Modular synthesis of bis(monoacylglycero)phosphate for convenient access to analogues bearing hydrocarbon and perdeuterated acyl chains of varying length

Article abstract of DOI:10.1016/j.tet.2009.06.085

Bis(monoacylglycero)phosphate is an important phospholipid that controls the structure of late endosomes as well as biological activities that occur there. The study of this lipid is complicated by the fact that acyl migrations are known to generate diffe

Full text of DOI:10.1016/j.tet.2009.06.085

Tetrahedron 65 (2009) 6844–6849
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Modular synthesis of bis(monoacylglycero)phosphate for convenient access to
analogues bearing hydrocarbon and perdeuterated acyl chains of varying length
*
Meng M. Rowland, Michael D. Best
Department of Chemistry, The University of Tennessee, Knoxville, TN 37996, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 April 2009
Received in revised form 17 June 2009
Accepted 19 June 2009
Available online 25 June 2009
Bis(monoacylglycero)phosphate is an important phospholipid that controls the structure of late endo-
somes as well as biological activities that occur there. The study of this lipid is complicated by the fact
that acyl migrations are known to generate different regioisomers. Herein, we describe a modular
synthesis of 3,30-BMP derivatives that allows for the incorporation of a range of different acyl chains at
a late stage. This approach was exploited to produce eight BMP analogues bearing normal saturated and
perdeuterated acyl chains of varying length.
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
Bis(monoacylglycero)phosphate
Lyso-bisphosphatidic acid
Phospholipids
Endosomes
1. Introduction
membranes was shown to induce the formation of complex mul-
tivesicular liposomes resembling those that exist in late endo-
Bis(monoacylglycero)phosphate (BMP), also referred to as lyso-
bisphosphatidic acid (LBPA), while a minimal component of cellular
phospholipid content, has emerged as an important lipid in the
regulation of biological pathways. This lipid primarily exists in late
endosomes, where it makes up w15% of the total membrane
composition,1 and w70% of the inner membranes of this organelle.2
As a result, the presence of this lipid has been found to affect the
trafficking and degradation of a number of molecules. For instance,
membranes presenting BMP are known to enhance the degradation
of sphingolipids including glucosylceramide,3 ceramide,4 GM1,5
and GM2.6 BMP also regulates cholesterol transport,7,8 which is
aberrant in Niemann-Pick type C disease, and the accumulation of
anti-BMP antibodies is known to alter cholesterol homeostasis.9 In
addition, BMP is a known antigen of antiphospholipid antibodies
associated with antiphospholipid syndrome.1 Finally, the transport
of stomatitus virus to late endosomes, the last step prior to cyto-
plasmic release of the nucleocapsid and infection, has been shown
to require the presence of BMP.10
somes.11 Since these initial studies, a number of techniques
including electron microscopy, differential scanning calorimetry,
fluorescence spectroscopy, and small- and wide-angle X-ray scat-
tering have been used to probe membrane structures and phase
transitions in different membranes containing BMP.12–14 While
these studies have provided insights such as the pH-dependence of
membrane morphology in different compositions containing BMP,
and the effect of BMP stereochemistry on membrane properties,
further analysis is needed to understand how BMP affects mem-
brane structure.
The molecular structure of BMP itself yields interesting issues as
different regioisomers (see (S,S)-3,30-BMP (1a), and (S,S)-2,20-BMP
(1b), Fig. 1) are possible depending upon the location of the acyl
chains. Understanding the precise composition of BMP in biological
systems is complicated by the fact that (S,S)-2,20-BMP is readily
converted to (S,S)-3,30-BMP via sn-2-to-sn-3 acyl chain migration
that occurs under a variety of conditions,13 including in mildly
acidic solution.15,16 This is important as the late endosomes in
which BMP exists are known to be acidic, and optimal BMP hy-
drolysis by the enzyme phospholipase A2 occurs under acidic
conditions.17 In addition, different stereoisomers have been ob-
served in nature. While the majority of BMP is believed to exist as
the somewhat unusual sn-1:sn-10 configuration,18–20,13 the sn-
3:sn-10 derivative has also been reported.21
Due to these implications, there has been great interest in un-
derstanding the molecular basis for the biological activity of BMP.
An important aspect of this is the effect that BMP has on the
structure of the inner endosomal membranes in which it exists.
This was spurred by studies in which the presence of BMP in model
Previously reported syntheses of BMPs initially utilized chemo-
enzymatic approaches through reaction of 2 equiv of diacylglycerol
with a phosphoryl dichloride, followed by acyl chain removal using
* Corresponding author. Tel.: þ1 865 974 8658; fax: þ1 865 974 9332.
E-mail address: mdbest@utk.edu (M.D. Best).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.06.085

M.M. Rowland, M.D. Best / Tetrahedron 65 (2009) 6844–6849
6845
O
P
O
The efficient production of a number of BMP analogues con-
taining diversity in the acyl chains would benefit from a modular
synthesis in which the lipid tails are introduced at a late stage. The
initial synthetic approach we developed for this purpose is in-
dicated in Scheme 1. This route commenced with commercially
available and enantiomerically pure (S)-acetonide-protected glyc-
erol (2), which contains the appropriate stereochemistry for the
targeted (S,S)-3,30-BMP derivatives. Protection of the hydroxyl
group of 2 as a para-methoxybenzyl (PMB) ether was followed by
acetonide deprotection to produce diol 3, as previously described.26
Next, the primary hydroxyl group of 3 was selectively protected as
a mono-methoxytrityl (MMT) ether to generate alcohol 4, and then
O
R
O
O
O
R
O
O
OH
HO
1a
(S,S)-3,3'-BMP
O
P
O
HO
O
O
OH
O
R
O
R
O
O
a
tert-butyldiphenylsilyl (TBDPS) group was installed at the
1b
remaining secondary hydroxyl to produce fully protected glycerol
derivative 5. The MMT protecting group was next removed and the
resulting alcohol, 6, was converted to phosphodiester 7 using
phosphoramidite chemistry. Finally, PMB-deprotection generated
diol 8 as a core scaffold for the production of a range of derivatized
BMPs.
