Efficient synthesis of the cholinephosphate phospholipid headgroup

Article abstract of DOI:10.1016/S0009-3084(00)00224-3

In search of an efficient method to prepare cholinephosphate headgroups in phospholipids under mild conditions (where the diacylglycerol moiety is not subject to oxidation), a method was developed for phosphorylation using a trialkyl phosphite and I₂. The active intermediate is a phosphoryl iodide formed by oxidation of the phosphite with I₂. 2-Bromoethanol, dimethyl chlorophosphite, and an alcohol (diglyceride) are converted to a phosphate triester in a one-pot reaction with high yield. In the second reaction, the phosphate triester is demethylated, and the ethyl bromide group is converted to choline by treatment with aqueous trimethylamine. This procedure is applied to the synthesis of hexadecylphosphocholine, and 1,2-didecanoyl-1-deoxy-1-thio-sn-glyceryo-3-phosphocholine.

Full text of DOI:10.1016/S0009-3084(00)00224-3

Chemistry and Physics of Lipids
09 (2001) 203–207
1
www.elsevier.com/locate/chemphyslip
Efficient synthesis of the cholinephosphate phospholipid
headgroup
Elizabeth K. Hendrickson, H. Stewart Hendrickson *
Department of Chemistry, Uni6ersity of Washington, Seattle, WA 98195-1700, USA
Received 05 July 2000; received in revised form 23 October 2000; accepted 23 October 2000
Abstract
In search of an efficient method to prepare cholinephosphate headgroups in phospholipids under mild conditions
where the diacylglycerol moiety is not subject to oxidation), a method was developed for phosphorylation using a
(
trialkyl phosphite and I . The active intermediate is a phosphoryl iodide formed by oxidation of the phosphite with
2
I2. 2-Bromoethanol, dimethyl chlorophosphite, and an alcohol (diglyceride) are converted to a phosphate triester in
a one-pot reaction with high yield. In the second reaction, the phosphate triester is demethylated, and the ethyl
bromide group is converted to choline by treatment with aqueous trimethylamine. This procedure is applied to the
synthesis of hexadecylphosphocholine, and 1,2-didecanoyl-1-deoxy-1-thio-sn-glyceryo-3-phosphocholine. © 2001
Published by Elsevier Science Ireland Ltd.
Keywords: 2-Bromoethanol; Cholinephosphate; Hexadecylphosphocholine; Phosphatidylcholine; Phosphite triester; Phospholipid;
Synthesis; Phosphoryl iodide
quent treatment with trimethylamine (Eibl and
1. Introduction
Woolley, 1988), or condensation with choline tosy-
late in the presence of 2,4,6-triisopropylbenzenesul-
fonyl chloride (Paltauf and Hermetter, 1991).
Alternatively, the diglyceride is phosphorylated
with 2-bromoethyl phosphoryldichloride (Hansen
et al., 1982), or 2-chloro-2-oxo-1,3,2-dioxaphos-
pholane (Chandrakumar and Hajdu, 1982; Fuji et
al., 1997) followed by treatment with trimethy-
lamine. A method based on phosphite chemistry
involves treatment of a diglyceride with methyl
dichlorophosphite followed by 2-bromoethanol,
oxidation of the phosphite diester with H O , and
Synthesis of the cholinephosphate headgroup in
glycerophospholipids has been accomplished by a
variety of methods (Bittman, 1993). Methods based
on phosphate chemistry, involve phosphorylation
of a diglyceride with phosphorus oxychloride, fol-
lowed by coupling of the diacylglycerophosphoryl
dichloride with choline tosylate (Brockerhoff and
Ayengar, 1979), or 2-bromoethanol, and subse-
*
Corresponding author. Tel.: +1-206-6164216; fax: 206-
2
2
6
85-8665.
quaternary amine formation with trimethylamine
(Martin et al., 1994).
E-mail address: hend@u.washington.edu (H.S. Hendrick-
son).
0
009-3084/01/$ - see front matter © 2001 Published by Elsevier Science Ireland Ltd.
PII: S0009-3084(00)00224-3

