Practical enantiospecific syntheses of lysobisphosphatidic acid and its analogues

Article abstract of DOI:10.1021/jo051894e

We describe a versatile, efficient, and practical method for the preparation of enantiomerically pure lysobisphosphatidic acid (LBPA), bisether analogues, and phosphorothioate analogues of LBPA from solketal. Phosphorylation of a protected sn-2-O-oleoyl g

Full text of DOI:10.1021/jo051894e

Practical Enantiospecific Syntheses of Lysobisphosphatidic Acid and
Its Analogues
Guowei Jiang, Yong Xu, and Glenn D. Prestwich*
Department of Medicinal Chemistry, The UniVersity of Utah, 419 Wakara Way, Suite 205,
Salt Lake City, Utah 84108-1257
gprestwich@pharm.utah.edu
ReceiVed September 8, 2005
We describe a versatile, efficient, and practical method for the preparation of enantiomerically pure
lysobisphosphatidic acid (LBPA), bisether analogues, and phosphorothioate analogues of LBPA from
solketal. Phosphorylation of a protected sn-2-O-oleoyl glycerol with 2-cyanoethyl bis(N,N-diisopropyl-
amino)phosphite, followed by oxidation and deprotection, generated the enantiomers of 2,2′-LBPA. The
corresponding phosphorothioate analogues were obtained by oxidation with sulfur. The (R,R) and (S,S)
enantiomers of both LBPA and phosphorothioate LBPA were synthesized from (S)- and (R)-solketal,
respectively. The ether analogue of (S,S)-lysobisphosphatidic acid (LBPA) and its enantiomer were
synthesized from the same enantiomer (S)-solketal by simply changing the sequence of deprotection
steps.
Introduction
is involved in cholesterol transport8 and receptor trafficking.7
LBPA has also been shown to be an antigen in the antiphos-
pholipid syndrome, a condition in which endosomal sorting and
multivesicular endosome formation is disrupted.9 The trafficking
defects observed in the cholesterol storage disorder Niemann-
Pick type C can be recapitulated by disruption of LBPA
function.10,11 As a result, versatile synthetic access to enantio-
merically and regiochemically defined LBPA and its analogues
will further physiological research in the treatment of antiphos-
pholipid syndrome lipid storage disorders.
Lysobisphosphatidic acid (LBPA) is a natural yet unusual
phospholipid found in most tissues and cell types.1,2 Although
LBPA represents less than 1% of the total phospholipid mass,3
increased LBPA titers have been found in several lipid disorders
and as the result of certain therapeutic drugs.4,5 Recent
biochemical and immunocytochemical studies have shown that
LBPA is highly enriched in late endosomes (≈15 mol %), but
is not detected in other subcellular compartments.6,7 This lipid
The naturally occurring LBPA has a peculiar and interesting
structurestwo acyl chains at each of the sn-2 positions of
* Address correspondence to this author. Phone: 801-585-9051. Fax: 801-
585-9053.
(1) Kobayashi, T.; Gu, F.; Gruenberg, J. J. Sem. Cell DeVel. Biol. 1998,
9, 517-526.
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G.; Gruenberg, J. Nature 1998, 392, 193-197.
(8) Kobayashi, T.; Beuchat, M.; Lindsay, M.; Frias, S.; Palmiter, R. D.;
Sakuraba, H.; Parton, R. G.; Gruenberg, J. Nature Cell Biol. 1999, 1, 133-
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(2) Amidon, B.; Brown, A.; Waite, M. Biochemistry 1996, 35, 13995-
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(4) Cochran, F. R.; Connor, J. R.; Roddick, V. L.; Waite, M. Biochem.
Biophys. Res. Commun. 1985, 130, 800-806.
(5) Brotherus, J.; Renkonen, O.; Fischer, W.; Hermann, J. Chem. Phys.
Lipids 1974, 13, 178-182.
(6) Kobayashi, T.; Beuchat, M.; Chevallier, J.; Makino, A.; Mayran, N.;
Escola, J.; Lebrand, C.; Cosson, P.; Kobayashi, T.; Gruenberg, J. J. Biol.
Chem. 2002, 277, 32157-32164.
(9) Matsuo, H.; Chevallier, J.; Mayran, N.; Blanc, I. L.; Ferguson, C.;
Faure, J.; Blanc, N. S.; Matile, S.; Dubochet, J.; Sadoul, R.; Parton, R. G.;
Vilbois, F.; Gruenberg, J. Science 2004, 303, 531-534.
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Mayran, N.; Faure, J.; Gruenberg, J. EMBO J. 2002, 21, 1289-1300.
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3253.
10.1021/jo051894e CCC: $33.50 © 2006 American Chemical Society
Published on Web 01/04/2006
934
J. Org. Chem. 2006, 71, 934-939

Syntheses of Lysobisphosphatidic Acid and Its Analogues
SCHEME 1. Synthesis of (R,R)-2,2′-Bisether-LBPA
glycerol backbones. However, this sn-2 acyl structure is very
labile during isolation or preparation conditions.12 Intramolecular
acyl migration, which is facilitated by both acidic and basic
conditions, results in an equilibrium between the 1-acyl- and
2-acyl-sn-glycerol 3-phosphates that favors the biologically
inactive 1-acyl isomer. The instability of 2-acyl-sn-glycerol thus
seriously compromises both isolation of the naturally occurring
species as well as purification of chemically synthesized
materials. The complex and fascinating structure and biology
of LBPA motivated the development of a facile and practical
synthetic route to LPBA and its analogues.
Replacement of acyl groups by alkyl chains in phospholipid
compounds has afforded many valuable analogues.13-15 For
example, we found that alkyl analogues of lysophosphatidic acid
(LPA) were equipotent with the natural acyl LPAs for three
G-protein coupled LPA receptors.16 Moreover, a strategic
substitution of acyl by alkyl chain can enhance biological
activity by altering pharmacokinetics and metabolism; the
resulting alkyl analogues are useful probes for determining the
mechanism of action. Since the alkyl chains cannot be hydro-
lyzed by phospholipase A,13 alkyl substitution can introduce
unexpected biological activity. Besides acyl chain hydrolysis,
the phosphate diester group at the LBPA could be hydrolyzed
in the presence of phosphatases.17 One common approach for
stabilization of phosphate diesters is the use of phosphothioate
analogues in place of the phosphates. Phosphorothioates thus
have potential applications for the selective manipulation of
fundamental cellular responses that could validate new thera-
peutic approaches for human diseases and elucidate cellular
signaling pathways.18 We decided to test the hypothesis that
bisether analogues of LBPA and phosphorothioate analogues
of LBPA might mimic the 2,2′-bisacyl-LBPA as a biological
ligand, but would lack the propensity to undergo intramolecular
acyl migration or phosphate hydrolysis.