(S,S)-2,2'-BMP
Figure 1. Generalized structures of regioisomers of sn-1:sn-10 bis(monoacylglycero)-
phosphate (BMP).
phospholipase A2.22,23 More recently, chemical synthesis has been
employed to access BMPs from simple glycerol precursors.16,24,15 In
these reports, the primary focus has been the synthesis of (S,S)-2,20-
BMP structures, in which methods were developed to minimize
acyl migration to successfully produce this target. In the generation
of (S,S)-3,30-BMPs, rationally induced acyl migration has been ef-
fective for the synthesis of this regioisomer from the corresponding
(S,S)-2,20-BMP structure.13
Compound 8 acts as a convenient modular precursor to BMP
analogues as different lipid tails can be appended to this diol, fol-
lowed only by global deprotection to the final targets (Scheme 2).
Here, coupling chemistry was used to introduce all saturated acyl
chains of even length between 12 and 18 carbons in both normal
saturated and perdeuterated form to produce protected (S,S)-3,30-
BMP phosphotriester intermediates 9a–h. These specific acyl chain
motifs were selected to study the effect of the length of the lipid tail
on biological and physiochemical properties, and because the cor-
responding perdeuterated precursors of these fatty acids are
commercially available. Finally, global deprotection using TBAF15
was achieved to access 10a–h, consisting of a number of (S,S)-3,30-
BMP probes with acyl chains of varying lengths composed of either
perdeuterated or natural hydrocarbon acyl chains. The final
deprotection step proceeded in high yield for the formation of
certain derivatives, such as bis-palmitoyl derivative 9c (91%).
However, this yield tended to decrease with compounds with
shorter acyl chains, likely due to problems in isolating these com-
pounds as a result of their amphiphilic nature. Other than this
challenge, the described synthesis is effective, with most steps
proceeding in w85 to 95% yield.
2. Results and discussion
Synthetic analogues of the different BMPs are invaluable as
chemical tools for understanding the biological and structural ef-
fects of these molecules. In the current study, we sought to develop
novel (S,S)-3,30-BMP analogues bearing labeled acyl chains for use
in characterizing the effect of this lipid on membrane structure. For
this purpose, a particularly advantageous structural design involves
the incorporation of perdeuterated acyl chains into the BMP
structure. Lipid probes with specifically deuterated domains are
beneficial for structural studies employing NMR and neutron
scattering, and have previously been synthesized.25 Furthermore,
since we are interested in determining how the identities of the
acyl chains affect packing within a membrane environment, we set
out to synthesize a number of (S,S)-3,30-BMP analogues bearing
different chain lengths of both normal saturated and perdeuterated
lipid tails.
Since BMPs are known to undergo acyl chain migrations, it is
important to confirm the identity of the products from the depro-
tection producing 10a–h. In previous reports of acyl chain migra-
tions, sn-2-to-sn-3 migration has primarily been noted, suggesting
that the primary driving force for this transformation is the relief of
1) NaH, DMF
2)
Cl
OCH3
Cl
TBDPSCl
Imidazole
HO
O
H3CO
PMBO
OH
PMBO
OMMT
PMBO
OMMT
OTBDPS
O
(PMBCl)
3) TsOH
92%
OH
OH
Pyridine
(MMTCl)
2
3
4
5
DMAP
Pyridine
94%
1)
N
P
N
O
P
O
O
O
P
O
O
DDQ
H2O
CSA
HO
TBDPSO
O
O
OH
OTBDPS
CH3OH
CN
PMBO
TBDPSO
O
OPMB
OTBDPS
PMBO
OH
OTBDPS
1H-Tetrazole
CH2Cl2
2) t-BuOOH
CH2Cl2
92%
CH2Cl2
87% (2 steps)
CN
CN
6
7
8
84%
Scheme 1. Synthetic route to modular BMP intermediate 8 for the installation of different derivatized acyl chains.

6846
M.M. Rowland, M.D. Best / Tetrahedron 65 (2009) 6844–6849
O
P
O
O
O
P
O
O
O
O
P
O
O
RCOOH
HO
TBDPSO
O
O
OH
OTBDPS
R
O
O
O
O
R
DCC, DMAP
TBAF
R
O
O
O
O
R
OTBDPS
TBDPSO
CH2Cl2
71-92%
H2O
19-91%
OH
HO
NH4
CN
CN
8
10a–h
9a–h
a: R = (CH2)16CH3
b: R = (CD2)16CD3
c: R = (CH2)14CH3
d: R = (CD2)14CD3
e: R = (CH2)12CH3
f: R = (CD2)12CD3
g: R = (CH2)10CH3
h: R = (CD2)10CD3
Scheme 2. Modular synthesis of BMP analogs containing several hydrocarbon and perderteraed acyl chains of varying length using scaffold 8.
steric strain upon the shift to the less substituted sn-3 position. In
fact, quantitative sn-2-to-sn-3 migration has been reported in the
synthesis of BMPs and other glycerolipids under certain condi-
tions.16,27 Based on this evidence, the reverse sn-3-to-sn-2 migra-
tion was not expected to be problematic in the deprotection of
9a–h. This was confirmed via 1H NMR analysis, as the typical
chemical shifts for the hydrogens on the (S,S)-3,30-BMP isomer
was added 4-methoxytrityl chloride (0.233 g, 0.754 mmol) and 4-
dimethylaminopyridine (0.005 g, 0.038 mmol). The solution was
stirred at rt for 24 h. The reaction mixture was then quenched with
methanol (0.5 mL), and the solvent was removed under reduced
pressure. The resulting residue was purified by column chroma-
tography with silica gel and a gradient solvent of 10–40% ethyl ac-
296K
etate/hexanes to yield 4 as a white solid (0.171 g, 94%). [
a
]
ꢀ0.3
D
(wd
3.9–
d
4.2) dominated the spectra of 10a–h (see 1H NMR spectra
(c 3.0, CH3OH/CHCl3 (1:1)); 1H NMR (300 MHz, CDCl3):
d
7.41 (d,
in Supplementary data), with minimal of the characteristic peaks of
J¼6.0 Hz, 4H), 7.16–7.30 (m, 10H), 6.83 (dd, 1J¼12.0 Hz, 2J¼9.0 Hz,
4H), 4.45 (s, 2H), 3.91–4.02 (m, 1H), 3.80 (s, 3H), 3.78 (s, 3H), 3.49–
3.60 (AB of ABX, JAX¼6 Hz, JAB¼12 Hz, JBX¼6 Hz, 2H), 3.15–3.24 (AB
the (S,S)-2,20-isomer (w
d 3.8, d 5.0), both of which have been pre-
viously described.16 Thus, this approach is effective for accessing
a range of analogues of the (S,S)-3,30-isomer of BMP.
