2,4-Dinitrophenol as an activating reagent in a facile preparation of cyclic phosphate triesters

Article abstract of DOI:10.1016/j.tetlet.2003.11.101

2,4-Dinitrophenol was employed with benzyloxy-bis-(diisopropylamino) phosphine to synthesise the cyclic phosphate derivatives of a series of alkane diols (HO-(CH₂)_n-OH, n=2-6) in good isolated yields. Tetrazole and DNP were compared

Full text of DOI:10.1016/j.tetlet.2003.11.101

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 1001–1004
2,4-Dinitrophenol as an activating reagent in a facile preparation
of cyclic phosphate triesters
Eric J. Amigues and Marie E. Migaud*
School of Chemistry, David Keir Building, QueenÕs University Belfast, Stranmillis Road, Belfast BT9 5AG, UK
Received 10 April 2003; revised 11 November 2003; accepted 21 November 2003
Abstract—2,4-Dinitrophenol was employed with benzyloxy-bis-(diisopropylamino)phosphine to synthesise the cyclic phosphate
P
derivatives of a series of alkane diols (HO–(CH2)n–OH, n ¼ 2–6) in good isolated yields. Tetrazole and DNP were compared by 31
NMR spectroscopy for their ability to catalyse the cyclisation at the P(III) stage. Investigation of the phosphate triester stability
under various oxidation and chromatographic conditions resulted in the optimisation of the isolation procedures of the chemically
unstable cyclic compounds. Conditions for debenzylation were developed to yield the corresponding cyclic phosphodiesters
quantitatively. The methodology was further applied to the preparation and isolation of the cyclic phosphate derivative of a car-
bohydrate.
Ó 2003 Elsevier Ltd. All rights reserved.
When compared to the vast number of biologically
active phosphate-containing compounds, cyclic phos-
phates are not so ubiquitous. Yet, two cyclic phosphate
diesters, derivatives of nucleosides, are secondary mes-
sengers regulating numerous intracellular signalling
events; cAMP and cGMP. Other cyclic phosphate
diesters of carbohydrates are known to be either reac-
tion intermediates during enzymatic catalysis or
important reaction products such as 20,30-cyclic phos-
phate terminated RNAs.1 In order to facilitate the
preparation of cyclic phosphate diesters of carbohy-
drates, we aimed at developing a method, which allowed
large-scale reaction, facile work-up and did not involve
chromatography on fully deprotected species, in par-
ticular, anion exchange-based purification techniques.
Consequently, we developed a method, which gave us
access to hydrolytically labile cyclic phosphate triesters,
which could be purified by chromatography and quan-
titatively converted to pure cyclic phosphate diesters by
hydrogenolysis.
methodology on solid support to access cyclic phos-
photriesters forced us to look for reaction conditions
that were suitable for solid-phase chemistry. A polymer-
supported reagent that incorporated immobilised p-hy-
droxybenzyl alcohol and that was used for the phos-
phorylation of carbohydrates has recently been reported
by Parang.2 Based on this precedent, we decided to
investigate (4-methoxy)benzyloxy- and benzyloxy-bis-
(diisopropylamino)phosphine (2) as phosphitylating
agents.
O
O
HO
BnO
P
O
HO
1. (BnO)P(NiPr )
2 2
2,4-DNP
2. t-BuOOH
O
O
BnO
BnO
BnO
BnO
OBn OBn
OBn OBn
1
The combination of phosphoramidites and azole-type
activators is amongst the most commonly used methods
in the synthesis of phosphates by the ÔphosphiteÕ
approach. Our interest in optimising a ÔphosphiteÕ
The syntheses of very few terminal cyclic phosphate
esters of sugars have been described in the literature.3–5
Two of these reported sugar phosphates were formed
from group migration due to an intramolecular trans-
esterification of an a-hydroxy-phosphate triester and
therefore were obtained in low yields. Isolation of pure
compounds required extensive anion exchange chro-
matography, a technique known to offer very variable
results depending on the nature of the compounds being
Keywords: Cyclic phosphite; Cyclic phosphate; Carbohydrate; Phos-
phoramidite.
* Corresponding author. Tel.: +44-289-27-4339; fax: +44-289-38-
2117; e-mail: m.migaud@qub.ac.uk
0040-4039/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2003.11.101

