Stereoselective formation of a P-P bond in the reaction of 2-alkoxy-2-thio-1,3,2-oxathiaphospholanes with O,O-dialkyl H-phosphonates and H-thiophosphonates

Article abstract of DOI:10.1039/c0ob00104j

A new method for the formation of organohypophosphates containing a P-P bond under mild conditions, based on the DBU-assisted reaction of 2-alkoxy-2-thio-1,3,2-oxathiaphospholanes with O,O-dialkyl H-phosphonates or H-thiophosphonates, has been elaborated. The resulting triesters of P ¹-thio- and P¹,P²-dithiohypophosphoric acids, respectively, having O-methyl or O-ethyl groups, can be selectively dealkylated to form the corresponding di- or monoesters. Appropriately protected 2′-deoxyguanosine-3′-O-(2-thio-1,3,2-oxathiaphospholane) was converted into the corresponding P¹-thio- and P¹,P ²-dithiohypophosphate esters in a highly stereoselective manner (98%+ and 90%+, respectively).

Full text of DOI:10.1039/c0ob00104j

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PAPER
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Stereoselective formation of a P–P bond in the reaction of
2-alkoxy-2-thio-1,3,2-oxathiaphospholanes with O,O-dialkyl H-phosphonates
and H-thiophosphonates†‡
Damian Błaziak, Piotr Guga, Agata Jagiełło, Dariusz Korczyn´ski, Anna Maciaszek, Anna Nowicka,
Aleksandra Pietkiewicz and Wojciech J. Stec*
Received 10th May 2010, Accepted 26th August 2010
DOI: 10.1039/c0ob00104j
A new method for the formation of organohypophosphates containing a P–P bond under mild
conditions, based on the DBU-assisted reaction of 2-alkoxy-2-thio-1,3,2-oxathiaphospholanes with
O,O-dialkyl H-phosphonates or H-thiophosphonates, has been elaborated. The resulting triesters of
P1-thio- and P1,P2-dithiohypophosphoric acids, respectively, having O-methyl or O-ethyl groups, can be
selectively dealkylated to form the corresponding di- or monoesters. Appropriately protected
2¢-deoxyguanosine-3¢-O-(2-thio-1,3,2-oxathiaphospholane) was converted into the corresponding
P1-thio- and P1,P2-dithiohypophosphate esters in a highly stereoselective manner (98%+ and 90%+,
respectively).
that effectively dehydrocouple primary and secondary phosphines
Introduction
via a s-bond metathesis mechanism.10 In general, such reactions
In spite of the wealth of chemical literature regarding the
require highly reactive substrates and rather harsh conditions.
synthesis of compounds containing a P–P bond,1,2 the number
Therefore, a method for making the P–P bond starting from less
of methods for the synthesis of mono-, di-, tri- and tetra-
reactive substrates under mild conditions would pave the way for
alkyl esters of hypophosphoric acids is limited. Monoesters of
synthesis of new compounds of biological importance. In the
hypophosphoric acid were obtained by Setondji by DCC-assisted
present paper we report on a method of synthesis of triesters
of P1-thio- and P1,P2-dithiohypophosphoric acid, i.e. compounds
condensation of inorganic hypophosphate with an appropriate
alcohol,3 while tetraalkyl hypophosphates are available either
possessing (S)P—P(O) or (S)P—P(S) moieties, respectively. This
by treatment of hypophosphoric acid with diazoalkanes,4 or by
condensation of sodium O,O-dialkyl phosphites with O,O-dialkyl
phosphorochloridates. However, the latter process is accompanied
by the formation of tetralkyl pyrophosphites, pyrophosphates
and PIII,PV–mixed anhydrides, which are not easily separable
from the desired hypophosphates.5,6 P1-Thio- and P1,P2-dithio-
congeners of tetraalkyl hypophosphates have been obtained by
condensation of O,O-dialkyl phosphorochloridothioates with
sodium O,O-dialkyl phosphites or thiophosphites, respectively,
but these reactions generate undesired side products.7,8 The
P–P bond can also be formed in the reaction of (RO)2P(O)Cl
with diesters of phosphonic acids activated with the 1,3,2-
dioxarsolan moiety ((RO)2P(O)—As(OCH2)2).9 In a recently
developed approach, zirconium complexes have been prepared
approach is based on a reaction of P-chiral 2-alkoxy-2-thio-1,3,2-
oxathiaphospholanes (including several nucleoside derivatives),
having a tetracoordinate phosphorus atom with O,O-dialkyl H-
phosphonates or H-thiophosphonates, respectively. The triesters
carrying two methoxy or ethoxy groups at the P2 atom can be
sequentially dealkylated to form the corresponding 1,2-diesters
and monoesters.
It should be mentioned that chirality of the P-atom in the
oxathiaphospholane ring gives rise to racemic mixtures of the
resulting esters of P1-thio- and P1,P2-dithio-hypophosphoric acid.
However, if the starting oxathiaphospholanes could be separated
into pure enantiomers or P-diastereomers, the resulting hypophos-
phate products would be formed in a highly stereoselective manner,
presumably with a stereoretentive outcome. This assumption is
based upon the stereospecificity of the oxathiaphospholane ring
opening condensation.11
The above mentioned method was used for the synthe-
sis of nucleoside 3¢-O-(P1-thiohypophosphate)s and (P1,P2-
dithiohypophosphate)s as well as their 5¢-O- regioisomers,
which can be further converted into corresponding nucleoside
hypophosphates–the analogues of nucleoside O-diphosphates.