of ABX, JAB¼9 Hz, JAX¼6 Hz, JBX¼6 Hz, 2H), 2.40 (d, J¼6.0 Hz,1H); 13
C
NMR (100.6 MHz, CDCl3):
d 159.2, 158.5, 144.3, 135.4, 130.3, 130.0,
129.3,128.3,127.8,126.9,113.7,113.1, 86.3, 72.9, 71.2, 69.9, 64.4, 55.2,
3. Conclusion
55.1; MALDI-HRMS [MþNa]þ calcd: 507.2142, found: 507.2153.
Bis(monoacylglycero)phosphates represent an important but
poorly understood class of phospholipid components of cellular
membranes. In order to advance studies of the biological and
structural ramifications of (S,S)-3,30-BMP lipids, we have developed
a high-yielding synthesis of analogues of this lipid. This synthesis
allows for modular introduction of various acyl chains at a late
stage, which we exploited for the synthesis of eight (S,S)-3,30-BMP
analogues. Currently, we are employing these compounds as
probes to understand the effect of these structures on membrane
properties using NMR and neutron scattering techniques, studies
which will be reported in due course.
4.3. 2-O-(tert-Butyldiphenylsilyl)-3-O-(4-methoxybenzyl)-1-
O-((4-methoxyphenyl)diphenylmethyl)-sn-glycerol (5)
Alcohol 4 (0.866 g, 2.89 mmol), tert-butyldiphenylsilyl chloride
(0.984 g, 3.58 mmol) and imidazole (0.393 g, 3.58 mmol) were
combined in dry pyridine (60 mL), and the reaction was allowed to
stir at rt for 24 h. The reaction mixture was then concentrated and
the resulting crude was purified by column chromatography with
silica gel and a gradient solvent of 5–15% ethyl acetate/hexanes to
yield crude 5 as a colorless oil. Compound 5 was generally passed
on to the next step without complete purification due to difficulties
in removing all byproducts. However, a sample of this compound
4. Experimental section
4.1. General experimental
was isolated to homogeneity for the purpose of characterization.
296K
[a
]
ꢀ0.85 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3):
d 7.64 (d,
D
J¼8 Hz, 2H), 7.59 (d, J¼8 Hz, 2H), 7.18–7.37 (m, 16H), 7.04 (d, J¼8 Hz,
2H), 6.76 (dd, 1J¼16 Hz, 2J¼8 Hz, 4H), 4.21–4.29 (AB, JAB¼12 Hz, 2H),
3.96–4.05 (m, 1H), 3.78 (s, 3H), 3.76 (s, 3H), 3.41–3.58 (AB of ABX,
JAB¼16 Hz, JAX¼8 Hz, JBX¼8 Hz, 2H), 3.13–3.22 (m, 2H), 1.02 (s, 9H);
Generally, reagents were purchased from Acros or Aldrich and
used as received. Perdeuterated fatty acids were purchased from
Cambridge Isotopes. Dry solvents were obtained from a Pure Solv
solvent delivery system purchased from Innovative Technology, Inc.
Column chromatography was performed using 230–400 mesh silica
gel purchased from Sorbent Technologies. NMR spectra were
obtained using a Bruker AC250 spectrometer updated with a TecMag
data collection system, a Varian Mercury 300 spectrometer, and
a Bruker Avance 400 spectrometer. Mass spectra were obtained with
either a JEOL DART-AccuTOF spectrometer or a Voyager DE MALDI-
TOF spectrometer, each with high resolution capabilities. Optical
rotation values were obtained using a Perkin–Elmer 241 polarimeter.
13C NMR (100.6 MHz, CDCl3):
d 158.9, 158.3, 144.6, 135.9, 135.8,
133.9, 130.6, 130.4, 129.4, 129.0, 128.5, 127.6, 127.4, 126.6, 113.5,
112.9, 86.1, 72.6, 72.0, 71.5, 65.0, 55.2, 55.1, 30.9, 27.0, 19.3; MALDI-
HRMS [MþNa]þ calcd: 745.3320, found: 745.3336.