A modular strategy for the expedient preparation of enan-
tiopure LBPA was described by using solketal and a protected
dichlorophosphite.12 This method demonstrated some advantages
of PIII relative to the PV chemistry commonly used in classical
phospholipid synthesis. In a preliminary communication, we
developed a general method for the preparation of migration-
resistant bisether LBPA analogues in which the commercially
available reagent 2-cyanoethyl bis(N,N-diisopropylamino)phos-
phite was employed for phosphorylation.19 Herein, we describe
the extension of this methodology to the multimilligram scale
synthesis of both enantiomers of 2,2′-bisacyl LPBA and the
corresponding phosphorothioate analogues. In addition, full
experimental details of the bisether LBPA analogues synthesis
are included.
the alkyl or acyl chains were installed early in the synthesis.
Second, a commercially available phosphatidylating reagent (2-
cyanoethyl-bis-N,N-diisopropylphosphordiamidite) was used to
introduce both glycerol backbones simultaneously. It would be
extremely difficult to install alkyl chain once the phosphate
moiety was constructed. Both enantiomers of LBPA and its
analogues could be synthesized from the same starting material,
S-solketal, by phosphorlyation of either the 1- or the 3-position
of glycerol backbone. Third, revealing the charged phosphodi-
ester of the enantiomeric LBPA and its analogues at the end of
the synthesis facilitated the purification of synthetic precursors.
Conventional silyl protection of the hydroxyl groups coupled
with the cyanoethyl ester protection of the phosphate was
selected as the most promising approach; all protecting groups
could thus be removed under mild conditions in a single final
step to give the desired LBPAs.
We selected S-solketal ((2S)-dimethyl-1,3-dioxolane-4-
methanol) as our chiral starting material. Using the phosphor-
amidite methodology, widely exploited in nucleic acid chem-
istry, the alcohol was efficiently phosphorylated by using a
variety of trivalent phosphorus reagents. The resulting phosphite
triester could be oxidized in situ to yield the corresponding
phosphate triester or phosphorothioate if desired. As shown in
Scheme 1, protection of (S)-1,2-O-isopropylidene-sn-glycerol
was performed with p-methoxybenzyl chloride (PMB-Cl) to give
PMB ether, which was transketalized (10 mol % of p-TsOH in
methanol) to the 1,2-diol in 83% isolated yield.20 After silylation
at the primary alcohol with tert-butyldimethylsilyl (TBDMS)
chloride,21 the secondary alcohol was alkylated with octadecenyl
((Z)-9-octadecen-1-yl) triflate in the presence of the hindered
base “proton sponge” (1,8-bis(dimethylamino)naphthalene)22 to
give ether 5. The octadecenyl triflate was prepared by a
Results and Discussion
The strategy for the synthesis of LBPA and its analogues
was designed on the basis of the following considerations. First,
(12) Chevallier, J.; Sakai, N.; Robert, F.; Kobayashi, T.; Gruenberg, J.;
Matile, S. Org. Lett. 2000, 2, 1859-1861.
(13) Tyagi, S. R.; Burnham, D. N.; Lambeth, J. D. J. Biol. Chem. 1989,
264, 12977-12982.
(14) Kondapaka, S. B.; Singh, S. S.; Dasmahapatra, G. P.; Sausville, E.
A.; Roy, K. K. Mol. Cancer Ther. 2003, 2, 1093-1103.
(15) Samadder, P.; Bittman, R.; Byun, H.; Arthur, G. J. Med. Chem.
2004, 47, 2710-2713.
(16) Xu, Y.; Tanaka, M.; Arai, H.; Aoki, J.; Prestwich, G. D. Bioorg.
Med. Chem. Lett. 2004, 14, 5323-5328.
(17) Lee, S.; Lynch, K. R. Biochem. J. 2005, 391, 317-323.
(18) Qian, L.; Xu, Y.; Hasegawa, Y.; Aoki, J.; Mills, G. B.; Prestwich,
G. D. J. Med. Chem. 2003, 46, 5575-5578.
(19) Jiang, G.; Xu, Y.; Falguie`res, T.; Gruenberg, J.; Prestwich, G. D.
Org. Lett. 2005, 7, 3877-3840.
(20) Chen, J.; Profit, A. A.; Prestwich, G. D. J. Org. Chem. 1996, 61, 1,
6305-6312.
(21) Qian, L.; Xu, Y.; Arai, H.; Aoki, J.; McIntyre, T. M.; Prestwich,
G. D. Org. Lett. 2003, 5, 4685-4688.
J. Org. Chem, Vol. 71, No. 3, 2006 935

Jiang et al.
SCHEME 3. Synthesis of (R,R)-2,2′-Bisoleoyl-LBPA
SCHEME 2. Synthesis of (S,S)-2,2′-Bisether-LBPA
modification to the literature protocol;23 specifically, the use of
2,6-lutidine in place of pyridine significantly increased the yield
by minimizing the N-alkylation of pyridine. Removal of the
PMB group with DDQ in wet CH2Cl2 (0.5% water in volume)
afforded the primary alcohol 6 in 65% yield without migration
of the alkyl group from the 2-position to the 3-position. Coupling
of two molecules of this alkyl glyceryl intermediate 6 with
2-cyanoethyl-bis-N,N-diisopropylphosphordiamidite in the pres-
ence of 1H-tetrazole followed by t-BuOOH oxidation gave the
fully protected LBPA precursor 7 in medium yield. However,
the use of the more reactive phosphatidylating reagent (2-
cyanoethyl-bis-N,N-diisopropylphosphordiamidite) gave a disap-
pointingly low yield (20%). The most frequently used reagent
for the deprotection of the TBS group is tetra(n-butyl)ammonium
fluoride, or TBAF. Since the cyanoethyl ester protective group
is base labile, the basicity of TBAF was harnessed to simulta-
neously deprotect the cyanoethyl ester and TBS groups. The
final deprotection was carried out in THF containing 10 equiv
of TBAF at room temperature overnight. The final product
(R,R)-2,2′-octadecenyl LBPA was readily purified on silica gel
with use of CH2Cl2 and methanol (10:1, v:v) as the eluent.
