Concise synthesis of ether analogues of lysobisphosphatidic acid

Article abstract of DOI:10.1021/ol051194w

(Chemical Equation Presented) We describe a versatile, efficient method for the preparation of ether analogues of (S,S)-lysobisphosphatidic acid (LBPA) and its enantiomer from (S)-solketal. Phosphorylation of a protected sn-2-O-octadecenyl glyceryl ether

Full text of DOI:10.1021/ol051194w

ORGANIC
LETTERS
2005
Vol. 7, No. 18
3837-3840
Concise Synthesis of Ether Analogues
of Lysobisphosphatidic Acid
Guowei Jiang,† Yong Xu,† Thomas Falguie`res,‡ Jean Gruenberg,‡ and
Glenn D. Prestwich*,†
Department of Medicinal Chemistry, The UniVersity of Utah, 419 Wakara Way,
Suite 205, Salt Lake City, Utah 84108-1257, and Department of Biochemistry,
Sciences II, 30 quai E. Ansermet, 1211-GeneVa-4, Switzerland
gprestwich@pharm.utah.edu
Received May 20, 2005
ABSTRACT
We describe a versatile, efficient method for the preparation of ether analogues of (S,S)-lysobisphosphatidic acid (LBPA) and its enantiomer
from (S)-solketal. Phosphorylation of a protected sn-2-O-octadecenyl glyceryl ether with 2-cyanoethyl bis-N,N-diisopropylamino phosphine
and subsequent deprotection generated the bisether LBPA analogues. By simply changing the sequence of deprotection steps, we obtained
the (R,R)- and (S,S)-enantiomers of 2,2
LBPAs were recognized with the same affinity as the natural 2,2
′
-bisether LBPA. An ELISA assay with anti-LBPA monoclonal antibodies showed that the bisether
-bisoleolyl LBPA.
′
Lysobisphosphatidic acids (LBPAs), known also as bis-
(monoacylglycerol)phosphates (BMPs), are natural phos-
pholipids with unusual, poorly understood structure and
activity.1,2 LBPA was first discovered in pig lung homogenate
in 1967 and has now been found in most tissues and cell
types. It usually represents less than 1% of the total
phospholipid mass,3 but increased LBPA titers have been
found in several lipidoses and in response to certain
pharmaceutical agents.4,5 Biochemical, immunocytochemical,
and labeling studies have shown that LBPA is a major
phospholipid of late endosomes (≈15 mol %), an obligatory
station in the pathway followed by all down-regulated
signaling receptors, and is not detected in other subcellular
compartments.6,7 This lipid is involved in cholesterol trans-
port8 and protein/receptor trafficking.6 Recent studies also
indicated that LBPA may regulate the sorting of the
multifunctional receptor (IGF2/MPR), which may influence
vesicular trafficking, lysosome biogenesis, cell growth, and
angiogenesis.6 LBPA has also been shown to be an antigen
in the antiphospholipid syndrome, which can lead to changes
in the endosomal sorting and multivesicular endosome
formation.9 Interfering with LBPA functions phenocopies the
(6) Kobayashi, T.; Stang, E.; Fang, K. S.; Moerloose, P. D.; Parton, R.
G.; Gruenberg, J. Nature 1998, 392, 193-197.
(7) 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.
† The University of Utah.
‡ Sciences II.
(1) Kobayashi, T.; Gu, F.; Gruenberg, J. J. Sem. Cell DeVel. Biol. 1998,
(8) Kobayashi, T.; Beuchat, M.; Lindsay, M.; Frias, S.; Palmiter, R. D.;
Sakuraba, H.; Parton, R. G.; Gruenberg, J. Nat. Cell Biol. 1999, 1, 133-
138.
9, 517-526.
(2) Amidon, B.; Brown, A.; Waite, M. Biochemistry 1996, 35, 13995-
14002.
(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.
(10) Lebrand, C.; Corti, M.; Goodson, H.; Cosson, P.; Cavalli, V.;
Mayran, N.; Faure, J.; Gruenberg, J. Embo. J. 2002, 21, 1289-1300.
(11) Mayran, N.; Parton, R. G.; Gruenberg, J. Embo. J. 2003, 22, 3242-
3253.
(3) Mason, R. J.; Stossel, T. P.; Vaughan, M. J. Clin. InVest. 1972, 51,
2399-2407.
(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.
10.1021/ol051194w CCC: $30.25
© 2005 American Chemical Society
Published on Web 08/02/2005

