A strategy for the stereoselective preparation of thymidine phosphorothioates with the (R) or the (S) configuration at the stereogenic phosphorus atom and their application to the synthesis of oligodeoxyribonucleotides with stereochemically pure phosphate

Article abstract of DOI:10.1002/ejoc.200600155

This paper describes a new method for the highly stereoselective preparation of dithymidine phosphorothioates (TpsT) with the (R) or the (S) configuration at the stereogenic phosphorus atom [(Rp)- or (Sp)-TpsT, respectively], together with the synthesis o

Full text of DOI:10.1002/ejoc.200600155

FULL PAPER
DOI: 10.1002/ejoc.200600155
A Strategy for the Stereoselective Preparation of Thymidine Phosphorothioates
with the (R) or the (S) Configuration at the Stereogenic Phosphorus Atom and
Their Application to the Synthesis of Oligodeoxyribonucleotides with
Stereochemically Pure Phosphate/Phosphorothioate Chimeric Backbones
Yoshihiro Hayakawa,*[a,b] Yoshiko Hirabayashi,[a] Mamoru Hyodo,[a] Satoko Yamashita,[a]
Tomoyuki Matsunami,[a] Dong-Mei Cui,[a] Rie Kawai,[a] and Hidehiko Kodama[b]
Keywords: Antisense agents / Synthetic methods / Diastereoselectivity / Oligonucleotides / Solid-phase synthesis
This paper describes a new method for the highly stereose- product, 4) stereospecific removal of the methyl group from
lective preparation of dithymidine phosphorothioates (TpsT)
with the (R) or the (S) configuration at the stereogenic phos-
phorus atom [(Rp)- or (Sp)-TpsT, respectively], together with
the synthesis of oligodeoxyribonucleotides with stereochemi-
cally pure phosphate/phosphorothioate mixed backbones
through the use of (Rp)- or (Sp)-TpsT as building blocks.
Stereochemically pure (Rp)- or (Sp)-TpsT was produced
the phosphorothioate moiety to give an allyl (Rp)-5Ј-O-di-
methoxytritylthymidine 3Ј-phosphorothioate diester, or stere-
ospecific removal of the allyl group from the phosphate moi-
ety to form a methyl (Sp)-5Ј-O-dimethoxytritylthymidine 3Ј-
phosphorothioate diester, and 5) stereospecific condensation
of these (Rp)- and (Sp)-diesters with a 5Ј-O-free thymidine in
a Mitsunobu reaction to produce the allyl (3Ј–5Ј)-linked (Sp)-
dithymidine phosphorothioate and the methyl (3Ј–5Ј)-linked
(Rp)-dithymidine phosphorothioate, respectively. The re-
sulting (Rp)- and (Sp)-dithymidine phosphorothioates were
converted into their 3Ј-phosphoramidites. Oligodeoxyribonu-
cleotides with stereochemically pure phosphate/phos-
phorothioate-mixed backbones were then synthesized by use
of these phosphoramidites as building blocks.
through
a
five-step process: 1) 1H-tetrazole-promoted
thermodynamic equilibration of a diastereomeric mixture of
allyl (Rp)- and (Sp)-thymidine 3Ј,5Ј-cyclic phosphate to give
the (Sp) isomer as the major component, 2) stereospecific sul-
furization of the allyl (Sp)-thymidine 3Ј,5Ј-cyclic phosphate to
afford an allyl (Rp)-thymidine 3Ј,5Ј-cyclic phosphorothioate,
3) regioselective and stereoselective methanolysis of the allyl
(Rp)-thymidine 3Ј,5Ј-cyclic phosphorothioate to provide an
allyl methyl (Rp)-thymidine 3Ј-phosphorothioate as the main
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2006)
as a drug. These phenomena included interaction with –
and, potentially, the abrogation of – the activity of heparin-
binding growth factors,[4] induction of immune stimulatory
effects in rodents,[5] complement activation and hypotension
in monkeys,[6] and induction of clotting abnormalities in
monkeys as a result of direct interactions with throm-
bin.[6a,6b]
In contrast, analogues with mixed phosphate/phos-
phorothioate backbones (PO/PS-chimeric oligonucleotides)
do not have these drawbacks even at high concentrations.[7]
Furthermore, PO/PS-chimeric oligonucleotides are ex-
pected to have greater binding affinity to the complemen-
tary nucleotides than the all-PS analogues and to show ac-
tivity similar to that of the all-PS derivatives.[8] Such PO/
PS-chimeric oligonucleotides are therefore attractive com-
pounds, and the development of efficient means to synthe-
size them is an important research subject.
Introduction
Oligodeoxyribonucleotides in which the backbones are
fully modified into phosphorothioate moieties (all-PS oligo-
nucleotides) are important molecules in antisense therapy.[1]
Some of them are in fact used as curatives for certain viral
diseases, but all-PS oligonucleotides have some drawbacks
in therapeutic use: they appear, for example, to bind non-
sequence-specifically to cellular proteins[2] and at high con-
centrations they furthermore competitively inhibit a variety
of nucleases and polymerases.[3] The high-concentration use
of an all-PS oligonucleotide also produced undesired phe-
nomena in laboratory work and in animal experiments per-
formed to assess the efficacy of the artificial oligonucleotide
[a] Graduate School of Information Science/Human Informatics,
Nagoya University,
Chikusa, Nagoya 464-8601, Japan
The phosphorothioate internucleotide linkage generates
asymmetry at the phosphorus atom, and the chirality of
this atom may affect the biological properties of PO/PS-
chimeric oligonucleotides, including transport through cell
membranes and/or interaction with intracellular biopoly-
Fax: +81-52-789-5646
E-mail: yoshi@is.nagoya-u.ac.jp
[b] CREST of JST, Nagoya University,
Chikusa, Nagoya 464-8601, Japan
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
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Thymidine Phosphorothioates with (R) or (S) Configurations at Phosphorus
FULL PAPER
mers such as proteins or nucleic acids, so it is important to cated that the product ratio of the nucleoside 3Ј-phos-
develop means to synthesize derivatives of these oligonucle- phorothioate to the nucleoside 5Ј-phosphorothioate was
otides with stereochemically pure phosphorothioate link- 90:10 and the isolated yield of 2 was 83%. The structure
1
ages. PO/PS-chimeric oligonucleotides have been prepared of this product was supported by the H NMR spectrum,
by several methods, most of which have allowed the synthe- showing a signal due to C(3Ј)-H of the sugar part at δ =
sis essentially to give mixtures of diastereomers in which the 4.16 ppm, and by the 31P NMR spectrum, showing a signal
stereogenic phosphorus atoms of phosphorothioate func- at δ = 66.2 ppm.
tions have (R) and (S) configurations [(Rp)- and (Sp)-phos-
phorothioates, respectively].[9] There has been only one ex-
ample of a stereocontrolled synthesis of PO/PS-chimeric
oligonucleotides: a study by Stec et al. employed the
oxathiaphosphorane method.[10] Here we present a novel,
facile method to synthesize stereochemically pure PO/PS-
chimeric oligodeoxyribonucleotides.
Compound 2 was subsequently converted into the (Rp)-
5Ј-O-p,pЈ-dimethoxytrityl (DMTr) derivative 3 in a 91%
yield by treatment with p,pЈ-dimethoxytrityl chloride
(1.4 equiv.) in a 1:5 (v/v) mixture of pyridine and DMF
(25 °C, 5 h). The phosphorothioate triester 3 was then
transformed into the allyl (Rp)-phosphorothioate diester 4
and the methyl (Sp)-phosphorothioate diester 5 by chemo-
selective and stereospecific removal of methyl and allyl
groups, respectively. Thus, treatment of 3 with tert-bu-
tylamine[17] at 35 °C (24 h) removed the methyl group selec-
tively in a stereoretentive manner to give the allyl (Rp)-
phosphorothioate diester 4 as the sole product. The struc-
ture of this product was confirmed by the spectroscopic
data: no signal due to a methyl group attached to the phos-
phate function was observed in the 1H NMR spectrum,
whilst the ESI mass spectrum indicated a molecular peak
at m/z 679.2713 [calcd. for C34H36N2O9PS [M–H]–
679.1885] and also supported the demethylated structure.
Furthermore, the stereochemical genuineness of the prod-
uct was confirmed by 31P NMR analysis, which indicated
one singlet at δ = 52.8 ppm. The yield of 4 was 92%.
Results and Discussion
The (Rp)- and (Sp)-thymidine-3Ј-phosphorothioic acid
diesters 4 and 5 were first prepared stereoselectively by the
route shown in Scheme 1. Thymidine (1.0 equiv.) was
treated with (allyloxy)[bis(diisopropylamino)]phosphane
(1.2 equiv.) with the assistance of 1H-tetrazole (3.0 equiv.)
in the presence of molecular sieves (4-Å mol. sieves)[11] in
acetonitrile at 25 °C for 4 h. The reaction mixture[12] was
heated at 55 °C for 36 h[13] and the resulting product was
treated with bis[3-(triethoxysilyl)propyl] tetrasulfide[14]
(TEST, 1.2 equiv., 25 °C, 2 h) to give the allyl (Rp)-thymid-
ine 3Ј,5Ј-cyclic phosphorothioate 1, contaminated with a
The stereospecific and chemoselective deallylation of 3
small amount of the (Sp) isomer.[15] The combined isolated (1.0 equiv.), on the other hand, was carried out by treat-
yield of the two isomers was 79% and the ratio of (Rp) and ment with a mixture of chloroform–tris(dibenzylidene-
(Sp) isomers in this product, estimated by 31P NMR, was acetone)dipalladium(0)
complex
[Pd2(dba)3·CHCl3]
98:2.
