Synthesis of a photoactivatable (2S,3R)-sphingosylphosphorylcholine analogue

Article abstract of DOI:10.1021/jo050513u

The receptor for the lipid mediator sphingosylphosphorylcholine (SPC) has not yet been identified. We describe here the synthesis of the first photoaffinity analogue of SPC. This probe, which contains a ¹⁴C- isotopic label in the choline methyl groups and a photoreactive benzophenone in the long-chain base, may be a useful tool in the identification of the G protein coupled receptors that have been postulated to interact directly and specifically with SPC and in the definition of the ligand-binding sites. The key steps in the synthesis are selective reduction of the triple bond in enyne 6 to install the 4E double bond, Suzuki coupling to incorporate the benzophenone photophore at the end of the sphingoid chain, and reduction of the 2-azidoethyl phosphate headgroup of 13 followed by N,N,N-trimethylation to introduce the radiolabel into the choline moiety. The synthesis was completed by the release of the amino group at C2 of the sphingoid base of SPC analogue 2.

Full text of DOI:10.1021/jo050513u

Synthesis of a Photoactivatable
(2S,3R)-Sphingosylphosphorylcholine Analogue
Xuequan Lu and Robert Bittman*
Department of Chemistry and Biochemistry, Queens College of The City University of New York,
Flushing, New York 11367-1597
robert_bittman@qc.edu
Received March 14, 2005
The receptor for the lipid mediator sphingosylphosphorylcholine (SPC) has not yet been identified.
We describe here the synthesis of the first photoaffinity analogue of SPC. This probe, which contains
a
14C-isotopic label in the choline methyl groups and a photoreactive benzophenone in the long-
chain base, may be a useful tool in the identification of the G protein coupled receptors that have
been postulated to interact directly and specifically with SPC and in the definition of the ligand-
binding sites. The key steps in the synthesis are selective reduction of the triple bond in enyne 6
to install the 4E double bond, Suzuki coupling to incorporate the benzophenone photophore at the
end of the sphingoid chain, and reduction of the 2-azidoethyl phosphate headgroup of 13 followed
by N,N,N-trimethylation to introduce the radiolabel into the choline moiety. The synthesis was
completed by the release of the amino group at C2 of the sphingoid base of SPC analogue 2.
Introduction
potential pathophysiological roles in angiogenesis (the
growth of new capillary blood vessels)7 and in enhance-
ment of the elasticity of human epithelial tumor cells.8
Several putative receptors for SPC have been sug-
gested.6,9 Several years ago, it was concluded that the
multiple cellular functions of SPC were directly trans-
duced via a G protein-coupled receptor called GPR4.10
However, this conclusion was retracted recently.11 Nev-
Sphingosylphosphorylcholine (SPC or lysosphingomy-
elin, 1) is formed by N-deacylation of sphingomyelin
(SPM).1 SPC is a natural component of blood plasma2 and
high-density lipoproteins.3 It accumulates in the brain
of patients with Niemann-Pick type A disease4 and in
the malignant ascites of patients with ovarian cancer.5
SPC participates in the regulation of many cellular
functions, including proliferation, cell migration, smooth
muscle contraction, and wound healing.6 SPC also has
(6) (a) Xu, Y. Biochim. Biophys. Acta 2002, 1582, 81-88. (b) Meyer
zu Heringdorf, D. M.; Himmel, H. M.; Jakobs, K. H. Biochim. Biophys.
Acta 2002, 1582, 178-189. (c) Kye, K. C.; Chae, E. K.; Piao, Y. J.; Park,
S.; Park, J. K.; Kim, C. D.; Lee, J. H.; Suhr, K. B. J. Invest. Dermatol.
2004, 122, 1365-1371.
(7) Kim, K.-S.; Ren, J.; Jiang, Y.; Ebrahem, Q.; Tipps, R.; Cristina,
K.; Xiao, Y.-J.; Qiao, J.; Taylor, K. L.; Lum, H.; Anand-Apte, B.; Xu,
Y. FASEB J. 2005, 19, 819-821.
* To whom correspondence should be addressed. Phone: (718) 997-
3279. Fax: (718) 997-3349.
(1) Ito, M.; Kita, K.; Kurita, T.; Sueyoshi, N.; Izu, H. Methods
Enzymol. 2000, 311, 297-303.
(2) Liliom, K.; Sun, G.; Bunemann, M.; Virag, T.; Nusser, N.; Baker,
D. L.; Wang, D. A.; Fabian, M. J.; Brandts, B.; Bender, K.; Eickel, A.;
Malik, K. U.; Miller, D. D.; Desiderio, D. M.; Tigyi, G.; Pott, L. Biochem.
J. 2001, 355, 189-197.
(8) Beil, M.; Micoulet, A.; von Wichert, G.; Paschke, S.; Walther, P.;
Omary, M. B.; Van Veldhoven, P. P.; Gern, U.; Wolff-Hieber, E.;
Eggermann, J.; Waltenberger, J.; Adler, G.; Spatz, J.; Seufferlein, T.
Nat. Cell Biol. 2003, 5, 803-811.
