Synthesis, chemical and enzymatic reactivity, and toxicity of dithymidylyl-3′,5′-phosphorofluoridate and -phosphorothiofluoridate

Article abstract of DOI:10.1016/S0968-0896(01)00025-6

Dithymidylyl-3′,5′-phosphorofluoridate and phosphorothiofluoridate were obtained by fluorinolysis of the P-Se bond in appropriate bisidmethoxytrityl selenomethyl esters. These compounds, which are hydrolytically unstable, are not inhibitors of snake venom, spleen phosphodiesterases and alkaline phosphatase. Neither compound is highly toxic. Copyright

Full text of DOI:10.1016/S0968-0896(01)00025-6

Bioorganic & Medicinal Chemistry 9 (2001) 1525–1532
Synthesis, Chemical and Enzymatic Reactivity, and Toxicity of
Dithymidylyl-30,50-phosphorofluoridate
and -phosphorothiofluoridate
Konrad Misiura,a,* Daria Szymanowicza and Halina Kusnierczykb
aDepartment of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences,
´
´
Sienkiewicza 112, 90-363 —odz, Poland
bDepartment of Tumour Immunology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences,
R. Weigla 12, 53-114 Wroc•aw, Poland
Received 21 December 2000; revised 15January 2001; accepted 29 January 2001
Abstract—Dithymidylyl-30,50-phosphorofluoridate and phosphorothiofluoridate were obtained by fluorinolysis of the P–Se bond in
appropriate bisdimethoxytrityl selenomethyl esters. These compounds, which are hydrolytically unstable, are not inhibitors of
snake venom, spleen phosphodiesterases and alkaline phosphatase. Neither compound is highly toxic. # 2001 Elsevier Science Ltd.
All rights reserved.
Introduction
their strong inhibition of acetylcholinesterases.8 Despite
the well-established chemistry of O,O-dialkyl phos-
phorofluoridates9 and phosphorothiofluoridates, the
corresponding dinucleoside analogues10 were obtained
only recently.
Oligonucleotide analogues possessing modified inter-
nucleotide phosphate moieties are extensively studied in
aspects of their synthesis1 and stereochemistry,2 stability
against nucleases3 and their biological activity4 (anti-
sense, antigene, and aptamer concepts).
The oxathiaphospholane method for the synthesis of
oligo(nucleoside phosphorothioate)s11 (S-Oligo) and its
dithiaphospholane modification leading to oligo(nu-
cleoside phosphorodithioate)s12 (S2-Oligo) have been
developed recently as the new strategy for the synthesis
of P-modified oligonucleotides where one or both non-
bridging oxygens at the internucleotide phosphate are
replaced by one or two different atoms. It was interest-
ing to examine if phospholane procedures can be
applied for the synthesis oligo(nucleoside phosphoro-
fluoridate)s (F-Oligo) and phosphorothiofluoridates
(SF-Oligo). To assess the potential utility of F-Oligo or
SF-Oligo for biological studies, the synthesis of model
fully deprotected dinucleotides has been developed and
their hydrolytic stability, chemical and enzymatic reac-
tivity, and toxicity in mice were studied.
The introduction of a fluorine atom attached to phos-
phorus may influence the specific chemical and biologi-
cal reactivity of such nucleoside phosphorofluoridates
and phosphorothiofluoridates. Although the attachment
of the fluorine to phosphorus involved in internucleo-
tide linkage eliminates the formal negative charge, the
analogue may still be involved in hydrophilic inter-
actions with other biomolecules due to the ability of
fluorine to form strong hydrogen bonds.5 Horner and
Gehring demonstrated6 that phosphoric acid diesters
containing a P(O)F moiety selectively react with HO
nucleophiles even in the presence of NH2 and SH
groups. Due to such chemoselectivity, O,O-diisopropyl
phosphorofluoridate could be used in the classical stu-
dies of the mechanism of serine proteases.7 Simple O,O-
dialkyl phosphorofluoridates are highly toxic due to
Results and Discussion
Our approach to the synthesis of F-Oligo and SF-Oligo
was based on the concept of the solid-phase method to
*Corresponding author. Tel.: +48-42-681-9744; fax: +48-42-681-
5483; e-mail: kmisiura@bio.cbmm.lodz.pl
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00025-6

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K. Misiura et al. / Bioorg. Med. Chem. 9(2001) 1525–1532
make oligonucleotide intermediates which, after release
from the support and deprotection of amino groups in
nucleobases, could be transformed into desired products.
Such a concept was supported by early observations13 of
Stec et al. that dithymidylyl-30,50-phosphorofluoridate
intermediate, formed in the reaction of appropriate
phosphorothioate with 2,4-dinitrofluorobenzene in pyri-
dine solution, reacted in situ with ethanol to give the
corresponding O-ethylphosphotriester. Also,0in our pre-
vious studies10a we proved that 30-O-acetyl-5 -O-DMTr-
dithymidylyl-30,50-phosphorofluoridate treated with
ammonia solution was completely hydrolysed within 15
min. Thus one could expect that exposure of F-Oligo or
SF-Oligo to this reagent commonly used in the solid-
phase synthesis of oligonucleotides for their release
from the solid support and nucleobase deprotection
may lead to the substantial hydrolytic loss of fluorine.
Our interest therefore focused on oligo(nucleoside
phosphoroselenoate)s (Se-Oligo) and phosphoro-
thioselenoates (SSe-Oligo) which could be directly
transformed into appropriate fluorinated derivatives or
could be methylated to form Se-methyl phosphoro-
selenoate and phosphorothioselenoate triesters. The
selenomethyl moiety is known to be a good leaving
group when a nucleophile attacks a phosphorus atom14
and the fluorine anion possesses strong nucleophilic
avidity towards P-IV compounds;15 such substitution
was expected to provide F-Oligo and SF-Oligo.
