Synthesis of phosphatidylcholines containing ricinoleic acid

Article abstract of DOI:10.1016/S0040-4020(01)01057-2

1,2-Diricinoleoyl- and 1-ricinoleoyl-2-oleoyl-sn-glycero-3-phosphocholine were synthesised with good yields. The synthesis started with the preparation of ricinoleic acid from castor oil. The choice of a suitable agent to protect the -OH group of ricinoleic acid was a key factor to afford the final products. Several protecting groups were assayed but only β-methoxyethoxymethyl chloride (MEMCl) and 2,2,2-trichloroethyl chloroformate (TRECCl) gave reasonable yields and good optical purities of the final products. The overall yields for 1,2-diricinoleoyl-sn-glycero-3-phosphocholine and 1-ricinoleoyl-2-oleoyl-sn-glycero-3-phosphocholine were 32.1% (with respect to ricinoleic acid methyl ester using TREC as protecting group) and 10.3% (with respect to 1-trityl-glycero-3-phosphocholine), respectively.

Full text of DOI:10.1016/S0040-4020(01)01057-2

TETRAHEDRON
Pergamon
Tetrahedron 57 02001) 10219±10227
Synthesis of phosphatidylcholines containing ricinoleic acid
Gianpietro Borsotti, Gianfranco Guglielmetti, Silvia Spera and Ezio Battistelp
EniChem s.p.a., Istituto G. Donegani, Via Fauser 4, 28100 Novara, Italy
Received 3 July 2001; revised 25September 2001; accepted 18 October 2001
AbstractÐ1,2-Diricinoleoyl- and 1-ricinoleoyl-2-oleoyl-sn-glycero-3-phosphocholine were synthesised with good yields. The synthesis
started with the preparation of ricinoleic acid from castor oil. The choice of a suitable agent to protect the ±OH group of ricinoleic acid was a
key factor to afford the ®nal products. Several protecting groups were assayed but only b-methoxyethoxymethyl chloride 0MEMCl) and
2,2,2-trichloroethyl chloroformate 0TRECCl) gave reasonable yields and good optical purities of the ®nal products. The overall yields for
1,2-diricinoleoyl-sn-glycero-3-phosphocholine and 1-ricinoleoyl-2-oleoyl-sn-glycero-3-phosphocholine were 32.1% 0with respect to
ricinoleic acid methyl ester using TREC as protecting group) and 10.3% 0with respect to 1-trityl-glycero-3-phosphocholine), respectively.
q 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
synthesise well de®ned oligoricinoleoyl oligoglycerols.
The secondary ±OH of ricinoleic acid was successfully
protected with t-butyl dimethylchlorosilane. It was then
possible to carry on the speci®c esteri®cation of 0oligo)-
glycerols with protected 0oligo)ricinoleic acids.
Ester derivatives of glycero-phosphocholines are key inter-
mediates of oil metabolism in plants. Ricinoleic acid is an
unsaturated hydroxylated acid which constitutes about
85±90% of the fatty acids fraction 0castor oil) stored in
mature Ricinus communis seeds.1 Although castor oil is
one of the most relevant plant commodities for non-food
application,2 ricinoleic acid in vivo metabolism is not yet
fully understood and metabolites are dif®cult to isolate.
Diricinoleoyl-glycero-3-phosphocholine is an obligatory
intermediate in oil formation in developing seeds, being
the substrate of important regulatory enzymes such as
speci®c endoplasmatic acyl- and phospho-transferases and
phospholipase A.3,4 1-Ricinoleoyl-2-oleoyl-glycero-3-phos-
phocholine, the actual substrate of the enzyme oleate-b-12-
hydroxylase,5 is the key enzyme of ricinoleic acid synthesis
in R. communis. The enzyme speci®cally promotes the
hydroxylation of the oleoyl moiety in position 2 of the
diacyl-glycero-3-phosphocholine, forming the correspond-
ing ricinoleoyl group. The aim of this contribution is to
de®ne a synthesis of those enzymatic substrates which
may be useful to biochemists to improve knowledge on
plant metabolic pathways of uncommon fatty acids.
Negelmann et al.7 proposed an elegant synthesis of hydroxy
fatty acid derivatives of glycero-phosphocholines. The
author used the same protecting group of the ricinoleic
È
±OH as Froling did. The protection successfully withstood
the alkaline conditions used to complete the synthesis of the
glycero-phosphocholines. However, the ®nal yield of the
diricinoleoyl-glycero-3-phosphocholine was low 09%).
Following previous ®ndings,8 the present work has
improved the method for preparing the diricinoleoyl-
glycero-3-phosphocholine 0®nal yield 32.1%) by changing
the protecting groups and the reaction conditions. For
instance, the hydrolysis of the ricinoleic methyl ester,
protected with a group sensitive to strong alkaline con-
ditions, was accomplished by enzymatic catalysis in mild
conditions 0neutral pH, 358C). Moreover, in this work
the synthesis of a mixed type of glycero-phosphocholine
01-ricinoleoyl-2-oleoyl-glycero-3-phosphocholine) it is
also presented, which, to our knowledge, has not been
reported yet.
A key step of the synthesis is the suitable protection of
6
È
the ricinoleic acid hydroxyl group. Froling was able to
2. Results and discussion
Keywords: ricinoleic acid; glycero-phosphocholine; OH-protection.
Corresponding author. Tel.: 139-321-4474444; fax: 139-321-447425;
p
The synthesis of 1,2-di-[0R)-12-hydroxy-octadec-9-enoyl])-
sn-glycero-3-phosphocholine
phosphocholine) is summarised in Scheme 1. The synthesis
of glycero-phosphocholines containing ricinoleoyl group0s)
has not been described in the literature until recently.7,8 The
dif®culty of synthesising these compounds is related to the
e-mail: ezio.battistel@enichem.it
Abbreviations: CDI, N,N-carbonyl diimidazole; DCC, N,N0-dicyclohexyl
carbodiimide; DEE, diethyl ether; DIPEA, N,N-diisopropyl-N-ethylamine;
EMK, ethylmethylketone; GPC, sn-glycero-3-phosphoryl choline;
MEMCl, b-methoxyethoxymethyl chloride; PPTS, pyridinium-p-toluen-
sulfonate; PPyr, pyrrolidinopyridine; pyr, pyridine; TRECCl, 2,2,2-
trichloroethyl-chloroformate; tritylCl, triphenylmethyl chloride.
0diricinoleoyl-glycero-3-
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020001)01057-2

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G. Borsotti et al. / Tetrahedron 57 ,2001) 10219±10227
Scheme 1. Synthesis of 1,2-di-[0R)-12-hydroxy-octadec-9-enoyl]-sn-glycero-3-phosphocholine.
presence of the 12-OH group of the ricinoleic acid. As a
matter of fact, direct reaction between l-glycero-3-phos-
phorylcholine 0GPC) and ricinoleic acid following the
procedure described in the literature for saturated fatty
acids,9 does not yield the desired product. Instead of
diricinoleoyl-glycero-3-phosphocholine only condensation
products between the 12-OH group and carboxylic acid
groups were observed. Therefore it was necessary to protect
the 12-OH group before the acylation of GPC.
exceed 90%. Further puri®cation 0.96%) was achieved
by extraction with a suitable solvent 0for instance diethyl
ether or THF), added to the dry low-purity product just
enough to form a paste. The [a]D agreed well with the
literature data.7 Complete removal of the residual impurities
was easily accomplished in the next synthetic step, the
methyl ester formation.
