The case for configurational stability of H-phosphonate diesters in the presence of diazabicyclo[5.4.0]undec-7-ene (DBU)

Article abstract of DOI:10.1016/S0968-0896(01)00140-7

Configurational stability of dinucleoside H-phosphonates and the stereochemical course of their sulfurisation in the presence of diazabicyclo[5.4.0]undec-7-ene (DBU) were investigated using ³¹ P NMR spectroscopy. It was found that under the rea

Full text of DOI:10.1016/S0968-0896(01)00140-7

Bioorganic & Medicinal Chemistry 9 (2001) 2315–2322
The Case for Configurational Stability of H-Phosphonate Diesters
in the Presence of Diazabicyclo[5.4.0]undec-7-ene (DBU)
Tommy Johanssona and Jacek Stawinskia,b,
*
aDepartment of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-10691 Stockholm, Sweden
bInstitute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland
Received 16 February 2001; accepted 10 April 2001
Abstract—Configurational stability of dinucleoside H-phosphonates and the stereochemical course of their sulfurisation in the
presence of diazabicyclo[5.4.0]undec-7-ene (DBU) were investigated using 31P NMR spectroscopy. It was found that under the
reaction conditions and irrespective of the type of protecting groups present in the nucleoside moieties, the H-phosphonate diesters
investigated did not undergo any detectable epimerisation at the phosphorus centre, and their sulfurisation with elemental sulfur in
the presence of DBU, proceeded stereospecifically. Thus, we could not confirm reports from another laboratory on a stereoselective
course of sulfurisation of H-phosphonate diesters and the corresponding acylphosphonates in the presence of DBU. # 2001
Elsevier Science Ltd. All rights reserved.
Introduction
advent of antisense11 and antigene12 techniques for
modulation of gene expression for therapeutical pur-
poses amplified problems connected with the presence
of stereogenic phosphorus centre in oligonucleotide
analogues,13 and caused high demand for products with
defined chirality13,14 at each phosphorus centre. Since
dinucleoside H-phosphonate and H-phosphonothioates
can be separated into diastereomers,7,15 they are of
potential interest as synthons for introduction of a
chiral phosphorus centre with known stereochemistry at
the predefined position in an oligonucleotide chain. A
prerequisite of such an approach is stereospecificity of
all transformation involving conversion of a chiral pre-
cursor into the final product at the oligonucleotide
level.5,16
In the last decade, the H-phosphonate methodology1,2
has emerged as a viable alternative to the well estab-
lished phosphite3 and phosphotriester4 approaches for
the preparation of biologically important phosphate
esters and their analogues. The growing interest in H-
phosphonate chemistry1,2,5 is probably due to the fact
that it combines synthetic advantages of other methods
based on P(III) and P(V) compounds, for example sta-
bility and ease of handling of starting materials,
experimental simplicity, high efficiency in the formation
of P–O bonds, and versatility in terms of phosphate
analogues that can be prepared from a common inter-
mediate. The tautomeric equilibrium of mono- or die-
sters of phosphonic acid, which is practically completely
shifted toward the H-phosphonate form, is most
favourable from a synthetic point of view and permits
synthesis of phosphorus compounds without recourse to
phosphate protecting groups.1
Tetracoordinated P(III) derivatives, for example simple
dioxaphosphinanes,17 phosphinate derivatives,18 dinu-
cleoside H-phosphonates7,16,19 are known to be config-
urationally stable at room temperature and their
conversion to tervalent species and vice versa occurs
without epimerisation at the phosphorus centre. This
constitutes the basis for synthetic applications of these
compounds as chiral precursors in various stereospecific
transformations. Since some of these reactions are also
frequently used in stereochemical correlation analysis
for establishing absolute configuration at the phospho-
rus centre, any departure from the established stereo-
chemical pattern requires meticulous scrutiny to pin-
point a possible source of a stereochemical variation.
Asynthesis area where H-phosphonate intermediates
proved to be most efficient is preparation of P-chiral
phosphorus compounds, for example nucleoside phos-
phorothioates,6,7 phosphoroselenoates,8,9 phosphoro-
selenothioates,9 phosphoramidates,10 and so forth. The
*Corresponding author. E-mail: js@mail1.organ.su.se
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00140-7

2316
T. Johansson, J. Stawinski / Bioorg. Med. Chem. 9 (2001) 2315–2322
In this respect, two reports by Hata et al.20 on stereo-
selective rather than stereospecific sulfurisation of H-
phosphonate diesters attracted our attention as excep-
tions from the commonly observed stereoretentive
course of sulfurisation of P(III) compounds. The
authors claimed that when a diastereomeric mixture of
acylphosphonate 3 was treated with elemental sulfur in
the presence of n-butylamine and DBU, the exclusive
formation of one diastereomer of phosphorothioate 2 (RP)
was observed, that is the transformation appeared to be
completely stereoselective.21 This unexpected course of the
reaction Hata et al.20 explained by DBU-mediated epi-
merisation of the intermediate H-phosphonate diesters 1,
formed via deacylation of acylphosphonate by n-butyl-
amine. According to this mechanism, DBU reacts with H-
phosphonate 1 forming a pentacoordinate intermediate
that undergoes epimerisation at the phosphorus centre and
the produced more thermodynamically stable 1 SP isomer
is oxidised with sulfur to afford RP phosphorothioate 2.22
side H-phosphonate 1 in pyridine using elemental sulfur
(15 equiv) in the presence of n-butylamine (10 equiv)
and DBU (0.5 equiv) (the reaction conditions from
Hata et al.20) did not reveal any stereoselectivity in the
formation of the corresponding phosphorothioate 2, as
judged form the same ratio of diastereomers in the
starting material 1 and the product 2. The analogous
reactions carried out on separate diastereomers of 1
showed that sulfurisation under these reaction condi-
tions was completely stereospecific. Varying amounts of
DBU and n-butylamine used for the reaction did not
produce any noticeable changes in product distribution
and the stereochemical outcome of the reaction
remained the same as that when only triethylamine was
used as a base.