Dithymidylyl-30,50-phosphoroselenoate (5) was then
used as a substrate for studies on the synthesis of
appropriate phosphorofluoridates. Phosphoroselenoate
5 reacted with 25% aq AgF in acetonitrile solution
providing dithymidylyl-30,50-phosphorofluoridate (7) as
the mixture of diastereoisomers in ca. 1:1 ratio, (31P
NMR control, dP -9.45( JP-F=981 Hz), dP -9.06 (JP-F
=
973 Hz)) in 100% yield in spite of the presence of water
introduced into the reaction mixture by AgF solution.
However, any attempts to isolate 7 from these reaction
mixtures by silica gel chromatography using several
eluting solutions failed, probably due to silica gel-assis-
ted hydrolysis of the desired product. Also attempts to
purify phosphorofluoridate 7 by its extraction from
aqueous solutions into chloroform or ethyl acetate were
unsuccessful since this compound is highly hydrophilic.
Recently, AgF solution was employed for efficient con-
version16 of simple phosphorothioates and phosphor-
oselenoates into appropriate phosphorofluoridates,
however these products were not isolated. Indepen-
dently, Stawinski described transformation of 5 into 7
by means of triethylamine trihydrofluoride and iodine in
a pyridine solution, but attempts to isolate this product
have also failed.17 This method was used for synthesis of
tetra-thymidine phosphorofluoridate18 but the product
hydrolysed during its purification by means of RP-
HPLC.
For the synthesis of Se-Oligo and SeS-Oligo, we devel-
oped a new method based on oxathia- and dithiapho-
spholane chemistry. To establish reaction conditions,
we started with the synthesis ‘in solution’ (Scheme 1).
50-O-DMTr-thymidine-30-O-(2-seleno-1,3,2-oxathiaphos-
pholane) (1) and -1,3,2-dithiaphospholane (2) were
obtained in reaction of 50-O-DMTr-thymidine with N,N-
diisopropylamino-1,3,2-oxathiaphospholane and -1,3,2-
dithiaphospholane in the presence of 1H-tetrazole, and
subsequent oxidation of P-III intermediates with ele-
mental selenium. Compounds 1 and 2 were condensed
with 30-O-acetylthymidine in the presence of 1,8-diaza-
bicyclo[4.3.0]undec-7-ene (DBU) to give 30-O-acetyl-50-
O-DMTr-thymidylyl-30,50-phosphoroselenoate (3) and
-30,50-phosphorothioselenoate (4), which were, without
isolation, deprotected by acetic acid followed by
ammonia treatment. Crude products were purified by
anion-exchange chromatography on DEAE-Sephadex.
The overall yield of dithymidylyl-30,50-phosphoro-
selenoate (5) and -phosphorothioselenoate (6) were 71
and 58%, respectively. Compounds 5 and 6 were obtained
as a mixture of diastereoisomers in nearly equimolar
ratio. Subsequently, the applicability of this method
under conditions of ‘solid-phase’ synthesis was examined.
50-O-DMTr-thymidine (1 mmol) anchored to controlled
pore glass support was detritylated and treated with a
mixture of 1 (0.2 M) and DBU (0.5M) in dry aceto-
nitrile. After 10 min, the reaction column was washed
with acetonitrile and, after detritylation, dithymidylyl-
30,50-phosphoroselenoate (5) was released from solid
support by ammonolysis. It was found that 5 was formed
in 97% yield (HPLC assay). In a similar experiment
starting from dithiaphospholane 2, dithymidylyl-30,50-
phosphorothioselenoate (6) was obtained in 96% yield.
Scheme 1. (a) (i) N,N-diisopropylamino-1,3,2-oxathia(dithia)phos-
pholane, 1H-tetrazole; (ii) Se8; (b) 30-O-acetylthymidine, DBU; (c) (i)
CH3COOH, (ii) NH4OH; (d) MeI; (e) AgF for 8, Et3NHF for 15.

K. Misiura et al. / Bioorg. Med. Chem. 9(2001) 1525–1532
1527
The failure to isolate phosphorofluoridate 7 formed
during direct PSe!PF transformation prompted us to
examine the possibility of synthesis of PF-Oligo via
selenomethyl intermediates. Phosphoroselenoate 5 was
methylated with methyl iodide providing the seleno-
methyl triester 8 in 81% yield. Similarly as for simple
dialkyl phosphoroselenoates, methylations occurred
exclusively at the selenium atom, as proved by 31P
NMR analysis of the reaction mixture. In our previous
studies,10a it was found that the fluoride anion can
smoothly substitute the MeSe group and that 30-O-ace-
tyl-50-O-DMTr-dithymidylyl-30,50-phosphorofluoridate,
isolated by extraction, was obtained in a good yield.
Indeed, reactions of Se-methyl phosphoroselenoate 8
with AgF in acetonitrile, pyridine, and DMSO solutions
were also fast and efficient, providing, after 1 h, phos-
phorofluoridate 7 in 100% yield (31P NMR assay) but
any attempts to isolate this compound failed.
(mixture of diastereoisomers ca. 1:1) was obtained with
85% yield. DMTr protecting groups were removed
from 11 by exposure to 3% p-toluenesulfonic acid pro-
viding the desired dithymidylyl-30,50-phosphoro-
fluoridate (7). Crude 7 was purified by fast column
chromatography on silica gel. Pure phosphorofluoridate
7 (mixture of diastereoisomers ca. 1:1) was obtained
with 53% yield.