1002
E. J. Amigues, M. E. Migaud / Tetrahedron Letters 45 (2004) 1001–1004
OH
4
a, n=1
b, n=2
c, n=3
d, n=4
e, n=5
OH
O2N
NO2
N
O
O
n
+
BnO
P
BnO
P
N
OH
n
2
"Ox"
5
a, n=1
b, n=2
c, n=3
d, n=4
e, n=5
O
O
O
P
BnO
n
Scheme 1. 2,4-Dinitrophenol-activated cyclic phosphate synthesis.
isolated. Furthermore, the purified compounds are often
isolated with high salt (i.e., NaCl) content, in concen-
trations sometimes unsuitable for biological evaluations.
Although numerous syntheses of carbohydrate and
nucleoside cyclic phosphates using tetrazole and bis-
aminophosphites have been reported,6–9 we were unable
to synthesise cyclic phosphate triester 1 in reasonable
yields with these reagents. Under standard conditions,
an excess of highly pure tetrazole was required and
contamination by such excess required extensive chro-
matography, leading to poor recovery due to decom-
position of the cyclic triester. In addition, excess of
phosphitylating reagent was required due to extended
reaction times and reagent degradation. We therefore
decided to investigate an alternative activator, 2,4-dini-
trophenol (2,4-DNP), which had been reported for the
preparation of nucleoside phosphites10 and to employ it
in combination with benzyloxy-bis-(diisopropyl-
amino)phosphine (Scheme 1) as alternative reagents to
the tri-(4-nitrophenol)phosphite that had been devel-
oped for the preparation of five-membered ring cyclic
phosphorothioates.11
the reaction mixture by aqueous extraction. As a con-
sequence, phosphitylation solvents could be selected
according to the diol solubility.
The mechanism of activation by 2,4-DNP and cyclisa-
tion is described in Scheme 2. The P(III) phosphoradi-
amidite 2 first reacted with 2,4-DNP to offer 3 in a
similar manner to that accepted for the activation of a
P(III) amidite by 2,4-DNP or tetrazole.10 Benzyloxy-2,4-
dinitrophenyloxy-(diisopropylamino)-phosphine (d ¼
152:5 ppm) 3 was the only intermediate accumulating
detectable by 31P NMR and was detected for all diols,
thus indicating that the subsequent reaction is the rate-
limiting step for the overall conversion. The activated
reagent 3 then reacted with one hydroxy group of the
diol to yield 7 (identified by 31P NMR (n ¼ 2;
P(III) d ¼ 148:0 ppm; n ¼ 3; P(III) d ¼ 147:7 ppm),
which subsequently cyclised to 4 after a second activa-
tion of the P(III) amidite with 2,4-DNP had taken
place.
Over time, reagent 2 hydrolysed to the hydrogen phos-
phonamidate 6 (d ¼ 13:8 ppm) at an identical rate
whether 2,4-DNP or tetrazole was used. Hydrolysis of 7
to 6 was also expected to take place and was one of the
reactions competing with the cyclisation of 7 to 4 as the
first steps of the conversion appeared to be reversible. In
the case of the medium size cyclic phosphites 4c–e, the
cyclisation failed to proceed to completion for either
activators, but much higher yields were obtained for the
2,4-DNP-catalysed-reactions (Table 1) for which the
ratio of cyclic phosphites 4 and therefore cyclic phos-
phates 5 to side product 6 was dramatically enhanced.
For the medium size ring formation reaction catalysed
by tetrazole, a large proportion of the hydrogen phos-
phonate 8 (Scheme 2) in addition to 6 was detected by
31P NMR and identified by 31P 1H coupled NMR. In the
case of the cyclisation reaction activated by 2,4-DNP,
little of 8 could be detected. Finally, once formed the
cyclic phosphites 4 did not hydrolyse to 8 in the presence
of 2,4-DNP. The enhanced rate of the second alk-
oxylation via the use of 2,4-DNP was therefore of pri-
mary importance for the formation of the cyclic
phosphite 4.
A systematic comparison of the ability of 2,4-DNP and
tetrazole to promote the formation of small to medium
size phosphate triester rings from diols and carbohy-
drates is reported here. The chemical sequence estab-
lishing the cyclisation mechanism and the nature of the
reaction intermediates is described. Conditions for
quantitative removal of the benzyl-protecting group to
yield the cyclic diesters are also discussed.
We first considered 2,4-DNP for its availability and ease
of use and purification. Unlike tetrazole, which involves
hazardous purification conditions and is only available
in solution, 2,4-DNP is purchased as an inexpensive
solid that can easily be dried and is easily removed from
2,4-DNP was purchased from Aldrich. Typically, 2,4-DNP (5–10 g)
was dried under high vacuum in the presence of P2O5 overnight after
azeotroping water with toluene (3x). Thus dried 2,4-DNP remained
suitable as an activating reagent for 3–4 weeks when stored at rt
under argon.