4.4. 2-O-(tert-Butyldiphenylsilyl)-3-O-(4-methoxybenzyl)-sn-
glycerol (6)
Crude compound 5 from the previous procedure and cam-
phorsulfonic acid (0.102 g, 0.44 mmol) were dissolved in 75 mL
methanol/methylene chloride mixture (v/v 2:1), and the solution
was allowed to stir at rt for 3 h. The solvent was then removed
under reduced pressure, and the residue was purified by column
chromatography with silica gel and a gradient solvent of 5–35%
4.2. 3-O-(4-Methoxybenzyl)-1-O-((4-methoxyphenyl)-
diphenylmethyl)-sn-glycerol (4)
Enantiomerically pure alcohol 2 was purchased from Acros Or-
ganics, converted to diol 3 using a literature procedure,25 and the
product matched previous characterizations.28,25 Diol 3 (0.080 g,
0.377 mmol) was then dissolved in dry pyridine (5 mL), to which
ethyl acetate/hexanes to yield 6 as a colorless oil (0.732 g, 87% from
296K
4). [
a]
þ28.6 (c 1.50, CHCl3); 1H NMR (300 MHz, CDCl3):
d 7.63–
D
7.69 (m, 4H), 7.32–7.41 (m, 6H), 7.10 (d, J¼9.0 Hz, 2H), 6.81 (d,

M.M. Rowland, M.D. Best / Tetrahedron 65 (2009) 6844–6849
6847
J¼9.0 Hz, 2H), 4.24–4.32 (AB, JAB¼12 Hz, 2H), 3.88–3.96 (m, 1H),
4.7.1. 2-Cyanoethyl bis-(2-O-(tert-butyldiphenylsilyl)-3-stearyl-sn-
glycer-1-yl) phosphate (9a)
Phosphotriester 8 (0.114 g, 0.147 mmol) and stearic acid (0.167 g,
3.78 (s, 3H), 3.61–3.65 (AB, JAB¼6 Hz, 2H), 3.38–3.53 (AB of ABX,
JAB¼9 Hz, JAX¼6 Hz, JBX¼6 Hz, 2H), 2.09 (t, J¼6.0 Hz, 1H), 1.06 (s,
296K
9H); 13C NMR (100.6 MHz, CDCl3):
d
159.1, 135.8, 133.4, 130.0, 129.9,
0.588 mmol) yielded 9a as a colorless oil (0.171 g, 89%). [a]
D
129.8, 129.2, 127.7, 127.6, 113.7, 72.9, 71.9, 71.3, 64.8, 55.2, 27.0, 19.3;
þ1.66 (c 9.4, CHCl3); 1H NMR (300 MHz, CDCl3):
d 7.60–7.70 (m, 8H),
7.34–7.47 (m, 12H), 3.87–4.12 (m, 12H), 2.51 (t, J¼6.0 Hz, 2H), 2.07–
MALDI-HRMS [MþNa]þ calcd: 473.2119, found: 473.2093.
2.18 (m, 4H), 1.46–1.55 (m, 4H), 1.25 (s, 56H), 1.05 (s, 18H), 0.88 (t,
4.5. 2-Cyanoethyl bis-(3-O-(4-methoxybenzyl)-2-O-(tert-
butyldiphenylsilyl)-sn-glycer-1-yl) phosphate (7)
J¼6.0 Hz, 6H); 13C NMR (100.6 MHz, CDCl3):
d 173.1, 135.6, 135.5,
132.9, 132.7, 129.8, 129.7, 127.6, 127.5, 115.8, 69.5, 67.9, 64.2, 61.5,
29.5, 29.1, 29.0, 26.6, 24.6, 22.5, 19.2, 19.1, 13.9; 31P NMR:
d
ꢀ0.759;
Alcohol 6 (1.19 g, 2.53 mmol) and bis-N,N-diisopropylamino
cyanoethyl phosphine (0.381 g, 1.27 mmol) were stirred in 10 mL
dry methylene chloride.1H-tetrazole (5.6 mL of a 0.45 M solution in
acetontrile, 2.52 mmol) was added and the solution was allowed to
stir at rt for 48 h. To the stirred reaction mixture, tert-butylhy-
droperoxide (0.50 mL, 5.06 mmol) was added. After 1 h, the reaction
was quenched by adding 50 mL of saturated sodium thiosulfate
aqueous solution. Next, the resulting solution was extracted with
methylene chloride (2ꢁ80 mL), and the organic layers were com-
bined and dried with magnesium sulfate. The solvent was then re-
moved under reduced pressure, and the resulting residue was
purified by column chromatography with silica gel and a gradient
MALDI-HRMS [MþNa]þ calcd: 1330.8237, found: 1330.8277.
4.7.2. 2-Cyanoethyl bis-(2-O-(tert-butyldiphenylsilyl)-3-stearyl-
D35-sn-glycer-1-yl) phosphate (9b)
Phosphotriester 8 (0.200 g, 0.258 mmol) and deuterated stearic
acid D35 (0.212 g, 0.663 mmol) yielded 9b as a colorless oil
296K
(0.280 g, 71%). [
a
]
þ1.44 (c 1.9, CHCl3); 1H NMR (300 MHz,
D
CDCl3):
d
7.66 (t, J¼6.0 Hz, 8H), 7.34–7.47 (m, 12H), 3.88–4.14 (m,
12H), 2.51 (t, J¼6.0 Hz, 2H), 1.05 (s, 18H); 13C NMR (100.6 MHz,
CDCl3):
d 173.4, 135.8, 135.7, 133.1, 132.9, 130.0, 129.9, 127.8, 127.7,
116.0, 69.8, 69.7, 68.1, 68.0, 64.3, 61.7, 28.6, 28.4, 28.2, 28.0, 27.8,
26.8, 19.4, 19.3; 31P NMR:
1401.2636, found: 1401.2641.
d
ꢀ0.752; MALDI-HRMS [MþNa]þ calcd:
solvent of 20–60% ethyl acetate/hexanes to yield 7 as a colorless oil
296K
(1.09 g, 84%). [
a
]
þ12.2 (c 7.25, CHCl3); 1H NMR (300 MHz,
D
CDCl3):
d
7.65 (d, J¼9.0 Hz, 8H), 7.30–7.43 (m,12H), 7.09 (d, J¼6.0 Hz,
4.7.3. 2-Cyanoethyl bis-(2-O-(tert-butyldiphenylsilyl)-3-palmitoyl-
sn-glycer-1-yl) phosphate (9c)
Phosphotriester 8 (0.200 g, 0.258 mmol) and palmitic acid
(0.265 g, 1.03 mmol) yielded 9c as a colorless oil (0.266 g, 82%).
