ј-H4Ј junction, which is consistent
well as reducing the required time and temperature, the use with a gauche(+) conformation of γ in both P-CNA moie-
of acetonitrile proved to be significantly more effective ties. The puckering of the 2Ј-deoxyribose moieties were as-
(Table 1, entries 7 and 8 vs. 5 and 6).
signed by examination of the sugar ring H/H coupling con-
stants (Table 2). The small JH3Ј/H4Ј (ca. 2 Hz) value mea-
sured for the upper nucleosides and the values of JH2Ј/H3Ј
and JH1Ј/H2Ј are close in each case to those found in the
standard C2Ј-endo conformation of the natural 2Ј-deoxyri-
bose. However, the south conformation of the upper nucle-
oside is enforced by the neutral phostone internucleotidic
linkage.
Table 1. Optimization of the Arbuzov reaction conditions with 8.
Entry Conditions
Solvent
Phostone
Yield
[%]
Activation[a] t [h] T [°C] SP/RP d.r[b]
1
2
3
4
5
6
7
8
9
10
dioxane
toluene
DMSO
pyridine
THF
CH2Cl2
MeCN
MeCN
MeCN
MeCN
–
–
–
–
10.0 130
20.0 130
14.0 130
1.6:1
1.7:1
–
–
–
20–65
15–70
0
0
0
8.0
0.5
0.5
0.5
1.5
140
110
110
110
105
105
90
Table 2. H/H coupling constants [Hz] in the 1H NMR spectra
(500 MHz) of (SC,SP)-α,β-P-CNA 13 and (SC,RP)-α,β-P-CNA 14.
MW
MW
MW
MW
MW, LiBr 1.0
MW, LiBr 3.0
–
0
1.8:1
2.2:1
2.7:1
1.9:1
35
55
40
85
Coupling constant J /Hz
J(1Ј,2Ј)
J(2Ј,3Ј) J(3Ј,4Ј) J(3Ј,P) J(5Ј,P)
13 upper nucleoside 8.0 6.0 6.0 2.0 2.0
lower nucleoside 7.0 7.0 6.0 4.0 3.0
14 upper nucleoside 8.5 5.5 5.5 1.5 1.5
lower nucleoside 8.0 6.0 6.0 2.5 2.5
6.5
–
4.5
–
–
Ͻ 1
–
[a] MW: microwave, 40–60 W. [b] Determined by 31P NMR spectro-
scopic analysis.
2.0
The first step in the Arbuzov reaction, i.e., attack of the
phosphorus electronic lone pair, is actually intramolecular
but the following halide-mediated dealkylation is not.
Therefore, we focused on the possibility of improving the
Arbuzov reaction by adding an external halide source such
as LiBr. Indeed, in combination with microwave activation
and a longer time period with a lower temperature, the yield
of phostone 11/12 reached 85%, with a diastereoisomeric
ratio of approximately 2:1. Interestingly, under these new
conditions, ethoxy or standard cyanoethoxy phosphites 8
and 9 with a tosylate as leaving group, proved to be good
substrates for the intramolecular Arbuzov reaction
(Scheme 3 and the Supporting Information).[14]
The structural analysis was corroborated by geometry
optimization procedures (see the Supporting Information
and Figure 1), showing that the difference in energy was ΔE
= 0.7 kcal (water and MeOH) between the two phostones
in chair conformations in favor of (SC,SP)-α,β-P-CNA (13),
which presents all the substituents in less sterically hindered
positions (apical upper nucleoside and equatorial lower nu-
cleoside).
Although surprising, with an initial equimolecular mix-
ture of phosphite, the observed diastereoselectivity can be
explained by phosphite epimerization[15] and equilibrium in
the Arbuzov reaction (see the Supporting Information).
The diastereoisomeric P-CNAs were separated by reverse
phase HPLC, and the remaining silyl protective group was
removed in good yield by treatment with fluoride ions, pro-
viding (SC,SP)-α,β-P-CNA (13) as the major isomer and
(SC,RP)-α,β-P-CNA (14) as the minor isomer.
Figure 1. Superimposition of α,β-P-CNA minimized structures.
Left and middle: (SC,SP)-13 and (SC,RP)-14 α,β-P-CNA with X-
ray structure of unmodified TpT,[17] respectively. Right: (SC,SP)-13
(blue) with (SC,RP)-14 (yellow) α,β-P-CNA.
Eur. J. Org. Chem. 2011, 6857–6863
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J.-M. Escudier et al.
