Total synthesis of two photoactivatable analogues of the growth-factor-like mediator sphingosine 1-phosphate: Differential interaction with protein targets

Article abstract of DOI:10.1021/jo034828q

The first synthesis of two photoreactive analogues of the lipid mediator and second messenger sphingosine 1-phosphate (S1P), [³²P]-labeled (2S,3R)-14-O-(4′-benzoylphenyl)- and (2S,3R)-14-O-((4′ -trifluoromethyldiazirinyl)phenyl)-(4E) -tetradece

Full text of DOI:10.1021/jo034828q

Tota l Syn th esis of Tw o P h otoa ctiva ta ble An a logu es of th e
Gr ow th -F a ctor -Lik e Med ia tor Sp h in gosin e 1-P h osp h a te:
Differ en tia l In ter a ction w ith P r otein Ta r gets
Xuequan Lu,† Sandor Cseh,‡ Hoe-Sup Byun,† Gabor Tigyi,‡ and Robert Bittman*,†
Department of Chemistry and Biochemistry, Queens College of The City University of New York,
Flushing, New York 11367-1597, and Department of Physiology, University of Tennessee Health Science
Center Memphis, Memphis, Tennessee 38163
robert_bittman@qc.edu
Received J une 13, 2003
The first synthesis of two photoreactive analogues of the lipid mediator and second messenger
sphingosine 1-phosphate (S1P), [32P]-labeled (2S,3R)-14-O-(4′-benzoylphenyl)- and (2S,3R)-14-O-
((4′-trifluoromethyldiazirinyl)phenyl)-(4E)-tetradecenyl-2-amino-3-hydroxy-1-phosphate, is described.
The interactions of these probes with the S1P type-1 receptor (S1P1) transfected into membranes
of rat hepatoma cells and with plasma proteins were analyzed. The S1P1 receptor interacted in a
specific manner with the benzophenone-containing ligand (KD ) 84 ( 10 nM vs KD for S1P ) 36 (
2 nM); in contrast, no saturable specific binding was found with the diazirine-containing ligand.
However, the same pattern was found for labeling of plasma proteins by both probes, indicating
that different parts of the S1P pharmacophore underlie the interaction of S1P with its receptor
and plasma carrier proteins.
In tr od u ction
of the substrate and the precise catalytic mechanism
remain to be elucidated.5 Elucidation of the S1P-mediated
receptor activation or of the enzymatic mechanisms
underlying S1P production are just two examples of
important molecular events that may benefit from the
availability of photoreactive S1P analogues. Binding of
S1P to serum proteins has been shown to attenuate its
biological activity.6 Identification of S1P binding proteins
in biological fluids and their binding domain could lead
to the development of synthetic peptides that offer
therapeutic applicability to block S1P-induced tumor
angiogenesis.
The benzophenone and trifluoromethylphenyldiazirine
chromophores have many useful features in common,
including high chemical stability in subdued ambient
light, photoactivation using near-UV light (λ > 350 nm),
and the ability to insert randomly into accessible C-H
bonds of amino acid residues to form photoadducts.7 In
the present study, we describe the total synthesis of two
new S1P derivatives that bear a benzophenone or a (4-
trifluoromethyl)phenoxydiazirine moiety in the sphingoid
Sphingosine 1-phosphate (S1P) is a member of the
lysophospholipid growth factor family with diverse ac-
tions in almost every cell type.1 S1P can act as an
extracellular mediator through the activation of any of
five G-protein-coupled plasma membrane receptors en-
coded by some of the endothelial differentiation gene
(EDG) family, which are named S1P(1-5).1c,2 Intracel-
lularly, when generated from sphingosine by sphingosine
kinases (SKs), S1P can also act as a second messenger
through a set of targets that are not yet fully character-
ized.3 Because of the many extra- and intracellular
targets that bind to S1P, photoactivatable analogues of
S1P would be of great practical importance provided that
the photoreactive moiety does not compromise the speci-
ficity and often high-affinity interaction of the ligand with
its binding protein. Ligand recognition of S1P by the S1P1
receptor has been partially mapped to a cluster of three
charged amino acids, but the position of the hydrophobic
tail remains unknown.4 Although residues involved in the
catalytic activity of SKs have been identified, the binding
(4) Parrill, A. L.; Wang, D.; Bautista, D. L.; Van Brocklyn, J . R.;
Lorincz, Z.; Fischer, D. J .; Baker, D. L.; Liliom, K.; Spiegel, S.; Tigyi,
G. J . Biol. Chem. 2000, 275, 39379-39384.
* To whom correpondence should be addressed. Phone: (718) 997-
3279. Fax: (718) 997-3349.
† Queens College of The City University of New York.
