First synthesis of natural phosphatidyl-β-d-glucoside

Article abstract of DOI:10.1016/j.tetlet.2008.04.036

Herein, we report the chemical synthesis of naturally occurring mammalian phosphatidyl-β-d-glucoside (PtdGlc), in order to confirm the proposed structure and to clarify its stereochemistry. We designed a convergent synthetic strategy, suitable to prepare

Full text of DOI:10.1016/j.tetlet.2008.04.036

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Tetrahedron Letters 49 (2008) 3562–3566
First synthesis of natural phosphatidyl-b-D-glucoside
Peter Greimel, Yukishige Ito *
RIKEN, The Institute of Physical and Chemical Research, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
Received 6 March 2008; revised 2 April 2008; accepted 7 April 2008
Available online 9 April 2008
Abstract
Herein, we report the chemical synthesis of naturally occurring mammalian phosphatidyl-b-D-glucoside (PtdGlc), in order to confirm
the proposed structure and to clarify its stereochemistry. We designed a convergent synthetic strategy, suitable to prepare sensitive
PtdGlc derivatives. As an initial demonstration of our strategy, we successfully prepared both PtdGlc diastereomers as well as its sen-
sitive arachidonyl analogue. The presence of both diastereomers in the natural sample was confirmed.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Phosphatidylglucoside; t-Butyl orthoester; H-phosphonate; Selective deprotection; Phospholipids
1. Introduction
anosyl bromide,5 trichloroacetimidate,6,7 or 1,2-(tert-Butyl
orthoacetate).7,8 On the other hand, Ramirez et al.9 took a
unique approach by first condensing benzylated glucose
with bis-(1,2-dimethylethenylene) pyrophosphate. Thus,
the resultant cyclic phosphate was linked with 1,2-di-O-
palmityl-sn-glycerol. However, the configuration of the
anomeric center was not determined and deprotection of
the final compound was not successful.
Enzymatic approaches have also been studied, using
phospholipase D from various vegetable10 or actinobacte-
ria.11,12 With vegetable enzyme, carbohydrates did not
act as acceptors. In contrast, phospholipase DA from Acti-
nomadura sp.11 catalysed transphosphorylation to various
carbohydrates, including glucose. Although the structural
analyses of the resulting carbohydrates have not been
reported, secondary evidence12 point toward primary
hydroxyl groups as phosphatidyl moiety acceptors.
Glucose containing phospholipids are rare in nature. In
1970, Short and White1 identified phosphatidylglucoside
(1, PtdGlc) in the lipid fraction of Staphylococcus aureus.
About 30 years later, the first isolation from mammalian
sources was reported by Nagatsuka et al.2,3 Recently,
PtdGlc was isolated from rat embryonic brain4 tissue, com-
prising a single molecular species containing exclusively
18:0/20:0 fatty acyl chains, rarely occurring in known
mammalian lipids. It was speculated, that this unusual
fatty acyl chain pattern might be responsible for its pres-
ence within distinct membrane microdomains on astroglial
cells. Furthermore, it was suggested that PtdGlc plays an
important role in glial cell development and differentiation.
However, the biological roles of PtdGlc in glial cells remain
largely unknown.
A variety of approaches toward chemical syntheses of
PtdGlc and its derivatives, which predominantly bear
palmityl (16:0) or stearyl (18:0) residues, have been
explored. Typically, the free acid or metal salt of the corre-
sponding phosphatidic acid derivative was condensed with
an activated carbohydrate moiety, such as a-D-glycopyr-
Our synthetic plan toward PtdGlc and its derivatives is
depicted in Scheme 1. In contrast to previous5–8
approaches, we decided to separately furnish the stereo-
chemically defined components, a b-glycopyranosyl H-
phosphonate unit and an enantiomerically pure glycerol
unit. The condensation of the glycerol and carbohydrate
units was expected to provide the desired phosphatidyl
glycerol. This approach was considered to be more suitable
to prepare sensitive PtdGlc derivatives. Since mammalian
*
Corresponding author. Tel.: +81 48 467 9430; fax: +81 48 462 4680.
