Further studies with adenosine 5′-dithiophosphoromorpholidate. A convenient preparation of nucleoside phosphorodithioates by a 'triester' approach

Article abstract of DOI:10.1016/j.tetlet.2004.01.135

Adenosine 5′-dithiophosphoromorpholidate 1c reacts with orthophosphate to give adenosine 5′-(α,α-dithio)diphosphate 3c but is not converted into adenosine 5′-phosphorodithioate 2c by acidic hydrolysis. A new approach to the synthesis of nucleoside phosphorodithioates is described.

Full text of DOI:10.1016/j.tetlet.2004.01.135

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 2653–2656
Further studies with adenosine 50-dithiophosphoromorpholidate.
A convenient preparation of nucleoside phosphorodithioates
by a ÔtriesterÕ approach
Colin B. Reese* and Hongbin Yan
Department of Chemistry, KingÕs College London, Strand, London WC2R 2LS, UK
Received 1 December 2003; revised 16 January 2004; accepted 23 January 2004
Abstract—Adenosine 50-dithiophosphoromorpholidate 1c reacts with orthophosphate to give adenosine 50-(a,a-dithio)diphosphate
3c but is not converted into adenosine 50-phosphorodithioate 2c by acidic hydrolysis. A new approach to the synthesis of nucleoside
phosphorodithioates is described.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Nucleoside 50-phosphoromorpholidates,1 such as the
adenosine derivative 1a, are important intermediates in
the synthesis of condensed phosphates (e.g., adenosine
50-di- and tri-phosphates1) and nucleotide coenzymes
(e.g., coenzyme A2); they also readily undergo acid-
catalysed hydrolysis1;3 to give the corresponding nucleo-
side 50-phosphates (e.g., adenosine 50-phosphate 2a). In
the same way, adenosine 50-thiophosphoromorphol-
idate3;4 1b reacts with orthophosphate to give adenosine
50-(a-thio)-diphosphate3 3b and undergoes acid-cataly-
sed hydrolysis to give adenosine 50-phosphorothioate3;4
2b. Several years ago, we were surprised to find that
adenosine 50-dithiophosphoromorpholidate4;5 1c under-
went hydrolysis in 95% acetic acid at room temperature
to give adenosine 4 as the sole nucleoside or nucleotide
product. Shortly after the publication of our study,4
Caruthers and his co-workers reported6 the first syn-
thesis of adenosine 50-phosphorodithioate 2c, and found
that it was readily converted into adenosine 4 under
mild conditions of acidic hydrolysis. We had originally
concluded4 that adenosine 50-dithiophosphoromorphol-
idate 1c was converted directly into adenosine 4 by
acidic hydrolysis. However, it was clearly possible that
acid-labile adenosine 50-phosphorodithioate 2c was an
intermediate, which, under the prevailing acidic condi-
tions, was rapidly converted into adenosine 4. This
possibility has prompted us to reconsider whether
adenosine 50-dithiophosphoromorpholidate 1c is a suit-
able starting material for the synthesis of condensed a,a-
dithiophosphates, such as adenosine 50-(a,a-dithio)-
diphosphate 3c.
Y
Y
P
X
Ade
O
N
P
X
O
Ade
HO
O
_
O
_
O
OH
HO
OH
HO
HO
1
2
O
P
O
Y
Ade
Ade
HO
O
P
X
O
HO
_
_
O
O
OH
HO
OH
3
4
Ade = adenin-9-yl
a; X = Y = O
b; X = S, Y = O
c; X = Y = S
We now report that adenosine 50-(a,a-dithio)diphos-
phate 3c was indeed obtained in ca. 25% yield when
adenosine 50-dithiophosphoromorpholidate 1c was
heated with an excess of mono-(tri-n-propylammonium)
phosphate in anhydrous pyridine solution.7 The modest
yield obtained may possibly have been due to the diffi-
culty encountered in excluding moisture in a relatively
small scale reaction. Although it would be premature to
conclude that this is a particularly satisfactory approach
* Corresponding author. Tel.: +44(0)20-7848-2260; fax: +44(0)20-
7848-1771; e-mail: colin.reese@kcl.ac.uk
0040-4039/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.01.135

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C. B. Reese, H. Yan / Tetrahedron Letters 45 (2004) 2653–2656
Scheme 1. Reagents and conditions: (i) (a) (Me3Si)2NH, CH2Cl2, rt, 24 h, (b) H2O, C5H5N, rt, 1 h, (c) (NH4)2CO3, MeOH, rt, 10 min; (ii) 7, 13, 14,
C5H5N, rt, 1 h; (iii) Me3SiCl, (MeN)2C ¼ NH (TMG), MeCN, 60 ꢁC, 6 h; (iv) NH3, H2O, MeCN, rt, 5 min.