Several ±OH protecting groups were assayed without
success. For example, chloroacetylchloride Cl±CH2±CO±
Cl was a promising group since activation of the free acid
with imidazole and protection afforded high yields 0.80%).
However, GPC acylation gave poor results because the
Ricinoleic acid 02) was obtained by alkaline hydrolysis of
castor oil 01). By carrying out the hydrolysis according to
standard procedures10 the purity of 2 does not usually

G. Borsotti et al. / Tetrahedron 57 ,2001) 10219±10227
10221
using the conditions described in the literature.11 Anhydride
fatty acid derivatives are able to acylate GPC with good
yields in the presence of 4-dimethylaminopyridine or
4-pyrrolidinopyridine. The drawbacks of this reaction are
that a large molar excess of the anhydride with respect to
the OH group is usually necessary and that half of the
anhydride group is lost as acid. A more convenient method
is that described by Warner and Benson.12 The fatty acid is
activated by preparing the corresponding imidazolyl deriva-
tive. Acylation is then carried out in the presence of NaCH2-
SOCH3 and DMSO with a slight molar excess of the
activated acid. However, also in this case there are some
disadvantages, such as the dif®culty of DMSO removal
0which may be accomplished at high temperatures where
product degradation may occur) and the presence of NaCH2-
SOCH3 which induces a complete destruction of the TREC
protection. Because of these dif®culties, the GPC acylation
reaction was further improved as follows: the reaction was
still carried out with the imidazolyl-derivative of the
O-protected ricinoleic acid, but DMF 0easier to remove)
was used as solvent instead of DMSO. Furthermore, the
reaction was optimised by adding anhydrous carbonate
such as Na2CO3, K2CO3 or Cs2CO3 0molar ratio carbon-
ate/GPC 5:1). These bases are mild, did not hydrolyse the
O-TREC group and gave the best yields 064%) also with the
O-MEM derivative.
Scheme 2. Transesteri®cation and transamidation within the O-TREC-
protected ricinoleic acid.
protecting group was partially removed under the reaction
conditions.
Chloromethyl-methylether Cl±CH2±OCH3 is another
protecting group which was supposed to be easily
removable. The protection was carried out as described in
the literature with formaldehyde dimethyl acetal in the
presence of P2O5.10 Unfortunately, the reaction yield was
low 0,30%).
A good yield 097%) of the protected ester was obtained with
2,2,2-trichloroethyl chloroformate 0TREC). Unfortunately,
in the following acylation step, performed in DMF in the
presence of an anhydrous carbonate, the acylation yield was
poor 0,2%) even if the protection was not removed. The
reason of such low yield was due to the concurrent trans-
esteri®cation and transamidation reaction within the
protected ricinoleic acid derivative, leading to the formation
of unreactive compound 8, as assessed by GC±MS and NMR
analysis 0Scheme 2). However, TREC can be still con-
veniently used if different acylation conditions are chosen.
The ®nal removal of ±OH protecting group is the last
critical step of the synthesis. MEM removal was the most
dif®cult to achieve. For example, the hydrolysis of MEM
with ZnBr2 or TiCl4 as catalysts13 afforded only degradation
by-products, all containing some halogen atoms. Better
results were obtained by using the complex of cathecol
with BBr3,14 but the overall yield was poor. The best way
to remove the MEM protecting group is the method
described by Monti et al.15 As suggested by the authors,
the use of pyridinium p-toluenesulfonate dissolved in ethyl-
methylketone 0EMK) was effective. Although the long reac-
tion time 040 h) at high temperature induced some
degradation of 7, nevertheless, after puri®cation, the ®nal
product 7 was recovered with a reasonable yield 058%) and
good optical purity. Unlike MEM, TREC protecting group
is more easily removable. The best method consisted on the
use of Zn in CH3COOH16 0reaction yield 84%). Compound
7 was obtained with high purity and could be used without
further puri®cation.
The best results were obtained with b-methoxyethoxy-
methyl chloride 0MEMCl) and TREC group 0if used with
speci®c acylation conditions), affording a reaction yield of
78 and 97%, respectively. The use of each protecting agent,
MEM or TREC, has both positive and negative aspects. The
O-MEM derivative is stable under strong alkaline condi-
tions. Consequently, the methyl ester 4a can be readily
hydrolysed with NaOH. On the other hand, MEM removal
at the end of the synthesis is dif®cult due to its intrinsic
stability and can be only accomplished over a long period
of time 0several hours) causing a concurrent partial degra-
dation of the compound of interest 07). A chromatographic
step is then needed to purify the remaining product. Conver-
sely, the TREC protecting group is not stable in strong alka-
line conditions. The hydrolysis of ester 4b cannot be done in
the presence of NaOH and has to be carried on with speci®c
non-chemical methods such as enzymatic catalysis. Several
hydrolytic enzymes were assayed but only the lipase from
Pseudomonas cepacea readily hydrolyses 4b with high
yields 098±99%). At the end of the synthesis, unlike
MEM, TREC can be easily removed without degradation
of the ®nal product 7. It should be noted that the ®nal yield
0with respect to ricinoleoyl methyl ester 3) of the `TREC
synthetic route' 0Scheme 1) is slightly higher 032.1%) than
the `MEM route' 027.2%).
The synthesis of 1-012-hydroxy-octadec-9-enoyl)-2-0octa-
dec-9-enoyl)-sn-glycero-3-phosphocholine 01-ricinoleoyl-
2-oleoyl-GP) is summarised in Scheme 3. The synthesis
follows the procedures described in the literature for fatty
acids without hydroxyl groups.17 A key point was the selec-
tion of a protecting group of the 1-OH of GPC. Since protec-
tion with MEM 0as suggested by the elegant synthesis of
phosphatidylcholines containing very long chain polyunsa-
turated fatty acids)18 was not satisfactory, triphenylmethyl
chloride 0tritylCl) was used as protecting agent of the
glycerol 1-OH group19 0reaction yield 51%). TritylCl is
large enough to react with only the 1-OH group of glycerol
without limiting the accessibility of the 2-OH to the incom-
ing activated oleic acid 0yield 62%). Moreover, the group
was ef®ciently removed since good yield 063%) of the
deprotection reaction was observed.
The GPC acylation with the protected acid was ®rst assayed

10222
G. Borsotti et al. / Tetrahedron 57 ,2001) 10219±10227
Scheme 3. Synthesis of 1-012-hydroxy-octadec-9-enoyl)-2-0octadec-9-enoyl)-sn-glycero-3-phosphocholine.