(9) (a) Xu, Y.; Zhu, K.; Hong, G.; Wu, W.; Baudhuin, L. M.; Xiao, Y.;
Damron, D. S. Nat. Cell Biol. 2000, 2, 261-267. (b) Ignatov, A.; Lintzel,
J.; Hermans-Borgmeyer, I.; Kreienkamp, H. J.; Joost, P.; Thomsen,
S.; Methner, A.; Schaller, H. C. J. Neurosci. 2003, 23, 907-914.
(10) Zhu, K.; Baudhuin, L. M.; Hong, G.; Williams, F. S.; Cristina,
K. L.; Kabarowski, J. H.; Witte, O. N.; Xu, Y. J. Biol. Chem. 2001,
276, 41325-41335.
(3) Nofer, J. R.; Geigenmuller, S.; Gopfert, C.; Assmann, G.; Bud-
decke, E.; Schmidt, A. Biochem. Biophys. Res. Commun. 2003, 310,
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10.1021/jo050513u CCC: $30.25 © 2005 American Chemical Society
Published on Web 05/06/2005
4746
J. Org. Chem. 2005, 70, 4746-4750

Synthesis of a (2S,3R)-Sphingosylphosphorylcholine Analogue
SCHEME 1. Retrosynthetic Plan
ertheless, very recently it was proposed that SPC-induced
angiogenesis in endothelial cells is mediated by GPR4.7
Thus, the molecular identity of the SPC receptor(s) is
unclear at present. The availability of a photoreactive
SPC analogue would provide the means for identifying
the SPC receptor and help elucidate the molecular
mechanisms underlying the normal and pathophysiologi-
cal functions of SPC.
Photolabeling techniques employ molecules that are
targeted to a biological system and, upon photolysis with
UV light, form short-lived, highly reactive intermediates
that form covalent bonds with adjacent molecules. Pho-
toactivatable analogues of phospholipids have been used
to identify lipid-binding membrane proteins.12 In a previ-
ous study of photoactivatable analogues of the lipid
mediator sphingosine 1-phosphate (S1P),13 we found that
a probe containing benzophenone was bound more tightly
to a S1P receptor than a similar S1P analogue bearing a
3-trifluoromethyl-3-aryldiazirine probe, perhaps because
of an electrostatic effect.14 Therefore, the benzophenone
photophore was selected for the present study. Among
its many useful features are (a) a high degree of hydro-
phobicity, thus inserting spontaneously into membrane
bilayers; (b) chemical stability with respect to many
solvents and reaction conditions, and stability in the
absence of light; (c) photoactivatability to a triplet state
with long-wavelength UV light (λ > 350 nm), thus mini-
mizing damage to proteins; and (d) the ability to undergo
photochemical reactions by random insertion into acces-
sible CR-H bonds of amino acid residues. A radiolabel
is generally incorporated into photoprobes to allow sensi-
tive detection and identification of proteins covalently
coupled to the probe. In the present study, we describe
an efficient synthesis of a photoactivatable analogue 2
that bears a benzophenone moiety in the sphingoid chain
and N-[14C]-methyl groups in the polar headgroup.
SCHEME 2. Synthesis of Alcohol 10 via
ω-Alkyn-1-ene 5a
a Reagents and conditions: (a) PPh3, I2, imidazole, Et2O/CH3CN
(3:1), 0-5 °C to rt, overnight; (b) TMS-acetylene, n-BuLi, HMPA,
THF, -78 °C; (c) 1 N NaOH, Et2O, rt, 1 h; (d) (S)-Garner aldehyde,
n-BuLi, HMPA, THF, -78 °C, 2.5 h; (e) Red-Al, Et2O, rt, 4 h; (f)
MOMCl, EtN(Pr-i)2, CH2Cl2, 0 °C to rt; (g) (i) 9-BBN, THF, 0 °C
to rt, (ii) Pd(PPh3)4, K3PO4, 4-bromobenzophenone, H2O, dioxane,
85 °C, overnight; (h) 80% HOAc, 80 °C, 5 h.
After Red-Al reduction of progarylic alcohol 6 and protec-
tion of the secondary alcohol, the benzophenone group
was installed by Suzuki coupling. The oxazolidine ring
was opened with retention of the N-Boc group to provide
alcohol 10. Reaction with 2-azidoethyl phosphorochlori-
date 1216 and introduction of radioactivity into the choline
moiety, followed by deprotection of the O-MOM and
N-Boc groups in a one-pot reaction, completed the
synthesis of 2.
Synthesis of Alcohol 10. 9-Decen-1-ol was converted
to iodide 3 as described previously17 (Scheme 2). Alkyn-
ylation with lithium trimethylsilylacetylene afforded
intermediate 4, and removal of the TMS group provided
ω-alkyn-1-ene 5 (82% yield for two steps). The acetylide
anion derived from 5 was coupled diastereoselectively
with (S)-Garner aldehyde in the presence of HMPA,
giving erythro isomer 6 in 76% yield.15 Reduction of
propargylic alcohol 6 with 2 equiv of Red-Al afforded (4E)-
diene 7 in moderate yield; 30% of starting material 6 was
Results and Discussion
Synthetic Plan. As illustrated in the retrosynthetic
analysis (Scheme 1), our synthesis of radiolabeled pho-
toactivatable analogue 2 started with the addition of the
acetylide ion derived from enyne 5 to N-Boc-N,O-isopro-
pylidene-L-serinal ((S)-Garner aldehyde).15 Use of non-
chelation-controlled conditions afforded the requisite
2S,3R stereochemistry of the sphingoid backbone of 6.