Since ribonucleoside 5¢-O-diphosphates are agonists of the P2Yn
class of receptors and also are substrates for ribonucleotide
reductase (RNR, an enzyme maintaining a proper level of 2¢-
deoxyribonucleotides in cells12), access to their analogues, which
may be used as molecular tools for studies in molecular and cell
biology, is of considerable interest.
Centre of Molecular and Macromolecular Studies, Polish Academy of
Sciences, Department of Bioorganic Chemistry, Sienkiewicza 112, 90-363,
Ło´dz´, Poland. E-mail: wjstec@bio.cbmm.lodz.pl; Fax: +48-42-6815483;
Tel: +48-42-6803248
† Electronic supplementary information (ESI) available: Synthesis of
3-O-benzoylpropyl ester of P2-O,O-diethyl-P1,P2-dithiohypophosphoric
acid, uridine 5¢-O-hypophosphate and N6-unprotected 5¢-O-(2-thio-1,3,2-
oxathiaphospholane)-2¢,3¢-O,O-diacetyladenosine, enzymatic phosphory-
lation of 5¢-O-(P1-thiohypophosphate) derivatives of nucleosides, mea-
surement of inhibitory activity of nucleoside hypophosphates towards
T7 RNA polymerase, calcium assay by fluorometric imaging plate reader
(FLIPR), 31P NMR proton decoupled spectra recorded for evaluation of
stereochemistry of a P–P bond formation. See DOI: 10.1039/c0ob00104j
‡ Dedicated to Prof. Andrzej Zwierzak on the occasion of his 75th
Birthday.
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diethyl-P1,P2-dithiohypophosphoric acid (3d, Scheme 1S in ESI†)
in 57% yield (by 31P NMR).
Results and discussion
In the course of the development of chemistry of P-
substituted 2-thio-1,3,2-oxathiaphospholanes11,13,14 it has been
found that the DBU-assisted reaction of 2-ethoxy-2-thio-1,3,2-
oxathiaphospholane (1a) with O,O-diethyl H-phosphonate (2)
in CH3CN (Scheme 1) provides P1-O-ethyl-P2-O,O-diethyl-P1-
thiohypophosphate (3a, R,R¢ = Et), which is converted into tri-
ethyl hypophosphate (4a) upon treatment with iodosobenzene.15,16
Finally, 4a was dealkylated with sodium iodide17 providing P1-O-
ethyl-P2-O-ethyl hypophosphate (5a).
In a typical workup, the compound 3b was purified by ion-
exchange chromatography on DEAE Sephadex A-25, eluted with
a gradient 0,05–0,3M triethylammonium bicarbonate (TEAB),
followed by conversion into sodium salt (DOWEX-Na+) and
lyophilization. Its identity was verified by 31P NMR and
FAB MS. Compound 3b upon treatment with t-butylamine20
(neat) was selectively demethylated to form P2-O-methyl-P1-
thiohypophosphate derivative 9b (Scheme 3). It was isolated
on DEAE Sephadex A-25, converted into a sodium salt and
lyophilized (total yield 25% calculated over 3b).
Scheme 3 Dealkylation of 3b with t-butylamine: R = CH2CH2CH2-
OC(O)Ph.
Following this model study we turned to the nucleoside
5¢-O-thiohypophosphate derivatives. Ebel and co-workers de-
scribed a DCC-based method of synthesis of adenosine 5¢-O-
hypophosphate3 (10, Scheme 4), which was proved in enzymatic
tests to have substrate affinity towards pyruvate kinase.21 This
compound was also shown to be translocated into mitochondria,
although it was not phosphorylated by the mitochondrial ATP
synthetase.22 In addition, attention has been paid to P2,P3-
hypophosphate analogues of nucleoside 5¢-O-triphosphates.23
Scheme 1 Synthesis of P1-O-alkyl-P2-O,O-dialkyl-P1-thiohypophosphates,
P1-O-ethyl-P2-O,O-diethyl hypophosphate and P1-O-ethyl-P2-O-ethyl
hypophosphate.
The molecular weight of 3a was documented by FAB MS, and
the presence of a P–P bond in 3a and 4a was confirmed by 31P
NMR spectra, which for each compound contained a pair of
doublets with spin–spin coupling constants of 466 and 650 Hz,
respectively.18,19 Apparently, the P–P bond was formed by attack
of the ambident, negatively charged phosphonyl ion (6, Scheme 2)
at the phosphorus atom in compound 1, followed by cleavage of
the endocyclic P–S bond and elimination of thiirane.