It should be noted that under the conditions 0boron tri-
¯uoride etherate in methylene chloride) described in the
literature,17 the deprotection reaction should have been
concurrent to the acylation reaction in the presence of
ricinoleic acid anhydride. Only deprotection was observed
and consequently the acylation of 11 was carried on with
DCC and pyrrolidinopyridine, as described.
generous gift from CHEMI s.p.a. 0Patrica, FR, Italy). Castor
oil and phospholipase A2 obtained from bee venom
01220 U/mg) were from Sigma 0Milano, Italy). Pseudo-
monas cepacea lipase PS 01000 U/mg) was from Amano
0Nagoya, Japan). Zinc powder was from Merck 0Italy).
Molecular mass analysis. Mass analysis was performed with
the Electron Spray 0ESI) technique by using a Finnigan
MAT LCQ spectrometer, ESI 0electron spray) ionisation
source: sheath gas ¯ow rate 50, spray voltage 5 kV, capil-
lary temperature 2108C, capillary voltage 26 and 235V
0positive and negative ions, respectively) tube lens offset
15and 245V 0positive and negative ions, respectively).
HRMS measurements were performed with a HR Finnigan
MAT FAB/MS instrument.
Since migration between position 1 and 2 may have
occurred in the deprotected sample, position 2 being
thermodynamically favoured with respect to 1, the isomer
distribution was controlled by selective hydrolysis by phos-
pholipase A2 of the ester in position 2. Good yield 0.95%)
of the correct isomer con®rmed the positional selectivity
afforded by the synthesis.
1
In conclusion, the synthesis of phosphocholines containing
ricinoleic acid was accomplished with reasonably good
yields, improving signi®cantly 0yield 32.1%) the literature
results 09%)7 in the case of diricinoleoyl-glycero-3-phospho-
choline. The choice of the protecting groups and the modula-
tion of the reaction media were the two main factors of the
yield improvements. These may be signi®cant enough to
prepare the radiolabelled counterparts of these compounds
for speci®c biological studies of castor oil metabolism.
NMR. H, 13C and 31P NMR spectra were recorded with
Varian VXR 300 instrument. The references for 13C and
1H CDCl3 were CDCl3 at 77.0 and 7.26 ppm, respectively.
The detailed assignment of all the hydrogen atoms was
completed by recording the COSY spectra. 13C NMR
spectra were well resolved, allowing the identi®cation of
the signal of all the 44 C atoms.
Polarimetry. The optical rotation 0a) was measured with a
Perkin±Elmer Polarimeter 241 at the sodium D-line.
Concentrations 0c) are given as g/100 mL solution.
3. Experimental
3.1. General methods
Gas chromatography. Gas chromatographic analysis was
performed with a HRGC 5300 gas chromatograph Carlo
Erba 0Milano, Italy) equipped with a Supelco SPB-5
column. Temperature gradient: from 80 to 3008C, 108C/min.
Chemicals. l-a-Glycerophosphorylcholine 0GPC) was a

G. Borsotti et al. / Tetrahedron 57 ,2001) 10219±10227
10223
3.2. Synthesis of 1,2-di[%R)-12-hydroxy-octadec-9-enoyl])-
sn-glycero-3-phosphocholine %Scheme 1)
C04±7)± CH2, C014±17)± CH2), 1.34±1.4502H, m,
C13±CH2), 1.45±1.65 02H, m, C3±CH2), 1.82±2.0502H,
m, C8±CH2), 2.17 02H, m, C11±CH2), 2.20 02H, t,
Jˆ8.5Hz, C2±CH 2), 3.29 03H, s, OCH3MEM), 3.56 03H, s,
OCH3), 3.40±3.68 04H, m, OCH2CH2O), 4.57±4.73 02H, m,
OCH2O), 5.20±5.45 02H, m, C9±CHv, C10±CHv).
3.2.1. %R)-12-Hydroxy-octadec-cis-9-enylic acid %ricino-
leic acid) %2). Two hundred grams of castor oil 01) were
dissolved in 400 mL of ethanol containing KOH 040 g),
re¯uxed for 15min and dried under vacuum. The solid
residue was ®nely dispersed and washed twice in diethyl
ether and then dissolved in 1 L of iced water acidi®ed
with HCl. The oil was extracted with hexane, treated with
Na2SO4 overnight at 48C and then dried under vacuum.
150 g 075% yield by weight) of ricinoleic acid 02) as colour-
less oil were obtained, 97% pure 0from GC analysis after
esteri®cation with diazomethane, the major contaminants
were: linoleic acid 0.3%, oleic acid 1.3%, stearic acid
3.2.4. %R)-12-O-TREC-octadec-cis-9-enylic acid methyl
ester %ricinoleic acid methyl ester-O-TREC) %4b). To
the reaction mixture, containing 3 023 g, 80.06 mmol),
diethyl ether 0DEE, 150 mL), pyridine 010 mL), 2,2,2-
trichloroethyl chloroformate 0TREC-Cl, 18 g, 84,9 mmol)
was slowly added in 2 h at 58C. After few hours at room
temperature, hexane 0100 mL) was added and the pyridine
HCl was ®ltered off. The product was dried under reduced
pressure and then under high vacuum at 508C. Pure product
4b 037.8 g, 77.7 mmol, yield 97.1%) was obtained as a
colourless viscous oil. [Found: C, 54.21; H, 7.77; Cl,
21.44. C22H37Cl3O5 requires C, 54.30; H, 7.67; Cl, 21.58].
20
0.4%). [a]D ˆ13.98 0cˆ1.0, CHCl3), data consistent
with literature 0[a]D ˆ13.88).7
20
3.2.2. %R)-12-Hydroxy-octadec-cis-9-enylic acid methyl
ester %ricinoleic acid methyl ester) %3). Into a solution of
2 0100 g, 335mmol) in methanol 0300 mL) at 0 8C, gaseous
HCl 030 g) was bubbled under stirring at room temperature.
After 30 min water 0500 mL) and then hexane 0200 mL)
were added. The extract phase was washed twice with
water, once with NaHCO3 010% w/v) and then treated
with Na2SO4. After drying the residue under vacuum,
106 g of 3 were obtained 0purity by GC: 96%). Further
puri®cation was achieved by silica gel chromatography. A
column 080£400 mm) was eluted with hexane/acetone
90:10. After evaporation of the solvent from the fractions
containing the methyl ester, 98.5g 0315mmol, yield 94.1%)
of pure 3 as a colourless oil were obtained 0purity: 99.9% by
GC). [Found: C, 73.30; H, 11.80. C19H36O3 requires C,
23
[a]D ˆ119.48 0cˆ4.4, CHCl3). nmax 0neat, ®lm) 3010
[0vCH)], 2925and 2860 [0CH , CH3)], 1742 [0CvO]ester
2
,
1755 [0CvO)]TREC, 1460, 1430, 1370, 1250 [0O±CO±O)],
1070, 1020, 820 [0CCl)3], 785[ p0O±CO2], 730 [0CCl)3]. dH
0300 MHz, CDCl3): 0.81 03H, t, Jˆ6.5Hz, C18±CH 3),
1.05±1.40 016H, m, C04±7)±CH2, C014±17)±CH2), 1.4±
1.68 04H, m, C3±CH2, C13±CH2), 1.85±2.07 02H, m,
C8±CH2), 2.23 02H, t, Jˆ7 Hz, C2±CH2), 2.31 02H, dd,
Jˆ13, 7 Hz, C11±CH2), 3.59 03H, s, OCH3), 4.60±4.80
03H, m, COCH21CH±C12), 5.20±5.50 02H, m, C9±
CHv, C10±CHv).
