Synthesis of 1-lyso-2-palmitoyl-rac-glycero-3-phosphocholine and its regioisomers and structural elucidation by NMR spectroscopy and FAB tandem mass spectrometry

Article abstract of DOI:10.1016/S0040-4020(03)00282-5

Three regioisomers of a naturally occurring lysophosphatidylcholine were chemically synthesized from glycerol. The phosphocholine moiety of the molecule was introduced by sequentially reacting with ethylene chlorophosphite, bromine, water, and trimethyl amine. Removal of a silyl protecting group of the hydroxyl group in the glycerol backbone was achieved without any accompanying acyl migration in the final stage of the synthesis by using NBS in a dimethyl sulfoxide-water cosolvent system. Structures of all regioisomers were compared by NMR spectroscopy and FAB tandem mass spectrometry.

Full text of DOI:10.1016/S0040-4020(03)00282-5

Tetrahedron 59 (2003) 2921–2928
Synthesis of 1-lyso-2-palmitoyl-rac-glycero-3-phosphocholine and
its regioisomers and structural elucidation by NMR spectroscopy
and FAB tandem mass spectrometry
a
Young-Ah Kim,a Myoung-Soon Park,a Young Hwan Kimb and So-Yeop Han
,
,
*
*
aDepartment of Chemistry, Division of Molecular Life Sciences, and Division of Nano Sciences, Ewha Womans University,
Seoul 120-750, Republic of Korea
bProteome Analysis Team, Korea Basic Science Institute, Daejeon 305-806, Republic of Korea
Received 4 November 2002; revised 21 February 2003; accepted 21 February 2003
Abstract—Three regioisomers of a naturally occurring lysophosphatidylcholine were chemically synthesized from glycerol. The
phosphocholine moiety of the molecule was introduced by sequentially reacting with ethylene chlorophosphite, bromine, water, and
trimethyl amine. Removal of a silyl protecting group of the hydroxyl group in the glycerol backbone was achieved without any
accompanying acyl migration in the final stage of the synthesis by using NBS in a dimethyl sulfoxide–water cosolvent system. Structures of
all regioisomers were compared by NMR spectroscopy and FAB tandem mass spectrometry. q 2003 Elsevier Science Ltd. All rights reserved.
Lysophospholipids can be produced through the action of
phospholipase on cell membranes and they function as key
intermediates in diverse biological processes. Recently,
lysophospholipids were reported to exist in the marine
sponge Spirastrella abata1 and deer antler.2 These com-
pounds display inhibition of cholesterol biosynthesis in
Chang liver cells and also exhibit antifungal activity. In
addition, lysophosphatidylcholine plays an important role in
signal transduction pathway by mediating protein kinase
C3,4 and inhibition of platelet aggregation.5 Lyso-
phosphatidylcholine also stimulates the release of arachi-
donic acid in endothelial cells6 and polyamine synthesis in
vascular smooth muscle cells.7 As diverse properties of
phospholipase A1 were investigated in bovine and rat
brains8,9 as well as fresh bonito white muscle,10 the role of
1-lysophosphatidylcholine has drawn considerable atten-
tion. The 1-lysophosphatidylcholine bound to albumin
could be an efficient delivery form of fatty acid to the
brain, compared with unesterified fatty acid.11 In particular,
1-lysophosphatidylcholine is preferentially transferred over
2-lysophosphatidylcholine in vivo.9 In addition, 1-lyso-
phosphatidylcholine is more prone to reacylation12 and
transacylation10 than 2-lysophosphatidylcholine, and this
result is in good agreement with the predominance of
2-lysophosphatidylcholine in cells. Due to the interesting
biological activity of lysophospholipids, extensive studies
have resulted in semi-synthetic and chemical procedures of
the 2-lysophospholipids13 as well as their derivatives.14
However, no such studies have been reported for 1-lyso-
phospholipids. In this study, we synthesized 1-lyso-2-
palmitoyl-rac-glycero-3-phosphocholine 1 (lysoPC 1) as
well as two regioisomers, 1-palmitoyl-2-lyso-rac-glycero-
3-phosphocholine 2 (lysoPC 2), and 1-lyso-3-palmitoyl-rac-
glycero-2-phosphocholine 3 (lysoPC 3), as shown in
Figure 1. After synthesis, the structures of 1–3 were
confirmed by NMR spectroscopy and FAB tandem mass
spectrometry. This study has demonstrated the development
of general synthetic protocols and structural elucidation of
Keywords: 1-lysophosphatidylcholine; acyl migration; regioisomer; NMR
(nuclear magnetic resonance); FAB-MS (fast atom bombardment mass
spectrometry); MS/MS (tandem mass spectrometry); CID (collision-
induced dissociation).
Figure 1. Structures of 1-lyso-2-palmitoyl-rac-glycero-3-phosphocholine 1
(lysoPC 1), 1-palmitoyl-2-lyso-rac-glycero-3-phosphocholine 2 (lysoPC
2), and 1-lyso-3-palmitoyl-rac-glycero-2-phosphocholine 3 (lysoPC 3)
with atom labeling used in NMR spectral data.