‡ University of Tennessee Health Science Center Memphis.
(1) (a) Pyne, S.; Pyne, N. J . Biochem. J . 2000, 349, 385-402. (b)
Spiegel, S.; Milstien, S. J . Biol. Chem. 2002, 277, 25851-25854. (c)
Hla, T. Pharmacol. Res. 2003, 47, 401-407.
(5) (a) Pitson, S. M.; Moretti, P. A.; Zebol, J . R.; Xia, P.; Gamble, J .
R.; Vadas, M. A.; D’Andrea, R. J .; Wattenberg, B. W. J . Biol. Chem.
2000, 275, 33945-33950. (b) Pitson, S. M.; Moretti, P. A.; Zebol, J . R.;
Vadas, M. A.; D’Andrea, R. J .; Wattenberg, B. W. FEBS Lett. 2001,
509, 169-173. (c) Pitson, S. M.; Moretti, P. A.; Zebol, J . R.; Zareie, R.;
Derian, C. K.; Darrow, A. L.; Qi, J .; D’Andrea, R. J .; Bagley, C. J .;
Vadas, M. A.; Wattenberg, B. W. J . Biol. Chem. 2002, 277, 49545-
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107-116. (b) Spiegel, S.; English, D.; Milstien, S. Trends Cell Biol.
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(3) (a) Maceyka, M.; Payne, S. G.; Milstien, S.; Spiegel, S. Biochim.
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Maceyka, M.; Milstien, S.; Spiegel, S. Prog. Nucleic Acid Res. Mol. Biol.
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(6) Murata, N.; Sato, K.; Kon, J .; Tomura, H.; Yanagita, M.;
Kuwabara, A.; Ui, M.; Okajima, F. Biochem. J . 2000, 352, 809-815.
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Dorma´n, G.; Prestwich, G. D. Trends Biotechnol. 2000, 18, 64-77.
10.1021/jo034828q CCC: $25.00 © 2003 American Chemical Society
Published on Web 08/12/2003
7046
J . Org. Chem. 2003, 68, 7046-7050

Synthesis of Analogues of Sphingosine 1-Phosphate
SCHEME 1. Syn th esis of ω-Hyd r oxysp h in gosin e 9a
SCHEME 2. Syn th esis of Com p ou n d 1a
a
Reagents and conditions: (a) DIAD, PPh3, 4-hydroxyben-
zophenone, CH2Cl2, 0 °C to rt; (b) 1 M NaOH, MeOH, rt; (c) 4 M
HCl, THF, rt; (d) sphingosine kinase, [γ-32P]ATP, pH 7.5, 37 °C, 1
h.
a
SCHEME 3. Syn th esis of Com p ou n d 2a
Reagents and conditions: (a) n-BuLi, THF, HMPA, (S)-Garner
aldehyde, -78 °C; (b) Li, EtNH2, THF, -78 °C; (c) Boc2O, Et3N,
dioxane; (d) Bz2O, DMAP, Py, rt; (e) TBAF, THF, rt.
chain of S1P (compounds 1 and 2, respectively) and the
characterization of their interaction with the S1P1 recep-
tor and S1P-binding proteins present in rat plasma.
These two new S1P analogues may facilitate the dissec-
tion of the molecular mechanisms involved in the specific
lipid-protein interaction taking place between S1P and
its protein targets.
Resu lts a n d Discu ssion
P r ep a r a tion of ω-Hyd r oxysp h in gosin e Der iva tive
9 (Sch em e 1). The hydroxy group of 10-undecyn-1-ol was
protected as its TBS ether 3 with TBSCl (1.5 equiv) and
imidazole (3 equiv) in CH2Cl2. The acetylide anion
derived from 3 was coupled diastereoselectively with (S)-
Garner aldehyde8 in the presence of HMPA,9 giving
erythro-isomer 4 in 67% yield and, in some reactions, a
trace amount of threo-isomer 5. Birch reduction of
erythro-4 afforded crude 2-amino-1,3-diol 6. To introduce
a benzophenone (Scheme 2) or (4-trifluoromethyl)phen-
oxydiazirinyl (Scheme 3) moiety at the terminal position
of the long-chain base, the hydroxy and amino groups
must be protected. As shown in Scheme 1, the amino
group was protected as an N-BOC carbamate to afford 7
in 84% yield (for two steps from 4). Then, the hydroxy
groups of 7 were protected as benzoyl esters, affording 8
in 91% yield. The TBS group of 8 was cleaved (TBAF,
THF) to give the ω-hydroxysphingosine intermediate 9
in 73% yield.