3.2.5. %R)-12-O-MEM-octadec-cis-9-enylic acid %ricino-
leic acid O-MEM) %5a). The product 4a 038 g,
94.9 mmol) was dissolved in methanol 0100 mL) and
NaOH±water 020 g, 500 mmol, in 100 mL). After few
hours the hydrolysis was complete. The reaction mixture
was diluted with water 0500 mL) and acidi®ed with HCl
020%). The product was extracted with hexane. The extract
was washed with water, treated with Na2SO4 and dried
under reduced pressure. Pure ricinoleic acid O-MEM-
protected 5a 034.1 g, 88.27 mmol, yield 92.99%) was
obtained 0purity 99% according to TLC and NMR analysis)
as a colourless oil. [Found: C, 68.21; H, 11.12. C22H42O5
requires C, 68.34; H, 10.96]. nmax 0neat liquid ®lm) 3400±
2500 br[0OH)ass)], 3009 [0vCH)], 2921 and 2861 [0CH2,
CH3)], 2818 [0OCH3)MEM], 1710 [0CvO)acid], 1410, 1130
and 1110 [0C±O±C)MEM], 1040 [0O±C±C)MEM], 850 [0C±
O±C)MEM], 723 [r0CH2)3 chain]. dH 0300 MHz, CDCl3): 0.88
03H, t, Jˆ7 Hz, C18±CH3), 1.05±1.38 016H, m, C04±7)±
CH2, C014±17)±CH2), 1.38±1.52 02H, m, C13±CH2),
1.52±1.72 02H, m, C3±CH2), 1.86±2.10 02H, m, C8±
CH2), 2.10±2.28 02H, m, C11±CH2), 2.30 02H, t,
23
73.01; H, 11.72]. [a]D ˆ13.4 0cˆ1.5, CHCl3), result
consistent with literature 0[a]D ˆ13.48).7 nmax 0liquid
20
®lm, neat) 3360 [0OH)ass], 3010 [0vCH)], 2924 and 2852
[0CH2, CH3)], 1743 [0CvO)], 1192 and 1168 [0C±O)ester],
724 [r0CH2)3 chain]. dH 0300 MHz, CDCl3): 0.850t,
Jˆ6.9 Hz, 3H, C18±CH3), 1.26±1.27 0m, 16H, 0CH2)8).
1.43 0m, 2H, C13±CH2). 1.62 0qui, Jˆ7.2 Hz, 2H, C3±
CH2). 2.01 0qua, Jˆ6.6 Hz, 2H, C8±CH2). 2.18 0t,
00
Jˆ6.7 Hz, 2H, C2±CH2). 2.27 0t, Jˆ7.5Hz, 2H, C ±
CH2). 3.58 0qui, Jˆ5.9 Hz, 1H, C12±CH). 3.63 0s, 3H,
CO2CH3). 5.39 0m, 1H, C9±CH). 5.51 0m, 1H, C10CH).
3.2.3. %R)-12-O-MEM-octadec-cis-9-enylic acid methyl
ester %ricinoleic acid methyl ester-O-MEM) %4a). To a
solution of 3 038 g, 121.6 mmol) and N,N-diisopropyl-N-
ethylamine 0DIPEA, 25mL, 137 mmol) in CH 2Cl2
0200 mL), MEMCl 020 g, 160 mmol) was slowly added at
08C in 2 h. The solution was stirred at room temperature
overnight. The reaction mixture was diluted with HCl
020% in water), washed with water and with NaHCO3
010% w/v). After the addition of Na2SO4, the product 4a
038 g, 94.9 mmol, yield 78.01%) was dried under reduced
pressure as a colourless oil. [Found: C, 68.81; H, 10.95.
Jˆ7.5Hz, C2±CH 2), 3.3503H, s, OCH ), 3.45±3.80 04H,
3
m, OCH2CH2O), 3.60 01H, m, C12±CH), 4.60±4.90 02H, m,
OCH2O), 5.25±5.50 02H, m, C9±CHv, C10±CHv).
23
3.2.6. %R)-12-O-TREC-octadec-cis-9-enylic acid %ricino-
leic acid O-TREC) %5b). The hydrolysis of the ester 4b was
accomplished by using enzymes as catalysts. The best
results were obtained with lipase PS from Pseudomonas
cepacea, a commercial product from Amano 0Japan).
Compound 4b 025g, 51.4 mmol) was suspended in
phosphate buffer 10 mM pH 7.3 0300 mL) under vigorous
C23H44O5 requires C, 68.95; H, 11.08]. [a]D ˆ121.3
0cˆ2.5, CH3COCH3). nmax 0neat liquid ®lm) 3010
[0vCH)], 2920 and 2860 [0CH2, CH3)], 2818 [0OCH3)MEM],
1742 [0CvO)], 1460, 1430, 1192 and 1168 [0C±O)ester],
1110 [0C±O±C)MEM], 1040 [0O±C±C)MEM], 850 [0C±O±
C)MEM], 723 [r0CH2)3 chain]. dH 0300 MHz, CDCl3): 0.78
03H, t, Jˆ6.5Hz, C18±CH 3), 1.02±1.34 016H, m,

10224
G. Borsotti et al. / Tetrahedron 57 ,2001) 10219±10227
agitation and lipase PS 04 g) was added. The reaction was
carried on for 24 h at 358C. The pH was kept constant by
means of the continuous addition of 0.5M NaOH in a auto-
matic titration device 0pH stat). The reaction mixture was
then acidi®ed with HCl 20% and the product was extracted
with hexane, washed with water, treated with Na2SO4 and
dried under reduced pressure. This procedure yielded 23.6 g
of 5b as a viscous oil 049.9 mmol, yield 97.2), containing
1±2% of the starting substrate 4b. The product may be used
without further puri®cation in the next synthetic step, other-
wise may be puri®ed to homogeneity by silica gel chromato-
graphy 0solvent for elution: hexane/acetone 85/15). [Found:
C, 53.51; H, 7.37; Cl, 21.99. C21H35Cl3O5 requires C, 53.37;
H, 7.47; Cl, 22.22]. nmax 0neat, ®lm) 3400±2500 [br0OH)ass],
3010 [0vCH)], 2920 and 2861 [0CH2, CH3)], 1755
[0CvO)TREC], 1710 [0CvO)acid], 1370, 1250 [0O±CO±O)
and 0C±O)acid], 820 [0CCl)3], 785[ p0O±CO2], 730 [0CCl)3].
dH 0300 MHz, CDCl3): 0.83 03H, t, Jˆ7 Hz, C18±CH3),
1.00±1.38 016H, m, C04±7)±CH2, C014±17)±CH2), 1.38±
1.69 04H, m, C3±CH2, C13±CH2), 1.88±2.03 02H, m, C8±
CH2), 2.12 02H, t, Jˆ7 Hz, C2±CH2), 2.15±2.52 02H, m,
C11±CH2), 4.51 02H, s, COCH2), 4.86 01H, m, CH±C12),
5.30±5.55 02H, m, C9±CHv, C10±CHv).