(11) Witte, O. N.; Kabarowski, J. H.; Xu, Y.; Le, L. Q.; Zhu, K.
Science 2005, 307, 206.
(12) (a) Brunner, J. Methods Enzymol. 1989, 172, 628-687. (b)
Dorma´n, G.; Prestwich, G. D. Trends Biotechnol. 2000, 18, 64-77.
(13) Lu, X.; Cseh, S.; Byun, H.-S.; Tigyi, G.; Bittman, R. J. Org.
Chem. 2003, 68, 7046-7050.
(14) Parrill, A. L.; Sardar, V. M.; Yuan, H. Seminars Cell Dev. Biol.
2004, 15, 467-476.
(15) (a) Garner, P.; Park, J. M. Org. Synth. 1991, 70, 18-27. (b)
Liang, X.; Andersch, J.; Bols, M. J. Chem. Soc., Perkin Trans. 1 2001,
2136-2157.
(16) Pfaendler, H. R.; Weimar, V. Synthesis 1996, 1345-1349.
(17) Wang, Y.; Dong, X.; Larock, R. C. J. Org. Chem. 2003, 68, 3090-
3098.
J. Org. Chem, Vol. 70, No. 12, 2005 4747

Lu and Bittman
SCHEME 3. Synthesis of SPC Photoprobe 2a
In summary, a photoactivatable analogue of SPC
bearing a benzophenone in the sphingoid base and
radioactivity in the polar headgroup was prepared in 12
steps and 7.6% overall yield starting from Garner alde-
hyde. Suzuki coupling was used to install the photophore,
and [14C]-N-methyl groups were introduced in the pen-
ultimate step. The specific activity of 2 was 3.2 mCi/
mmol. It is anticipated that the availability of [14C]-2 will
help unravel the identity of the SPC receptor(s), the
nature of which has been elusive until now.11
Experimental Section
The general methods have been described previously.21
1-Dodecen-11-yne (5). To a solution of trimethylsilylacetyl-
ene (0.75 g, 7.5 mmol) in dry THF (20 mL) at -78 °C was
added n-BuLi (2.89 M in hexanes, 2.6 mL, 7.5 mmol) over 5
min. After 30 min, HMPA (5 mL) was added. A solution of
iodide 3 (1.33 g, 5.0 mmol) in dry THF (5 mL) was added
dropwise, the cold bath was removed, and stirring was
continued overnight at room temperature. The reaction was
quenched with saturated aqueous NH4Cl solution (20 mL), the
layers were separated, the aqueous layer was extracted with
EtOAc (3 × 10 mL), and the combined organic extracts were
washed with brine, dried (MgSO4), and evaporated to give
crude TMS-enyne 4. To a solution of crude 4 in 15 mL of Et2O
was added 15 mL of aqueous 1 N NaOH solution. The mixture
was stirred at room temperature for 1 h and then neutralized
with 15 mL of 1 M HCl. The organic layer was isolated, and
the aqueous layer was extracted with EtOAc (3 × 10 mL). The
combined organic extracts were washed with brine, dried
(MgSO4), and evaporated. Flash chromatography (elution with
hexane) gave ω-alkyn-1-ene 5 (0.67 g, 82%) as a colorless oil:
Rf 0.80 (hexane); 1H NMR (CDCl3) δ 1.24-1.56 (m, 12H), 1.93
(t, 1H, J ) 2.8 Hz), 2.04 (m, 2H), 2.17 (m, 2H), 4.94 (m, 2H),
5.80 (m, 1H); 13C NMR (CDCl3) δ 18.4, 28.5, 28.7, 28.9, 29.1,
29.3, 29.7, 33.8, 68.4, 84.8, 114.1, 139.2.
a Reagents and conditions: (a) NaN3, n-Bu4NBr, 15 h, 110 °C;
(b) POCl3, 0 °C; (c) (i) 10, pyridine, Et2O, rt (30 min), reflux (3 h),
(ii) H2O; (d) HS(CH2)3SH, Et3N, MeOH, rt; (e) (i) [14C]MeI,
NaHCO3, MeOH, 50 °C, pressure tube, 3 h, (ii) MeI (excess), 50
°C, pressure tube, 3 h; (f) 3 M HCl/THF (1:1), 70 °C, 3 h.
recovered. Use of longer reaction times or additional Red-
Al resulted in oxazolidine ring opening. Protection of the
hydroxy group of 7 as a MOM ether furnished 8, which
was subjected to hydroboration with 9-BBN (0 °C,
overnight). After unreacted 9-BBN was destroyed, Suzuki
coupling18 with 4-bromobenzophenone afforded benzo-
phenone-labeled sphingosine analogue 9 in 73% yield.