Since compounds 3a and 4a have poor UV-chromophores,
their analysis by TLC and, especially, by HPLC techniques is
cumbersome. Therefore, in next experiments we used a model
substrate 1b having the benzoyl moiety (Scheme 1). Compound 1b
was obtained by monobenzoylation of 1,3-propandiol in pyridine,
followed by phosphitylation of the isolated material with 2-chloro-
1,3,2-oxathiaphospholane (1.2 eq.) in the presence of elemental
Scheme 4 Synthesis of adenosine 5¢-O-hypophosphate by Setondji.3
Since Setondji’s method is not suitable for synthesis of
nucleoside 5¢-O-(P1-thiohypophosphates) we have used the oxathi-
aphospholane approach to obtain nucleoside 5¢-O-(P1-thiohypo-
phosphates) and 5¢-O-hypophosphates. As a model, 5¢-O-(2-thio-
1,3,2-oxathiaphospholane)-2¢,3¢-O,O-diisopropoxyacetyl-uridine
(11, Scheme 5) was treated with 7. The resulting 5¢-O-(P2-
O,O-dimethyl-P1-thiohypophosphate) derivative (12) upon
ammonolytic removal of isopropoxyacetyl groups yielded uridine
5¢-O-(P2-O,O-dimethyl-P1-thiohypophosphate) (13) in 38% yield
(by 31P NMR), further converted by treatment with t-butylamine
into uridine 5¢-O-(P2-O-methyl-P1-thiohypophosphate) (14) in
virtually quantitative yield. Independently, treatment of 12 with
trimethylsilyl bromide24 and hydrolytic removal of trimethylsilyl
and acyl groups with aqueous ammonia furnished uridine
1
sulfur, and its identity was confirmed by H NMR, 31P NMR
and FAB MS. The reaction of 1b with an equimolar amount
of (MeO)2P(O)H (7) and DBU (1.1 equiv.) in any of four anhy-
drous solvents, acetonitrile, diethyl ether, benzene, or dimethyl-
formamide, after 3–12 h provided the expected P1-O-(3-O-
benzoylpropyl)-P2-O,O-dimethyl-P1-thiohypophosphate (3b) in
64–90% yield (by 31P NMR). Similar reaction of 1b with 2
in acetonitrile furnished P2-O,O-diethyl-P1-thiohypophosphoric
derivative (3c) in 45% yield. Condensation of 1b with (EtO)2P(S)H
(8) in acetonitrile provided 3-O-benzoylpropyl ester of P2-O,O-
Scheme 2 Suggested mechanism of formation of the P–P bond.
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pure 3¢-O-(1,3,2-oxathiaphospholane) derivatives of nucleo-
sides (e.g. 19, Scheme 7) were successfully used for synthe-
sis of stereodefined oligonucleoside phosphorothioates (with-
out epimerization at phosphorus),14 we checked the stereo-
selectivity of the oxathiaphospholane ring opening in 19
with a negatively charged phosphonyl ion 6 and its phos-
phonothioyl counterpart. We prepared diastereomerically en-
riched (fast (SP):slow (RP) 100 : 44, by 31P NMR, Figure 1S
in the ESI†) 5¢-O-DMT-NiBu,ODPC-deoxyguanosine-3¢-O-(2-thio-
1,3,2-oxathiaphospholane) (19, DPC = diphenylcarbamoyl).
Scheme 5 Synthesis of uridine 5¢-O-(P1-thiohypophosphate) deriva-
tives. X = isopropoxyacetyl. Reagents: (i) (MeO)2P(O)H; (ii) DBU; (iii)
NH4OHaq.; (iv) t-Bu-NH2; (v) TMS-Br.
Scheme 7 Synthesis of stereodefined nucleoside 3¢-O-(P1-thiohypo-
phosphate) or 3¢-O-(P1,P2-dithiohypophosphate). Gua¢ = GuaiBu,DPC
;
Reagents: X = O, (i) (EtO)2P(O)H; X = S, (ii) (EtO)2P(S)H. (The major SP
isomer of 19 is shown.).
5¢-O-(P1-thiohypophosphate) (15). Next, N6-unprotected 5¢-O-
(2-thio-1,3,2-oxathiaphospholane)-2¢,3¢-O,O-diacetyladenosine
(16, Scheme 6, synthesis is described in the ESI†) was converted
into adenosine 5¢-O-(P1-thiohypophosphate) (18). Oxidation of
18 with iodoxybenzene provided adenosine 5¢-O-hypophosphate
(10), identical to the compound described by Setondji.3
This diastereomeric ratio range was selected because the content
of the minor component is high enough to observe the resonances
of interest for the P1 atoms in both P-diastereomers of each
product 20 and 21 (a pair of doublets, the resonances split due
to the direct 31P–31P spin–spin coupling), and the presence of four
signals provides evidence for sufficient resolution of the spectrum.
Substrate 19 was reacted with 2 in the presence of DBU to give
20. Its 31P NMR spectrum (Fig. 1) contained resonances for P1
atoms at d 54.0 ppm (d, 1JP–P = 469 Hz, CD3CN) and d 54.4 ppm
1
(d, JP–P = 466 Hz) at relative intensity 100 : 43, i.e. close to the
diastereomeric ratio 100 : 44 of the substrate 19.
Scheme 6 Synthesis of adenosine 5¢-O-(P1-thiohypophosphate) and
hypophosphate derivatives. Reagents: (i) (MeO)2P(O)H; (ii) DBU; (iii)
TMS-Br.; (iv) NH4OHaq; (v) PhIO2.
Fig. 1 A region of the 31P NMR spectrum for the P1 atoms in both
P-diastereomers of 5¢-O-DMT-NiBu,ODPC-deoxyguanosine-3¢-O-(P2-O,O-
diethyl-P1-thiohypophosphate) (20). The vertical numbers represent inte-
gration data.