2
04
E.K. Hendrickson, H.S. Hendrickson / Chemistry and Physics of Lipids 109 (2001) 203–207
Phosphorylation based on phosphite chemistry
Beaucage and Iyer, 1992) has proven more reli-
ported here and shown in Scheme 1. 2-Bro-
moethanol, and dimethyl chlorophosphite are
converted to the phosphate triester (3a, and b) in
a one-pot reaction with high yield. In the second
reaction, the phosphate triester is demethylated
and the ethyl bromide group is converted to
choline by treatment with aqueous trimethy-
lamine. This procedure is applied to the synthesis
of hexadecylphosphocholine and 1,2-didecanoyl-
1-deoxy-1-thio-sn-glyceryo-3-phosphocholine
(Hendrickson and Hendrickson, 1990).
(
able and gives higher yields than methods based
on phosphate chemistry. Watanabe et al. (1993)
reported a method involving activation of tri-
alkylphosphites with pyridinium bromide perbro-
mide in the presence of triethylamine. Later,
Stowell and Widlanski (1995) published a method
for phosphorylation using a trialkyl phosphite
and I . They reported near-quantitative yields un-
2
der exceptionally mild conditions. Since, the ac-
tive intermediate is a phosphoryl iodide formed
by oxidation of the phosphite with I , this method
2
avoids subjecting the diacylglycerol moiety to oxi-
dizing conditions. We have successfully used this
method, to synthesize phosphate monoesters of
inositol (Hendrickson and Hendrickson, 1998),
and Gefflaut et al., 1997 used this method in an
efficient synthesis of bromoacetol phosphate, and
dihydroxyacetone phosphate. In search of an effi-
cient method to prepare choline phosphate head-
groups in phospholipids under mild conditions
2. Experimental
2.1. Materials and analytical procedures
1,2-Didecanoyl-1-deoxy-1-thio-sn-glycerol was
synthesized according to the procedure of Hen-
drickson and Hendrickson (1990). Dimethylchlor-
ophosphite was synthesized from phosphorus
trichloride, and trimethylphosphite according
to Nagai et al. (1989). 2-Bromoethanol, hexade-
(
where the diacylglycerol moiety is not subject to
oxidation), we have developed the procedure re-
canol, and trimethylamine (40% in H O)
2
Scheme 1. Synthesis of cholinephosphate diesters via phosphite triester and I2.

E.K. Hendrickson, H.S. Hendrickson / Chemistry and Physics of Lipids 109 (2001) 203–207
205
were obtained from Aldrich Chemical Co., St.
Louis, MO. Methylene chloride and pyridine were
dried by distillation from calcium hydride.
Column chromatography was performed on silica
gel 60 (75–150 mm; Analtech, Newark, DE), and
40% aqueous trimethylamine. The reaction was
stirred at room temperature overnight. TLC (sil-
ica gel; CHCl :CH OH:H O, 65:35:3) indicated
3
3
2
the reaction was complete. The solvent was re-
moved in vacuo, and the residue was shaken with
a mixture of 9 ml H O, 9 ml CHCl , and 12 ml
1
TLC on silica gel plates. H NMR spectra were
2
3
determined at 200 MHz.
CH OH. The CHCl layer was separated and the
3 3
aqueous layer again extracted with CHCl . The
3
2
.2. Hexadecyl methyl 2-bromoethylphosphate
combined CHCl3 extracts were dried over