Although these results clearly showed that that sulfur-
isation of H-phosphonate diesters in the presence of
DBU occurred stereospecifically, there was still a possi-
bility that P-chiral H-phosphonates may undergo epi-
merisation at the phosphorus centre when exposed for a
sufficiently long time to DBU.26 Since sulfurisation of 1
in the presence of DBU in our hands was complete
within 5–10 min (31P NMR) while that reported by
Hata et al. required 1 h, we investigated, if treatment of
separate diastereomers of H-phosphonate diester 1 in
pyridine with DBU for at least 1 h would cause their
epimerisation.
Although the proposed mechanism via a pseudorotation
pathway seems to be unlikely,23 the epimerisation of 1
might occur in some other way and thus, the claimed
stereoselectivity could point towards general configura-
tional instability of H-phosphonate diesters in the pre-
sence of DBU. The latter aspects is of crucial
importance since most oxidative transformations of H-
phosphonates require strong bases, and for some of
them, for example the formation of P–C bonds24 via
H-phosphonate intermediates, DBU was found to be
most efficient.
Stability of dinucleoside H-phosphonate 1 in the presence
of DBU. First, chemical stability of dinucleoside H-
phosphonate 1 in pyridine in the presence of DBU was
investigated. Unfortunately, the addition of 2 equiv of
DBU to a pyridine solution of H-phosphonate 1 caused
large broadening of the 31P NMR resonances, which
prevented any reliable analysis using this spectroscopic
method. Since we knew that sulfurisation of 1 under
these conditions occurred stereospecifically (vide supra),
we decided to make use of this finding to infer the
composition of the reaction mixture resulted from
treatment of dinucleoside H-phosphonate 1 with DBU.
In this paper, we address the problem of possible con-
figurational instability of H-phosphonate diesters in the
presence of DBU in light of our 31P NMR spectroscopic
studies.
Results and Discussion
To find out if DBU can cause epimerisation of P-chiral
H-phosphonate diesters we investigated (i) the stereo-
chemical course of DBU-catalysed sulfurisation of
separate diastereomers of dinucleoside H-phosphonate
1, (ii) the configurational stability of dinucleoside H-
phosphonate 1 in the presence of DBU, and (iii) the
stereochemical course of sulfurisation of separate dia-
stereomers of dinucleoside acylphosphonate 3 in the
presence of n-butylamine and DBU.
To this end, a 1:1 diastereomeric mixture of 1 and 2
equiv of DBU were dissolved in pyridine, and after 2 h
excess of elemental sulfur was added (Scheme 1). The
31P NMR spectroscopy revealed27 that the reaction
mixture contained several products. These were identi-
fied as diastereomeric dinucleoside phosphorothioates 2
(dP=57.32 and 57.37 ppm, 1:1 ratio, ca. 42%), symme-
trical dinucleoside phosphorothioates 4 (dP=56.9 ppm,
ca. 21%) and 5 (dP=58.3 ppm, ca. 21%), and two iso-
For these studies, we chose dinucleoside H-phospho-
nates 1 with the 50-O-dimethoxytrityl and the 30-O-(1,3-
benzodithiol-2-yl) protecting groups since it was
claimed20 that presence of these bulky, lipophilic
groups, together with a sequence of bases was essential
for the observed stereoselectivity of sulfurisation of 1 in
the presence of DBU (Chart 1).
meric nucleoside H-phosphonate monoesters
6
(dP=3.1 ppm, ca. 7%) and 7 (dP=4.7 ppm, ca. 7%).28
The ratio of the products varied slightly from experi-
ment to experiment, however, a general tendency
observed was that the amount of symmetrical phos-
phorothioates (4+5) formed paralleled those of H-
phosphonate monoesters (6+7). Essentially the same
results were obtained when the reaction was carried out
in dichloromethane.
Sulfurisation of dinucleoside H-phosphonate 1 in the
presence of DBU
As we reported in a preliminary account of this work,25
sulfurisation of mixture of diastereomers of dinucleo-
These results indicated that probably, in the presence of
DBU, a significant degradation of dinucleoside H-

T. Johansson, J. Stawinski / Bioorg. Med. Chem. 9 (2001) 2315–2322
2317
phosphonate 1 occurred, and that prior to the addition
of sulfur, this reaction mixture contained along with 1, a
significant proportion of symmetrical dinucleoside H-
phosphonates 8, 9, and nucleoside H-phosphonate
monoesters 6, 7 (Scheme 2). This degradation of 1 was,
most likely, due to adventitious water, which in the
presence of a very strong base, DBU, caused partial
hydrolysis of the parent dinucleoside H-phosphonate 1,
followed by its transesterification with the produced
nucleosides. Although, one can envisage the formation
of symmetrical H-phosphonates 8 and 9 even under
strictly anhydrous via a ligand exchange mechanism,29
this possibility seemed less likely in light of the obser-
vation (vide supra) that amounts of phosphorothioates
4 and 5 (formed most likely from H-phosphonates 8 and
9, respectively) after sulfurisation paralleled those of H-
phosphonates 6 and 7. The hydrolytic pathway of 1 in
the presence of DBU, however, had to be significantly
slower than the sulfurisation reaction, since no degra-
dation products could be observed when DBU was
added to the reaction mixture containing H-phospho-
nate 1 and elemental sulfur.
