A modular synthesis of alkynyl-phosphocholine headgroups for labeling sphingomyelin and phosphatidylcholine

Article abstract of DOI:10.1021/jo901824h

(Figure Presented) A general route to phospho- and sphingolipids that incorporate an alkyne in the phosphocholine headgroup is described. The strategy preserves the ammonium functionality of the phosphocholine and can be easily modified to introduce desir

Full text of DOI:10.1021/jo901824h

pubs.acs.org/joc
A Modular Synthesis of Alkynyl-Phosphocholine Headgroups for
Labeling Sphingomyelin and Phosphatidylcholine
Mahendra S. Sandbhor, Jessie A. Key, Ileana S. Strelkov, and Christopher W. Cairo*
Alberta Ingenuity Centre for Carbohydrate Science, Department of Chemistry, University of Alberta,
Edmonton, Alberta T6G 2G2, Canada
ccairo@ualberta.ca
Received August 24, 2009
A general route to phospho- and sphingolipids that incorporate an alkyne in the phosphocholine
headgroup is described. The strategy preserves the ammonium functionality of the phosphocholine
and can be easily modified to introduce desired functional groups at the N-acyl chain. The targets
accessible with this strategy provide a bioorthogonal handle for postsynthetic introduction of
fluorophores or other labeling agents with aqueous phase chemistry. We report the synthesis of
sphingomyelin derivatives that incorporate a fluorophore and an alkyne. The modified sphingolipids
retain activity as substrates for sphingomyelinase, making these compounds viable probes of
enzymatic activity. Importantly, the strategy allows modification of the lipid across the phospho-
diester, making the alkyne a potential probe of sphingomyelinase activity.
Introduction
may help elucidate their function in live cells.7 Therefore,
synthetic strategies to introduce bioorthogonal labels into
these molecules are needed.8
Sphingomyelin is a critical component of the plasma
membrane bilayer in mammalian cells. Importantly, the
molecule is a substrate for several enzymes, known as
sphingomyelinases (SMase), such as the acidic and neutral
sphingomyelinases.1-3 These enzymes convert sphingomye-
lin to the signaling molecule ceramide upon cleavage of the
phosphodiester.3,4 Synthesis of various sphingomyelin ana-
logues has been reported previously in the literature.5,6 The
subcellular activity of acidic sphingomyelinases (ASM) is
poorly understood, and new fluorescent derivatives that can
be used to examine the location and rate of enzyme activity
Although stereoselective routes to the core of the lipid are
known,9,10 modifications tonaturallyisolated lipids havebeen
more frequently applied in labeling strategies. Existing stra-
tegies capable of generating fluorescently modified sphingo-
myelin include modification of the aminoacyl chain11,12 and
cross-metathesis.13,14 Recently, Lampkins et al. have demon-
strated the applicability of labeling in diacylglycerol lipids by
installing an azide handle at the acyl chain of the lipid,
followed by attachment of alkynyl fluorophores.15 To the
*To whom correspondence should be addressed. Phone: 780 492 0377.
Fax: 780 492 8231.
(7) Ghidoni, R.; Sala, G.; Giuliani, A. Biochim. Biophys. Acta, Mol. Cell
Biol. Lipids 1999, 1439, 17–39.
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Schafer, R.; Sickman, A.; Schmidt, W. E.; Schroder, J. M.; Meyer, H. E.;
Sandhoff, K.; Kronke, M. J. Biol. Chem. 2000, 275, 7641–7647.
(2) Chudakova, D. A.; Zeidan, Y. H.; Wheeler, B. W.; Yu, J.; Novgorodov,
S. A.; Kindy, M. S.; Hannun, Y. A.; Gudz, T. I. J. Biol. Chem. 2008, 283,
28806–28816.
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(9) Solladiecavallo, A.; Koessler, J. L. J. Org. Chem. 1994, 59, 3240–3242.
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311–322.
(12) Dagan, A.; Wang, C.; Fibach, E.; Gatt, S. Biochim. Biophys. Acta,
Mol. Cell Biol. Lipids 2003, 1633, 161–169.
(4) Liao, J.; Tao, J.; Lin, G.; Liu, D. Tetrahedron 2005, 61, 4715–4733.
(5) Taguchi, M.; Sugimoto, K.; Goda, K.; Akama, T.; Yamamoto, K.;
Suzuki, T.; Tomishima, Y.; Nishiguchi, M.; Arai, K.; Takahashi, K.; Kobori,
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(14) Yamamoto, T.; Hasegawa, H.; Hakogi, T.; Katsumura, S. Chem.
Lett. 2008, 37, 188–189.
(6) Yamamoto, T.; Hasegawa, H.; Ishii, S.; Kaji, S.; Masuyama, T.;
Harada, S.; Katsumura, S. Tetrahedron 2008, 64, 11647–11660.
