A versatile one-pot procedure to phosphate monoesters and pyrophosphates using Di(p-methoxybenzyl)-N,N-diisopropylphosphoramidite

Article abstract of DOI:10.1021/ol801608j

(Chemical Equation Presented) A one-pot procedure for the preparation of phosphoramidates, phosphorothioates, pyrophosphates, phosphodiesters, and phosphofluoridates has been devised using di(p-methoxybenzyl)-N,N- diisopropylphosphoramidite as the common phosphitylating reagent.

Full text of DOI:10.1021/ol801608j

ORGANIC
LETTERS
2008
Vol. 10, No. 20
4461-4464
A Versatile One-Pot Procedure to
Phosphate Monoesters and
Pyrophosphates Using
Di(p-methoxybenzyl)-N,N-diisopropylphosphoramidite
Gerbrand J. van der Heden van Noort, Carlo P. Verhagen, Maarten G. van der
Horst, Herman S. Overkleeft, Gijsbert A. van der Marel,* and Dmitri V. Filippov*
Leiden Institute of Chemistry, Leiden UnVersity, PO Box 9502, 2300 RA Leiden, The
Netherlands
marel_g@chem.leidenuniV.nl; filippoV@chem.leidenuniV.nl
Received July 15, 2008
ABSTRACT
A one-pot procedure for the preparation of phosphoramidates, phosphorothioates, pyrophosphates, phosphodiesters, and phosphofluoridates
has been devised using di(p-methoxybenzyl)-N,N-diisopropylphosphoramidite as the common phosphitylating reagent.
Phosphorus-containing functional groups not only are es-
sential elements of nucleic acids but also are present in other
naturally occurring compounds such as proteins,1 carbohy-
drates,2 and lipids.3 Among these biomolecules phospho-
romono- and diesters,1,2 pyro- and triphosphates,4,5 phos-
phoramidates,6 and C-phosphonates7 can be discerned. The
pivotal role that phosphorus-containing molecules play in a
broad palette of biological processes has stimulated research
into the synthesis of these classes of compounds.8,9 In
addition, the pharmacological potential of phosphorus com-
pounds10-12 led to the development of artificial phosphate
derivatives such as phosphorothioates13 and phosphofluori-
dates.14 Initially developed to meet the needs of automated
nucleic acid synthesis, the phosphoramidite approach is the
synthetic methodology most applied toward phosphodiester-
containing molecules.15-17 Introduction of phosphate mo-
noesters is generally attained with the aid of amidites
provided with two protecting groups, which are often base-
(8) Kraszewski, A.; Stawinski, J. Pure Appl. Chem. 2007, 79, 2217–
2227
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S. V.; Wong, C. H. Bioorg. Med. Chem. 2000, 8, 1937–1946
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10.1021/ol801608j CCC: $40.75
Published on Web 09/19/2008
2008 American Chemical Society