To find out if DBU can cause epimerization of H-
phosphonate diesters at the phosphorus centre, separate
RP and SP diastereomers of dinucleoside H-phospho-
nate 1 in pyridine were treated with DBU (2 equiv) for
2 h and then sulfurized with elemental sulfur. The 31P
Chart 1.
Scheme 1.

2318
T. Johansson, J. Stawinski / Bioorg. Med. Chem. 9 (2001) 2315–2322
NMR spectroscopy revealed that the same type of pro-
ducts as in the reactions with diastereomeric mixture
(1:1) of 1 were formed (phosphorothioates 2, sym-
metrical phosphorothioates 4 and 5, and nucleoside
H-phosphonate monoesters 6 and 7), but the ratio
of diastereomeric phosphorothioates 2RP/2SP varied
depending on which diastereomer of H-phosphonate 1
was used for the reaction. Thus, treatment of 1 RP with
DBU followed by sulfurisation, produced 2 RP/ 2 SP in
a ratio of ca. 2:3, while from H-phosphonate 1 SP, this
ratio was ca. 6:1.30
It was gratifying to find, that presence of TMS-Cl (1
equiv) in the reaction mixture completely eliminated the
formation of hydrolysis products (compounds 6 and 7)
upon addition of DBU (1 equiv) to a pyridine solution
of separated RP and SP diastereomers of H-phospho-
nate 1, and also preserved their stereochemical integrity.
No hydrolysis or P-epimerization of dinucleoside H-
phosphonate 1 was observed upon addition of more
DBU (total 6 equiv). On this basis we could tentatively
conclude that H-phosphonate diesters of type 1 are
chemically and configurationally stable in the presence
of DBU, provided that the hydrolytic path of their
decomposition is eliminated.
Formation of both diastereomers of phosphorothioate 2
from diastereomerically pure RP and SP H-phospho-
nates 1 at first sight might have suggested a DBU-
mediated epimerization of H-phosphonate diester 1.
However, by considering compositions of the whole
reaction mixtures, a more likely explanation was that
stereochemistry of H-phosphonate 1 diastereomers used
for the reaction was eroded due to transesterification
with nucleosides, formed as products of hydrolysis the
starting material 1. This was in line with the observation
that the ratio between phosphorothioate 2 diaster-
eomers varied and approached value 1:1 with the
increasing degree of hydrolysis of dinucleoside H-phos-
phonate 1.
Stereochemical course of sulfurisation of dinucleoside
benzoylphosphonate 3
The stereochemistry of sulfurisation of acylphosphonate
3 with elemental sulfur in the presence of n-butylamine
and DBU was investigated to find out if there is any
mechanism operating under the reaction conditions that
may lead to epimerisation at the phosphorus centre of
acylphosphonate 3 and result in stereoselective forma-
tion of only RP diastereomers of phosphorothioate 2.20
To this end, a 1:1 mixture of acylphosphonate 3 dia-
stereomers (dP=À1.35 and À1.56 ppm)32 pyridine was
treated with elemental sulfur in the presence of n-butyl-
amine (10 equiv) and DBU (0.5 equiv). The reaction
was fast and both 31P NMR spectroscopy and TLC
analyses indicated the formation of two diastereomers
of phosphorothioate 2 in a ratio of ca. 1:1. Thus, we
could not confirm the results reported by Hata et al.,20
that sulfurisation of acylphosphonate 3 under the above
conditions occurred stereoselectively (Scheme 3).
However, since DBU exhibits a pronounced nucleophi-
licity towards phosphorus centre,31 we could not reject a
possibility, that P-epimerization of 1 in the above reac-
tion was due (at least partly) to a DBU-catalyzed ligand
exchange.29 To find out if DBU by itself can promote
epimerization of H-phosphonate
1 via a ligand
exchange mechanism, we had to completely eliminate
the hydrolytic path of decomposition of 1. Since all
technical measure to eliminate the presence of adventi-
tious water failed, we tried to use trimethylsilyl chloride
(TMS-Cl) to secure the anhydrous reaction conditions.
As to a possible influence of the amounts of n-butyla-
mine and DBU on the stereochemical outcome of the
above transformation, additional experiments were
Scheme 2.

T. Johansson, J. Stawinski / Bioorg. Med. Chem. 9 (2001) 2315–2322
2319
acylphosphonate 3 in the presence of n-butylamine and
DBU.
Conclusions
In conclusion, we have shown that sulfurisation of H-
phosphonate diester 1 in the presence of n-butylamine
and DBU as well as the generation this species from the
corresponding benzoylphosphonate 3 occurred stereo-
specifically, most likely with retention of configuration.
Thus, we tentatively conclude that H-phosphonate die-
sters of type 1 are chemically and configurationally
stable in the presence of DBU, provided that the
hydrolytic path of their decomposition is eliminated.