As indicated earlier, the thiohypophosphate compounds 3
and 9 have a chiral phosphorus atom derived from their
oxathiaphospholane precursor. Since the P-diastereomerically
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To assess precisely the stereoselectivity of the reaction, the same
experiment was executed using diastereomerically pure 19 (100%
fast-isomer) and the relevant region of the 31P NMR spectrum
recorded on a 500 MHz spectrometer (Figure 2S, see ESI†) did
not contain any measurable signals of the epimerized product (to
be seen as a doublet centered at d 54.4 ppm (d, 1JP–P = 466 Hz). This
result indicates that the DBU-assisted ring-opening condensation
of 2-alkoxyl-2-thio-1,3,2-oxathiaphospholanes with O,O-dialkyl
H-phosphonates is highly stereoselective (>98%). The presumed
stereoretentive outcome (SP-19→RP-20/21) needs to be confirmed
by X-ray or additional NMR measurements.
pyrimidinergic P2Y receptor. The assay consisted of measure-
ments of intracellular calcium ion concentrations in mammalian
cells (1321N1 astrocytoma cell line) that stably express the human
receptor. Both compounds fully activated the P2Y6 receptor (the
thio analogue was slightly less potent than the oxo analogue,
EC50 = 16 mM and 9 mM, respectively), although less potent
than the endogenous agonist UP3U, for which EC50 = 0.4 mM
(Calcium assay description and data given in ESI†).28 One can
argue that 40- and 22-fold reductions in activity, respectively,
are discouraging, especially when a remarkable family of UDP
derivatives and analogues were synthesized and evaluated at P2Y2,
P2Y4, and P2Y6 subtypes of the P2Y receptor. For example, a
large phenacyl substituent at N3 of UDP was well tolerated by
the P2Y6 receptor, yielding a potent and selective P2Y6 receptor
agonist (EC50 = 70 nM).29 However, there could be an advantage
of the hypophosphate analogues for their use in pharmacology
experiments due to significantly higher nucleolytic stability of the
P–P bond over the P–O–P bond while the unchanged nucleoside
moieties may interact with proteins in virtually unaltered way. It
was found that 10 was refractory to several hydrolases as bacterial
alkaline phosphatase (phosphate-monoester phosphohydrolase,
EC 3.1.3.1), lupin apyrase (ATP diphosphatase, EC 3.6.1.5)
and snake venom phosphodiesterase (EC 3.1.15.1). Only snake
venom 5¢-nucleotidase (5¢-ribonucleotide phosphohydrolase, EC
3.1.3.5) slowly hydrolyzed this compound, releasing adenosine and
hypophosphate anion.30
An analogous reaction of 19 (the mixture 100 : 44) with 8 yielded
P2-O,O-diethyl-P1,P2-dithiohypophosphate derivative 21 and the
relevant region of the 31P NMR spectrum (Figure 3S, see ESI†)
contained resonances for the P1 atoms at d 58.5 ppm (d) and d
1
58.9 ppm (d), JP–P = 352 Hz. However, their relative intensity
100 : 53, indicates that the reaction was slightly less stereoselective
(ca. 90%).
When diastereomerically pure (or highly enriched) 5¢-O-(2-
thio-1,3,2-oxathiaphospholane) derivatives of nucleosides become
available, it can be assumed that the similar stereochemistry
and stereoselectivity will be observed leading to the desired
stereodefined thiohypophosphate congeners of NDPs.
For preliminary biological evaluation, 5¢-O-(P1-thiohypo-
phosphate) derivatives of adenosine (18), uridine (15) and cytidine
(obtained analogously to the uridine derivative 15) were used
as mixed diastereoisomers. Only the adenosine derivative was
accepted as a substrate by the purine nucleotide preferring25
phosphoenolpyruvate/pyruvate kinase system (see ESI†) to form
the hypo-analogue of ATPaS (22) (Scheme 8). Moreover, from the
two P-diastereomers of adenosine 5¢-O-(P1-thiohypophosphate)
(separated by RP-HPLC) only the fast-eluting diastereomer was
transformed into the corresponding adenosine 5¢-O-(P1-thio-
P1,P2-hypotriphosphate), i.e. P1,P2-hypophosphate analogue of
ATPaS.26 Its identity was confirmed by MALDI TOF mass
spectrometry.
Conclusion
In this report we present an extension of the oxathiaphos-
pholane method to P–P bond formation under relatively mild
conditions, and have applied it to the synthesis of hypophos-
phorylated alcohols (including nucleosides) and their P1-thio-
and P1,P2-dithio-congeners in moderate yield. The process of
DBU-assisted oxathiaphospholane ring opening by O,O-dialkyl
H-phosphonates or O,O-dialkyl H-thiophosphonates is highly
stereoselective. Enhanced stability of the P–P bond in nucleo-
side hypophosphates and their P1-thiohypophosphate congeners
makes them potentially interesting models for studies on the
biochemistry of nucleoside polyphosphates.
Scheme
8
Enzymatic conversion of adenosine 5¢-O-(P1-thiohypo-
Experimental section
phosphate) or 5¢-O-hypophosphate into the corresponding P1,P2-hypotri-
phosphate analogues (22 or 23) of ATPaS or ATP, respectively. PEP/PK,
phosphoenolpyruvate/pyruvate kinase.