*
Corresponding authors. Tel.: þ82-2-3277-2382; fax: þ82-2-3277-2384;
e-mail: syhan@ewha.ac.kr; yhkim@kbsi.re.kr
0040–4020/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4020(03)00282-5

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Y.-A. Kim et al. / Tetrahedron 59 (2003) 2921–2928
all three possible regioisomers of lysophosphatidylcholine
and thus provides the tools for clear assignment of these
biologically important organic lysophosphatidylcholines.
1. Results and discussion
1.1. Synthesis
At first we envisioned synthesis of lysoPC 1 as one of three
target molecules. The key intermediates, 7a and 7b, were
prepared as outlined in Scheme 1. Synthesis began with
commercially available glycerol. The 1,3-diol of glycerol
was protected as 1,3-benzylideneacetal 4 by treatment with
p-TsOH and benzaldehyde. The remaining hydroxyl group
of 4 was treated with palmitic acid in the presence of DCC
and DMAP to afford ester 5, which was then deprotected by
using H2/Pd/C in EtOAc over the course of 3 days. The
resulting diol 6 was selectively monobenzylated with BnBr
and Ag2O15 to give two benzylated products, C2-hexa-
decanoate 7a and C3-hexadecanoate 7b. These regio-
isomers were chromatographically separated and the
starting material 6 was recovered. Structure of the desired
C2-hexadecanoate 7a was clearly distinguished from its
C2,C3-acyl group migrated byproduct 7b by comparison of
the chemical shifts at C1 and C3 of the glycerol backbone in
1H NMR spectra, as we previously reported for a
monoacetyldiglyceride system.16
Scheme 2.
identical with those of 2-lysophosphatidylcholine purchased
from Sigma.
To prevent acyl migration, we next chose a silyl protecting
group in place of a benzyl group at the C1 position
(Scheme 3). Diol 6 was selectively monosilylated by
treatment with TBDPSCl in the presence of imidazole to
afford compound 9 in 70% yield. The reaction was
conducted carefully and monitored closely by TLC in
order to obtain C1-monosilylated derivative as the major
product. Introduction of the phosphocholine moiety suc-
cessfully furnished 10 in good yield. We found that TBDMS
protecting group was not suitable for our synthesis, because
it was removed in the presence of aqueous trimethylamine
during phosphocholine formation. Desilylation method of
10 was investigated in depth to avoid acyl migration. It is
noteworthy to mention that desilylation was only successful
with NBS in DMSO/H2O to provide the desired compound,
lysoPC 1, according to the procedure reported by Johnson.18
The trace amount of byproduct could be removed success-
fully by silica-gel flash column chromatography. In
comparison, we found that the use of TBAF or HF–
pyridine to deprotect the TBDPS group gave exclusively
lysoPC 2.
Insertion of the phosphocholine moiety was achieved using
a four step sequence as shown in Scheme 2.17 Compound 7a
was treated with ethylene chlorophosphite in the presence of
DIEA in THF at 2208C to produce cyclic phosphite
intermediate. After ring opening with bromine and sub-
sequent hydrolysis, the resulting phosphoethanolamine was
quaternized with 40% aqueous trimethylamine in CHCl3/
iPrOH/CH3CN to afford compound 8 in good yield (78%
from 7a). Interestingly, we found that catalytic hydrogen-
ation of compound 8 proceeded exclusively with
unexpected acyl migration. Thus, our second target, lysoPC
2, was obtained instead of lysoPC 1 in 97% yield from 8.
Both the proton and carbon NMR spectra of lysoPC 2 were
Scheme 3.
Scheme 1.
Scheme 4.

Y.-A. Kim et al. / Tetrahedron 59 (2003) 2921–2928
2923
The third target, lysoPC 3, was prepared from 7b via the
same synthetic procedure as for lysoPC 2 (Scheme 4).
Sequential reaction of 7b with ethylene chlorophosphite,
bromine, and water, followed by aqueous trimethylamine
gave 11 without accompanying phosphoryl migration.19
Subsequently, palladium-catalyzed hydrogenolysis of 11
afforded lysoPC 3.
1.2. Structural elucidation
We have previously determined the structure of the
2-lysophosphatidylcholine isolated from deer antler using
a combination of NMR spectroscopic and FAB tandem
mass spectrometric analysis.2 However, several spectral
analyses were not sufficient to provide a complete assign-
ment of carbons on the glycerol backbone. Thus, all three
regioisomers of lysoPCs 1–3 have been synthesized and
1
1
characterized by H, 13C, DEPT 135, H–1H COSY, and
1H–13C COSY NMR as well as FAB tandem mass
spectrometric methods. These data were used to establish
a relationship to the regiospecificity of the fatty acyl group
and the polar head group on the glycerol backbone of
lysophosphatidylcholine.