4H), 6.81 (d, J¼6.0 Hz, 4H), 4.22–4.29 (m, 4H), 3.88–4.06 (m, 8H),
3.78 (s, 6H), 3.34–3.44 (m, 4H), 2.43 (t, J¼6.0 Hz, 2H), 1.05 (s, 18H);
13C NMR (100.6 MHz, CDCl3):
d
159.2, 136.0, 133.7, 133.3, 130.0,
296K
129.3, 127.8 127.7, 113.8, 73.0, 71.0, 70.9, 70.3, 70.0, 69.0, 68.9, 61.6,
[a
]
þ0.14 (c 13.3, CHCl3); 1H NMR (300 MHz, CDCl3):
d 7.67 (t,
D
60.5, 55.4, 27.0, 19.4, 14.3; 31P NMR:
d
ꢀ0.534; MALDI-HRMS
J¼6.0 Hz, 8H), 7.34–7.48 (m, 12H), 3.82–4.14 (m, 12H), 2.51 (t,
J¼6.0 Hz, 2H), 2.06–2.19 (m, 4H), 1.46–1.57 (m, 4H), 1.26 (s, 48H),
1.05 (s, 18H), 0.88 (t, J¼6.0 Hz, 6H); 13C NMR (100.6 MHz, CDCl3):
[MþNa]þ calcd: 1308.4168, found: 1308.4135
4.6. 2-Cyanoethyl bis-(2-O-(tert-butyldiphenylsilyl)-sn-glycer-
d 173.8, 136.3, 136.2, 133.6, 133.5, 133.4, 130.5, 130.4, 128.3, 128.2,
1-yl) phosphate (8)
116.6, 70.3, 70.1, 68.6, 68.5, 64.9, 64.8, 62.2, 30.2, 30.0, 29.9, 29.8,
29.6, 27.3, 25.3, 23.2, 19.8, 14.6; 31P NMR:
d
ꢀ0.752; MALDI-HRMS
Phosphotriester 7 (1.09 g, 1.07 mmol) was dissolved in methy-
lene chloride (20 mL) and water (1 mL), and 2,3-dichloro-5,6-
dicyano-1,4-benzoquinone (DDQ, 1.21 g, 5.35 mmol) was added.
The reaction was then allowed to stir at rt for 12 h. Next, 60 mL of
10% sodium bicarbonate solution was added, which was extracted
with methylene chloride (2ꢁ80 mL). The organic layers were then
combined, concentrated, and the residue was purified by column
chromatography with silica gel and a gradient solvent of 5–20%
[MþNa]þ calcd: 1274.7611, found: 1274.7509.
4.7.4. 2-Cyanoethyl bis-(2-O-(tert-butyldiphenylsilyl)-3-palmitoyl-
D31-sn-glycer-1-yl) phosphate (9d)
Phosphotriester 8 (0.125 g, 0.161 mmol) and deuterated paltimic
acid D31 (0.116 g, 0.403 mmol) yielded 9d as a colorless oil (0.180 g,
296K
85%). [
a
]
þ1.56 (c 6.2, CHCl3); 1H NMR (300 MHz, CDCl3):
d 7.66
D
(t, J¼6.0 Hz, 8H), 7.34–7.45 (m, 12H), 3.87–4.15 (m, 12H), 2.51 (t,
acetone/methylene chloride to give 8 as a colorless oil (0.762 g,
J¼6.0 Hz, 2H), 1.05 (s, 18H); 13C NMR (100.6 MHz, CDCl3):
d 173.6,
296K
92%). [
d
a]
ꢀ12.6 (c 13.4, CHCl3); 1H NMR (300 MHz, CDCl3):
136.1, 136.0, 133.3, 133.2, 133.1, 130.2, 128.0, 116.3, 70.0, 69.9, 68.3,
D
7.63–7.68 (m, 8H), 7.32–7.48 (m, 12H), 3.95–4.15 (m, 6H), 3.85 (t,
64.6, 64.5, 62.0, 61.9, 28.6, 28.5, 28.4, 28.3, 27.0, 19.7, 19.6, 19.5; 31P
J¼6.0 Hz, 2H), 3.48–3.58 (m, 4H), 2.57 (t, J¼6.0 Hz, 2H), 2.49 (br s,
NMR:
d
ꢀ0.752; MALDI-HRMS [MþNa]þ calcd: 1337.1168, found:
1H),1.06 (s,18H); 13C NMR (100.6 MHz, CDCl3):
d
135.9,135.8,133.2,
1337.1210.
130.2, 128.1, 128.0, 116.4, 71.9, 71.8, 67.6, 67.5, 62.5, 62.0, 27.1, 19.4;
31P NMR:
798.2960.
d
0.378; MALDI-HRMS [MþNa]þ calcd: 798.3018, found:
4.7.5. 2-Cyanoethyl bis-(2-O-(tert-butyldiphenylsilyl)-3-myristoyl-
sn-glycer-1-yl) phosphate (9e)
Phosphotriester 8 (0.128 g, 0.165 mmol) and myristic acid
4.7. General procedure for the synthesis of compounds
of type 9
(0.151 g, 0.660 mmol) yielded 9e as a colorless oil (0.177 g, 90%).
þ1.64 (c 2.1, CHCl3); 1H NMR (300 MHz, CDCl3):
d 7.66 (t,
296K
[a
]
D
J¼6.0 Hz, 8H), 7.33–7.47 (m, 12H), 3.89–4.16 (m, 12H), 2.52 (t,
J¼6.0 Hz, 2H), 2.12–2.16 (m, 4H), 1.50–1.54 (m, 4H), 1.26 (s, 40H),
1.05 (s, 18H), 0.88 (t, J¼6.0 Hz, 6H); 13C NMR (100.6 MHz, CDCl3):
Fatty acid (4 equiv for normal fatty acid; 2.5 equiv for deuterated
fatty acid) was dissolved in methylene chloride. N,N0-Dicyclohexyl-
carbodiimide (4 equiv for normal fatty acid or 3 equiv for deuterated
fatty acid) and 4-dimethylaminopyridine (4 equiv for normal fatty
acid or 3 equiv for deuterated fatty acid) were added to the solution.
