An optimized method for the synthesis of amino-functionalized phosphatidylcholine

Article abstract of DOI:10.1016/j.tetlet.2011.11.100

Phosphatidylcholine analogues were synthesised as affinity ligands for the capture of membrane proteins. Several protecting group strategies were investigated to synthesize the amino-functionalized phosphatidylcholine: 11-aminoundecyl 2-(trimethylammonio)ethyl phosphate (4). The acid-mediated deprotection of the Boc group generated a mixture of the target products which could only be purified by HPLC. However, an alternative strategy, using the hydrazine-labile phthalimide group route, followed by a gel filtration step proved straightforward to afford the desired amino-functionalized phosphatidylcholine product in high yield and purity.

Full text of DOI:10.1016/j.tetlet.2011.11.100

Tetrahedron Letters 53 (2012) 546–549
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
An optimized method for the synthesis of amino-functionalized
phosphatidylcholine
⇑
⇑
Fanzhi Kong , Goreti Ribeiro Morais, Robert A. Falconer, Chris W. Sutton
Institute of Cancer Therapeutics, School of Life Sciences, University of Bradford, Bradford BD7 1DP, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Phosphatidylcholine analogues were synthesised as affinity ligands for the capture of membrane pro-
teins. Several protecting group strategies were investigated to synthesize the amino-functionalized phos-
phatidylcholine: 11-aminoundecyl 2-(trimethylammonio)ethyl phosphate (4). The acid-mediated
deprotection of the Boc group generated a mixture of the target products which could only be purified
by HPLC. However, an alternative strategy, using the hydrazine-labile phthalimide group route, followed
by a gel filtration step proved straightforward to afford the desired amino-functionalized phosphatidyl-
choline product in high yield and purity.
Received 28 September 2011
Revised 31 October 2011
Accepted 18 November 2011
Available online 26 November 2011
Keywords:
Phosphatidylcholine (PC)
Boc deprotection
Ó 2011 Elsevier Ltd. All rights reserved.
Phthalimide deprotection
Phospholipid purification
Cytochrome P450s (CYPs) are a superfamily of mixed-function
oxidases, members of which are present in virtually all living
organisms. CYPs are characterized into families and sub-families
by their sequence similarities. There are 103 isoforms identified
in mouse and 57 in humans, predominantly expressed in the liver,
but also specific forms occurring in other tissues, including
tumours. Each member can also be characterized by their substrate
(endogenous and foreign metabolites) specificity. CYPs are thought
to have evolved, in part, as a protective adaptive response against
the toxic effects of environmental chemicals.1 They are the most
important drug metabolizing enzymes in mammals, and, in
humans, are responsible for the phase I metabolism of 70–80% of
all clinically used drugs.2 Essentially, by monitoring the changes
in the protein expression profile of CYPs, the effect of drugs on tar-
get tissues can be determined, and hypotheses proposed as to the
likely mechanisms of drug action. In addition to their detoxifica-
tion role, CYPs are also responsible for the conversion of chemical
toxins and procarcinogens into their toxic or carcinogenic forms.3
The ability of CYPs to activate chemical toxins has been exploited
in cancer chemotherapy where several anticancer drugs, including
cyclophosphamide, tamoxifen and banoxantrone, are known to
require metabolic CYP activation in order to exert their cytotoxic
effects.4 Because of their ability to metabolize drugs, CYPs have
huge significance in pharmaceutical research.5 The action of CYPs
is one of the major causes of adverse drug reactions to many
marketed drugs and drug-combination therapies, and many fail-
ures of novel drugs during their development have been attributed
to their interactions with this class of enzyme. For example, drugs
may be metabolized too rapidly by CYPs before they have had time
to be effective, or they may be metabolized into smaller molecules
which are toxic beyond their site of proposed action. Certain drugs
may also inhibit the activity of a CYP enzyme which is involved in
the metabolism of another drug that, given at the same time, can
become elevated in the patient to levels which can cause side ef-
fects or become dangerous. There is now the potential to use the
CYP profile of a subject to develop personalized medicines. A sub-
ject’s genotype may impact on the pharmacodynamics (drug con-
centration vs time vs pharmacological effect), pharmacokinetics
(absorption, distribution, metabolism and excretion) and/or the
incidence of adverse events. The use of pharmacogenetic tests to
determine this genetic variation can facilitate correct drug selec-
tion for treatment efficacy and minimize adverse side effects.
In the course of our research into the affinity purification of
membrane proteins such as the CYP450s, we required a route to
synthesize the amino-functionalized phosphatidylcholine: 11-
aminoundecyl 2-(trimethylammonio)ethyl phosphate (4), which
would be immobilized onto epoxy-activated softgel agarose to
facilitate affinity enrichment-based proteomics experiments. There
have been many papers and patents published outlining the
synthesis of phosphatidylcholine derivatives,6 but each of these
methods has drawbacks. Some processes are lengthy and overly
complex,7 others use expensive and toxic reagents,8 some require
heavy metals during purification, or result in poor yields.9 To
address these issues, we report two new and simple methods for
the synthesis and purification of phosphatidylcholine derivatives
in excellent yields, utilizing relatively cheap, commercially avail-
able starting materials.
⇑
Corresponding authors. Tel.: +44 (0) 1274 235894.
E-mail addresses: F.Kong@bradford.ac.uk (F. Kong), C.W.Sutton@bradford.ac.uk
(C.W. Sutton).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.11.100