FULL PAPER
12.9, J2Ј–1Ј = 5.4 Hz, 1 H, H2Ј], 1.84 (d, J7–6 = 1.2 Hz, 3 H, H7),
1.83 [B of an ABX(Y), 2J2ЈЈ–2Ј = 12.9, 3J2ЈЈ–1Ј = 9.3, 3J2ЈЈ–3Ј = 5.1 Hz,
1 H, H2ЈЈ], 1.51–1.29 (m, 4 H, H6Ј and H7Ј), 1.08 (s, 9 H, tBu),
–0.09 (s, 9 H, SiMe3) ppm. 13C NMR (CDCl3, 75 MHz): δ = 163.5
(C4), 150.3 (C2), 144.9 (CqS, Ts), 136.1 (C6), 135.8 and 135.7 (4 ϫ
CH, Ph), 133.4 and 133.0 (2 ϫ Cq, Ph), 133.1 (CqCH3, Ts), 130.2
and 130.1 (2 ϫ CH, Ph), 129.9 (2 ϫ CH, Ts), 128.0 (4 ϫ CH, Ph),
127.9 (2 ϫ CH, Ts), 110.9 (C5), 88.9 (C4Ј), 85.0 (C1Ј), 76.1 (C3Ј),
72.4 (C5Ј), 70.1 (C8Ј), 40.7 (C2Ј), 30.0 (C7Ј), 26.9 (CMe3), 24.9 (C6Ј),
21.7 (CH3, Ts), 19.1 (CMe3), 12.5 (C7), 0.2 (SiMe3) ppm.
C39H52N2O8SSi2 (765.08): calcd. C 61.23, H 6.85, N 3.66; found C
59.99, H 6.96, N 3.77.
3
4
Overall, these elements allow us to assign the conforma-
tion of the backbone torsion angles α, β, and γ as (α,β,γ) =
(g+, t, g+) for (SC,SP)-α,β-P-CNA (13) and (α,β,γ) = (a–/t, t,
g+) for (SC,RP)-α,β-P-CNA (14).
Conclusions
An improvement to the classical Arbuzov reaction by ap-
plying microwave activation and addition of a salt has al-
lowed the synthesis of six-membered cyclic-phosphonate
(phostone) as an internucleotidic linkage with defined re-
(5ЈS)-C-(Bromopropyl)-3Ј-O-(tert-butyldiphenylsilyl)-5Ј-O-(tri-
strain on the α and β torsion angles. The dioxaphosphorin- methylsilyl)thymidine (5): To a solution of dried LiBr (360 mg,
4.15 mmol) at room temp. under argon in a mixture of anhydrous
acetone/dimethylformamide (4:1; 28 mL, 0.15 m), was added a solu-
tion of 4 (2.12 g, 2.77 mmol) in the same solvent mixture (7 mL,
0.4 m). The reaction mixture was heated to reflux for 45 min, then
the acetone was evaporated and saturated NaHCO3 (50 mL) was
added. The aqueous phase was extracted with EtOAc, then the
organic phase was washed three times with water and brine, dried
with MgSO4, and the solvent was evaporated under vacuum to give
ane-constrained nucleic acid (D-CNA) approach has al-
ready provide us with constrained dinucleotides exhibiting
promising properties,[18] the phostone-constrained nucleic
acid (P-CNA) provides access to new constrained dinucleo-
tides featuring atypical α/β values (a–/t, t).
With the need to develop functional DNA and RNA mo-
lecules,[19] new nucleotide analogue building blocks featur-
ing preorganized structures should be useful for the elabo-
ration of synthetic, distorted nucleic acids with improved
1
5 (1.85 g, quant.) as a white foam. H NMR (CDCl3, 300 MHz): δ
4
= 9.71 (s, 1 H, NH), 7.76 (d, J6–7 = 1.2 Hz, 1 H, H6), 7.65–7.60
3
stability and folding ability. The extended CNA family of- (m, 4 H, Ph), 7.47–7.36 (m, 6 H, Ph), 6.60 (dd, J1Ј–2ЈЈ = 9.0,
3
3J1Ј–2Ј = 5.1 Hz, 1 H, H1Ј), 4.21 (d, J = 5.1 Hz, 1 H, H3Ј), 3.81 (s,
fers interesting elements that can be used to elaborate and
study unusually shaped nucleic acids. Incorporation of these
new P-CNA conformationally constrained building units
into selected unpaired nucleic acid secondary structures will
be published in due course.
3
1 H, H4Ј), 3.24 (t, J8Ј–7Ј = 6.0 Hz, 2 H, H8Ј), 2.99 (m, 1 H, H5Ј),
2
3
2.31 [A of an ABX(Y), J2Ј–2ЈЈ = 13.2, J2Ј–1Ј = 5.4 Hz, 1 H, H2Ј],
1.90–1.81 (m, 4 H, H2ЈЈ and H7), 1.72–1.41 (m, 4 H, H6Ј and H7Ј),
1.07 (s, 9 H, tBu), 0.08 (s, 9 H, SiMe3) ppm. 13C NMR (CDCl3,
75 MHz): δ = 164.1 (C4), 150.7 (C2), 136.2 (C6), 135.8 and 135.7
(4 ϫ CH, Ph), 133.5 and 133.0 (2 ϫ Cq, Ph), 130.2 and 130.1 (2 ϫ
CH, Ph), 128.0 (4 ϫ CH, Ph), 110.0 (C5), 88.9 (C4Ј), 85.1 (C1Ј),
76.1 (C3Ј), 72.0 (C5Ј), 40.8 (C2Ј), 33.3 (C6Ј), 32.6 (C7Ј), 28.6 (C8Ј),
27.0 (CMe3 ), 19.1 (CMe3 ), 12.6 (C7 ), 0.2 (SiMe3 ) ppm.