(15) Lampkins, A. J.; O’Neil, E. J.; Smith, B. D. J. Org. Chem. 2008, 73,
6053–6058.
DOI: 10.1021/jo901824h
r
Published on Web 10/27/2009
J. Org. Chem. 2009, 74, 8669–8674 8669
2009 American Chemical Society

Sandbhor et al.
JOCArticle
best of our knowledge, the only example that uses a biocom-
patible label to modify a lipid headgroup is the introduction of
an azide at the sn-1 position of diacylglycerol; however, this
method has not yet been adapted to phosphocholyl lipids or
sphingolipids.16 Therefore, thereare no existing strategies that
allow for labeling of phosphocholyl lipids under aqueous
conditions or in the presence of other biomolecules. We
propose that strategies which introduce a chemical handle at
the cholyl headgroup could overcome this limitation and will
be of great utility in biological systems where the cleavage of
this group is of interest.
SCHEME 1. Synthesis of Model Alkyne Ammonium Analogues
General methods for the modification of the sphingolipid
headgroup are relatively sparse. Sphingomyelin analogues
that contain modified headgroups have been produced as
secondary amines,17 and related derivatives are commer-
cially available. At least one strategy has been able to
generate a modification of the quaternary ammonium group
found in the native ligand.18 We chose to pursue a general
method for producing sphingomyelin analogues that contain
a label in the phosphocholine headgroup while preserving
the ammonium group of the native compound.
Since the phosphocholine group found in sphingomyelin is
also a component of phosphatidylcholine choline lipids,
strategies for generating analogues of this headgroup should
prove instructive. Previous methods to modify the cholyl
group have relied on phosphoramidate chemistry.19 Alky-
lammonium labels have also been introduced to phosphati-
dylcholyl analogues using enzyme-catalyzed methods.20 We
sought to develop an alternative modular strategy for gener-
ating a modified trialkylammonium phosphocholine group
amenable to postsynthetic modification using fluorophores or
labeling reagents. A critical goal of our strategy is to develop
compounds which exploit bioorthogonal reactions compati-
ble with aqueous phase chemistry.8 We have developed a
phosphorylchloride strategy that provides access to trialky-
lammonium sphingolipids incorporating alkyne functionality
at the lipid headgroup. This strategy allows for reaction of the
headgroup alkyne with an azide via a click reaction. The
strategy is general and has been applied to both sphingolipids
and phospholipids, and we demonstrate routes to the corre-
sponding sphingomyelin and phosphatidylcholine choline
analogues. A fluorophore group was introduced into a sphin-
gomyelin analogue to demonstrate the feasibility of the ap-
proach. We also confirm that these modified derivatives retain
substrate activity for a sphingomyelinase enzyme.
a modified alkylammonium group of a phospho- or sphingo-
lipid substrate (Scheme 1).21 We envisioned these derivatives
could be made through a modification of known methods
that exploit cyclic chlorophosphate (2-chloro-2-oxo-1,2,3-
dioxaphospholane) 1 via phosphorylation and subsequent
nucleophilic opening of the cyclic phosphate triester inter-
mediate 2.22-25 Our initial attempts to generate an azido-
phosphocholine were impeded by the instability of the N,N-
dimethylazidoethylamine nucleophile, 3. Elevated tempera-
ture and activation with TMSOTf only provided minor
quantities of the desired product, 4. This observation is
perhaps unsurprising considering that 3 can be used as a
hypergolic fuel.26 The purification of the minor product was
difficult because of high concentrations of triflate salt impu-
rities. We attempted to avoid this issue by first converting the
azidoamine to the corresponding triazole 5 (see the Support-
ing Information), followed by subsequent amination. Unfor-
tunately, no product was observed from this reaction,
probably due to the increased steric bulk of nucleophile 5.
Therefore we changed our strategy by introducing the alkyne
handle instead of azide for use as a click reaction substrate
(Scheme 1d).
Results and Discussion
Tokeep perturbationsof the nativestructure toa minimum,
we considered small functional groups that could be used to
introduce a desired label. We considered alkyne-azide cou-
pling using copper-catalyzed Huisgen 1,3-dipolar addition to
Nucleophilic opening of the phosphate triester 2 by N,N-
dimethylpropargylamine, 6, gave the desired phosphodiester, 7,
in good yield. We then confirmed that copper-catalyzed reaction
with benzylazide could provide the 1,2,3-triazole derivative, 8, in
excellent yield. On the basis of this validation of our strategy,
we turned our attention to the synthesis of alkyne-modified
(16) Smith, M. D.; Gong, D.; Sudhahar, C. G.; Reno, J. C.; Stahelin,
R. V.; Best, M. D. Bioconjugate Chem. 2008, 19, 1855–1863.
(17) Bushnev, A. S.; Liotta, D. C. In Sphingolipid Metabolism and Cell
Signaling, Part A; Academic Press Inc: San Diego, CA, 2000; Vol. 311, pp
535-547.
