A new method for the synthesis of nucleoside 2′,3′-O,O-cyclic phosphorodithioates via aryl cyclic phosphites as intermediates

Article abstract of DOI:10.1016/S0040-4039(01)01695-1

The reaction of 5′-O-protected ribonucleosides with tri(4-nitrophenyl) phosphite in the presence of pyridine furnished rapid formation of the corresponding 4-nitrophenyl 2′,3′-O,O-cyclic phosphites which upon sulfhydrolysis, followed by sulfurization of t

Full text of DOI:10.1016/S0040-4039(01)01695-1

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 8055–8058
A new method for the synthesis of nucleoside 2%,3%-O,O-cyclic
phosphorodithioates via aryl cyclic phosphites as intermediates
Małgorzata Wenska,a Jadwiga Jankowska,a Michał Sobkowski,a Jacek Stawin´skia,b and
Adam Kraszewskia,*
aInstitute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan´, Poland
bDepartment of Organic Chemistry, Stockholm University, Arrhenius Laboratory, S-106 91 Stockholm, Sweden
Received 5 June 2001; revised 16 August 2001; accepted 7 September 2001
Abstract—The reaction of 5%-O-protected ribonucleosides with tri(4-nitrophenyl) phosphite in the presence of pyridine furnished
rapid formation of the corresponding 4-nitrophenyl 2%,3%-O,O-cyclic phosphites which upon sulfhydrolysis, followed by sulfuriza-
tion of the resultant cyclic H-phosphonothioate and removal of the 5%-O-protecting groups, afforded nucleoside 2%,3%-O,O-cyclic
phosphorodithioates in high yields. © 2001 Elsevier Science Ltd. All rights reserved.
Nucleoside 2%,3%-O,O-cyclic phosphates have been
exploited for years in the biochemistry and molecular
biology of nucleic acids as indispensable tools for estab-
lishing substrate specificity of various nucleases and in
investigations of chemical and enzymatic aspects of
ribonuclease catalysed reactions.1 Although their bio-
logical significance is less clear than that of nucleoside
3%,5%-cyclic phosphates, recent studies implicate the
involvement of five-membered cyclic phosphodiesters in
various metabolic pathways.2,3 Rapidly growing interest
in self-splicing of RNA4 and in the development of
ribozymes for therapeutical purposes5 again places
nucleoside 2%,3%-O,O-cyclic phosphates in the forefront
of synthetic and mechanistic chemistry,6,7 with the aim
to provide chemical support for diverse biological stud-
ies involving these compounds.
nucleoside 2%,3%-O,O-cyclic phosphorothioates. This
method was based on phosphonylation of suitably 5%-
O-protected nucleosides with diphenyl H-phosphonate
to produce nucleoside 2%,3%-O,O-cyclic H-phosphonates,
which via oxidation with elemental sulfur provided the
corresponding nucleoside 2%,3%-O,O-cyclic phosphoroth-
ioates in high yield.9
As part of this program, we became interested in a
largely unexplored class of nucleotide analogues,
nucleoside 2%,3%-O,O-cyclic phosphorodithioates for
which only one representative, uridine 2%,3%-O,O-cyclic
phosphorodithioate, was known. This compound was
obtained from 5%-O-acetyluridine and P2S5 at elevated
temperature,10 but the reaction produced a complex
mixture of products from which the desired cyclic phos-
phorodithioate was isolated in low yield (ca. 28%) after
tedious ion-exchange chromatography on DEAE-
cellulose.10
Due to the importance of nucleotide analogues in eluci-
dation of mechanisms of enzymatic reactions,8 we have
initiated a program on the chemistry and biochemistry
of nucleoside 2%,3%-O,O-cyclic phosphates and their ana-
logues to study the effects of modification at the phos-
phorus centre on the conformational equilibria and
flexibility of the fused five-membered rings in these
compounds in the context of their interaction with
RNases. In connection with these, we recently devel-
oped a new efficient method for the synthesis of
Inspired by the ease of formation of 2%,3%-O,O-cyclic
H-phosphonates using diphenyl H-phosphonate in the
synthesis of nucleoside 2%,3%-O,O-cyclic phosphoroth-
ioates,9 we attempted the same strategy for the prepara-
tion of 2%,3%-O,O-cyclic phosphorodithioates, by making
use of diphenyl H-phosphonothioate 2 to generate the
2%,3%-O,O-cyclic H-phosphonothioates 3 as a key inter-
mediate (Scheme 1). Although the desired H-phospho-
nothioate 3a always constituted the major product of
the reaction of 1 and 2, and could be easily converted
into cyclic phosphorodithioate 4a by treatment with
elemental sulfur (vide infra), a parallel disproportiona-
Keywords: nucleosides; nucleotides; cyclic phosphates; dithiophos-
phates.
* Corresponding author. Fax: +48-61 82 05 32; e-mail: akad@
ibch.poznan.pl
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)01695-1