The thio-analogue of 7 was synthesised by the same
method starting from 50-O-DMTr-thymidine-30-O-(2-
seleno-1,3,2-dithiaphospholane) (2) (Scheme 2). The
only difference was that triethylammonium fluoride was
used during the fluorination step of 30,50-O,O-bis-DMTr-
dithymidylyl-30,50-Se-methylphosphorothioselenoate (12)
because AgF caused rapid oxidation of thio compound
13 into oxo congener 11. Dithymidylyl-30,50-phosphoro-
thiofluoridate (14) (mixture of diastereoisomers in ca.
1:1 ratio) was obtained with 12% total yield. Com-
pound 14 was synthesized earlier19 by us using direct
fluorination of dithymidylyl-30,50-Se-methyl phosphoro-
thioselenoate (15) but it could not be obtained in a pure
form.
Problems with purification of dithymidylyl-30,50-phos-
phorofluoridate (7) mentioned above suggested that, for
the isolation of F-Oligo, the presence of fluoride anions
during the silica gel purification should be avoided.
Thus, it was decided to modify the synthetic procedure
by keeping the DMTr protection group of the 30- and
50-hydroxyls during PSeMe!PF transformation. The
presence of two lipophilic groups should enable good
solubility of the compound in organic solvents like
chloroform and allow its purification by means of
extraction. So oxathiaphospholane 1 reacted with
30-O-DMTr-thymidine in a presence of DBU giving 30,50-
O,O-bis-DMTr-dithymidylyl-30,50 -phosphoroselenoate
(9), which was, without, isolation methylated with
methyl iodide providing selenomethyl triester 10 (mix-
ture of diastereoisomers in ca. 1:1 ratio) with 68% yield
(Scheme 2). Compound 10 reacted with 25% aq AgF in
acetonitrile solution and, after 1 h, 30,50-O,O-bis-DMTr-
dithymidylyl-30,50-phosphorofluoridate (11) was formed
in 100% yield (31P NMR assay). Crude 11 was dissolved
in chloroform and washed with brine to remove an
excess of silver fluoride. Pure phosphorofluoridate 11
Hydrolytic lability of phosphorofluoridate 7 and phos-
phorothiofluoridate 14 leading to dithymidylyl-30,50-
phosphate (16) or dithymidylyl-30,50-phosphorothioate
(17), respectively, was examined by means of 31P NMR
and HPLC (Scheme 3). Compounds 7 and 14 (both
2 mM) in 100 mM Tris–HCl, pH 7.5were hydrolysed at
room temperature after 1 h in 77 and 26%, respectively,
with complete hydrolysis after 2 h for 7 and 12 h for 14.
Compound 7 (0.1 mM) in the same buffer was com-
pletely hydrolysed after 5min at 37 ꢀC whereas 1 h was
required to hydrolyse 14. Compounds 7 and 14 (both
2 mM) were hydrolysed in less then 5min ( 31P NMR
assay) under alkaline conditions in 14% NH4OH. A
great acceleration of the rate of hydrolysis of O,O-dii-
sopropyl phosphorofluoridate in alkaline conditions
versus neutral one (k(OH)/k(H2O)=8.2ꢁ106) had been
observed earlier by Hudson.20 The only nucleoside
phosphorofluoridate diester reported21 to be stable to
the ammonia solution was 20-deoxycytidine cyclic 30,50-
phosphorofluoridate (single diastereoisomer). The most
probable explanation for this unusual hydrolytic stabi-
lity is a ring strain effect operating during formation of
the P-V intermediate possessing a six-membered ring in
a diaxial orientation. On the other hand, no difference
in the rates of hydrolysis between O,O-diethyl phos-
phorofluoridate and cyclic 2-fluoro-1,3,2-dioxapho-
sphinane 2-oxide were found.22 So one can speculate
that the bicyclic ring system present in 20-deoxycytidine
cyclic 30,50-phosphorofluoridate should be responsible
for this unusual stability. Compounds 7 and 14 also
underwent fast methanolysis leading to the appropriate
methyl esters 18 and 19 when dissolved in methanol in
the presence of bases like triethylamine or pyridine. In
the presence of DBU an intramolecular cyclisation of 7
and 14 occurred giving compounds 20 and 21. 31P NMR
analysis of 20 and 21 revealed the presences of only two
signals (dꢂ3.17, ꢂ4.62 (ratio 1:2), d 66.30, 63.66
(ratio 3:1), respectively) suggesting that the cyclisation is
50-regioselective and stereoselective.
Scheme 2. (a) (i) 30-O-DMTr-thymidine, DBU; (ii) MeI; (b) AgF for
10, Et3NHF for 12; (c) p-toluenesulfonic acid.

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K. Misiura et al. / Bioorg. Med. Chem. 9(2001) 1525–1532
Scheme 3. (a) Tris–HCl pH8 or NH4OH; (b) MeOH, Et3N; (c) DBU.
The high reactivity of phosphorofluoridate 7 and phos-
phorothiofluoridate 14 towards OH nucleophiles and
the known inhibitory effect of O,O-dialkyl phosphoro-
fluoridates of serine proteases7 prompted us to examine
the inhibition of certain phosphodiesterases by 7 or 14.