(18) Gaudino, J. J.; Bjergarde, K.; Chan-Hui, P. Y.; Wright, C. D.;
Thomson, D. S. Bioorg. Med. Chem. Lett. 1997, 7, 1127–1132.
(19) Ferguson, C. G.; Bigman, C. S.; Richardson, R. D.; van Meeteren,
L. A.; Moolenaar, W. H.; Prestwich, G. D. Org. Lett. 2006, 8, 2023–2026.
(20) Rose, T. M.; Prestwich, G. D. Org. Lett. 2006, 8, 2575–2578.
(21) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed.
2001, 40, 2004–2021.
(22) Chandrakumar, N. S.; Hajdu, J. J. Org. Chem. 1982, 47, 2144–2147.
(23) Gadek, T. R. Tetrahedron Lett. 1989, 30, 915–918.
(24) Tegoulia, V. A.; Rao, W. S.; Kalambur, A. T.; Rabolt, J. R.; Cooper,
S. L. Langmuir 2001, 17, 4396–4404.
(25) Weis, A. L. Chem. Phys. Lipids 1999, 102, 3–12.
(26) Mellor, B. Proceedings of the 2nd International Conference on
Green Propellants for Space Propulsion (ESA SP-557), June 7-8, 2004;
pp 22.1-22.6.
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Sandbhor et al.
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SCHEME 2. Synthesis of Alkyne-Modified Phosphatidylcho-
line
with acyl protecting groups.29 The protected sphingosine, 11,
was reacted under similar conditions to the diacylglycerol
derivative used in Scheme 2 for both phosphorylation and
amination (Scheme 3). The protected phosphodiester, 12, was
obtained in moderate yield. Intermediate 12 is a versatile starting
material that can now be selectively modified at the N-acyl chain
and the alkynylammonium group. Initial attempts to reduce the
azide of 12 to an amine were not successful with hydrogen sulfide
or Staudinger reduction conditions.30 We found the best condi-
tions for reducing the azide were zinc and acetic acid.31,32
Subsequent acylation with an NHS-activated ester of palmitic
acid provided compound 13 in good yield. The deprotection of
the PMB ether with ceric ammonium nitrate was incomplete
even after extended reaction times. However, we observed
quantitative conversion of 13 in the presence of trifluoroacetic
acid to give the desired alkynyl-sphingomyelin, 14, in 53% yield
over 5 steps from the protected sphingosine, 11.33
To demonstrate the feasibility of introducing a fluoro-
phore label using the alkyne handle, we chose to attach a
benzoxadiazole fluorophore. Benzoxadiazole derivatives are
commonly used for labeling of lipids.34 Their small size and
environmental sensitivity are desirable attributes for these
applications.35 Introduction of the fluorophore using a
Sharpless-Meldal reaction can be performed in aqueous
media with excellent yield.36 A variety of conditions are
known for this reaction, varying in the choice of copper
source, reducing agent, base, ligand, and solvent.36,37 In our
hands, reaction of fluorescent benzoxadiazole azide, 15, with
alkyne 14 provided the fluorescent triazole 16 in quantitative
yield with copper sulfate and ascorbic acid conditions.
With sphingomyelin derivatives 14 and 16 in hand, we pro-
ceeded to test the activity of these compounds as substrates for a
sphingomyelinase enzyme. Previous reports of sphingomyelin
analogues modified at the alkylammonium group have observed
that these compounds remain substrates for the enzyme.18
Several methods are known for the assay of sphingomyelinase
activity, which include the use of radioactivity,18,38 coupled
enzyme assays,39 or thin-layer chromatography (TLC).40 Experi-
ments using commercially available coupled assays that rely on
phospho- and sphingolipids. We began the synthesis of alkynyl-
phosphatidylcholine, 10, from commercially available diacyl-
glycerol 9 (Scheme 2). Phosphorylation and amination of the
alcohol as before provided the desired alkyne-modified phos-
pholipid, 10, in moderate yield over two steps. Although this
strategy requires no protecting group manipulations, introduc-
tion of modified acyl chains in 10 would likely require protection
of one of the glycerol side chains.
We proceeded to test a modification of this strategy that
allows for elaboration of the acyl chain using sphingolipids.
Beginning from a known protected sphingosine base, 11,27 we
planned to first install the alkyne-modified phosphocholine.