(0.05 equiv.) and triphenylphosphane (0.25 equiv.) in the
Treatment of 1 (1.0 equiv.) with sodium methoxide presence of butylammonium formate (20 equiv.) in THF
(3.0 equiv.) in methanol (25 °C, 24 h) opened the cyclic (25 °C, 2 h)[18] to give 5, which showed a single signal at
phosphorothioate ring in a highly stereoselective and re- δ = 54.3 ppm in its 31P NMR spectrum. This observation
gioselective manner to give the allyl methyl (Rp)-thymidine confirmed that this product was stereochemically pure, and
1
3Ј-phosphorothioate 2 as a major product.[16] In this meth- the H NMR spectrum also indicated the stereochemical
anolysis, small amounts of the corresponding (Sp) isomer singleness of the product. The molecular peak at
(Ͻ3%) and allyl methyl thymidine 5Ј-phosphorothioate m/z 653.2584 in the ESI mass spectrum was consistent with
were obtained as by-products. The 31P NMR analysis indi- 5 [calcd. for C32H34N2O9PS [M–H]– 653.1728].
Scheme 1. Reagents and conditions: a) 1) 1H-tetrazole, CH2CH=CH2OP[N(i-C3H7)2]2, CH3CN, mol. sieves (4 Å), 25 °C, 2) 55 °C, 36 h,
3) TEST, 25 °C, 1 h. b) CH3ONa, CH3OH, 25 °C, 24 h. c) DMTrCl, DMF/pyridine 5:1 (v/v), 25 °C, 7 h. d) 1) t-C4H9NH2, 35 °C, 24 h,
2) (C2H5)3NH+HCO3 /H2O. e) 1) Pd2(dba)3·CHCl3, (C6H5)3P, n-C4H9NH2, HCOOH, THF, 25 °C, 2 h, 2) (C2H5)3NH+HCO3 /H2O.
–
–
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Y. Hayakawa et al.
FULL PAPER
Scheme 2. Reagents and conditions: a) DEAD, (C6H5)3P, THF, 55 °C, 3 h. b) CH3ONa, CH3OH, 25 °C, 4 h. c) 1) Pd2(dba)3·CHCl3,
(C6H5)3P, n-C4H9NH2, HCOOH, THF, 25 °C, 3 h, 2) CH3COOH, 60 °C, 4 h.
Scheme 3. Reagents and conditions: a) DEAD, (C6H5)3P, THF, 55 °C, 3 h. b) CH3ONa, CH3OH, 25 °C, 4 h. c) 1) t-C4H9NH2, 35 °C,
12 h, 2) CH3COOH, 60 °C, 4 h.
The nucleoside phosphorothioate diesters 4 and 5 were mate (25 °C, 3 h) and subsequent removal of the 5Ј-O-
converted into the corresponding (Sp)- and (Rp)-dithymid- DMTr protector by treatment with acetic acid (60 °C, 4 h).
ine phosphorothioates 7 and 10, respectively, in a stereo- Compound 11, on the other hand, was converted into 12
specific manner through Mitsunobu reactions[19] with the by successive treatment with tert-butylamine (35 °C, 12 h)
5Ј-O-free thymidine 6 (Scheme 2 and Scheme 3). Treatment to remove the methyl protector on the internucleotide link-
of 4 or 5 (1.0 equiv.) with 6 (2.0 equiv.) in the presence of age and acetic acid (60 °C, 4 h) to eliminate the 5Ј-O-DMTr
diethyl azodicarboxylate (DEAD) and triphenylphosphane group. The resulting phosphorothioate diesters 9 and 12
(1.3 equiv.) in THF at 55 °C for 3 h gave stereochemically were subjected to enzyme reactions with nuclease P1 in the
pure 7 or 10, respectively.[20] Each reaction also afforded a presence of a Zn2+ salt in a Tris·HCl buffer solution[8b,21]
considerable amount of the dithymidine thiaphosphate 13 and with snake venom phosphodiesterase in the presence of
as a by-product.
a Mg2+ salt in a Tris·HCl buffer solution.[8b,22] The reaction
with nuclease P1 digested 9 but did not hydrolyze 12 at all,
whilst in contrast the reaction with snake venom phospho-
diesterase degraded 12 but left 9 intact. These results indi-
cated that 9 and 12 are phosphorothioates with (S) and (R)
configurations, respectively.
Subsequently, we carried out solid-phase syntheses of
four kinds of PO/PS-chimeric oligodeoxyribonucleotides –
(Rp,Rp)-T[TpsT]T7[TpsT]T9 (20), (Rp)-G[TpsT]CAT (21),
(Sp,Sp)-T[TpsT]T7[TpsT]T9 (22), and (Sp)-G[TpsT]CAT
(23) – by the phosphoramidite method, using the dithymid-
ine phosphorothioates 8 and 11 as building blocks for the
The stereochemistry of 7 and 10 was confirmed as fol- construction of the phosphorothioate components. First,
lows. The 3Ј-O-acetyl protectors of 7 and 10 were first re- the phosphoramidites 14 and 15, which are requisite build-
moved with sodium methoxide in methanol (25 °C, 4 h) to ing blocks for the synthesis, were prepared from 8 and 11,
give 8 and 11, respectively. Subsequently, 8 was converted respectively. Compound 8 was converted into 14 in an 85%
into 9 by stereospecific, stereoretentive removal of the allyl yield by treatment with (allyloxy)[bis(diisopropylamino)]-
group on the phosphorothioate moiety in a reaction cata- phosphane in the presence of diisopropylammonium tetra-
lyzed by a chloroform–tris(dibenzylideneacetone)dipalla- zolide in acetonitrile containing mol. sieves (3 Å, 25 °C,
dium(0) complex in the presence of butylammonium for- 3 h), whilst 15 was prepared in a similar way from 11 in a
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Thymidine Phosphorothioates with (R) or (S) Configurations at Phosphorus
FULL PAPER
78% yield. The solid-phase synthesis was carried out on an 2 h) and to remove the methyl protectors (55 °C, 6 h) in the
Applied Biosystems Model 392 DNA/RNA synthesizer synthesis of 20 and 21. The yields of products estimated by
with the nucleoside allyl phosphoramidites 16–19 and the conventional trityl assay were 85% for 20, 87% for 21, 82%
dinucleoside phosphoramidites 14 or 15 as building units for 22, and 86% for 23.
by the standard procedure with a slight modification, in
The product structures were confirmed by the enzymatic
which the phosphoramidite coupling (0.2 min) and subse- treatment. The cases of 20 and 22, with (Rp) and (Sp) con-
quent waiting (5.0 min) were carried out once in the cases figurations at all phosphorothioate functions, respectively,
of 16, 17, or 19 as the phosphoramidite whilst this treat- are representatively described. Treatment of 20 with nucle-
ment was repeated twice in the cases of 14, 15, or 18 as the ase P1 in Tris·HCl (37 °C, 24 h) did not cleave two of the
phosphoramidite (Table 1). Here, the synthesis of oligonu- phosphorothioate parts and gave pTpsT (see part A in Fig-
cleotides with only (S) configurations at the phosphorothio- ure 1). In contrast, in the digestion of 20 with snake venom
ate components was performed with 14 and 16–19 as build- phosphodiesterase in Tris·HCl (37 °C, 24 h), the phos-
ing units, whilst the synthesis of derivatives incorporating phorothioate parts were hydrolyzed and no formation of
phosphorothioate moieties with only (R) configurations, on pTpsT was observed (Figure 1, B). These results suggested
the other hand, employed 15 and 16–19 as phosphoramid- that two phosphorothioate functions in 20 have (R) config-
ites. After chain elongation was complete, products were urations. In the case of 22, on the other hand, its two phos-
deprotected and detached in all cases by successive treat- phorothioate bonds were completely hydrolyzed by nucle-
ment with the chloroform–tris(dibenzylideneacetone)dipal- ase P1 in Tris·HCl (37 °C, 24 h) (Figure 1, C), but were not
ladium(0) complex in the presence of butylammonium for- decomposed by snake venom phosphodiesterase in
mate (50 °C, 14 h) to remove the allyl protectors, followed Tris·HCl (37 °C, 24 h), thus giving pTpsT (Figure 1, D).
by treatment with concentrated aqueous ammonia to de- These results confirmed that the absolute configurations of
tach the target compound from the solid support (25 °C, the two phosphorothioate moieties in 22 are both (S). In a
similar manner, the stereochemistry of the phosphorothio-
ate in 21 and 23 was confirmed.
Table 1. Reaction sequence of the solid-phase synthesis.
Conclusions
We have demonstrated a new methodology for the stereo-
regulated synthesis of PO/PS-chimeric oligodeoxyribonucle-
otides, the keystone of which is a novel method for prepar-
ing the stereochemically pure (Rp)- and (Sp)-dithymidine
phosphorothioates 7 and 10, achieved by a combination of
stereocontrolled reactions. These involve: 1) 1H-tetrazole-
promoted condensation of thymidine and (allyloxy)[bis(di-
isopropylamino)]phosphane followed by thermal equilibra-
tion of the resulting product, affording an (Sp)-thymidine
3Ј,5Ј-cyclic phosphate[23] as the major component, 2)
stereospecific sulfurization of this major product, giving an
allyl (Rp)-thymidine 3Ј,5Ј-cyclic phosphorothioate, 3) re-
[a] In the cases of 16, 17, or 19 as the amidite component this
gioselective and stereoselective methanolysis of the allyl
treatment is conducted once (n = 1). In the cases of 14, 15, or 18
(Rp)-thymidine 3Ј,5Ј-cyclic phosphorothioate, producing an
allyl methyl (Rp)-thymidine 3Ј-phosphorothioate as the
as the amidite component this treatment is conducted twice (n =
2).
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Y. Hayakawa et al.