Nuclear magnetic resonance spectra (31P NMR) were recorded
on a Bruker AC-200 instrument (200 MHz for 1H), or DRX-500
(500 MHz); 85% H3PO4 was used as an external standard. All
chemical shifts are measured in units of ppm. FAB-MS spectra
(13keV, Cs+) were recorded on a Finnigan MAT 95 spectrometer,
negative ion MALDI TOF mass spectra were recorded on a
Voyager-Elite instrument (PerSeptive Biosystems Inc., Framing-
ham, MA). HPLC analyses were performed using a binary HPLC
system (ProStar, Varian Inc.). Anhydrous acetonitrile and 1,4-
diazabicyclo[5.4.0]undec-7-ene (DBU) were supplied by Fluka and
Acros Organics, respectively. O,O-Dialkyl H-phosphonates were
purchased from Aldrich. 5¢-O-(2-Thio-1,3,2-oxathiaphospholane)
derivatives of N-protected nucleosides were synthesized as de-
scribed earlier.31 Nucleoside oxathiaphospholanes 19 were syn-
thesized according to published methods.14 Iodosobenzene was
Diastereomerically pure 18-fast, 18-slow, 22 (derived from 18-
fast) and P-achiral adenosine 5¢-O-(P1,P2-hypotriphosphate) (23,
obtained from 10 analogously to 22) were screened for their
inhibitory activity towards T7 RNA polymerase (see ESI†). For
the transcription reaction, the inhibitory concentrations IC50 of
2.90, 2.36, 0.58 and 0.55 mM, respectively, were determined, thus
P1,P2-hypotriphosphate analogues of ATP and ATPaS were the
most potent inhibitors of virtually the same activity.27
Interesting preliminary results were also obtained on the inter-
actions of the sodium salt of uridine 5¢-O-hypophosphate (synthe-
sis is described in ESI†) and uridine 5¢-O-(P1-thiohypophosphate)
15 with the P2Y6 receptor, which is a subtype of the human
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a gift from Dr A. Łopusin´ski. Iodoxybenzene was synthesized
according to the published procedure.32
salt (DOWEX, Na+) and lyophilized (21 mg, 53%). MALDI TOF
MS m/z 353, M-, m/z 375, [M+Na+]-.
3-Hydroxypropyl benzoate: To a cooled solution (0 ◦C) of 1,3-
propandiol (1.89 mL; 0.026 mol) in pyridine (5 mL) benzoyl
chloride (3 mL; 1 eq) was added dropwise. The mixture was stirred
overnight at room temperature and the product isolated on a silica
gel column eluted with CHCl3:acetone (9 : 1, v/v) (1.79 g, 40%).
1H NMR d 1,97 (quintet, 2H); 3.09 (s, 1H); 3.74 (t, 2H); 4.43 (t,
2H); 7.21–8.54 (m, 5H).
P1 -O-(3-O-Benzoylpropyl)-P2 -O,O-diethyl-P1 -thiohypopho-
sphate (3c): To a solution of 2-(3-O-benzoylpropyloxy)-2-thio-
(1,3,2-oxathiaphospholane) (1b, 68 mg; 0.21 mmol) in dry MeCN
(1 mL), (EtO)2P(O)H (2, 30 mL, 0.21 mmol) and DBU (34 mL,
0.23 mmol) were added. After 12 h the 31P NMR spectrum showed
the presence of the product 3c (45%, not isolated). d 31P NMR
(CH3OD) 53.53 (d), 14.39 (d), 1JP–P = 475 Hz.
2-(3-O-Benzoylpropyloxy)-2-thio-(1,3,2)-oxathiaphospholane
(1b): Into a solution of 3-hydroxypropyl benzoate (1.79 g;
0.010 mol) in anhydrous pyridine (10 mL), elemental sul-
fur was added (0.64 g; 2 eq.) followed by 2-chloro-1,3,2-
oxathiaphospholane (1 mL; 1.2 eq., dropwise). The mixture was
stirred overnight at room temperature and the product was isolated
(1.58 g, 51%) on a silica gel column eluted with a chloroform/
acetone mixture (9 : 1, v/v). d 31P NMR (CDCl3) 104.19 (s) FAB
MS C12H15O4PS2, calc. 318, [M - H]- found m/z 317.
Uridine 5¢-O-(P2-O,O-dimethyl-P1-thiohypophosphate) (13):
To a solution of 5¢-O-(2-thio-1,3,2-oxathiaphospholane)-2¢,3¢-
O,O-diisopropoxyacetyluridine (11, 29 mg, 0.05 mmol) in 0.5 mL
of dry MeCN, (MeO)2P(O)H (7, 5.5 mg, 0.05 mmol) and DBU
(7.8 mL, 0.055 mmol) were added. After 2.5 h, the reaction mixture
was evaporated, the residue was dissolved in 3 mL of concentrated
NH4OHaq and kept at room temperature. After 1 h, the mixture
was evaporated to dryness. The product 13 (triethylammonium
salt) was isolated using ion-exchange chromatography on DEAE
Sephadex A-25, with a linear gradient 0.1 to 0.6 M TEAB buffer
(pH 7.5), (5 mg, 20%). d 31P NMR (D2O) 55.18 (d), 15.65 (d),
1JP–P = 501 Hz; MALDI TOF MS m/z 431.0, M-.