C32H45BrN2O5Si2 (673.79): calcd. C 57.04, H 6.73, N 4.16; found
C 56.82, H 6.77, N 4.21.
Experimental Section
General: Products were purified by medium pressure liquid
chromatography with a Jobin et Yvon Modoluprep apparatus by
using Amicon 6–35 μm or Merck 15 μm silica. NMR spectra were
recorded with a Bruker AC-250, Avance-300, or Avance 500 spec-
trometer (250, 300, or 500 MHz for 1H and 63, 75, or 125 MHz
for 13C). Chemical shifts are referenced to tetramethylsilane. Mass
spectra were recorded with a Nermag R10-10 or with a Perkin–
Elmer API 365. Microwave-assisted reactions were performed with
a CEM Discover apparatus. All solvents were distilled and dried
before use. Compounds 1–3 have been described previously.[11]
(5ЈS)-C-(Bromopropyl)-3Ј-O-(tert-butyldiphenylsilyl)thymidine (6):
To a solution of 5 (1.69 g, 2.51 mmol) in methanol (12 mL, 0.2 m),
p-toluenesulfonic acid (95 mg, 0.50 mmol) was added. After 1 h
stirring at room temp., NaHCO3 (42 mg, 0.50 mmol) was added
and the reaction mixture was diluted with EtOAc (150 mL). The
organic phase was washed twice with water and brine, dried with
MgSO4, and the solvent was evaporated to give 6 (1.05 g, 70%)
after isolation by silica gel chromatography using dichloromethane/
ethyl acetate (4:1) as eluents. 1H NMR (CDCl3, 300 MHz): δ =
3Ј-O-(tert-Butyldiphenylsilyl)-5Ј-O-(trimethylsilyl)-(5ЈS)-C-(tosyl-
oxypropyl)thymidine (4): To a solution of 3 (1.47 g, 2.41 mmol) at 8.24 (br. s, 1 H, NH), 7.67–7.61 (m, 4 H, Ph), 7.48–7.38 (m, 6 H,
4
3
0 °C under argon in freshly distilled chloroform (8 mL), pyridine
(5 mL) and tosyl chloride (688 mg, 3.61 mmol) were added. The
reaction was stirred at room temp. for 16 h then quenched with
saturated aqueous NH4Cl (40 mL). The mixture was diluted with
ethyl ether (100 mL) and washed with brine. The organic layer was
dried with MgSO4 and concentrated in vacuo. The crude product
was deposited on a silica gel column and eluted with diethyl ether/
Ph), 7.25 (d, J6–7 = 1.2 Hz, 1 H, H6), 6.11 (dd, J1Ј–2ЈЈ = 8.4,
3J1Ј–2Ј = 6.0 Hz, 1 H, H1Ј), 4.45 [X of an ABX(Y), J3Ј–2ЈЈ = 4.8,
3
3
3
3
3J3Ј–2Ј = 2.4, J3Ј–4Ј = 2.1 Hz, 1 H, H3Ј], 3.77 (t, J4Ј–3Ј = J4Ј–5Ј
=
2.1 Hz, 1 H, H4Ј), 3.32 (t, J8Ј–7Ј = 6.6 Hz, 2 H, H8Ј), 3.03 (br. s, 1
3
H, H5Ј), 2.35 [A of an ABX(Y), J2ЈЈ–2Ј = 13.5, J2ЈЈ–1Ј = 8.4,
2
3
3J2ЈЈ–3Ј = 4.8 Hz, 1 H, H2ЈЈ], 2.23 [B of an ABX(Y), J2Ј–2ЈЈ = 13.5,
2
3
4
3J2Ј–1Ј = 6.0, J2Ј–3Ј = 2.4 Hz, 1 H, H2Ј], 1.88 (d, J7–6 = 1.2 Hz, 3
petroleum ether (7:3) to give 4 (1.60 g, 87%) as a white foam after
H, H7), 1.84–1.68 (m, 2 H, H7Ј), 1.51–1.42 (m, 2 H, H6Ј), 1.09 (s,
1
removal of the solvent. H NMR (CDCl3, 300 MHz): δ = 8.07 (s, 9 H, tBu) ppm. 13C NMR (CDCl3, 75 MHz): δ = 164.1 (C4), 150.5
1 H, NH), 7.77 (A of an AB, 3J = 8.4 Hz, 2 H, Ts), 7.71 (br. d,
(C2), 137.8 (C6), 135.8 and 135.7 (4 ϫ CH, Ph), 133.4 and 133.1
(2 ϫ Cq, Ph), 130.2 and 130.1 (2 ϫ CH, Ph), 128.0 and 127.9 (4 ϫ
CH, Ph), 110.0 (C5), 89.7 (C4Ј), 88.1 (C1Ј), 74.4 (C3Ј), 70.1 (C5Ј),
39.5 (C2Ј), 35.6 (C6Ј), 32.7 (C7Ј), 29.0 (C8Ј), 27.0 (CMe3), 19.1
4J6–7 = 1.2 Hz, 1 H, H6), 7.64–7.60 (m, 4 H, Ph), 7.46–7.39 (m, 6
3
3
H, Ph), 7.34 (B of an AB, J = 8.4 Hz, 2 H, Ts), 6.55 (dd, J1Ј–2ЈЈ
= 9.3, J1Ј–2Ј = 5.4 Hz, 1 H, H1Ј), 4.18 (d, J3Ј–2ЈЈ = 5.1 Hz, 1 H,
3
3
H3Ј), 3.91 (t, 3J8Ј–7Ј = 6.0 Hz, 2 H, H8Ј), 3.77 (s, 1 H, H4Ј), 2.96 (m, (CMe3), 12.5 (C7) ppm. C29H37BrN2O5Si (601.61): calcd. C 57.90,
2
2 H, H5Ј), 2.44 (s, 3 H, MeTs), 2.28 [A of an ABX(Y), J2Ј–2ЈЈ
=
H 6.20, N 4.66; found C 57.63, H 6.27, N 4.70.
