A practical selective synthesis of mixed short/long chains glycerophosphocholines

Article abstract of DOI:10.1016/j.chemphyslip.2007.03.008

Glycerophosphorylcholine (GPC) is transformed into the cyclic stannylene derivatives, which are selectively acylated to 1-acyl-2-lyso-glycerophosphocholines. The reaction is effective using C-2 to C-16 acid chlorides in 2-propanol. After solvent replacement the lyso-phospholipid (lyso-PL) is subjected to a second acylation using acid anhydrides in methylene chloride. A series of 1(2)-short-2(1)-long-diacyl-glycerophosphocholines are obtained in high yields and selectivity. No diacylation product was detected. In order to detect mixed-chain lipids with inverted disposition of acyl chains, the long chain was introduced first and the thus resulting isomeric compounds compared by NMR. An NMR method was developed in order to determine the positional purity of the isomeric compounds.

Full text of DOI:10.1016/j.chemphyslip.2007.03.008

Chemistry and Physics of Lipids 147 (2007) 113–118
Short communication
A practical selective synthesis of mixed short/long
chains glycerophosphocholines
a,∗
1
b
a
Paola D’Arrigo , Ezio Fasoli , Giuseppe Pedrocchi-Fantoni , Cristina Rossi ,
a
a
a
Caterina Saraceno , Davide Tessaro , Stefano Servi
Dipartimento di Chimica, Materiali e Ingegneria Chimica G. Natta, Politecnico di Milano,
via Mancinelli 7, 20131 Milano, Italy
a
b
ICRM-CNR via Mario Bianco 9, 20131 Milano, Italy
Received 21 December 2006; received in revised form 28 March 2007; accepted 28 March 2007
Available online 5 April 2007
Abstract
Glycerophosphorylcholine (GPC) is transformed into the cyclic stannylene derivatives, which are selectively acylated to 1-acyl-
-lyso-glycerophosphocholines. The reaction is effective using C-2 to C-16 acid chlorides in 2-propanol. After solvent replacement
2
the lyso-phospholipid (lyso-PL) is subjected to a second acylation using acid anhydrides in methylene chloride. A series of 1(2)-
short-2(1)-long-diacyl-glycerophosphocholines are obtained in high yields and selectivity. No diacylation product was detected. In
order to detect mixed-chain lipids with inverted disposition of acyl chains, the long chain was introduced first and the thus resulting
isomeric compounds compared by NMR. An NMR method was developed in order to determine the positional purity of the isomeric
compounds.
©
2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Glycerophosphorylcholine; Lyso-phospholipid; Mixed-chains; Diacylglycerophosphocholine; Dibutyltin oxide
1
. Introduction
2002; Lindberg et al., 2002; Paltauf and Hermetter, 1994;
Hajdu, 1999).
Development of new synthetic methods for the prepa-
Specifically, while phospholipids have long been
known as constituents of membrane bilayers (Barenholz
and Cevc, 2002), they were recently recognized as
essential molecules for regulation of cell functions by
hormones, neurotransmitters, growth factors and inflam-
matory cytokines.
ration of biologically active phospholipids (PLs) is
important in solving problems of membrane-chemistry
and biochemistry (Vares et al., 2003; Vance and Vance,
Membrane glycerophospholipids are precursors of
lipid metabolites with second messenger functions, serv-
ing as substrates for phospholipases, lipid kinases and
phosphatases that generate signaling lipid molecules.
Elucidation of the mechanistic details involved in
the enzymological, cell-biological, and membrane-
biophysical roles of phospholipids relies on the
availability of structurally diverse phospholipids (Goetzl
Abbreviations: GPC, glycerophosphorylcholine; PL, phospho-
lipid; TEA, triethylamine; DMAP, 4-dimethylamino pyridine; PC,
diacylated-glycerophosphocholine; DBTO, dibutytin oxide; lyso-PL,
lyso-phospholipid; PLA2, phospholipase A2
Corresponding author. Tel.: +39 0223993075;
fax: +39 0223993080.
∗
E-mail address: paola.d’arrigo@polimi.it (P. D’Arrigo).
Current address: University of Puerto Rico at Humacao, Depart-
1
ment of Chemistry, CUH Station, Humacao 00791, Puerto Rico.
0
009-3084/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2007.03.008