FULL PAPER
Figure 1. Reversed-phase HPLC profiles: A) treatment of 20 with nuclease P1, B) treatment of 20 with svPDE, C) treatment of 22 with
nuclease P1, D) treatment of 22 with svPDE. Conditions: COSMOSIL 5C18-AR-II column (4.6×250 mm); buffer A, 0.1  triethylammo-
nium acetate in H2O; buffer B, CH3CN/H2O, 4:1; gradient, linear 0–20% B in A for 60 min; detection; 254 nm; flow rate, 1.0 mL·min–1;
temperature, 40 °C.
major product, 4) after dimethoxytritylation of the free 5Ј- (R) and (S) configurations at the stereogenic phosphorus
hydroxy group in the allyl methyl (Rp)-thymidine 3Ј-phos- atom. In contrast, all the existing methods for the stereodef-
phorothioate, stereospecific removal of the methyl group ined synthesis of these two dithymidine phosphorothioates
from the phosphorothioate triester moiety by treatment require the preparation, as precursors, of two kinds of suit-
with tert-butylamine, giving an allyl (Rp)-5Ј-O-dimeth- able thymidine 3Ј-phosphorus compound, one with the
oxytritylthymidine 3Ј-phosphorothioate diester, or stere- (Rp) configuration and the other with the (Sp) configura-
ospecific removal of the allyl group from the phosphate tion, in pure forms. Moreover, the acquisition of these
moiety in an organopalladium-catalyzed reaction, produc- monomer units is troublesome: the methods of Stec, Beauc-
ing a methyl (Sp)-5Ј-O-dimethoxytritylthymidine 3Ј-phos- age, and Stawinski, for example, require the separation of
phorothioate, and 5) stereospecific condensation of the allyl nearly 1:1 mixtures of (Rp)- and (Sp)-thymidine 3Ј-phos-
(Rp)-thymidine 3Ј-phosphorothioate or the methyl (Sp)- phorus compounds such as thymidine 3Ј-O-(2-thio-spiro-
thymidine 3Ј-phosphorothioate with a 5Ј-O-free thymidine 4,4-pentamethylene-1,3,2-oxathiaphosphorane) (Stec),
a
derivative in Mitsunobu reactions, which results in retention thymidine 3Ј-(cyclic phosphoramidite) based on N-fluo-
of the stereochemistry of the phosphorothioate function to roacetyl-2-aminoethanol (Beaucage), and a thymidine 3Ј-[1-
provide an allyl (3Ј–5Ј)-linked (Sp)-dithymidine phos- oxido-2-picolyl-(9-fluorenemethyl) phosphorothioate] (Sta-
phorothioate or a methyl (3Ј–5Ј)-linked (Rp)-dithymidine winski). Meanwhile, the methods of Agrawal, Just, and
phosphorothioate, respectively. This preparation of 7 and Wada require the preparation of enantiomerically or dia-
10 has remarkable advantages not found in existing meth- stereomerically pure chiral amino alcohols: namely (R)- and
ods for the stereodefined preparation of related dinucleo- (S)-2-(hydroxymethyl)pyrrolidines (Agrawal), 1,2-di-O-cy-
side phosphorothioates, including those developed by clopentylidene-5-isopropylamino--xylofuranose and its 
Stec,[8b,10,24] Beaucage,[25] Agrawal,[26] Just,[27] Wada,[28] and isomer (Just), (R)- and (S)-2-(indolyl)ethanol derivatives
Stawinski.[29] The most remarkable advantage of this method (Just), or (R)- and (S)-2-methylamino-1-phenylethanols
is that both the (Rp)- and the (Sp)-dithymidine phos- (Wada), all of these serving as components of their nucleo-
phorothioates can be synthesized from a common intermedi- side 3Ј-phosphoramidite units. In these methods, the stereo-
ate, the corresponding (Rp)-thymidine 3Ј,5Ј-cyclic phosphate. chemically pure chiral amino alcohols have to be synthe-
In other words, this method requires only one synthetic pre- sized individually by two pathways starting from two kinds
cursor for either kind of dithymidine phosphorothioate with of suitable optically pure substances. In contrast, our
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Thymidine Phosphorothioates with (R) or (S) Configurations at Phosphorus
FULL PAPER
Figure 2. Three typical modes of synthetic strategies.
conditions were carried out under argon in flasks dried by heating
at 400 °C under 133–400 Pa, or by washing with a 5% solution of
dichlorodimethylsilane in dichloromethane followed by anhydrous
dichloromethane, and then heating at 100 °C.
method does not require any chiral compounds other than
a nucleoside for the preparation of the key building unit 1
with the pure (Rp) configuration, which is another remark-
able advantage of our method. To illustrate these matters,
three types of strategies for the stereodefined synthesis of
(Rp)- and (Sp)-dinucleoside phosphorothioates, as outlined
in Figure 2, have so far been developed. The Stec and Be-
aucage syntheses were achieved by the first type of strategy,
whilst the syntheses carried out by Agrawal, Just, and Wada
used the second type. Meanwhile, the current synthesis is
the only use of the third type to have been reported. Among
these three strategies, the third is undoubtedly superior in
operational simplicity, which is essential in organic synthe-
sis. Accordingly, this approach may serve as a highly useful
tool for the synthesis of stereodefined PO/PS-chimeric oli-
gonucleotides.
Materials and Solvents: Compounds 6[30] and 16–19,[31]
CH2=CHCH2OP[N(i-C3H7)2]2,[18b]
Pd2[(C6H5CH=CH)2CO]3·
CHCl3,[18b] and a 1.0  tert-butyl hydroperoxide/toluene solution[32]
were prepared by the reported methods. Bis[3Ј-(triethoxysilyl)pro-
pyl] tetrasulfide (TEST)[14] was purchased from Shin-etsu (Japan).
1H-Tetrazole was used after sublimation of material purchased
from Dozindo (Japan). Triphenylphosphane was recrystallized
from hexane before use. THF was used after drying by reflux over
sodium/benzophenone ketyl. Acetone, acetonitrile, and dichloro-
methane were distilled from calcium hydride. Deoxyribonucleosides
(Yamasa), sodium methoxide (Nakalai Tesque), p,pЈ-dimethoxytri-
tyl chloride (Tokyo Kasei), dichloroacetic acid (Tokyo Kasei), tri-
ethylamine (Kishida), tert-butylamine (Nakalai Tesque), a 40% di-
ethyl azodicarboxylate/toluene solution (Tokyo Kasei), snake
venom phosphodiesterase (Funakoshi), Sma I (Takara Bio), nucle-
ase P1 (Yamasa), and a 1  Tris·HCl buffer solution (Nakalai
Tesque) were used as commercially supplied without any purifica-
tion. Solid and amorphous organic substances were used after dry-
ing at 50–60 °C for 8–12 h under 133–400 Pa. Powdery mol. sieves
(3 Å) were employed after the commercially supplied (Nakalai
Tesque) product had been dried at 200 °C for 12 h under 133–
400 Pa.
Experimental Section
General Methods: UV spectra were measured on a JASCO V-500
spectrometer. NMR spectra were taken in CDCl3, unless otherwise
1
noted, on a JEOL JNM-α400 or a ECA-500 instrument. The H,
13C, and 31P NMR chemical shifts are given as δ values in ppm
relative to (CH3)4Si for 1H and 13C NMR spectra, 85% H3PO4 was
used as reference for 31P NMR spectra. ESI-TOF high-resolution
mass (HRMS) spectra were obtained with an Applied Biosystems
Mariner or a QStar spectrometer. HPLC analysis was carried out
with a COSMOSIL 5C18-MS column (Nacalai Tesque, ODS-
5 mm, 4.6×250 mm) on a Waters 2695 Separations Module chro-
matograph with a Waters 2996 Photodiode Array detector. Column
chromatography was performed on Nacalai Tesque silica gel 60
(neutral, 75 µm). Unless otherwise noted, synthetic reactions were
carried out at ambient temperature. Reactions requiring anhydrous
Synthesis of Allyl (Rp)-Thymidine 3Ј,5Ј-Cyclic Phosphorothioate (1):
A mixture of thymidine (4.84 g, 20.0 mmol) and 1H-tetrazole
(4.20 g, 60.0 mmol) in acetonitrile (250 mL) containing powdered
mol. sieves (4 Å) was stirred at 25 °C for 30 min. To this mixture
was slowly added, at 0 °C, a solution of CH2=CHCH2OP[N(i-
C3H7)2]2 (7.67 mL, 7.29 g, 24.0 mmol) in dichloromethane
(25.0 mL). The resulting mixture was stirred at 0 °C for 30 min, at
25 °C for 4 h, and at 55 °C for 24 h. After the mixture had cooled
to 25 °C, TEST (11.8 mL, 13.0 g, 24.0 mmol) was added to it, and
Eur. J. Org. Chem. 2006, 3834–3844
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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3839

Y. Hayakawa et al.
the resulting mixture was stirred at the same temperature for 2 h. 2.64 (m, 1 H), 3.45–3.46 (m, 2 H), 3.69 (d, J = 13.7 Hz, 3 H), 3.80
FULL PAPER
The reaction mixture was passed through a Celite 545 pad to re-
move the mol. sieves and the filtrate was concentrated. The residue
was dissolved in ethyl acetate (200 mL), and the resulting organic
solution was washed with an aqueous sodium hydrogen carbonate
solution (200 mL). The aqueous layer was extracted with ethyl ace-
tate (200 mL, 2×), and the combined organic solutions were
washed with brine (300 mL), dried with Na2SO4 (ca. 150 g), and
concentrated to give a viscous oil. This crude material was sub-
jected to silica gel column chromatography with a 1:2 mixture of
ethyl acetate and hexane as eluent to afford the desired product 1
(5.68 g, 79%) in a pure form: TLC: Rf = 0.41 (ethyl acetate/hexane,
(s, 6 H), 4.27 (d, J = 1.9 Hz, 1 H), 4.55 (dd, J = 5.6 and 10.2 Hz,
2 H), 5.27–5.40 (m, 3 H), 5.88–5.98 (m, 1 H), 6.44 (dd, J = 5.4 and
8.5 Hz, 1 H), 6.84–6.86 (m, 4 H), 7.23–7.41 (m, 9 H), 7.56 (s, 1 H),
8.25 (br. s, 1 H) ppm. 13C NMR: δ = 11.7, 39.2 (d, J = 5.7 Hz),
54.6 (d, J = 5.8 Hz), 55.3, 63.3, 69.0 (d, J = 5.0 Hz), 78.8 (d, J =
4.2 Hz), 150.1, 158.8, 163.3 ppm. 31P NMR: δ = 68.5 ppm. HRMS
(ESI+): m/z calcd. for C35H39N2O9PSNa [M+Na]+: 717.2006;
found 717.2423.
