One-pot synthesis of organophosphate monoesters from alcohols

Article abstract of DOI:10.1016/j.tetlet.2013.01.059

A one-pot procedure for the phosphorylation of alcohols provides the corresponding phosphate monoesters in improved yields. The protocol features the use of tetrabutylammonium hydrogen phosphate and trichloroacetonitrile, followed by purification of the crude product by flash chromatography on silica gel. The final step, cation exchange chromatography, affords the organophosphates as ammonium salts that are usually required for biochemical applications. The mechanism appears to be phosphate rather than alcohol activation by trichloroacetonitrile.

Full text of DOI:10.1016/j.tetlet.2013.01.059

Tetrahedron Letters 54 (2013) 1690–1692
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Tetrahedron Letters
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One-pot synthesis of organophosphate monoesters from alcohols
Lucas M. Lira a,b, Dimitar Vasilev b, Ronaldo A. Pilli a, Ludger A. Wessjohann b,
⇑
a Institute of Chemistry, University of Campinas, 13083-970 Campinas, SP, Brazil
b Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A one-pot procedure for the phosphorylation of alcohols provides the corresponding phosphate monoes-
ters in improved yields. The protocol features the use of tetrabutylammonium hydrogen phosphate and
trichloroacetonitrile, followed by purification of the crude product by flash chromatography on silica gel.
The final step, cation exchange chromatography, affords the organophosphates as ammonium salts that
are usually required for biochemical applications. The mechanism appears to be phosphate rather than
alcohol activation by trichloroacetonitrile.
Received 2 November 2012
Revised 9 January 2013
Accepted 14 January 2013
Available online 21 January 2013
Keywords:
Alcohol
Ó 2013 Elsevier Ltd. All rights reserved.
Phosphorylation
Monophosphates
Organophosphates
One-pot synthesis
Prenylphosphates
The condensation of a phosphate residue with hydroxyl groups
present in biogenic alcohols like carbohydrates, proteins, and other
biomolecules to form the corresponding phosphate ester is called
phosphorylation. This transformation can significantly change the
physical, chemical, and biological properties of the substrate due
to change in its solubility, electric charge, conformational proper-
ties, and protein recognition, among others. Phosphorylation of
biological systems is usually carried out by ATP and is catalyzed
by kinases, while phosphatases catalyze the removal of phosphate
groups in biological systems. These two families of enzymes are
key players in cell signaling and in the regulation of signal trans-
duction cascades involved in cell metabolism. Disruption of their
proper functioning has been related to several diseases such as dia-
betes, cancer, and others.1
avoid such side products, the use of organic phosphate triesters
containing two cleavable ester residues has been established as a
valuable method for the preparation of phosphate monoesters.
Evans,6 Johns,7 and Miyashita8 have reported methodologies
wherein phosphorous trichloride, diethylchlorophosphite, and
phosphoryl chloride, respectively, are used to obtain the aforemen-
tioned phosphate esters. Some of these reagents are not suitable at
all for acid sensitive alcohols, for example, those leading to higher
prenylphosphates.
A more direct approach to phosphate monoesters is the phos-
phorylation of an alcohol substrate with an activated form of phos-
phoric acid under mild conditions. The pioneering work by Cramer
in 1959 set the stage for direct phosphorylation of alcohols.9
An enormous variety of biomolecules is encountered in the
form of phosphate monoesters such as nucleotides, proteins, and
also a great number of natural products arising from the secondary
metabolism of, for example, fungi such as (À)-calyculin A (1),
cytostatin (2), and fostriecin (3) (Fig. 1).2
OH
O
O
MeO
(Me)2N
N
H
OH
N
N
C
H2O3PO
O
Phosphate monoesters are important in medicinal chemistry,
for example, as water soluble prodrugs and for biological studies.3
Several approaches to their preparation have been reported but
some of them are plagued by the competitive formation of di-
and tri-substituted esters or by the formation of the corresponding
pyrophosphates; or they require the utilization of a great excess of
either the alcohol or the phosphorylation reagent.4,5 In order to
O
OH OH OMe
OH
1
(-)-Calyculin (
)
M e
H2O3PO
OH
OH
Me
Cytostatin (
Figure 1. Examples of phosphate monoester natural products.
H2O3PO
OH
O
O
O
O
Me OH
M e
2
3
)
)
Fostriecin (
⇑
Corresponding author. Tel.: +49 (0) 345 5582 1301; fax: +49 (0) 5582 1309.
E-mail address: wessjohann@ipb-halle.de (L.A. Wessjohann).
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.01.059