Snake venom and spleen phosphodiesterases were cho-
sen for those studies since both kinetic and stereo-
chemical analysis23 proved formation of a covalent
enzyme substrate complex during catalysed reactions,
indicating the presence of hydroxy amino acids in the
active centres of these enzymes. HPLC analysis of
hydrolysis of dithymidylyl-30,50-phosphate (16) by both
snake venom and spleen phosphodiesterases performed
in the presence of phosphorofluoridate 7 or phosphoro-
thiofluoridate 14 in concentrations up to 200 nM
showed that no inhibitory effect was observed. Earlier,
we also proved24 that 7 and 14 are not substrates for
these enzymes. Similarly, 7 and 14 were not inhibitors of
alkaline phosphatase,25 which is known to possess ser-
ine residue in its active centre. Most probably, phos-
fluoridates cannot be explained by an anchimeric assis-
tance of 50- or 30-hydroxyl groups since no major
differences in the rate of hydrolysis between 7 and its
30-acetyl-50-DMTr protected analogue were observed.
Most probably this reactivity is caused by the hydro-
philic nature of nucleoside residues. In our opinion, such
hydrolytic instability precludes the use of F-Oligo and
SF-Oligo in biochemical and biological studies.
Experimental
Methylene chloride, acetonitrile, tetrahydrofuran, and
trietylamine were dried by theirs distillation from cal-
cium hydride. Dry DBU was obtained by its distillation
from calcium hydride under reduced pressure. Com-
mercial 25% solution of AgF was used. 1 M TEAF in
dry THF was obtained by neutralisation of triethyla-
mine trihydrofluoride with two equivalents of dry Et3N.
Snake venom and spleen phosphodiesterases (svPDE
and sPDE) were from Sigma and alkaline phosphatase
(AP) from Boehringer Mannheim.
phorofluoridate
7 and phosphorothiofluoridate 14
undergo hydrolysis faster than they react with a
hydroxy group of amino acid residue present in active
centres of these enzymes. Lack of inhibition of these
enzymes can also suggest low reactivity of 7 and 14
towards cholinoesterases. Indeed, it was found that 7
and 14 were not toxic to mice at the concentration up to
10 mg/kg, which is a much higher level than observed8
for phosphorofluoridate nerve agents (LD50 in a range
of 0.01–1 mg/kg). The observed lack of toxicity of 7 and
14 can be also explained by fast hydrolysis of these
compounds in biological fluids.
Unless stated otherwise, the progress of each reaction
was monitored on TLC. HPLC analyses were per-
formed on LDC Analytical using Econoshere C-18
(Alltech) reverse-phase column (flow 1 ml/min) and
gradient 0!60% B (A, 0.1 M CH3COONH4, B, 0.02 M
CH3COONH4 in 80% acetonitrile) in 15min. 1H and
31P NMR spectra were recorded on a Brucker AC-200.
Mass spectrometry analyses were performed using a
Finnigan MAT 95apparatus.
The high hydrolytic reactivity of phosphorofluoridate 7
as compared to other simple O,O-dialkyl phosphoro-

K. Misiura et al. / Bioorg. Med. Chem. 9(2001) 1525–1532
1529
50-O-DMTr-thymidine-30-O-(2-seleno-1,3,2-oxathiaphos-
pholane) (1). To a stirred solution of 50-O-DMTr-thy-
midine (2.72 g, 5.0 mmol) and 1H-tetrazole (0.42 g,
6 mmol) in anhydrous methylene chloride (20 mL)
was added 2-N,N-diisopropylamino-1,3,2-oxathiapho-
spholane11b (1.24 g, 6 mmol). After 1 h, selenium (0.60 g,
7.5mmol) was added to the reaction mixture. P-III
Intermediate (dP 172.9, 171.8) was oxidized within 12 h
(31P NMR assay). The reaction mixture was filtered and
the filtrate was washed with water (2ꢁ25mL). The
organic layer was dried and filtered and the solvent was
evaporated. Crude product was purified by silica-gel
column chromatography eluted with methylene chloride
containing 0–5% of ethanol. Product 1 was obtained as
solid foam (2.80 g, 77% yield): TLC (methylene chloride/
ethanol 19:1) Rf 0.46; 1H NMR (CDCl3) d 1.41, 1.42 (pair
of d, J=1.1 Hz, J=1.0Hz, 3H), 2.57 (m, 2H), 3.48 (m,
4H), 3.79 (s, 6H), 4.41 (m, 1H), 4.50 (m, 2H), 5.60 (m, 1H),
6.45(dd, J=8.8Hz, J=5.7 Hz, 1H), 6.84 (d, J=9.0 Hz,
4H), 7.27 (m, 9 H), 7.60, 7.61 (pair of q, J=1.1 Hz,
J=1.0 Hz, 1H), 8,45(s, 1H); 31P NMR (CDCl3) d: 99.0
(JP-Se=953 Hz), 99.2 (JP-Se=952 Hz); MS (ꢂFAB) m/z
729.0 (MꢂH), (+FAB) m/z 731.3 (M+H); elemental
analysis for C33H35O8N2PSSe: calcd/found C 54.32/54.42,
H 4.83/4.91, N 3.84/3.80, P 4.25/4.58, S 4.39/4.33.
ammonium hydroxide (25mL). After 16 h, the reaction
mixture was concentrated and the residue dissolved in
water (100 mL). Obtained suspension was filtered, and
the filtrate was evaporated. Crude product was purified
on DEAE-Sephadex column eluting with 0.02–0.8 M
TEAB, pH 7.0. Product 5 was obtained as solid foam
(1.26 g, 71% yield): HPLC tR 8.42, 8.57; 1H NMR
(D2O) d: 1.18 (t, J=7.3 Hz, 9H), 1.79 (d, J=1.0 Hz,
3H), 1.84, 1.85(pair of d, J=1.0 Hz, 3H), 2.27 (m, 4H),
3.10 (q, J=7.3 Hz, 6H), 3.75(m, 2H), 4.10 (m, 4H), 4.49
(m, 1H), 4.92 (m, 1H), 6.13 (t, J=6.8 Hz, 1H), 6.23 (t,
J=6.8 Hz, 1H), 7.58 (q, J=1.0 Hz), 7.65(q, J=1.0 Hz,
1H); 31P NMR (D2O) d 49.8 (JP-Se=777 Hz), 49.1
(JP-Se=775Hz); MS ( ꢂFAB) m/z 609.1 (MꢂH).