6860
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Eur. J. Org. Chem. 2011, 6857–6863

Phostone-Constrained Nucleic Acid Dinucleotides
1
3Ј-O-(tert-Butyldiphenylsilyl)-(5ЈS)-C-(tosyloxypropyl)thymidine
(7): To a solution of 4 (1.2 g, 1.62 mmol) in methanol (8 mL, 0.2 m),
p-toluenesulfonic acid (27 mg) was added. After 1 h stirring at
room temp., an aqueous solution of NaHCO3 (10 mL) was added
and the methanol was removed under vacuum before the reaction
mixture was diluted with EtOAc (120 mL). The organic phase was
washed twice with water and brine, dried with MgSO4, and the
solvent evaporated. Compound 7 (1.01 g, 90%) was isolated by sil-
ica gel chromatography using dichloromethane/ethyl acetate (4:1)
as eluents. 1H NMR (CDCl3, 300 MHz): δ = 8.14 (br. s, 1 H, NH),
reagent (10.0 mL of 5-ethylthiotetrazol in acetonitrile; 0.25 m). H
and 13C NMR spectra were highly complex due to the diastereoiso-
meric mixture of thymidine dinucleotide analogues. 31P NMR
(C6D6, 100 MHz): δ = 142.4, 140.8 ppm. MS: m/z = 1374.4 [M +
K+].
3Ј-O-(tert-Butyldiphenylsilyloxy)-(SC,SP) (α,β-P-CNA; 11): Dried
LiBr (10 equiv.) and phosphites 8, 9, or 10 (1 equiv.) were dissolved
under argon in degassed anhydrous acetonitrile (30 mg/mL) in a
closed microwave flask and the mixture was stirred at 90 °C under
microwave irradiation (30–50 W) for 4 h (phosphites 8 and 9) or
8 h (phosphite 10). The solution was then diluted with EtOAc,
washed with water and brine, dried with MgSO4, and the solvent
was evaporated. The crude product was treated with 3% trifluoro-
acetic acid in dichloromethane for 1–4 h (the reaction may be fol-
lowed on RP-HPLC). The acid was subsequently neutralized with
saturated NaHCO3, and the organic phase was washed with brine,
dried with MgSO4 and the solvent was evaporated to yield a mix-
ture of the two diastereoisomers 11 and 12 (80–90%) in a diastereo-
isomeric ratio of approximately 2:1. The two diastereoisomers were
then separated using reverse phase HPLC (80% MeOH in water).
1H NMR (CDCl3, 500 MHz): δ = 7.55–7.45 (m, 8 H, H6a, H6b,
3
7.76 (A of an AB, J = 8.4 Hz, 2 H, Ts), 7.65–7.60 (m, 4 H, Ph),
3
7.46–7.37 (m, 6 H, Ph), 7.33 (B of an AB, J = 8.4 Hz, 2 H, Ts),
4
3
3
7.24 (d, J6–7 = 1.2 Hz, 1 H, H6), 6.09 (dd, J1Ј–2ЈЈ = 8.4, J1Ј–2Ј
=
=
6.0 Hz, 1 H, H1Ј), 4.43 [X of an ABX(Y), J3Ј–2ЈЈ = 5.4, J3Ј–4Ј
3
3
2.4, J3Ј–2Ј = 2.1 Hz, 1 H, H3Ј], 3.97 (A of an ABX2, J8Ј–8ЈЈ = 9.6,
3
2
3J8Ј–7Ј = 6.0 Hz, 1 H, H8Ј), 3.92 (B of an ABX2, J8ЈЈ–8Ј = 9.6,
2
3J8ЈЈ–7Ј = 6.0 Hz, 1 H, H8ЈЈ), 3.72 (t, J4Ј–3Ј = J4Ј–5Ј = 2.4 Hz, 1 H,
3
3
H4Ј), 2.98 [X of an ABX(Y), J5Ј–6Ј = 8.7, J5Ј–6ЈЈ = 5.1, J5Ј–4Ј
=
3
3
3
2.4 Hz, 1 H, H5Ј], 2.44 (s, 3 H, MeTs), 2.33 [A of an ABX(Y),
2J2ЈЈ–2Ј = 13.2, J2ЈЈ–1Ј = 8.4, J2ЈЈ–3Ј = 5.4 Hz, 1 H, H2ЈЈ], 2.20 [B of
3
3
an ABX(Y), 2J2Ј–2ЈЈ = 13.2, 3J2Ј–1Ј = 6.0, 3J2Ј–3Ј = 2.1 Hz, 1 H, H2Ј],