F. Kong et al. / Tetrahedron Letters 53 (2012) 546–549
547
In our initial efforts (Scheme 1), we prepared the Boc protected
long chain alcohol 2 through the selective reduction of commer-
cially available acid 1 resulting in a 95% isolated yield of alcohol
2 as a white powder after flash silica gel chromatography10
(CH3)3N was added to the reaction mixture. The mixture was
stirred at 60–65 °C for 5 h in the presence of trimethylsilyl trifluo-
romethanesulfonate (TMSOTf) under a condenser charged with
dry ice/acetone. 31P NMR spectroscopy11 confirmed that 30%,
50% and 70% yields of the target compound 4 had been formed
in the presence of 0.5, 1.0 and 2.0 equiv of TMSOTf, respectively.
Further attempts to optimize the yield of this reaction proved
unsuccessful. Hence, it was difficult to obtain pure phosphate 4
even after employing repeated gel filtration with a Sephadex
LH-20 column.
(methanol:dichloromethane = 5:95, Rf = 0.4). Alcohol
2 reacted
with 2-chloro-2-oxo-1,3,2-dioxaphospholane to give phosphate 3
with 31P NMR spectroscopy indicating the reaction yield was
97%. After removing the solvent in vacuo, the residue was used
without further purification. In a glass Schlenk tube, phosphate
3
was dissolved in CH3CN, cooled to À78 °C, and 1 mL of
O
O
BH3·THF
O
N
H
OH
RT, 2 h
95%
1
O
O
Cl
P
O
O
O
O
N
OH
O
H
DIPEA
toluene 97%
2
O
Me3N
O
O
N
H
P
TMSOTf
70%
O
3
O
O
N
H2N
O
P
O
4
Scheme 1. Synthesis of terminal amine phosphatidylcholine (PC) analogue 4 via the acid-mediated deprotection method.
O
O
O
N
H
OH
1
O
O
Me3N
O
O
N
O
P
H
RT, 24 h
98%
O
3
O
O
O
N
O
N
O
P
H
O
5
O
O
10% TFA
P
N
H3N
O
CH2Cl2
100%
CF3COO
O
6
Scheme 2. Synthesis of terminal amine phosphatidylcholine (PC) analogue 6 via the two step acid-mediated deprotection method.