1.2.1. NMR spectroscopic analysis. The 1H and 13C NMR
spectra of lysoPCs 1–3 are shown in Figure 2. LysoPC 1
contains palmitic acid at the C2 position and phospho-
choline at the C3 position of the glycerol backbone, whereas
lysoPC 2 and lysoPC 3 contain palmitic acid at C1 in
addition to phosphocholine at C3 and C2, respectively. The
fatty acyl group could be readily distinguished as being in
1
the upfield region in the H (Fig. 2(a)–(c)) and 13C NMR
(Fig. 2(d)–(f)) spectra on the basis of previous studies.2,20
Additionally, lysoPCs 1–3 displayed several characteristic
signals, belonging to the glycerol backbone and polar head
group. As a representative example, the methyl protons Hf
on the polar head group of lysoPC 1 displayed a signal at
3.23 ppm, whereas the methylene protons Hd and He were
observed at 4.28 and 3.67 ppm. For the remaining peaks, we
assumed that the peaks at 3.67 and 3.96 ppm correspond to
the methylene protons, Ha and Hc, and the peak at 5.00 ppm
to the methine proton Hb on the glycerol backbone
(Fig. 2(a)). Three carbons Cd, Ce, and Cf attached directly
to the polar head group protons displayed carbon signals at
60.7, 67.7, and 55.0 ppm based on the 1H–13C COSY
experiment. The methine carbon Cb at 74.9 ppm was
correlated to the proton Hb at 5.00 ppm. The two remaining
peaks at 61.5 and 65.0 ppm were assigned as the backbone
carbons, Ca and Cc, which were associated with the protons,
Ha and Hc, at 3.67 and 3.96 ppm, respectively (Fig. 2(d)).
Further analysis of the 13C NMR spectrum of lysoPC 1 in
comparison with those of its regioisomers, lysoPCs 2 and 3,
in detail enabled us to fully assign all carbon signals
corresponding to protons. The 1H and 13C NMR data of their
representative regions of lysoPCs 1–3 are listed in Tables 1
and 2, respectively. An important issue in the NMR analyses
was the effect of the specific position of the fatty acid on the
glycerol backbone on chemical shift in order to confirm the
structures of synthetic lysoPCs. In the 1H NMR of lysoPC 1,
the fatty acyl chain at the C2 position caused the downfield
shift of Hb from 3.97 to 5.00 ppm compared with that of
lysoPC 2, as well as the upfield shift of Ha from 4.14 to
3.69 ppm (Table 1). In addition, we found that structural
Figure 2. 1H NMR spectra (250 MHz, CD3OD) of (a) lysoPC 1 (b) lysoPC
2 (c) lysoPC 3, and 13C NMR spectra (63 MHz, CD3OD) of (d) lysoPC 1
(e) lysoPC 2 (f) lysoPC 3.
1
Table 1. H NMR spectral data for the representative regions on lysoPCs
1–3 (250 MHz, CD3OD)
Assignment
Chemical shifts (d)
1
2
3
a
b
c
d
e
f
3.67
5.00
3.96
4.28
3.67
3.23
4.14
3.97
3.89
4.30
3.65
3.23
3.70
4.31
4.26
4.31
3.70
3.23
Structures are drawn in Figure 1.

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Y.-A. Kim et al. / Tetrahedron 59 (2003) 2921–2928
Table 2. 13C NMR spectral data for the representative regions on lysoPCs
1–3 (63 MHz, CD3OD)
existence of carbon–nitrogen coupling, thus distinguishing
the carbon Ce from the carbon Cd. The 13C NMR spectra of
lysoPCs 1 and 2 showed identical coupling patterns.
However, careful investigation of the spectra suggested
that the relative changes of chemical shifts in lysoPC 1 and
lysoPC 2 demonstrated the deshielding contribution22 from
the fatty acyl groups at C1 and C2 positions. The 13C NMR
spectrum of lysoPC 1 showed the signal of the backbone
carbon Cb at 74.9 ppm shifted downfield in comparison with
that of lysoPC 2 at 70.1 ppm. Therefore, by combining this
information we could confirm the structures of lysoPCs
1–3.
Assignment
Chemical shifts (d, mult.a, Jb)
1
2
3
a
b
c
d
e
f
61.5 (s)
66.5 (s)
63.3 (d, 4.4)
76.1 (d, 6.3)
64.9 (d, 5.0)
60.8 (d, 5.0)
67.8 (m)
74.9 (d, 8.2)
65.0 (d, 5.0)
60.7 (d, 4.4)
67.7 (m)
70.1 (d, 7.6)
68.1 (d, 5.0)
60.7 (d, 4.4)
67.7 (m)
55.0 (m)
55.0 (m)
55.0 (m)
Structures are drawn in Figure 1 and spectra in Figure 3.
a
Singlet, s; doublet, d; multiplet, m.
The C–P coupling constants.
b
Based on these observations, we suggest that the substituent
pattern affects the 1H and 13C chemical shifts of the glycerol
backbone, but not the 13C chemical shifts of the polar head
group.
changes in carbon skeleton of the glycerol backbone did not
affect the chemical shifts of the polar head group protons
(Hd, He and Hf) as they consistently appeared in the spectra
of compounds 1–3 (Table 1).