P1-O-ethyl-P2-O,O-diethyl-P1-thiohypophosphate (3a): To a so-
lution of 2-ethoxy-2-thio-1,3,2-oxathiaphospholane (1a) (38 mg,
0.21 mmol) in dry MeCN (1 mL), (EtO)2P(O)H (2, 13 mL,
0.10 mmol) and DBU (46 mL, 0.31 mmol) were added. After 8 h the
mixture was evaporated to dryness and the product 3a (DBUH+
salt, M.W. 414) was isolated on a silica gel column eluted with
a chloroform–methanol mixture (1 : 1, v/v), (17 mg, 0.041 mmol,
19%). d 31P NMR (D2O) 50.31 (d), 12.08 (d), 1JP–P = 466 Hz; FAB
MS m/z 261, [M - H]-.
Uridine 5¢-O-(P2-O-methyl-P1-thiohypophosphate) (14): Uri-
dine 5¢-O-(P2-O,O-dimethyl-P1-thiohypophosphate) (13, 2.5 mg,
0.005 mmol) was dissolved in 0.5 mL of t-butylamine. After
4 d, t-butylamine was evaporated under high vacuum and the
product 14 was obtained in near quantitative yield. d 31P NMR
(D2O) 65.11 (d), 9.81 (d), 1JP–P = 531 Hz; MALDI TOF MS m/z
416.9 M-.
Uridine 5¢-O-(P1-thiohypophosphate) (15): To a solution of
5¢-O-(2-thio-1,3,2-oxathiaphospholane)-2¢,3¢-O,O-diisopropoxy-
acetyluridine (12, 29 mg, 0.05 mmol) in 0.5 mL of MeCN,
(MeO)2P(O)H (7, 5.5 mg, 0.05 mmol) and DBU (7.8 mL,
0.055 mmol) were added. After 2.5 h, the reaction mixture was
cooled to -40 ◦C and trimethylsilyl bromide (26 mL, 0.2 mmol)
added dropwise with a gas-tight syringe. The mixture was gradu-
ally warmed up to a room temperature and stirred overnight. Then,
the mixture was evaporated and the residue was dissolved in 3 mL
of concentrated NH4OHaq. After 1 h, solvent was evaporated and
the product 15 (containing three triethylammonium counterions)
was isolated using ion-exchange chromatography on DEAE
Sephadex A-25 with a linear gradient 0.1 to 0.6 M TEAB buffer,
followed by gel filtration on Sephadex LH-20 (3.6 mg, 14%). d 31P
NMR (D2O) 66.37 (d), 7.64 (d), 1JP–P = 543 Hz; MALDI TOF MS
m/z 403.0 M-.
Triethyl hypophosphate (4a): To a solution of P1-O-ethyl-P2-
O,O-diethyl-P1-thiohypophosphate (3a, 13 mg, 0.031 mmol) in
1 mL of MeCN, iodoxybenzene (PhIO2, 7 mg, 0.031 mmol) was
added. After 48 h, the reaction mixture was filtered and the filtrate
was evaporated to dryness to yield 4a quantitatively. d 31P NMR
(D2O) 15.80 (d), -3.17 (d), 1JP–P = 650 Hz.
P1-O-ethyl-P2-O-ethyl-hypophosphate (5a): To a solution of
crude triethyl hypophosphate (4a, 12 mg, 0.03 mmol) in 1 mL of
acetone, sodium iodide (4.7 mg, 0.031 mmol) was added. Reaction
mixture was refluxed for 24 h and cooled down. The crystalline
product was washed with acetone and dried (5 mg, 61%). d 31P
NMR (D2O) 9.39 (s); FAB MS m/z 217.1.
P1-O-(3-O-Benzoylpropyl)-P2-O,O-dimethyl-P1-thiohypopho-
sphate (3b): To a solution of 2-(3-O-benzoylpropyloxy)-2-thio-
1,3,2-oxathiaphospholane (1b, 67 mg; 0.21 mmol) in dry MeCN
(1 mL), (MeO)2P(O)H (7, 19 mL, 0.21 mmol) and DBU (34 mL,
0.23 mmol) were added. After 3 h the reaction mixture was
concentrated to dryness and the product 3b was isolated on a
DEAE Sephadex A-25 column eluted with a triethylammonium
bicarbonate buffer (TEAB) using a gradient 0.05–0.3 M. The
product was >92% pure by 31P NMR. d (CH3OD) 54.63 (d),
Adenosine 5¢-O-(P1-thiohypophosphate) (18): To
a solu-
tion of N6-unprotected 2¢,3¢-O,O-diacetyladenosine-5¢-O-(2-thio-
1,3,2-oxathiaphospholane) (16, 98 mg, 0.2 mmol, see ESI†) in
3 mL of dry MeCN, (MeO)2P(O)H (7, 22 mg, 0.2 mmol) and DBU
(33 mL, 0.22 mmol) were added. After 2 h, the reaction mixture was
1
16.31 (d), JP–P = 496 Hz; FAB MS m/z 367, [M-1]-. Finally, the
◦
product was converted into the sodium salt (DOWEX, Na+) and
lyophilized (37 mg, 45%).
cooled to -40 C and trimethylsilyl bromide (105 mL, 0.8 mmol)
was added dropwise via gas-tight syringe. The mixture was
gradually warmed up to room temperature and stirred overnight.