Scheme 3.
carried out. It was found that irrespective of the ratio of
n-butylamine and DBU used for the reaction, the sul-
furisation invariably led to the same equimolar amounts
of phosphorothioate 2 diastereomers. When the reac-
tion was carried out in pyridine in the presence of DBU
alone (10 equiv) or if DBU was added to the reaction
mixture before n-butylamine, the formation of phos-
phorothioate 2 was significantly suppressed due to
initiation of an alternative reaction pathway.33
Experimental
The 31P NMR spectra were recorded on Jeol GSX-270
FT or Varian 300 MHz spectrometer at 25 ꢀC in 5 mm
tubes using 25 mmols of phosphorus-containing com-
pounds in 0.5 mL of CDCl3. The spectra were refer-
enced by 2% H3PO4 in D2O as external standard
(coaxial inner tube). Pyridine (LabScan, Ireland) was
stored over molecular sieves (4 A), and pivaloyl chloride
(Aldrich, Sweden) and diazabicyclo[5.4.0]undec-7-ene
(DBU, from Aldrich), were freshly distilled. Starting
materials, 50-O-dimethoxytritylthymidine 30-H-phos-
phonate34 and 30-O-(1,3-benzodithiol-2-yl)thymidine35
were obtained according to published procedures.
Although these experiments showed that the conversion
of acylphosphonate 3 into the corresponding phos-
phorothioate 2 was not stereoselective, we could not
exclude a possibility of epimerization of acylpho-
sphonate 3 occurring in the presence of n-butylamine
and DBU. This point called for clarification since epi-
merisation, if it occurred, might under certain circum-
stances lead to
a
diastereomer of 1, and thus be a potential source of
preferential formation of one
The reference compounds used for the assignment of 31
P
NMR resonances, were obtained as follows: 50-O-dime-
thoxytritylthymidine 30-H-phosphonate 6 and 30-O-(1,3-
benzodithiol-2-yl)thymidine 50-H-phosphonate 7, by the
reaction of the appropriate nucleosides with diphenyl
H-phosphonate;34 bis(50-O-dimethoxytritylthymidin-30-
yl) H-phosphonate 8 and bis(30-O-(1,3-benzodithiol-2-
yl)thymidin-50-yl) H-phosphonate 9, by treatment of the
appropriate nucleoside (2 equiv) with diphenyl H-phos-
phonate in pyridine; bis(50-O-dimethoxytritylthymidin-
stereoselectivity in this type of reactions.
To investigate this problem, we needed separate RP and
SP diastereomers of acylphosphonate 3. Unfortunately,
in our hands acylphosphonate 3 proved to be much
more labile than expected on the basis of literature
reports,20 and the attempted separation of its diaster-
eomers failed due to extensive decomposition of 3 dur-
ing silica gel chromatography. However, we found that
benzoylation of the separate diastereomers of H-phos-
phonate 1 with benzoyl chloride in methylene chloride
produced the desired RP and SP diastereomers of 3, that
after purification via precipitation from petroleum
ether, gave only one signal in the 31P NMR spectrum.
30-yl) phosphorothioate
4
and bis(30-O-(1,3-benzo-
dithiol-2-yl)thymidin-50-yl) H-phosphonate 5, by sulfur-
isation of the corresponding H-phosphonate precursors,
8 and 9, respectively.
The assignments of signals in the 31P NMR spectra to
particular products or intermediates were done on the
basis of their chemical shifts, multiplicity of the signals
in 1H-coupled and 1H-decoupled spectra, by spiking the
reaction mixtures with appropriate species and, if pos-
sible, by isolation of the compound in question from
reaction mixtures. The assignment of proton and car-
bon resonances of 1–3 was done on the basis of known
Using these compounds we found that sulfurisation of a
diastereomer of acylphosphonate 3 resonating at higher
field in 31P NMR (dP=À1.56, probably SP) with ele-
mental sulfur in the presence of n-butylamine and DBU
produced only the RP diastereomer of 2 (dP=57.37
ppm), while the other isomer of 3 (resonating at lower
field, dP=À1.35, probably RP), gave exclusively SP
phosphorothioate 2 (dP=57.472 ppm). Thus, these
experiments established the conversion of acylpho-
sphonates 3 into the corresponding phosphorothioates 2
as stereospecific and showed that no epimerization
occurred during generation of H-phosphonates 1 from
or expected chemical shifts in conjunction with H–1H,
1H–13C, and DEPT correlated NMR spectroscopy.
1
50-O-Dimethoxytritylthymidin-30-yl
thiol-2-yl)thymidin-50-yl H-phosphonate 1. To a solution
of 50-O-dimethoxytritylthymidine 30-H-phosphonate 6
30-O-(1,3-benzodi-

2320
T. Johansson, J. Stawinski / Bioorg. Med. Chem. 9 (2001) 2315–2322
(triethylammonium salt, 2.11 mmol) and 30-O-(1,3-ben-
zodithiol-2-yl)thymidine (2.11 mmol, 1.00 equiv) in pyr-
idine (100 mL) was added pivaloyl chloride (4.23 mmol,
2.00 equiv). When the reaction was complete (ꢁ1 h,
TLC analysis), the mixture was quenched with metha-
nol (5 mL), partitioned between 5% aq NaHCO3
(100 mL) and CH2Cl2 (100 mL). The organic layer was
dried with Na2SO4, evaporated and purified by silica gel
chromatography using a stepwise gradient of ethyl ace-
tate (0–100%) in CH2Cl2. Yield: 90%. The RP and SP
diastereomers of 1 were separated using the same chro-
matographic system.
yl units, respectively) 163.93, 163.82 (2ꢂC4), 158.92 (2C
of DMT), 150.74, 150.43 (2ꢂC2), 144.13 (1C of DMT),
135.70, 135.43 (2C of BDT), 135.36 (Cb6), 135.21 (Ca6),
135.12, 135.10 (2C of DMT), 130.20, 130.18, 128.23,
128.17 (8C of DMT), 127.42 (1C of DMT), 126.02 (2C
of BDT), 122.31, 122.14 (2C of BDT), 113.46 (4C of
DMT), 111.90, 111.68 (2ꢂC5), 89.41 (SCHS), 87.40
(CDMT), 85.13 (Cb10), 84.85 (Ca40), 84.37 (Ca10), 82.36
(d, J=6.1 Hz, Cb40), 77.16 (Ca30), 73.66 (Cb30), 64.79
(d, J=6.1 Hz, Cb50), 63.17 (Ca50), 55.37 (2ꢂCH3O),
39.30 (Ca20), 38.43 (Cb20), 12.56 (Cb5–CH3), 11.81 (Ca5–
CH3).