1.2.2. FAB tandem mass spectrometric analysis. The
FAB tandem mass spectra provide useful structural
information based on understandable fragmentation
patterns. LysoPC 1 was subjected to FAB-MS in combi-
nation with tandem mass spectrometry (MS/MS) and
compared to that of regioisomers, lysoPCs 2 and 3. FAB
mass spectrum showed the prominent peak at m/z 518
corresponding to the sodium-adducted molecule [MþNa]þ
In the 13C NMR spectra, characteristic couplings between
carbon and phosphorus as well as carbon and nitrogen were
observed. The corresponding segments of spectra are shown
in Figure 3. Analysis of these coupling patterns played a
crucial role in the structural elucidation. For lysoPCs 1 and 2
(Fig. 3(a) and (b)), the two-bond carbon–phosphorus
couplings involved the methylene carbon Cc of the glycerol
backbone and the methylene carbon Cd of the polar head
group. On the other hand, the backbone carbon Cb and the
polar head group carbon Ce exhibited the three-bond
carbon–phosphorus couplings. For lysoPC 3 (Fig. 3(c)),
the methine carbon Cb of the glycerol backbone and the
methylene carbon Cd of the polar head group exhibited the
two-bond carbon–phosphorus couplings, whereas the back-
bone carbons, Ca and Cc, and the polar head group carbon Ce
displayed the three-bond carbon–phosphorus couplings.
Therefore, lysoPCs 1 and 2 exhibited a sharp singlet for
carbon Ca in 13C NMR spectra. In comparison, lysoPC 3
showed a doublet for carbon Ca with a 3Jcp coupling value of
4.4 Hz. The Jcp coupling constants are given in Table 2. In
2
case of lysoPCs 1 and 2, the Jcp coupling values were
3
smaller than the Jcp, in agreement with the literature
values.21 An extraordinary coupling pattern of the carbon Ce
in the polar head group of lysoPCs 1–3 demonstrated the
Figure 3. 13C NMR spectra (63 MHz, CD3OD) of the glycerol backbone
and phosphocholine region of (a) lysoPC 1 (b) lysoPC 2 (c) lysoPC 3.
Figure 4. Positive-ion FAB tandem mass spectra of [MþNa]þ ion of
(a) lysoPC 1 (b) lysoPC 2 (c) lysoPC 3, observed at m/z 518.

Y.-A. Kim et al. / Tetrahedron 59 (2003) 2921–2928
2925
(not shown). It was proven that the collision-induced
dissociation (CID) of this sodium-adducted molecule
generated a wide variety of product ions. These ions
provide information about the regiospecificity of fatty acyl
linkages on the glycerol backbone, as well as on fatty acid
composition and polar head group.23 The tandem mass
spectra (Fig. 4) of fragment ions produced by CID of
[MþNa]þ ions provided significant information about the
structures of lysoPCs 1–3. The product ions appearing
below m/z 250 are formed by the fragmentation of the
phosphocholine head group, supporting the presence of
phosphocholine moiety. There are [CH2vNMe2]þ (m/z 58),
[CH2vCHNMe3]þ (m/z 86), [HOCH2CH2NMe3]þ (m/z
104), and [HOP(O)(ONa)OCH2CH2NMe3]þ (m/z 206). An
intense peak was also observed at m/z 147, which
corresponds to the sodiated five-membered cyclo-
phosphane. In addition, the neutral losses of trimethylamine,
dehydrocholine and choline resulted in the ge0ne0rations of
sodiated 1-acyl- or 2-acyl-sn-glycero-3-cyclo(1 ,2 -ethylene
glycol)phosphodiester (i.e. [MþNa259]þ at m/z 459),
sodiated 1-acyl- or 2-acyl-sn-glycerol-3-phosphate (i.e.
[MþNa285]þ at m/z 433) and sodiated monoacyl glycero-
phosphodiester (i.e. [MþNa2103]þ at m/z 415), respect-
ively. These product ions were also observed in their
electrospray ionization (ESI) tandem mass spectra reported
previously.24 The structure of the fatty acyl group can be
determined from analysis of the spectral pattern of the
homologous ions formed by charge-remote fragmentation
occurring along the long chain of fatty acyl group.23 The
palmitoyl group was confirmed by separation of 14 Da
between the neighboring peaks due to the neutral loss of
CnH2nþ2 along the fatty acyl chain from the alkyl-terminus.
lysoPC from glycerol. During the synthesis of the first target
molecule, lysoPC 1, we were unexpectedly confronted with
acyl migration within the molecule. It was found that the
benzyl protecting group was not suitable for the C1
hydroxyl group of the glycerol backbone because catalytic
hydrogenolysis of the benzyl moiety resulted in only
unexpected acyl-migrated product 2. Thus, the second
target molecule, lysoPC 2, was easily obtained from
glycerol in nine steps and 12% overall yield. However,
this acyl migration was eliminated by use of TBDPS
protecting group as well as careful deprotection of the silyl
group with NBS in DMSO/H2O in the last step. This
afforded lysoPC 1 in nine steps and overall 15% yield.
Therefore, the choice of protecting group played a major
role in the synthetic strategy. The third target molecule,
lysoPC 3, was also synthesized in a similar manner in
overall 9% yield. Structures of all three isomers were
confirmed by spectroscopic analysis. We determined the
carbon–phosphorus and carbon–nitrogen coupling patterns
were important criteria for complete assignment of the 13C
NMR spectra. The two and three bond C–P coupling and
the shield effect both contributed to the coupling pattern and
chemical shift differences between lysoPC 1 and lysoPC 2.
Additionally, the FAB tandem mass spectra were highly
informative for structural elucidation. In particular, the CID
of sodium-adducted molecules generated product ions,
which were informative about the regiospecificity of the
fatty acyl group on the glycerol backbone.