1
14
P. D’Arrigo et al. / Chemistry and Physics of Lipids 147 (2007) 113–118
Scheme 1. Chemo-enzymatic transformations of natural lecithins into 1-acyl-sn-2-lysophospholipids.
and An, 1998; Sciorra and Morris, 2002; Bradshaw et al.,
achieved with different fatty acid derivatives, but up to
12% of 1,2-diacyl-phosphatidylcholine was formed as a
by-product in the reaction.
1
999; Muresan et al., 2001; Vance, 2002).
The synthesis of mixed-chain 1,2-diacyl-3-phospho-
glycerols is a challenging task requiring in prin-
ciple regioselective incorporation of three different
substituents at the three glycerol positions involv-
ing protection–deprotection sequences of the hydroxyl
groups and subsequent acylation/phosphorylation while
acyl migration during these transformations must be pre-
vented. Moreover the presence of a chiral center requires
a suitable starting material (Massing and Eibl, 1995;
Chevallier et al., 2000; Wang et al., 1997; Rosseto and
Hajdu, 2005; Stamatov et al., 2005; Rosseto et al., 2006).
The use of glycerophospholipids derived from
deacylation of natural lecithins as starting material
for the synthesis of structured lipids has proven to
be a very effective method for this purpose through
the chemo-enzymatic sequence GPC ⇒ diacylated-
glycerophosphocholine (PC) ⇒ phospholipase A2
Among biologically active phospholipids, 2-acyl-
1-lyso-sn-glycerophosphocholines are considered of
special interest for the in vivo delivery of chains in
position sn-2. However chain migration to the more sta-
ble sn-1 position makes these compounds non-available
in vivo. It has recently been discovered that phospho-
lipids bearing a short acyl chain in position sn-1 behave
like the analogues 2-acyl-1-lyso-compounds with added
in vivo stability (Bayon et al., 1997). These com-
pounds should be accessible via the pathways described
above. However those methods suffer from the forma-
tion of considerable amounts of diacylated products
in the form of a mixture of diastereomers originated
from acyl migration prior to the second acylation
step.
We have recently disclosed a new effective procedure
for the mono-acylation of glycero-phospholipids in high
yields and selectivity: the tin-mediated synthesis of lyso-
phospholipids (Fasoli et al., 2006). The method consists
of the formation of a stannylene derivative in 2-propanol
and subsequent acylation with a stoichiometric amount
of acylating agent, resulting in the exclusive formation
of 1-acyl-2-lyso-phospholipids (compounds 1, 8, 10, 12,
14, 16, 18; Scheme 2 and Table 1).
(
PLA2) catalysed hydrolysis ⇒ reacylation at position
sn-2 (Scheme 1).
In a consecutive two-step chemo-enzymatic trans-
formation, the 1-acyl-sn-2-lyso-phosphatidylcholine is
obtained from GPC (Baer and Kates, 1948), a fatty
acid derivative and an appropriate enzyme in microaque-
ous system. With a lipase preparation from Rhizomucor
miehei or from Candida antarctica (Adlerkreutz and
Wetije, 2004) relatively high yields of the product
can be obtained. The second step of the reaction is
carried out by mixing the purified 1-acyl-defined-2-lyso-
phosphatidylcholine with an activated fatty acid donor,
such as fatty acid anhydride or acyl chloride, in an
organic solvent in the presence of a chemical catalyst to
obtain the appropriate 1,2-diacyl-phosphatidylcholine.
With this procedure conversions around 70% were
We propose a synthesis of 1,2-mixed chains-
glycerophosphocholines (compounds 2–7, 9, 11,
13, 15, 17, 19; see Table 1) through the sequence
GPC ⇒ [stannylene ketal with in situ mono-
acylation] ⇒ second acylation at position sn-2
(Scheme 2). The all procedure can be performed as a
one-pot reaction requiring only removal/replacement of
solvents. High yields and selectivity are observed.