a
Reagents and conditions: (a) DIAD, PPh3, 4-CF3COC6H4OH
(22), CH2Cl2, rt; (b) NH2OH, Py, EtOH, 50-60 °C; (c) p-TsCl, NEt3,
DMAP, CH2Cl2, 0 °C to rt; (d) liquid NH3; (e) 1 N NaOH, MeOH,
rt; (f) I2, Et3N, MeOH, rt; (g) 4 M HCl, THF, rt; (h) sphingosine
kinase, [γ-32P]ATP, pH 7.5, 37 °C, 1 h.
diate 9, which afforded coupling product 10 in 76% yield.
After the benzoyl esters of 10 were hydrolyzed (1 M
NaOH in MeOH) and the N-BOC group of 11 was
removed by treatment with 4 M HCl in THF, sphingosine
analogue 12 was obtained in 80% yield.
Syn th esis of th e (4-Tr iflu or om eth yld ia zir in yl)-
p h en oxy S1P An a logu e, Com p ou n d 2. Compound 2
was prepared as shown in Scheme 3. Mitsunobu reaction
(DIAD/Ph3P) of 4-hydroxytrifluoroacetophenone (22) (pre-
pared by trifluoroacetylation of the Grignard reagent of
4-bromoanisole with N-(trifluoroacetyl)piperidine (20),
followed by O-demethylation of 21 with LiCl in DMF)
with 9 gave trifluoroacetophenone ether 13 in 74% yield.
The trifluoroacetyl group of 13 was converted to the (4-
Syn th esis of th e Ben zop h en on e S1P An a logu e,
Com p ou n d 1. Compound 1 was synthesized as shown
in Scheme 2. The key step is the Mitsunobu reaction
(DIAD/Ph3P)10 of 4-hydroxybenzophenone with interme-
(8) (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.
(10) For a review of the Mitsunobu reaction, see: Hughes, D. L. Org.
React. 1992, 42, 335-656.
(9) Herold, P. Helv. Chim. Acta 1988, 71, 354-362.
J . Org. Chem, Vol. 68, No. 18, 2003 7047

Lu et al.
trifluoromethyl)phenoxydiazirinyl moiety by using stan-
dard procedures.11 Briefly, a mixture of (E/Z)-oximes 14
derived from 13 was treated with p-TsCl and triethyl-
amine in the presence of a catalytic amount of DMAP in
CH2Cl2 to afford tosylates 15. Tosylates 15 were con-
verted to diaziridine 16 with liquid ammonia, and base-
catalyzed hydrolysis of benzoyl ester 16 with 10% meth-
anolic NaOH gave diol 17. Oxidation of 17 (I2, Et3N,
MeOH) gave the desired diazirine 18. Finally, the N-BOC
group of 18 was removed to give sphingosine analogue
19.
Ra d iola belin g of S1P a n d th e Sp h in gosin e P h o-
toa ctiva ta ble An a logu es. The reaction buffer contained
20 mM Tris, pH 7.5, 12 mM MgCl2, 2 mM DTT, 0.25 mM
EDTA, 5 mM NaF, 12 mM â-glycerophosphate, 1 mM
sodium pyrophosphate, 5% glycerol, and 1% protease
inhibitor cocktail. A 65 nmol sample of sphingosine or
its analogue was incubated in 1 mL of reaction buffer
containing 100 µg of protein from the cell lysate of
RH7777 cells that had been transfected with SK and 250
µCi of [γ-32P]ATP (30 Ci/mmol) at 37 °C for 1 h. The
product was extracted with a mixture of 1.6 mL of CHCl3/
MeOH/HCl (100:200:1), 1 mL of 2 M KCl, and 1 mL of
CHCl3. The organic phase was extracted again with 2 mL
of MeOH, 1 mL of CHCl3, 2 mL of 2 M KCl, and 100 µL
of NH4OH. The aqueous phase was extracted with 3 mL
of CHCl3 and 200 µL of 14 N HCl. The organic extract
was dried under a stream of N2, and the residue was
redissolved in 0.5 mL of PBS containing 1 mM BSA.