L. M. Lira et al. / Tetrahedron Letters 54 (2013) 1690–1692
1691
1. Cl3CCN
2. (n-Bu)4NH2PO4, CH3CN
NH4HCO3 buffer solution, a process which also helps to remove
dissolved silica gel.13
O
P
R
OH
R
O
O NH4
OH
3. DOWEX 50WX8, NH4HCO3
The ammonium phosphate monoesters were characterized by
ESI-MS and 1H-, 13C-, and 31P NMR analyses. In the 1H NMR
spectra of the allylic and benzylic monoesters the coupling con-
stant 3J (1H–31P) is evident (4.5–6.0 Hz) while in the 13C NMR
spectra the 13C–31P coupling is generally observed for the carbi-
Scheme 1. General phosphorylation procedure.
nolic carbon (2J = 4.0–7.5 Hz) and for the carbon
a to the carbi-
Table 1
nolic center (3J = 3.0–9.0 Hz). In the 31P NMR spectra the 31P
signal appears at d 1.69–4.95 ppm with the aliphatic phosphate
monoesters appearing upfield and the allylic ones downfield in
the above region (H3PO4 was used as an internal standard).
When allyl alcohol was employed, we were able to detect
minuscule formation of the corresponding pyrophosphate mono-
ester which displays lower field signals in the 1H- and 13C NMR
spectra when compared to the corresponding phosphate mono-
ester. In the 31P NMR spectrum, the phosphate monoester signal
appears at d 4.5 ppm while the signals for the corresponding
pyrophosphate are found at d 5,63 and 9,51 ppm (2JP–P = 21 Hz)
The described method was applied to a series of alcohols with
moderate to good results (Table 1). The list of phosphate mono-
esters comprises those derived from primary and secondary ali-
phatic alcohols, including benzylic and allylic ones. However, the
phosphorylation of tert-butyl alcohol and phenol could not be
carried out under these conditions.
Alcohols phosphorylated according to Scheme 1, and yields of purified organic
monophosphates
Entry
Starting alcohol
Isolated yield of phosphate (%)
OH
1
2
86
75
OH
3
4
5
6
66
50
43
59
OH
OH
OH
OH
OH
7
70
O
All yields were calculated based on the amount of starting
alcohol and have proved to be superior to those reported in
the literature by the previously described methods. Furthermore,
the formation of byproducts such as pyrophosphates was absent
with all alcohols employed, with the exception of the very reac-
tive allyl alcohol (Table 1, entry 8) showing some minor double
activation.
A proposed mechanism for this reaction is shown in Scheme 2,
where trichloroacetonitrile reacts with tetrabutylammonium
dihydrogenphosphate to give the corresponding reactive phos-
8
9
76
57
OH
OH
10
11
67
52
OH
H
OH
phorylated trichloroacetimidate,
vated phosphate, followed by nucleophilic attack by the alcohol
to yield the desired phosphorylated alcohol and
trichloroacetamide.
a mixed anhydride-like acti-
However, unsatisfactory yields were achieved with this original
procedure. A few years later, Danilov published a modification of
the Cramer procedure with good yields, but with a long and te-
dious work-up of the crude reaction mixture.10 It is also known
from the work published by Keller in 1993 that isoprenoid diphos-
phates can be purified by standard flash silica chromatography.11
More recently, Ishihara12 and Pascal5 have shown improvements
with direct phosphorylation.
Based on these preceding developments of the phosphorylation
reaction, we describe herein a straightforward method for the
preparation of phosphate monoesters directly from alcohols. The
commercially available tetrabutylammonium dihydrogenphos-
phate is used as the phosphate donor in combination with trichlo-
roacetonitrile9d as a mild esterification agent (Scheme 1).
The reaction was monitored by TLC and a mixture of isopropa-
nol/NH4OH/H2O 7:2:1 was used as the eluent. After consumption
of the alcohol, the product was purified by flash silica column chro-
matography using the above solvent mixture and it was finally
converted into the corresponding ammonium salt by percolation
through a DOWEX 50WX8 ion-exchange column with a 0.025 M
The proposed mechanism is supported by 13C NMR experi-
ments carried out under the experimental conditions described
above: no reaction was observed upon either mixing of 4-
methoxybenzyl trichloroacetimidate with tetrabutylammonium
dihydrogenphosphate, or by pre-mixing of trichloroacetonitrile
with geraniol. However, when geraniol, trichloroacetonitrile,
and tetrabutylammonium dihydrogenphosphate have been suc-
cessively mixed, the disappearance of the 13C signals of trichlo-
roacetonitrile and the appearance of the signals corresponding
to the phosphorylated alcohol were observed. The proposed
mechanism is supported by a previously reported synthesis of
deuterated dimethylallyl diphosphate by treatment of a solution
of (R)-[1-2H]3-methyl-2-butenol in trichloroacetonitrile with bis-
triethylammonium phosphate in acetonitrile to afford the corre-
sponding diphosphate with retention of configuration.14
The method described herein is a straightforward approach to
phosphate monoesters in a one-pot reaction procedure and should
be applicable to a wide range of primary and secondary alcohols.
+NBu4
H
N-
O
P
OH
O
P
OH
N
R-OH
Cl3C
O- +NBu4
+
Cl3CCONH2
RO
+
Cl3C
HO
O- +NBu4
O
O
P
OH
O
Scheme 2. Proposed mechanism for the formation of phosphate monoester using trichloroacetonitrile and dihydrogenphosphate via a reactive ‘mixed anhydride’.