E. J. Amigues, M. E. Migaud / Tetrahedron Letters 45 (2004) 1001–1004
1003
OH
N
HO
NO2
OH
N
P
BnO
O
2 N
N
n
NO2
O
BnO
P
BnO
P
O
N
2
3
7
n
OH
NO2
OH
NO2
O2N
N
P
NO2
BnO
H
O
O
O
6
BnO
P
O
P
n
BnO
NO2
O
4 a, n=1
b, n=2
c, n=3
d, n=4
e, n=5
OH
H2
O
n
OH
O
P
n
BnO
H
8
O
Scheme 2. Proposed chemical sequence yielding to cyclic phosphites 4.
Table 1. Activator catalysed cyclisation reactions of diol a–e with reagent 2
Entry
Activator
31P(III) 4
Reaction time
Ratio 4/3a
31P(V) 5
Isolated yield 5 Isolated yield 5
silica (%)
HPLC (%)
a
b
c
d
e
DNP
Tetrazole
DNP
135.7
131.2
134.8
133.9
140.3
2 h
10/1
18.3
)6.7
3.9
21
56
2.5/1
3.5/1
3.5/1
4.0/1
1.5/1
2 h
50
43
36
––
62
52
44
54
Tetrazole
DNP
2 h
Tetrazole
DNP
18 h
18 h
)0.2
)0.2
Tetrazole
DNP
Tetrazole
1/1
3/1
1/1
a Based on 31P NMR and 1H NMR.
The ratio of cyclic phosphates 5 to hydrogen phosphon-
amidate 6 remained constant after oxidation with
t-BuOOH in decane to that of the cyclic phosphites 4–6.
However, this ratio decreased when H2O2 or t-BuOOH
in water was used instead of t-BuOOH in decane.
Hydrolysis of cyclic phosphites and phosphates could
take place at )78 °C and as expected, the rate was
dependent on the ring size. Reversed-phase chroma-
tography (C18-RP Supelcosyl HPLC column; acetoni-
trile–H2O) was the only suitable method of purification
of most cyclic phosphates, especially 5a as less than 50%
recovery could be obtained from silica chromatography
columns (Table 1).
first high-yielding conversion of a carbohydrate terminal
diol to a cyclic phosphate triester, using that same
reagent but with 2,4-DNP as activator. The conversion
of the diol to the cyclic phosphite with 2 in the presence
of 2,4-DNP was quantitative, while similar results to
those observed by Schweda3 were obtained when tetra-
zole was used. Subsequent oxidation with t-BuOOH in
decane yielded 1 quantitatively.12
Since the ultimate aim of this work is to access cyclic
phosphate diesters in high yields and high degrees of
purity, we investigated means to remove selectively the
benzyl group from the highly labile cyclic phosphate of
triesters 5 without formation of any ring-opened side-
products. While the cyclic phosphate triesters 5b–e could
be easily deprotected quantitatively using hydrogenoly-
sis conditions (H2, Pd/C), 5a was rapidly hydrolysed and
subsequently deprotected to yield 2-hydroxyethyl phos-
Having optimised the reaction and isolation conditions
for simple diols, we turned our attention to the prepa-
ration of 1. Attempts had been made to prepare a cyclic
phosphate of a manno-heptoside using reagent 2 with
tetrazole, however this strategy was reported to be
unsuitable.3–5 While the synthesis of the diol precursor
to 1 will be reported in due course, we wish to report the
1
phate monoester (structure confirmed by H, 13C and
31P NMR). It was postulated that a catalytic amount of
acid generated in the early stages of the hydrogenolysis