labile.18,19 Upon activation with weakly acidic azole the
amidite reagent reacts with an alcohol to give a phosphite
triester. Subsequent oxidation to the phosphate triester and
final deprotection furnishes the target phosphate.20 As part
of our efforts21-23 aimed at the synthesis of naturally
occurring phosphates and derivatives thereof via the phos-
phoramidite approach, we embarked on the exploration of
the phosphitylation properties of methoxybenzyl-N,N-diiso-
propylphosphoramidites 1 and 2 (Figure 1). At the outset
Scheme 1. Synthesis of Phosphate 5 and Phosphorothioate 6
Figure 1. Methoxybenzyl phosphoramidites.
(162 MHz, δ 141.8, acetone-d6 capillary in MeCN), the
formation of phosphite triester 4 as the major product
together with some minor unidentified phosphonates (δ 14.1,
9.1). In the subsequent steps 4 was used without isolation,
since silica gel column chromatography yielded only 29%
of 4, presumably due to its instability. Addition of t-BuO2H
to oxidize phosphite 4, followed by p-methoxybenzyl depro-
tection using 3% TFA in DCM and chromatographic
purification yielded phosphate 5 (31P NMR, 162 MHz, δ 1.6,
CDCl3/MeOD-d4) in 78% yield. In contrast, phosphitylation
of 3 using agent 2 under identical conditions failed to yield
the intended phosphite triester but instead produced an
unidentified C- and H-phosphonate in a 1:1 ratio (31P NMR,
162 MHz, δ 31.9, 14.5, acetone-d6 capillary in MeCN).
Having demonstrated the effectiveness of phosphitylating
agent 1 to introduce phosphate monoesters under mild acidic
(deprotection) conditions, the susceptibility of di(p-meth-
oxybenzyl) protected phosphite triester 4 to undergo an
Arbuzov reaction was explored. Thymidine 3 was converted
into phosphite triester 4 as described above, but now the
reaction mixture was treated with diphenyldisulfide at the
time the transformation of 3 to 4 was complete (as revealed
by NMR), leading to phosphorothioate diester 6 in 79% yield
(Scheme 1). To our surprise we observed complete in situ
cleavage of both p-methoxybenzyl protecting groups when
the reaction was monitored by 31P NMR spectroscopy, where
we expected to see the corresponding triester still carrying
one p-methoxybenzyl group. This one-pot procedure thus
allows an immediate access to S-phenyl phosphorothioates,
known precursors26 in the synthesis of pyrophosphates.
we deemed these reagents to provide attractive entries to
phosphomonoesters, since we envisaged that phosphitylation
of an alcohol of choice with di(p-methoxybenzyl)-N,N-
diisopropyl phosphoramidite (1) followed by oxidation and
mild acidic removal of both p-methoxybenzyl groups would
readily provide the corresponding phosphomonoester. The
reagent 2 (Figure 1) equipped with 2,4-dimethoxybenzyl
groups would conceivably lead to a phosphotriester moiety
convertible to a phosphomonoester under even milder
conditions.
An additional useful feature of reagents 1 and 2 is the
potential of methoxybenzyl substituents to engage in Arbu-
zov-type reactions.24 This would open the way to the
synthesis of modified phosphates. Phosphoramidites 1 and
2 were synthesized in good yields via a procedure reported
for a related compound,25 comprising treatment of p-
methoxybenzylalcohol and 2,4-dimethoxybenzylalcohol, re-
spectively, with N,N-diisopropylaminophosphorodichloridite
in the presence of triethylamine.
Reaction of reagent 1 (1.2 equiv) with thymidine 3 (1.0
equiv) using dicyanoimidazole (DCI) (1.2 equiv) as activator
(Scheme 1) gave, as monitored by 31P NMR spectroscopy
(15) Nifantiev, E. E.; Grachev, M. K.; Burmistrov, S. Y. Chem. ReV.
2000, 100, 3755–3799.
(16) Beaucage, S. L.; Iyer, R. P. Tetrahedron 1993, 49, 6123–6194.
(17) Beaucage, S. L.; Iyer, R. P. Tetrahedron 1992, 48, 2223–2311.
(18) Ausin, C.; Grajkowski, A.; Cieslak, J.; Beaucage, S. L. Org. Lett.
2005, 7, 4201–4204.
(19) Lartia, R.; Asseline, U. Tetrahedron. Lett. 2004, 45, 5949–5952.
(20) Nurminen, E.; Lonnberg, H. J. Phys. Org. Chem. 2004, 17, 1–17.
(21) Ghisaidoobe, A. B. T.; de Koning, M. C.; Duynstee, H. I.; Ten
Kortenaar, P. B. W.; Overkleeft, H. S.; Filippov, D. V.; van der Marel,
Guided by these results we were eager to find out whether
we could extend the one-pot procedure we discovered to
other Arbuzov-type reactions. It occurred to us that using
iodine as the oxidation reagent for 4 would allow us to
introduce a range of nucleophilic atoms at the phosphorus
center via the putative phosphoryliodide.27 We first focused
G. A. Terahedron Lett. 2008, 49, 3129–3132
(22) Kriek, N.; Meeuwenoord, N. J.; van den Elst, H.; Heus, H. A.; van
der Marel, G. A.; Filippov, D. V. Org. Biomol. Chem. 2006, 4, 3576–3586
(23) Filippov, D.; Timmers, C. M.; Roerdink, A. R.; van der Marel,
G. A.; van Boom, J. H. Terahedron Lett. 1998, 39, 4891–4894
.
.
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(24) Meyer, A.; Morvan, F.; Vasseur, J. J. Terahedron Lett. 2004, 45,
3745–3748.
(25) Dreef, C. E.; Tuinman, R. J.; Lefeber, A. W. M.; Elle, C. J. J.; van
der Marel, G. A.; van Boom, J. H. Tetrahedron 1991, 47, 4709–4722.
(26) Shuto, S.; Shirato, M.; Sumita, Y.; Ueno, Y.; Matsuda, A.
Terahedron Lett. 1998, 39, 7341–7344.
4462
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on phosphoromorpholidates, the most well-known intermedi-
ates en route to pyrophosphates.28
The solution-phase synthesis of thymidine derivative 8 was
undertaken by adaptation of an iodine-mediated procedure
to create phosphoromorpholidates on a solid support.29
Thymidine 7 (Scheme 2) was phosphitylated with 1 in the
DAST as fluoride source to the reaction mixture after iodine-
mediated oxidation resulted in the formation of phosphof-
luoridate 10 (80%). Similarly, addition of the sodium salt
of p-nitrophenol to the oxidized reaction mixture resulted in
formation of the corresponding phosphodiester 11 (44%).
Surprisingly, addition of water did not result in the formation
of the expected phosphate monoester but furnished the
symmetric pyrophosphate 12 (78%). All reactions described
above were conducted under the same conditions, and the
formation of the pyrophosphate 12 upon addition of water
as well as the fact that no special treatment is needed to
remove the second p-methoxybenzyl group in the remaining
reactions (Scheme 2) put forward the question of the identity
of the reactive intermediates in the reaction sequence
accompanying our one-pot procedure. On the basis of the
data that resulted from the analysis of selected reactions by
31P NMR spectroscopy, we propose the following mechanism
(Scheme 3).
Scheme 2
.
Synthesis of Phosphate Monoesters 8-11 and
Symmetric Pyrophosphate 12
Scheme 3. Proposed Reaction Mechanism
Activation of amidite 1 (δ: 148.5) with DCI and subse-
quent coupling with alcohol 3 gave the phosphite triester 4
(31P NMR, 162 MHz, δ 141.8, acetone-d6 capillary in MeCN,
see Scheme 1). Treatment of the phosphite triester 4 with
iodine in pyridine (Scheme 3) would lead to phosponium
species A and the subsequent phosphoryliodide B, having
resonances around δ -44, as described.27 However, these
resonances could not be detected in our experiments, but
instead a signal at δ -11.2 was observed. This indicates that
the putative phosphoryliodide B was transformed im-
mediately into another intermediate with concomitant cleav-
age of the second p-methoxybenzyl group (PMB). This loss
of the protective group can be explained by nucleophilic
attack of iodide generated in step C on the benzylic position
of the PMB group. On the basis of literature data that
comparable phosphorazolides have 31P NMR resonances at
δ -12.228 (162 MHz, pyridine/DMSO-d6) and -11.2431 (40
MHz, pyridine-d5) we tentatively assign the resonance at δ
-11.24 to the dicyanoimidazolide species D. Subsequent
addition of a nucleophile leads to displacement of dicy-
anoimidazole to give the corresponding phosphate derivative.
The proposed dicyanoimidazolide species D as ultimate
reactive intermediate explains not only the removal of both
presence of DCI under the same conditions as described
above, and the obtained di(p-methoxybenzyl)phosphite tri-
ester was oxidized with iodine in pyridine and subsequently
treated with morpholine for 1 h. After silica gel column
chromatography morpholidate 8 was isolated in 86% yield.30
Following an analogous procedure using iso-butylamine
instead of morpholine as nucleophile led to the clean
formation of the protected phosphoramidate 9 (79%).30
To broaden the scope of the one-pot procedure, the use
of non-nitrogen nucleophiles was investigated. Addition of
(27) Nielsen, J.; Caruthers, M. H. J. Am. Chem. Soc. 1988, 110, 6275–
6276.
(28) Wittmann, V.; Wong, C. H. J. Org. Chem. 1997, 62, 2144–2147.
(29) Cooke, L. A.; Frauendorf, C.; Gilea, M. A.; Holmes, S. C.; Vyle,
J. S. Terahedron Lett. 2006, 47, 719–722.
(30) It became apparent that under the influence of morpholine or
isobutylamine the benzoyl protecting group on the nucleobase was removed.
This problem was nonexistent when the more stable POM group was used
for protection of the thymine base.
Org. Lett., Vol. 10, No. 20, 2008
4463