4
1.87 (d, J7–6 = 1.2 Hz, 3 H, H7), 1.64–1.29 (m, 4 H, H6Ј and H7Ј),
3
3
Ar), 6.43 (A of an AX2, J1Јa–2Јa = 7.5, J1Јa–2ЈЈa = 6.0 Hz, 1 H,
1.00 (s, 9 H, tBu) ppm. 13C NMR (CDCl3, 75 MHz): δ = 164.1
(C4), 150.5 (C2), 144.8 (CqS, Ts), 137.9 (C6), 135.7 (4 ϫ CH, Ph),
133.3 (2 ϫ Cq, Ph), 133.0 (CqCH3, Ts), 130.2 (2 ϫ CH, Ph), 130.1
(2 ϫ CH, Ts), 128.0 (4 ϫ CH, Ph), 127.9 (2 ϫ CH, Ts), 110.0 (C5),
89.7 (C4Ј), 88.0 (C1Ј), 74.4 (C3Ј), 70.4 (C5Ј), 70.3 (C8Ј), 39.4 (C2Ј),
30.0 (C7Ј), 26.9 (CMe3), 25.3 (C6Ј), 21.7 (CH3, Ts), 19.0 (CMe3),
12.4 (C7) ppm. C36H44N2O8SSi (692.89): calcd. C 62.40, H 6.40, N
4.04; found C 61.97, H 6.51, N 4.14.
H1Јa), 6.37 (A of an AX2, J1Јb–2Јb = 9.5, J1Јb–2ЈЈb = 5.5 Hz, 1 H,
3
3
H1Јb), 5.09 [A of an AX2(Y), J3Јa–P = 7.5, J3Јa–2ЈЈa = 6.0, J3Јa–2Јa
3
3
3
3
=
3J3Јa–4Јa = 3.0 Hz, 1 H, H3Јa], 4.36 (d, J3Јb–2Јb = 5.5 Hz, 1 H,
H1Јb), 3.82 (m, J4Јb–P = 5.0 Hz, 1 H, H4Јb), 3.78 (q, J4Јa–3Јa
=
4
3
3J4Јa–5Јa
=
3J4Јa–5ЈЈa = 3.0 Hz, 1 H, H4Јa), 3.59 (A of an ABX,
2J5Јa–5ЈЈa = 12.0, J5Јa–4Јa = 3.0 Hz, 1 H, H5Јa), 3.54 (B of an ABX,
3
2J5ЈЈa–5Јa = 12.0, J5ЈЈa–4Јa = 3.0 Hz, 1 H, H5ЈЈa), 3.51–3.49 (m, 2 H,
3
H5Јb), 2.44 [A of an ABX(Y), J2ЈЈa–2Јa = 14.0, J2ЈЈa–1Јa = 6.0,
2
3
3J2ЈЈa–3Јa = 3.5 Hz, 1 H, H2ЈЈa], 2.32 (m, 2 H, H2Јa, H2Јb), 2.07 (m,
Ethyl [(5ЈS)-5Ј-C-(3-Bromopropyl)-3Ј-O-(tert-butyldiphenylsilyl-
oxy)thymidine-5Ј-O-yl][(5Ј-O-dimethoxytrityl)thymidine-3ЈO-yl]
Phosphate (8): At room temp., under argon, 6 (200 mg, 0.33 mmol),
thymidine ethyl phosphoramidite (250 mg, 0.35 mmol), and the ac-
tivation reagent (7.6 mL of 5-ethylthiotetrazole in acetonitrile;
0.25 m) were added to a flask. The mixture was stirred for 45 min,
then degassed EtOAc (50 mL) was added. The organic phase was
washed with cooled 10% aqueous Na2CO3 (20 mL) and dried with
MgSO4. The solvent was evaporated under vacuum and the solid
residue was isolated by silica gel chromatography using petroleum
ether/ethyl acetate (7:3 + triethylamine) as eluents. Compound 8
(373 mg, 92%, 1:1 mixture of isomers) was obtained as a white
foam. 1H and 13C NMR spectra were highly complex due to the
2 H, H2ЈЈb, H8Јb), 1.96 (d, J7a–6a = 1.0 Hz, 3 H, H7a), 1.95 (d,
4
4J7b–6b = 1.0 Hz, 3 H, H7b), 1.73–1.57 (m, 3 H, H6Јb, H8Јb), 1.42–
1.39 (m, 2 H, H7Јb) ppm. 13C NMR (CDCl3, 125 MHz): δ = 163.9,
163.8, 150.7 and 150.5 (C2a, C4a, C2b and C4b), 135.8 and 135.7
(C6a and C6b), 133.4 and 132.5 (CqPh), 130.4, 130.3, 128.1 (Ph),
112.0 and 111.5 (C5a and C5b), 88.5 (C4Јb), 85.5 (C4Јa), 85.7 (C1Јa),
85.2 (C1Јb), 81.3 (C5Јb), 75.2 (C3Јa), 75.1 (C3Јb), 61.5 (C5Јa), 39.9
(C2Јb), 39.0 (C2Јa), 27.7 (C6Јb), 26.8 [C(CH3)3], 23.0 (C8Јb), 20.6
(C7Јb), 12.7 and 12.4 (C7a and C7b), 1.0 [C(CH3)3] ppm. 31P NMR
(MeOD, 100 MHz): δ = 25.1 ppm. MS (Maldi-Tof): m/z = 809.2
[M + H+], 831.1 [M + Na+], 847.1 [M + K+].