Ra d ioliga n d Bin d in g Assa y. The rat hepatoma
RH7777 cell line was obtained from ATCC commercially
and was cultured in DMEM medium supplemented with
10% fetal bovine serum (FBS). This cell line does not
express endogenous S1P1 and is nonresponsive to S1P.4
Binding of 32P-labeled S1P and the photoactivatable
analogues to intact cells was carried out in subdued light
after transient transfection of the S1P1 receptor into
RH7777 cells as described earlier.12 Scatchard analysis
of the binding curves was carried out using the Ka-
leidograph software. Each binding experiment was car-
ried out on triplicate samples and was repeated at least
three times using a new transfection of cells.
P h otoa ffin ity La belin g of P la sm a . Rat plasma
diluted to 5% (v/v) in PBS was mixed with 5 nM
photoaffinity analogue on ice. The sample was irradiated
at 365 nm on ice for 10 min using a UV lamp. Aliquots
of 10 µL were loaded on reducing 12.5% SDS-PAGE gels.
The gels were silver stained using the SilverQuest kit
and exposed to a phosphorimager screen for 24 h.
Liga n d Bin d in g P r op er ties of Com p ou n d s 1 a n d
2 on S1P 1 Recep tor s. The RH7777 cell line does not
express endogenous S1P receptors and has been used for
the heterologous expression of the S1P1 receptor.4 Bind-
ing of the S1P photoactivatable analogues to S1P1 was
tested in the dark after transient transfection of the
receptor into RH7777 cells. The dose-response curves
of S1P (Figure 1A) and compound 1 (Figure 1B) showed
saturable binding, whereas that of compound 2 did not
(Figure 1C), even though both probes were tethered to
F IGURE 1. Binding of S1P and photoactivatable analogues
to RH7777 cells transfected with S1P1 receptor: (A) concentra-
tion dependence of specific binding of S1P (the inset shows
the Scatchard plot); (B) concentration dependence of specific
binding of compound 1 (the inset is the Scatchard plot); (C)
concentration dependence of specific binding of compound 2.
Note that the binding of S1P and compound 1 shows satura-
tion, whereas the binding of compound 2 does not.
the same position of the sphingoid base. Scatchard
analysis yielded KD values of 36 ( 2 and 84 ( 10 nM for
S1P and compound 1, respectively, indicating that com-
pound 1 closely mimics the binding properties of the
endogeneous ligand for S1P1. Because of the lack of
saturable binding, the KD value could not be determined
for compound 2. These results indicate that compound 2
does not interact specifically with the high-affinity bind-
ing pocket of the S1P1 receptor. These binding studies
(11) Li, G.; Samadder, P.; Arthur, G.; Bittman, R. Tetrahedron 2001,
57, 8925-8932.
(12) Wang, D. A.; Lorincz, Z.; Bautista, D. L.; Liliom, K.; Tigyi, G.;
Parrill, A. L. J . Biol. Chem. 2001, 276, 49213-49220.
7048 J . Org. Chem., Vol. 68, No. 18, 2003

Synthesis of Analogues of Sphingosine 1-Phosphate
likely to fuel future studies aimed at the identification
of the binding sites of the biological targets of S1P at the
molecular level, which will also provide important infor-
mation for the synthesis of drugs selectively targeting
proteins that bind S1P and regulate its biological activity.
Exp er im en ta l Section
11-[(ter t-Bu tyld im eth ylsilyl)oxy]-1-u n d ecyn e (3). This
compound was prepared by a modification of a reported
procedure.14 To a solution of tert-butyldimethylsilyl chloride
(4.52 g, 30 mmol) in dry CH2Cl2 (20 mL) was added imidazole
(4.08 g, 60 mmol) at rt under a N2 atmosphere. The mixture
was stirred for 1 h before a solution of 10-undecyn-1-ol (3.36
g, 20 mmol) in dry CH2Cl2 (10 mL) was added. The mixture
was stirred overnight, and then quenched with water (10 mL).