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M. Wenska et al. / Tetrahedron Letters 42 (2001) 8055–8058
HO
DMTO
DMTO
B
B
B
CH3COOH
O
P
S8
S
O
P
O
P
PhO P OPh
O
-S
O
S
O
H
O
S
O
-S
O
S
2
H
DMTO
3
5
B
4
O
OH OH
H2S
B
1
1a, 3a-7a; B = uracil-1-yl
DMTO
OAr
1b, 3b-7b; B = adenin-9-yl
1c, 3c-7c; B = cytosin-1-yl
1d, 3d-7d; B = guanin-9-yl
O
ArO P
OAr
6
O
O
P
O
NO2
DMT = 4,4'-dimethoxytrityl
Ar = 4-nitrophenyl
7
Scheme 1.
ical reactivity) to that produced from nucleoside 1a and
diphenyl H-phosphonothioate (nucleoside 2%,3%-O,O-H-
phosphonothioate 3a, vide supra).14
tion of diphenyl H-phosphonothioate
2
towards
H-phospho-
triphenyl
phosphite
and
phenyl
nodithioate,11 together with side-reactions involving 3a,
lowered the yield and made chromatographic purifica-
tion of 4a (or 5a) troublesome.
As the only side
product in this reaction, we observed the formation of
variable amounts (10–15%) of the corresponding
nucleoside 2%,3%-O,O-cyclic H-phosphonate (lP=23.22
and 27.22 ppm, 1JHP=659.0 and 1JHP=659.0 Hz),9
which we assumed to be due to the presence of adventi-
tious water in the reaction mixture. To remedy this
problem, we carried out the sulfhydrolysis of cyclic
phosphite 7a in the presence of trimethylsilyl chloride
(TMSCl, 15 molar equiv.). This completely eliminated
the interfering hydrolysis of the intermediate 7 and
nucleoside 2%,3%-O,O-cyclic H-phosphonothioate 3a
could be produced as the sole nucleotidic species (31P
NMR). Addition of elemental sulfur (3 molar equiv.)
directly to the reaction mixture furnished rapid (ca. 5
min, 31P NMR) and clean sulfurisation to produce
2%,3%-O,O-phosphorodithioate 4a (31P NMR). Because
phosphorodithioate 4a was always the sole nucleotidic
product present in the reaction mixture (ca. 90% of
total phosphorus species, 31P NMR), the final deprotec-
tion (80% acetic acid, 20 min)15 was performed without
prior purification. The unprotected uridine 2%,3%-O,O-
cyclic phosphorodithioate 5a was isolated by simple
silica gel chromatography and obtained as a white
amorphous solid after freeze-drying (95% yield).
To overcome these problems, we searched for another
route to produce cyclic H-phosphonothioate intermedi-
ates of type 3 and considered sulfhydrolysis of 4-nitro-
phenyl cyclic phosphite 7 as a viable alternative for this
purpose. We found that the required phosphite interme-
diate 7 can be efficiently produced by reacting 5%-O-
protected nucleosides of type 1 with a crystalline
reagent, tri(4-nitrophenyl) phosphite 6 (prepared in
large scale by modification12 of the Walsh method13).
31P NMR spectroscopy showed that formation of phos-
phite 7a from uridine derivative 1a and reagent 6 (1.1
equiv.) in DMF/pyridine (9:1, v/v) was rapid (<3 min)
and clean. It was apparent from the presence in the
spectrum of only two resonances in the range of chem-
ical shifts for cyclic phosphite derivatives [lP=140.90
3
ppm (s) and 146.40 ppm (t, JHP=10.2 Hz)] that two
diastereoisomers of 7 had been formed. To ensure that
the use of triaryl phosphite 6 is compatible with the
presence of unprotected amino functions in nucleosidic
substrates, in separate experiments we treated 3%,5%-
O,O-di(t-butyldimethylsilyl) 2%-deoxyadenosine, 2%-
deoxycytidine and 2%-deoxyguanosine with an excess (3
molar equiv.) of tris(4-nitrophenyl) phosphite 6. No
signals which could be assigned to N- or O-phosphity-
lated species produced from nucleobases and reagent 6
were observed within 20 min by 31P NMR
spectroscopy.
When the reaction conditions developed for the synthe-
sis of uridine derivative 5a were applied to the other
5%-O-(4,4%-dimethoxytrityl)nucleosides 1b–d, the corre-
sponding 2%,3%-O,O-cyclic phosphorodithioates 5b–d
were also obtained in high yields (84–90%). In all
instances investigated, high efficiency of the individual
steps eliminated the need for chromatographic purifica-
tion of the intermediates involved (compounds of type
3, 4 or 7) and the final products, nucleoside 2%,3%-O,O-
cyclic phosphorodithioates of type 5, could be isolated
in excellent yields using silica gel chromatography (vide
supra).
Next, the susceptibility of intermediate 7 to sulfhydroly-
sis was investigated. To this end, nucleoside 4-nitro-
phenyl 2%,3%-O,O-cyclic phosphite 7a (produced in situ
from nucleoside 1a and 6 as described above) was
treated with an excess of H2S (5 molar equiv., 1 M
stock solution in dioxane). The reaction was rapid (<3
min, 31P NMR) and afforded a new compound (ca.
90%) [lP=87.70 ppm (1JHP=661.9 Hz, brd) and 91.16
A typical procedure for the preparation of nucleoside
2%,3%-O,O-cyclophosphodithioates 5: A solution of tri(4-
nitrophenyl) phosphite 6 (1.1 molar equiv.) in DMF/
3
ppm (1JHP=659.0 Hz, JHP=10.2 Hz, dd)], which was
identical (chemical shifts, coupling constants and chem-