3Ј-O-(tert-Butyldiphenylsilyloxy)-(SC,RP) (α,β-P-CNA; 12): 1H
NMR (CDCl3, 500 MHz): δ = 7.67–7.61 (m, 4 H, Ar), 7.47–7.36
diastereoisomeric mixture of thymidine dinucleotide analogues. 31
P
NMR (C6D6, 60 MHz): δ = 142.5, 141.6 ppm. MS: m/z = 1243.4
(m, 8 H, H6a, H6b, Ar), 6.47 (A of an AX2, J1Јb–2Јb = J1Јb–2ЈЈb
=
=
=
=
3
3
[M + Na+].
5.5 Hz, 1 H, H1Јb), 6.09 (A of an AX2, J1Јa–2Јa = 7.0, J1Јa–2ЈЈa
3
3
6.5 Hz, 1 H, H1Јa), 5.22 (m, 1 H, H3Јa), 4.42 (br. d, J3Јb–4Јb
3
Ethyl [(5ЈS)-5Ј-C-(Tosyloxypropyl)-3Ј-O-(tert-butyldiphenylsilyl-
oxy)thymidine-5Ј-O-yl][(5Ј-O-dimethoxytrityl)thymidine-3ЈO-yl]
Phosphite (9): Compound 9 (1:1 mixture of diastereoisomers,
250 mg, 66%) was obtained by using an identical procedure to that
described for 8, starting from 7 (200 mg, 0.29 mmol), thymidine
ethylphosphoramidite (250 mg, 0.35 mmol), and activation reagent
(7.6 mL of 5-ethylthiotetrazol in acetonitrile 0.25 m). 1H and 13C
NMR spectra were highly complex due to the diastereoisomeric
mixture of thymidine dinucleotide analogues. 31P NMR (C6D6,
60 MHz): δ = 143.1, 142.2 ppm. MS: m/z = 1349.6 [M + K+].
5.5 Hz, 1 H, H3Јb), 4.11 [A of a AX(Y), J4Јb–3Јb = 5.5, J4Јb–5Јb
3
3
2.5 Hz, 1 H, H4Јb], 3.88 (A of an ABX, J5Јa–5ЈЈa = 12.0 Hz, 1 H,
H5Јa), 3.82 (B of an ABX, 2J5ЈЈa–5Јa = 12.0 Hz, 1 H, H5ЈЈa), 3.76 (m,
2
1 H, H4Јa), 3.71 (m, 1 H, H5Јb), 2.34–1.34 (m, 10 H, H2Јa, H2Јb
,
H6Јb, H7Јb, H8Јb), 1.90 (m, 6 H, H7a, H7b) ppm. 13C NMR (CDCl3,
125 MHz): δ = 164.0, 163.9, 150.4 and 150.3 (C2a, C4a, C2b and
C4b), 135.8 and 135.7 (C6a and C6b), 133.2 and 132.7 (CqPh), 130.2,
128.1, 128.0 (Ph), 111.2 and 111.0 (C5a and C5b), 88.6 (C4Јb), 85.7
(C4Јa), 85.2 (C1Јa), 84.8 (C1Јb), 79.6 (C5Јb), 76.5 (C3Јb), 74.6 (C3Јa),
61.9 (C5Јa), 40.4 (C2Јb), 39.1 (C2Јa), 28.1 (C6Јb), 26.9 [C(CH3)3], 23.9
(C8Јb), 20.5 (C7Јb), 12.8 and 12.6 (C7a and C7b), 1.0 [C(CH3)3] ppm.
31P NMR (MeOD, 100 MHz): δ = 29.8 ppm. MS (MALDI-TOF):
m/z = 831.1 [M + Na+], 847.1 [M + K+].