The enantiomeric (S,S)-2,2′-octadecenyl LBPA was prepared
from intermediate 5 as shown in Scheme 2. First, TBAF was
used to remove the TBS group and gave the 2R configuration
primary alcohol 9. The 2R configuration alcohol 9 reacted with
2-cyanoethyl-bis-N,N-diisopropylphosphordiamidite in the pres-
ence of 1H-tetrazole, and subsequently was oxidized by tert-
butyl hydrogen peroxide to give the fully protected (S,S)-LBPA
10 in high yield. Next, DDQ in wet CH2Cl2 (overnight, room
temperature) completely removed both PMB protective groups
to give the primary alcohol 11. Under basic aprotic conditions
in the presence of N,O-bis(trimethylsilyl)trifluoroacetamide,
deprotection of cyanoethyl ester occurred at room temperature
and without any side reactions to yield the final bisether LBPA
analogue 12.24 Both natural and unnatural enantiomers of LBPA
can thus be obtained in optically pure form from the (S)-solketal.
The routes are short and efficient and proceed in good overall
yields.
acylated with oleic acid in the presence of the DCC and DMAP
to give ester 13. Removal of the PMB group with DDQ in wet
CH2Cl2 afforded the primary alcohol 14 in 93% yield without
migration of the acyl group. Rapid elution with silica gel flash
chromatography was necessary. Although the sn-2 acyl glycerol
14 was reasonably stable, extended contact with silica gel
facilitated acyl chain migration. Coupling of two molecules of
this acyl glyceryl intermediate 14 with 2-cyanoethyl-bis-N,N-
diisopropylphosphordiamidite in the presence of 1H-tetrazole
followed by t-BuOOH oxidation gave the fully protected LBPA
precursor 15 in 63% yield.25
Since the migration of the acyl group from the 2-position to
the 3-position is facilitated under basic conditions, TBAF
neutralized with HOAc was employed for deprotection. As
expected, the TBS group and cyanoethyl ester protective group
were both removed. The final (R,R)-2,2′-bisoleoyl-LBPA 16 was
readily purified on silica gel with cold (0 °C) CH2Cl2:methanol
(6:1, v:v) as the eluent. No acyl migration was detectable by
proton NMR. Importantly, the LBPA generated and purified in
this fashion was the tetra-n-butylammonium salt, which was
stable to purification on silica gel without acyl migration. The
tetra-n-butylammonium LBPA salt was then converted to the
sodium or ammonium salt by passage through a Dowex sodium
or ammonium ion exchange column. The sodium and am-
monium LBPA salts were stable for further NMR analysis, and
optimal for biological studies. In this way, we have circumvented
the instability12 of acyl LBPA during its deprotection and
purification. The product was soluble in CH3OH and showed
1
clear H NMR signals, consistent with those reported previ-
ously.12 The acyl chain migration can be monitored and
determined accurately by proton NMR. For 2,2,-bisoleyl-LBPA,
the sn-2 proton has the distinct resonance at 4.9 ppm. If acyl
chain migration had occurred, the chemical shift of sn-2 proton
would move to 4.0 ppm. Similarly, the chemical shift of sn-1
proton would change significantly if acyl chain migration had
occurred.12
Coupling of two molecules of this acyl glyceryl intermediate
14 with 2-cyanoethyl-bis-N,N-diisopropylphosphordiamidite in
the presence of 1H-tetrazole followed by elemental sulfur
oxidation gave the fully protected phosphorothioate LBPA
precursor 17 in 75% yield.21 The phosphorothioate LBPA was
obtained in 63% yield after deprotection of TBS and cyanoethyl
groups by TBAF. The tetra-n-butylammonium salt was then
converted to the ammonium salt by ion exchange for spectro-
scopic and biological studies.
The syntheses of (S,S)-2,2′-bisoleoyl LBPA and its analogues
were designed by using the same strategy as the synthesis of
bisether LBPA. The acyl chain was installed instead of an alkyl
chain on the 3-O-TBS-1-O-PMB-sn-glycerol building block. As
shown in Scheme 3, 3-O-TBS-1-O-PMB-(2S)-sn-glycerol 4 was
(22) Heyes, J. A.; Niculescu-Duvaz, D.; Cooper, R. G.; Springer, C. J.
J. Med. Chem. 2002, 45, 99-114.
(23) Aoki, T.; Poulter, C. D. J. Org. Chem. 1985, 50, 5634-5636.
(24) Heeb, N. V.; Nambiar, K. P. Tetrahedron Lett. 1993, 34, 6193-
6196.
(25) Sekine, M.; Tsuruoka, H.; Iimura, S.; Kusuoku, H.; Wada, T. J.
Org. Chem. 1996, 61, 4087-4100.
936 J. Org. Chem., Vol. 71, No. 3, 2006

Syntheses of Lysobisphosphatidic Acid and Its Analogues
SCHEME 4. Synthesis of the Phosphorothioate Analogue 18
of (R,R)-2,2′-Bisoleoyl-LBPA
In summary, we have described a general and practical
enantiospecific method for the preparation of LBPA, its bisether
analogues, and its phosphorothioate analogue from commercially
available reagents. Our synthetic strategy utilized TBAF for
deprotection and neutralization of the final product, thereby
solving the problem of obtaining and purifying LBPA without
acyl migration. In addition, both enantiomers of bisether LBPA
were synthesized from the same starting material, S-solketal,
by phosphorlyation of either the 1- or the 3-position of the
glycerol backbone.
Experimental Section
SCHEME 5. Synthesis of the (S,S)-2,2′-Bisoleoyl-LBPA
3-O-tert-Butyldimethylsilyl-1-O-methoxybenzyl-(2S)-glyc-
erol (4). To an ice-cooled solution of 1.36 g (6.41 mmol) of 3-O-
methoxybenzyl-(2S)-glycerol and 0.98 g (14.2 mmol) of imidazole
in 5 mL of DMF was added 1.17 g (7.45 mmol) of tert-
butyldimethylsilyl chloride in 3 mL of DMF over 1 h. Then the
reaction mixture was kept at 4 °C overnight and stirred at room
temperature for 8 h. Next, 5 mL of water was added to quench the
reaction, and the mixture was extracted with 100 mL of ethyl
acetate/hexane ) 1/4 and 80 mL of diethyl ether. The combined
organic extracts were washed with ice-cold 0.5 N HCl, saturated
NaHCO3 solution, followed by a saturated NaCl solution. Solvent
was removed at reduced pressure and residue was purified by FC
(ethyl acetate/ hexane, 1/5, v/v) to afford a colorless oil 4 (1.52 g,
73%): Rf 0.20 (ethyl acetate/hexane, 1/5, v/v); [R]20D -6.25 (c 3.04,
MeOH); 1H NMR (CDCl3) δ 7.20 (m, 2H), 6.82 (m, 2H), 4.42 (s,
2H), 3.66-3.80 (m, 4H), 3.60 (m, 2H), 3.42 (m, 2H), 2.44 (s, 1H),
0.83 (s, 9H), 0.01 (s, 6H); 13C NMR (CDCl3) δ 159.2 (s), 130.1
(s), 129.3 (d, J ) 91.6 Hz), 113.8 (s), 73.1 (s), 70.7 (s), 70.6 (s),
64.0 (s), 55.2 (s), 25.83 (s), 18.24 (s), -5.45 (s); MS (CI) m/z 325.2
(M+ - 1). HRMS (CI) for C17H30O4Si (M+): found 326.1936, calcd
326.1913.