Selective deprotection of 9 with 80% HOAc at 80 °C
yielded primary alcohol 10 with retention of the N-Boc
group.19
N-tert-Butoxycarbonyl (4S,1′R)-2,2-Dimethyl-4-(1′-hy-
droxy-2′-dodecyn-11′-enyl)oxazolidine (6). To a solution
of alkyne 5 (0.51 g, 3.1 mmol) in dry THF (50 mL) was added
n-BuLi (2.5 M in hexane, 1.24 mL, 3.1 mmol) at -78 °C under
N2. The mixture was stirred for 2 h before HMPA (45 mg, 44
mL, 0.25 mmol) was added. After the mixture was stirred for
30 min, a solution of (S)-Garner aldehyde (0.57 g, 2.5 mmol)
in 5 mL of dry THF was added slowly. The solution was stirred
at -78 °C for 2.5 h and then quenched with aqueous saturated
NH4Cl solution (40 mL). The mixture was extracted with Et2O
(3 × 50 mL), and the combined organic phases were washed
with brine (100 mL) and dried (MgSO4). The crude oil was
purified by flash chromatography (EtOAc/hexane 1:3) to afford
6 (0.77 g, 76%) as a colorless oil: Rf 0.59 (EtOAc/hexane 1:3);
[R]25D -34.1 (c 1.59, CHCl3); 1H NMR (CDCl3) δ 1.24-1.65 (m,
27H), 2.04 (m, 2H), 2.20 (m, 2H), 3.91 (m, 1H), 4.11 (m, 2H),
4.52 (m, 1H), 4.94 (m, 2H), 5.80 (m, 1H); 13C NMR (CDCl3) δ
18.8, 25.8, 28.4, 28.6, 29.1, 29.4, 33.8, 60.4, 62.8, 64.2, 77.9,
81.2, 86.6, 94.9, 114.2, 139.2, 154.1; HR-MS (FAB, MNa+) m/z
calcd for C23H39NO4Na+ 416.2771, found 416.2777.
N-tert-Butoxycarbonyl (4S,1′R)-2,2-Dimethyl-4-(1′-hy-
droxy-2′,11′-dodecadienyl)oxazolidine (7). To a solution of
6 (0.68 g, 1.67 mmol) in anhydrous Et2O (20 mL) at 0 °C was
added a solution of Red-Al (0.96 mL, 70% in toluene, 3.34
mmol) dropwise. After 10 min, the cooling bath was removed,
and the reaction mixture was stirred at room temperature for
4 h. An aqueous saturated solution of NH4Cl (2 mL) was slowly
added (Caution! very exothermic). The resulting white slurry
was diluted with Et2O (10 mL), 1 N NaOH (5 mL), and water
(5 mL), and the layers were separated. The aqueous phase was
re-extracted with Et2O (3 × 5 mL), and the combined organic
Synthesis of SPC Analogue 2. To prepare 2-azido-
ethyl chlorophosphate (12),16 2-bromoethanol was reacted
with sodium azide, and the resulting 2-azidoethanol
(11)16 was added to phosphorus oxychloride (Scheme 3).
Phosphorylation was carried out by adding a solution of
alcohol 10 in Et2O to 12 in the presence of pyridine,
providing 2-azidoethyl phosphate ester 13 in good yield.
Reduction of the azido group to an amino group with
PPh3 or polymer-supported PPh3 failed but reduction
with 1,3-propanedithiol20 in the presence of dry triethyl-
amine afforded amine 14 in 85% yield. The radiolabel
was introduced into the polar headgroup of 2 by N,N-
dimethylation of 2-aminoethyl phosphate ester 14 with
∼2.5 equiv of [14C]methyl iodide in the presence of
NaHCO3 and MeOH at 50 °C in a pressure tube for 3 h.
After complete N-methylation with a large excess (20
equiv) of unlabeled methyl iodide, the solvent and excess
methyl iodide were evaporated, and the residue was
purified on a short silica gel column to provide [14C]-
phosphocholine 15 in 78% yield. Deprotection of the
N-Boc and O-MOM groups at the same time with 3 N
HCl at 70 °C gave final product 2 in 75% yield.
(18) For reviews of the Suzuki reaction, see: (a) Chemler, S. R.;
Trauner, D.; Danishefsky, S. J. Angew. Chem., Int. Ed. Engl. 2001,
40, 4544-4568. (b) Kotha, S.; Lahiri, K.; Kashinath, D. Tetrahedron
2002, 58, 9633-9695. (c) Bellina, F.; Carpita, A.; Rossi, R. Synthesis
2004, 2419-2440.
(19) Khiar, N.; Singh, K.; Garcia, M.; Martin-Lomas, M. Tetrahedron
Lett. 1999, 40, 5779-5782.
(20) Bayley, H.; Standring, D. N.; Knowles, J. R. Tetrahedron Lett.
1978, 19, 3633-3634.
(21) Chun, J.; Li, G.; Byun, H.-S.; Bittman, R. J. Org. Chem. 2002,
67, 2600-2605.