These results provide valuable information for the
preparation and structural elucidation of the lyso-
phosphatidylcholine as well as other lysophospholipids in
order to study their biochemical mechanism of action.
On the other hand, several product ions informative on the
regiospecificity of the fatty acyl group on the glycerol
backbone were observed in their tandem mass spectra.
Especially, the ratios of the peak intensities of specific
product–ion pairs from lysoPC 1 and lysoPC 2 were
significantly different (Fig. 4(a) and (b)). For example, the
peak intensity ratio of product ions at m/z 104 and 147
differed by about tenfold in individual regioisomers.
Furthermore, the product ions observed at m/z 415 and
433 in the MS/MS spectra of sodiated lysoPC 1 were less
abundant than that from sodiated lysoPC 2. These results are
consistent with previous data obtained from low-energy
CID of sodiated lysophosphocholine regioisomers produced
by electrospray ionization.24 In addition, in the MS/MS
spectrum of lysoPC 3 (Fig. 4(c)), two characteristic peaks
corresponding to [CH2vCHP(O)(ONa)OCH2CH2NMe3]þ
(m/z 232) and the fragment ion after the neutral loss of
palmitic acid (m/z 262) have greater abundance than those
from other regioisomers, lysoPCs 1 and 2.
3. Experimental
3.1. General
Melting points were determined on Fisher–Johns melting
point apparatus and are uncorrected. Optical rotation was
determined on a Jasco DIP-140 Digital Polarimeter. All
anhydrous reaction performed in the oven-dried glassware
under Ar condition. THF was distilled from sodium
benzophenone ketyl, while MeOH, CH2Cl2 and EtOAc
were distilled from calcium hydride. Benzaldehyde and
DMF were vacuum distilled. Ethylene chlorophosphite and
40% aqueous trimethylamine were obtained from Aldrich
Chemical Co. Br2 was purchased from Junsei Chemical Co.
Merck silica gel 60 (230–400 mesh) was used for flash
column chromatography. TLC was performed on Merck
silica gel 60 F-254 plates (0.25 mm). NMR spectra were
recorded on a Bruker DPX 250 FT NMR and chemical shifts
are expressed in parts per million (d) relative to trimethyl-
silane (TMS, 0 ppm), chloroform (CHCl3, 7.24 ppm for 1H
and 77.0 ppm for 13C) or methanol (CH3OH, 4.8 ppm for 1H
and 49.0 ppm for 13C) as internal reference. Peak assign-
Thus, the regiospecificity of the fatty acyl linkage in the
lysoPCs 1–3 can be easily determined from the ratios of the
peak intensities of these product ions observed in CID
spectra of sodium-adducted molecules of individual lyso-
phosphocholine regioisomers.
1
1
ments based on DEPT 135, H–1H COSY and H–13C
COSY experiments. IR spectra were recorded on a Bio-Rad
FTS 165 spectrometer. Fast atom bombardment mass
spectra and tandem mass spectra were obtained on JMS-
HX110/110A tandem mass spectrometer (JEOL, Tokyo,
Japan), a four-sector instrument of E1B1E2B2 configuration
2. Conclusions
We efficiently synthesized all three regioisomers of racemic

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Y.-A. Kim et al. / Tetrahedron 59 (2003) 2921–2928
described previously,25 using the positive-ion mode with a
3-nitrobenzyl alcohol (NBA) as the matrix. CID of the
sodium-adducted molecules selected with the first mass
spectrometer occurred in the collision cell located between
B1 and E2 and floated at 3.0 kV. The resultant product ions
were analyzed by the B/E linked scan method in the second
mass spectrometer. The collision gas, N2, was introduced
into the collision chamber at a pressure sufficient to reduce
the precursor ion signal by 70%.
silver(I) oxide (1.72 g, 5.46 mmol) and dropwise benzyl-
bromide (0.7 mL, 5.46 mmol) at room temperature. After
5 h, the mixture was filtered through a pad of celite and the
filtrate was concentrated. Chromatography on silica gel
(n-hexane/EtOAc, 5:1!4:1) gave 0.480 g (44%) of product
7a and 0.360 g (33%) of product 7b as a colorless oil.
1
Compound 7a. Rf 0.26 (n-hexane/EtOAc, 3:1); H NMR
(250 MHz, CDCl3) d 0.87 (m, 3H), 1.25 (m, 24H), 1.61 (m,
2H), 2.15 (m, 1H), 2.32 (t, J¼7.5 Hz, 2H), 3.69 (m, 3H),
4.23 (d, J¼7.5 Hz, 2H), 4.64 (m, 2H), 7.34 (m, 5H); 13C
NMR (63 MHz, CDCl3) d 14.8, 23.4, 25.6, 29.8, 30.0, 30.1,
30.2, 30.3, 30.4, 32.6, 34.9, 35.0, 62.7, 63.4, 72.8, 128.5,
128.6, 129.2, 138.5, 174.5; FTIR (KBr, cm21) 3448, 2926,
2854, 1739, 1457, 1114, 736; FABHRMS [MþNa]þ m/z
calcd for C26H44O4Na 443.3240, found 443.3132.