Cyanoethyl [(5ЈS)-3Ј-O-(tert-Butyldiphenylsilyloxy)-5Ј-C-(tosyloxy-
propyl)thymidine-5Ј-O-yl][(5Ј-O-dimethoxytrityl)thymidine-3ЈO-yl]
Phosphate (10): Compound 10 (1:1 mixture of diastereoisomers,
340 mg, 60%) was obtained by using an identical procedure to that
described for 8, starting from 7 (290 mg, 0.42 mmol), thymidine
cyanoethylphosphoramidite (330 mg, 0.44 mmol), and activation
(SC,SP) α,β-P-CNA (13): To a solution of 10 (22.6 mg, 28.0 μmol)
in distilled THF (1 mL), cooled to 0 °C, a solution of TBAF (1 m
Eur. J. Org. Chem. 2011, 6857–6863
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6861

J.-M. Escudier et al.
FULL PAPER
in THF, 40 μL, 40 μmol) was added. The mixture was stirred for
1 h at room temp., then the THF was evaporated and the crude
product was filtered through silica gel using EtOAc/MeOH (80:20)
as solvent. The solvent was evaporated to yield 13 (15.3 mg, 93%)
as a white foam. 1H NMR (MeOD, 500 MHz): δ = 7.80 (d,
isomerization, 31P NMR spectra obtained during the Arbuzov re-
action.
Acknowledgments
4
4J6b–7b = 1.0 Hz, 1 H, H6b), 7.69 (d, J6a–7a = 1.5 Hz, 1 H, H6a),
3
6.39 (dd, J1Јb–2ЈЈb
=
3J1Јb–2Јb = 7.0 Hz, 1 H, H1Јb), 6.36 (dd,
This work was granted access to the HPC resources of CALMIP
under the allocation 2011-p1102.
3J1Јa–2ЈЈa = 8.0, 3J1Јa–2Јa = 6.0 Hz, 1 H, H1Јa), 5.18 [X of an ABX(Y),
3J3Јa–P = 7.0, 3J3Јa–2Јa = 6.0, 3J3Јa–4Јa = 3J3Јa–2ЈЈa = 2.0 Hz, 1 H, H3Јa],
3
4.59 (m, J5Јb–P Ͻ 0.5 Hz, 1 H, H5Јb), 4.51 [X of an ABX(Y),
3J3Јb–2ЈЈb = 6.0, J3Јb–2Јb = 4.0, J3Јb–4Јb = 3.0 Hz, 1 H, H3Јb], 4.25
3
3
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3
3
(dt, J4Јa–5Јa
=
3J4Јa–5ЈЈa = 3.0, J4Јa–3Јa = 2.0 Hz 1 H, H4Јa), 3.91
(dd, 4J4Јb–P = 4.0, J4Јb–3Јb = 3.0, 3J4Јb–5Јb = 2.5 Hz, 1 H, H4Јb),3.84
3
(A of an ABX, J5Јa–5ЈЈa = 15.5, J5Јa–4Јa = 3.0 Hz, 1 H, H5Јa), 3.81
2
3
(B of an ABX, 2J5ЈЈa–5Јa = 15.5, 3J5ЈЈa–4Јa = 3.0 Hz, 1 H, H5ЈЈa), 2.56
2
3
3
[A of an ABX(Y), J2ЈЈa–2Јa = 14.0, J2ЈЈa–1Јa = 8.0, J2ЈЈa–3Јa
=
2.0 Hz, 1 H, H2ЈЈa], 2.46 [B of an ABX(Y), J2Јa–2ЈЈa = 14.0,
3J2Јa–1Јa = 6.0, 3J2Јa–3Јa = 6.0 Hz, 1 H, H2Јa], 2.29 [A of an ABX(Y),
2J2ЈЈb–2Јb = 19.5, 3J2ЈЈb–1Јb = 7.0, 3J2ЈЈb–3Јb = 6.0 Hz, 1 H, H2ЈЈb], 2.26
[B of an ABX(Y), 2J2Јb–2ЈЈb = 19.5, 3J2Јb–1Јb = 7.0, 3J2Јb–3Јb = 4.0 Hz,
1 H, H2Јb], 2.24 (m, 1 H, H7Јb), 2.19 (m, 1 H, H8Јb), 1.96 (m, 1 H,
H7Јb), 1.93 (d, 4J7a–6a = 1.5 Hz, 3 H, H7a), 1.91 (d, 4J7b–6b = 1.0 Hz,
3 H, H7b), 1.87 (m, 2 H, H6Јb), 1.80 (m, 1 H, H8Јb) ppm. 13C NMR
(MeOD, 125 MHz): δ = 165.0, 164.9, 151.0 and 150.9 (C2a, C4a,
C2b and C4b), 136.5 and 136.2 (C6a and C6b), 110.8 and 110.5 (C5a
and C5b), 87.6 (C4Јb), 85.7 (C4Јa), 84.7 (C1Јa), 84.6 (C1Јb), 81.7 (C5Јb),
76.0 (C3Јa), 71.3 (C3Јb), 61.0 (C5Јa), 39.3 (C2Јb), 38.4 (C2Јa), 27.6
2
(C6Јb), 22.0 (C8Јb), 20.5 (C7Јb), 11.2 and 11.1 (C7a and C7b) ppm. 31
P
NMR (MeOD, 100 MHz): δ = 26.9 ppm. C23H31N4O11P (570.49):
calcd. C 48.42, H 5.48, N 9.82; found C 46.87, H 5.70, N 9.49 (+
H2O).