p-methoxybenzyl groups but also the formation of the
pyrophosphate 12. Addition of water will partly and slowly
hydrolyze dicyanoimidazolide species D to give the corre-
sponding monophosphate, which in turn will react more
quickly than water with D to give pyrophosphate (for
example 12).
To further establish the applicability of our one-pot
procedure using di(p-methoxybenzyl)-N,N-diisopropylphos-
phoramidite 1, the synthesis of asymmetric pyrophosphate32
14 and the phosphoramidon precursor33 16 (Scheme 4) were
influence of DCI, subsequent iodine/pyridine oxidation, and
finally addition of thymidine phosphate 5. RP-HPLC and gel
filtration purification yielded homogeneous pyrophosphate
14 in moderate yield (27%). Similarly phosphoramidon
precursor 16 was synthesized by phosphitylating ꢀ-rhamnose-
triacetate 15 with 1, iodine oxidation and addition of the
N-terminal unprotected Leu-Trp ethyl ester in a moderate
yield (19%). Such modest yields, compared to synthesis of
compounds 8-12 are probably caused by the losses in the
purification process, as no major side reactions have been
observed.
In conclusion, we have presented an efficient one-pot
procedure to phosphoramidates, phosphorothioates, pyro-
phosphates, and phosphofluoridates by a sequence of reac-
tions comprising phosphitylation of an alcohol with di(p-
methoxybenzyl)-N,N-diisopropylphosphoramidite 1, oxidation
with iodine, and treatment with the nucleophile of choice.
The nature of the p-methoxybenzyl groups allows the use
of reagent 1 to introduce phosphate monoesters under
relatively mild acidic conditions.
Scheme 4
.
Asymmetric Pyrophosphate 14 and Phosphoramidon
Precursor 16
Acknowledgment. This work was funded by The Neth-
erlands Organization for Scientific Research (NWO).
Supporting Information Available: Spectroscopic data
and experimental procedures. This material is available free
of charge via the Internet at http://pubs.acs.org.
OL801608J
undertaken. Crude pyrophosphate 14 was obtained by
phosphitylation of protected adenosine 13 with 1 under the
(32) Ohkubo, A.; Aoki, K.; Seio, K.; Sekine, M. Terahedron Lett. 2004,
45, 979–982.
(31) Chandrasegaran, S.; Murakami, A.; Kan, L. S. J. Org. Chem. 1984,
49, 4951–4957.
(33) Donahue, M. G.; Johnston, J. N. Bioorg. Med. Chem. Lett. 2006,
16, 5602–5604.
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