548
F. Kong et al. / Tetrahedron Letters 53 (2012) 546–549
O
NK
O
Br
OH
88%
7
O
O
Cl
P
O
O
N
OH
O
DIPEA
/toluene
98%
O
O
8
O
O
Me3N
90%
P
N
O
O
9
O
O
MeNH2-NH2
NMe3
O
P
O
N
95%
O
O
10
4
O
NMe3
H2N
O
P
O
O
Scheme 3. Synthesis of terminal amine phosphatidylcholine (PC) analogue 4 via the base-mediated deprotection method.
An alternative two-step preparation of phosphatidylcholine 6
showed that 90% of product 10 had been formed. The solvents
were removed under reduced pressure, the mixture passed first
through a Sephadex LH-20 size exclusion column (CHCl3/MeOH;
from phosphate 3 (Scheme 2) was also performed. Phosphatidyl-
choline 6 is the trifluoroacetate salt form of the desired compound
4 and could easily be transformed into the amine 4 during the suc-
ceeding agarose bead coupling reaction. In a glass pressure tube,
phosphate 3 was reacted with neat (CH3)3N which was added to
the reaction mixture from a cylinder with stirring at room temper-
ature for 24 h. 31P NMR spectroscopic data indicated that 5 had
been formed in a 98% yield. After fully removing the excess
(CH3)3N by evaporation, crude phosphatidylcholine 5 was dis-
solved in 10% trifluoroacetic acid in dichloromethane and stirred
at room temperature for 5 h, affording the phosphatidylcholine 6,
quantitatively. Following in vacuo removal of the solvent, phos-
phatidylcholine 6 was obtained in high purity (NMR acceptable
product) after three successive gel filtration steps using a Sephadex
LH-20 column. Unfortunately, this two-step procedure proved to
be troublesome and non-reproducible: often four or five gel filtra-
tion steps were still insufficient to yield pure compound 6. A
further drawback of this method was that 6 still needed to be
de-salted into 4 before coupling to agarose beads.
50:50),
followed
by
silica
column
chromatography
(CH2Cl2:MeOH:H2O; 65:25:4) to give the product phosphatidyl-
choline 10 as a white powder. Compound 10 underwent base-pro-
moted deprotection to give the final product 4. A simple gel
filtration through Sephadex LH-20 yielded compound 4 in high
yield and purity.
In conclusion, we have developed simple, robust and reproduc-
ible routes to 11-aminoundecyl 2-(trimethylammonio)ethyl phos-
phate (4) via methyl hydrazine promoted deprotection as the key
step to obtain the phosphatidylcholine in high purity.13
Acknowledgments
This synthetic work was supported by Yorkshire Cancer
Research and EPSRC.
Supplementary data
While the two-step Boc deprotection sequence worked occa-
sionally, a more robust and reproducible synthesis was required.
We next examined the methyl hydrazine promoted deprotection
of a phthalimide group (Scheme 3). We prepared the phthalimide
protected long chain amino alcohol 8 from commercially available
bromo alcohol 7 in an 88% isolated yield after recrystallization
from methanol following the method of Terashima et al.12 Phthal-
imide protected alcohol 8 was reacted with 2-chloro-2-oxo-1,3,2-
dioxaphospholane to give phosphate 9 in a 98% yield (as calculated
by 31P NMR spectroscopy). After removing the solvent in vacuo, the
residue was used in the amination without further purification. In
a glass pressure tube, phosphate 9 was dissolved in CH3CN, cooled
to À78 °C, and (CH3)3N was added to the mixture. The mixture was
stirred at room temperature for 48 h. 31P NMR spectroscopy
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2011.11.100.
References and notes
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through a Sephadex LH-20 size exclusion chromatography column (CHCl3/
MeOH; 50:50), followed by silica column chromatography (CH2Cl2:MeOH:H2O;
65:25:4) to give the final product 10 (210 mg) as a white powder. A small
analytical sample was obtained via preparative inverse phase C18 HPLC
purification or by preparative TLC (65:25:4; CH2Cl2:MeOH:H2O).
Phosphatidylcholine 10 (1.85 mmol) was dissolved in absolute EtOH (20 mL)
and cooled to 0 °C. MeNH2NH2 (27.75 mmol) was added and the mixture
stirred at 50 °C for 3 d. After removal of the solvent, the mixture was passed
through a Sephadex LH-20 size exclusion chromatography column (CHCl3/
MeOH; 50:50), followed by silica column chromatography (65:25:4;
CH2Cl2:MeOH:H2O) to give the final product 4 (200 mg) as a white powder.
31P NMR spectroscopy indicated the reaction yield to be approx. 95%. 1H NMR
(250 MHz, CDCl3): d 1.27–1.38 (m, 10H), 1.50–1.68 (m, 4H), 2.86–2.88 (m, 2H),
3.06–3.08 (m, 2H), 3.20 (s, 9H), 3.28–3.31 (m, 2H), 3.60–3.64 (m, 2H), 3.88–
3.90 (m, 2H), 4.26–4.34 (m, 2H); 13C NMR (62.9 MHz, CD3OD): d 21.8, 24.2,
25.2, 26.1, 28.8, 28.9, 29.1, 30.5, 39.5, 52.1, 53.2 (2C), 59.0, 59.2, 66.0, 66.2. MS
(ES+) C16H37N2O4P (352) m/z (%) 353 [M+H]+.
11. Meers, P. R.; Feigenson, G. W. J. Lipid Res. 1985, 26, 882–888.
12. Terashima, T.; Mes, T.; De Greef, T. F. A.; Gillissen, M. A. J.; Besenius, P.;
Palmans, A. R. A.; Meijer, E. W. J. Am. Chem. Soc. 2011, 133, 4742–4745.
13. The optimized procedure for the synthesis of 11-aminoundecyl 2-
(trimethylammonio)ethyl phosphate (4): Under an atmosphere of Ar,
phthalimide protected alcohol
8 (500 mg, 1.742 mmol) was dissolved in
toluene (25 mL). DIPEA (3.484 mmol) was added and the solution turned
pale yellow. 2-Chloro-2-oxo-1,3,2-dioxaphospholane (2.09 mmol) was
subsequently added and the reaction solution turned brown. The reaction
was stirred for 24 h, then another aliquot of phosphorylation reagent was
added and the mixture stirred for another 24 h. The solvent was removed
under reduced pressure giving
a
yield (as determined by 31P NMR
spectroscopy) of 98%. The crude phosphate 9 was dissolved in CH3CN (2 mL)
and cooled to À78 °C. Pre-cooled (on ice) (CH3)3N (1 mL) was then added to the
reaction mixture. The mixture was stirred at room temperature for 48 h, then
the solvents were removed under reduced pressure. The mixture was passed