(SC,RP) α,β-P-CNA (14): Compound 14 (13 mg, 90 %) was ob-
tained by using an identical procedure as described for 13, starting
from 12 (20 mg, 24.7 μmol), and TBAF solution (40 μL, 40 μmol).
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4
1H NMR (MeOD, 500 MHz): δ = 7.82 (d, J6b–7b = 1.0 Hz, 1 H,
4
H6b), 7.68 (d, J6a–7a = 1.0 Hz, 1 H, H6a), 6.37 (dd, 3J1Јb–2ЈЈb = 8.0,
3
3
3J1Јb–2Јb = 6.0 Hz, 1 H, H1Јb), 6.29 (dd, J1Јa–2ЈЈa = 8.5, J1Јa–2Јa
=
5.5 Hz, 1 H, H1Јa), 5.27 [X of an ABX(Y), J3Јa–2ЈЈa = 5.5, J3Јa–P
3
3
3
= 4.5, J3Јa–4Јa
AX2(Y), J5Јb–6Јb,ax = 11.0, J5Јb–6Јb,ec
=
3J3Јa–2Јa = 1.5 Hz, 1 H, H3Јa], 4.67 [A of an
3
3
3
=
3J5Јb–4Јb = 2.5, J5Јb–P
Ͻ
Páv, I. Barvík, M. Budesˇínský, M. Masojídková, I. Rosenberg,
˘
Org. Lett. 2007, 9, 5469–5472.
2.0 Hz, 1 H, H5Јb], 4.46 [X of an ABX(Y), J3Јb–2ЈЈb = 6.0, J3Јb–4Јb
3
3
3
3
3
= J3Јb–2Јb = 2.5 Hz, 1 H, H3Јb], 4.22 (td, J4Јa–5Јa = J4Јa–5ЈЈa = 3.0,
[9] a) M. Bosco, P. Bisseret, J. Eustache, Tetrahedron Lett. 2003,
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4
3
3J4Јa–3Јa = 1.5 Hz, 1 H, H4Јa), 3.97 (td, J4Јb–P = 4.0, J4Јb–3Јb
=
3J4Јb–5Јb = 2.5 Hz, 1 H, H4Јb), 3.83 (A of an ABX, J5Јa–5ЈЈa = 13.5,
2
3J5Јa–4Јa = 3.0 Hz, 1 H, H5Јa), 3.80 (B of an ABX, J5ЈЈa–5Јa = 13.5,
2
3J5ЈЈa–4Јa = 3.0 Hz, 1 H, H5ЈЈa), 2.46 [A of an ABX(Y), J2Јa–2ЈЈa
=
2
14.5, J2Јa–1Јa = 5.5, J2Јa–3Јa = 1.5 Hz, 1 H, H2Јa], 2.39 [B of an
3
3
2
3
3
ABX(Y), J2ЈЈa–2Јa = 14.5, J2ЈЈa–1Јa = 8.5, J2ЈЈa–3Јa = 5.5 Hz, 1 H,
H2ЈЈa], 2.29 (m, 1 H, H7Јb), 2.27 [A of an ABX(Y), 2J2Јb–2ЈЈb = 14.0,
3J2Јb–1Јb = 6.0, 3J2Јb–3Јb = 2.5 Hz, 1 H, H2Јb], 2.18 [B of an ABX(Y),
2J2ЈЈb–2Јb = 14.0, 3J2ЈЈb–1Јb = 8.0, 3J2ЈЈb–3Јb = 6.0 Hz, 1 H, H2ЈЈb], 2.12
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(m, 1 H, H8Јb), 2.07 (m, 1 H, H7Јb), 1.95 (d, J7a–6a = 1.0 Hz, 3 H,
4
H7a), 1.93 (m, 2 H, H6Јb), 1.89 (m, 1 H, H8Јb), 1.88 (d, J7b–6b
=
4
1.0 Hz, 3 H, H7b) ppm. 13C NMR (CDCl3, 125 MHz): δ = 165.3,
165.2, 151.2 and 150.9 (C2a, C4a, C2b and C4b), 136.2 and 136.0
(C6a and C6b), 110.8 and 110.5 (C5a abd C5b), 87.8 (C4Јb), 86.0
(C4Јa), 85.1 (C1Јb), 84.4 (C1Јa), 80.8 (C5Јb), 78.3 (C3Јa), 71.7 (C3Јb),
61.3 (C5Јa), 39.7 (C2Јb), 39.1 (C2Јa), 27.7 (C6Јb), 22.8 (C8Јb), 20.1
(C7Јb), 11.6 and 11.1 (C7a and C7b) ppm. 31P NMR (MeOD,
100 MHz): δ = 31.3 ppm. C23H31N4O11P (570.49): calcd. C 48.42,
H 5.48, N 9.82; found C 47.01, H 5.62, N 9.49 (+H2O).
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cle): Calculation procedure, putative mechanism for phosphite
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© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Received: July 20, 2011
Published Online: October 12, 2011
Eur. J. Org. Chem. 2011, 6857–6863
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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