The reaction mixture was allowed to stir for 10 min, followed by the
addition of phosphotriester 8. The solution was then allowed to stir
for 12 h at rt, at which point the precipitate that formed was filtered
off and the solvent was removed under reduced pressure. The residue
was purified by column chromatography with silica gel and a gradi-
ent solvent of 30–40% ethyl acetate/hexanes to yield 9a–h.
d
173.3, 135.8, 135.7, 132.9, 130.0, 129.9, 127.8, 127.7, 116.1, 69.8, 69.6,
68.1, 68.0, 64.4, 64.3, 61.7, 33.9, 31.9, 29.1, 26.8, 24.8, 22.7, 19.3, 14.1;
31P NMR:
found: 1218.6965.
d
ꢀ0.752; MALDI-HRMS [MþNa]þ calcd: 1218.6985,
4.7.6. 2-Cyanoethyl bis-(2-O-(tert-butyldiphenylsilyl)-3-myristoyl-
D27-sn-glycer-1-yl) phosphate (9f)
Phosphotriester 8 (0.120 g, 0.155 mmol) and deuterated myristic
acid D27 (0.099 g, 0.387 mmol) yielded 9f as a colorless oil (0.164 g,

6848
M.M. Rowland, M.D. Best / Tetrahedron 65 (2009) 6844–6849
296K
85%). [
a
]
þ1.46 (c 1.6, CHCl3); 1H NMR (300 MHz, CDCl3):
d
7.66 (t,
d
d
3.78–4.28 (m, 10H), 1.27 (s, 7H), 0.83–0.92 (m, 2H); 31P NMR:
D
J¼6.0 Hz, 8H), 7.33–7.47 (m, 12H), 3.86–4.15 (m, 12H), 2.51 (t,
2.813; MALDI-HRMS [C42H12D70O10PNaþNa]þ calcd: 893.9848,
J¼6.0 Hz, 2H), 1.06 (s, 18H); 13C NMR (100.6 MHz, CDCl3):
d
173.6,
found: 893.9797.
136.1, 136.0, 133.3, 133.2, 133.1, 130.3, 130.2, 128.0, 116.3, 70.0, 69.9,
68.3, 64.5, 62.0, 34.2, 28.3, 25.2, 19.7, 19.6, 19.5; 31P NMR:
d
ꢀ0.759;
4.8.3. Bis-(3-palmitoyl-sn-glycer-1-yl) phosphate ammonium
salt (10c)
Phosphotriester 9c (0.266 g, 0.213 mmol) was treated with tet-
rabutylammonium fluoride trihydrate (1.00 g, 3.19 mmol) in 5 mL
MALDI-HRMS [MþNa]þ calcd: 1273.0084, found: 1,273.0082.
4.7.7. 2-Cyanoethyl bis-(2-O-(tert-butyldiphenylsilyl)-3-lauroyl-
sn-glycer-1-yl) phosphate (9g)
THF and yielded phosphate 10c as a white powder (0.143 g, 91%).
296K
Phosphotriester 8 (0.122 g, 0.157 mmol) and lauric acid (0.126 g,
[a
]
þ0.36 (c 1.4, MeOH/CHCl3); 1H NMR (300 MHz, CD3OD/
D
296K
0.629 mmol) yielded 9g as a colorless oil (0.158 g, 88%). [
a]
CDCl3): d 3.70–4.30 (m, 10H), 2.28–2.42 (m, 4H), 1.55–1.68 (m, 4H),
D
þ1.86 (c 5.3, CHCl3); 1H NMR (300 MHz, CDCl3):
d
7.66 (t, J¼6.0 Hz,
1.27 (s, 48H), 0.80–0.95 (m, 6H); 13C NMR (100.6 MHz, CD3OD/
8H), 7.32–7.44 (m, 12H), 3.87–4.16 (m, 12H), 2.51 (t, J¼6.0 Hz, 2H),
CDCl3):
d 174.0, 66.5, 66.4, 64.7, 33.7, 31.7, 29.5, 29.3, 29.1, 29.0, 28.7,
2.06–2.18 (m, 4H),1.50–1.54 (m, 4H), 1.26 (s, 32H),1.05 (s, 18H), 0.88
24.6, 22.4, 13.6; 31P NMR:
d
2.806; MALDI-HRMS [C38H75O10PþNa]þ
(t, J¼6.0 Hz, 6H); 13C NMR (100.6 MHz, CDCl3):
d
173.6, 136.1, 136.0,
calcd: 745.4990, found: 745.4944.
133.3, 133.1, 130.3, 130.2, 128.0, 116.3, 70.0, 69.9, 68.3, 64.6, 62.0,
61.9, 29.9, 29.7, 29.6, 29.5, 29.4, 27.0, 25.0, 22.9, 19.5, 14.4; 31P NMR:
4.8.4. Bis-(3-palmitoyl-D31-sn-glycer-1-yl) phosphate ammonium
salt (10d)
d
ꢀ0.759; MALDI-HRMS [MþNa]þ calcd: 1162.6359, found:
1162.6335.
Phosphotriester 9d (0.180 g, 0.137 mmol) was treated with tetra-
butylammoniumfluoridetrihydrate(0.648 g, 2.05 mmol)in5 mLTHF
296K
4.7.8. 2-Cyanoethyl bis-(2-O-(tert-butyldiphenylsilyl)-3-lauroyl-
D23-sn-glycer-1-yl) phosphate (9h)
Phosphotriester 8 (0.122 g, 0.157 mmol) and deuterated lauric
acid D23 (0.088 g, 0.393 mmol) yielded 9h as a colorless oil (0.172 g,
and yielded phosphate 10d as a white powder (0.051 g, 46%). [a]
D
þ0.27(c4.1, MeOH/CHCl3); 1HNMR(300 MHz,CD3OD/CDCl3):
d3.85–
4.28 (m, 10H), 1.18–1.35 (m, 2H); 31P NMR:
d 2.483; MALDI-HRMS
[C38H12D62O10PNaþNa]þ calcd: 829.8367, found: 829.8643.
296K
92%). [
a
]
þ1.60(c 1.9, CHCl3); 1H NMR (300 MHz, CDCl3):
d 7.66 (t,
D
J¼6.0 Hz, 8H), 7.33–7.46 (m, 12H), 3.86–4.18 (m, 12H), 2.51 (t,
4.8.5. Bis-(3-myristoyl-sn-glycer-1-yl) phosphate ammonium
salt (10e)
J¼6.0 Hz, 2H), 1.05 (s, 18H); 13C NMR (100.6 MHz, CDCl3):
d 173.6,
136.1,136.0,133.1, 130.2,128.0,116.3, 70.0, 69.9, 68.3, 64.5, 62.0, 61.9,
Phosphotriester 9e (0.177 g, 0.148 mmol) was treated with
tetrabutylammonium fluoride trihydrate (0.700 g, 2.22 mmol) in
34.2, 28.5, 28.3, 28.0, 27.0,19.7,19.6,19.5; 31P NMR:
d
ꢀ0.759; MALDI-
HRMS [MþNa]þ calcd: 1208.9000, found: 1208.9042.