Dithymidylyl-30,50-phosphorothioselenoate (6). To a stir-
red solution of 50-O- DMTr-thymidine-30-O-(2-seleno-
1,3,2-dithiaphospholane) (1) (1.84 g, 2.5mmol) and 3 0-
O-acetylthymidine (0.86g, 3.0 mmol) in anhydrous aceto-
nitrile (25mL) was added DBU (0.45mL, 3.0 mmol).
The reaction was completed after 15min providing 3 0-O
-acetyl-50 -O-DMTr-dithymidylyl-30,50 -phosphorothio-
selenoate (4) (dP 103.1 (JP-Se=782 Hz), 103.4 (JP-Se
=
783 Hz)) with 96% yield. The mixture was concentrated
and the residue was treated with 80% acetic acid
(25mL) for 6 h. Then the solution was concentrated.
Obtained residue was dissolved in acetonitrile (25mL)
and concentrated ammonium hydroxide (25mL). After
16 h, the reaction mixture was concentrated and dis-
solved in water (100 mL). Obtained suspension was fil-
tered and the filtrate was evaporated. Crude product
was purified on DEAE-Sephadex column eluting with
0.025–1.0 M TEAB, pH 7.0. Product 6 was obtained as
50 -O-DMTr-thymidine-30 -O-(2-seleno-1,3,2-dithiaphos-
pholane) (2). To a stirred solution of 50-O-DMTr-thy-
midine (2.72 g, 5mmol) and 1 H-tetrazole (0.38 g,
5.5 mmol) in anhydrous methylene chloride (20 mL)
was added 2-N,N-diisopropylamino-1,3,2-oxathiaphos-
pholane26 (1.23 g, 5.5 mmol). After 1 h selenium (0.55 g,
6.9 mmol) was added to reaction mixture. The P-III
Intermediate (dP 152.4) was oxidized within 12 h (31P
NMR assay). The reaction mixture was filtered and the
filtrate was washed with water (2ꢁ25mL). The organic
layer was dried, filtered and the solvent was evaporated.
Crude product was purified by silica-gel column chro-
matography eluted with methylene chloride containing
0–5% of ethanol. Product 2 was obtained as solid foam
(2.76 g, 74% yield): TLC (methylene chloride/ ethanol
1
solid foam (1.05g, 58% yield): HPLC tR 8.47, 8.59; H
NMR (D2O) d 1.29 (t, J=7.3 Hz, 9H), 1.89 (s, 3H), 1.98
(s, 3H), 2.38 (m, 4H), 3.22 (q, J=7.3 Hz, 6H), 3.88 (m,
2H), 4.22 (m, 4H), 4.62 (m, 1H), 5.20 (m, 1H), 6.25 (t,
J=6.8 Hz, 1H), 6.34 (t, J=6.9 Hz, 1H), 7.7 (s, 1H), 7.8
(s, 1H); 31P NMR (D2O) d 102.2 (JP-Se=753 Hz),
102.5( JP-Se=754 Hz); MS (ꢂFAB) m/z 625.6 (MꢂH),
(+FAB) m/z 627.8 (M+H).
1
19:1) Rf 0.46; H NMR: (CD3Cl) d: 1.44 (d, J=1.1 Hz,
3H), 2.61 (m, 2H), 3.59 (m, 6H), 3.79 (s, 6H), 4.32 (m,
1H), 5.60 (dd, J=15Hz, J=5.9 Hz, 1H), 6.46 (dd,
J=8.8 Hz, J=5.5 Hz, 1H), 6.84 (d, J=8.9, 4H), 7.28
(m, 9H), 7.60 (q, J=1.1 Hz), 8.61 (s, 1H); 31P NMR
(CDCl3) d 106.0 (JP-Se=884 Hz); MS (-FAB) m/z 745.2
(M-H), (+FAB) m/z 747.5(M+H); elemental analysis
for C33H35O7N2PS2Se: calc./found C 53.15/53.48, H
4.73/4.83, N 3.76/3.73, P 4.15/4.22, S 8.60/8.44.
Solid-phase synthesis of dithymidylyl-30,50-phosphoro-
selenoate (5) and dithymidylyl-30,50-phosphorothioselenoate
(6). 50-O-DMTr-thymidine (1 mmol) bound to the stan-
dard LCA CPG solid support was detritylated with 3%
solution of dichloroacetic acid in methylene chloride
(3 mL) within 3 min. Then the column was dried (water
pump vacuum) and washed with dry acetonitrile
(10 mL), dried again and filled with a mixture of 1
(29 mg, 0.04 mmol) and dry DBU (36 mL, 0.2 mmol ) in
dry acetonitrile (150 mL) and left for 10 min. Column
was washed with dry acetonitrile (10 mL), 3% dichloro-
acetic acid in methylene chloride (3 mL) and acetonitrile
(10 mL) again. The support was dried and the product
was released by washing the column with 30% ammonia
(1 mL) within 1 h. The solution was concentrated on a
Speed-Vac. The residue was dissolved in water (1 mL)
and filtered. The solution of 5 was analysed by RP-
HPLC.