3.1.1. 1,3-Benzylideneglycerol (4). p-Toluenesulfonic acid
(2.06 g, 10.9 mmol) was added to the solution of glycerol
(10.0 g, 109 mmol) in benzaldeyde (110 mL, 1.09 mol). The
solution was stirred at room temperature for 48 h and
benzaldehyde was removed by distillation under reduced
pressure. The residue was purified by flash column
chromatography on silica gel (n-hexane/EtOAc, 7:1!6:1)
to gave 8.50 g (46%) of product 4 as a white solid: mp 70–
1
Compound 7b. Rf 0.31 (n-hexane/EtOAc, 3:1); H NMR
1
718C; Rf 0.32 (n-hexane/EtOAc, 1:1); H NMR (250 MHz,
(250 MHz, CDCl3) d 0.88 (m, 3H), 1.26 (m, 24H), 1.56 (m,
2H), 2.27 (t, J¼7.5 Hz, 2H), 3.28 (d, J¼2.2 Hz, 1H), 3.46
(m, 2H), 3.97 (m, 1H), 4.13 (m, 2H), 4.49 (s, 2H), 7.28 (m,
5H); 13C NMR (63 MHz, CDCl3) d 14.6, 23.1, 25.3, 29.6,
29.7, 29.8, 29.9, 30.1, 30.1, 30.1, 32.4, 34.5, 65.7, 69.1,
71.4, 73.8, 127.2, 128.2, 128.8, 138.2, 174.2; FTIR (KBr,
cm21) 2925, 2854, 1738, 1457, 1112, 735; FABHRMS
[MþH]þ m/z calcd for C26H45O4 421.3318, found
421.3320.
CDCl3) d 3.13 (d, J¼8.8 Hz, 1H), 3.62 (m, 1H), 4.15 (m,
4H), 5.55 (s, 1H), 7.38 (m, 3H), 7.49 (m, 2H); 13C NMR
(63 MHz, CDCl3) d 63.9, 72.2, 101.6, 125.8, 128.3, 129.1,
137.8; FTIR (KBr, cm21) 3273, 2860, 1158, 1092, 1015,
737.
3.1.2. 2-Palmitoyl-1,3-benzylideneglycerol (5). To a
solution of compound 4 (8.45 g, 49.8 mmol) in CH2Cl2
(250 mL) was added palmitic acid (15.3 g, 59.7 mmol) and
then was cooled to 08C. To this was added 1,3-dicyclo-
hexylcarbodiimide (12.3 g, 59.7 mmol) and 4-(N,N-
dimethylamino)pyridine (1.20 g, 9.95 mmol). The reaction
mixture was stirred at 08C for 1 h followed by stirring at
room temperature for 3 h. The insoluble material was
filtered and the filtrate was concentrated. Chromatography
on silica gel (n-hexane/EtOAc, 14:1!10:1) gave 16.7 g
(82%) of product 5 as a white solid: mp 53–548C; Rf 0.41
(n-hexane/EtOAc, 3:1); 1H NMR (250 MHz, CDCl3) d 0.88
(m, 3H), 1.25 (m, 24H), 1.67 (m, 2H), 2.44 (t, J¼7.5 Hz,
2H), 4.23 (dd, J1¼12.4 Hz, J2¼30.7 Hz, 4H), 4.72 (s, 1H),
5.56 (s, 1H), 7.38 (m, 3H), 7.51 (m, 2H); 13C NMR
(63 MHz, CDCl3) d 14.1, 22.7, 24.9, 29.1, 29.2, 29.3, 29.5,
29.6, 29.7, 31.9, 34.4, 65.7, 69.1, 101.2, 126.0, 128.3, 129.1,
137.8, 173.9; FTIR (KBr, cm21) 3302, 2919, 2851, 1731,
1470, 1180, 1048, 720; FABHRMS [MþNa]þ m/z calcd for
C26H42O4Na 441.3083, found 441.2982.
3.1.5. 1-Benzyl-2-palmitoyl-rac-glycero-3-phospho-
choline (8). To a solution of compound 7a (0.400 g,
0.702 mmol) in THF (14 mL) at 2208C was added N,N-
diisopropylethylamine (0.5 mL, 2.81 mmol) and ethylene
chlorophosphite (0.2 mL, 2.11 mmol). The mixture was
stirred for 1 h and then Br2 (0.1 mL, 2.11 mmol) was added
at 2208C. After 15 min, H2O (2.5 mL) was added at room
temperature and then stirred for 1 h. To this was added NaCl
and then separated. The organic layer was dried over
Na2SO4 and concentrated. A solution of the residue in
CHCl3/iPrOH/CH3CN (v/v, 3/5/5, 14 mL) at 08C was added
40% aqueous trimethylamine (8.0 mL, 135 mmol). The
reaction mixture was stirred at 08C for 1 h followed by
stirring at room temperature for 12 h. The mixture was
concentrated, and lyophilized. Chromatography on silica gel
(CH2Cl2/MeOH, 3:1!0:1) gave 0.420 g (78% from 7a) of
product 8 as a white solid: Rf 0.31 (CH2Cl2/MeOH/H2O,
2:1:0.2); 1H NMR (250 MHz, CD3OD) d 0.90 (m, 3H), 1.28
(m, 24H), 1.58 (m, 2H), 2.32 (t, J¼7.3 Hz, 2H), 3.14 (s, 9H),
3.52 (m, 2H), 3.87 (m, 1H), 4.00 (m, 2H), 4.21 (m, 3H), 4.34
(m, 1H), 4.67 (s, 2H), 7.32 (m, 5H); 13C NMR (63 MHz,
CD3OD) d 14.8, 24.1, 26.3, 30.5, 30.7, 30.8, 30.9, 31.1,
33.4, 35.3, 54.9, 60.7 (d, Jcp¼5.0 Hz), 64.7, 66.0 (d,
Jcp¼5.6 Hz), 67.7, 73.4, 78.0 (d, Jcp¼8.0 Hz), 129.1,
129.4, 129.7, 140.1, 175.5; FTIR (KBr, cm21) 3394,
2928, 2856, 1732, 1468, 1266, 1090, 970, 743; FABHRMS
[MþH]þ m/z calcd for C31H57NO7P 586.3872, found
586.3869.