C032±35)±CH2), 1.35±1.46 04H, m, C13±CH2, C31±CH2),
1.46±1.6504H, m, C3±CH 2, C21±CH2), 1.73±2.08 04H, m,
C8±CH2, C26±CH2), 2.08±2.36 08H, m, C2±CH2, C20±
CH2,C11±CH2, C29±CH2), 3.30 06H, s, ±OCH3), 3.31
09H, s, N0CH3)3), 3.40±3.70 08H, m, OCH2CH2O), 3.55
02H, m, C12±CH, C30±CH), 3.8502H, m, CH ±N), 3.92
2
02H, m, glycerol-C3), 4.0501H, dd, Jˆ12, 7 Hz, glycerol-
C1±Hb), 4.24 02H, m, PO±OCH2), 4.3501H, dd, Jˆ12,
2.5Hz glycerol-C1±H a), 4.60±4.80 04H, m, O±CH2±O),
5.15 01H, m, glycerol-C2±H), 5.25±5.50 04H, m, C9±
CHv, C10±CHv, C27±CHv, C28±CHv). m/z 1016
[MNa]1 0100%), 2009 [2M1Na]1, 957 [MNa2N0CH3)3]1;
m/z 978 0[M2CH3]2 100%), 907 [0M2CH3)2 C4H9N]2,
385[Ricinol. O-MEM±H ac.]2.
3.3.2. Synthesis of 6a with CDI. The reaction was
performed after activation of the acid with N,N-carbonyl-
diimidazole 0CDI), as follows. 5a 08 g, 20.70 mmol) was
dissolved in anhydrous THF 020 mL) under a gentle stream
of nitrogen. CDI 06.6 g, 40.7 mmol) was then added slowly,
allowing the temperature to rise. After the completion of the
reaction 0end of CO2 production), THF was evaporated
under reduced pressure. To the residue, the `activated'
ricinoleic acid O-protected 09 g, 20.58 mmol, yield 99%),
GPC 02,57 g, 10 mmol), DMF 020 mL) and K2CO3 04 g)
were added. The mixture of reactants and solvent was stirred
for 24 h at room temperature. Diethyl ether was added to
dilute the suspension, then the salts were ®ltered off and the
products were dried under reduced pressure and high
vacuum. The residue was dissolved in chloroform and puri-
®ed as described earlier 0Section 3.3.1). After drying the
fractions containing the product, 6.6 g 06.54 mmol, yield
3.3. 1,2-Di-%O-MEM-octadec-9-enoyl)-sn-glycero-3-
phosphocholine %6a)
The acylation of sn-glycero-3-phosphorylcholine 0GPC)
was assayed by using two methods: the ®rst method
consisted on forming in situ an anhydride intermediate
with N,N0-dicyclohexylcarbodiimide 0DCC), whereas the
second one consisted on the activation of the ester with
N,N-carbonyldiimidazole 0CDI).
22
64.2%) of pure 6a were obtained. [a]D ˆ120.2 0cˆ1.75,
CHCl3).
3.3.1. Synthesis of 6a with DCC. Under nitrogen
atmosphere, methanol 050 mL), 5a 015.5 g, 40.12 mmol)
and GPC 02,57 g, 10.0 mmol) were mixed until complete
dissolution was obtained. Methanol was removed under
reduced pressure and the residue heated several hours at
658C. Chloroform 030 mL) was added and then a solution
of 4-pyrrolidinopyridine 0PPyr, 6 g, 40.4 mmol in 20 mL
chloroform) and DCC 08.3 g, 40.2 mmol) was added slowly
030 min) at 458C. The reaction was allowed to proceed over-
night under stirring. The mixture was diluted with diethyl
ether, the salt ®ltered and the solvent evaporated under
reduced pressure. The residue was dissolved in chloro-
form/methanol 1:1 and the solution was puri®ed with a
column of Amberlist 15to eliminate 4-pyrrolidinopyridine.
The product was dried and dissolved in chloroform and
puri®ed by silica gel chromatography: the product was
eluted ®rst with CHCl3/CH3OH/H2O 65:25:2 and then by
gradually increasing the water content from 2 to 4. After
drying the fractions containing the product, 4.6 g
04.63 mmol, yield 23.0%) of pure 6a as white waxy paste
were obtained. [Found: C, 62.70; H, 10.29; N, 1.37; P, 3.06.
C52H100NO14P requires C, 62.80; H, 10.14; N, 1.41; P, 3.12].
3.3.3. 1,2-Di-%12-O-TREC-octadec-9-enoyl)-sn-glycero-
3-phosphocholine %6b). Acylation of GPC with O-TREC
ricinoleic acid was assayed only with the method of
anhydride formed in situ, as described in Section 3.3.1 in
the case of the O-MEM derivative 0the method with DMF
and K2CO3 induced the unwanted rearrangement shown in
Scheme 2). The reaction was carried on by mixing GPC
01.8 g, 7.0 mmol), adduct 5b 03.3 g, 6.78 mmol), 4-pyrroli-
dinopyridine 0PPyr, 4.15g, 28 mmol) and DCC 05.8 g,
28.1 mmol) in chloroform. After puri®cation by silica gel
chromatography, 3.2 g 02.74 mmol, yield 40.5%) of 6b as
white waxy paste were obtained. [Found: C, 51.60; H, 7.31;
N, 1.37; P, 2.60; Cl, 17.85. C50H86NO14Cl6P requires C,
51.48; H, 7.44; N, 1.20; P, 2.66; Cl, 18.00]. [a]D
23
ˆ
120.98 0cˆ1.2, CHCl3). nmax 0neat) 3010 [0vCH)],
2925and 2860 [0CH 2, CH3)], 1750 [0CvO)TREC and
0CvO)ester]overlap, 1465, 1390, 1240 [0PvO)], 1280 [0O±
CO±O)TREC], 1175[0C±O) ester], 1090 and 1060 [0P±O±
C)], 970 [r0CH3)choline], 725[0CCl) 3]. dH 0300 MHz,
CDCl3): 0.84 06H, t, Jˆ6.5Hz, C18±CH 3, C36±CH3),
1.05±1.45 032H, m, C04±7)±CH2, C014±17)±CH2, C022±
25)±CH2, C032±35)±CH2), 1.45±1.73 08H, m, C13±CH2,
C31±CH2, C3±CH2, C21±CH2), 1.88±2.12 04H, m, C8±
CH2, C26±CH2), 2.17±2.28 04H, m, C20±CH2, C2±CH2),
2.28±2.48 04H, m, C11±CH2, C29±CH2), 3.35ppm 09H, s,
N0CH3)3), 3.79 ppm 02H, m, glycerol-C3), 3.90 02H, m,
CH2±N), 4.09 ppm 01H, dd, Jˆ12, 7 Hz, glycerol-C1±
Hb), 4.28 ppm 02H, m, PO±OCH2), 4.37 ppm 01H, dd,
22
[a]D ˆ120.28 0cˆ1.75, CHCl3). nmax 0neat) 3009
[0vCH)], 2926 and 2855 [0CH2, CH3)], 1742 and 1730
[0CvO)overlap], 1466, 1365, 1240 [0PvO)], 1199 and 1171
[0C±O)ester], 1135[C±O±C) MEM], 1091 and 1045[0P±O±
C)choline and 0O±C±C)MEM], 970. dH 0300 MHz, CDCl3):
0.82 06H, t, Jˆ7 Hz, C18±CH3, C36±CH3), 1.03±1.35
032H, m, C04±7)±CH2, C014±17)± CH2, C022±25)±CH2,

G. Borsotti et al. / Tetrahedron 57 ,2001) 10219±10227
10225
Jˆ12, 2.5Hz, glycerol-C1±H a), 4.75ppm 02H, m, C12±
CH, C30±CH), 4.73 ppm 04H, s, CH2CCl3), 5.16 ppm
01H, m, glycerol-C2±H), 5.24±5.55 04H, m, C27±CHv,
C9±CHv, C28±CHv, C10±CHv). m/z 1188 [MNa]1,
1190 0100%), 1192, 1194, 1196, 2353 [2M1Na]1, 1129
[MNa2N0CH3)3]1; m/z 1150 [M2CH3]2, 1152 0100%),
1154, 1156, 1158, 471 [ricinol. ac. O-TREC±H]2.