5 mL THF and yielded phosphate 10e as a white powder (0.046 g,
296K
45%). [
a
]
þ0.96 (c 1.2, MeOH/CHCl3); 1H NMR (300 MHz,
D
4.8. General procedure for the synthesis of compound 10
CD3OD/CDCl3):
d
3.85–4.18 (m, 10H), 2.36 (t, J¼9.0 Hz, 4H), 1.55–1.70
(m, 4H), 1.27(s, 40H), 0.89 (t, J¼6.0 Hz, 6H); 13C NMR (100.6 MHz,
Phosphotriester 9 (1 equiv) and tetrabutylammonium fluoride
trihydrate (TBAF, 15–20 equiv) were dissolved in 5 mL of THF, and
the reaction was allowed to stir at rt overnight. The solvent was
then removed under reduced pressure, and the resulting residue
was dissolved in 100 mL of methanol/chloroform mixture (v/v 1:4),
and extracted with 40 mL of 8 mM aqueous ammonium acetate
solution. Next, the organic layer was concentrated, and the result-
ing residue was purified by column chromatography with 20 g
silica gel and a gradient solvent of 5–16% methanol/methylene
chloride to yield a yellowish solid as the crude product. The crude
product was then washed with water and then methanol to provide
a white solid, which was dissolved in methanol/chloroform mixture
(v/v 1:4) and filtered. The filtrate was then concentrated under
reduced pressure to yield 9 as a white powder.
CD3OD/CDCl3): d 174.4, 68.7, 66.7, 65.0, 34.1, 32.0, 29.6, 29.3, 24.9,
22.7, 14.0; 31P NMR:
d
ꢀ1.804; MALDI-HRMS [C34H67O10PþNa]þ
calcd: 689.4370, found: 689.4360.
4.8.6. Bis-(3-myristoyl-D27-sn-glycer-1-yl) phosphate ammonium
salt (10f)
Phosphotriester 9f (0.265 g, 0.212 mmol) was treated with
tetrabutylammonium fluoride trihydrate (0.969 g, 3.07 mmol) in
5 mL THF and yielded phosphate 10f as a white powder (0.030 g,
296K
19%). [
a
]
þ0.31 (c 2.3, MeOH/CHCl3); 1H NMR (300 MHz,
D
CD3OD/CDCl3):
d
d
3.85–4.25 (m, 10H), 1.20–1.36 (m, 5H); 31P NMR:
ꢀ1.931; MALDI-HRMS [C34H12D54O10PNaþNa]þ calcd: 765.7282,
found: 765.7543.
4.8.7. Bis-(3-lauroyl-sn-glycer-1-yl) phosphate ammonium
salt (10g)
Phosphotriester 9g (0.158 g, 0.139 mmol) was treated with
tetrabutylammonium fluoride trihydrate (0.875 g, 2.77 mmol) in
4.8.1. Bis-(3-stearyl-sn-glycer-1-yl) phosphate ammonium
salt (10a)
Phosphotriester 9a (0.094 g, 0.0719 mmol) was treated with
tetrabutylammonium fluoride trihydrate (0.340 g, 1.08 mmol) in
5 mL THF and yielded phosphate 10g as a white powder (0.039 g,
296K
296K
5 mL THF and yielded 10a as a white powder (0.046 g, 80%). [
a
]
45%). [
a
]
þ0.23 (c 2.0, MeOH/CHCl3); 1H NMR (300 MHz,
D
D
þ0.33 (c 1.2, MeOH/CHCl3); 1H NMR (300 MHz, CD3OD/CDCl3):
CD3OD/CDCl3):
d
3.82–4.25 (m, 10H), 2.36 (t, J¼9.0 Hz, 4H), 1.55–1.70
d
3.75–4.35 (m, 10H), 2.30–2.40 (m, 4H), 1.58–1.66 (m, 4H), 1.27 (s,
(m, 4H), 1.28 (s, 32H), 0.89(t, J¼6.0 Hz, 6H); 13C NMR (100.6 MHz,
56H), 0.83–0.92 (m, 6H); 13C NMR (100.6 MHz, CD3OD/CDCl3):
CD3OD/CDCl3): d 173.9, 68.2, 66.2, 64.6, 33.6, 31.5, 29.1, 28.8, 24.4,
d
174.1, 68.4, 66.6, 64.7, 33.8, 31.7, 29.5, 29.4, 29.3, 29.1, 29.0, 24.6,
22.2, 13.3; 31P NMR:
d
2.413; MALDI-HRMS [C30H58O10PNaþNa]þ
22.4, 13.7; 31P NMR:
d
ꢀ2.483; MALDI-HRMS [C42H83O10PþNa]þ
calcd: 655.3558, found: 655.3541.
calcd: 801.5616, found: 801.5597.
4.8.8. Bis-(3-lauroyl-D23-sn-glycer-1-yl) phosphate ammonium
salt (10h)
Phosphotriester 9h (0.260 g, 0.219 mmol) was treated with tet-
rabutylammonium fluoride trihydrate (1.38 g, 4.38 mmol) in 5 mL
4.8.2. Bis-(3-stearyl-D35-sn-glycer-1-yl) phosphate ammonium
salt (10b)
Phosphotriester 9b (0.062 g, 0.0450 mmol) was treated with
tetrabutylammonium fluoride trihydrate (0.213 g, 0.675 mmol) in
THF and yielded phosphate 10d as a white powder (0.029 g, 20%).