SCHEME 6. Synthesis of (S,S)-2,2′-Bisoleoyl-LBPA 23 and
Its Phosphorothioate Analogue 24
3-O-tert-Butyldimethylsilyl-1-O-methoxybenzyl-(2S)-O-((Z)-
9-octadecen-1-yl)-sn-glycerol (5). To a mixture of 4 (800 mg, 2.45
mmol), serachyl triflate (3.00 g, 7.20 mmol) and 1,8-bis(dimethyl-
amino)naphthalene (proton sponge, 1.54 g, 7.20 mmol) under argon
was added anhydrous distilled CH2Cl2 (12 mL). The yellow solution
was refluxed under argon for 48 h. CH2Cl2 was then removed under
reduced pressure to give dark brown oil, and hexane was added
(60 mL). Then, the mixture was sonicated to ensure dissolution of
product and filtered through a 1-in. bed of Celite 521, and the filtrate
was evaporated under reduced pressure to yield orange oil. Silica
gel FC (ethyl acetate/hexane, 1/100, v/v) gave 5 as a colorless oil
Initially, we attempted to synthesize the enantiomeric (S,S)-
2,2′-bisoleyl LBPA from intermediate 13 as shown in Scheme
5. First, TBAF was used to remove the TBS group and gave
the 2R configuration primary alcohol 19. The 2R configuration
alcohol 19 reacted with 2-cyanoethyl-bis-N,N-diisopropylphos-
phordiamidite in the presence of 1H-tetrazole, and subsequently
was oxidized by tert-butyl hydrogen peroxide to give the fully
protected (S,S)-LBPA 20 in high yield. The protective groups
were removed sequentially by TEA followed by DDQ. Unfor-
tunately, the deprotection of PMB with DDQ in wet CH2Cl2
proved problematic, as substantial acyl chain migration occurred
indicated by both TLC and NMR analysis. Deprotection of the
PMB group with BBr3 or TMSBr at low temperature (-78 to
-20 °C) failed and afforded a complicated mixture.26,27 From
these results, we determined that it was unsuitable for prepara-
tion of 2,2′-bisoleoyl-LBPA. Therefore, we circumvented this
problem for synthesis of (S, S)-2,2′-bisoleoyl-LBPA by starting
from (R)-solketal and following the same procedure as for (R,R)-
2,2′-bisoleoyl-LBPA.
(925 mg, 66%): Rf 0.23 (ethyl acetate/hexane, 1/15, v/v); [R]20
D
1
-1.28 (c 0.57, MeOH); H NMR (CDCl3) δ 7.21 (m, 2H), 6.82
(m, 2H), 5.32 (m, 2H), 4.42 (m, 2H), 3.75 (s, 3H), 3.60 (d, J ) 4.8
Hz, 2H), 3.52 (m, 3H), 3.42 (m, 2H), 1.96 (m, 4H), 1.50 (m, 2H),
1.16-1.36 (m, 24H), 0.83 (m, 12H), 0.01 (m, 6H); 13C NMR
(CDCl3) δ 159.1 (s), 130.5 (s), 129.9 (s), 129.8 (s), 129.2 (s), 113.7
(s), 79.5 (s), 73.0 (s), 70.6 (s), 69.7 (s), 70.6 (s), 62.9 (s), 55.2 (s),
35.6 (s), 31.9 (s), 30.1 (s), 29.8 (s), 29.7 (s), 29.6 (s), 29.5 (s), 29.5
(s), 29.3 (s), 29.3 (s), 29.2 (s), 27.2 (s), 26.1 (s), 25.9 (s), 22.7 (s),
18.2 (s), 14.1 (s), -5.4 (s), -5.5 (s); MS (CI) m/z 577.5 (M+ + 1).
HRMS (CI) for C35H64O4Si (M+ + 1): found 577.4631, calcd
577.4652.
3-O-tert-Butyldimethylsilyl-(2S)-O-((Z)-9-octadecen-1-yl)-sn-
glycerol (6). To a solution of glyceryl ether 5 (364 mg, 0.612 mmol)
in 10 mL of wet CH2Cl2 was added DDQ (290 mg, 1.27 mmol).
The mixture was stirred at room temperature overnight, diluted with
CH2Cl2, washed with 10% NaHCO3, dried over Na2SO4, concen-
trated, and purified on silica gel FC (ethyl acetate/hexane, 1/10,
Both the (S,S)-LBPA and phosphorothioate (S,S)-LBPA were
prepared following the same procedures but starting from
R-solketal as outlined in Scheme 6. The availability of both the
natural S,S (as the biologically active configuration) and the
unnatural R,R enantiomers (as experimental control) is crucial
for in vitro structure-function studies with LBPA.
(26) Imai, H.; Uehara, H.; Inoue, M.; Oguri, H.; Oishi, T.; Hirama, M.
Tetrahedron Lett. 2001, 2001, 6219-6222.
(27) Wakabayashi, T.; Mori, K.; Kobayashi, S. J. Am. Chem. Soc. 2001,
123, 1372-1375.
J. Org. Chem, Vol. 71, No. 3, 2006 937

Jiang et al.
v/v) to give compound 6 (188 mg, 65%): Rf 0.18 (ethyl acetate/
hexane, 1/10, v/v); [R]20D +1.11 (c 0.48, MeOH); 1H NMR (CDCl3)
δ 5.28 (m, 2H), 3.66 (m, 2H), 3.53 (m, 3H), 3.32-3.47 (m, 2H),
2.14 (s, 1H), 1.94 (m, 4H), 1.48 (m, 2H), 1.16-1.32 (m, 24H),
0.81 (m, 12H), 0.01 (s, 6H); 13C NMR (CDCl3) δ 129.9 (d, J ) 13
Hz), 79.7 (s), 70.4 (s), 63.0 (s), 62.8 (s), 31.9 (s), 30.1 (s), 29.8 (s),
29.7 (s), 29.7 (s), 29.6 (s), 29.5 (s), 29.5 (s), 29.4 (s), 29.3 (s), 29.2
(s), 27.2 (s), 26.1 (s), 25.8 (s), 22.7 (s), 18.1 (s), 14.1 (s), -5.5 (s),
-5.5 (s); MS (CI) m/z 457.2 (M+ + 1). HRMS (CI) for C27H56O3-
Si (M+): found 456.3983, calcd 456.3999.