1. (RP) (faster moving isomer). Yield 35%, white solid.
Purity >98% (1H NMR spectroscopy).
50-O-Dimethoxytritylthymidin- 30-yl 30-O-(1,3-benzodi-
thiol-2-yl)thymidin-50-yl phosphorothioate 2, triethylam-
monium salts. To a solution of separate RP and SP
diastereomers of 1 (0.102 mmol) in pyridine (2 mL) was
added n-butylamine (1.02 mmol, 10 equiv), DBU
(0.053 mmol, 0.52 equiv) and elemental sulfur
(1.53 mmol, 15.0 equiv). The reaction was complete
within 5 min (TLC analysis) but it was left for 1 h,
(according to Hata’s procedure) before the mixture was
partitioned between 5% aq NaHCO3 (10 mL) and
CH2Cl2 (10 mL), The organic layer was dried with
Na2SO4, evaporated and purified by silica gel chromato-
graphy using CH2Cl2/methanol 9:1.
1
31P NMR:. (d in ppm CDCl3) 7.24 (d, JPH=717 Hz).
1H NMR. (d in ppm CDCl3. Subscripts ‘a’ and ‘b’
denote resonances in the thymidyl 30-yl and thymidyl 50-
yl units, respectively) 9.36 (s, 1H, NH), 9.34 (s, 1H,
NH), 7.54 (s, 1H, Ha-6), 7.40–7.10 (m, 13H, ArH), 7.07
(s, 1H, Hb-6), 6.84 (d, 4H, J=9 Hz, CH3OCH¼CH),
6.77 (d, 1H, J=718 Hz, P-H), 6.75 (s, 1H, SCHS), 6.43
(m, 1H, Ha-10), 6.11 (t, 1H, J=7 Hz, Hb-10), 5.22 (m,
1H, Ha-30), 4.27–4.09 (m, 5H, Hb-30, 2ꢂH-40, Hb 50),
3.79 (s, 6H, 2ꢂCH3O), 3.44 (m, 2H, Ha-50), 2.60–2.42
(m, 2H, Ha-20), 2.50–2.19 (m, 2H, Hb-20), 1.81 (s, 3H,
C5b–CH3), 1.40 (s, 3H, C5a–CH3).
2. (SP) (faster moving isomer), obtained form 1 (RP).
Yield 88%, white solid. Purity >98% (1H NMR spec-
troscopy).
13C NMR. (d in ppm CDCl3. Subscripts ‘a’ and ‘b’
denote resonances in the thymidyl 30-yl and thymidyl 50-
yl units, respectively) 163.91, 163.90 (2ꢂC4), 158.94 (1C
of DMT), 150.76, 150.43 (2ꢂC2), 144.08 (1C of DMT),
135.81 (Cb6), 135.72, 135.48 (2C of BDT), 135.20 (Ca6),
135.07 (2C of DMT), 130.21, 130.18, 128.22, 128.18 (8C
of DMT), 127.43 (1C of DMT), 125.96 (2C of BDT),
122.39, 122.23 (2C of BDT), 113.47 (4C of DMT),
111.95, 111.52 (2ꢂC5), 89.54 (SCHS), 87.45 (CDMT),
85.70 (Cb10), 84.61 (d, J=6.9 Hz, Ca40), 84.38 (Ca10),
82.35 (d, J=6.9 Hz, Cb40), 77.62 (Ca30), 74.10 (Cb30),
64.57 (Cb50), 63.31 (Ca50), 55.39 (2ꢂCH3O), 39.53
(Ca20), 38.26 (Cb20), 12.53 (Cb5–CH3), 11.84 (Ca5–CH3).
31P NMR. (d in ppm CDCl3) 57.42.
1H NMR. (d in ppm CDCl3. Subscripts ‘a’ and ‘b’
denote resonances in the thymidyl 30-yl and thymidyl 50-
yl units, respectively) 7.62–7.03 (m, 15H, 2ꢂH6, ArH),
6.82 (d, 4H, J=9 Hz, CH3OCH¼CH), 6.62 (s, 1H,
SCHS), 6.43 (m, 1H, Ha-10), 6.25 (m, 1H, Hb-10), 5.32
(m, 1H, Ha-30), 4.30 (m, 1H, Hb-30), 4.22 (m, 1H, Ha-40),
4.13 (m, 1H, Hb-40), 3.93 (m, 2H, Hb-500), 3.77 (s, 6H,
2ꢂCH3O), 3.42 (m, 2H, Ha-50), 2.63 (q, 6H, J=7 Hz, N-
CH2), 2.67–2.27 (m, 2H, Ha-20), 2.35–1.96 (m, 2H, Hb-
20), 1.93 (s, 3H, C5b-CH3), 1.38 (s, 3H, C5a-CH3), 1.08 (t,
9H, J=7 Hz, N-C-CH3).