The mixture was evaporated and the residue containing diacylated
monoester intermediate 17 dissolved in 3 mL of concentrated
NH4OHaq. After 2 h, solvent was evaporated and the product
18 (containing three triethylammonium counterions) was isolated
using ion-exchange chromatography on DEAE Sephadex A-25
with a linear gradient 0.1 to 0.6 M TEAB buffer (35 mg, 24%).
P1 -O-(3-O-Benzoylpropyl)-P2 -O-methyl-P1 -thiohypopho-
sphate (9b): Sodium salt of P1-O-(3-O-benzoylpropyl)-P2-O,O-
dimethyl thiohypophosphate (3b, 37 mg, 0.095 mmol) was dis-
solved in t-BuNH2 (1 mL) and kept for 72 h at 45 ◦C. The reaction
mixture was analyzed by 31P NMR (C6D6) d 64.86 (d), 10.18 (d),
1JP–P = 537 Hz (62% by 31P NMR). The product 9b was isolated
on a DEAE Sephadex A-25 column eluted with TEAB (a gradient
0.05–0.3 M). Finally, the product was converted into the disodium
d
31P NMR (D2O) 68.22 (d), 68.05 (d) (resonances for a pair of
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Org. Biomol. Chem., 2010, 8, 5505–5510 | 5509
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1
P-diastereoisomers), 7.64 (d), JP–P = 530 Hz; MALDI TOF MS
3 J. Setondji, P. Remy, G. Dirheimer and J. P. Ebel, Biochim. Biophys.
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4 M. Baudler, Z. Anorg. Allg. Chem., 1956, 288, 171.
5 J. Michalski and A. Zwierzak, Bull. Acad. Polon. Sci. Ser. Sci. Chim.,
1965, 13, 253–259.
6 W. Stec, A. Zwierzak and J. Michalski, Bull. Acad. Polon. Sci. Ser. Sci.
Chim., 1970, 18, 23–29.
7 L. Almasi and L. Paskucz, Chem. Ber., 1963, 96, 2024–2028.
8 J. Michalski, W. Stec and A. Zwierzak, Bull. Acad. Polon. Sci. Ser. Sci.
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9 R. C. Mehrotra, G. Srivastava and P. N. Nagar, Phosphorus Sulfur
Relat. Elem., 1983, 18, 145–148.
10 R. Waterman, Organometallics, 2007, 26, 2492–2494.
11 W. J. Stec, B. Karwowski, M. Boczkowska, P. Guga, M. Koziołkiewicz,
M. Sochacki, M. Wieczorek and J. Błaszczyk, J. Am. Chem. Soc., 1998,
120, 7156–7167.
12 J. Herrick and B. Sclavi, Mol. Microbiol., 2007, 63, 22–34.
13 P. Guga, A. Okruszek, W. J. Stec, Topics in Current Chemistry. Vol. 220,
Springer Verlag, 2002, J. P. Majoral, Ed., pp. 169-200.
14 P. Guga, W. J. Stec,(2003) Synthesis of Phosphorothioate Oligonu-
cleotides with Stereodefined Phosphorothioate Linkages. In Current
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m/z 426.0, M-.
Adenosine 5¢-O-hypophosphate (10): To a stirred solution of
adenosine 5¢-O-(P1-thiohypophosphate) (18) (22 mg, 0.05 mmol)
in water (0.8 mL), iodoxybenzene (PhIO2, 6 mg, 0.025 mmol)
was added. After 10 min, the mixture was diluted with water
(9 mL) and extracted with chloroform. The aqueous layer was
concentrated and the product 10 was isolated using ion-exchange
chromatography on DEAE Sephadex A-25, with a linear gradient
0.05 to 0.6 M TEAB buffer. Finally, the product was converted
into the sodium salt (DOWEX Na+) and lyophilized (9 mg, three
sodium counterions, 63%). d 31P NMR (D2O) 17.09 (d), 4.93 (d),
1JP–P = 660 Hz; MALDI TOF MS m/z 410.0 M-.
5¢-O-DMT-NiBu,ODPC -2¢-deoxyguanosine-3¢-O-(2-thio-1,3,2-
oxathiaphospholane) (19): the compound was synthesized and
enriched in the fast diastereomer (final ratio fast:slow 100 : 44 or
100% fast) by means of chromatography on a silica gel column,
according to the published method.14
5¢-O-DMT-NiBu,ODPC -2¢-deoxyguanosine-3¢-O-(P2 -O,O-die-
thyl-P1-thiohypophosphate) (20): To a solution of 5¢-O-DMT-
15 A. Łopusin´ski and G. Mielniczak, Synlett., 2001, 4, 505–
508.
N
iBu,ODPC-2¢-deoxyguanosine-(2-thio-1,3,2-oxathiaphospholane)
16 B. Nawrot, B. Re˛bowska, O. Michalak, M. Bulkowski, D. Błaziak, P.
Guga and W. J. Stec, Pure Appl. Chem., 2008, 80, 1859–1871.
(19, 40 mg, 0.04 mmol, fast:slow 100 : 44 or 100% fast) in 0.5 mL
of anhydrous MeCN, (EtO)2P(O)H (2, 5mL, 0.04 mmol) and DBU
(8 mL, 0.045 mmol) were added. After 12 h, the reaction mixture
17 J. Michalski, W. Stec and A. Zwierzak, Chem.
& Ind., 1966,
856.