to the synthesis of the dithio-diphosphate 3c and related
a,a-dithio condensed phosphates, this result led us to
re-examine whether adenosine 50-phosphorodithioate 2c
was indeed an intermediate in the acid-catalysed con-
version of adenosine 50-dithiophosphoromorpholidate
1c into adenosine 4. In order to further investigate this
matter, we required a sample of adenosine 50-phosphoro-
dithioate 2c. Although several procedures have been
described in the literature for the preparation of nucleo-
side phosphorodithioates,6;8–10 none of them appeared to
us to be particularly convenient experimentally. For this
reason, we set out to develop an alternative approach
involving triester intermediates.
oxyacetyl)thymidine22 10 was converted (Scheme 1c)
into the intermediate triester 11 in almost quantitative
yield. Following the same deblocking procedure, thy-
midine 30-phosphorodithioate 12 was obtained25 as its
TMGHþ salt, also in high yield.
With adenosine 50-phosphorodithioate 2c now in hand, a
study of its decomposition both in acetic acid–water (95:5
v/v) (the medium in which the acid-catalysed hydrolysis of
adenosine 50-dithiophosphoromorpholidate 1c was orig-
inally examined4) and acetic acid–water (5:95 v/v)
(pH 2.3) was undertaken. The decomposition reactions
were monitored first by HPLC at 25 ꢁC, and adenosine 4
was found to be the sole nucleoside or nucleotide product.
The half-times for the conversion of the phosphorodi-
thioate 2c into adenosine 4 were found to be ca. 24 and
40 min, respectively, under these conditions. The
decomposition reactions of adenosine 50-phosphorodi-
thioate 2c were then monitored by 31P NMR spectros-
copy both in CD3CO2D–D2O (95:5 v/v) and CD3CO2D–
D2O (5:95 v/v) solution at 23 ꢁC. In 95% CD3CO2D, the
substrate 2c (dP 86.6) was converted first into dithio-
phosphate 15 (dP 68.1),26 which was itself then converted
into thiophosphate 16 (dP 38.1).26 Integration of the res-
onance signals suggested that t1=2 of the substrate 2c under
these conditions was ca. 9 min. After 50 min, thiophos-
phate 16 was virtually the sole phosphorus-containing
species present in the products. Decomposition in 5%
CD3CO2D occurred more slowly: t1=2 of the substrate 2c
Ammonium bis-S-(2-cyanoethyl) phosphorodithioate 7
was obtained11 (Scheme 1a) as a pure crystalline solid in
81% isolated yield by allowing ammonium phosphinate
6 to react with N-[(2-cyanoethyl)sulfanyl]phthalimide12
5 in the presence of hexamethyldisilazane. Treatment of
6-N,20-O,30-O-tri(phenoxyacetyl)adenosine13 8 with the
phosphorodithioate salt 7, 2,4,6-triisopropylbenzenesulfon-
yl chloride 13 and 3-nitro-1,2,4-1H-triazole 14 in pyri-
dine solution16 (Scheme 1b) gave the intermediate
triester 9 in high yield.17 When this product 9 was
treated first with an excess each of chlorotrimethylsilane
and 1,1,3,3-tetramethylguanidine (TMG)18;19 in aceto-
nitrile solution at 60 ꢁC to remove both 2-cyanoethyl
protecting groups and then with aqueous ammonia to
remove the phenoxyacetyl protecting groups,20 adeno-
sine 50-phosphorodithioate 2c was obtained21 as its
1,1,3,3-tetramethylguanidinium (TMGHþ) salt in vir-
tually quantitative yield. In the same way, 50-O-(phen-
(dP 102.4) appeared to be more than 30 min, and the 31
P
chemical shifts of dithio- and thio-phosphate (15 and 16,
respectively), were found to be 92.9 and 47.3 ppm.

C. B. Reese, H. Yan / Tetrahedron Letters 45 (2004) 2653–2656
2655
S
P
S
Ade
O
O
N
Ade
HO
O
_
O
?