3.1.3. 2-Palmitoylglycerol (6). To a solution of compound
5 (3.90 g, 9.56 mmol) in EtOAc was added 10% Pd–C
(0.800 g) and the mixture was stirred at a room temperature
for 72 h under a hydrogen atmosphere. The reaction mixture
was filtered through celite and concentrated to gave 2.74 g
(98%) of product 6 as a white solid: mp 59–608C; Rf 0.4
(n-hexane/EtOAc, 1:2); 1H NMR (250 MHz, CDCl3) d 0.88
(m, 3H), 1.26 (m, 24H), 1.66 (m, 2H), 2.06 (t, J¼6.1 Hz,
2H), 2.38 (t, J¼7.5 Hz, 2H), 3.84 (m, 4H), 4.93 (m, 1H); 13C
NMR (63 MHz, CDCl3) d 14.1, 22.7, 25.0, 29.1, 29.2, 29.3,
29.4, 29.6, 29.7, 31.9, 34.3, 62.6, 75.0, 174.1; FABHRMS
[MþNa]þ m/z calcd for C19H38O4Na 353.2770, found
353.2666.
3.1.6. 1-Palmitoyl-2-lyso-rac-glycero-3-phosphocholine
(2). To a solution of compound 8 (0.160 g, 0.270 mmol)
in MeOH (3.0 mL) was added 10% Pd–C (32.0 mg) and the
mixture was stirred at a room temperature for 13 h under a
hydrogen atmosphere. The reaction mixture was filtered
through celite and concentrated. Chromatography on silica
3.1.4. 1-Benzyl-2-palmitoyl-rac-glycerol (7a) and 1-ben-
zyl-3-palmitoyl-rac-glycerol (7b). To a solution of com-
pound 6 (1.45 g, 4.96 mmol) in CH2Cl2 (50 mL) was added

Y.-A. Kim et al. / Tetrahedron 59 (2003) 2921–2928
2927
gel (CH2Cl2/MeOH, 5:1!1:3!0:1) gave 0.520 g (97%) of
product 2 as an white solid: Rf 0.21 (CH2Cl2/MeOH/H2O,
2:1:0.2); 1H NMR (250 MHz, CD3OD) d 0.90 (m, 3H), 1.28
(m, 24H), 1.60 (m, 2H), 2.35 (t, J¼7.4 Hz, 2H), 3.23 (s, 9H),
3.65 (m, 2H), 3.89 (m, 2H), 3.97 (m, 1H), 4.14 (m, 2H), 4.30
(m, 2H); 13C NMR (63 MHz, CD3OD) d 14.8, 24.0, 26.3,
30.5, 30.8, 30.9, 31.1, 33.4, 35.2, 35.4, 55.0, 60.7 (d,
Jcp¼4.4 Hz), 66.5, 67.7, 68.1 (d, Jcp¼5.0 Hz), 70.1 (d,
Jcp¼7.6 Hz), 175.6; FTIR (KBr, cm21) 3242, 2918, 2851,
1736, 1468, 1241, 1092, 970; FABHRMS [MþH]þ m/z
calcd for C24H51NO7P 496.3403, found 496.3401.
3.1.10. 1-Benzyl-3-palmitoyl-rac-glycero-2-phospho-
choline (11). Compound 11 was prepared under the
procedure outlined above for compound 8 and was obtained
as a white solid (0.189 g, 46%): Rf 0.31 (CH2Cl2/MeOH/H2-
1
O, 2:1:0.2); H NMR (250 MHz, CD3OD) d 0.90 (m, 3H),
1.28 (m, 24H), 1.57 (m, 2H), 2.30 (t, J¼7.4 Hz, 2H), 3.14 (s,
9H), 3.50 (m, 2H), 3.68 (d, J¼5.2 Hz, 2H), 4.24 (m, 2H),
4.30 (m, 2H), 4.48 (m, 1H), 4.55 (s, 2H), 7.32 (m, 5H); 13C
NMR (63 MHz, CD3OD) d 14.7, 24.0, 26.3, 30.5, 30.7,
30.7, 30.9, 31.1, 33.4, 35.2, 55.0, 60.7 (d, Jcp¼5.0 Hz), 65.1
(d, Jcp¼5.0 Hz), 67.6, 71.3 (d, Jcp¼5.3 Hz), 74.2 (d,
Jcp¼5.5 Hz), 74.7, 129.1, 129.4, 129.8, 139.9, 175.4;
FTIR (KBr, cm21) 3423, 2924, 2853, 1735, 1458, 1233,
1088, 969, 749; FABHRMS [MþH]þ m/z calcd for
C31H57NO7P 586.3872, found 586.3873.