E-mail address: yukito@riken.jp (Y. Ito).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.04.036

P. Greimel, Y. Ito / Tetrahedron Letters 49 (2008) 3562–3566
3563
prepared in an identical manner from 8 via 9 and 10. Diac-
ylglycerol derivatives are prone to ester migration during
prolonged reaction as well as during slow chromatographic
purification.14 Thus, short reaction times and rapid flash
chromatography over neutral silica gel were essential to
successfully suppress the acyl migration.
O
O
O
CH2
CH3
16
OH
O
O
P
O
HO
HO
O
CH2
CH3
18
O
O
Na
OH
1
O
OAc
O
Initially, preparation of arachidonate carrying glycerol
14 was attempted starting from 7. However, this approach
was abandoned, because conjugated double bonds were
not tolerant to DDQ treatment and TBDPS-protected 12
was employed as the starting material. After sequential
esterification, the TBDPS group was removed at 0 °C
within 1 h to give 14 (46% yield), with 50% of the starting
material recovered. More forcing conditions increase the
risk of the above-described ester migration considerably.
The glucose moiety 2 was prepared via a two-step
sequence from the corresponding peracetate 15 (Scheme
3). Firstly, 15 was converted to t-butyl orthoester 3 under
Lewis acid mediated conditions.15 The use of DMAP
instead of 2,6-lutidine gave superior yields of 3. Subse-
quently, autocatalytic reaction with phosphonic acid gave
the desired b-phosphonate 2.16
Linkage of the carbohydrate and glycerol moieties was
furnished in a unified manner with a ‘one-pot’ procedure
through pivalyl chloride-mediated H-phosphonate activa-
tion and subsequent oxidation.17 Iodine was used as an oxi-
dant, which was shown to be well-compatible with the
conjugated double bonds of the arachidonic acid residue,
giving 21 in a reasonable yield.
O
O
CH2 16 CH3
AcO
AcO
O
O
PH
O
OAc
O
CH2 18 CH3
HNEt3
OH
2
4
OAc
O
AcO
AcO
OH
O
O
O
O
3
5
O
Scheme 1. Synthetic strategy towards PtdGlc (1) and its derivatives.
PtdGlc was initially proposed to have a multiple unsatu-
rated arachidonic acid substituent, we selected an acetyl
(Ac) group for the protection of the hydroxyl groups.
Herein, we report the first unambiguous syntheses as
well as the structural confirmation of naturally occurring
mammalian PtdGlc.2–4
2. Syntheses
To begin with, glycerol moiety 4 was synthesized from
(S)-(+)-2,2-dimethyl-1,3-dioxolane-4-methanol (5; Scheme
2). Thus, protection with p-methoxybenzyl (PMB) group
was followed by the removal of isopropylidene group to
give 6.13 Compound 6 was esterified with stearic acid using
DCC to give monostearate 7 (63%) together with its regio-
isomer (8%) and distearate (10%). Further esterification
with arachidic acid, followed by DDQ-mediated removal
of the PMB protecting group, under carefully controlled
conditions, gave glycerol moiety 4. Its enantiomer 11 was
The final deprotection to yield compounds 17, 19, and
22 required the selective removal of acetyl groups under
conservation of the fatty acid esters. This seemingly chal-