2-Cyanoethyl Bis(3-O-tert-butyldimethylsilyl-(2R)-O-((Z)-9-
octadecen-1-yl)-sn-glycer1-yl) Phosphate (7). To a solution of
2-cyanoethyl-bis(N,N-diisopropylamino)phosphine (51 mg, 0.167
mmol, 1.0 equiv) and 1H-tetrazole (70 mg 1.0 mmol, 6.0 equiv) in
dry CH2Cl2 was added a solution of ether 6 (168 mg, 0.367 mmol,
2.2 equiv) in CH2Cl2. The mixture was stirred under argon for 48
h. Then t-BuOOH (0.075 mL, 0.67 mmol, 4.0 equiv) was added,
and the reaction was stirred for 1 h. The reaction mixture was diluted
with CH2Cl2, poured into saturated Na2S2O3, and extracted with 2
× 20 mL of CH2Cl2. The combined organic phases were dried (Na2-
SO4) and concentrated in vacuo, and the crude product was purified
on silica gel FC (ethyl acetate/hexane, 1/5, v/v) to give 70 mg (0.068
t-BuOOH (0.148 mL, 1.32 mmol, 4.0 equiv) was added, and the
reaction was stirred for a further 1 h. The reaction was diluted with
CH2Cl2, poured into saturated Na2S2O3, and extracted with 2 × 40
mL of CH2Cl2. Combined organic phases were dried (Na2SO4) and
concentrated in vacuo, and the crude product was purified on silica
gel FC (ethyl acetate/hexane, 1/3, v/v) to give 142 mg (0.137 mmol,
42%) of phosphate 10. Rf 0.41 (ethyl acetate/hexane, 1/1, v/v);
[R]20D +15.14 (c 0.12, MeOH); 1H NMR (CDCl3) δ 7.18 (m, 4H),
6.80 (m, 4H), 5.29 (m, 4H), 4.39 (m, 4H), 4.00-4.15 (m, 6H),
3.73 (s, 6H), 3.40-3.65 (m, 10H), 2.60 (t, J ) 6.4 Hz, 2H), 1.92
(m, 8H), 1.47 (t, J ) 6.4 Hz, 4H), 1.18-1.22 (m, 44H), 0.81 (t, J
) 6.8 Hz, 6H); 13C NMR (CDCl3) δ 159.2 (s), 130.4 (s), 130.2
(s), 130.0 (s), 129.9 (s), 129.8 (s), 129.3 (s), 116.3 (s), 113.7 (s),
73.1 (s), 70.6 (s), 68.4 (s), 67.3 (s), 61.7 (s), 61.6 (s), 55.2 (s), 32.6
(s), 31.9 (s), 30.0 (s), 29.7 (s), 29.7 (s), 29.7 (s), 29.6 (s), 29.5 (s),
29.5 (s), 29.4 (s), 29.3 (s), 29.2 (s), 29.1 (s), 27.2 (s), 26.0 (s), 22.6
(s), 19.5 (s), 19.4 (s), 14.1 (s); 31P NMR (CDCl3) -0.39 (s). HRMS
(MALDI) for C61H102NNaO10P (M+ + Na): found 1062.7120, calcd
1062.7139.
2-Cyanoethyl Bis(3-hydroxy-(2S)-O-((Z)-9-octadecen-1-yl)-sn-
glycer-1-yl) Phosphate (11). To a solution of 10 (110 mg, 0.106
mmol) in 2 mL of wet CH2Cl2 was added DDQ (120 mg, 0.529
mmol). The mixture was stirred at room temperature overnight,
diluted with CH2Cl2, washed with 10% NaHCO3, dried over Na2-
SO4, concentrated, and purified on silica gel FC (ethyl acetate/
hexane, 2/1, v/v followed by CH2Cl2/MeOH, 25/1, v/v) to give
mmol, 41%) of phosphate 7. Rf 0.33 (ethyl acetate/hexane, 1/5,
1
v/v); [R]20 -5.71 (c 0.41, MeOH); H NMR (CDCl3) δ 5.33 (m,
D
4H), 4.20 (m, 4H), 4.05 (m, 2H), 3.37-3.67 (m, 10H), 2.72 (t, J )
6 Hz, 2H), 1.97 (m, 8H), 1.54 (m, 4H), 1.25 (m, 44H), 0.85 (m,
24H), 0.04 (s, 12H); 13C NMR (CDCl3) δ 129.8 (d, J ) 11.5 Hz),
116.2 (s), 78.7 (s), 78.6 (s), 70.7 (s), 67.1 (s), 66.2 (s), 61.8 (s),
61.7 (s), 51.6 (s), 51.0 (s), 31.9 (s), 30.2 (s), 29.8 (s), 29.7 (s), 29.7
(s), 29.5 (s), 29.5 (s), 29.3 (s), 27.2 (s), 26.1 (s), 25.8 (s), 22.7 (s),
19.6 (s), 18.2 (s), 14.1 (s), -5.4 (s); 31P NMR (CDCl3) -0.91 (s);
HRMS (MALDI) for C57H114NNaO8PSi2 (M+ + Na): found
1050.7713, calcd 1050.7718.
compound 11 (57 mg, 67%): Rf 0.34 (CH2Cl2/MeOH, 15/1, v/v);
1
[R]20 +3.80 (c 0.36, MeOH); H NMR (CDCl3) δ 5.28 (m, 4H),
D
4.10-4.23 (m, 6H), 3.40-3.70 (m, 10H), 2.71 (t, J ) 6.0 Hz, 2H),
1.94 (m, 8H), 1.49 (m, 4H), 1.20 (m, 44H), 0.81 (t, J ) 5.6 Hz,
6H); 13C NMR (CDCl3) δ 129.95 (s), 129.77 (s), 116.28 (s), 81.15
(s), 77.85 (s), 70.59 (s), 66.12 (s), 66.06 (s), 61.99 (s), 60.78 (s),
60.75 (s), 31.88 (s), 29.94 (s), 29.74 (s), 29.68 (s), 29.50 (s), 29.43
(s), 29.30 (s), 29.26 (s), 29.19 (s), 26.01 (s), 22.66 (s), 19.67 (s),
19.60 (s), 14.10 (s); 31P NMR (CDCl3) 0.35 (s). HRMS (MALDI)
for C45H86NNaO8P (M+ + Na): found 822.6010, calcd 822.5989.