After the mixture was extracted with CH2Cl2 (3 × 25 mL), the
combined organic phases were washed with brine (40 mL) and
dried (MgSO4). The residue was purified by flash chromatog-
raphy to afford 3 (5.53 g, 98%) as a colorless oil: Rf 0.95
(EtOAc/hexane, 1:3); 1H NMR δ 0.01 (s, 6H), 0.85 (s, 9H), 1.23-
1.53 (m, 14H), 1.87 (s, 1H), 2.12 (m, 2H), 3.55 (m, 2H); 13C
NMR δ -5.25, 18.2, 18.3, 25.6, 25.7, 28.4, 28.5, 28.7, 29.0, 29.1,
29.3, 29.4, 29.5, 32.8, 63.1, 68.0, 84.8.
F IGURE 2. Photoaffinity labeling of normal Sprague-Dawley
and analbuminemic Nagase rat plasma with compounds 1 and
2. The labeled plasma was separated on a 12.5% SDS-PAGE
gel, silver-stained (A), and exposed to a phosphorimager screen
(B). Lanes 1 and 5: normal rat plasma. Lanes 2 and 6: normal
rat plasma labeled in the presence of 20 µM S1P. Lanes 3 and
7: analbuminemic rat plasma. Lanes 4 and 8: analbuminemic
rat plasma labeled in the presence of 20 µM S1P competitor.
suggest a highly restricted interaction between S1P1 and
its ligand that has been compromised for compound 2 but
was relatively unaffected for compound 1.
N-ter t-Bu toxyca r bon yl (4S,1′R)-2,2-Dim eth yl-4-(1′-h y-
d r oxy-12′-ter t-b u t yld im et h ylsilyloxy-2′-d od ecyn yl)ox-
a zolid in e (4). To a solution of alkyne 3 (5.08 g, 18.0 mmol)
in dry THF (150 mL) was added n-BuLi (2.5 M in hexane, 6.6
mL, 1.65 mmol) at -78 °C under a N2 atmosphere. The
mixture was stirred for 2 h before HMPA (116 mg, 114 µL,
0.65 mmol) was added. After the mixture was stirred for
another 30 min, a solution of Garner aldehyde (3.45 g, 15.0
mmol) in 15 mL of dry THF was added slowly. The solution
was stirred at -78 °C for 2.5 h before being 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 to afford
P h otoa ffin ity La belin g of Ra t P la sm a w ith Com -
p ou n d s 1 a n d 2. To map the S1P interactions with
plasma proteins, the photoactivatable analogues were
used to label normal and analbuminemic rat plasma
samples (Figure 2). The analbuminemic Nagase rats,
which were derived from the Sprague-Dawley strain,
contain reduced concentrations of albumin (19.4 mg/mL
vs 0.4 mg/mL) because of a defect in albumin synthesis
by the liver.13 Both compounds gave an identical footprint
of labeling of plasma proteins. Besides albumin, another
protein with an apparent molecular weight of 25000 was
labeled. The addition of S1P at 20 µΜ competed for the
labeling of this protein, indicative of the specific nature
of the interaction. Labeling of albumin was not affected
by 20 µΜ S1P, possibly because of the high-affinity
binding and the high concentration of albumin (∼14.5
µM) in the plasma samples.
4 (4.34 g, 67%) as a colorless oil: [R]25 -33.2° (c 5.0, CHCl3);
D
1
Rf 0.61 (EtOAc/hexane, 1:3); H NMR δ 0.01 (s, 6H), 0.85 (s,
9H), 1.23-1.53 (m, 29H), 2.15 (m, 2H), 3.54 (t, 2H, J ) 6.4
Hz), 3.89 (s, 1H), 4.08 (m, 2H), 4.51 (s, 1H); 13C NMR δ -5.25,
18.3, 18.8, 25.8, 28.3, 28.4, 28.5, 29.1, 29.4, 29.5, 32.9, 62.9,
63.3, 64.2, 65.1, 81.2, 86.6, 94.9, 99.4, 154.1; HR-MS [FAB,
MNa+] m/z calcd for C28H53NO5SiNa 534.3591, found 534.3561.