lenging strategy was chosen in order to approach 22, which
corresponds to the initially proposed structure of PtdGlc,
carrying an arachidonic acid residue.2 In addition, b-phos-
phate diesters are poorly tolerant to acidic conditions. Ini-
tial attempts to employ a catalytic amount of sodium
O
O
OH
OPMB
OH
OH
OH
O
CH2 16 CH3
CH2 18 CH3
4
b
b
b
a
a
e
c, d
c, d
f, g
O
CH2
CH3
CH3
CH3
16
16
16
O
O
O
O
O
O
O
O
O
OH
OH
OH
PMBO
PMBO
OH
OH
OH
OH
PMBO
PMBO
5
6
6
7
O
O
O
O
OH
OPMB
OH
CH2 16 CH3
CH2 18 CH3
11
O
CH2
OH
OH
OH
8
9
9
10
O
O
O
O
OH
OTBDPS
OH
CH2 16 CH3
CH2 CH2CH=CH
O
CH2
OH
OH
TBDPSO
OH
2
4
TBDPSO
5
12
12
13
O
14
Scheme 2. Syntheses of asymmetric glycerol units 4, 11 and 14. Reagents and conditions: (a) (1) PMBCl, NaH, TBAI, DMF; (2) Amberlite H+, CHCl3/
MeOH = 2:1 (60% for 6, 54% for 9); (b) DCC, DMAP, stearic acid, DCM (63% for 7, 67% for 10, 61% for 13); (c) DCC, DMAP, arachidic acid, DCM
(82% on route to 4, 93% on route to 11); (d) DDQ, DCM/H2O = 18:1 (88% for 4, 83% for 11); (e) (1) TBDPSCl, imidazole, DCM; (2) Amberlite H+,
CHCl3/MeOH = 2:1 (60%); (f) DCC, DMAP, arachidonic acid, DCM (94%); and (g) TBAF, AcOH, THF, 0 °C (46%).

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P. Greimel, Y. Ito / Tetrahedron Letters 49 (2008) 3562–3566
OAc
O
OAc
O
a
O
O
AcO
AcO
AcO
OAc
O
O
O
CH2 16 CH3
CH2 18 CH3
15
AcO3
a
OR
O
OAc
2 + 11
O
P
O
O
RO
RO
O
O
O
R'
OAc
O
OR
O
AcO
AcO
b
O
18 R=Ac; R'=HNEt3
19 R=H; R'=HNEt3
20 R=H; R'=Na
PH
b
c
OAc
HNEt3
2
O
O
c
O
O
CH2 16 CH3
CH2 CH3
O
a
OR
O
O
P
O
2 + 14
RO
RO
O
O
O
CH2 16 CH3
CH2 CH2CH=CH
O
O
18
OR
O
O
P
OR
R'
RO
RO
O
O
O
2
4
16 R=Ac; R'=HNEt3
17 R=H; R'=HNEt3
1 R=H; R'=Na
d
e
OR
O
HNEt3
21 R=Ac
22 R=H
b
Scheme 3. Syntheses of carbohydrate unit 2, linkage and selective
deprotection to yield PtdGlc (1). Reagents and conditions: (a) AlCl3,
DMAP, t-BuOH, CHCl3 (89%); (b) (i) H3PO3, THF, (ii) NEt3, 4 °C (55%);
(c) (i) 4, PivCl, pyridine, THF, 0 °C?rt, (ii) I2, pyridine/H2O (76%); (d)
N2H4/HOAc = 4:1, CHCl3/MeOH = 1:2.5 (42%); and (e) Dowex 50WX8,
CHCl3/MeOH = 2:1 (94%).
Scheme 4. Linkage and selective deprotection to yield PtdGlc derivatives
20 and 22. Reagents and conditions: (a) (i) PivCl, pyridine, THF, 0 °C?rt,
(ii) I2, pyridine/H2O (96% for 18, 51% for 21); (b) N2H4/HOAc = 4:1,
CHCl3/MeOH = 1:2.5 (67% for 19, 67% for 22); and (c) Dowex 50WX8,
CHCl3/MeOH = 2:1 (94%).
methoxide were futile, showing poor selectivity, while gua-
nidine/guanidinium nitrate18 exhibited somewhat better
selectivity. Most satisfactory was a modified hydrazine
mediated deacetylation protocol5a yielding the desired
products within 5 min in 42–67% yield. The exchange of
counter ion from triethylammonium to sodium via Dowex
50WX8 Na+ form column gave the desired 1 and its stereo-
isomer 20 in good yields. PtdGlc (119) and its derivatives
(17, 19, 20,20 and 2221) exhibited poor solubility in any
organic solvents, for example, chloroform, methanol,
DMSO, or ether, as well as in water, thus enabling NMR
measurements only in very dilute solutions (Scheme 4).