Subsequent reduction of an azide would then provide a free
amine for acylation with any desired lipid chain. The protected
sphingosine base, 11, was synthesized from commercially avail-
able D-erythro-sphingosine, using the method of Du et al. with
only minor modification (see the Supporting Information).27
Briefly, sphingosine was converted to 2-azidosphingosine using
imidazole-1-sulfonyl azide hydrochloride,28 followed by protec-
tion of the primary hydroxyl group using trityl chloride and
protection of the secondary hydroxyl as a p-methoxybenzyl
(PMB) ether. Cleavage of the trityl ether with BF3OEt2 provided
compound 11. We chose to employ the PMB protecting group
to avoid migration side reactions often observed in sphingosines
SCHEME 3. Alkyne- and Triazole-Modified Sphingomyelin
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Sandbhor et al.
JOCArticle
used to introduce fluorescent groups into the lipid. Impor-
tantly, modification of the headgroup with an alkyne or
triazole moiety does not prevent cleavage of the phospho-
diester by sphingomyelinase. This strategy is unique in that it
allows for postsynthetic modification of the lipid through the
aqueous phase-exposed headgroup. Current work in our
group is aimed at developing fluorogenic versions of com-
pound 16 containing both a fluorophore and an intramolecular
quencher group. These routes will exploit compound 12 as the
key intermediate.
Experimental Section
(2-Azidoethyl)[2-(hydroxyoctyloxyphosphoryloxy)ethyl]dimethyl-
ammonium (4). NEt3 (0.36 mL, 2.6 mmol) was added slowly under
stirring to an ice cold solution of 2-chloro[1,3,2]dioxaphospholane
2-oxide 1(285 mg, 2 mmol) and freshly distilled 1-octanol (260 mg, 2
mmol) in anhydrous toluene (5 mL). The reaction was stirred at
room temperature for 4 h. The solution was filtered through a pad
FIGURE 1. Enzymatic cleavage of sphingomyelin analogues.
SMase substrates sphingomyelin (a), 14 (b), and 16 (c) were
incubated for 7 or 24 h with the enzyme. The region of the TLC
plate shown is at the Rf of the expected product of SMase hydrolysis,
ceramide (Rf 0.33). Lanes on the plate were substrate in buffer (1);
substrate, buffer, and enzyme at 7 h (2); substrate, buffer, and
enzyme at 24 h (3); buffer (4); and ceramide (5). The intensities of the
spots in lane 3 relative to the control (a) were 50% (b) and 70% (c),
respectively.
of Celite to remove the NEt3 HCl salts and the solvent was
3
evaporated under reduced pressure. The colorless oily substance 2
was obtained. 1H NMR (400 MHz, CDCl3) δ 0.87 (t, J = 6.8 Hz,
3H), 1.2-1.42 (m, 10H), 1.64-1.74 (m, 2H), 4.09-4.16 (m, 2H),
4.30-4.48 (m, 4H). 31P NMR (162 MHz, CDCl3) δ 18.69.
Compound 2 was immediately dissolved in anhydrous CH2Cl2
(10 mL) and TMSOTf (0.7 mL, 4 mmol) and 2-azidoethyldi-
methylamine (900 mg, 10 mmol) were added at 0 °C. The reaction
mixture was allowed to warm to room temperature and main-
tainedthere for12 h. The reactionmixture was dilutedwithCHCl3
(10mL) and neutralized with sat. NaHCO3. The organic layer was
choline oxidase activity gave inconsistent results in our hands,
presumably due to the structural modifications of the choline
group. We therefore employed a TLC-based assay. Cleavage of
the native sphingomyelin, alkyne 14, and triazole 16 was com-
pared over a 7-24 h period (Figure 1). The intensity of the spots
indicating product formation from compounds 14 and 16 is 0.5
and 0.7, relative to the native sphingomyelin substrate. These
results strongly support that both modified sphingomyelin deri-
vatives are substrates for the enzyme, although they appear to be
less active than the native substrate. Future work will determine
the kinetic parameters of these substrates and test the viability
of the substrates for sphingomyelinase enzymes from other
sources.
1
dried with Na2SO4 and concentrated, giving a sticky solid. H
NMR (400 MHz, CD3OD) δ 0.89 (t, J = 7.2 Hz, 3H), 1.26-1.44
(m, 10H), 1.58-1.67 (m, 2H), 3.22 (s, 6H), 3.62-3.72 (m, 4H),
3.81-3.90 (m, 2H), 3.91-4.10 (m, 2H), 4.03-4.11 (m, 2H). EIMS
351 (Mþ þ H), 373 (Mþ þ Na).
[2-(Hydroxyoctyloxyphosphoryloxy)ethyl]dimethylprop-2-ynyl-
ammonium (7). Compound 2 (47 mg, 0.2 mmol) was dissolved in
CH2Cl2 (10 mL) and TMSOTf (0.07 mL, 0.4 mmol) and N,N-
dimethylpropargylamine 6 (33 mg, 0.4 mmol) were added at 0 °C.