(2S,3R)-2-N-(ter t-Bu toxyca r bon yla m id o)-14-O-(ter t-bu -
tyld im eth ylsilyl)-(4E)-tetr a d ecen e-1,3,14-tr iol (7). To a
blue solution prepared from lithium (1.40 g, 200 mmol) and
dry EtNH2 (50 mL) was added a solution of 4 (5.12 g, 10 mmol)
in THF (10 mL) at -78 °C. The reaction mixture was stirred
for 4 h at -78 °C and then quenched with solid NH4Cl. After
EtNH2 was removed under reduced pressure, the aqueous
solution was extracted with CH2Cl2 (3 × 10 mL). The organic
phase was washed with water, dried (MgSO4), and concen-
trated. To the residue were added dioxane (60 mL), H2O (20
mL), Et3N (5 mL), and Boc2O (2.62 g, 12 mmol). The solution
was stirred overnight at rt before being quenched with
saturated aqueous NH4Cl solution (15 mL). The mixture was
extracted with Et2O (3 × 50 mL), and the combined organic
phases were washed with brine (100 mL), dried (MgSO4), and
concentrated. The residue was purified by chromatography
(elution with EtOAc/hexane, 1:1), providing 7 (3.97 g, 84%) as
a colorless wax: [R]25D -1.33° (c 11.7, CHCl3); Rf 0.43 (EtOAc/
Con clu sion s
A convenient method has been demonstrated for the
first synthesis of two photoactivatable S1P analogues.
This method can be readily modified to prepare a range
of compounds carrying linkers with different chain
lengths to the photoreactive moiety at the terminal
position of the long-chain base. The application of these
photoactivatable S1P agents in the present study sug-
gests that their interactions with the S1P1 receptor,
albumin, and an unidentified molecular weight 25000
novel plasma protein are affected by different parts of
the S1P pharmacophore. With respect to S1P1 recogni-
tion, we found that a benzophenone-containing derivative
of S1P but not a diazirine analogue showed competitive
displacement of S1P with nanomolar affinity from the
ligand-binding site. In contrast, similar patterns of
labeled proteins were obtained for both analogues in rat
plasma, where albumin was the predominant carrier of
the S1P analogues. The availability of these probes is
1
hexane, 1:1); H NMR δ 0.00 (s, 6H), 0.85 (s, 9H), 1.12-1.47
(m, 23 H), 1.98 (q, 2H, J ) 7.2 Hz), 3.55 (t, 2H, J ) 6.4 Hz),
3.56-3.62 (m, 2H), 3.73 (m, 1H), 3.83 (m, 2H), 4.19 (m, 1H),
5.43 (t, 1H, J ) 6.4 Hz), 5.47 (d, 1H, J ) 6.4 Hz), 5.75 (m,
1H); 13C NMR δ -5.26, 18.3, 25.8, 26.0, 28.4, 29.1, 29.4, 29.6,
(13) Nagase, S.; Shimamune, K.; Shumiya, S. Science 1979, 205,
590-591.
(14) Kalivretenos, A.; Stille, J . K.; Hegedus, L. S. J . Org. Chem.
1991, 56, 2883-2894.
J . Org. Chem, Vol. 68, No. 18, 2003 7049

Lu et al.
32.3, 32.8, 55.5, 62.3, 63.3, 74.1, 79.6, 129.2, 133.7, 156.3; MS
6.93 (d, 2H, J ) 7.2 Hz), 7.46 (t, 2H, J ) 8.0 Hz), 7.57 (t, 1H,
J ) 7.2 Hz), 7.74 (d, 2H, J ) 6.8 Hz), 7.80 (d, 2H, J ) 7.6 Hz);
13C NMR δ 24.0, 28.4, 29.0, 29.2, 29.3, 29.4, 29.5, 32.3, 55.4,
62.6, 68.3, 74.6, 79.7, 114.0, 124.0, 128.1, 129.0, 129.5, 129.7,
129.8, 131.9, 132.6, 133.8, 138.3, 156.3, 162.9, 195.7; MS (ESI)
m/z 540.3 (MH+).
(ESI) m/z 474.3 (MH+).