Synthesis of Allyl (Rp)-5Ј-O-(p,pЈ-Dimethoxytrityl)thymidin-3Ј-yl
Phosphorothioate Triethylammonium Salt (4): A solution of the
phosphorothioate triester 3 (1.00 g, 1.44 mmol) in tert-butylamine
(25.0 mL) was stirred at 35 °C for 24 h. Evaporation of the reaction
mixture gave a yellow oil, which was dissolved in dichloromethane
(20 mL). The solution was washed with an aqueous triethylammo-
nium hydrogen carbonate solution (0.1 , 10 mL, 2×), the organic
layer was dried with magnesium sulfate (ca. 20 g) and concentrated,
and the resulting residue was subjected to silica gel column
chromatography with a 1:9 methanol/dichloromethane mixture.
Fractions including the desired compound were combined and con-
centrated to give a residual material, which was again dissolved in
dichloromethane (5.0 mL). The solution was shaken with an aque-
1
1:2). H NMR: δ = 1.96 (s, 3 H), 2.53–2.65 (m, 2 H), 3.94 (dt, J =
4.4 and 10.4 Hz, 1 H), 4.45 (dt, J = 2.0 and 9.6 Hz, 1 H), 4.53–
4.72 (m, 3 H), 4.80 (dd, J = 8.4 and 17.6 Hz 1 H), 5.34 (dd, J =
0.8 and 10.4 Hz, 1 H), 5.45 (dd, J = 1.6 and 17.6 Hz, 1 H), 5.97–
6.10 (m, 2 H), 6.98 (d, J = 1.2 Hz, 1 H), 9.05 (br., 1 H) ppm. 13C
NMR: δ = 12.5, 35.2 (d, J = 8.2 Hz), 69.2 (d, J = 4.2 Hz), 70.0 (d,
J = 11.5 Hz), 73.9 (d, J = 7.4 Hz), 77.2 (d, J = 8.2 Hz), 86.1, 112.0,
119.4, 132.0 (d, J = 7.4 Hz), 136.2, 149.8, 163.4 ppm. 31P NMR: δ
= 61.0 ppm. HRMS (ESI+): m/z calcd. for C13H17N2O6PSNa
[M+Na]+: 383.0437; found 383.0663.
Synthesis of Allyl Methyl (Rp)-Thymidin-3Ј-yl Phosphorothioate (2): ous solution of triethylammonium hydrogen carbonate (0.1 ,
A solution of sodium methoxide in methanol (1.0 , 15.0 mL) was
slowly added at 0 °C to a stirred solution of 1 (1.80 g 5.00 mmol)
in methanol (50.0 mL). The reaction mixture was stirred at 25 °C
for 24 h, and ethyl acetate (500 mL) was added. The organic solu-
tion was washed with an ammonium chloride-saturated aqueous
solution (100 mL), the aqueous layer was extracted with ethyl ace-
tate (200 mL, 2×), and the combined organic layers were washed
10 mL, 2×) to convert the phosphorothioic acid completely into its
triethylammonium salt. The resulting organic solution was dried
with magnesium sulfate (ca. 20 g) and concentrated, the residue
was dissolved in dichloromethane (2.0 mL), and the solution was
poured into petroleum ether (200 mL). The precipitates were col-
lected by filtration and dried to give the phosphorothioate diester
4 (1.04 g, 92% yield): TLC Rf = 0.37 (methanol/dichloromethane,
1
with brine (300 mL), dried with Na2SO4 (ca. 150 g), and concen- 1:9). H NMR ([D6]DMSO): δ = 1.17 (t, J = 7.2 Hz, 9 H), 1.34 (s,
trated. The resulting crude product was subjected to silica gel col-
umn chromatography with elution with a 1:2 mixture of ethyl ace-
tate and hexane to afford 2 (1.63 g, 83% yield): TLC Rf = 0.37
3 H), 2.24–2.40 (m, 2 H), 3.05–3.15 (m, 7 H), 3.28–3.34 (m, 1 H),
3.74 (s, 6 H), 4.16–4.24 (m, 3 H), 5.03 (dd, J = 1.7 and 10.5 Hz, 2
H), 5.19 (ddd, J = 2.0, 3.9, and 17.3 Hz, 1 H), 5.82–5.91 (m, 1 H),
6.20 (dd, J = 5.6 and 8.5 Hz, 1 H), 6.88–6.90 (m, 4 H), 7.22–7.40
(m, 9 H), 7.49 (s, 1 H), 9.23 (br. s, 1 H), 11.32 (s, 1 H) ppm. 13C
NMR: δ = 8.56, 11.5, 39.5 (d, J = 3.3 Hz), 45.6, 55.2, 64.0, 67.0
(d, J = 5.8 Hz), 76.7 (d, J = 4.9 Hz), 84.7, 85.6 (d, J = 5.8 Hz),
87.0, 111.1, 113.3, 116.2, 127.0, 128.2, 130.2, 134.6 (d, J = 8.2 Hz),
135.4, 135.6, 135.9, 144.4, 150.4, 158.7, 163.7 ppm. 31P NMR ([D6]-
1
(ethyl acetate/hexane, 1:2). H NMR: δ = 1.93 (s, 3 H), 2.49 (dd, J
= 4.4 and 7.2 Hz, 1 H), 2.58 (br. s, 1 H), 3.78 (d, J = 13.6 Hz, 3
H), 3.89–3.99 (m, 2 H), 4.24 (dd, J = 2.4 and 5.2 Hz, 1 H), 4.58
(ddt, J = 1.2, 2.4, and 6.8 Hz, 2 H), 5.18–5.23 (m, 1 H), 5.29 (dd,
J = 1.2 and 10.5 Hz, 1 H), 5.39 (ddd, J = 1.5, 2.9, and 17.3 Hz, 1
H), 5.90–5.99 (m, 1 H), 6.20 (t, J = 6.8 Hz, 1 H), 7.45 (d, J =
1.2 Hz, 1 H), 8.61 (br. s, 1 H) ppm. 13C NMR: δ = 12.5, 38.2 (d, J
= 5.7 Hz), 54.7 (d, J = 5.7 Hz), 62.1, 69.1 (d, J = 4.9 Hz), 78.2 (d,
J = 4.9 Hz), 85.5 (d, J = 5.7 Hz), 86.5, 111.3, 118.8, 132.1 (d, J =
7.4 Hz), 136.6, 150.4, 163.7 ppm. 31P NMR: δ = 66.2 ppm. HRMS
(ESI+): m/z calcd. for C14H21N2O7PSNa [M+Na]+: 415.0699;
found 415.0911.
DMSO):
δ
=
52.8 ppm. HRMS (ESI–): m/z calcd. for
C34H36N2O9PS [M–H]–: 679.1885; found 679.2713.
Synthesis of Methyl (Sp)-5Ј-O-(p,pЈ-Dimethoxytrityl)thymidin-3Ј-yl
Phosphorothioate Triethylammonium Salt (5): Triphenylphosphane
(6.56 mg, 25.0 µmol) and Pd2[(C6H5CH=CH)2CO]3·CHCl3
(5.18 mg, 5.00 µmol) were added to a solution of the phos-
phorothioate triester 3 (65.9 mg, 100 µmol) in THF (1.00 mL), and
Dimethoxytritylation of Allyl Methyl (Rp)-Thymidin-3Ј-yl Phos-
phorothioate (2) to Provide Allyl Methyl (Rp)-5Ј-O-(p,pЈ-Dimeth- the resulting mixture was stirred at 25 °C for 30 min. To this mix-
oxytrityl)thymidin-3Ј-yl Phosphorothioate (3): A solution of 2
(834 mg, 2.12 mmol) in a 5:1 mixture of N,N-dimethylformamide
(20.0 mL) and pyridine (4.00 mL) was chilled to 0 °C, and small
portions of p,pЈ-dimethoxytrityl chloride (1.01 g, 2.97 mmol) were
added. The resulting homogeneous mixture was stirred at 0 °C for
30 min and then at 25 °C for 7 h. Ethyl acetate (100 mL) was added
to the reaction mixture, and the resulting organic solution was
washed with a sodium hydrogen carbonate-saturated aqueous solu-
tion (50 mL). The aqueous layer was extracted with ethyl acetate
(100 mL, 2×), and the organic layer was washed with brine
(50 mL), dried with Na2SO4 (ca. 30 g), and concentrated to give an
oily residue. This crude product was purified by silica gel column
ture were added, at 0 °C, butylamine (19.0 µL, 14.1 mg, 200 µmol)
and formic acid (7.50 µL, 9.18 mg, 200 µmol). The reaction mixture
was stirred at 25 °C for 2 h and was then concentrated to afford a
yellow oil. This material was dissolved in dichloromethane (5.0 mL)
and washed with a solution of triethylammonium hydrogen carbon-
ate in water (0.1 , 5.0 mL, 2×). The organic layer was dried with
magnesium sulfate (ca. 10 g) and concentrated, the residue was sub-
jected to silica gel column chromatography with a 1:9 methanol/
dichloromethane mixture, collected fractions containing the desired
product were concentrated, and the resulting residue was dissolved
in dichloromethane (5.0 mL). In order to convert the phos-
phorothioic acid completely into the triethylammonium salt, the
chromatography with a 1:1 mixture of ethyl acetate and hexane as solution was again treated with an aqueous triethylammonium hy-
eluent to afford 3 (1.34 g, 91% yield): TLC Rf = 0.43 (ethyl acetate/
drogen carbonate solution (0.1 , 5.0 mL, 2×). The organic layer
was dried with magnesium sulfate (ca. 10 g) and concentrated, the
hexane, 1:1). 1H NMR: δ = 1.45 (s, 3 H), 2.36–2.43 (m, 1 H), 2.59–
3840
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 3834–3844

Thymidine Phosphorothioates with (R) or (S) Configurations at Phosphorus
FULL PAPER
residue was dissolved in dichloromethane (1.0 mL) and poured into
petroleum ether (100 mL), and the precipitates were collected by
filtration and dried to give the target compound 5 (66.5 mg, 88%
saturated aqueous solution (50 mL). The aqueous layer was ex-
tracted with ethyl acetate (100 mL, 2×), and the organic layers were
collected and washed with brine (50 mL). After drying, concentra-
tion of the organic solution afforded a residual material, which was
subjected to silica gel column chromatography with elution with a
1:9 mixture of methanol and dichloromethane followed by a
1:30:10 mixture of methanol, ethyl acetate, and hexane to give 10
(389 mg, 62% yield): TLC Rf = 0.60 (methanol/dichloromethane,
1
yield): TLC Rf = 0.21 (methanol/dichloromethane, 1:9). H NMR
([D6]DMSO): δ = 1.17 (t, J = 7.2 Hz, 9 H), 1.37 (s, 3 H), 2.25–2.38
(m, 2 H), 3.07 (q, J = 7.3 Hz, 6 H), 3.15 (dd, J = 2.7 and 10.2 Hz,
1 H), 3.27–3.29 (m, 1 H), 3.74 (s, 6 H), 4.11 (d, J = 2.4 Hz, 1 H),
5.00–5.03 (m, 1 H), 6.18 (dd, J = 5.9 and 8.3 Hz, 1 H), 6.88–6.90
(m, 4 H), 7.22–7.41 (m, 9 H), 7.48 (s, 1 H), 9.49 (br. s, 1 H), 11.32
1
1:9), Rf = 0.38 (methanol/ethyl acetate/hexane, 1:30:10). H NMR:
(s, 1 H) ppm. 13C NMR: δ = 8.62, 11.6, 39.8 (d, J = 4.1 Hz), 45.7, δ = 1.46 (s, 3 H), 1.95 (s, 3 H), 2.11 (s, 3 H), 2.16–2.25 (m, 1 H),
52.9 (d, J = 6.5 Hz), 55.2, 63.9, 76.5 (d, J = 4.9 Hz), 84.7, 85.1 (d,
J = 5.8 Hz), 87.0, 111.1, 113.3, 127.0, 128.2, 130.1, 135.4, 135.6,
135.8, 144.4, 150.4, 158.7, 163.8 ppm. 31P NMR ([D6]DMSO): δ =
54.3 ppm. HRMS (ESI–): m/z calcd. for C32H34N2O9PS [M–H]–:
653.1728; found 653.2584.