The reaction mixture was allowed to warm to room temperature
and maintained there for 12 h. The reaction mixture was diluted
with CHCl3, neutralized, and extracted with H2O (10 mL). The
organic layer was dried with Na2SO4 and evaporated under
reduced pressure to an oil. The crude product was purified by
column chromatography with Iatrobeads (CH2Cl2/MeOH, 4:1
then CH2Cl2/MeOH/H2O, 55:45:3) to obtain 7 (54 mg, 85%) as a
colorless oil. IR (KBr neat) 3380, 3310, 3255, 2926, 2855, 2121,
Conclusions
We describe here the development of a general route for
sphingomyelin analogues that contain modifications of the
N-acyl lipid chain and an N,N-dimethyl-N-propargylammo-
nium headgroup. As shown in Scheme 3, this method can be
1
1653, 1468, 1235, 1072 cm-1. H NMR (400 MHz, CD3OD) δ
0.88 (t, J = 7.2 Hz, 3H), 1.20-1.40 (m, 10H), 1.55-1.66 (m, 2H),
3.25 (s, 6H), 3.52 (t, J = 2.8 Hz, 1H), 3.69-3.74 (m, 2H), 3.85 (dt,
J = 6.8, 6.4 Hz, 2H), 4.22-4.30(m, 2H), 4.42 (d, J = 2.8 Hz, 2H).
13C NMR (100 MHz, CD3OD) δ 14.4, 23.7, 26.9, 30.41, 30.43,
31.9 (d, JC-P = 7.3 Hz), 33.0, 52.0, 56.4, 60.1 (d, JC-P = 4.6 Hz),
(27) Du, W. J.; Gervay-Hague, J. Org. Lett. 2005, 7, 2063–2065.
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129, 7690–7701.
67.0 (d, JC-P = 6.1 Hz), 65.3 (d, JC-P = 6.9 Hz), 72.4, 83.2. 31
P
NMR (162 MHz, CD3OD) δ 1.13. ESIMS calcd for C15H31NO4P
[M]þ 320.1985, found 320.1984.
(1-Benzyl-1H-[1,2,3]triazol-4-ylmethyl)[2-(hydroxyoctyloxy-
phosphoryloxy)ethyl]dimethylammonium (8). Alkyne 7 (20 mg,
0.06 mmol) and benzyl azide (9.9 mg, 0.07 mmol) were dissolved
in CH3CN:H2O (4 mL, 1:1). CuI (2.3 mg, 0.01 mmol) and N,N-
diisopropylethylamine (16 mg, 0.12 mmol) were added. The
reaction mixture was stirred for 6 h at room temperature. The
reaction mixture was then diluted with CHCl3 (5 mL); the organic
layer was separated, dried with Na2SO4, and concentrated under
reduced pressure. Purification by column chromatography
was performed with Iatrobeads (CH2Cl2/MeOH, 4:1 then
(33) Yan, L.; Kahne, D. Synlett 1995, 523–524.
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Dyes Pigm. 2009, 82, 196–203.
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Sandbhor et al.
JOCArticle
CH2Cl2/MeOH/H2O, 60:40:3), giving 8 (27 mg, 100%) as a color-
less oil. IR (KBr neat) 3352, 2922, 2855, 1600, 1431, 1051 cm-1. 1H
NMR (500 MHz, CD3OD) δ 0.88 (t, J = 6.8 Hz, 3H), 1.22-1.40
(m, 10H), 1.55-1.63 (m, 2H), 3.17 (s, 6H), 3.54-3.60 (m, 2H),
3.82-3.90 (m, 2H), 4.24-4.34 (m, 2H), 4.73 (s, 2H), 5.66 (s, 2H),
7.35-7.40 (m, 5H), 8.35 (s, 1H). 13C NMR (125 MHz, CD3OD) δ
14.4, 23.7, 26.9, 30.40, 30.42, 31.8 (d, JC-P = 6.7), 31.9, 32.9, 52.1,
55.2, 60.2, 60.4, 64.9, 67.0, 129.3, 129.7, 129.8, 130.1, 136.4, 137.1.