2.19 04H, t, Jˆ7 Hz, C11±CH2, C29±CH2,), 2.27 02H, t,
Jˆ7.5Hz, C20±CH 2), 2.29 02H, t, Jˆ7 Hz, C2±CH2),
3.33 09H, s, N0CH3)3), 3.49±3.67 02H, m, C12±CH, C30±
CH), 3.67±3.84 02H, m, CH2±N), 3.84±4.03 02H, m,
glycerol-C3±Ha,b), 4.11 01H, dd, Jˆ12.2, 7.3 Hz, glycerol-
C1±Hb), 4.19±4.34 02H, m, PO±CH2), 4.37 01H, dd,
Jˆ12.2, 3 Hz, glycerol-C1±Ha), 5.18 01H, m, glycerol-
C2±H), 5.30±5.60 04H, m, C27±CHv, C9±CHv, C28±
CHv, C10±CHv). d13C 075.42 MHz, CDCl3):13.96 0C18,
C36) 02CH3), 22.502), 24.70, 24.76, 25.6102), 27.2502),
28.90, 28.9604), 29.0202), 29.2502), 29.4602) 019CH2),
31.7302), 33.96, 34.13, 35.2502), 36.7202) 08CH2),
54.19 ppm 03C), 59.21 0CH±OH), 62.87 0±CH2±N), 63.29
0glycerol), 66.17 0glycerol), 70.42 0glycerol), 71.26 0PO±
CH2±), 125.40, 125.43, 132.69, 132.72 CH09)vCH010),
CH027)vCH028),173.06, 173.42 CO01), CO013). d31P
081.0 MHz, CDCl3)ˆ0.95. m/z 840 [MNa]10100%), 1657
[2M1Na]1, 781 [MNa2N0CH3)3]1; m/z802 [M2CH3]2
0100%), 731 [0M2CH3)2C4H9N]2, 297 [ricinoleic ac.-
H]2. HRMS 03-NBA) MH1, found 818.5930;
C44H85NO10P requires 818.5911.
3.3.4.
1,2-Di-[%R)-12-hydroxy-octadec-cis-9-enoyl]-sn-
glycero-3-phosphocholine %7). Deprotection of the deriva-
tive 6a containing MEM protecting group. The reactants, 6a
02 g, 2.01 mmol) and pyridinium p-toluenesulfonate 0PPTS,
3 g, 11.9 mmol) were dissolved in ethylmethylketone
0EMK) 045mL) and the solution was re¯uxed for 42 h.
After dilution with diethyl ether, the precipitated material
was ®ltered. The products were recovered after evaporation
of the solvent under reduced pressure, dissolved in chloro-
form and separated with silica gel chromatography. The
column was ®rst eluted with CHCl3, then with a stepwise
increase of CH3OH 0up to 30% v/v) and ®nally with addition
of water 0up to 4%). The fractions containing the product
were dried and the residue dissolved in diethyl ether and
washed with NaHCO3 010% w/v). After the addition of
Na2SO4 and evaporation of the solvent, 0.96 g
01.17 mmol, yield 58.4%) of pure 7 as a waxy solid were
obtained.
3.4. Synthesis of 1-%12-hydroxy-octadec-9-enoyl)-2-%octa-
dec-9-enoyl)-sn-glycero-3-phosphocholine %Scheme 3)
3.4.1. 1-Trityl-glycero-phosphocholine %1-trityl-GPC) %9).
sn-Glycero-3-phosphorylcholine 0GPC, 26 g, 101 mmol)
and ZnCl2 014 g, 102 mmol) were dissolved in 220 mL of
anhydrous DMF and stirred for 3 h at room temperature.
Triphenylmethyl chloride 0tritylCl, 28.5g, 102 mmol) was
then added at 58C. The reaction was stirred overnight and
diluted with 1 L of diethyl ether. The organic phase was
dissolved in CHCl3/isobuthanol 2:1 and washed with
NH4OH 04%). After adding Na2SO4, the mixture was
®ltered and concentrated down to 400 mL. After addition
of diethyl ether 02 L), 1-trityl-GPC 09) precipitated from
solution 025.1 g, 51.8 mmol, yield 51.2%) as a white
powder. [Found: C, 66.72; H, 7.45; N, 2.77; P, 6.35.
C27H35NO5P requires C, 66.91; H, 7.28; N, 2.89; P, 6.40].
The overall yields were 27.2% 0from ricinoleic acid methyl
ester via DMF±carbonate route) and 9.7% 0via DCC and
23
pyrrolidinopyridine route), respectively. [a]D ˆ17.38
0cˆ1.5, CHCl3).
3.3.5.
1,2-Di-[%R)-12-hydroxy-octadec-cis-9-enoyl]-sn-
glycero-3-phosphocholine %7). Deprotection of the deriva-
tive 6b containing TREC protecting group. The compound
6b 01.5g, 1.28 mmol) was dissolved in acetic acid 015mL)
and water 01.5mL). Powdered Zn 01.5g washed with HCl
5% 0in order to remove inert oxide), water, methanol, ether
and then dried under vacuum) was added in small aliquots
for 10 min, keeping the temperature as low as 10±158C. The
mixture was stirred for 2 h at room temperature, poured in
water 050 mL) and adjusted to neutrality by adding NaHCO3
010% w/v). The product was extracted with diethyl ether and
then recovered after addition of Na2SO4 and evaporation of
the solvent. The white solid was then further puri®ed as
described in Section 3.3.4. After the addition of Na2SO4,
and evaporation of the solvent from the fraction of the chro-
matography column, 0.88 g 01.076 mmol, yield 84.09%) of
pure 7, a waxy white solid, was obtained. On the assumption
that the unreacted protected acid 0two fold molar excess of
the stoichiometric quantity) used in the acylation reaction is
fully recovered, the overall was yield 32.1% with respect to
ricinoleic acid methyl ester. [Found: C, 64.68; H, 10.21; N,
1.60; P, 3.69. C44H84NO10P requires C, 64.58; H, 10.35; N,
23
[a]D ˆ210.58 0cˆ2.5, CH3OH). dH 0300 MHz, CDCl3):
3.16 09H, s, N0CH3)31), 3.16 02H, m, glycerol-C3), 3.48±
3.6502H, m, ±CH ±N), 3.83±4.00 02H, m, glycerol-C1),
2
4.00±4.10 01H, m, glycerol-C2), 4.14±4.30 02H, m, PO±
OCH2), 7±7.50 015H, 0C6H5)3).