4748 J. Org. Chem., Vol. 70, No. 12, 2005

Synthesis of a (2S,3R)-Sphingosylphosphorylcholine Analogue
phase was dried (MgSO4) and concentrated. The residue was
purified by chromatography (EtOAc/hexane 1:3) to afford 396
mg (58%) of diene 7 as a colorless oil: Rf 0.54 (EtOAc/hexane
132.1, 135.0, 137.0, 138.0, 148.2, 156.0, 196.5; HR-MS (FAB,
MNa+
) m/z calcd for C35H51NO6Na+ 604.3609, found 604.3586.
2-Azidoethyl Phosphorochloridate (12). To a 50-mL
flask containing 8.86 g (58 mmol) of POCl3 was added 2.5 g
(28.7 mmol) of 11 (see the Supporting Information) dropwise
at 0 °C. The mixture was heated at 70 °C for 20 h, and the
remaining POCl3 was evaporated at room temperature (1 Torr,
2 days) to give crude 12, which was used without further
purification.
1:3); [R]25 -22.3 (c 1.51, CHCl3); 1H NMR (CDCl3) δ 1.24-
D
1.57 (m, 27H), 2.04 (m, 4H), 3.91 (m, 1H), 4.02 (m, 1H), 4.11
(m, 1H), 4.20 (m, 1H), 4.94 (m, 2H), 5.48 (m, 1H), 5.80 (m,
2H); 13C NMR (CDCl3) δ 18.7, 26.2, 28.6, 28.9, 29.1, 29.2, 29.4,
29.7, 32.4, 33.8, 60.4, 62.3, 64.9, 74.0, 81.0, 94.4, 114.1, 128.2,
133.3, 139.2, 154.1; HR-MS (FAB, MNa+) m/z calcd for C23H41-
NO4Na+ 418.2928, found 418.2926.
(2S,3R)-1-O-[2′-Azidoethyl(hydroxy)phosphoryl]-2-N-
(tert-butoxycarbonylamido)-3-O-methoxymethyl-15-(4′-
benzoylphenyl)-(4E)-pentadecene-1,3-diol (13). To a well-
dried 50-mL flask containing 202 mg (1.0 mmol) of crude 12
in 15 mL of anhydrous Et2O was added 0.16 mL (2.0 mmol) of
anhydrous pyridine. After 30 min of stirring, a solution of 200
mg (0.34 mmol) of alcohol 10 in 2 mL of Et2O was added
dropwise. The reaction mixture was stirred at room temper-
ature for 30 min, and then was heated at reflux for about 3 h
until the starting material (alcohol 10) disappeared. Water (2
mL) was added at 0 °C, and stirring was continued at room
temperature overnight. The solvent was removed, and the
residue was purified by chromatography (CHCl3/MeOH 9:1,
N-tert-Butoxycarbonyl (4S,1′R)-2,2-Dimethyl-4-(1′-meth-
oxymethoxy-2′,11′-dodecadienyl)oxazolidine (8). To a
solution of 409 mg (1.0 mmol) of alcohol 7 in 15 mL of
anhydrous CH2Cl2 were added 269 mL (1.55 mmol) of (i-
Pr)2NEt and 118 mL (1.55 mmol) of MOMCl at 0 °C. After 10
min, the cooling bath was removed, and the reaction mixture
was stirred overnight at room temperature. The reaction
mixture was poured into H2O (70 mL) and extracted with CH2-
Cl2 (3 × 50 mL). The combined organic extracts were dried
(Na2SO4) and concentrated. Purification by flash chromatog-
raphy (hexane/EtOAc 6:1) gave 417 mg (92%) of ether 8 as a
colorless oil: Rf 0.82 (EtOAc/hexane 1:3); [R]25D -73.8 (c 1.50,
CHCl3); 1H NMR (CDCl3) δ 1.24-1.60 (m, 27H), 2.04 (m, 4H),
3.36 (s, 3H), 3.92 (m, 2H), 4.07 (m, 1H), 4.28 (m, 1H), 4.51 (d,
1H, J ) 6.4 Hz), 4.73 (d, 1H, J ) 6.4 Hz), 4.94 (m, 2H), 5.32
(m, 1H), 5.69 (m, 1H), 5.80 (m, 1H); 13C NMR (CDCl3) δ 22.7,
24.9, 26.2, 28.4, 29.0, 29.4, 32.4, 33.8, 55.8, 60.3, 64.6, 76.3,
79.9, 93.7, 94.3, 114.1, 126.7, 136.8, 139.2, 152.4; HR-MS (FAB,
MNa+) m/z calcd for C25H45NO5Na+ 462.3190, found 462.3202.