Dithymidylyl-30,50-phosphoroselenoate (5). To a stirred
solution of 50-O-DMTr-thymidine-30-O-(2-seleno-1,3,2-
oxathiaphospholane) (1) (1.82 g, 2.5mmol) and 3 0-O-
acetylthymidine (0.86 g, 3.0 mmol) in anhydrous aceto-
nitrile (25mL) was added DBU (0.45mL, 3.0mmol). The
reaction was completed after 15min providing 3 0-O-acetyl-
50-O-DMTr-dithymidylyl-30,50-phosphoroselenoate
(3)
(dP 50.6 (JP-Se=813 Hz), 50.1 (JP-Se=814 Hz)) with
100% yield. The mixture was concentrated and the
residue was treated with 80% acetic acid (25mL) for
6 h. Then the solution was concentrated. The residue
was dissolved in acetonitrile (25mL) and concentrated
Starting from 2, and using the same procedure, product
6 was obtained.

1530
K. Misiura et al. / Bioorg. Med. Chem. 9(2001) 1525–1532
Dithymidylyl-30,50-Se-methyl phosphoroselenoate (8). To
stirred solution of dithymidyl-30,50-phosphoro-
(3.7 mL, 0.57 mmol). After 5 min, 10% solution of pyr-
idine in chloroform (0.48 mL, 0.6 mmol) was added to
the reaction mixture to neutralise the acid. The crude
product was purified by silica-gel column chromato-
graphy eluted with chloroform containing 0–15% etha-
nol. Product 7 was obtained as solid foam (0.11 g, 53%
a
selenoate (5) (0.71 g, 1.0 mmol) dissolved in a mixture of
solvents CH3CN/H2O/0.5M TEAB (pH 7.0) in a ratio
2.5:1.5:1 (10 mL) was added methyl iodide (0.12 mL,
2.0 mmol). After 2 h, reaction mixture was concentrated
and the residue was dissolved in acetonitrile and con-
centrated again. The residue was twice dissolved in
ethanol and concentrated. The crude product was
purified by silica-gel column chromatography eluted
with chloroform containing 0–15% ethanol. Product 8
was obtained as solid foam (0.52 g, 81% yield): TLC
1
yield): TLC (chloroform/ethanol 4:1) Rf 0.36; H NMR
(DMSO-d6) d: 1.78 (s, 6H), 2.15(m, 4H), 3.60 (m, 2H),
4.10 (m, 2H), 4.24 (m, 1H), 4.36 (m, 2H), 5.16 (m, 1H),
5.26 (m, 1H), 5.50 (d, J=4.4 Hz, 1H), 6.21 (m, 2H),
7.46 (s, 1H), 7.66 (s, 1H), 11.33 (s, 1H), 11.37 (s,
1H); 31P NMR (d6-DMSO) d: ꢂ9.2 (JP-F=977 Hz),
ꢂ9.5( JP-F=981 Hz); 19F NMR (d6-DMSO) d: ꢂ77.6
(JP-F=976 Hz), ꢂ77.9 (JP-F=981 Hz); MS (ꢂFAB) m/z
547.3 (MꢂH), (+FAB), m/z 549.5 (M+H).
30,50-O,O-Bis-DMTr-dithymidylyl -30,50-Se-methyl phos-
phorothioate (12). To a stirred solution of 50-O-DMTr-
thymidine-30-O-(2-seleno-1,3,2-dithiaphospholane) (2)
(2.3 g, 3.08 mmol) and 30-O-DMTr-thymidine (1.34 g,
2.46 mmol) in anhydrous acetonitrile (21 mL) was added
DBU (0.44 mL, 3.0 mmol). The 31P NMR spectrum
showed the completion of the reaction in 1 h. The mix-
ture was treated with methyl iodide (0.23 mL,
3.7 mmol). Progress of the methylation was observed by
31P NMR, which showed disappearence of substrate
after 1 h. The reaction mixture was concentrated and
crude product was purified by silica-gel column chro-
matography eluted with chloroform containing 0–5%
ethanol. The product was obtained as solid foam
(2.89 g, 75% yield): TLC (chloroform/ethanol 19:1) Rf
0.46, 31P NMR (CD3Cl) d 88.8 (JP-Se=492 Hz), 88.2
(JP-Se=492 Hz); MS (ꢂFAB) m/z (MꢂH), (+FAB) m/z
(M+H).
1
(chloroform/ethanol 4:1) Rf 0.36; H NMR (DMSO-d6)
d 1.77 (m, 6H), 2.18 (d, J=13.9 Hz, 3H), 2.38 (m, 4H),
3.32 (m, 4H), 3.62 (m, 2H), 4.25(m, 2H), .523 (t,
J=4.1 Hz, 1H), 5.45, 5.47 (pair of d, J=1.3 Hz, 1H),
6.21 (m, 2H), 7.48, 7.50 (pair of d, J=1.2 Hz, 1H), 7.67,
7.69 (pair of d, J=1.2 Hz, 1H), 11.35(m, 2H); 31P NMR
(d6-DMSO) d: 21.8, 21.9 (JP-Se=496 Hz); MS (ꢂFAB)
m/z 623.9 (MꢂH), (+FAB) m/z 625.8 (M+H).