Scheme 1. Alkyl Analogues of LPA and LBPA
trafficking defects observed in the cholesterol storage
decided to test the hypothesis that bisether analogues of
LBPA might mimic 2,2′-bisacyl-LBPA as a biological ligand
but would lack the propensity to undergo intramolecular acyl
migration.
disorder Niemann-Pick type C.8,10,11 Therefore, LBPA
analogues with agonistic effects may have potential thera-
peutic effects in treating antiphospholipid syndrome and
possibly storage disorders, or analogues with antagonistic
effects could be useful in cancer therapy.
We describe herein a flexible, modular strategy for the
expedient preparation of enantiopure alkyl LBPA analogues
from a common intermediate (Scheme 2). The strategy for
the synthesis of (S,S)-2,2′-bisether LBPA analogues was
designed on the basis of the following considerations. First,
the alkyl chains were installed early in the synthesis. Second,
a commercially available phosphatidylating reagent (bis-N,N-
diisopropylamino-cyanoethylphosphine) was used to intro-
duce both glycerol backbones simultaneously. Both enanti-
omers of alkyl LBPA 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 phosphodiester of the enantiomeric 2,2′-bisether
LBPA analogues at the end of the synthesis facilitated the
purification of synthetic precursors. Conventional silyl
protection of the hydroxyl groups coupled with the cyano-
ethyl 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 bisether LBPAs.
The naturally occurring LBPA has a peculiar and interest-
ing structure: two acyl chains at each of the sn-2 positions
of glycerol backbones. However, this sn-2 acyl structure is
very labile during isolation and structural determination
conditions.12 Intramolecular acyl migration, which is facili-
tated by both acidic and basic conditions, affords an
equilibrium mixture of 1-acyl- and 2-acyl-sn-glycerol 3-phos-
phates that favors the 1-acyl isomer (Scheme 1). The
instability of 2-acyl-sn-glycerol thus seriously compromises
both isolation of the naturally occurring species and deter-
mination of the activating ligand in structure-activity studies.
Replacement of acyl groups by alkyl chains in phospho-
lipid compounds has afforded many valuable analogues.13-15
For example, we found that alkyl analogues of lysophos-
phatidic acid (LPA) were equipotent with the natural acyl
LPAs for three G-protein-coupled LPA receptors.16 More-
over, 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 hydrolyzed by phospholipase A,13 alkyl
substitution can introduce unexpected biological activity. We
We selected (S)-solketal ((2S)-dimethyl-1,3-dioxolane-4-
methanol) as our chiral starting material. Using the phos-
phoramidite methodology, widely exploited in nucleic acid
chemistry, the alcohol was efficiently phosphorylated using
Scheme 2. Common Intermediate for the Enantiomeric LBPA Ethers
3838
Org. Lett., Vol. 7, No. 18, 2005