18 R. K. Harris, J. R. Woplin and W. J. Stec, J. Chem. Soc. Chem. Commun.,
was evaporated and the residue was dissolved in CD3CN. The 31
P
1970, 1391.
NMR spectrum (500 MHz instrument) contained resonances for
P1 atoms at d 54.0 (d, JP–P = 467 Hz, CD3CN) and d 54.4 (d,
19 W. J. Stec, J. R. Van Wazer and N. Goddard, J. Chem. Soc., Perkin
Trans. 2, 1972, 463.
20 M. D. M. Gray and D. J. H. Smith, Tetrahedron Lett., 1980, 21, 859–
860.
21 J. Setondji, P. Remy, G. Dirheimer and J. P. Ebel, Biochim. Biophys.
Acta, Nucleic Acids Protein Synth., 1971, 232, 585–594.
22 P. V. Vignais, J. Setondji and J. P. Ebel, Biochimie, 1971, 53, 127–
129.
1
1JP–P = 466 Hz) at relative intensity 100 : 40 (only one doublet at d
54.4 (1JP–P = 470 Hz) for the diastereomerically pure substrate).
5¢-O-DMT-NiBu,ODPC -2¢-deoxyguanosine-3¢-O-(P2 -O,O-die-
thyl-P1,P2-dithiohypophosphate) (21): To a solution of 5¢-O-
DMT-NiBu,ODPC -2¢-deoxyguanosine-(2-thio-1,3,2-oxathiaphos-
pholane) (19, 40 mg, 0.04 mmol, fast:slow 100 : 44) in 0.5 mL of
anhydrous MeCN, (EtO)2P(S)H (8, 6 mL, 0.04 mmol) and DBU
(8 mL, 0.045 mmol) were added. After 12 h, the reaction mixture
was evaporated and the residue dissolved in CD3CN. The relevant
31P NMR spectrum (500 MHz) showed the presence of 21 in 17%
23 (a) P. Remy, G. Dirheimer and J. P. Ebel, Biochim. Biophys. Acta, Gen.
Subj., 1967, 136, 99–107; (b) P. Remy, J. Setondji, G. Dirheimer and
J. P. Ebel, Biochim. Biophys. Acta, Nucleic Acids Protein Synth., 1970,
204, 31–38; (c) M. K Kukhanova, N. F Zakirova, A. V. Ivanov, L. A.
Alexandrova, M. V. Jasco and A. R. Khomutov, Biochem. Biophys. Res.
Commun., 2005, 338, 1335–1341.
24 J. Chojnowski, M. Cypryk and J. Michalski, Synthesis, 1978, 777–
1
1
779.
yield; d 58.5 (d, JP–P = 354 Hz) and d 58.9 (d, JP–P = 352 Hz),
25 J. Bao and D. D. Y. Ryu, Biotechnol. Bioeng., 2007, 98, 1–11.
26 Analogous P-diastereoselectivity was observed in phosphorylation of
a mixture of isomers of ADPaS with pyruvate kinase, which yielded
only one P-diastereomer of ATPaS. F. Eckstein and R. S. Goody,
Biochemistry, 1976, 15, 1685–1691.
relative intensity 100 : 53.
Acknowledgements
27 For the T7 RNA polymerase/ATP transcription system (pH 7.9), Km
values of 0.0095 mM and 0.14 mM were reported in the literature:
(a) D. Imburgio, M. Anikin and W. T. McAllister, J. Mol. Biol., 2002,
319, 37–51; (b) D. Patra, E. M. Lafer and R. Sousa, J. Mol. Biol., 1992,
224, 307–318.
28 K. A. Jacobson (Laboratory of Bioorganic Chemistry & Molecular
Recognition Section, NIH, Bethesda, MD – unpublished results.
29 A. El-Tayeb, A. Qi and C. E. Muller, J. Med. Chem., 2006, 49, 7076–
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30 A. Guranowski, A. M. Wojdyła, J. Zimny, A. Wypijewska, J. Kowalska,
M. Łukaszewicz, J. Jemielity, E. Darz˙ynkiewicz, A. Jagiełło and P.
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31 K. Misiura, D. Szymanowicz and W. J. Stec, Org. Lett., 2005, 7, 2217–
2220.
This work was financially supported by Polish Ministry of
Science and Higher Education, grant 3 T09A 059 28 (to WJS).
Authors are indebted to Prof. K.A. Jacobson of NIH, Bethesda,
MD., for sharing with us the results of preliminary studies on
interaction of nucleoside hypophosphates with P2Y6 receptor.
Critical comments of Prof. G. M. Blackburn on the manuscript
are highly appreciated.
Notes and references
1 A. H. Cowley, Chem. Rev., 1965, 65, 617–634.
2 I. F. Lutsenko and M. V. Proskurnina, Russ. Chem. Rev., 1978, 47,
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32 P. Kaz´mierczak, L. Skulski and Ł. Kraszkiewicz, Molecules, 2001, 6,
881–891.
5510 | Org. Biomol. Chem., 2010, 8, 5505–5510
This journal is
The Royal Society of Chemistry 2010
©