3. Structure confirmation
The presence of an arachidonyl moiety (22) in the natu-
ral sample, as initially proposed,2 could be ruled out, due to
the absence of vinylic proton signals around 5.4 ppm. In
the case of PtdGlc (1) and its diastereomer 20, proton
Figure 1. Comparison of NMR data from synthetic PtdGlc diastereomers 1 and 20, as well as from natural source4 (spectrum at the bottom)
in methanol-d4.

P. Greimel, Y. Ito / Tetrahedron Letters 49 (2008) 3562–3566
3565
6. (a) Schmidt, R. R.; Stumpp, M.; Michel, J. Tetrahedron Lett. 1982,
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2008–2013.
NMR spectra are virtually identical. The methylene group
bearing the primary fatty acid residue, designated sn-1/3b
and sn-1/3a, exhibited the most prominent difference in
the chemical shift of about 0.02 ppm, while the difference
between the anomeric signals was less substantial. Upon
comparison (see Fig. 1) with the previously published nat-
ural PtdGlc,4 similarity of the major constituent from the
natural sample with the synthetic compound 1 was readily
observed, confirming its stereochemistry. Intriguingly, the
minor constituent of the natural sample, about 15%, coin-
cides well with synthetic diastereomer 20, supporting the
hypothesis4 that natural PtdGlc is a mixture of both
diastereomers.
10. (a) Dawson, R. M. C. Biochem. J. 1967, 102, 205–210; (b) Yang, S. F.;
Freer, S.; Benson, A. A. J. Biol. Chem. 1967, 242, 477–484.
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Biotechnol., Biochem. 1993, 57, 1302–1305.
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1987, 28, 199–202; (b) Nagao, A.; Ishida, N.; Terao, J. Lipids 1991,
26, 390–394.
4. Conclusion
13. Schlueter, U.; Lu, J.; Fraser-Reid, B. Org. Lett. 2003, 5, 255–
257.
Diastereomerically pure PtdGlc and its derivatives were
prepared by linkage of b-configured H-phosphonate and
asymmetric diacyl glycerol via a longest linear sequence
of 7 steps. The preparation of b-H-phosphonate was con-
ducted by the direct conversion of the corresponding
orthoester. Further investigations to diversify the carbohy-
drate as well as the glycerol moiety are currently underway
and will be reported in due course.
14. (a) Gaffney, P. R. J.; Reese, C. B. Tetrahedron Lett. 1997, 38, 2539–
2542; (b) Xu, Y.; Lee, S. A.; Kutateladze, T. G.; Sbrissa, D.; Shisheva,
A.; Prestwich, G. D. J. Am. Chem. Soc. 2006, 128, 885–897.
15. Bochkov, A. F.; Sokolovskaya, T. A.; Kochetkov, N. K. Izv. Akad.
Nauk SSSR Khim. 1968, 7, 1570–1575.
16. Utkina, N. S.; Mal’tsev, S. D.; Danilov, L. L.; Shibaev, V. N. Bioorg.
Khim. 1995, 21, 376–381.
17. Nikolaev, A. V.; Ivanov, I. A.; Shibaev, V. N.; Ignatenko, A. V.
Bioorg. Khim. 1991, 17, 1550–1561.
Comparison of 1H NMR data of PtdGlc (1) and its dia-
stereomer 20 suggested the presence of both diastereomers
in the natural sample, obtained from rodent brain.4 As
expected, sn-1,2-di-O-acyl PtdGlc (1) represents the major
compound in the natural sample.