1. (SP) (slower moving isomer). Yield 40%, white solid.
Purity >98% (1H NMR spectroscopy).
13C NMR. (d in ppm CDCl3. Subscripts ‘a’ and ‘b’
denote resonances in the thymidyl 30-yl and thymidyl 50-
yl units, respectively) 164.10, 164.03 (2ꢂC4), 158.58 (2C
of DMT), 150.62, 150.52 (2ꢂC2), 144.30 (1C of DMT),
135.97, 135.52, 135.43, 135.38, 135.31, 135.24 (2C of
BDT, 2C of DMT, 2ꢂC60), 130.08, 128.13, 127.92 (8C
of DMT), 127.02 (1C of DMT), 125.61, 125.56 (2C of
BDT), 122.37, 122.29 (2C of BDT), 113.21 (4C of
DMT), 111.17 (2ꢂC5), 89.45 (SCHS), 86.95 (CDMT),
85.47 (Ca40), 84.71 (Cb10), 84.58 (Ca10), 82.36 (d,
J=8.4 Hz, Cb40), 77.53 (Ca30), 77.06 (Cb30), 64.89 (Cb50),
63.96 (Ca50), 55.20 (2ꢂCH3O), 45.87 (3ꢂCH2 of TEA),
39.17 (Ca20), 37.94 (Cb20), 12.39 (Cb5–CH3), 11.54 (Ca5–
CH3), 9.46 (3ꢂCH3 of TEA).
1
31P NMR. (d in ppm CDCl3) 8.38 (d, JPH=724 Hz)
1H NMR. (d in ppm CDCl3. Subscripts ‘a’ and ‘b’
denote resonances in the thymidyl 30-yl and thymidyl 50-
yl units, respectively) 9.04 (s, 1H, NH), 8.97 (s, 1H,
NH), 7.53 (s, 1H, Ha-6), 7.38–7.10 (m, 13H, ArH), 6.98
(s, 1H, Hb-6), 6.83 (d, 4H, J=9 Hz, CH3OCH¼CH),
6.80 (d, 1H, J=717 Hz, P-H), 6.75 (s, 1H, SCHS), 6.43
(m, 1H, Ha-10), 6.13 (t, 1H, J=7 Hz, Hb-10), 5.24 (m,
1H, Ha-30), 4.21 (m, 1H, Ha-40), 4.15–4.08 (m, 4H, Hb-
30, Hb-40, Hb 50), 3.78 (s, 6H, 2ꢂCH3O), 3.44 (m, 2H,
Ha-50), 2.57–2.42 (m, 2H, Ha-20), 2.50–2.07 (m, 2H, Hb-
20), 1.81 (s, 3H, C5b–CH3), 1.40 (s, 3H, C5a–CH3).
2. (RP) (slower moving isomer), obtained form 1 (SP).
Yield 88%, white solid. Purity >98% (1H NMR spec-
troscopy).
13C NMR. (d in ppm CDCl3. Subscripts ‘a’ and ‘b’
denote resonances in the thymidyl 30-yl and thymidyl 50-

T. Johansson, J. Stawinski / Bioorg. Med. Chem. 9 (2001) 2315–2322
2321
31P NMR. (d in ppm CDCl3) 57.37.
benzoyl), 129.27, 129.23 (2C of benzoyl), 127.40 (1C of
DMT), 125.99, 125.95 (2C of BDT), 122.33, 122.26 (2C
of BDT), 113.49 (4C of DMT), 111.96, 111.57 (2ꢂC5),
89.55 (SCHS), 87.46 (CDMT), 85.44 (Cb10), 84.85 (Ca40),
84.37 (Ca10), 82.36 (d, J=6.1 Hz, Cb40), 79.49 (Ca30),
74.28 (Cb30), 66.58 (Cb50), 63.46 (Ca50), 55.38
(2ꢂCH3O), 39.87 (Ca20), 38.30 (Cb20), 12.42 (Cb5–CH3),
11.86 (Ca5–CH3).
1H NMR. (d in ppm CDCl3. Subscripts ‘a’ and ‘b’
denote resonances in the thymidyl 30-yl and thymidyl 50-
yl units, respectively) 7.52–6.98 (m, 15H, 2ꢂH6, ArH),
6.75 (d, 4H, J=9 Hz, CH3OCH¼CH), 6.57 (s, 1H,
SCHS), 6.35 (m, 1H, Ha-10), 6.18 (m, 1H, Hb-10), 5.22
(m, 1H, Ha-30), 4.40 (m, 1H, Hb-30), 4.28 (m, 1H, Ha-40),
4.13 (m, 1H, Hb-40), 4.00 (m, 2H, Hb-50), 3.70 (s, 6H,
2ꢂCH3O), 3.37 (m, 2H, Ha-50), 2.68 (q, 6H, J=7 Hz, N-
CH2), 2.49–2.23 (m, 2H, Ha-20), 2.39–2.05 (m, 2H, Hb-
20), 1.83 (s, 3H, C5b–CH3), 1.29 (s, 3H, C5a–CH3), 1.07
(t, 9H, J=7 Hz, N–C–CH3).
3. (SP), obtained form 1 (SP). Yield 88%, white solid.
Purity >90% (1H NMR spectroscopy).
3
31P NMR. (d in ppm CDCl3) À1.56 (q, JPH=7 Hz);
MS (-FAB) m/z 1088.5 (MÀH).