M. Wenska et al. / Tetrahedron Letters 42 (2001) 8055–8058
8057
pyridine (9:1, v/v) (10 mL; sometimes gentle heating
was necessary to dissolve 6) was added to 5%-O-(4,4%-
dimethoxytrityl)nucleoside of type 1 (1 mmol; made
anhydrous by repeated evaporation of added anhy-
drous pyridine). The aryl nucleoside cyclic phosphite of
type 7 formed was treated after 5 min with a mixture of
H2S (5 molar equiv., 1 M solution in dioxane) and
trimethylsilyl chloride (15 molar equiv.) and then (after
another 5 min) elemental sulfur (3 molar equiv.) was
added. When the sulfurisation was complete (ca. 5 min,
31P NMR and TLC), the reaction mixture was neu-
tralised with 5% aq. NaHCO3 (5 mL) and the solvents
were removed by evaporation under reduced pressure.
The residue was dissolved in methylene chloride con-
taining triethylamine (1%, v/v; 30–40 mL), washed with
5% aq. NaHCO3 (3×10 mL) and the organic layer
evaporated. The removal of the 5%-O-dimethoxytrityl
group was effected by treatment of the oily residue with
80% aq. acetic acid (10 mL) during 20 min. After
evaporation of acetic acid, the crude product was dis-
solved in a minimum volume of methylene chloride/
methanol (4:1, v/v) and applied on a silica gel column
pre-equilibrated with methylene chloride/triethylamine
(99:1, v/v). Chromatography was performed using a
stepwise gradient (0–10%, v/v) of methanol in methyl-
ene chloride containing triethylamine (1%, v/v). The
fractions containing pure product were collected, evap-
orated and freeze-dried from benzene/methanol (4:1,
v/v). Cyclic phosphorodithioates 5 (triethylammonium
salts) were obtained as white amorphous solids (purity
of the produced cyclic H-phosphonothioate 3 with ele-
mental sulfur. All transformations involved can be car-
ried out as ‘a one-pot reaction’, and are compatible
with nucleosidic substrates bearing unprotected amino
functions. The method is very efficient and experimen-
tally simple.
Acknowledgements
Financial support from the State Committee for Scien-
tific Research, Republic of Poland and the Swedish
Natural Science Research Council is gratefully
acknowledged.
References
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1
>98%, H NMR). Yields: 5a 95%, 5b 85%, 5c 90%, 5d
84%.†
7. Ora, M.; Peltomaki, M.; Oivanen, M.; Lonnberg, H. J.
Org. Chem. 1998, 63, 2939–2947.
In conclusion, we have developed a new, general proto-
col for the preparation of nucleoside 2%,3%-O,O-cyclic
phosphorodithioates of type 5. The method relies on
8. Eckstein, F.; Gish, G. TIBS 1989, 97–100.
9. Jankowska, J.; Wenska, M.; Popenda, M.; Stawinski, J.;
Kraszewski, A. Tetrahedron Lett. 2000, 41, 2227–2229.
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11. Kers, A.; Kers, I.; Stawinski, J.; Sobkowski, M.;
Kraszewski, A. Tetrahedron 1996, 52, 9931–9944.
12. 4-Nitrophenol (4.2 g, 30 mmol; rendered anhydrous by
repeated addition and evaporation of excess acetonitrile)
and phosphorus trichloride (1.4 g, 10 mmol) were
refluxed in acetonitrile (50 mL) for 3 days under slightly
reduced pressure to evacuate the HCl evolved. After