18. Ellervik, U.; Magnusson, G. Tetrahedron Lett. 1997, 38, 1627–1628.
19. Spectroscopic properties of PtdGlc (1): 1H NMR (600 MHz, MeOH-
d4, 35 °C): d = 5.25–5.22 (m, 1H; sn-2), 4.84 (dd, 3J (H,P) = 7.6 Hz,
J = 7.6 Hz, 1H; H-1), 4.46 (dd, J = 3.0 Hz, J = 12.1 Hz, 1H; sn-1b),
4.18 (dd, J = 6.6 Hz, J = 12.1 Hz, 1H; sn-1a), 4.08 (ddd, J = 5.7 Hz,
3J (H,P) = 5.7 Hz, J = 11.3 Hz, 1H; sn-3b), 4.04 (ddd, J = 5.5 Hz, 3J
(H,P) = 5.5 Hz, J = 11.1 Hz, 1H; sn-3a), 3.84 (dd, J = 2.0 Hz,
J = 12.1 Hz, 1H; H-6b), 3.64 (dd, J = 6.0 Hz, J = 12.1 Hz, 1H; H-
6a), 3.37 (dd, J = 9.1 Hz, J = 9.1 Hz, 1H; H-3), 3.35–3.34 (m, 1H; H-
5), 3.26 (dd, J = 9.6 Hz, J = 9.6 Hz, 1H; H-4), 3.22 (dd, J = 8.1 Hz,
J = 9.1 Hz, 1H; H-2), 2.32 (t, J = 7.3 Hz, 2H; Stea-2), 2.30 (t,
J = 7.6 Hz, 2H; Ara-2), 1.63–1.56 (n.r., 4H; Stea-3, Ara-3), 1.28 (s,
60H; Stea-4-17, Ara-4-19), 0.89 (t, J = 7.1 Hz, 6H; Stea-18, Ara-20);
31P NMR (240 MHz, MeOH-d4, 35 °C): d = À0.70 (1P; P); HRMS
(ESI-TOF, neg) calcd for C47H90O13NaP [MÀNa]À: 893.6119, found:
893.6075.
20. Spectroscopic properties of 20: 1H NMR (600 MHz, MeOH-d4,
35 °C): d = 5.25–5.22 (m, 1H; sn-2), 4.84 (dd, 3J (H,P) = 7.1 Hz,
J = 7.6 Hz, 1H; H-1), 4.44 (dd, J = 3.0 Hz, J = 12.1 Hz, 1H; sn-3b),
4.20 (dd, J = 7.1 Hz, J = 12.1 Hz, 1H; sn-3a), 4.07 (ddd, J = 5.7 Hz,
3J (H,P) = 5.7 Hz, J = 11.6 Hz, 1H; sn-1b), 4.04 (ddd, J = 5.7 Hz, 3J
(H,P) = 5.7 Hz, J = 11.3 Hz, 1H; sn-1a), 3.84 (dd, J = 2.0 Hz,
J = 12.1 Hz, 1H; H-6b), 3.65 (dd, J = 5.5 Hz, J = 12.1 Hz, 1H; H-
6a), 3.37 (dd, J = 9.1 Hz, J = 9.1 Hz, 1H; H-3), 3.34–3.34 (m, 1H; H-
5), 3.25 (dd, J = 9.6 Hz, J = 9.8 Hz, 1H; H-4), 3.22 (dd, J = 8.1 Hz,
J = 9.1 Hz, 1H; H-2), 2.32 (t, J = 7.1 Hz, J = 7.6 Hz, 2H; Stea-2),
2.30 (t, J = 7.1 Hz, 2H; Ara-2), 1.62–1.57 (n.r., 4H; Stea-3, Ara-3),
1.28 (s, 60H; Stea-4-17, Ara-4-19), 0.89 (t, J = 6.8 Hz, 6H; Stea-18,
Ara-20); 31P NMR (240 MHz, MeOH-d4, 35 °C): d = À0.70 (1P; P);
HRMS (ESI-TOF, neg) calcd for C47H90O13NaP [MÀNa]À:
893.6119, found: 893.6117.
Acknowledgments
This research was supported by JSPS and a Grant-in-
Aid from the Ministry of Education, Science, Culture,
Sports, and Technology in Japan. The authors thank Ms.
Akemi Takahashi for technical assistance and Dr. Hiro-
yuki Koshino for acquiring high field NMR.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tetlet.2008.
04.036.