13C NMR. (d in ppm CDCl3. Subscripts ‘a’ and ‘b’
denote resonances in the thymidyl 30-yl and thymidyl 50-
yl units, respectively) 164.20, 164.12 (2ꢂC4), 158.76 (2C
of DMT), 150.80, 150.62 (2ꢂC2), 144.45 (1C of DMT),
136.16, 135.78, 135.51, 135.43, 135.38 (2C of BDT, 2C
of DMT, 2ꢂC60), 130.24, 128.28, 128.08 (8C of DMT),
127.16 (1C of DMT), 125.78, 125.73 (2C of BDT),
122.49, 122.35 (2C of BDT), 113.39 (4C of DMT),
111.38 (2ꢂC5), 89.61 (SCHS), 87.13 (CDMT), 85.21
(Ca40), 84.74 (Ca10), 84.63 (Cb10), 83.56 (Cb40), 77.18
(Ca30), 76.87 (Cb30), 65.26 (Cb50), 64.11 (Ca50), 55.33
(2ꢂCH3O), 45.94 (3ꢂCH2 of TEA), 39.86 (Ca20), 38.12
(Cb20), 12.49 (Cb5–CH3), 11.71 (Ca5–CH3), 9.81
(3ꢂCH3 of TEA).
1H NMR. (d in ppm CDCl3) 8.82 (s, 1H, NH), 8.74 (s,
1H, NH), 8.17 (d, 2H, J=7 Hz, benz-HA), 7.66 (t, 1H,
J=7 Hz, benz-HC), 7.52–6.91 (m, 17H, 2ꢂH6, ArH),
6.81 (d, 4H, J=9 Hz, CH3OCH¼CH), 6.70 (s, 1H,
SCHS), 6.41 (m, 1H, Ha-10), 6.05 (t, 1H, J=7 Hz, Hb-
10), 5.30 (m, 1H, Ha-30), 4.26–4.06 (m, 5H, Hb-30, 2ꢂH-
40, Hb 50), 3.76 (s, 6H, 2ꢂCH3O), 3.44 (m, 2H, Ha-50),
2.65–2.25 (m, 2H, Ha-20), 2.45–2.15 (m, 2H, Hb-20), 1.76
(s, 3H, C5b–CH3), 1.39 (s, 3H, C5a–CH3).
13C NMR. 196.90 (d, J=173.2, C¼O), 163.79, 163.66
(2ꢂC4), 158.92 (2C of DMT), 150.52, 150.18 (2ꢂC2),
144.18 (1C of DMT), 135.57, 135.40, 135.28, 135.16 (2C
of BDT, 2C of DMT, 1C of benzoyl, 2ꢂC60), 130.20,
128.23, 128.20 (8C of DMT), 129.96 (2C of benzoyl),
129.21 (2C of benzoyl), 127.42 (1C of DMT), 126.02,
125.98 (2C of BDT), 122.28 (2C of BDT), 113.48 (4C of
DMT), 111.80, 111.52 (2ꢂC5), 89.45 (SCHS), 87.42
(CDMT), 85.12 (Cb10), 84.96 (Ca40), 84.47 (Ca10), 82.41
(Cb40), 79.96 (Ca30), 73.96 (Cb30), 66.21 (Cb50), 63.22
(Ca50), 55.38 (2ꢂCH3O), 39.33 (Ca20), 38.41 (Cb20),
12.51 (Cb5–CH3), 11.82 (Ca5–CH3).
50-O-Dimethoxytritylthymidin- 30-yl 30-O-(1,3-benzodi-
thiol-2-yl)thymidin-50-yl benzoylphosphonate 3. To a
solution of separate RP and SP diastereomers of 1
(0.203 mmol) in CH2Cl2 (4 mL ) were added, con-
secutively,
N,N-diethylaniline
(0.409 mmol,
2.0
equiv), benzoyl chloride (0.809 mmol, 4.0 equiv) and
bis(trimethyl)acetamide BSA(0.607 mmol/3.0 equiv).
After ca. 2 h (31P NMR) the mixture was added to pet-
roleum ether (150 mL) and the white solid was filtered
off and washed with several portions of petroleum ether.
The precipitation from petroleum ether was repeated.
Acknowledgements
3. (RP), obtained form 1 (RP). Yield 82%, white solid.
Purity >90% (1H NMR spectroscopy).
The authors are indebted to Professor Per J. Garegg for
interest and helpful discussions. Financial support from
the Swedish Natural Science Research Council is grate-
fully acknowledged.
3
31P NMR. (d in ppm CDCl3) À1.35 (q, JPH=7 Hz) );
MS (-FAB) m/z 1088.3 (MÀH).
1H NMR. (d in ppm CDCl3) 8.94 (m, 2H, 2ꢂNH), 8.12
(d, 2H, J=7 Hz, benz-HA), 7.65 (t, 1H, J=7 Hz, benz-
HC), 7.55 (s, 1H, Ha-6) 7.47 (t, 2H, J=7 Hz, Benz-HB),
7.35–7.05 (m, 14H, Hb-6, ArH), 6.81 (d, 4H, J=9 Hz,
CH3OCH¼CH), 6.72 (s, 1H, SCHS), 6.46 (m, 1H, Ha-
10), 6.09 (t, 1H, J=7 Hz, Hb-10), 5.29 (m, 1H, Ha-30),
4.45–4.13 (m, 5H, Hb-30, 2ꢂ H-40, Hb 50), 3.76 (s, 6H,
2ꢂ CH3O), 3.45 (m, 2H, Ha-50), 2.65–2.38 (m, 2H, Ha-
20), 2.49–2.10 (m, 2H, Hb-20), 1.76 (s, 3H, C5b–CH3),
1.42 (s, 3H, C5a–CH3).