30,50-O,O-Bis-DMTr-dithymidylyl-30,50-Se-methyl phos-
phoroselenoate (10). To a stirred solution of 50-O-
DMTr-thymidine-30-O-(2-seleno-1,3,2-oxathiaphospho-
lane) (1) (1.46 g, 2.0 mmol) and 30-O-DMTr-thymidine27
(0.86 g, 1.6 mmol) in anhydrous acetonitrile (12 mL) was
added DBU (0.24 mL, 1.6 mmol). The reaction was
completed after 15min ( 31P NMR (CH3CN) d 50.38
(J=809.7 Hz)) and the obtained mixture was treated
with methyl iodide (0.12 mL, 2.0 mmol). Progress of the
methylation was observed by 31P NMR, which showed
disappearance of the substrate after 2 h. The reaction
mixture was concentrated and the crude product was
purified by silica-gel column chromatography eluted
with chloroform containing 0–5% ethanol. Product 10
was obtained as solid foam (1.12 g, 68% yield): TLC
(chloroform/ethanol 19:1) Rf 0.45; 1H NMR (CDCl3) d:
1.58 (m, 6H), 1.82 (m, 4H), 2.00 (d, J=14.3 Hz, 3H),
3.45(m, 2H), 3.69 (m, 1H), 3.77 (s, 6H), 3.78 (s, 6H),
3.94 (m, 2H), 4.25(m, 2H), 6.10 (m, 1H), 6.36 (m, 2H),
6.83 (d, J=8.7 Hz, 8H), 7.30 (m, 18H), 7.42 (m, 2H),
8.16 (s, 1H), 8.22 (s, 1H); 31P NMR (CDCl3) d 22.1
(JP-Se=503 Hz), 22.0 (JP-Se=506 Hz); MS (ꢂFAB) m/z
1227.9 (MꢂH).
30,50-O,O-Bis-DMTr-dithymidylyl-30,50-phosphorofluori-
date (11). To a stirred solution of 30,50-O,O-Bis-DMTr-
dithymidylyl-30,50-Se-methyl phosphoroselenoate (10)
(0.162 g, 0.13 mmol) in acetonitrile (6 mL) was added
25% aqueous solution of silver fluoride (79 ml,
0.2 mmol). The 31P NMR spectrum showed the reaction
was completed after 1 h. Chloroform (10 mL) was added
to the reaction mixture and resulting mixture was
extraction with brine twice. The organic fraction was
dried, filtered and the solvent was evaporated. Product 11
was obtained as solid foam (0.13 g, 85% yield): 31P NMR
(CD3Cl) d ꢂ9.2 (JP-Se=979 Hz), ꢂ9.6 (JP-Se=985Hz);
MS (ꢂFAB) m/z 1152.8 (MꢂH).
30,50 -O,O-Bis-DMTr-dithymidylyl-30,50 -phosphorothio-
fluoridate (13). To a stirred solution of 30,50-O,O-bis-
DMTr-dithymidylyl-30,50-Se-methyl phosphoroseleno-
thioate (0.5g, 0.4 mmol) in acetonitrile (8 mL) was
added 1 M TEAF in dry THF (0.87 mL, 0.87 mmol) and
DBU (0.07 mL, 0.45mmol). 31P NMR spectrum showed
the reaction was completed after 0.5h. The reaction
mixture was concentrated and crude product was
purified by silica-gel column chromatography eluted
with methylene chloride containing 0–5% methanol.
The product was obtained as solid foam (0.21 g, 56%
yield): TLC (methylene chloride/methanol 19:1) Rf
0.38; 31P NMR (CH3Cl) d 61.8 (JP-F=1086 Hz), 61.1
(JP-F=1096 Hz); 19F NMR, purity 92%, d: ꢂ41.2
(JP-F=1085Hz), ꢂ42.1 (JP-F=1096 Hz), MS (ꢂFAB)
m/z 1152.8 (MꢂH).
Dithymidylyl-30,50-phosphorothiofluoridate (14). To a
stirred solution of 30,50-O,O-bis-DMTr-dithymidylyl-
30,50-phosphorothiofluoridate (0.136 g, 0.11 mmol) in
chloroform (2.8 mL) was added 3% p-toluenesulfonic
acid (1.18 mL, 0.19 mmol). The reaction was completed
after 5min (TLC assay). A 10% solution of pyridine in
chloroform (0.15mL, 0.18 mmol) was added to the
reaction mixture to neutralise the acid. The crude pro-
duct was purified by silica-gel column chromatography
eluted with chloroform containing 0–15% ethanol.
Product 14 was obtained as solid foam (0.025g, 38%
Dithymidylyl-30,50-phosphorofluoridate (7). To a stirred
solution of 30,50-O,O-Bis-DMTr-dithymidylyl-30,50-
phosphorofluoridate (11) (0.426 g, 0.37 mmol) in
chloroform (9 mL) was added 3% p-toluenesulfonic acid

K. Misiura et al. / Bioorg. Med. Chem. 9(2001) 1525–1532
1531
1
1
yield): TLC (chloroform/ethanol 4:1) Rf 0.58; H NMR
(DMSO-d6) d: 1.78 (s, 6H), 2.14 (m, 4H), 3.61 (m, 2H),
.3.97 (m, 1H), 4.11 (m, 1H), 4.26 (m, 1H), 4.46 (m, 2H),
5.02 (m, 1H), 5.28 (m, 1H), 5.50 (d, J=4.0 Hz, 1H), 6.21
(m, 2H), 7.43 (s, 1H), 7.67 (s, 1H), 11.34 (s, 2H); 31P
NMR (CD3Cl/C2H5OH 3/1) d 61.3 (JP-F=1081 Hz),
60.7 (JP-F=1093 Hz); 19F NMR d ꢂ42.8 (JP-F=1081 Hz),
ꢂ43.1 (JP-F=1093 Hz); MS (ꢂFAB) m/z 563.1 (MꢂH),
(+FAB) m/z 565.1 (M+H).