3.4.2. 1-Trityl-2-%octadec-cis-9-enoyl)-sn-glycero-3-phos-
phocholine %10). The reaction was carried on after activa-
tion of oleic acid with N,N-carbonyldiimidazole 0CDI).
Oleic acid 05.64 g, 19.9 mmol) was dissolved in THF and
then CDI 03.95g, 24.3 mmol) was slowly added. After stir-
ring few hours, the solvent was evaporated and the residue
suspended in DMSO 025mL) containing compound
9
05.1 g, 10.53 mmol). Na 00.69 g, 30 mmol, in 55 mL
DMSO) was added and the mixture was stirred for 30 min
at 58C. Acetic acid 063 mL, 1000 mmol) was added to the
solution which was further diluted with CHCl3/CH3OH 2:1
01.0 L), washed with acetic acid/water 0250 mL, 1:1) and
concentrated NH4OH 02.5mL). The formation of two well
separated phases was favoured by methanol 0200 mL). The
phase containing chloroform was distilled after dilution
with toluene and methanol. The residue was dried under
vacuum for several hours, dissolved in CH3Cl and puri®ed
by silica gel chromatography. The product was eluted ®rst
23
1.71; P, 3.79]. [a]D ˆ17.38 0cˆ1.5in CHCl 3). nmax 0neat)
3600±3100 [0OH)ass], 3010 [0vCH)], 2921 and 2852 [0CH2,
CH3)], 1742 and 1730 [0CvO)ester], 1234 [0PvO)], 1162
[0C±O)ester], 1088 and 1057 [0P±O±C)], 964 [r0CH3)choline].
dH 0300 MHz, CDCl3): 0.88 06H, t, Jˆ7 Hz, C18±CH3,
C36±CH3), 1.19±1.40 032H, m, C04±7)± CH2, C014±
17)±CH2, C022±25)±CH2, C032±35)±CH2), 1.40±1.51
04H, m, C13±CH2, C31±CH2), 1.51±1.65 04H, m, C3±
CH2, C21±CH2), 1.98±2.10 04H, m, C8±CH2, C26±CH2),

10226
G. Borsotti et al. / Tetrahedron 57 ,2001) 10219±10227
with CHCl3/CH3OH/NH3 70:30:2 and then with CHCl3/
CH3OH/NH3 50:50:2. The fractions containing the product
were dried under reduced pressure and the adduct 10 04.9 g,
6.54 mmol, yield 62.1%) was recovered as a colourless
viscous residue. [Found: C, 72.01; H, 9.26; N, 1.75; P,
4.35. C45H67NO6P requires C, 72.15; H, 9.02; N, 1.87; P,
4.14]. dH 0300 MHz, CDCl3): 0.8503H, t, Jˆ6 Hz, C36±
CH3), 1.05±1.45 020H, m, C022±25)±CH 2, C030±35)±
CH2), 1.45±1.73 02H, m, C21±CH2), 1.82±2.10 04H, m,
C26±CH2, C29±CH2), 2.30 02H, t, Jˆ8 Hz, C20±CH2),
3.20 09H, s, N0CH3)31), 3.20 02H, m, glycerol-C1), 3.5±
3.7502H, m, ±CH 2±N), 3.96 02H, t, Jˆ6 Hz, glycerol-
C3), 4.05±4.25 02H, m, PO±OCH2), 5.25 01H, m,
glycerol-C2), 5.28±5.45 02H, m, C27±CHv, C28±
CHv), 7±7.50 015H, 0C6H5)3). m/z 786 [MNa]10100%),
product were dried and passed through a silica gel column
eluted with the same solvent. After evaporation of the
solvent, 1-0O-MEM)ricinoleoyl-2-oleoyl-GPC 012) was
recovered 01 g, 1.12 mmol, yield 58.6%) as a white waxy
paste. [Found: C, 64.51; H, 10.62; N, 1.72; P, 3.65.
C48H93NO11P requires C, 64.67; H, 10.52; N, 1.57; P,
3.48]. nmax 0neat) 3006 [0vCH)], 2926 and 2855 [0CH2,
CH3)], 1741 and 1732 [0CvO)ester and 0CvO)ester overlap,
]
1466, 1241 [0PvO)], 1172 [0C±O)ester], 1090 and 1044
[0P±O±C)choline and 0O±C±C)MEM], 970 [r0CH3)choline]. dH
0300 MHz, CDCl3): 0.84 06H, t, Jˆ6 Hz, C18±CH3, C36±
CH3), 1.00±1.40 036H, m, C04±7)± CH2, C014±17)± CH2,
C022±25)± CH2, C030±35)± CH2), 1.40±1.60 06H, m, C3±
CH2, C13±CH2, C21±CH2), 1.87±2.12 06H, m, C8±CH2,
C26±CH2, C29±CH2), 2.12±2.38 06H, t, C2±CH2, C11±
1549
[2M1Na]1,
727
[MNa2N0CH3)3]1ˆ243
CH2, C20±CH2), 3.20±3.45012H, s, N0CH ) 11OCH3),
3 3
[C0C6H5)3]1. m/z 748 [M2CH3]2 0100%), 677
[0M2CH3)2C4H9N]2.
3.45±3.75 04H, m, OCH2CH2O), 3.58 01H, m, C12±CH),
3.75±3.85 02H, m, glycerol-C3), 3.85±4.00 02H, m, ±CH 2±
N), 4.07 01H, dd, Jˆ12, 7 Hz, glycerol-C1±Hb), 4.20±4.35
02H, m, PO±OCH2), 4.3501H, dd, Jˆ12, 3 Hz, glycerol-
C1±Ha), 4.65±4.83 02H, m, O±CH2±O), 5.15 01H, m,
glycerol-C2), 5.25±5.50 04H, m, C27±CHv, C28±CHv,
C9±CHv, C10±CHv). m/z 912 [MNa]1, 1801
[2M1Na]1, 853 [MNa2N0CH3)3]1; m/z 874 [M2CH3]2,
803 [0M2CH3)2 C4H9N]2, 385[ricinol. O-MEM ac.-H]2.
3.4.3.