P. D’Arrigo et al. / Chemistry and Physics of Lipids 147 (2007) 113–118
115
Scheme2. Thetwo-stepsynthesisofmixedchainsglycerophosphocholines1–19:tin-mediatedmonoacylationofglycerophosphorylcholinefollowed
by a second acylation.
2
. Experimental procedures
.1. Materials
starting from 60% A, 40% B ramping to 100% B in
6 min. The 20 ␮l samples of a CH2Cl2/CH3OH solu-
tion of 1 mg/ml were injected into the column at a mobile
phase flow rate of 1.5 ml/min.
1
2
All Chemicals were purchased from Sigma–Aldrich.
All solvents were of analytical grade.
2.2. Synthesis
The ion exchange resin is a cation exchange resin
+
H -form Fluka 44443, 20–50 mesh.
The synthetic methodology for the preparation of the
two isomeric compounds 4 and 13 is described with the
two different approaches A and B.
1
H NMR spectra were recorded on a 400 MHz Var-
ian EMX instrument. Chemical shifts were reported in
δ units (ppm) relative to tetramethylsilane (TMS) as
internal standard and all spectra recorded in CDCl3 or
CDCl3/CD3OD as indicated.
2.2.1. 1-Palmitoyl-2-butanoyl-sn-glycero-3-
phosphocholine (4) (method
Mass spectra were recorded on a ESI/MS Bruker
Esquire 3000.
Silica gel 60 F254 plates (Merck) were used for ana-
lytical TLC in solvent system of CHCl3/CH3OH/NH3 in
different ratios. Detection has been performed with UV
light followed by I2 staining.
A)
GPC (1 g, 3.9 mmol) and dibutyltin oxide (DBTO)
(1.1 g, 3.9 mmol) were suspended in 2-propanol (40 ml)
and refluxed for 1 h. The mixture was cooled to 25 C
◦
and treated with TEA (0.65 ml, 4.7 mmol) and palmi-
toyl chloride (1.28 g, 4.7 mmol). The formation of
the lysoPC was followed by HPLC. After 15 min the
reaction mixture contains 97% of 1-palmitoyl-2-lyso-
sn-glycero-3-phosphocholine 1 and 3% of GPC as the
only phospholipids. The solution was then treated with
water (40 ml) and extracted with heptane (40 ml). The
water-alcohol solution was extracted three times with
heptane (3× 40 ml) and evaporated in vacuo; the crude
residue was dissolved in ethanol (10 ml) and precip-
HPLC analysis were performed on a Agilent 1100
series apparatus fitted with a LiChrospher 100 diol
5
␮m column, length/ internal diameter 125/4 and an
evaporative light scattering detector (ELSD) (Sedex
◦
model 75 Sedere, 41 C). The operating temperature
◦
and pressure were 55 C and 3.5 bar. A binary sol-
vent system of solvent A: hexane/2-propanol/acetic
acid/triethylamine (815/170/15/0.8, v/v/v/v) and sol-
vent B: 2-propanol/water/acetic acid/triethylamine
◦
itated with acetone (40 ml) at −10 C to give 1.5 g
of 1 as a white powder (80%). 1H NMR spectrum
(
837/140/15/0.8, v/v/v/v) was used in a gradient mode

1
16
P. D’Arrigo et al. / Chemistry and Physics of Lipids 147 (2007) 113–118
Table 1
Yields and HPLC retention times of compounds 1–19
Compound
Namea
Methodb
Yieldc
HPLC (tR, min)
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
C(16:0)lysoPtdCho
C(16:0)/(2:0)PtdCho
C(16:0)/(3:0)PtdCho
C(16:0)/(4:0)PtdCho
C(16:0)/(5:0)PtdCho
C(16:0)/(6:0)PtdCho
C(16:0)/(9:0)PtdCho
C(2:0)lysoPtdCho
C(2:0)/(16:0)PtdCho
C(3:0)lysoPtdCho
C(3:0)/(16:0)PtdCho
C(4:0)lysoPtdCho
C(4:0)/(16:0)PtdCho
C(5:0)lysoPtdCho
C(5:0)/(16:0)PtdCho
C(6:0)lysoPtdCho
C(6:0)/(16:0)PtdCho
C(9:0)lysoPtdCho
C(9:0)/(16:0)PtdCho
80
52
48
69
60
66
63
54
30
64
25
82
87
80
85
75
86
66
58
6.24
5.30
4.58
4.43
4.33
4.20
3.61
14.41
5.18
12.74
4.68
11.17
4.35
10.10
3.83
9.06
3.94
8.10
3.51
A
A
A
A
A
A
B
B
B
B
B
B
1
1
1
1
1
1
1
1
1
1
a
C(16:0)lysoPtdCho is 1-palmitoyl-2-lyso-sn-glycero-3-phosphocholine; C(16:0)/(2:0)PtdCho is 1-palmitoyl-2-acetyl-sn-glycero-3-
phosphocholine.
b
Method A: isolation of lyso-PL and subsequent acylation. Method B: procedure without isolation of the intermediate.
Isolated yield of purified product from GPC.
c
has been already reported in literature (Fasoli et al.,
sion was filtered and the solution was concentrated under
+
+
2
006). ESI/MS: [M + H] = 496.3; [M + Na] = 518.2. tR
vacuo without heating. The residue was suspended in
dry dichloromethane (10 ml) and treated with DMAP
(0.335 g, 2.74 mmol) and palmitic anhydride (1.36 g,
2.74 mmol). The reaction mixture was stirred for 24h
at r.t., then concentrated under vacuo. The crude product
was then dissolved in heptane (10 ml) and water (10 ml).
After centrifugation the organic phases were evaporated
to give a residue which is dissolved in dichloromethane
(
HPLC) = 6.24 min.
To a solution of 1 (50.0 mg, 0.101 mmol) in
dry dichloromethane (2 ml) was added DMAP
0.037 g, 0.303 mmol) and butyric anhydride (49.5 ␮l,
.303 mmol). The reaction mixture was stirred for 20 h
(
0
at r.t., then vacuum distilled. The crude product was
then dissolved in dichloromethane and stirred at room
temperature for 1 h with Dowex 50 × 8 cation exchange
◦
(2 ml) and precipitated in acetone (30 ml) at −20 C.
+
resin H form. The suspension was then filtered and
Solid 13 (511 mg, 87%) is recovered by filtration and
1
concentrated. The residue was dissolved again in CHCl3
dried under vacuo. H NMR (CDCl3/CD3OD): δ 5.19
◦
(
1 ml) and precipitated in acetone (20 ml) at −20 C
(m, 1H), 4.37 (m, 1H), 4.25 (m, 2H), 4.11 (m, 1H),
3.97 (m, 2H), 3.61 (m, 2H), 3.22 (s, 9H), 2.31 (m, 4H),
1.61 (m, 4H), 1.23 (m, 24H), 0.87 (m, 6H). ESI/MS:
1
to give 49 mg of compound 4 (86 % yield). H NMR
CDCl3): δ 5.19 (m, 1H), 4.35 (m, 1H), 4.28 (m, 2H),
(
+
4
2
6
.10 (m, 1H), 3.98 (m, 2H), 3.69 (m, 2H), 3.25 (s, 9H),
.26 (m, 4H), 1.63 (m, 4H), 1.22 (m, 24H), 0.93 (m,
H). ESI/MS: [M + H] = 566.3; [M + Na] = 588.3. tR
[M + Na] = 588.3. tR (HPLC) = 4.354 min.
+
+
3. Results and discussion
(
HPLC) = 4.434 min.
GPC is a readily available compound obtainable from
natural lecithins. It is the ideal starting material for
the preparation of PLs of various type since it has the
same chirality that PLs have in nature. However the low
solubility inorganicsolvents hashampered its useinsyn-
thesis. Although the corresponding Cd-complex (Chada,
1970) can be used in DMF or other polar organic sol-
vents, it appears not suitable for the larger scale synthesis
2
.2.2. 1-Butanoyl-2-palmitoyl-sn-glycero-3-
phosphocholine (13) (method B)
GPC (250 mg, 0.97 mmol) and DBTO (275 mg,
.1 mmol) were suspended in 2-propanol (10 ml) and
1
◦
refluxed for 1 h. The mixture was cooled to 25 C.
Then DMAP (143 mg, 1.2 mmol) and butanoyl chloride
(
121.5 ␮l, 1.2 mmol) are added. After 48 h, the suspen-