1004
E. J. Amigues, M. E. Migaud / Tetrahedron Letters 45 (2004) 1001–1004
5. Burger, A.; Tritsch, D.; Biellmann, J.-F. Carbohydr. Res.
2001, 332, 141–149.
6. Pavey, J. B. J.; Cosstick, R.; OÕNeill, I. Tetrahedron Lett.
1998, 39, 8923–8924.
7. Strasser, M.; Ugi, I. Acta Chem. Scand. 1993, 47, 125–130.
8. Riley, A. M.; Guedat, P.; Schlewer, G.; Spiess, B.; Potter,
B. V. L. J. Org. Chem. 1998, 63, 295–305.
9. Sutherland, R. S.; Kogan, B. A.; Piechota, H. J.; Bredt,
D. S. J. Urol. 1997, 157, 1109–1116.
is enough to catalyse the ring opening of the remaining
cyclic phosphate triesters present in solution. Conse-
quently, half an equivalent amount of solid K2CO3 was
added to the THF solution containing 5a. Under such
conditions, 5a was deprotected to yield the cyclic
phosphate diester in quantitative yields. The purity of
the samples thus obtained was confirmed by 1H, 13C and
31P NMR and no purification by chromatography was
required on the cyclic phosphate diesters.
10. Dabkowski, W.; Tworowska, I.; Michalski, J.; Cramer, F.
Tetrahedron Lett. 2000, 41, 7535–7539.
11. Wenska, M.; Jankowska, J.; Sobkowski, M.; Stawinski, J.;
Kraszewski, A. Tetrahedron Lett. 2001, 42, 8055–8058.
12. A dry DCM solution of the diol, 2 and 2,4-DNP
(equimolar amounts) was stirred at rt until only one peak
Acknowledgements
corresponding to the cyclic phosphite was observed by 31
P
NMR. The oxidation, carried out by addition of
We are grateful to the Engineering and Physical Sciences
Research Council (grant # R89257/01) for financial
support of this work.
t-BuOOH at )78 °C, was stopped by addition of sodium
1
thiosulfate to afford 1. H NMR (CDCl3) d 7.38–7.26 (m,
25H), 5.12 (d, JPOC ¼ 4:9 Hz, 2H), 5.04 (d, J ¼ 10:9 Hz,
1H), 5.00 (d, J ¼ 8:1 Hz, 1H), 4.84 (d, J ¼ 12:0 Hz, 1H),
4.81 (d, J ¼ 11 Hz, 1H), 4.74 (d, J ¼ 12:1 Hz, 1H), 4.70 (s,
1H), 4.61 (d, J ¼ 13:6 Hz, 1H), 4.70–4.50 (m, 1H), 4.58 (d,
J ¼ 11:7 Hz, 1H), 4.50 (d, J ¼ 11:9 Hz, 1H), 4.12 (dd,
J ¼ 7:2, 14.3 Hz, 1H), 4.07 (dd, J ¼ 8:7, 9.4 Hz, 1H), 3.98
(dd, J ¼ 1:7, 10.5 Hz, 1H), 3.50 (ddd, J ¼ 7:6, 8, 15.6 Hz,
1H), 4.47 (dd, J ¼ 3:7, 9.6 Hz, 1H), 3.20 (dd, J ¼ 8:6,
10.4 Hz, 1H). 13C NMR (CDCl3) d 138.5, 137.8, 137.3,
136.7, 135.8, 129.0, 128.8, 128.7, 128.6, 128.5, 128.4, 128.3,
128.0, 127.9, 127.7, 127.6, 127.0, 94.2, 82.4, 79.7, 76.3,
75.8, 75.5, 74.4, 72.7, 70.1, 69.5, 69.3, 68.8. 31P NMR
(CDCl3) d 17.4. HRMS (FABþ) Calcd for C42H44O9P
(M+Hþ) 723.2723, found 723.2702.
References and notes
1. Vyle, J.; Williams, N.; Grasby, J. Tetrahedron Lett. 1998,
39, 7975–7978.
2. Parang, K. Bioorg. Med. Chem. Lett. 2002, 12, 1863–
1866.
3. Stewart, A.; Bernlind, C.; Martin, A.; Oscarson, S.;
Richards, J. C.; Schweda, E. K. Carbohydr. Res. 1998,
313, 193–202.
4. DÕSouza, F. W.; Benenson, Y.; Baasov, T. Bioorg. Med.
Chem. Lett. 1997, 7, 2457–2462.