2.37–2.50 (m, 2 H), 2.58 (dd, J = 5.6 and 14.0 Hz, 1 H), 3.45 (br.,
2 H), 3.69 (d, J = 14.0 Hz, 3 H), 3.80 (s, 6 H), 4.18–4.23 (m, 2 H),
4.28–4.38 (m, 2 H), 5.28–5.30 (m, 1 H), 5.35–5.39 (m, 1 H), 6.34–
6.41 (m, 2 H), 6.85 (d, J = 8.8 Hz, 4 H), 7.23–7.70 (m, 11 H), 8.65
(s, 1 H), 8.73 (s, 1 H) ppm. 13C NMR: δ = 11.8, 12.5, 20.9, 37.2,
39.0 (d, J = 5.8 Hz), 54.8 (d, J = 4.9 Hz), 55.3, 63.3, 67.7 (d, J =
5.8 Hz), 74.4, 79.7 (d, J = 3.3 Hz), 82.7 (d, J = 9.1 Hz), 84.4, 84.6
(d, J = 6.6 Hz), 84.8, 87.3, 111.7 (2 C), 113.4, 127.2, 128.1, 130.1,
135.0 (2 C), 135.1, 135.2, 144.1, 150.5, 150.6, 158.8, 163.8 (2 C),
170.6 ppm. 31P NMR: δ = 68.0 ppm. HRMS (ESI+): m/z calcd. for
C44H49N4O14PSNa [M+Na]+: 943.2596; found 943.3270.
Synthesis of 3Ј-O-(Acetyl)thymidin-5Ј-yl Allyl (Sp)-5Ј-O-(p,pЈ-Di-
methoxytrityl)thymidin-3Ј-yl Phosphorothioate (7): A solution of
DEAD in toluene (40%, 0.35 mL, 0.77 mmol) was added at 0 °C
to a solution of triphenylphosphane (202 mg, 0.77 mmol) in THF
(20 mL). After the mixture had been stirred at 0 °C for 30 min, 4
(460 mg, 0.59 mmol) and the nucleoside 6 (335 mg, 1.18 mmol)
were added at the same temperature and the reaction mixture was
stirred at 60 °C for 3 h. According to 31P NMR analysis, this reac-
tion mixture included not only the desired compound 13 but also
Preparation of Allyl (Sp)-5Ј-O-(p,pЈ-Dimethoxytrityl)thymidin-3Ј-yl
Thymidin-5Ј-yl Phosphorothioate (8) by Deacetylation of (Sp)-3Ј-O-
(Acetyl)thymidin-5Ј-yl Allyl 5Ј-O-(p,pЈ-Dimethoxytrityl)thymidin-3Ј-
yl Phosphorothioate (7): Sodium methoxide in methanol solution
(1.0 , 55.0 µL, 55.0 µmol) was slowly added at 0 °C to a solution
of 7 (103 mg, 109 µmol) in methanol (4.00 mL). The reaction mix-
ture was warmed up to 25 °C and stirred at this temperature for
4 h, diluted with ethyl acetate (20 mL), and washed with an ammo-
nium chloride-saturated aqueous solution (5.0 mL). The aqueous
layer was extracted with ethyl acetate (20 mL, 2×), and the com-
bined organic layers were washed with brine (10 mL), dried with
Na2SO4 (ca. 30 g), and concentrated to give an oily product. This
crude product was purified by silica gel column chromatography
with a 1:9 mixture of methanol and dichloromethane as eluent to
afford 8 (86.8 mg, 88% yield): TLC Rf = 0.53 (methanol/dichloro-
a considerable amount (ca. 42%) of the by-product 13 (R1
=
CH2=CHCH2), showing a 31P NMR signal at δ = 26.5 ppm. The
reaction mixture was diluted with ethyl acetate (50 mL) and washed
with an ammonium chloride-saturated aqueous solution (20 mL).
The aqueous layer was extracted with ethyl acetate (50 mL, 2×),
and the collected organic solutions were washed with brine
(20 mL), dried with Na2SO4 (ca. 50 g), and concentrated. The re-
sulting residue was subjected to column chromatography on silica
gel with a 1:9 mixture of methanol and dichloromethane, followed
by a 1:30:10 mixture of methanol, ethyl acetate, and hexane as el-
uents to afford 7 (301 mg, 54% yield): TLC Rf = 0.57 (methanol/
dichloromethane, 1:9), Rf = 0.43 (methanol/ethyl acetate/hexane,
1
1:30:10). H NMR: δ = 1.47 (s, 3 H), 1.94 (s, 3 H), 2.10–2.19 (m, methane, 1:9). 1H NMR: δ = 1.48 (s, 3 H), 1.92 (s, 3 H), 2.10–2.17
4 H), 2.35–2.44 (m, 2 H), 2.60 (dd, J = 5.6 and 12.8 Hz, 1 H), 3.46
(m, 1 H), 2.35–2.45 (m, 2 H), 2.60 (dd, J = 5.6 and 13.2 Hz, 1 H),
(d, J = 2.4 Hz, 2 H), 3.80 (s, 6 H), 4.13–4.35 (m, 4 H), 4.60 (dd, J 3.46 (d, J = 2.4 Hz, 2 H), 3.80 (s, 6 H), 4.08–4.32 (m, 4 H), 4.46
= 5.6 and 10.8 Hz, 2 H), 5.21–5.41 (m, 4 H), 5.88–5.97 (m, 1 H),
6.36 (dd, J = 5.6 and 9.2 Hz, 1 H), 6.41 (dd, J = 5.2 and 8.4 Hz, 1
H), 6.85 (d, J = 8.8 Hz, 4 H), 7.22–7.40 (m, 10 H), 7.58 (s, 1 H),
(t, J = 2.8 Hz, 1 H), 4.59 (dd, J = 5.6 and 10.8 Hz, 2 H), 5.26–5.40
(m, 3 H), 5.87–5.97 (m, 1 H), 6.32 (t, J = 6.4 Hz, 1 H), 6.41 (dd, J
= 5.2 and 8.8 Hz, 1 H), 6.85 (d, J = 9.2 Hz, 4 H), 7.22–7.40 (m, 10
8.81 (s, 1 H), 8.85 (s, 1 H) ppm. 13C NMR: δ = 11.8, 12.6, 21.0, H), 7.56 (d, J = 1.2 Hz, 1 H), 9.36 (s, 1 H), 9.53 (s, 1 H) ppm. 13C
37.1, 39.1 (d, J = 5.8 Hz), 55.3, 63.3, 67.5 (d, J = 5.8 Hz), 69.4 (d,
J = 4.9 Hz), 74.4, 79.6 (d, J = 4.1 Hz), 82.6 (d, J = 9.1 Hz), 84.4,
84.6, 84.7 (d, J = 5.8 Hz), 87.3, 111.6, 111.9, 113.4, 119.5, 127.2,
NMR: δ = 11.8, 12.6, 39.0 (d, J = 4.9 Hz), 40.1, 55.3, 63.4, 67.6
(d, J = 6.6 Hz), 69.3 (d, J = 4.1 Hz), 71.3, 79.6 (d, J = 4.9 Hz),
84.5, 84.6 (d, J = 8.2 Hz), 84.7 (d, J = 4.1 Hz), 85.1, 87.3, 111.5,
128.1, 130.1, 131.7 (d, J = 7.4 Hz), 134.8, 135.1 (2 C), 135.2, 144.1, 111.9, 113.4, 119.3, 127.2, 128.1, 130.1, 131.8 (d, J = 6.9 Hz), 135.1,
150.4, 150.5, 158.8, 163.6, 163.7, 170.4 ppm. 31P NMR: δ =
67.0 ppm. HRMS (ESI+): m/z calcd. for C46H51N4O14PSNa
[M+Na]+: 969.2752; found 969.3589.
135.2, 135.6, 144.2, 150.7, 150.9, 158.8, 164.0, 164.2 ppm. 31P
NMR:
65.7 ppm. HRMS (ESI+): m/z calcd. for
C44H49N4O13PSNa [M+Na]+: 927.2647; found 927.3500.