P. D’Arrigo et al. / Chemistry and Physics of Lipids 147 (2007) 113–118
117
Fig. 1. 1H NMR spectra (from 5.5 to 3.5 ppm) of compounds 4 and 13.
of derivatives (Ichihara et al., 2005). We have recently
discovered that mono- and regio-selective acylation of
GPC can be readily achieved with tin technology if per-
formed in 2-propanol, a solvent which will not compete
with the much more reactive tin ketal obtained from GPC
and alkyl-tin oxides. With this method high yields of
the range 5.5–3.5 ppm. Signals in this portion of spec-
trum are attributed to protons belonging to the glycerol
backbone and to the polar head of the compounds. Fig. 1
comparesthespectruminthatregionforthetwoisomeric
butanoyl-palmitoyl PCs 4 and 13. The signal attributed
+
to the –CH –N (CH3)3 in compound 4 (3.69 ppm), is
2
1
-acyl-2-lyso-glycero-3-phosphocholines or glycerol-3-
shifted upfield (3.61 ppm) when the palmitic chain is in
phosphates have been prepared in good yields (Fasoli
et al., 2006). We have extended this procedure to the
preparation of mixed chains-glycero-3-phosphocholines
alternating a short acyl residue (C-2 to C-9) to a long one
position sn-2 in compound 13. Moreover O-CH signals
2
in sn-1 position and O-CH ␣- to the phosphate on the
2
polar head are well resolved in spectrum b in compound
13 while they overlap partially when the palmitic chain
is in sn-1 position (compound 4, spectrum a).
Similar observation can be done for all the other iso-
meric mixed chains PCs prepared. The method seems to
be useful for the evaluation of the positional purity of
these compounds.
In conclusion we have described the preparation
of mixed chains PLs employing fewer synthetic steps
in comparison with known methods. This synthetic
approach also shows the versatility of GPC as starting
material for the preparation of an all set of glycerophos-
pholipids.
(
C-16).
In the first procedure (method A) GPC is transformed
into the cyclic tin ketal by treatment with DBTO in
-propanol at reflux. The mixture is then treated at
2
room temperature with triethylamine (TEA) and the acid
chloride. The formation of the lyso-PC was practically
complete in 15 min. Selective extraction allowed the
removal of the tin compounds and subsequent precip-
itation of the product. The lyso-PL was then dissolved
in dry dichloromethane and acylated with the acid anhy-
dride. Yields (see Table 1) from GPC were in the range
of 52–69%.
A direct method without the isolation of the lyso-PL
(
method B) consists in the formation of the tin ketal as
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