3.1.7. 1-tert-Butyldiphenylsilyl-2-palmitoyl-rac-glycerol
(9). To a solution of compound 6 (0.540 g, 1.85 mmol) in
DMF (20 mL) was added imidazole (0.300 g, 4.07 mmol)
and tert-butyldiphenylsilylchloride (0.5 mL, 2.04 mmol) at
08C, and the mixture was stirred for 15 min. Then it was
diluted with ether and washed with 5% HCl, brine and dried
over Na2SO4. After evaporation of the solvent, the residue
was purified by flash column chromatography on silica gel
(n-hexane/EtOAc, 14:1!12:1) to give gave 0.300 g (70%)
of product 9 as a colorless oil: Rf 0.39 (n-hexane/EtOAc,
5:1); 1H NMR (250 MHz, CDCl3) d 0.87 (m, 3H), 1.06 (m,
9H), 1.26 (m, 24H), 1.60 (m, 2H), 2.31 (m, 3H), 3.82 (m,
4H), 4.99 (m, 1H), 7.39 (m, 6H), 7.67 (m, 4H); 13C NMR
(63 MHz, CDCl3) d 14.1, 19.1, 22.7, 24.9, 26.7, 29.1, 29.2,
29.3, 29.4, 29.6, 29.7, 31.9, 34.3, 62.5, 63.0, 74.5, 127.7,
129.8, 132.9, 135.5, 173.7; FTIR (KBr, cm21) 3651, 2927,
2856, 1737, 1428, 1112, 740; FABHRMS [MþH]þ m/z
calcd for C35H57O4Si 569.4026, found 569.4028.
3.1.11. 1-Lyso-3-palmitoyl-rac-glycero-2-phospho-
choline (3). Compound 3 was prepared under the procedure
outlined above for compound 2 and was obtained as a white
solid (0.120 g, 90%): Rf 0.21 (CH2Cl2/MeOH/H2O,
1
2:1:0.2); H NMR (250 MHz, CD3OD) d 0.88 (m, 3H),
1.29 (m, 24H), 1.61 (m, 2H), 2.35 (t, J¼7.3 Hz, 2H), 3.23 (s,
9H), 3.66 (m, 2H), 3.70 (m, 2H), 4.26 (m, 2H), 4.31 (m, 3H);
13C NMR (63 MHz, CD3OD) d 14.8, 24.0, 26.3, 30.5, 30.7,
30.9, 31.1, 33.4, 35.2, 55.0, 60.8 (d, Jcp¼5.0 Hz), 63.3 (d,
Jcp¼4.4 Hz), 64.9 (d, Jcp¼5.0 Hz), 67.8, 76.1 (d,
Jcp¼6.3 Hz), 175.4; FTIR (KBr, cm21) 3400, 2921, 2844,
1736, 1463, 1227, 1065, 949; FABHRMS [MþH]þ m/z
calcd for C24H51NO7P 496.3403, found 496.3414.
3.1.8. 1-tert-Butyldiphenylsilyl-2-palmitoyl-rac-glycero-
3-phosphocholine (10). Compound 10 was prepared
under the procedure outlined above for compound 8 and
was obtained as a white solid (0.402 g, 78%): Rf 0.50
(CH2Cl2/MeOH/H2O, 2:1:0.2); 1H NMR (250 MHz,
CD3OD) d 0.79 (m, 3H), 0.94 (s, 9H), 1.16 (m, 24H), 1.49
(m, 2H), 2.22 (m, 2H), 3.09 (s, 9H), 3.50 (m, 2H), 3.77 (d,
J¼4.7 Hz, 2H), 3.94 (m, 2H), 4.11 (m, 2H), 5.08 (m, 1H),
7.33 (m, 6H), 7.58 (m, 4H); 13C NMR (63 MHz, CD3OD) d
14.8, 20.4, 24.0, 26.3, 27.6, 30.5, 30.7, 30.8, 30.9, 31.1,
31.1, 33.4, 35.5, 54.9, 60.7 (d, Jcp¼5.0 Hz), 64.3, 65.3 (d,
Jcp¼5.3 Hz), 67.6, 75.0 (d, Jcp¼8.3 Hz), 129.2, 131.3,
134.6, 137.0, 175.1; FTIR (KBr, cm21) 3346, 2927, 2856,
1735, 1429, 1247, 1069, 969; FABHRMS [MþH]þ m/z
calcd for C40H69NO7PSi 734.4581, found 734.4581.
Acknowledgements
We are grateful to the KOSEF (R14-2002-015-01002-0) and
Ministry of Health & Welfare (01-PJ1-PG3-21500-0042)
for financial support of this research. Y.-A. Kim and M.-S.
Park are recipients of the graduate fellowship of Brain
Korea 21 program.
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