(chloroform/ethanol 4:1) Rf 0.80, H NMR (CD3CN) d
1.28 (s, 6H), 2.53 (m, 4H), 3.50 (m, 1H), 4.02 (m, 2H),
4.32 (m, 1H), 4.54 (m, 1H), 4.92 (m,1H), 6.23 (m, 2H),
7.38 (m,2H); 31P NMR (CD3CN) d 66.30, 63.66 (2.9:1),
MS (ꢂFAB) m/z 543.4 (MꢂH).
Enzyme inhibition studies. Dithymidylyl-30,50-phosphate
(16) (0.1 mM) and svPDE (0.005U/mL) in 0.1 M Tris–
HCl pH 8.0 containing 20 mM MgCl2, with or without 7
or 14 (both 0.05, 0.1, or 0.2 mM) added (total volume
1000 mL), were incubated at 37 ꢀC for 20, 40, and 60
min. After each time samples were analysed on HPLC.
In an analogous way, experiments with sPDE (0.15U/
mL) in 0.05M acetate buffer pH 5.0 were performed.
Dithymidylyl-30,50-O-methyl phosphate (18). To a solu-
tion of dithymidylyl-30,50-phosphorofluoridate (7)
(0.020 g, 0.036 mmol) in methanol (0.5mL) was added
triethylamine (0.025mL, 0.182 mmol). 31P NMR spec-
trum showed the reaction was completed after 1 h. The
crude product was purified by preparative TLC (devel-
oping system: chloroform/ethanol, 4:1 (v/v)). Product
18 was obtained with 65% yield (0.013 g): TLC
Adenosine 50-O-monophosphate (0.1 mM) and AP
(150 U/mL) in 0.05 M Tris–HCl pH 8.0 containing
5mM MgCl 2 with or without 7 or 14 (both 0.05, 0.1, or
0.2 mM) added (total volume 1000 mL), were incubated
at 37 ꢀC for 20, 40, and 60 min. After each time samples
were analysed on HPLC.
1
(chloroform/ethanol 4:1) Rf 0.45, H NMR (CD3OD) d
1.88 (s, 3H), 1.90 (d, J=1.1 Hz, 3H), 2.28 (m, 4H), 3.78
(m, 2H), 3.84, 3.85(pair of d, J=11.4 Hz, 11.3 Hz, 3H),
4.33 (m, 4H), 4.40 (m, 1H), 5.06 (m, 1H), 6.28 (m, 2H)
7.54 (m, 1H), 7.78 (m, 1H); 31P NMR (CH3OD): 0.22,
0.06; MS (ꢂFAB) m/z 559.3 (MꢂH)
Dithymidylyl-30,50-O-methyl phosphorothioate (19). To a
solution of dithymidylyl-30,50-phosphorothiofluoridate
(14) (0.037 g, 0.07 mmol) in methanol (0.6 mL) was
added triethylamine (0.05mL, 0.36 mmol). 31P NMR
spectrum showed the reaction was completed after 2.5h.
The crude product was purified by preparative TLC
(developing system: chloroform: ethanol, 4:1 (v/v)).
Product 19 was obtained with 45% yield (0.018 g): TLC
Mice toxicity studies. Compounds 7 and 14 were dis-
solved and diluted in the Hanks buffer immediately
before injections. Doses 2.5, 5.0 and 10.0 mg/kg were
given intraperitoneally to male mice CD2F1 (groups of
5). No mortality was observed during 3 weeks. In trea-
ted mice the loss of weight was dose-dependent and was
not more then 10% in the highest dose.
Acknowledgements
1
(chloroform/ethanol 4:1) Rf 0.75, H NMR (CD3OD) d
The authors thank Professor Wojciech J. Stec for valu-
able discussions. The work presented here was finan-
cially assisted by the State Committee for Scientific
Research (KBN) Grant No. 4 P05F 006 17.
1.88 (s, 3H), 1.90 (d, J=1.1 Hz, 3H), 2.28 (m, 4H), 3.78
(m, 2H), 3.84, 3.85(pair of d, J=11.4 Hz, 11.3 Hz, 3H),
4.33 (m, 4H), 4.40 (m, 1H), 5.06 (m, 1H), 6.28 (m, 2H)
7.54 (m, 1H), 7.78 (m, 1H); 31P NMR (CD3OD): 70.01,
69.89; MS (ꢂFAB) m/z 575.0 (MꢂH), (+FAB) m/z
577.6 (M+H)
References
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added DBU (0.027 mL, 0.182 mmol). 31P NMR spec-
trum showed completition of the reaction after 1 h. The
crude product was purified by preparative TLC (devel-
oping system: chloroform/ethanol, 4:1 (v/v)) Product 20
was obtained with 52% yield (0.010 g): TLC (chloro-
form/ethanol 4:1) Rf 0.51, 1H NMR (CD3CN) d 1.26 (s,
6H), 2.50 (m, 4H), 3.52 (m, 1H), 4.01 (m, 2H), 4.35 (m,
1H), 4.54 (m, 1H), 4.92 (m,1H), 6.21 (m, 2H), 7.36
(m,2H); 31P NMR (CD3CN) d ꢂ3.17, ꢂ4.62 (1:2.2), MS
(ꢂFAB) m/z 527.3 (MꢂH).
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