δ
=
Synthesis of 3Ј-O-(Acetyl)thymidin-5Ј-yl (Rp)-5Ј-O-(p,pЈ-Dimeth- Preparation
of (Rp)-5Ј-O-(p,pЈ-Dimethoxytrityl)thymidin-3Ј-yl
Methyl Thymidin-5Ј-yl Phosphorothioate (11) by Deacetylation of
toluene solution (40%, 0.40 mL, 0.88 mmol) was added at 0 °C (Rp)-3Ј-O-(Acetyl)thymidin-5Ј-yl 5Ј-O-(p,pЈ-Dimethoxytrityl)thymi-
oxytrityl)thymidin-3Ј-yl Methyl Phosphorothioate (10): A DEAD/
with stirring to
a
solution of triphenylphosphane (231 mg,
din-3Ј-yl Methyl Phosphorothioate (10): Deacetylation of 10, afford-
ing 11, was carried out in a manner similar to that described above
for preparation of 8 from 7. From 19.0 mg (21.0 µmol) of 10,
15.5 mg of 11 (84% yield) was produced: TLC Rf = 0.54 (methanol/
0.88 mmol) in THF (23 mL). Stirring at this temperature was con-
tinued for 30 min. To this mixture, at the same temperature, were
added the phosphorothioate 5 (510 mg, 0.68 mmol) and the nucleo-
side 6 (387 mg, 1.36 mmol). The reaction mixture was stirred at
60 °C for 3 h. 31P NMR analysis showed that this reaction mixture
included the desired compound 10 and a considerable amount (ca.
33%) of the by-product 13 (R1 = CH3), showing a 31P NMR signal
at δ = 27.1 ppm. Ethyl acetate (100 mL) was added to this reaction
mixture, and the mixture was washed with an ammonium chloride-
1
dichloromethane, 1:9). H NMR: δ = 1.50 (s, 3 H), 1.92 (s, 3 H),
2.16–2.23 (m, 1 H), 2.31–2.38 (m, 1 H), 2.44 (ddd, J = 3.4, 5.8 and
9.8 Hz, 1 H), 2.68 (dd, J = 4.9 and 13.6 Hz, 1 H), 3.40 (dd, J = 3.4
and 10.7 Hz, 1 H), 3.69 (d, J = 6.8 Hz, 3 H), 3.79 (s, 6 H), 4.13–
4.15 (m, 1 H), 4.24–4.35 (m, 3 H), 4.52–4.55 (m, 1 H), 5.26–5.29
(m, 1 H), 6.29–6.33 (m, 2 H), 6.85 (d, J = 8.8 Hz, 4 H), 7.22–7.41
Eur. J. Org. Chem. 2006, 3834–3844
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3841

Y. Hayakawa et al.
FULL PAPER
(m, 10 H), 7.58–7.59 (m, 1 H), 9.22 (s, 1 H), 9.83 (s, 1 H) ppm. 13
NMR: δ = 11.9, 12.4, 39.4 (d, J = 3.3 Hz), 40.5, 54.8 (d, J =
5.8 Hz), 55.3, 63.4, 67.4 (d, J = 6.6 Hz), 71.0, 80.2 (d, J = 3.3 Hz),
C
shown in Table 1, with the use of suitable phosphoramidites 16–19
as building units. Subsequently, the trityl-on or trityl-off product
was subjected to deprotection and detachment from the solid sup-
84.5 (d, J = 8.2 Hz), 84.6 (d, J = 7.4 Hz), 84.8, 85.3, 87.4, 111.1, port as follows to give the desired oligonucleotide.
112.0, 113.4, 127.3, 128.4, 130.1, 134.9, 135.1 (2 C), 144.1, 150.6,
(Rp,Rp)-T[TpsT]T7[TpsT]T9 (20): After the nucleotide chain elong-
ation was complete, the trityl-on protected oligonucleotide bound
on CPG supports was washed with acetonitrile and dried in vacuo.
The resulting solid material was mixed with triphenylphosphane
151.2, 158.8, 163.8, 163.9 ppm. 31P NMR: δ = 68.1 ppm. HRMS
(ESI+): m/z calcd. for C42H47N4O13PSNa [M+Na]+: 901.2490;
found 901.2828.
(59.0 mg, 0.23 mmol), butylamine (0.60 mL, 444 mg, 6.0 mmol),
formic acid (0.23 mL, 281 mg, 6.0 mmol), and Pd2[(C6H5CH=CH)
2CO]3·CHCl3 (23.3 mg, 22.5 µmol) in THF (5 mL), and the re-
sulting mixture was vigorously shaken until the THF phase had
changed from a dark violet color to yellow/orange. The hetero-
geneous mixture was then stirred at 50 °C for 14 h. After the super-
natant fluid had been decanted, the resulting CPG supports were
washed successively with THF (1 mL, 2×) and acetone (1 mL, 2×).
Subsequently, the CPG supports were stirred in an aqueous sodium
N,N-diethyldithiocarbamate (ddtc) solution (0.1 , 1 mL) for
15 min. The mixture was filtered to remove the aqueous solution,
and the solid material was then washed with acetone (1 mL, 3×)
and water (1 mL, 2×). The resulting CPG supports were again sub-
jected to the treatment with the sodium ddtc solution described
above and were then washed with acetone and water. The solid
supports were stirred in an aqueous concentrated NH4OH solution
(2 mL) at 25 °C for 2 h, and the supernatant fluid was separated
by decantation and then heated at 55 °C for 6 h. The resulting solu-
tion was freeze-dried to give a residual material, which was dis-
solved in a solution of triethylammonium acetate in water (0.1 ,
2 mL). The solution was subjected to HPLC purification on COS-
MOSIL 5C18-AR-II (4.6×250 mm) under the following conditions
(buffer mobile phase: A = 0.1  triethylammonium acetate in water,
B = H2O/acetonitrile (1:4), linear gradient of 0–100% B in A for
60 min; temperature: 40 °C; flow rate: 1.000 mL·min–1; detection:
UV 254 nm) (see Supporting Information). Fractions containing
the desired 5Ј-O-dimethoxytritylated oligonucleotide were collected
and concentrated. The resulting gummy product was dissolved in
an aqueous solution of acetic acid (0.4 , 2 mL), and the solution
was stirred at 60 °C for 6 h. The reaction mixture was concentrated
to furnish the target PO/PS-chimeric oligonucleotide 20, showing
a single peak by HPLC analysis [column: COSMOSIL 5C18-AR-
II (4.6×250 mm); mobile phase: A = 0.1  triethylammonium ace-
tate in water, B = H2O/acetonitrile (1:4), linear gradient of 0–20%
B in A for 60 min; temperature: 40 °C; flow rate: 1.000 mL·min–1;
detection: UV 254 nm] (see Supporting Information).
Preparation of the Phosphoramidite 14 from 8: Diisopropylammo-
nium 1H-tetrazolide (281 mg, 1.64 mmol) was added to a solution
of 14 (738 mg, 0.82 mmol) in acetonitrile (27.0 mL) containing
powdery mol. sieves (3 Å), and the resulting solution was stirred at
25 °C for 30 min. CH2=CHCH2OP[N(i-C3H7)2]2 (0.39 mL, 350 mg,
1.23 mmol) was slowly added at the same temperature to this solu-
tion, and the mixture was stirred for an additional 3 h. The reaction
mixture was filtered to remove the mol. sieves, and the filtrate was
concentrated. The residue was dissolved in ethyl acetate (150 mL)
and washed with an aqueous sodium hydrogen carbonate solution
(100 mL), the aqueous layer was extracted with ethyl acetate
(150 mL, 2×), and the combined organic solutions were washed
with brine (150 mL), dried with Na2SO4 (ca. 100 g), and concen-
trated to provide a residual product, which was dissolved in dichlo-
romethane (5.0 mL) and poured into petroleum ether (500 mL).
The precipitates were collected by filtration and dried in a vacuum
to give the phosphoramidite 14 (765 mg, 85% yield) as a mixture
of two diastereomers: TLC Rf = 0.39 (ethyl acetate/hexane, 2:1).
1H NMR: δ = (mixture of two diastereomers) 1.16 (d, J = 6.8 Hz,
12 H), 1.43 (s, 3 H), 1.91 (s, 3 H), 2.05–2.14 (m, 1 H), 2.36–2.49
(m, 2 H), 2.56–2.61 (m, 1 H), 3.40–3.47 (m, 2 H), 3.55–3.63 (m, 2
H), 3.79 (s, 6 H), 4.06–4.24 (m, 6 H), 4.45–4.61 (m, 3 H), 5.12 (d,
J = 10.4 Hz, 1 H), 5.23–5.39 (m, 4 H), 5.87–5.96 (m, 2 H), 6.31 (t,
J = 6.4 Hz, 1 H), 6.41 (t, J = 7.2 Hz, 1 H), 6.84 (d, J = 8.8 Hz, 4
H), 7.22–7.40 (m, 10 H), 7.58 (s, 1 H), 8.92 (br. s, 2 H) ppm. 13C
NMR: δ = (mixture of two diastereomers) 11.7, 12.6, 24.5 (3 C),
24.6 (3 C), 24.7, 39.2 (2 C), 39.5 (2 C), 43.1, 43.2, 55.3, 63.4, 64.2
(2 C), 64.4 (2 C), 67.3, 69.3 (2 C), 72.6, 72.8, 73.0, 79.4, 79.5, 83.7,
83.8 (2 C), 83.9, 84.1, 84.2 (2 C), 84.3, 84.4, 84.7, 84.8, 85.0 (2 C),
87.3, 111.4, 111.7, 113.4, 115.8, 116.0, 119.3 (2 C), 127.2, 128.1 (2
C), 130.1, 131.7, 131.8 (2 C), 131.9, 135.1 (2 C), 135.2, 135.3 (4 C),
135.4, 144.1,150.4, 150.5, 158.8, 163.9 ppm. 31P NMR: δ = (mixture
of two diastereomers) 67.4, 147.5, 147.9.