Bis(3-hydroxy-(2S)-O-((Z)-9-octadecen-1-yl)-sn-glycer-1-yl)
Phosphate (12). To a solution of 11 (12 mg, 0.015 mmol) in 0.5
mL of CH3CN was added 0.1 mL of BSTFA, followed by 0.5 mL
of triethylamine. The mixture was stirred at room temperature
overnight. Solvent was removed at reduced pressure. Then 0.5 mL
of MeOH/H2O (20/1, v/v) was added, and the mixture was stirred
for 30 min, concentrated, and purified on silica gel (200-300 mesh,
10 g) FC (ethyl acetate, followed by CH2Cl2/MeOH, 5/1, v/v) to
give a colorless oil. The oil was purified on H+ Dowex ion-
exchange resin (5 g) to give compound 12: Rf 0.17 (CH2Cl2/MeOH,
5/1, v/v); [R]20D -2.36 (c 0.17, MeOH); 1H NMR (CD3OD) δ 5.33
(m, 4H), 3.90-4.10 (m, 4H), 3.40-3.70 (m, 10H), 2.02 (m, 8H),
1.56 (m, 4H), 1.26 (m, 44H), 0.89 (t, J ) 6.8 Hz, 6H); 13C NMR
(CD3OD) δ 128.9 (s), 128.8 (s), 78.6 (s), 78.6 (s), 69.5 (s), 63.4
(s), 63.3 (s), 59.9 (s), 31.1 (s), 29.3 (s), 28.9 (s), 28.9 (s), 28.8 (s),
28.1 (s), 28.5 (s), 28.4 (s), 28.4 (s), 26.2 (s), 26.2 (s), 25.3 (s), 21.8
(s), 12.5 (s); 31P NMR (CD3OD) -1.01 (s). HRMS (MALDI) for
C42H83NaO8P (M+ + Na): found 769.5718, calcd 769.5723.
3-O-tert-Butyldimethysilyl-1-O-methoxybenzyl-(2S)-O-oleoyl-
sn-glycerol (13). To a solution of 3-O-tert-butyldimethysilyl-1-O-
methoxybenzyl-2(S)-glycerol 4 (190 mg, 0.58 mmol) and oleic acid
(0.22 mL, 0.70 mmol) in dry CH2Cl2 (1 mL) was added a solution
of DCC (157 mg, 0.75 mmol) and DMAP (91 mg, 0.75 mmol) in
dry CH2Cl2 (4 mL). The solution was stirred for 16 h at room
temperature, filtered, and concentrated in vacuo, and the residue
was purified by FC (ethyl acetate:hexane, 1:100, v:v) to afford 325
mg of 13 (95%) as a colorless oil: Rf 0.20 (ethyl acetate:hexane,
1:20, v:v); [R]20D -8.96 (c 0.44, MeOH); 1H NMR (CDCl3) δ 7.10
(m, 2H), 6.83 (m, 2H), 5.30 (m, 2H), 5.00 (m, 1H), 4.42 (m, 2H),
3.76 (s, 3H), 3.69 (d, J ) 4.8 Hz, 2H), 3.54 (m, 2H), 2.27 (t, J )
7.2 Hz, 2H), 1.96 (m, 4H), 1.58 (m, 2H), 1.16-1.36 (m, 20H),
0.83 (m, 12H), 0.01 (m, 6H); 13C NMR (CDCl3) δ 173.3 (s), 159.2
(s), 130.1 (s), 130.0 (s), 129.7 (s), 129.2 (s), 113.7 (s), 72.9 (s),
Bis(3-Hydroxy-(2R)-O-((Z)-9-octadecen-1-yl)-sn-glyceryl) Phos-
phate (8). To a solution of protected 7 (50 mg, 0.0486 mmol) in
anhydrous THF (10 mL) was added TBAF (90 mg, 0.285 mmol, 6
equiv). The reaction mixture was stirred at room temperature
overnight. Concentration and silica gel FC (CH2Cl2:/MeOH, 6/1,
v/v) furnished colorless oil. The oil was purified on H+ Dowex
ion-exchange resin to give compound 8 (31 mg, 85%): Rf 0.17
(CH2Cl2/MeOH, 5/1, v/v); [R]20D +2.33 (c 0.14, MeOH); 1H NMR
(CD3OD) δ 5.26 (m, 4H), 3.97 (m, 4H), 3.40-3.60 (m, 10H), 1.93
(m, 8H), 1.48 (m, 4H), 1.21 (m, 44H), 0.80 (t, J ) 7.2 Hz, 6H);
13C NMR (CD3OD) δ 128.9 (s), 128.8 (s), 78.6 (s), 78.6 (s), 69.5
(s), 63.4 (s), 63.3 (s), 59.9 (s), 31.1 (s), 29.3 (s), 28.9 (s), 28.9 (s),
28.8 (s), 28.1 (s), 28.5 (s), 28.4 (s), 28.4 (s), 26.2 (s), 26.2 (s), 25.3
(s), 21.8 (s), 12.5 (s); 31P NMR (CD3OD) 0.55 (s). HRMS (MALDI)
for C42H83NaO8P (M+ + Na): found 769.5745, calcd 769.5723.
1-O-Methoxybenzyl-(2R)-O-((Z)-9-octadecen-1-yl)-sn-glyc-
erol (9). To a solution of protected 5 (420 mg, 0.739 mmol) in
anhydrous THF (20 mL) was added TBAF‚3H2O (690 mg, 2.22
mmol). The reaction mixture was stirred at room temperature
overnight. Concentration and silica gel flash chromatography (ethyl
acetate/hexane, 1/5, v/v) furnished 9 (320 mg, 95%): Rf 0.10 (ethyl
1
acetate/hexane 1/5, v/v); [R]20 +1.44 (c 0.49, MeOH); H NMR
D
(CDCl3) δ 7.18 (m, 2H), δ 6.81 (m, 2H), 5.27 (m, 2H), 4.10 (m,
2H), 3.73 (s, 3H), 3.40-3.65 (m, 7H), 1.94 (m, 4H), 1.50 (t, J )
7.2 Hz, 2H), 1.18-1.22 (m, 22H), δ 0.81 (t, J ) 6.8 Hz, 3H); 13
C
NMR (CDCl3) δ 159.1 (s), 129.9 (s), 129.8 (s), 129.3 (s), 113.8
(s), 78.4 (s), 73.1 (s), 73.4 (s), 69.4 (s), 62.9 (s), 55.2 (s), 31.9 (s),
30.0 (s), 29.7 (s), 29.7 (s), 29.5 (s), 29.5 (s), 29.4 (s), 29.3 (s), 29.2
(s), 27.2 (s), 26.1 (s), 22.7 (s), 14.1 (s). HRMS (MALDI) for C29H50-
NaO4 (M+ + Na): found 485.3601, calcd 485.3607.