then 9:2) to give 206 mg (82%) of 13 as a wax: Rf 0.48 (CHCl3/
1
MeOH 9:2); [R]25 -24.4 (c 6.40, CHCl3/MeOH 1:1); H NMR
D
(CDCl3) δ 1.26-1.65 (m, 25H), 2.03 (m, 2H), 2.68 (t, 2H, J )
7.6 Hz), 3.36 (s, 3H), 3.48 (m, 2H), 3.85 (m, 1H), 4.09 (m, 5H),
4.53 (m, 1H), 4.67 (m, 1H), 5.33 (m, 1H), 5.71 (m, 1H), 7.28 (d,
2H, J ) 7.2 Hz), 7.47 (t, 2H, J ) 7.2 Hz), 7.56 (m, 1H), 7.74
(d, 2H, J ) 7.6 Hz), 7.78 (d, 2H, J ) 7.2 Hz); 13C NMR (CDCl3)
δ 28.4, 29.1, 29.3, 29.4, 29.5, 29.6, 31.2, 32.4, 36.0, 51.3, 54.0,
55.7, 64.8, 65.3, 79.4, 93.9, 125.9, 127.9, 128.2, 128.3, 130.0,
130.3, 132.1, 135.0, 137.4, 138.0, 148.2, 155.9, 196.5; 31P NMR
(CDCl3) δ -0.28; HR-MS (FAB, MNa+) m/z calcd for C37H55N4O9-
PNa+ 753.3599, found 753.3567.
N-tert-Butoxycarbonyl (4S,1′R)-2,2-Dimethyl-4-(1′-meth-
oxymethoxy-2′-dodecene-13′-benzoylphenyl)oxazoli-
dine (9). To a solution of 363 mg (0.80 mmol) of 8 in 5 mL of
dry THF was added 1.8 mL (0.90 mmol) of a 0.5 M solution of
9-BBN in THF. The solution was stirred overnight until 8 had
completely disappeared (TLC, EtOAc/hexane 1:6). Unreacted
9-BBN was destroyed by adding 2 drops of H2O with stirring
for 10 min. To this reaction mixture was added a solution of
209 mg (0.80 mmol) of 4-bromobenzophenone in 4 mL of
dioxane, followed by Pd(PPh3)4 (28 mg, 0.024 mmol) and K3-
PO4 (0.92 g, 40 mmol). The reaction mixture was heated
overnight at reflux (85 °C). After the solvents were removed,
the residue was purified by chromatography (EtOAc/hexane
1:6), providing 9 (363 mg, 73%) as a colorless oil: Rf 0.51
(2S,3R)-1-O-[2′-Aminoethyl(hydroxy)phosphoryl]-2-N-
(tert-butoxycarbonylamido)-3-O-methoxymethyl-15-(4′-
benzoylphenyl)-(4E)-pentadecene-1,3-diol (14). To a so-
lution of azide 13 (197 mg, 0.27 mmol) in dry MeOH (5 mL)
were added dry Et3N (0.14 mL, 1.0 mmol) and 1,3-pro-
panedithiol (0.10 mL, 1.0 mmol). The solution was stirred
overnight at room temperature. A white precipitate formed,
which was removed by filtration, and the filtrate was concen-
trated. The residue was purified by chromatography (CHCl3/
MeOH 2:1) to give 162 mg (85%) of 14 as a wax: Rf 0.47
(CHCl3/MeOH 2:1); [R]25D -36.4 (c 0.78, CHCl3/MeOH 1:1); 1H
NMR (CDCl3) δ 1.26-1.65 (m, 25H), 2.03 (m, 2H), 2.68 (t, 2H,
J ) 7.6 Hz), 3.17 (m, 2H), 3.36 (s, 3H), 3.77 (m, 1H), 3.97-
4.10 (m, 5H), 4.50 (m, 1H), 4.67 (m, 1H), 5.33 (m, 1H), 5.71
(m, 1H), 7.27 (d, 2H, J ) 7.2 Hz), 7.47 (t, 2H, J ) 7.2 Hz),
7.56 (m, 1H), 7.74 (d, 2H, J ) 8.0 Hz), 7.78 (d, 2H, J ) 6.8
Hz), 8.51 (br s, 2H); 13C NMR (CDCl3) δ 28.5, 29.2, 29.4, 29.5,
29.6, 31.2, 32.4, 36.0, 40.3, 54.2, 55.6, 62.1, 64.7, 79.0, 93.7,
126.4, 127.9, 128.2, 128.3, 130.0, 130.1, 130.3, 132.1, 135.1,
137.2, 138.0, 148.2, 155.8, 196.5; 31P NMR (CDCl3) δ 0.61; HR-
MS (FAB, MNa+) m/z calcd for C37H57N2O9PNa+ 727.3694,
found 727.3690.
(2S,3R)-1-O-[2′-[14C]Trimethylaminoethyl(hydroxy)-
phosphoryl]-2-N-(tert-butoxycarbonylamido)-3-O-meth-
oxymethyl-15-(4′-benzoylphenyl)-(4E)-pentadecene-1,3-
diol (15). To a solution of 12 mg (0.017 mmol) of 14 in 2 mL
of dry MeOH in a pressure tube with a stirring bar were added
6 mg (0.043 mmol, ∼2.5 equiv) of [14C]MeI (2.0 mCi, specific
activity, 47.0 mCi/mmol) and 72 mg (0.85 mmol) of anhydrous
NaHCO3. After the tip of the tube containing [14C]MeI was
broken, the contents were transferred to the pressure tube and
the vial was washed with MeOH (3 × 0.5 mL). The pressure
tube was sealed, the contents were heated to 50 °C (no higher
than 65 °C) in an oil bath for 3 h and then cooled to 0 °C in an
ice bath, and 50 mg (0.35 mmol) of unlabeled MeI was added.