Preparation of the Phosphoramidite 15 from 10: According to the
procedure used to produce 14, 15 (343 mg, 78% yield) was prepared
as a mixture of two diastereomers on the basis of the reaction of 10
(358 mg, 0.41 mmol) and CH2=CHCH2OP[N(i-C3H7)2]2 (0.20 mL,
(Rp)-G[TpsT]CAT (21): The trityl-off protected oligonucleotide
bound on CPG supports was washed with acetonitrile and dried
under reduced pressure. A mixture of the resulting solid material,
triphenylphosphane (21.0 mg, 88.0 µmol), butylamine (240 µL,
178 mg 2.4 mmol), formic acid (90 µL, 110 mg, 22.4 mmol), and
Pd2[(C6H5CH=CH)2CO]3·CHCl3 (9.1 mg, 8.8 µmol) in THF
(2 mL) was vigorously shaken. When the original dark violet color
of the THF layer had changed to yellowish orange, the mixture
was warmed up to 50 °C and stirred at the same temperature for
14 h. The supernatant fluid was removed by decantation, and the
CPG supports were washed with THF (1 mL, 2×) followed by ace-
tone (1 mL, 2×), and were then treated with a sodium ddtc aqueous
solution (0.1 , 1 mL) for 15 min. After the sodium ddtc solution
had been removed by filtration, the CPG supports were washed
with acetone (1 mL, 3×) and water (1 mL, 2×). Treatment with the
sodium ddtc solution and the succeeding washing were repeated.
The resulting CPG supports were treated with an aqueous concen-
1
179 mg, 0.62 mmol): TLC Rf = 0.54 (ethyl acetate/hexane, 2:1). H
NMR: δ = (mixture of two diastereomers) 1.17 (d, J = 3.4 Hz, 6
H), 1.19 (d, J = 3.9 Hz, 6 H), 1.44 (s, 3 H), 1.93 (s, 3 H), 2.14–2.23
(m, 1 H), 2.35–2.60 (m, 3 H), 3.44 (s, 2 H), 3.56–3.68 (m, 5 H),
3.79 (s, 6 H), 4.05–4.32 (m, 6 H), 4.50–4.54 (m, 1 H), 5.13–5.16 (m,
1 H), 5.26–5.29 (m, 1 H), 5.34–5.38 (m, 1 H), 5.88–5.99 (m, 1 H),
6.27–6.31 (m, 1 H), 6.39 (dd, J = 5.9 and 8.8 Hz, 1 H), 6.84 (d, J
= 8.8 Hz, 4 H), 7.24–7.39 (m, 10 H), 7.55 (s, 1 H), 8.37 (br. s, 2
H) ppm. 13C NMR: δ = (mixture of two diastereomers) 11.7, 12.8,
24.4, 24.5, 24.6, 24.7, 39.0, 39.1, 39.5 (3 C), 39.6, 43.1, 43.2, 54.7,
54.8, 55.3, 63.3, 64.2, 64.4, 67.3, 67.4 (2 C), 67.5, 72.6, 72.8 (2 C),
72.9, 79.4 (2 C), 83.8, 84.2 (2 C), 84.3, 84.6 (2 C), 85.2, 85.3, 87.3,
111.3, 111.7, 113.4, 115.8, 116.0, 127.2, 128.1, 130.1, 135.0, 135.1,
135.2, 135.3 (2 C), 135.4, 135.5, 135.6 ppm. 31P NMR: δ = (mixture
of two diastereomers) 68.7, 147.6, 148.0.
Solid-Phase Synthesis of PO/PS-Chimeric Oligodeoxyribonucleo- trated NH4OH solution (2 mL) at 25 °C for 2 h, and the superna-
tides: The nucleotide chain was elongated by the reaction cycle tant fluid was separated by filtration. The GPG supports were
3842
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 3834–3844

Thymidine Phosphorothioates with (R) or (S) Configurations at Phosphorus
FULL PAPER
washed with a small amount of water and the aqueous filtrates
were combined and heated at 55 °C for 6 h. Freeze-drying of the
resulting solution afforded a gummy material, which was dissolved
in a solution of triethylammonium acetate in water (0.1 , 2 mL).
The resulting solution was purified by HPLC as described in the
preparation of 20, and the reaction mixture was concentrated to
give 21 (see Supporting Information for details about the purity of
this product).
Supporting Information Available (see also the footnote on the first
1
page of this article): H, 13C, and 31P NMR spectra and ESI-TOF
1
high-resolution mass spectra of 1–5, 7, 8, 10, and 11; H, 13C, and
31P NMR spectra of 14 and 15; RP-HPLC profiles of the mixtures
obtained by the digestion of 9 and 12 with svPDE and with nucle-
ase P1. ESI-TOF high-resolution mass spectra of 20–23; RP-HPLC
profiles of crude 20–23 and purified 20–23; RP-HPLC profiles of
the mixture obtained by the digestion of 20–23 with svPDE and
with nuclease P1; and the program for the solid-phase synthesis of
20–23 on an Applied Biosystems 392 DNA/RNA Synthesizer.
This material is available free of charge through the Internet at
http://www.eurjoc.org.
(Sp,Sp)-T[TpsT]T7[TpsT]T9 (22): The CPG supports binding the
trityl-on protected oligonucleotide were washed with acetonitrile
and dried in vacuo. The resulting solid material was mixed with
triphenylphosphane (65.6 mg, 0.25 mmol), butylamine (0.60 mL,
444 mg, 6.0 mmol), formic acid (0.23 mL, 281 mg, 6.0 mmol), and
Pd2[(C6H5CH=CH)2CO]3·CHCl3 (25.9 mg, 25.0 µmol) in THF
(5.0 mL), the resulting mixture was vigorously shaken until the
solution had changed from a dark violet color to yellow/orange,
and the mixture was then heated with stirring at 50 °C for 14 h.
After the supernatant fluid had been removed by decantation, the
resulting CPG supports were washed successively with THF (1 mL,
2×) and acetone (1 mL, 2×), and were then treated with a solution
of sodium ddtc in water (0.1 , 1 mL) for 15 min. The solid sup-
ports were separated by filtration and washed with acetone (1 mL,
3×) followed by water (1 mL, 2×). The sodium ddtc treatment and
succeeding washing were repeated once again. The CPG supports
were stirred in an aqueous concentrated NH4OH solution (2 mL)
at 25 °C for 2 h, and the aqueous layer was separated by filtration
and concentrated by freeze-drying. The resulting residual material
was treated with aqueous acetic acid (0.4 ) at 60 °C for 6 h. Con-
centration of the whole reaction mixture afforded 22 (for details
about the purity of this product, see Supporting Information).
Acknowledgments
This study was partly supported by Grants-in-Aid for Scientific
Research (No. 16011223) and the 21st Century COE Program (Es-
tablishment of COE of Materials Science: Elucidation and Creation
of Molecular Functions) from the Ministry of Education, Culture,
Science, Sports, and Technology of Japan. This work was also sup-
ported by CREST of JST (Japan Science and Technology).
[1] a) E. Uhlmann, A. Peyman, Chem. Rev. 1990, 90, 543–584; b)
J. F. Milligane, M. D. Matteucci, J. C. Martin, J. Med. Chem.
1993, 36, 1923–1937; c) R. S. Varma, Synlett 1993, 621–637; d)
W. J. Stec, A. Wilk, Angew. Chem. Int. Ed. Engl. 1994, 33, 709–
722; e) Antisense Therapeutics (Ed.: S. Agrawal), Humana, To-
towa, 1996.
[2] a) C. A. Stein, A. Cleary, L. Yakubov, S. Lederman, Antisense
Res. Dev. 1993, 3, 19–31; b) L. Yakubov, Z. Khaled, L.-M.
Zhang, A. Truneh, V. Vlassov, C. A. Stein, J. Biol. Chem. 1993,
268, 18818–18823.
(Sp)-G[TpsT]CAT (23): The trityl-off product attached to CPG
supports was washed with acetonitrile and dried in vacuo. This
dried solid was mixed with triphenylphosphane (26.2 mg,
0.10 mmol), butylamine (240 µL, 178 mg, 2.4 mmol), formic acid
(90 µL, 110 mg, 2.4 mmol), and Pd2[(C6H5CH=CH)2CO]3·CHCl3
(10.4 mg, 10 µmol) in THF (2 mL), and the mixture was vigorously
shaken to change the dark violet color of the THF layer to light
orange. The heterogeneous mixture was heated at 50 °C and stirred
at this temperature for 14 h, the supernatant fluid was decanted,
and the resulting CPG supports were washed successively with
THF (1 mL, 2×) and acetone (1 mL, 2×). Subsequently, the solid
material was stirred with an aqueous sodium ddtc solution (0.1 ,
1 mL, 15 min). The aqueous phase was removed by filtration, and
the solid material was washed with acetone (1 mL, 3×) and water
(1 mL, 2×). This sodium ddtc solution treatment and succeeding
washings were repeated again. The resulting CPG supports were
stirred in a concentrated aqueous NH4OH solution (2 mL) at 25 °C
for 2 h. The solid material was removed by filtration, and the fil-
trate was freeze-dried to furnish a residual product. Concentration
of the whole reaction mixture gave 23 (for details about the purity
of this product, see Supporting Information).
[3] a) W.-Y. Gao, F.-S. Han, C. Storm, W. Egan, Y. C. Cheng, Mol.
Pharmacol. 1991, 41, 223–230; b) W.-Y. Gao, F.-S. Han, C.
Storm, W. Egan, Y.-C. Cheng, Molecules Molecul. Pharmacol.
1992, 41, 223–229; c) R. M. Crooke, M. J. Graham, M. E. Co-
oke, S. T. Crooke, J. Pharmacol. Exp. Ther. 1995, 275, 462–473;
d) S. T. Crooke, K. L. Lemonidis, L. Neilson, R. Griffey, E. A.
Lesnik, B. P. Monia, Biochem. J. 1995, 312, 599–608.
[4] M. A. Guvakova, L. A. Yakubov, I. Voldavlsky, J. L. Tonkin-
son, C. A. Stein, J. Biol. Chem. 1995, 270, 2620–2627.