Na SO , and then rotovaped. The crude product
(
3a)
2
4
was dissolved in CHCl :CH OH (10:1), and chro-
3
3
2
-Bromoethanol (redistilled, 0.37 g, 3 mmol)
matographed on a silica gel column with increas-
ing amounts of CH OH in CHCl , followed by
was added to a solution of 1.26 g of dry pyridine
and 30 ml of dry CH Cl with stirring in an ice
3
3
CHCl –CH OH–H O. Pure (by TLC) 4a (0.16 g,
2
2
3
3
2
bath. Dimethylchlorophosphite (0.40 g. 3.2 mmol)
80% yield), eluted with CHCl :CH OH:H O,
65:35:4. H NMR (CDCl /CD OD) l 0.88 (t,
3 3
3 3 2
1
in 2 ml of CH Cl2 was added drop-wise, and
2
stirring was continued for 10 min. A stoichiomet-
J=6.5 Hz, 3H), 1.35–1.45 (m, 26H), 1.75–1.85
(m, 2H), 3.22 (s, 9H), 4.0–4.1 (m, 2H), 4.15–4.25
(m, 2H).
ric amount of I (about 0.55 g, titrated to just
2
colorless by a small addition of dimethylphos-
phite, if necessary) was added. A copious precipi-
tate formed, and after a few min the reaction
mixture was filtered at room temperature and
returned to an ice bath. Hexadecanol (0.36 g, 1.5
mmol) in 2 ml of CH Cl and 0.2 ml of pyridine
2.4. Methyl 1,2-didecanoyl-1-deoxy-1-thio-sn-
glycero-3-phospho-2-bromoethanol (3b)
2
2
was immediately added drop-wise, and the reac-
tion was followed by, TCL (silica gel; hexane-ace-
tone, 7:3). The reaction was complete after about
Compound 3b was synthesized in 64% yield
from 2-bromoethanol and 1,2-didecanoyl-1-de-
oxy-1-thio-sn-glycerol by the same procedure as
for 3a. Pure (by TLC) 3b eluted from a silica gel
45 min. The reaction mixture was diluted with an
1
equal volume of CH Cl . washed with 10%
column with 13% acetone. H NMR (CDCl3/
2
2
NaHSO (a small amount of methanol was added
CD OD) l 0.88 (t, J=7.0 Hz, 6H), 1.2–1.3 (m,
4
3
to help break the emulsion), 10% sodium phos-
phate buffer (pH 7), dried over Na SO , and
rotovaped to dryness with additions of absolute
ethanol to remove any water. The crude product
was dissolved in hexane and chromatographed on
a silica gel column with increasing amounts of
24H), 1.6–1.7 (m, 4H), 2.32 (t, J=7.5 Hz, 2H),
2.56 (t, J=7.5 Hz, 2H), 3.01–3.34 (m, 2H), 3.54
(t, J=6.6 Hz, 2H), 3.81 (dd, J=11.3, 2.6 Hz,
3H), 4.13–4.20 (m, 2H), 4.26–4.38 (m, 2H), 5.08–
5.19 (m, 1H).
2
4
acetone in hexane. Pure (by TLC) 3a (0.54 g, 82%
1
yield) eluted with 8% acetone. H NMR (CDCl /
2.5. 1,2-Didecanoyl-1-deoxy-1-thio-sn-glycero-
3-phosphocholine (4b)
3
CD OD) l 0.88 (t, J=6.7 Hz, 3H), 1.24–1.27 (m,
3
28H), 1.6–1.7 (m, 2H), 3.5–3.6 (m, 2H), 3.79 (d,
J=11.4 Hz, 3H), 4.0–4.1 (m, 2H), 4.25–4.35 (m,
Compound 4b was synthesized in 71% yield
from 3b by the same procedure as for 4a. Pure (by
2H).
TLC; R identical to same compound synthesized
f
2.3. Hexadecylphosphocholine (4a)
earlier, Hendrickson and Hendrickson, 1990)
4
b
eluted from
a
silica gel column with
Hexadecyl methyl 2-bromoethylphosphate (3a)
CHCl :CH OH:H O, 65:35:2. The NMR spec-
3
3
2
(
3
0.22 g, 0.5 mmol) was dissolved in 2 ml CH Cl ,
.3 ml acetonitrile, 3.3 ml isopropanol, and 5 ml
trum was identical (under the same conditions), to
that of the same compound synthesized earlier
2
2