2-Cyanoethyl Bis(1-O-methoxybenzyl-(2S)-O-((Z)-9-octade-
cen-1-yl)-sn-glycer-1-yl) Phosphate (10). To a solution of 2-cya-
noethyl bis(N,N-diisopropylamino) phosphine (370 mg, 0.80 mmol,
2.4 equiv) and 1H-tetrazole (139 mg 1.98 mmol, 6.0 equiv) in 1.5
mL of CH3CN was added a solution of ether 9 (100 mg, 0.33 mmol,
1.0 equiv). The mixture was stirred under argon for 48 h. Then
938 J. Org. Chem., Vol. 71, No. 3, 2006

Syntheses of Lysobisphosphatidic Acid and Its Analogues
72.8 (s), 68.0 (s), 61.6 (s), 55.2 (s), 34.4 (s), 31.9 (s), 29.7 (s), 29.7
(s), 29.5 (s), 29.3 (s), 29.2 (s), 29.1 (s), 27.2 (s), 27.2 (s), 25.8 (s),
25.0 (s), 22.7 (s), 18.2 (s), 14.1 (s), -5.5 (s), -5.5 (s); MS (ESI)
129.1 (s), 72.8 (s), 71.5 (d, J ) 7.7 Hz), 68.1 (s), 63.9 (s), 34.1 (s),
31.7 (s), 29.6 (s), 29.4 (s), 29.2 (s), 29.1 (s), 29.0 (s), 27.1 (d, J )
1.5 Hz), 24.8 (s), 22.5 (s), 13.9 (s); 31P NMR (CDCl3/CD3OD) δ
2.46 (s); MS (ESI) m/z 775.67 (M+ + 1).
m/z 591.59 (M+ + 1). HRMS (MALDI) for C35H62NaO5Si (M+
+
Na): found 613.4259, calcd 613.4264.
2-Cyanoethyl Bis(3-O-tert-butyldimethysily-(2R)-O-oleoyl-sn-
glycer-1-yl) Phosphorothioate (17). To a solution of 2-cyanoethyl
bis(N,N-diisopropylamino) phosphite (98 µL, 0.30 mmol, 1.0 equiv)
and 1H-tetrazole (170 mg, 2.4 mmol, 8.0 equiv) in 0.5 mL of dry
CH3CN was added a solution of ester 16 (300 mg, 0.63 mmol, 2.1
equiv) in 1.5 mL of dry CH3CN. The mixture was stirred under
argon for 48 h. Then sulfur (48 mg, 1.5 mmol, 5.0 equiv) and CS2/
pyridine (75 µL, 1:1, v/v) were added, and the reaction was stirred
for an additional 2 h. The reaction mixture was filtered and the
filtrate was washed with brine, dried over Na2SO4, and concentrated
in vacuo, and the crude product was purified by FC (ethyl acetate:
hexane, 1:15, v:v) to give 242 mg of phosphorothioate 17 (75%).
3-O-tert-Butyldimethysilyl-(2S)-O-oleoyl-sn-glycerol (14). To
a solution of glyceryl ester 13 (420 mg, 0.713 mmol) in 20 mL of
wet CH2Cl2 was added DDQ (247 mg, 1.09 mmol). The mixture
was stirred at room temperature overnight, diluted with CH2Cl2,
washed with 10% NaHCO3, dried over Na2SO4, concentrated, and
purified by FC quickly (ethyl acetate:hexane, 1:15, v:v) to give
311 mg of compound 14 (93%): Rf 0.12 (ethyl acetate:hexane, 1:10,
1
v:v); [R]20 -7.06 (c 0.14, MeOH); H NMR (CDCl3) δ 5.32 (m,
D
2H), 4.86 (m, 1H), 3.72-3.87 (m, 4H), 2.32 (t, J ) 7.2 Hz, 2H),
2.00 (m, 4H), 1.59 (m, 2H), 1.20-1.35 (m, 20H), 0.85 (m, 12H),
0.05 (s, 6H); 13C NMR (CDCl3) δ 173.7 (s), 129.9 (d, J ) 28 Hz),
74.2 (s), 62.8 (s), 62.5 (s), 34.4 (s), 31.9 (s), 29.8 (s), 29.7 (s), 29.5
(s), 29.3 (s), 29.2 (s), 29.18 (s), 27.2 (s), 27.2 (s), 25.8 (s), 24.9
(s), 22.7 (s), 18.2 (s), 14.1 (s), -5.5 (s), -5.6 (s); MS (ESI) m/z
471.56 (M+ + 1). HRMS (CI) for C27H55O4Si (M+ + 1): found
471.3862, calcd 471.3869.
Rf 0.50 (ethyl acetate:hexane, 1:5, v/v); [R]20 -2.43 (c 2.33,
D
1
MeOH); H NMR (CDCl3) δ 5.30 (m, 4H), 4.99 (m, 2H), 4.11-
4.30 (m, 6H), 3.69 (d, J ) 6.8 Hz, 4H), 2.70 (t, J ) 6.0 Hz, 2H),
2.29 (dt, J ) 2.8 Hz, J ) 6.8 Hz, 4H), 1.97 (m, 8H), 1.58 (m, 4H),
1.16-1.32 (m, 40H), 0.84 (m, 24H), 0.03 (s, 12H); 13C NMR
(CDCl3) δ 172.9 (s), 129.8 (d, J ) 27 Hz), 71.9 (s), 66.5 (s), 62.2
(s), 60.9 (s), 60.8 (s), 34.2 (s), 31.9 (s), 29.7 (s), 29.5 (s), 29.3 (s),
29.2 (s), 29.1 (s), 27.1 (s), 25.7 (s), 24.8 (s), 22.6 (s), 18.1 (s), 14.1
(s), -5.5 (s); 31P NMR (CDCl3) δ 70.00 (s); MS (ESI) m/z 1072.2
(M+). HRMS (ESI) for C57H111NO9PSSi2 (M+ + 1): found
1072.7240, calcd 1072.7250.