The reaction mixture was again heated to 50 °C in an oil bath
for 3 h. The reaction mixture was cooled to room temperature,
and the contents of the tube were transferred to a 25-mL
(EtOAc/hexane 1:6); [R]25 -40.6 (c 0.83, CHCl3); 1H NMR
D
(CDCl3) δ 1.27-1.65 (m, 31H), 2.13 (m, 2H), 2.68 (t, 2H, J )
7.6 Hz), 3.36 (s, 3H), 3.92 (m, 2H), 4.07 (m, 1H), 4.28 (m, 1H),
4.51 (d, 1H, J ) 6.4 Hz), 4.73 (d, 1H, J ) 6.4 Hz), 5.30 (m,
1H), 5.69 (m, 1H), 7.29 (d, 2H, J ) 6.4 Hz), 7.47 (t, 2H, J )
7.2 Hz), 7.56 (m, 1H), 7.73 (d, 2H, J ) 8.0 Hz), 7.78 (d, 2H, J
) 7.2 Hz); 13C NMR (CDCl3) δ 25.6, 28.4, 29.1, 29.4, 29.5, 31.2,
36.0, 55.8, 60.3, 64.6, 76.3, 79.9, 93.7, 94.3, 126.7, 128.2, 128.3,
130.0, 132.1, 135.0, 136.8, 137.9, 148.2, 152.4, 196.4; HR-MS
(FAB, MNa+) m/z calcd for C38H55NO6Na+ 644.3922, found
644.3951.
(2S,3R)-2-N-(tert-Butoxycarbonylamido)-3-O-methoxy-
methyl-15-(4′-benzoylphenyl)-(4E)-pentadecene-1,3-diol
(10). Oxazolidine 9 (311 mg, 0.50 mmol) was dissolved in acetic
acid (0.8 mL) and water (0.2 mL), and the mixture was stirred
at 80 °C for 5 h. The mixture was concentrated and coevapor-
ated with heptane (2 × 1 mL) to provide a residue that was
purified by chromatography (hexane/EtOAc 1:1), affording
N-Boc alcohol 10 (256 mg, 88%) as a colorless oil: Rf 0.18
(EtOAc/hexane 1:3); [R]25 -38.4 (c 0.90, CHCl3); 1H NMR
D
(CDCl3) δ 1.27-1.65 (m, 25H), 2.04 (m, 2H), 2.68 (t, 2H, J )
7.6 Hz), 2.80 (br s, 1H), 3.36 (s, 3H), 3.68 (m, 2H), 3.93 (m,
1H), 4.24 (m, 1H), 4.51 (d, 1H, J ) 6.4 Hz), 4.66 (d, 1H, J )
6.4 Hz), 5.26 (m, 1H), 5.36 (dd, 1H, J ) 8.0, 15.6 Hz), 5.73 (m,
1H), 7.29 (d, 2H, J ) 8.0 Hz), 7.47 (t, 2H, J ) 7.2 Hz), 7.57
(m, 1H), 7.73 (d, 2H, J ) 8.0 Hz), 7.78 (d, 2H, J ) 7.2 Hz); 13
C
NMR (CDCl3) δ 26.2, 26.4, 28.4, 29.0, 29.3, 29.6, 31.2, 32.3,
36.0, 55.7, 61.6, 62.4, 78.5, 79.5, 93.9, 126.0, 128.2, 128.3, 130.0,
J. Org. Chem, Vol. 70, No. 12, 2005 4749

Lu and Bittman
round-bottom flask. The tube was washed with MeOH (3 × 1
mL), and the washings were transferred to the flask. The
solvent and excess MeI were removed by evaporation. The
residue was dissolved in 10 mL of CH2Cl2/ H2O (1:1), and the
solution was transferred to a separatory funnel. The organic
layer was collected, and the aqueous layer was washed with
CH2Cl2 (3 × 3 mL). The combined CH2Cl2 layers were washed
with brine, water, dried (Na2SO4), and concentrated. The
residue was purified on a short silica gel column (3 cm); elution
was first with 20 mL of CHCl3/MeOH 9:1 (to remove an
impurity), and then with 50 mL of CHCl3/MeOH/H2O 65:25:4
(to collect the product), affording compound 15 (10 mg, 78%)
as a white wax. To ensure that the product had been eluted
completely, the fraction that was UV active and had Rf 0.23
(developed with CHCl3/MeOH/H2O 65:25:4) was monitored by
TLC. For unlabeled 15: Rf 0.23 (CHCl3/MeOH/H2O 65:25:4);
dried under vacuum. Dry MeOH (5 mL) was added with
stirring, which dissolved the product, leaving some NH4Cl
remaining as a solid. The mixture was filtered through filter
paper, which was washed with 3 mL of dry MeOH. The
combined MeOH solutions were collected, and the solvent was