OH
HO
OH
HO
1c
4
O
_
Ade
Ade
HO
S
P
S
O
O
P
S
O
O
O
_
_
_
S
OH
S
P
OH
16
OH
_
OH
OH
HO
HO
15
2c
4
Scheme 2. Decomposition of 1c and 2c in CD3CO2D–D2O (95:5 v/v) at 23 ꢁC.
The decomposition of adenosine 50-dithiophosphoro-
morpholidate 1c in 95% acetic acid was then re-exam-
ined. Our previous conclusion4 that adenosine 4 was the
sole nucleoside or nucleotide product and that adenosine
50-phosphorodithioate 2c was not an intermediate was
confirmed first by reversed phase HPLC: t1=2 of the
substrate 1c was found to be between 10 and 15 min at
25 ꢁC. 31P NMR spectroscopic studies in CD3CO2D–
D2O (95:5 v/v) at 23 ꢁC were then repeated: t1=2 of sub-
strate (dP 119.9) appeared to be between 15 and 20 min
and it was clear that adenosine 50-phosphorodithioate 2c
(dP 86.6) was not an intermediate in its decomposition.
What are believed to be dithio- and thio-phosphate (15
and 16, respectively; dP 68.1 and 38.1 ppm) appeared to
be the main phosphorus-containing products28 and, after
80 min, thiophosphate 16 was confirmed4 to be virtually
the sole phosphorus-containing product. Adenosine 50-
dithiophosphoromorpholidate 1c was found to decom-
pose very slowly indeed in CD3CO2D–D2O (5:95 v/v) at
23 ꢁC. The decomposition reactions of adenosine 50-di-
thiophosphoromorpholidate 1c and adenosine 50-phos-
phorodithioate 2c in 95% CD3CO2D are summarised in
Scheme 2. The nature of the initial phosphorus-con-
taining decomposition product (or products) of the
dithiophosphoromorpholidate 1c in 95% CD3CO2D has
not yet been elucidated. The most surprising and as yet
unexplained conclusion of this study is that although the
substrate 1c reacts with orthophosphate to give adeno-
sine 50-(a,a-dithio)diphosphate 3c, albeit in modest yield,
it does not undergo hydrolysis in 95% acetic acid to give
adenosine 50-phosphorodithioate 2c.
3. Richards, K. H. Ph.D. Thesis, London University, 1987,
pp 52–76.
4. Reese, C. B.; Shek, L. H. K.; Zhao, Z. Tetrahedron Lett.
1994, 35, 5085–5088.
5. Reese, C. B.; Shek, L. K. H.; Zhao, Z. J. Chem. Soc.,
Perkin Trans. 1 1995, 3077–3084.
6. Seeberger, P. H.; Yau, E.; Caruthers, M. H. J. Am. Chem.
Soc. 1995, 117, 1472–1478.
7. A solution of mono-(tri-n-propylammonium) phosphate
(0.141 g, 0.58 mmol) and tri-n-propylammonium adeno-
sine
50-dithiophosphoromorpholidate
1c
(0.040 g,
0.068 mmol) in dry pyridine (10 mL) was concentrated to
small volume and was then re-dissolved in dry pyridine
(3 mL). The resulting solution was heated at 50 ꢁC for 6.5 h
and was then cooled to room temperature. Ethyl acetate
(30 mL) was added and the precipitate obtained was
collected by centrifugation and washed with ethyl acetate
(30 mL). Reversed phase HPLC of this material revealed a
major component (ca. 25%) that was isolated by
preparative HPLC; dH (D2O) includes the following
signals: 8.70 (1H, s), 8.18 (1H, s), 6.08 (1H, d, J 5.9),
4.51 (1H, m), 4.36 (1H, m), 4.24 (2H, m); dP (D2O) )10.50
(d, J 33.1), 100.40 (d, J 33.7); FAB-MS: calcd for
C10H14N5O8P2S2 [M)H]ꢀ, 458.0; found, 458.0.
8. Okruszek, A.; Olesiak, M.; Krajewska, D.; Stec, W. J.
J. Org. Chem. 1997, 62, 2269–2272.
ꢀ
ꢀ
9. Jankowska, J.; Cieslak, J.; Kraszewski, A.; Stawinski, J.
Tetrahedron Lett. 1997, 38, 2007–2010.
ꢀ
10. Jankowska, J.; Sobkowska, A.; Cieslak, J.; Sobkowski,
M.; Kraszewski, A.; Stawinski, J.; Shugar, D. J. Org.