2-Cyanoethyl Bis(3-O-tert-butyldimethysilyl-(2R)-O-oleoyl-sn-
glycer-1-yl) Phosphate (15). To a solution of 2-cyanoethyl bis-
(N,N-diisopropylamino) phosphite (84 mg, 0.28 mmol, 1.0 equiv)
and 1H-tetrazole (160 mg, 2.28 mmol, 8.0 equiv) in 0.5 mL of dry
CH3CN was added a solution of ester 14 (291 mg, 0.62 mmol, 2.2
equiv) in 1 mL of dry CH3CN. The mixture was stirred under argon
for 48 h. Then, t-BuOOH (0.126 mL, 1.12 mmol, 4.0 equiv) was
added, and the reaction was stirred for 1 h. The reaction was diluted
with CH2Cl2, poured into saturated Na2S2O3, and extracted with 2
× 30 mL of CH2Cl2. The combined organic phases were dried (Na2-
SO4) and concentrated in vacuo, and the crude product was purified
by FC (ethyl acetate:hexane, 1:5, v:v) to give 181 mg of phosphate
15 (61%). Rf 0.18 (ethyl acetate:hexane, 1:3, v:v); [R]20D -9.21 (c
2-Bis(3-hydroxyl-(2R)-O-oleoyl-sn-glycer-1-yl)phosphorothio-
ate (18). A solution of protected 7 (52 mg, 0.052 mmol, 1 equiv)
in anhydrous THF (1 mL) was treated successively with acetic acid
(59 µL, 1.04 mmol, 20 equiv) and TBAF (329 mg, 1.04 mmol, 20
equiv) in 3.5 mL of THF. The reaction mixture was stirred at room
temperature for 12 h. After concentration in vacuo, the residue was
dissolved in 40 mL of CHCl3, transferred to separatory funnel, and
treated with MeOH (10 mL) and 8 mM acetate ammonium solution
(20 mL). After mixing, the lower layer was concentrated in vacuo.
The residue was loaded onto silica gel (200-300 mesh, 15 g) and
eluted quickly with hexane:acetone (2:1 to 1:2, v:v, with a drop of
2.0 M ammonium hydroxide) solution (precooled to 0 °C) to furnish
26 mg of a colorless oil 18 (63%): Rf 0.20 (hexane:acetone, 1:1,
v:v); [R]20D 6.72 (c 0.08, MeOH); 1H NMR (CDCl3/CD3OD) δ 5.17
(m, 4H), 4.80 (m, 2H), 3.92 (m, 4H), 3.57 (m, 4H), 2.18 (t, J )
7.2 Hz, 4H), 1.84 (m, 8H), 1.44 (m, 4H), 1.21 (m, 40H), 0.71 (t, J
) 6.4 Hz, 6H); 13C NMR (CDCl3/CD3OD) δ 173.6 (s), 129.7 (d, J
) 29 Hz), 72.5 (s), 62.9 (s), 59.4 (s), 58.4 (s), 33.9 (s), 31.6 (s),
29.4 (s), 29.2 (s), 29.0 (s), 28.8 (s), 26.9 (s), 24.5 (s), 22.4 (s), 13.7
(s); 31P NMR (CDCl3/CD3OD) δ 60.54 (s); MS (ESI) m/z 789.5
(M+ - 1). HRMS (MALDI) for C42H79O9PS (M+): found
790.5141, calcd 790.5132.
1
0.47, MeOH); H NMR (CDCl3) δ 5.31 (m, 4H), 5.01 (m, 2H),
4.13-4.28 (m, 6H), 3.70 (m, 4H), 2.73 (t, J ) 6.4 Hz, 2H), 2.29
(dt, J ) 2.0 Hz, 7.6 Hz, 4H), 1.98 (m, 8H), 1.58 (m, 4H), 1.16-
1.32 (m, 40H), 0.85 (m, 24H), 0.03 (s, 12H); 13C NMR (CDCl3) δ
173.0 (s), 129.9 (d, J ) 13 Hz), 79.7 (s), 70.4 (s), 63.0 (s), 62.8
(s), 31.9 (s), 30.1 (s), 29.8 (s), 29.7 (s), 29.7 (s), 29.6 (s), 29.5 (s),
29.5 (s), 29.4 (s), 29.3 (s), 29.2 (s), 27.2 (s), 26.1 (s), 25.8 (s), 22.7
(s), 18.1 (s), 14.1 (s), -5.5 (s), -5.5 (s); 31P NMR (CDCl3) δ -0.42
(s); MS (ESI) m/z 1056.81 (M+ + 1). HRMS (ESI) for C57H111
NO10PSi2 (M+ + 1): found 1056.7483, calcd 1056.7479.
-
2-Bis(3-hydroxyl-(2R)-O-oleoyl-sn-glycer-1-yl) Phosphate (16).12
A solution of protected 15 (14 mg, 0.0133 mmol, 1 equiv) in
anhydrous THF (1 mL) was treated successively with acetic acid
(15.1 µL, 0.265 mmol, 20 equiv) and TBAF (84 mg, 0.265 mmol,
20 equiv) in 3 mL of THF. The reaction mixture was stirred at
room temperature overnight. After concentration in vacuo, the
residue was dissolved in 30 mL of CHCl3 and transferred to a
separatory funnel, and MeOH (20 mL) and water (10 mL) were
added. After mixing, the lower layer was concentrated in vacuo.
The residue was loaded onto silica gel (200-300 mesh, 10 g) and
eluted quickly with CH2Cl2: MeOH (8:1 then 6:1, v:v) solution
(precooled to 0 °C) to furnish 8 mg of a colorless oil 16 (78%): Rf
0.19 (CH2Cl2/MeOH, 5/1, v/v); 1H NMR (CDCl3 /CD3OD) δ 5.28
(m, 4H), 4.89 (m, 2H), 3.95 (m, 4H), 3.65 (m, 4H), 2.25 (t, J )
7.6 Hz, 4H), 1.94 (m, 8H), 1.53 (m, 4H), 1.21 (m, 40H), 0.81 (t, J
) 6.8 Hz, 6H); 13C NMR (CDCl3/CD3OD) δ 173.6 (s), 129.6 (s),
Acknowledgment. We thank the NIH (NS29632 and
HL070231) for financial support of this work.
Supporting Information Available: NMR spectra for the
synthetic molecules, experimental procedures, and compound
characterization for the synthesis of (S,S)-2,2′-oleoyl-LBPA and
its phosphorothioate analogue. This material is available free of
charge via the Internet at http://pubs.acs.org.
JO051894E
J. Org. Chem, Vol. 71, No. 3, 2006 939