31P NMR (202 MHz, CDCl3) δ 3.7 (m). ESIMS calcd for
C22H37N4O4PNa [M þ Na]þ 475.2444, found 475.2448.
in three portions during the 24 h reaction time. After complete
conversion observed by TLC (CH2Cl2/MeOH/H2O, 55:45:3), a
new spot was observed at Rf 0.1, then the reaction mixture was
filtered through Celite and concentrated in vacuo. The remaining
AcOH was removed by azeotrope with toluene (three times). The
crude compound was dissolved in CH2Cl2 (10 mL). DMAP
(catalytic) and the NHS ester of palmitic acid (33 mg, 0.09 mmol)
were added and the solution was allowed to stir at room
temperature for 12 h; the mixture was then concentrated in vacuo
and purified by column chromatography with Iatrobeads
(CH2Cl2/MeOH, 9:1 then CH2Cl2/MeOH/H2O, 60:40:3) to give
13 (38 mg, 76% from 12). [R]25D -14.52 (c 0.9, CHCl3). IR (KBr
neat) 3291, 2953, 2917, 2850, 2124, 1644, 1515, 1467, 1247, 1071
cm-1. 1H NMR (600 MHz, CDCl3) δ 0.88 (t, J = 7.2 Hz, 6H),
1.20-1.42 (m, 46H), 1.48-1.60 (m, 2H), 2.0-2.16 (m, 4H), 2.80
(s, 1H), 3.28 (br s, 6H), 3.72 (br s, 2H), 3.78 (s, 3H), 3.90 (dd, J =
8.4, 7.8 Hz, 1H), 3.96-4.02 (m, 1H), 4.08-4.18 (m, 2H),
4.18-4.28 (m, 3H), 4.48 (d, J = 11.4 Hz, 1H), 4.54 (br s, 2H),
5.38 (dd, J = 15.6, 8.4 Hz, 1H), 5.67 (dt, J = 15.6, 6.6 Hz, 1H),
6.72 (d, J = 7.8 Hz, 1H, NH), 6.84 (d, J = 8.4 Hz, 2H), 7.22 (d,
J = 8.4 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 14.3, 22.9, 26.1,
29.5, 29.6, 29.7, 29.8, 29.9, 30.0, 32.1, 32.6, 37.2, 51.8, 53.0 (d,
{2-[(2,3-Bis(tetradecanoyloxy)propoxy)hydroxyphosphoryloxy]-
ethyl}dimethylprop-2-ynylammonium (10). A solution of freshly
distilled 2-chloro-1,3,2-dioxaphospholane 2-oxide 1 (56 mg,
0.39 mmol) in dry toluene (1 mL) was added to 1,2-dimyristoyl-
sn-glycerol 9 (68 mg, 0.13 mmol) and DMAP (catalytic) in dry
toluene (5 mL). The reaction mixture was cooled to 0 °C. NEt3
(40 mg, 0.39 mmol) was added dropwise to the reaction mixture
and allowed to stir at room temperature for 12 h. TLC (hexane/
EtOAc, 3:1) showed complete disappearance of starting material
and formation of a new spot at Rf 0.07. The reaction mixture was
filtered through a pad of Celite to remove salts and concentrated
under reduced pressure. The crude compound was dissolved in dry
CH2Cl2 (10 mL) followed by addition of 1-dimethylamino-2-
propyne 6 (110 mg, 1.3 mmol) in CH2Cl2 (2 mL). The reaction
was cooled to 0 °C and TMSOTf (117 mg, 0.52 mmol) was added
dropwise over 5 min, then the reaction mixture was allowed to stir
at room temperature for 12 h. TLC (CH2Cl2/MeOH/H2O,
70:30:1) showed the disappearance of the starting material and
formation of a new spot at Rf 0.1. Concentration in vacuo and
purification by column chromatography with Iatrobeads
(CH2Cl2/MeOH, 9:1 then CH2Cl2/MeOH/H2O, 70:30:1) gave 10
JC-P = 5.3 Hz), 55.5, 59.2 (d, JC-P = 3.7 Hz), 64.3 (d, JC-P =
4.2 Hz), 64.5 (d, JC-P = 5.0 Hz), 70.0, 72.0, 79.9, 81.4, 113.9,
127.5, 129.4, 131.2, 137.0, 159.2, 173.2. 31P NMR (162 MHz,
CDCl3) δ 1.14. ESIMS calcd for C49H87N2O7PNa [M þ Na]þ
869.6143, found 869.6144.
{2-[(2-Hexadecanoylamino-3-hydroxyoctadec-4-enyloxy)-
hydroxyphosphoryloxy]ethyl}dimethylprop-2-ynylammonium (14).
Compound 13 (11 mg, 0.013 mmol) was stirred in TFA:CH2Cl2/1:9
(5 mL) for 1 h at 0 °C. TLC (CH2Cl2/MeOH/H2O, 55:45:3) shows
complete disappearance of starting material and formation of
product at Rf 0.4. The reaction mixture was neutralized with
NH4OH and concentrated in vacuo to give 14 (9.4 mg, 100%).