ꢀ
Chem. 1998, 63, 8150–8156.
11. Ammonium phosphinate 6 (1.66 g, 20.0 mmol), hexamethyl-
disilazane (12.66 mL, 60.0 mmol), N-[(2-cyanoethyl)sul-
fanyl]phthalimide12
5
(13.94 g, 60 mmol) and dry
dichloromethane (80 mL) were stirred together at room
temperature, in an atmosphere of argon, for 24 h. The
products were evaporated under reduced pressure and
pyridine–water (1:9 v/v, 100 mL) was added. The stirring
was continued for a further period of 1 h and the products
were evaporated under reduced pressure. Water (100 mL)
was then added, and the resulting mixture was stirred for
30 min and filtered. The residue was washed with water
(20 mL), and the combined filtrate and washings were
concentrated under reduced pressure. The residue was
dissolved in methanol (50 mL) and ammonium carbonate
(2.0 g) was added. After 10 min, the products were
evaporated to dryness under reduced pressure. Crystalli-
sation of the residue from propan-2-ol gave ammonium
bis-S-(2-cyanoethyl) phosphorodithioate 7 as colourless
crystals (4.10 g, 81%) (Found: C, 28.55; H, 4.9; N, 16.4.
C6H12N3O2PS2 requires: C, 28.45; H, 4.78; N, 16.59%),
Acknowledgements
We thank Avecia Ltd for generous financial support.
References and notes
1. Moffatt, J. G.; Khorana, H. G. J. Am. Chem. Soc. 1961,
83, 649–658.
2. Moffatt, J. G.; Khorana, H. G. J. Am. Chem. Soc. 1961,
83, 663–675.

2656
C. B. Reese, H. Yan / Tetrahedron Letters 45 (2004) 2653–2656
mp 133–134 ꢁC; dC [(CD3)2SO] 18.97, 27.80, 120.21; dP
[(CD3)2SO] 28.77.
12. Klose, J.; Reese, C. B.; Song, Q. Tetrahedron 1997, 53,
(0.100 g, 0.13 mmol) in acetonitrile (1.0 mL). The reaction
solution was heated at 60 ꢁC in a sealed vessel for 6 h.
Aqueous ammonia (d 0.88, 1.0 mL) was added to the
cooled products. After 5 min, the products were concen-
trated under reduced pressure and co-evapoarated with
absolute ethanol–7 M methanolic ammonia (99:1 v/v;
2 · 2 mL). Diethyl ether (30 mL) was added to a solution
of the residue in methanol (1.5 mL). The solid precipitate
was collected by centrifugation and re-precipitated by
adding ethyl acetate (30 mL) to its solution in methanol
(1.5 mL) to give the 1,1,3,3-tetramethylguanidinium salt of
adenosine 50-phosphorodithioate as a colourless hygro-
scopic solid (0.060 g); dH [D2O] 2.84 (12H, s), 4.07 (2H, m),
4.35 (1H, m), 4.49 (1H, m), 4.74 (1H, m), 6.04 (1H, d, J
6.1), 8.12 (1H, s), 8.73 (1H, s); dP [D2O] 89.9.
14411–14416.
13. 6-N,20-O,30-O-Tri-(phenoxyacetyl)adenosine
8
was
obtained in two steps (acylation with phenoxyacetic
anhydride in the presence of triethylamine and DMAP
in dichloromethane, followed by treatment with dichloro-
acetic acid and pyrrole14 in dichloromethane) and in 70%
overall yield from 50-O-(4,40-dimethoxytrityl)adenosine;15
dH [(CD3)2SO] 3.73 (2H, m), 4.32 (1H, m), 4.68 (1H, d, J
16.9), 4.77 (1H, d, J 16.9), 4.90 (1H, d, J 16.8), 4.97 (1H, d,
J 16.8), 5.05 (2H, s), 5.46 (1H, t, J 5.5), 5.72 (1H, dd, J 3.0
and 5.4), 6.11 (1H, t, J 6.0), 6.37 (1H, d, J 6.5), 6.80–7.00
(9H, m), 7.19 (2H, m), 7.31 (4H, m), 8.73 (1H, s), 8.75 (1H,
s), 11.01 (1H, s).