[5] a) D. S. Pisetsky, C. F. Reich, Life Sci. 1993, 54, 101–109; b) J.
Messina, G. Gilkeson, D. Piesetsky, Cell Immunol. 1993, 147,
148–157. See also; c) Q. Zhao, J. Temsamani, P. L. Iadarola, Z.
Jiang, S. Agrawal, Biochem. Pharmacol. 1996, 51, 173–182.
[6] a) K. G. Cornish, P. Iversen, L. Smith, D. Becker, C. B. Meyer,
Pharmacol. Res. Commun. 1993, 3, 239–244; b) W. M. Gal-
braith, W. C. Hobson, D. C. Giclas, P. J. Schechter, S. Agrawal,
Antisense Res. Dev. 1994, 4, 201–211. See also; c) P. L. Iversen,
K. G. Cornish, L. J. Iversen, J. E. Mata, D. B. Bylund, Toxicol.
Appl. Pharmacol. 1999, 160, 289–296.
[7] For a pioneering work on synthesis and properties of PO/PS-
chimeric oligonucleotides, see B. T. Monia, J. F. Johnston, H.
Sasmor, L. L. Cummins, J. Biol. Chem. 1996, 271, 14533–
14540.
Treatment of a PO/PS-Chimeric Oligonucleotide with an Enzyme
and HPLC Analysis of the Resulting Product: A mixture of the PO/
PS-chimeric oligonucleotide and a suitable enzyme, out of snake
venom phosphodiesterase (svPDE), Serratia marcescens (Sma I),
and nuclease P1, was incubated at 37 °C for 24 h. The reaction mix-
ture was heated at 100 °C for 3 min to inactivate the enzyme and
then subjected to RP-HPLC analysis on COSMOSIL 5C18-AR-II
(4.6×250 mm) under the following conditions: buffer mobile
phase: A = 0.1  triethylammonium acetate in water, B = H2O/
acetonitrile (1:4), linear gradient of 0–20% B in A for 60 min; tem-
perature: 40 °C; flow rate: 1.000 mL·min–1; detection: UV 254 nm.
[8] a) C. A. Stein, C. Subasinghe, K. Shinozuka, J. S. Cohen, Nu-
cleic Acids Res. 1988, 16, 3209–3221; b) W. J. Stec, A. Grajkow-
ski, A. Kobylanska, B. Karwowski, M. Koziolkiewicz, K. Mis-
iura, K. Okruszek, A. Wilk, P. Guga, M. Bockowska, J. Am.
Chem. Soc. 1995, 117, 12019–12029.
[9] For representative synthesis: a) B. A. Connolly, F. Eckstein, A.
Pingoud, J. Biol. Chem. 1984, 259, 10760–10763; b) J. A. Fid-
anza, H. Ozaki, L. W. McLaughlin, J. Am. Chem. Soc. 1992,
114, 5509–5517; c) D. R. Lesser, A. Grajkowski, M. R. Kur-
pirwski, M. Koziolkiewocz, W. J. Stec, L. Jen-Jacobson, J. Biol.
Chem. 1992, 267, 24810–24818; d) S. V. Patil, R. B. Mane,
M. M. Salunkhe, Bioorg. Med. Chem. Lett. 1994, 4, 2663–2666.
Eur. J. Org. Chem. 2006, 3834–3844
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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Y. Hayakawa et al.
FULL PAPER
[10] W. J. Stec, B. Karwowski, M. Boczkowska, P. Guga, M. Koziol-
kiewiz, M. Sochacki, M. W. Wieczorek, J. Blaszczyk, J. Am.
Chem. Soc. 1998, 120, 7156–7167.
[11] Y. Hayakawa, A. Hirata, J. Sugimoto, R. Kawai, A. Sakakura,
M. Kataoka, Tetrahedron 2001, 57, 8823–8826.
[21] B. V. L. Potter, B. A. Connolly, F. Eckstein, Biochemistry 1983,
22, 1369–1377.
[22] F. R. Bryant, S. J. Benkovic, Biochemistry 1979, 18, 2825–2828.
[23] a) K. A. Nelson, A. E. Sopchik, W. G. Bentrude, J. Am. Chem.
Soc. 1983, 105, 7752–7754; b) N. L. Broeders, A. P.
van der Heiden, J. A. Kanters, A. Schouten, Can. J. Chem.
1993, 71, 855–863. See also; c) K. Taira, W. L. Mock, D. G.
Gorenstein, J. Am. Chem. Soc. 1984, 106, 7831–7835.
[24] a) W. S. Zielinski, W. J. Stec, J. Am. Chem. Soc. 1977, 99, 8365–
8366; b) J. Baraniak, W. J. Stec, J. Chem. Soc., Perkin Trans. 1
1987, 1645–1656; c) W. J. Stec, A. Grajkowski, A. Kobylanska,
B. Karwowski, M. Koziolkiewicz, K. Misiura, A. Okruszek, A.
Wilk, P. Guga, M. Boczkowska, J. Am. Chem. Soc. 1995, 117,
12019–12029; d) L. A. Wozniak, M. Góra, M. Bukowiecka-
Matusiak, S. Mourgues, G. Pratviel, B. Meunier, W. J. Stec,
Eur. J. Org. Chem. 2005, 2924–2930; e) B. Nawrot, B. Re˛bow-
ska, K. Cies´lin´ska, W. J. Stec, Tetrahedron Lett. 2005, 46, 6641–
6644.
[25] A. Wilk, A. Grajkowski, T. E. Bull, A. M. Dixon, D. I. Freeb-
erg, S. L. Beaucage, J. Am. Chem. Soc. 2002, 124, 1180–1181.
[26] a) R. P. Iyer, M.-J. Guo, D. Yu, S. Agrawal, Tetrahedron Lett.
1998, 39, 2491–2494; b) D. Yu, E. R. Kandimalla, A. Roskey,
Q. Zhao, L. Chen, J. Chen, S. Agrawal, Bioorg. Med. Chem.
2000, 8, 275–284.
[27] a) Y. Jin, G. Biancotto, G. Just, Tetrahedron Lett. 1996, 37,
973–976; b) Y. Jin, G. Just, J. Org. Chem. 1998, 63, 3647–3654;
c) Y. Lu, G. Just, Angew. Chem. Int. Ed. 2000, 39, 4521–4524;
d) Y. Lu, G. Just, Tetrahedron 2000, 56, 4355–4365; e) Y. Lu,
G. Just, Tetrahedron 2001, 57, 1677–1687.
[12] In all cases, the ratio of the (Rp) isomer to the (Sp) isomer in
the product before heating was approximately 1:1.
[13] According to the 31P NMR analysis, the combined yield of the
(Rp) and (Sp) isomers was unchanged before and after heating,
indicating that the increase in the ratio of the (Rp) isomer in
the nucleoside cyclic phosphate is caused by equilibration and
is not the result of a decrease in the (Sp) isomer through its
selective decomposition.
[14] M. Hyodo, Y. Sato, S. Yamashita, A. Hattori, E. Kambe, M.
Kataoka, Y. Hayakawa, Tetrahedron 2005, 61, 965–970.
[15] This strategy could be generally applied to the stereoselective
synthesis of 2Ј-deoxyribonucleoside 3Ј,5Ј-cyclic (Rp)-phos-
phorothioates. Thus, allyl (Rp)-deoxyadenosine 3Ј,5Ј-cyclic
phosphorothioate, allyl (Rp)-deoxycytidine 3Ј,5Ј-cyclic phos-
phorothioate, and allyl (Rp)-deoxyguanosine 3Ј,5Ј-cyclic phos-
phorothioate were stereoselectively prepared from the corre-
sponding parent nucleosides. For details on the preparation of
these compounds, see Supporting Information.
[16] a) N. L. Broeders, A. P. van der Heiden, I. Peeters, H. M.
Janssen, L. H. Koole, J. Am. Chem. Soc. 1992, 114, 9624–9633;
b) J. Varila, T. Hankamaki, M. Oivanen, L. H. Koole, H.
Lonnberg, J. Org. Chem. 1997, 62, 893–898; c) S. Mehdi, J. A.
Coderre, J. A. Gerlt, Tetrahedron 1983, 39, 3483–3492; d) M. I.
Elzagheid, K. Mattila, M. Oivanen, B. C. N. M. Jones, R. Cos-
stick, H. Lonnberg, Eur. J. Org. Chem. 2000, 62, 893–898.
[17] a) M. D. M. Gray, D. J. H. Smith, Tetrahedron Lett. 1980, 21,
859–860; b) D. J. H. Smith, K. K. Ogilvie, M. F. Gillen, Tetra-
hedron Lett. 1980, 21, 1987–1991.
[28] a) N. Oka, T. Wada, K. Saigo, J. Am. Chem. Soc. 2002, 124,
4962–4963; b) N. Oka, T. Wada, K. Saigo, J. Am. Chem. Soc.
2003, 125, 8307–8317.
[29] H. Almer, T. Szabo, J. Stawinski, Chem. Commun. 2004, 290–
291.
[18] a) Y. Hayakawa, H. Kato, M. Uchiyama, H. Kajino, R.
Noyori, J. Org. Chem. 1986, 51, 2400–2402; b) Y. Hayakawa,
S. Wakabayashi, H. Kato, R. Noyori, J. Am. Chem. Soc. 1990,
112, 1691–1696.
[30] L. H. Koole, M. H. P. van Genderen, H. M. Buck, J. Am.
Chem. Soc. 1987, 109, 3916–3921.
[31] S. B. Heidenhain, Y. Hayakawa, Nucleosides Nucleotides 1999,
[19] For a comprehensive review: O. Mitsunobu, Synthesis 1981, 1–
18, 1771–1787.
28.
[32] Y. Hayakawa, M. Uchiyama, R. Noyori, Tetrahedron Lett.
1986, 27, 4191–4194.
[20] Similarly, (Rp)-ApsA, (Rp)-CpsC, (Sp)-ApsA, and (Sp)-CpsC
could be prepared from suitable building blocks. For details on
the synthesis of these compounds, see Supporting Information.
Received: February 23, 2006
Published Online: June 21, 2006
3844
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 3834–3844