cooling to rt and concentration to half of the initial
volume, white crystals of product 6 precipitated and were
filtered off and dried. Yield 3.5 g (80%). 31P NMR lP
(DMF) 126.17 ppm; m/z 445.0301, calculated for
C18H12N3O9P 445.0311; mp=174–176°C.
13. Walsh, E. N. J. Am. Chem. Soc. 1959, 81, 3023–3026.
14. In separate experiments we showed that the integrity of
the phospholane ring in 3a remained intact upon pro-
longed (3–4 h) treatment with hydrogen sulfide (31P
NMR).
15. On this occasion we also studied the stability of the
phosphorodithioates moiety in 2%,3%-cyclic phosphates
under acidic conditions [see Ora et al. (Ref. 16) on the
pH-dependent desulfurisation of nucleoside phospho-
rodithioates and their 2%,3%-O,O-phosphorodithioates].
When uridine 2%,3%-O,O-cyclic phosphorodithioate 5a
sulfhydrolysis of 4-nitrophenyl cyclic phosphite
7
[accessible from ribonucleosides using the stable, crys-
talline, and readily available phosphitylating agent,
tris(4-nitrophenyl) phosphite 6], followed by oxidation
† Chemical identity of compounds 5a–d was confirmed by, 1H, 31P
NMR and HRMS. 5a, lH (D2O) 1.32 (9H, t, J=7.5 Hz, CH2CH3),
3.22 (6H, q, J=7.5 Hz, CH2CH3), 3.79 (1H, m, 5%, 5¦-H2), 4.42 (1H,
m, 4%-H), 4.96 (1H, m, 3%-H), 5.07 (1H, m, 2%-H), 5.68 (1H, d, J=7.5
Hz, 5-H), 6.06 (1H, d, J=2.4 Hz, 1%-H), 7.78 (1H, d, J=7.5 Hz,
6-H); lP (D2O) 138.22 (dd, 3JHP=10.1 and 7.3 Hz); m/z 336.9740,
calculated for [C9H10N2O6PS2]− 336.9718. 5b, lH (D2O) 1.09 (9H, t,
J=7.5 Hz, CH2CH3), 2.97 (6H, q, J=7.5 Hz, CH2CH3), 3.74 (2H,
m, 5%, 5¦-H2), 4.44 (1H, m, 4%-H), 5.12 (1H, m, 3%-H), 5.40 (1H, m,
2%-H), 6.29 (1H, d, J=4.2 Hz, 1%-H), 8.06 (1H, s, 2-H), 8.16 (1H, s,
8-H); lP (D2O) 137.65 (dd, 3JHP=11.9 and 5.5 Hz); m/z 359.9992,
calculated for [C10H11N5O4PS2]− 359.9990. 5c, lH (D2O) 1.30 (9H,
t, J=7.5 Hz, CH2CH3), 3.19 (6H, q, J=7.5 Hz, CH2CH3), 3.78
(2H, m, 5%, 5¦-H2), 4.44 (1H, m, 4%-H), 4.98 (1H, m, 3%-H), 5.03 (1H,
m, 2%-H), 5.95 (1H, d, J=7.5 Hz, 5-H), 6.05 (1H, d, J=2.4 Hz,
3
1%-H), 7.81 (1H, d, J=7.5 Hz, 6-H); lP (D2O) 137.10 (dd, JHP
=
11.9 and 6.4 Hz); m/z 335.9852, calculated for [C9H11N3O5PS2]−
335.9878. 5d, lH (D2O) 1.30 (9H, t, J=7.3 Hz, CH2CH3), 3.22 (6H,
q, J=7.3 Hz, CH2CH3), 3.80 (2H, m, 5%, 5¦-H2), 4.49 (1H, m, 4%-H),
5.18 (1H, m, 3%-H), 5.38 (1H, m, 2%-H), 6.24 (1H, d, J=3.9 Hz,
1%-H), 7.93 (1H, s, 8-H); lP (D2O) 136.91 (t, 3JHP=8.7 Hz); m/z
375.9947, calculated for [C10H11N5O5PS2]− 375.9939.

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M. Wenska et al. / Tetrahedron Letters 42 (2001) 8055–8058
was kept in 80% aqueous acetic acid for 8 h at 25°C,
the dimethoxytrityl group from 4 (80% aq. acetic acid,
rt, 20 min) were not detrimental to 2%,3%-O,O-cyclic
phosphorodithioates of type 5.
31P NMR spectroscopy and TLC analysis did not show
the formation of any products of potential degradation
of 5a (desulfurisation, dephosphorylation, etc.). This
indicated that the conditions used for the removal of
16. Ora, M.; Jarvi, J.; Oivanen, M.; Lonnberg, H. J. Org.
Chem. 2000, 65, 2651–2657.