[R]25D -34.51 (c0.5, CHCl3). IR (KBr neat) 3376, 3268, 2956, 2918,
(60 mg, 65%from9) as a colorless oil. [R]25 þ4.12 (c 0.5, CHCl3).
D
IR (KBr neat) 3398, 3250, 2918, 2850, 2127, 1738, 1467, 1255, 1065
cm-1. 1H NMR (500 MHz, CDCl3) δ 0.88 (t, J = 7.0 Hz, 6H),
1.25-1.45 (m, 40H), 1.55-1.64 (m, 4H), 1.85 (br s, 4H), 2.28 (t,
J = 7.5 Hz, 2H), 2.30 (t, J = 7.5 Hz, 2H), 2.86 (t, J = 2.5 Hz, 1H),
3.44 (s, 6H), 3.90 (br s, 2H), 3.91-4.03 (m, 2H), 4.15 (dd, J = 12.0,
7.0 Hz, 1H), 4.35 (br s, 2H), 4.41 (dd, J = 12.0, 3.0 Hz, 1H), 4.68
(br s, 2H), 5.20-5.26 (m, 1H). 13C NMR (125 MHz, CDCl3) δ
14.1, 22.7, 24.9, 25.0, 29.21, 29.23, 29.3, 29.4, 29.5, 29.6, 29.7, 32.0,
34.1, 34.2, 34.4, 52.0, 55.3 59.1 (d, JC-P = 5.4 Hz), 63.0, 63.5, 64.5,
70.6 (d, JC-P = 4.3), 71.8, 81.2, 173.3, 173.6. 31P NMR (202 MHz,
CDCl3) δ 0.77 (m). ESIMS calcd for C38H72NO8PNa [M þ Na]þ
724.4887, found 724.4884.
1
2850, 2125, 1660, 1468, 1240, 1089 cm-1. H NMR (600 MHz,
CDCl3) δ 0.88 (t, J = 7.2 Hz, 6H), 1.20-1.40 (m, 46H), 1.50-1.64
(m, 2H), 1.94-2.02 (m, 2H), 2.10-2.20 (m, 3H), 2.97 (s, 1H), 3.38
(s, 6H), 3.80-4.0 (m, 4H), 4.08 (dd, J = 7.2, 6.6 Hz, 1H), 4.16-4.4
(m, 3H), 4.61 (br s, 2H), 5.46 (dd, J = 15.6, 7.2 Hz, 1H), 5.66 (dt,
J = 15.6, 6.6 Hz, 1H), 6.72 (br s, 1H, NH). 13C NMR (100 MHz,
CDCl3) δ 14.1, 22.7, 25.9, 26.0, 29.2, 29.3, 29.4, 29.53, 29.58, 29.6,
29.7, 29.8, 29.82, 32.0, 32.6, 36.9, 51.6, 54.5 (JC-P = 3.3 Hz), 55.3,
59.2 (JC-P = 5.4 Hz), 64.4 (JC-P = 4.2 Hz), 65.0, 71.4, 72.0, 81.5,
129.7, 133.6, 173.2. 31P NMR (162 MHz, CDCl3) δ 1.37 (m).
ESIMS calcd for C41H79N2O6PNa [M þ Na]þ 749.5568, found
749.5572.
(1-Benzo[1,2,5]oxadiazol-5-ylmethyl-1H-[1,2,3]triazol-4-ylmethyl)-
{2-[(2-hexadecanoylamino-3-hydroxyoctadec-4-enyloxy)hydroxy-
phosphoryloxy]ethyl}dimethylammonium (16). To the solution of
alkyne 14 (3.5 mg, 0.005 mmol) in EtOH:H2O (4 mL, 1:1) were
added the azide 15 (1.68 mg, 0.01 mmol), CuSO4 (0.15 mg, 0.001
mmol), and ascorbic acid (0.25 mg, 0.0014 mmol). After 10 min
formation of precipitate was observed in the reaction mixture.