2
06
E.K. Hendrickson, H.S. Hendrickson / Chemistry and Physics of Lipids 109 (2001) 203–207
1
(
Hendrickson and Hendrickson, 1990). H NMR
Acknowledgements
(
CDCl /CD OD) l 0.88 (t, J=6.9 Hz, 6H), 1.2–
3
3
1.3 (m, 24H), 1.5–1.7 (m, 4H), 2.30 (t, J=7.3 Hz,
2H), 2.54 (t, J=7.5 Hz, 2H), 2.9–3.0 (m, 1H),
3.23 (s, 9H), 3.25–3.35 (m, 1H), 3.5–3.6 (m, 2H),
4.2–4.3 (m, 2H), 5.0–5.1 (m, 1H).
This work was supported by a grant (MCB-
9619859) from the National Science Foundation.
References
3
. Results and discussion
Beaucage, S.L., Iyer, R.P., 1992. Advances in the synthesis of
oligonucleotides by the phosphoramidite approach. Tetra-
hedron 48, 2223–2311.
2-Bromoethanol, dimethyl chlorophosphite,
and an alcohol (hexadecanol or 1,2-didecanoyl-1-
deoxy-1-thio-sn-glycerol) were converted in high
yield, in a one-pot reaction, to the corresponding
methyl alkyl 2-bromoethylphosphate. Subsequent
treatment of this product with aqueous trimethy-
lamine, resulted in both demethylation of the
methylphosphate and conversion of the 2-bro-
moethyl moiety to choline. The conditions of the
first reaction are very mild and do not subject the
diglyceride moiety to oxidative conditions. This is
important in the synthesis of the thiolester analog
Bittman, R., 1993. In: Cevc, G. (Ed.), Phospholipids Hand-
book. Marcel Dekker, New York, pp. 141–232.
Brockerhoff, H., Ayengar, N.K., 1979. Improved synthesis of
choline phospholipids. Lipids 14, 88–89.
Chandrakumar, N.S., Hajdu, J., 1982. Syntheses of enzyme-in-
hibitory phospholipid analogues. Stereospecific synthesis of
2
-amidophosphatidylcholines and related derivatives. J.
Org. Chem. 47, 2144–2147.
Eibl, H., Woolley, P., 1988. A general synthetic method for
enantiomerically pure ester and ether lysophospholipids.
Chem. Phys. Lipids 47, 63–68.
Fuji, M., Watanabe, F., Fujii, Y., Hashizume, H., Okuno, T.,
Shirahase, K., Teshirogi, I., Ohtani, M., 1997. A stereose-
lective and highly practical synthesis of cytosolic phospho-
3b, which is subject to oxidation at the sulfur
lipase
-S-arachidonyl-1-O-hexadecyl-sn-2-thioglycero-3-O-phos-
phocholine. J. Org. Chem. 62, 6804–6809.
A2
substrate,
atom and labile to hydrolytic cleavage. Conver-
sion of the methyl alkyl 2-bromoethylphosphate
2
(
3a, and b) to the alkyl cholinephosphate (4a, and
Gefflaut, T., Lemaire, M., Valetin, M.-L., Bolte, J., 1997. A
novel efficient synthesis of dihydroxyacetone phosphate
and bromoacetol phosphate. J. Org. Chem. 62, 5920–5922.
Hansen, W.J., Ramaswamy, M., Wedmid, Y., Baumann, W.J.,
b) (see Woolley and Eibl, 1988), which involves
both demethylation and quaternary amine forma-
tion, is accomplished in a single reaction with
trimethylamine. The yield of hexadecylphospho-
choline was high, whereas the yield of the thi-
olester analog (4b) was somewhat lower due to
hydrolysis of the thiolester and ester groups under
the basic aqueous conditions of the reaction. Nev-
ertheless, the overall yield of 4b was much higher
than that achieved in the previous method with
POCl3 and choline tosylate (Hendrickson and
Hendrickson, 1990).
This method can also be used for the synthesis
of other phospholipids, such as, phosphatidyl–
ethanolamine, and -serine, using suitably-pro-
tected alcohols in place of 2-bromoethanol. A
simple high-yield synthesis of 1,2-dialkylphos-
phatidylmethanol was accomplished (Ghomaschi
F., unpublished results), by reacting 1,2-dialkyl-
1
982. An improved procedure for the synthesis of choline
phospholipids via 2-bromoethyl dichlorophosphate. Lipids
7, 453–459.
1
Hendrickson, H.S., Hendrickson, E.K., 1990. A facile asym-
metric synthesis of glycerol phospholipids via tritylglycidol
prepared by the asymmetric epoxidation of allyl alcohol.
Thiolester and thioether analogs of phosphatidylcholine.
Chem.Phys.Lipids 53, 115–120.
Hendrickson, H.S., Hendrickson, H.S., 1998. A thiophosphate
analog of dimyristoylphosphatidylinositol-4-phosphate is a
substrate for mammalian phosphoinositide-specific phos-
pholipase C. Bioorg. Med. Chem. Lett. 8, 1057–1060.
Martin, S.F., Josey, J.A., Wong, Y.L., Dean, D.W., 1994.
General-method for the synthesis of phospholipid deriva-
tives of 1,2-O-diacyl-sn-glycerols. J. Org. Chem. 59, 4805–
4
820.
Nagai, H., Fujiwara, T., Fujii, M., Sekine, M., Hata, T., 1989.
Reinvestigation of deoxyribonucleoside phosphorothioites:
synthesis and properties of deoxyribonucleoside-3%
dimethyl phosphites. Nucleic Acids Res. 17, 8581–8593.
Paltauf, F., Hermetter, A., 1991. Preparation of alkyl ether
and vinyl ether substrates for phospholipases. Methods
Enzymol. 197, 134–148.
glycerol with trimethylphosphite and I , followed
2
by monodemethylation of the dimethyl alkylphos-
phate with LiBr.

E.K. Hendrickson, H.S. Hendrickson / Chemistry and Physics of Lipids 109 (2001) 203–207
207
Stowell, J.K., Widlanski, T.S., 1995. A new method for the
phosphorylation of alcohols and phenols. Tetrahedron
Lett. 36, 1825–1826.
Watanabe, Y., Inada, E., Jinno, M., Ozaki, S., 1993. Phospho-
nium salt methodology for the synthesis of phosphoric
monoesters and diesters, and its application to selective
phosphorylation. Tetrahedron Lett. 34, 497–500.
Woolley, P., Eibl, H., 1988. Synthesis of enantiomerically pure
phospholipids including phosphatidylserine and phos-
phatidylglycerol. Chem. Phys. Lipids 47, 55–62.
.