2-%Octadec-cis-9-enoyl)-sn-glycero-3-phospho-
choline %11). According to literature, deprotection and
acylation with O-MEM±ricinoleic acid anhydride could
be obtained in a one pot reaction.17 2.7 g 03.60 mmol) of
10 and 5.5 g 07.3 mmol) of O-MEM±ricinoleic acid
anhydride 0prepared from 5.6 g 014.5 mmol) of O-MEM±
ricinoleic acid 5a and 1.5g 07.3 mmol) of DCC in 50 mL of
CCl4) in CH2CL2 090 mL) were treated at 08C with BF3
etherate 03.8 mL). After 1 h the solution was neutralised
with 10 g of NaHCO3 suspended in 20 mL of water. Metha-
nol 040 mL) was added and the organic phase separated and
evaporated to dryness. The residue was dissolved in CHCl3
and puri®ed by silica gel chromatography. The product was
eluted ®rst with CHCl3/CH3OH/H2O 60:40:2 and then the
eluent composition was changed to 50:50:4. The recovered
product 0as a white waxy viscous paste) were 1.2 g
02.29 mmol, yield 63.6%) of 2-oleoyl-GPC 011) suggesting
that in these conditions deprotection was not followed by
the acylation reaction. [Found: C, 59.61; H, 10.39; N, 2.59;
P, 5.75. C26H53NO7P requires C, 59.73; H, 10.23; N, 2.68; P,
3.4.5. 1-%12-hydroxy-octadec-9-enoyl)-2-%octadec-cis-9-
enoyl)-sn-glycero-3-phosphocholine %13). The reaction
solution containing 12 01 g, 1.12 mmol) and pyridinium
p-toluenesulfonate 0PPTS, 1 g, 3.98 mmol) dissolved in
ethylmethylketone 0EMK 20 mL) was re¯uxed for 42 h.
After dilution with diethyl ether, the precipitated material
was ®ltered. The products were recovered after evaporation
of the solvent under reduced pressure, dissolved in chloro-
form and separated with silica gel chromatography. The
column was ®rst eluted with CHCl3/CH3OH/H2O 65:25:2
and then the water content was doubled. After evaporation
of the solvent, pure 1-ricinoleyl-2-oleoyl-GP 013), a waxy
white solid, was obtained 00.4 g, 0.498 mmol, yield 44.4%).
The overall yield was 10.3% from trityl-GPC 9. [Found: C,
64.68; H, 10.76; N, 1.68; P, 3.79. C44H8523NO9P requires C,
65.79; H, 10.67; N, 1.74; P, 3.86]. [a]D ˆ15.1 0cˆ0.63,
CHCl3). nmax 0neat) 3600±3100 [0OH)ass], 3006 [0vCH)],
2926 and 2854 [0CH2, CH3)], 1740 and 1731 [0CvO)ester
5.93]. nmax 0neat) 3005[0 vCH)], 2925and 2854 [0CH
,
2
CH3)], 1731 [0CvO)ester], 1467, 1232 [0PvO)], 1086 and
1059 [0P±O±C)], 970 [r0CH3)choline]. dH 0300 MHz,
CDCl3): 0.8503H, t, Jˆ6 Hz, C36±CH3), 1.05±1.45 020H,
m, C022±25)± CH2, C030±35)± CH2), 1.45±1.70 02H, m,
and 0CvO)ester overlap, 1466, 1242 [0PvO)], 1174 [0C±
O)ester], 1090 and 1066 [0P±O±C)choline and 0O±C±C)MEM],
969 [r0CH3)choline].
]
C21±CH2), 1.82±2.1504H, m, C26±CH , C29±CH2), 2.27
2
02H, t, Jˆ7.5Hz, C20±CH 2), 3.32 09H, s, N0CH3)31), 3.60±
3.86 02H, m, ±CH2±N), 3.86±3.90 02H, m, glycerol-C1),
4.00±4.1502H, m, glycerol-C3), 4.15±4.38 02H, m, PO±
OCH2), 4.38±4.60 01H, m, glycerol-C2), 5.20±5.45 02H, m,
C27±CHv, C28±CHv). m/z 544 [MNa]1, 1065
[2M1Na]1, 485[MNa 2N0CH3)3]1; m/z 506 [M2CH3],
435[0M 2CH3)2C4H9N]2.
3.4.4. 1-%12-O-MEM-octadec-9-enoyl)-2-%octadec-cis-9-
enoyl)-sn-glycero-3-phosphocholine %12). The reaction
mixture containing 11 01 g, 1.91 mmol), MEM-protected
ricinoleic acid 5a 02.5g, 6.5mmol), 4-pyrrolidinopyridine
01 g, 6.7 mmol) and DCC 01.35g, 6.55mmol, added slowly
in 30 min) in CH2Cl2 020 mL) was stirred for 6 h at room
temperature. After dilution with hexane, the suspension was
®ltered and the solvent evaporated. The residue was
dissolved in CHCl3/CH3OH/H2O 60:30:4 and puri®ed on a
column of Amberlist 15. The fractions containing the
dH 0300 MHz, CDCl3): 0.87 06H, t, Jˆ6.5Hz, C18±CH 3,
C36±CH3), 1.12±1.39 036H, m, C04±7)± CH2, C014±17)±
CH2, C022±25)± CH2, C030±35)± CH2), 1.39±1.49 02H, m,
C13±CH2), 1.49±1.68 04H, t, C3±CH2, C21±CH2), 1.87±
2.10 06H, m, C8±CH2, C26±CH2, C29±CH2), 2.18 02H, t,
Jˆ6.5Hz, C11±CH 2), 2.26 02H, t, Jˆ7 Hz, C20±CH2),
2.28 02H, t, Jˆ7 Hz, C2±CH2), 3.32 09H, s, N0CH3)3),

G. Borsotti et al. / Tetrahedron 57 ,2001) 10219±10227
10227
3.52±3.66 01H, m, C12±CH), 3.66±3.81 02H, m, CH2±N),
References
3.81±4.02 02H, m, glycerol-C3±Ha,b), 4.11 01H, dd, Jˆ12,
7 Hz, glycerol-C1±Hb), 4.18±4.33 02H, m, PO±CH2), 4.33
01H, dd, Jˆ12, 3 Hz, glycerol-C1±Ha); 5.18 01H, m,
glycerol-C2±H), 5.24±5.36 02H, m, C27±CHv, C28±
CHv), 5.36±5.62 02H, m, C9±CHv, C10±CHv). d13C
075.42 MHz, CDCl3) 14.06, 14.07 0C18, C36), 22.59,
22.64, 24,8502), 25.72, 27.17, 27.19, 27.31 08CH 2), 28.97,
29.01, 29.07, 29.13, 29.16, 29.22, 29.28, 29.29, 29.35,
29.50, 29.52, 29.7302) 013CH2), 31.83, 31.87, 34.09,
34.23, 35.36, 36.83 06CH2) 54.38 ppm 03), 59.34 0CH±
OH), 62.96 0CH2±N), 63.37 0glycerol), 66.350glycerol),
70.52 0glycerol), 71.35 0PO±CH2±), 125.50, 132.89
CH09)vCH010), 129.98, 129.66 CH027)vCH028),
173.14, 173.50 CO01), CO013). d31P 081.0 MHz, CDCl3)ˆ
0.74. m/z 824 [MNa]10100%), 1625[2M 1Na]1, 802
[MH]1, 765[MNa 259]1 059ˆN0CH3)3); m/z 800
[M2H]2, 786 [M2CH3]2, 715[R 2HPO4]2 0100%).
HRMS 03-NBA) MH1, found 803.6060; C44H86NO9P
requires 803.6040.
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Acknowledgements
This work has been carried out with the ®nancial support of
Á
the Ministero dell'Universita e della Ricerca Scienti®ca e
Tecnologica, Progetto Biogene.