296K
296K
5 mL THF and yielded 10b as a white powder (0.034 g, 87%). [
a
]
[a]
þ0.60 (c 2.0, MeOH/CHCl3); 1H NMR (300 MHz, CD3OD/
D
D
þ2.03 (c 3.2, MeOH/CHCl3); 1H NMR (300 MHz, CD3OD/CDCl3):
CDCl3): d d 2.490;
3.85–4.25 (m, 10H), 1.20–1.38 (m, 15H); 31P NMR:

M.M. Rowland, M.D. Best / Tetrahedron 65 (2009) 6844–6849
6849
MALDI-HRMS [C30H12D46O10PNaþNa]þ calcd: 701.6450, found:
8. Chevallier, J.; Chamoun, Z.; Jiang, G. W.; Prestwich, G.; Sakai, N.; Matile, S.;
Parton, R. G.; Gruenberg, J. J. Biol. Chem. 2008, 283, 27871–27880.
9. Delton-Vandenbroucke, I.; Bouvier, J.; Makino, A.; Besson, N.; Pageaux, J. F.;
Lagarde, M.; Kobayashi, T. J. Lipid Res. 2007, 48, 543–552.
701.6435.
10. Le Blanc, I.; Luyet, P. P.; Pons, V.; Ferguson, C.; Emans, N.; Petiot, A.; Mayran, N.;
Demaurex, N.; Faure, J.; Sadoul, R.; Parton, R. G.; Gruenberg, J. Nat. Cell Biol.
2005, 7, 653–664.
Acknowledgements
11. Matsuo, H.; Chevallier, J.; Mayran, N.; Le Blanc, I.; 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.
12. Holopainen, J. M.; Soderlund, T.; Alakoskela, J. M.; Sailya, M.; Eriksson, O.;
Kinnunen, P. K. J. Chem. Phys. Lipids 2005, 133, 51–67.
MDB acknowledges funding from the University of Tennessee. In
addition, we acknowledge our collaborator Dr. Gail E. Fanucci
(University of Florida) for helpful discussions.
13. Hayakawa, T.; Hirano, Y.; Makino, A.; Michaud, S.; Lagarde, M.; Pageaux, J. F.;
Doutheau, A.; Ito, K.; Fujisawa, T.; Takahashi, H.; Kobayashi, T. Biochemistry
2006, 45, 9198–9209.
Supplementary data
14. Hayakawa, T.; Makino, A.; Murate, M.; Sugimoto, I.; Hashimoto, Y.; Takahashi,
H.; Ito, K.; Fujisawa, T.; Matsuo, H.; Kobayashi, T. Biophys. J. 2007, 92, L13–L15.
15. Jiang, G. W.; Xu, Y.; Prestwich, G. D. J. Org. Chem. 2006, 71, 934–939.
16. Chevallier, J.; Sakai, N.; Robert, F.; Kobayashi, T.; Gruenberg, J.; Matile, S. Org.
Lett. 2000, 2, 1859–1861.
This includes 1H, 13C and 31P NMR spectra of new compounds.
Supplementary data associated with this article can be found in the
online version, at doi:10.1016/j.tet.2009.06.085.
17. Ito, M.; Tchoua, U.; Okamoto, M.; Tojo, H. J. Biol. Chem. 2002, 277, 43674–43681.
18. Brotheru, J.; Renkonen, O. Chem. Phys. Lipids 1974, 13, 178–182.
19. Joutti, A.; Brotherus, J.; Renkonen, O.; Laine, R.; Fischer, W. Biochim. Biophys.
Acta 1976, 450, 206–209.
References and notes
20. Joutti, A.; Renkonen, O. J. Lipid Res. 1979, 20, 840–847.
21. Joutti, A. Biochim. Biophys. Acta 1979, 575, 10–15.
1. Kobayashi, T.; Stang, E.; Fang, K. S.; de Moerloose, P.; Parton, R. G.; Gruenberg, J.
Nature 1998, 392, 193–197.
22. Dang, Q. Q.; Rogalle, P.; Salvayre, R.; Dousteblazy, L. Lipids 1982, 17, 798–802.
23. Dang, Q. Q.; Rogalle, P.; Salvayre, R.; Dousteblazy, L. Biochim. Biophys. Acta 1985,
834, 124–129.
24. Jiang, G. W.; Xu, Y.; Falguieres, T.; Gruenberg, J.; Prestwich, G. D. Org. Lett. 2005,
7, 3837–3840.
2. Kobayashi, T.; Beuchat, M. H.; Chevallier, J.; Makino, A.; Mayran, N.; Escola, J. M.;
Lebrand, C.; Cosson, P.; Gruenberg, J. J. Biol. Chem. 2002, 277, 32157–32164.
3. Wilkening, G.; Linke, T.; Sandhoff, K. J. Biol. Chem. 1998, 273, 30271–30278.
4. Linke, T.; Wilkening, G.; Sadeghlar, F.; Mozcall, H.; Bernardo, K.; Schuchman, E.;
Sandhoff, K. J. Biol. Chem. 2001, 276, 5760–5768.
25. Perly, B.; Dufourc, E. J.; Jarrell, H. C. J. Labelled Compd. Radiopharm. 1984, 21, 1–13.
26. Qin, D. H.; Byun, H. S.; Bittman, R. J. Am. Chem. Soc. 1999, 121, 662–668.
27. Smith, M. D.; Gong, D. H.; Sudhahar, C. G.; Reno, J. C.; Stahelin, R. V.; Best, M. D.
Bioconjugate Chem. 2008, 19, 1855–1863.
5. Wilkening, G.; Linke, T.; Uhlhorn-Dierks, G.; Sandhoff, K. J. Biol. Chem. 2000,
275, 35814–35819.
6. Werth, N.; Schuette, C. G.; Wilkening, G.; Lemm, T.; Sandhoff, K. J. Biol. Chem.
2001, 276, 12685–12690.
28. De Medeiros, E. F.; Herbert, J. M.; Taylor, R. J. K. J. Chem. Soc., Perkin Trans. 11991,
2725–2730.
7. Kobayashi, T.; Beuchat, M. H.; Lindsay, M.; Frias, S.; Palmiter, R. D.; Sakuraba, H.;
Parton, R. G.; Gruenberg, J. Nat. Cell Biol. 1999, 1, 113–118.