the yield by minimizing the N-alkylation of pyridine.
Removal of the PMB group with DDQ in wet CH2Cl2 (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 bis-N,N-diisopropylamino-
cyanoethyl-phosphine in the presence of 1H-tetrazole fol-
lowed by t-BuOOH oxidation gave the fully protected LBPA
precursor 7 in medium yield. It is worth noting that the use
of the more reactive phosphatidylating reagent (cyanoeth-
yldichlorophosphine) gave a disappointingly low yield (20%).
The most frequently used reagent for the deprotection of 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 simultaneously 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′-octade-
cenyl LBPA was readily purified on silica gel using CH2Cl2
and methanol (10:1, v:v) as the eluent.
Scheme 3. Synthesis of (R,R)-2,2′-Bisether-LBPA
The enantiomeric (S,S)-2,2′-octadecenyl LBPA was pre-
pared from intermediate 5 as shown in Scheme 4. First,
TBAF was used to remove the TBS group and gave the 2R-
configured primary alcohol 9. The 2R-configured alcohol 9
reacted with bis-N,N-diisopropylamino cyanoethyl phosphine
in the presence of 1H-tetrazole and was subsequently
oxidized by tert-butyl hydrogen peroxide to give the fully
protected (S,S)-LBPA 10 in high yield. Next, DDQ in wet
CH2Cl2 (overnight, rt) 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 12.21 Both natural and
unnatural enantiomers of LBPA can thus be obtained in
optically pure form from (S)-solketal. The routes are short
and efficient and proceed in good overall yields.
a variety of trivalent phosphorus reagents. The resulting
phosphite triester could be oxidized in situ to yield the
corresponding phosphate triester. Furthermore, this approach
allows the introduction of a phosphorothioate moiety if
desired. As shown in Scheme 3, protection of (S)-1,2-O-
isopropylidene-sn-glycerol was performed with p-methoxy-
benzyl chloride (PMB-Cl) to give PMB ether, which was
transketalized (10 mol % p-TsOH in methanol) to the 1,2-
diol in 83% isolated yield.17 After silylation at the primary
alcohol with tert-butyldimethylsilyl (TBDMS) chloride,18 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)19 to
give ether 5. The octadecenyl triflate was prepared by a
modification to the literature protocol;20 specifically, the use
of 2,6-lutidine in place of pyridine significantly increased
Previous results indicated that LBPA was one of the
physiological antigens that is recognized by sera from
patients with antiphospholipid syndrome, which suggests that
Scheme 4. Synthesis of (S,S)-2,2′-Bisether-LBPA
Org. Lett., Vol. 7, No. 18, 2005
3839

Figure 1. Recognition of bisether LBPAs and natural LBPA by anti-LBPA antiserum.
these antibodies exert some pathological effects intracellu-
larly by altering endosomal sorting and/or trafficking of
IGF2/MPR.6 As IGF2/MPR is multifunctional, changes in
its transport cycle may have multiple effects, including in
vesicular traffic, lysosome biogenesis,22 cell growth,23 and
angiogenesis.24 The antiphospholipid syndrome, particularly
fetal loss, may be at least partly related to the role of IGF2/
MPR in endothelial cell migration and neovascularization.24
To determine whether the synthetic bisether LBPA ana-
logues possessed the same physical and biological activities
as natural LBPA, we initially tested these compounds using
TLC analysis and ELISA. Purified LBPA was chromato-
graphed on silica gel 60 HPTLC plates with chloroform/
methanol/32% ammonia (65/35/5, v/v) as the developing
solvent. The TLC analysis showed that alkyl-LBPAs and
natural LBPA had the same Rf values (data not shown),
which indicates that they share similar physical properties.
Both (R,R)- and (S,S)-bisether analogues 8 and 12 were
highly immunoreactive toward the 6C4 murine monoclonal
antibody using ELISA. The comparative ELISA assay
showed that analogues 8 and 12 had essentially equivalent
immunoreactivity relative to natural (S,S)-2,2′-bisoleoyl
LBPA (Figure 1).
In summary, we have described a general and efficient
method for the preparation of LBPA bisether analogues from
a common intermediate. The resulting compounds had
comparable immunoreactivity relative to natural bisoleoyl
LBPA. The evaluation of the bisether LBPA analogues in
rescuing cells with a cholesterol storage disorder will be
described elsewhere in due course.
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Acknowledgment. We thank Dr. H. Zhang (University
of Utah) for many helpful discussions. We acknowledge the
NIH (NS29632 to G.D.P.) and the Swiss National Science
Foundation (3100A0-104251 to J.G.) for financial support
of this work. T.F. was supported by a fellowship from the
Fondation pour la Recherche Me´dicale.
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Supporting Information Available: Experimental pro-
cedures and characterization for all new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
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