References and Notes
1. Stawinski, J. In Handbook of Organophosphorus Chem-
istry; Engel, R., Ed.; Marcel Dekker: New York, 1992; pp
377–434.
2. Stawinski, J.; Stromberg, R. Trends Org. Chem. 1993, 4, 31.
3. Beaucage, S. L.; Iyer, R. P. Tetrahedron 1993, 49, 6123.
4. Narang, S. A. Tetrahedron 1983, 39, 3.
5. Kers, A.; Kers, I.; Kraszewski, A.; Sobkowski, M.; Szabo,
T.; Thelin, M.; Zain, R.; Stawinski, J. Nucleosides Nucleotides
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6. Froehler, B. C.; Ng, P. G.; Matteucci, M. D. Nucleic Acids
Res. 1986, 14, 5399. Agrawal, S.; Tang, J.-Y. Tetrahedron Lett.
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7. Almer, H.; Stawinski, J.; Stromberg, R.; Thelin, M. J. Org.
Chem. 1992, 57, 6163.
13C NMR. 197.00 (d, J=173.2 Hz, C¼O), 163.77,
163.73 (2ꢂC4), 158.93 (2C of DMT), 150.62, 150.34
(2ꢂC2), 144.20 (1C of DMT), 135.72, 135.55, 135.41,
135.18 (2C of BDT, 2C of DMT, 1C of benzoyl,
2ꢂC60), 130.21, 128.20 (8C of DMT), 129.93 (2C of

2322
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8. Lindh, I.; Stawinski, J. J. Org. Chem. 1989, 54, 1338.
9. Stawinski, J.; Thelin, M. J. Org. Chem. 1994, 59, 130.
10. Froehler, B. C. Tetrahedron Lett. 1986, 27, 5575. Let-
singer, R. L.; Zhang, G.; Sun, D. K.; Ikeuchi, T.; Sarin, P. S.
Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 6553.
11. Uhlmann, E.; Peyman, A. Chem. Rev. 1990, 90, 543.
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25. Johansson, T.; Bollmark, M.; Stawinski, J. In Collection
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26. At the initial stages of this project, there was a remote
possibility that the reported stereoselectivity during sulfurisa-
tion of 1 could be due to configurational instability of phos-
phorothioates 2 in the presence of DBU. In a separate
experiment, we found that this was not the case.
27. Before analysis, the reaction mixture was partitioned
between dichloromethane and 0.3 M TEAB buffer, to secure
good resolution of the 31P NMR resonances from diastere-
omeric dinucleoside phosphorothioates 2.
13. Stec, W. J.; Wilk, A. Angew. Chem., Int. Ed. Engl. 1994,
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21. In the original publications the authors erroneously refer-
red to this and the related reactions as stereospecific instead of
stereoselective ones.
28. The species were identified on the basis of their distinctive
chemical shifts, multiplicity of the signals in 31P–1H coupled
spectra, and by comparison with authentic samples, prepared
as described in the Experimental.
29. Zain, R.; Stawinski, J. J. Org. Chem. 1996, 61, 6617.
30. The degree of hydrolysis of H-phosphonate 1, as judged
from the sum of H-phosphonate monoesters 6 and 7 formed
under the reaction conditions, was higher for diastereomer 1
RP (ca. 20%) than for 1 SP (ca. 10%).
31. Kers, A.; Kers, I.; Stawinski, J. J. Chem. Soc., Perkin
Trans. 2, 2071. Kers, A.; Kers, I.; Bollmark, M.; Stawinski, J.
Acta Chem. Scand. 1998, 52, 1405.
32. Acylphosphonate diesters resonate in 31P NMR downfield
from the corresponding H-phosphonates (usually at À1 to
À3 ppm; see, e.g., de Vroom, E.; Spierenburg, M. L.; Dreef, C.
E.; van der Marel, G. A.; van Boom, J. H. Recl. Trav. Chim.
Pays-Bas 1987, 106, 65) and thus the chemical shift of purified
acylphosphonate 3 reported in ref 20 (dP=7.54 and 8.31 ppm)
is probably erroneous and refers most likely to its decomposi-
tion product, i.e., H-phosphonate 1 diastereomers.
22. The reaction occurs with retention of configuration at the
phosphorus center, but the priority rules of the CIP notation
of absolute configuration lead in this instance to a formal
change of the sense of chirality at the P-atom.
33. Under such conditions, the major (or the sole) product of
the reaction was apparently the corresponding tetranucleoside
1-(phosphinoxy)benzylphosphonate formed via the reaction of
H-phosphonate
1 (generated from 3) with benzoylpho-
23. This problem will be addressed separately on another
occasion.
24. Kers, A.; Stawinski, J.; Dembkowski, L.; Kraszewski, A.
Tetrahedron 1997, 53, 12691. Kers, A.; Stawinski, J. Tetra-
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Kraszewski, A.; Stawinski, J. Heteroatom Chem. 1999, 10, 492.
sphonate 3, followed by the rearrangement of the produced
symmetrical tetranucleoside bisphosphonate. See also: Fitch,
S. J.; Moedritzer, K. J. Am. Chem. Soc. 1962, 84, 1876.
34. Jankowska, J.; Sobkowski, M.; Stawinski, J.; Kraszewski,
A. Tetrahedron Lett. 1994, 35, 3355.
35. Sekine, M.; Hata, T. J. Am. Chem. Soc. 1983, 105, 2044.