TLC (CH2Cl2/MeOH/H2O, 55:45:3) showed that both the start-
ing material and the UV active product appear at the same Rf
(0.4). Therefore, the reaction was allowed to stir for 12 h to ensure
complete conversion. The product was collected by filtration and
further purified by column chromatography with Iatrobeads
(CH2Cl2/MeOH, 9:1 then CH2Cl2/MeOH/H2O, 55:45:3) to ob-
tain 16 (4.55 mg, 100%) as a white solid, mp 118-120 °C. IR
(KBr neat) 3364, 3123, 3076, 2925, 2109, 1637, 1541, 1289, 1008,
881, 798, 756 cm-1. 1H NMR (500 MHz, CD3OD) δ 0.90 (t, J =
7.0 Hz, 6H), 1.20-1.40 (m, 46H), 1.48-1.64 (m, 3H), 2.01 (dt,
J = 7.0, 6.5 Hz, 2H), 2.12-2.22 (m, 2H), 3.20 (s, 6H), 3.54-3.68
(m, 4H), 3.80-3.88 (m, 1H), 3.90-4.14 (m, 4H), 4.33 (br s, 2H),
4.78 (s, 2H), 5.44 (dd, J = 15.0, 7.5 Hz, 1H), 5.7 (dt, J = 15.0,
7.0 Hz, 1H), 6.82-6.88 (br s, 1H, NH), 7.49 (d, J = 9.5 Hz, 1H),
(2-{[2-Azido-3-(4-methoxybenzyloxy)octadec-4-enyloxy]hydroxy-
phosphoryloxy}ethyl)dimethylprop-2-ynylammonium (12). Com-
pound 12 was prepared from azido sphingosine 11 (172 mg, 0.38
mmol) employing the same procedure as described for 10. Purifica-
tion by column chromatography with Iatrobeads (CH2Cl2/MeOH,
9:1 then CH2Cl2/MeOH/H2O, 55:45:3) gave 12 (168 mg, 70% from
the azido sphingosine 11). [R]25D -22.78 (c 2.6, CHCl3). IR (KBr
neat) 3389, 2924, 2853, 2101, 1613, 1514, 1467, 1249, 1073 cm-1. 1H
NMR (500 MHz, CDCl3) δ 0.87 (t, J = 7.0 Hz, 3H), 1.20-1.35 (m,
20H), 1.34-1.44 (m, 2H), 2.08 (apparent q, J= 6.5 Hz, 2H), 2.91 (s,
1H), 3.33 (s, 6H), 3.66-3.72 (m, 1H), 3.76-3.72 (m, 6H), 4.0-4.08
(m, 2H), 4.22-4.34 (m, 3H), 4.49 (d, J = 11.5 Hz, 1H), 4.61 (br s,
2H), 5.37 (dd,
J
=
15.5, 7.5 Hz, 1H), 5.71 (dt,
J = 15.5, 6.5 Hz, 1H), 6.84 (d, J = 8.5 Hz, 2H), 7.21 (d, J = 8.5
Hz, 2H). 13C NMR (125 MHz, CDCl3) δ 14.1, 22.7, 29.1, 29.3, 29.4,
29.5, 29.7, 29.72, 31.9, 32.4, 51.4, 55.2, 55.3, 59.2 (JC-P = 3.7 Hz),
63.9 (JC-P = 4.5 Hz), 65.0 (JC-P = 4.1 Hz), 65.4 (d, J = 7.0 Hz),
69.5, 72.2, 79.4, 81.2, 113.8, 125.8, 129.3, 130.1, 138.1, 159.1. 31P
NMR (201 MHz, CDCl3) δ 0.54. ESIMS calcd for C33H55N4O6P-
Na [M þ Na]þ 657.3751, found 657.3745.
(2-{[2-Hexadecanoylamino-3-(4-methoxybenzyloxy)octadec-4-
enyloxy]hydroxyphosphoryloxy}ethyl)dimethylprop-2-ynylammo-
nium (13). To the solution of azide 12 (40 mg, 0.06 mmol) in
AcOH:H2O/5:1 (5 mL) was added zinc powder (50 mg, 0.75 mmol)
J. Org. Chem. Vol. 74, No. 22, 2009 8673

Sandbhor et al.
JOCArticle
7.85 (s, 1H), 7.93 (d, J = 9.5 Hz, 1H), 8.50 (s, 1H). 13C NMR
(100 MHz, CDCl3) δ 14.4, 23.7, 27.1, 30.41, 30.5, 30.6, 30.7, 30.8,
33.1, 33.4, 37.4, 52.2, 54.4 (JC-P = 3.3 Hz), 55.3, 60.4 (JC-P =
Science (AICCS), and the University of Alberta. We would
like to thank M. Richards and N. Tulsi for editorial assis-
tance, W. Moffat and the University of Alberta Spectral
Services for support, and the Canadian Foundation for
Innovation for infrastructure support.
5.4 Hz), 71.5, 72.6, 116.3, 118.3, 130.4, 131.2, 133.2, 135.1, 137.3,
141.0, 150.1, 176.0, 31P NMR (162 MHz, CDCl3) δ 1.04.
ESIMS calcd for C48H84N7O7PNa [M þ Na]þ 924.6062, found
924.6060.
Supporting Information Available: Experimental proce-
dures for the preparation of compounds 5, 11, and 15, support-
ing spectral data, and details of enzymatic experiments. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Acknowledgment. This work was funded by the Natural
Sciences and Engineering Research Council of Canada
(NSERC), the Alberta Ingenuity Centre for Carbohydrate
8674 J. Org. Chem. Vol. 74, No. 22, 2009