1692
L. M. Lira et al. / Tetrahedron Letters 54 (2013) 1690–1692
8. Miyashita, K.; Ikejiri, M.; Kawasaki, H.; Maemura, S.; Imanishi, T. J. Am. Chem.
Acknowledgments
Soc. 2003, 125, 8238–8243.
9. (a) Cramer, F.; Böhm, W. Angew. Chem. 1959, 71, 775; (b) Cramer, F. Preparation
of Esters, Amides and Anhydrides of Phosphoric Acid In Newer methods of Organic
Chemistry; Academic Press: New York, 1964; Vol. 3, p 319; (c) Cramer, F. Liebigs
Ann. Chem. 1962, 654, 180–188; (d) Cramer, F.; Weimann, G. Chem. Ber. 1961,
94, 996–1007; (e) Cramer, F.; Rittersdorf, W. Tetrahedron 1967, 23, 3015–3022.
10. Danilov, L. L.; Druzhinina, T. N.; Kalinchuck, N. A.; Maltsev, S. D.; Shibaev, V. N.
Chem. Phys. Lipid 1989, 51, 191–203.
We thank IPB-Halle and the SAW-Leibniz Project ‘Proteinmus-
ter’, FAPESP (research Grant 2009/51602-5) and Institute of Chem-
istry/UNICAMP for financial and academic support.
Supplementary data
11. Keller, R. K.; Thompson, R. J. Chromatogr. 1993, 645, 161–167.
12. Sakakura, A.; Katsukawa, M.; Ishihara, K. Org. Lett. 2005, 7, 1999–2002.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2013.01.
059.
13. Typical experimental procedure: To
a solution of the alcohol (1 mmol) in
acetonitrile (1 mL), trichloroacetonitrile (2.4 mmol) is added, followed by
dropwise addition of tetrabutylammonium dihydrogenphosphate (2.0 mmol)
in acetonitrile (5 mL). The reaction mixture is stirred at room temperature for
two hours, or until all the starting material is consumed. The solvent is
removed in vacuo and the crude material is pre-purified by flash
chromatography on silica gel (isopropanol/conc. aq NH4OH/H2O 7:2:1). The
column fractions which show the presence of phosphate on TLC (e.g., with
molybdate reagent) are combined and concentrated under reduced pressure. A
Dowex 50WX8 ion-exchange column of 8 cm tall and 1 cm diameter is loaded
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The silica column residue is percolated through the DOWEX column with the
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