References and notes
1. Short, S. A.; White, D. C. J. Bacteriol. 1970, 104, 126–132.
2. Nagatsuka, Y.; Kasama, T.; Ohashi, Y.; Uzawa, J.; Ono, Y.; Shimizu,
K.; Hirabayashi, Y. FEBS Lett. 2001, 497, 141–147.
3. Nagatsuka, Y.; Hara-Yokoyama, M.; Kasama, T.; Takekoshi, M.;
Maeda, F.; Ihara, S.; Fujiwara, S.; Ohshima, E.; Ishii, K.; Kobayashi,
T.; Shimizu, K.; Hirabayashi, Y. PNAS 2003, 100, 7454–7459.
4. Nagatsuka, Y.; Horibata, Y.; Yamazaki, Y.; Kinoshita, M.; Shinoda,
Y.; Hashikawa, T.; Koshino, H.; Nakamura, T.; Hirabayashi, Y.
Biochemistry 2006, 45, 8742–8750.
5. (a) Verheij, H. M.; Smith, P. F.; Bonsen, P. P. M.; Van Deenen, L. L.
M. Biochim. Biophys. Acta 1970, 218, 97–101; (b) Volkova, L. V.;
Luchinskaya, M. G.; Karimova, N. M.; Evstigneeva, R. P. Zh.
Obshch. Khim. 1972, 42, 1405–1408.
21. Spectroscopic properties of 22: 1H NMR (600 MHz, CDCl3/MeOH-
d4 = 2:1, 25 °C): d = 5.42–5.32 (n.r., 8H; Ara-DB), 5.28–5.25 (n.r.,
1H; sn-2), 4.85 (dd, J = 7.6 Hz, 3J (H,P) = 7.6 Hz, 1H; H-1), 4.46 (dd,
J = 3.0 Hz, J = 12.1 Hz, 1H; sn-1b), 4.20 (dd, J = 6.6 Hz,
J = 12.1 Hz, 1H; sn-1a), 4.09–4.04 (m, 2H; sn-3a/b), 3.88 (dd,
J = 2.5 Hz, J = 12.1 Hz, 1 H; H-6b), 3.67 (dd, J = 6.0 Hz,
J = 12.1 Hz, 1H; H-6a), 3.43 (dd, J = 9.1 Hz, J = 9.1 Hz, 1H; H-3),

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P. Greimel, Y. Ito / Tetrahedron Letters 49 (2008) 3562–3566
3.39 (ddd, J = 2.5 Hz, J = 6.6 Hz, J = 9.4 Hz, 1H; H-5), 3.31 (dd,
1.62–1.58 (m, J = 7.1 Hz, 2H; Stea-3), 1.40–1.35 (m, J = 7.3 Hz, 2H;
Ara-17), 1.33 (t, J = 7.6 Hz, 9H; NEt3), 1.27 (n.r., 32H; Stea-4-17,
Ara-18-19), 0.90 (t, J = 7.1 Hz, 3H; Ara-20), 0.89 (t, J = 7.1 Hz, 3H;
Stea-18); 31P NMR (240 MHz, CDCl3/MeOH-d4 = 2:1, 25 °C):
J = 9.1 Hz, J = 9.6 Hz, 1H; H-4), 3.30 (dd, J = 8.1 Hz, J = 9.1 Hz,
1H; H-2), 3.14-3.13 (n.r., 6H; NEt3), 2.86–2.81 (m, 6H; Ara-7,Ara-
10,Ara-13), 2.36 (t, J = 7.6 Hz, 2H; Ara-2), 2.31 (t, J = 7.6 Hz, 2H;
Stea-2), 2.13 (dt, J = 6.6 Hz, J = 7.1 Hz, 2H; Ara-4), 2.07 (dt,
J = 7.1 Hz, J = 7.6 Hz, 2H; Ara-16), 1.74–1.67 (m, 2H; Ara-3),
d = À0.66 (1P; P); HRMS (ESI-TOF, neg) calcd for C53H98NO13
P
[MÀHNEt3]À: 885.5493, found: 885.5463.