removed by vacuum evaporation. The residue was dissolved
in CHCl3/MeOH/H2O (65:25:4) and loaded onto a TLC plate,
which was developed with CHCl3/MeOH/H2O (65:25:4). The
UV-active band (Rf 0.2) was scraped from the plate and
extracted with CHCl3/MeOH/H2O (65:25:4). The solution was
passed through a Cameo syringe filter (elution with CHCl3/
MeOH 65:25) to remove traces of suspended silica gel. The
solvents were removed and the residue was dried under
vacuum to give product 2 as a white solid (6.0 mg, 75%): Rf
0.20 (CHCl3/MeOH/H2O 65:25:4); mp 178.5 °C-182.3 °C; [R]25
D
-5.4 (c 0.36, CHCl3/MeOH 1:1); 1H NMR (CD3OD) δ 1.26-
1.39 (m, 14H), 1.63 (m, 2H), 2.13 (m, 2H), 2.74 (t, 2H, J ) 7.6
Hz), 3.26 (s, 9H), 3.33 (m, 1H), 3.69 (m, 2H), 4.11 (m, 2H),
4.31 (m, 3H), 5.50 (dd, 1H, J ) 8.0, 15.6 Hz), 5.88 (m, 1H),
7.37 (d, 2H, J ) 8.0 Hz), 7.55 (t, 2H, J ) 8.0 Hz), 7.66 (m,
1H), 7.74 (d, 2H, J ) 8.0 Hz), 7.77 (d, 2H, J ) 7.2 Hz); 13C
NMR (CD3OD) δ 30.2, 30.3, 30.5, 30.6, 30.7, 32.4, 33.4, 36.9,
54.7, 57.5, 60.7, 62.8, 63.6, 67.7, 70.7, 128.3, 129.5, 129.6, 130.9,
131.4, 133.7, 136.3, 137.2, 139.2, 149.9, 198.5; 31P NMR (CD3-
OD) δ -0.38; HR-MS (FAB, MNa+) m/z calcd for C33H51N2O6-
PNa+ 625.3377, found 625.3403. Radioactivity was determined
on a liquid-scintillation counter. The specific activity of [14C]-
labeled probe 2 was determined to be 3.2 mCi/mmol.
[R]25 -23.6° (c 0.75, CHCl3:MeOH 1:1); 1H NMR (CDCl3) δ
D
1.26-1.39 (m, 23H), 1.63 (m, 2H), 2.03 (m, 2H), 2.68 (t, 2H, J
) 7.6 Hz), 3.34 (s, 3H), 3.39 (s, 9H), 3.76 (m, 1H), 3.86 (m,
2H), 3.99 (m, 1H), 4.10 (m, 2H), 4.36 (m, 2H), 4.50 (d, 1H, J )
6.4 Hz), 4.67 (d, 1H, J ) 6.4 Hz), 5.33 (dd, 1H, J ) 8.0, 15.6
Hz), 5.56 (m, 1H), 5.70 (m, 1H), 7.27 (d, 2H, J ) 7.2 Hz), 7.47
(t, 2H, J ) 7.2 Hz), 7.56 (m, 1H), 7.74 (d, 2H, J ) 8.0 Hz),
7.78 (d, 2H, J ) 6.8 Hz); 13C NMR (CDCl3) δ 28.5, 29.1, 29.3,
29.5, 29.6, 31.2, 32.4, 36.0, 54.5, 55.7, 59.4, 64.2, 66.4, 78.9,
93.8, 126.2, 128.2, 128.3, 130.0, 130.1, 130.3, 132.2, 135.1,
137.2, 138.0, 148.2, 155.8, 196.6; 31P NMR (CDCl3) δ 0.61; HR-
MS (FAB, MNa+) m/z calcd for C40H63N2O9PNa+ 769.4163,
found 769.4159.
(2S,3R)-1-O-[2′-[14C]Trimethylaminoethyl(hydroxy)-
phosphoryl]-2-amino-15-(4′-benzoylphenyl)-(4E)-penta-
decene-1,3-diol (2). (Unlabeled 2 is needed for competitive
binding studies with putative membrane proteins.) For unla-
beled probe 2: A solution of 15 (10 mg, 0.013 mmol) in 5 mL
of THF and 5 mL of 3 M HCl in a 25-mL round-bottom flask
was heated at 70 °C in an oil bath for 5 h. After the solution
was cooled to room temperature, the solvents were removed
by vacuum evaporation. The residue was dried, and a drop of
concentrated NH4OH was added to neutralize the residue.
After about 5 min, NH4OH was removed, and the residue was
Acknowledgment. This work was supported in part
by National Institutes of Health Grant No. HL 16660.
Supporting Information Available: Preparation of com-
1
pound 11; H and 13C NMR spectra for compounds 2, 5-10,
and 13-15; and 31P NMR spectra for compounds 2 and 13-
15. This material is available free of charge via the Internet
at http://pubs.acs.org.
JO050513U
4750 J. Org. Chem., Vol. 70, No. 12, 2005