22. 50-O-(Phenoxyacetyl)thymidine23 10 was prepared by the
action of phenoxyacetyl chloride on thymidine in the
presence of 2,6-lutidine in acetonitrile solution, according
to the procedure reported24 for the preparation of 50-O-(4-
chloro-phenoxyacetyl)thymidine; it was obtained as a
colourless crystalline solid, mp 133–134 ꢁC, in 86% yield;
dH [(CD3)2SO] 1.75 (3H, d, J 1.0), 2.09 (2H, m), 3.95 (1H,
m), 4.21 (1H, m), 4.32 (1H, m), 4.81 (1H, d, J 6.6), 4.88
(1H, d, J 6.6), 5.42 (1H, d, J 4.3), 6.20 (1H, t, J 7.0), 6.94
(3H, m), 7.28 (2H, m), 7.45 (1H, m), 11.34 (1H, br s).
23. Baker, B. R.; Neenan, J. P. J. Med. Chem. 1972, 15, 940–
944.
14. Reese, C. B.; Serafinowska, H. T.; Zappia, G. Tetrahedron
Lett. 1986, 27, 2291–2294.
15. Song, Q.; Wang, W.; Fischer, A.; Zhang, X.; Gaffney, B.
L.; Jones, R. A. Tetrahedron Lett. 1999, 40, 4153–4156.
16. Reese, C. B.; Zhang, P.-Z. J. Chem. Soc., Perkin Trans. 1
1993, 2291–2301.
17. 2,4,6-Triisopropylbenzenesulfonyl chloride 13 (0.442 g,
1.46 mmol) was added to a stirred solution of 6-N,20-
O,30-O-tri-(phenoxyacetyl)adenosine
8
(0.390 g, 0.58
mmol), ammonium bis-S-(2-cyanoethyl) phosphorodithio-
ate 7 (0.221 g, 0.87 mmol) and 3-nitro-1,2,4-1H-triazole 14
(0.166 g, 1.46 mmol) in dry pyridine (10 mL) at rt. After
1 h, methanol–water (1:1 v/v, 1 mL) was added and the
products were worked-up and chromatographed on silica
gel to give the triester 9 as a colourless froth (0.45 g, 87%);
dH [(CD3)2SO] 2.90 (4H, m), 3.10 (4H, m), 4.53 (3H, m),
4.77 (1H, d, J 16.9), 4.83 (1H, d, J 17.0), 4.84 (1H, d, J
17.0), 4.93 (1H, d, J 16.8), 5.05 (2H, s), 5.89 (1H, m), 6.21
(1H, t, J 5.5), 6.41 (1H, d, J 5.2), 6.92 (9H, m), 7.22 (2H,
m), 7.31 (4H, m), 8.70 (1H, s), 8.73 (1H, s), 11.03 (1H, s);
dP [(CD3)2SO] 55.8.
24. Arentzen, R.; Reese, C. B. J. Chem. Soc., Perkin Trans. 1
1977, 445–460.
25. dH [D2O] 1.89 (3H, s), 2.26 (1H, m), 2.42 (1H, m), 2.77 (ca.
24H, s), 3.69 (1H, dd, J 4.9 and 12.6), 3.75 (1H, dd, J 3.2
and 12.6), 4.06 (1H, m), 4.83 (1H, m), 6.16 (1H, t, J 6.8),
7.52 (1H, s); dP [D2O] 89.8.
26. The chemical shifts of the 31P resonance signals of
trisodium dithio- and thio-phosphates in 10% aqueous
sodium sulfide are reported27 to be 61 and 32 ppm,
respectively. However, the 31P chemical shifts of dithio-
and thio-phosphoric acids would be expected to be pH and
solvent dependent.
18. Evans, D. A.; Gage, R. G.; Leighton, J. L. J. Org. Chem.
1992, 57, 1964–1966.
19. Gaffney, P. R. J.; Reese, C. B. J. Chem. Soc., Perkin Trans.
1 2001, 192–205.
27. Maier, L.; Van Wazer, J. R. J. Am. Chem. Soc. 1962, 84,
3054–3058.
20. Reese, C. B.; Stewart, J. C. M. Tetrahedron Lett. 1968,
4273–4276.
21. Tetramethylguanidine (TMG) (0.099 mL, 0.79 mmol) and
chlorotrimethylsilane (0.073 mL, 0.58 mmol) were added
to a dry solution of the fully-protected intermediate 9
28. We are unable to explain why what is believed to be
dithiophosphate 15 (dP 68.1) was not observed as an
intermediate decomposition product in the original study4
relating to the action of CD3CO2D–D2O (95:5 v/v) on the
dithiophosphoromorpholidate 1c.