(2S,3R)-1,3-O-Diben zoyl-2-N-(ter t-bu toxyca r bon yla m i-
d o)-14-O-(ter t-bu tyld im eth ylsilyl)-(4E)-tetr a d ecen e-1,3,-
14-tr iol (8). (Dimethylamino)pyridine (137 mg, 0.55 mmol)
was added to a solution of 7 (1.37 g, 2.89 mmol) and benzoic
anhydride (2.61 g, 11.6 mmol) in pyridine (30 mL), and the
mixture was stirred at rt overnight. Volatiles were evaporated
under vacuum, and the residue was purified by chromatog-
raphy (elution with EtOAc/hexane, 1:6), providing 8 (1.80 g,
(2S,3R)-2-Am in o-14-O-(4′-b en zoylp h en yl)-(4E)-t et r a -
d ecen e-1,3,14-tr iol (12). To a solution of 4 M HCl (10 mL)
and THF (10 mL) was added 11 (42 mg, 0.080 mmol). The
solution was stirred at rt overnight and then neutralized with
4 M NaOH (10 mL). The product was extracted with EtOAc
(3 × 15 mL), and the combined organic layers were washed
with brine and dried (Na2SO4). The volatiles were evaporated
under vacuum, and the residue was purified by chromatog-
raphy (elution with CHCl3/MeOH/concd NH4OH, 130:25:4),
providing 12 (28 mg, 80%) as a white solid: mp 79.5-80.8 °C;
[R]25D -6.24° (c 2.43, CHCl3); Rf 0.30 (CHCl3/MeOH/concd NH4-
91%) as a colorless oil: [R]25 -4.14° (c 5.6, CHCl3); Rf 0.61
D
(EtOAc/hexane, 1:3); 1H NMR δ 0.00 (s, 6H), 0.84 (s, 9H), 1.20-
1.37 (m, 21H), 1.43 (m, 2H), 2.01 (q, 2H, J ) 7.2 Hz), 3.54 (t,
2H, J ) 6.8 Hz), 4.46 (m, 3H), 4.94 (d, 1H, J ) 9.6 Hz), 5.54
(m, 1H), 5.63 (m, 1H), 5.88 (m, 1H), 7.37 (m, 4H), 7.50 (m,
2H), 7.99 (m, 4H); 13C NMR δ -5.26, 18.4, 25.8, 26.0, 28.3,
28.7, 29.2, 29.4, 29.5, 32.3, 32.9, 52.3, 63.3, 63.6, 75.1, 79.8,
124.0, 128.3, 128.4, 129.5, 129.7, 130.0, 132.8, 133.1, 137.2,
155.3, 165.4, 166.4; MS (ESI) m/z 699.3 (MNH4+).
1
OH, 130:25:4); H NMR δ 1.26-1.50 (m, 12H), 1.79 (m, 2H),
(2S,3R)-1,3-O-Diben zoyl-2-N-(ter t-bu toxyca r bon yla m i-
d o)-(4E)-tetr a d ecen e-1,3,14-tr iol (9). A solution of TBAF (1
M, 7.2 mL) in THF (1.88 g, 7.20 mmol) was added to a solution
of 8 (1.65 mg, 2.42 mmol) in THF (80 mL). After the mixture
was stirred at rt overnight, the volatiles were evaporated
under vacuum. The residue was purified by chromatography
(elution with EtOAc/hexane, 1:6), providing 9 (1.00 g, 73%) as
2.04 (q, 2H, J ) 7.2 Hz), 3.02 (s, 1H), 3.45 (s, 4H), 3.71 (m,
2H), 4.02 (t, 2H, J ) 6.4 Hz), 4.20 (m, 1H), 5.47 (dd, 1H, J )
6.4, 15.6 Hz), 5.78 (dt, 1H, J ) 8.0, 15.6 Hz), 6.93 (d, 2H, J )
7.2 Hz), 7.46 (t, 2H, J ) 8.0 Hz), 7.57 (t, 1H, J ) 7.2 Hz), 7.74
(d, 2H, J ) 6.8 Hz), 7.80 (d, 2H, J ) 7.6 Hz); 13C NMR δ 26.0,
29.1, 29.2, 29.3, 29.4, 29.5, 32.3, 56.4, 62.4, 68.3, 73.8, 114.0,
124.0, 128.1, 128.3, 129.7, 129.8, 131.9, 132.6, 134.7, 138.3,
162.9, 195.7; HR-MS [DCI, MH+] m/z calcd for C27H38NO4
440.2801, found 440.2795.
a colorless oil: [R]25 -5.85° (c 4.80, CHCl3); Rf 0.39 (EtOAc/
D
hexane, 1:1); 1H NMR δ 1.20-1.58 (m, 23H), 2.04 (q, 2H, J )
7.2 Hz) 3.62 (t, 2H, J ) 6.8 Hz), 4.46 (m, 3H), 4.90 (d, 1H, J )
9.2 Hz), 5.54 (dd, 1H, J ) 7.2, 15.2 Hz), 5.67 (m, 1H), 5.89 (td,
1H, J ) 6.4, 15.6 Hz), 7.41 (m, 4H), 7.57 (m, 2H), 8.03 (m,
4H); 13C NMR δ 25.7, 28.3, 28.7, 29.0, 29.2, 29.3, 29.4, 32.3,
32.8, 52.4, 63.0, 63.6, 75.1, 79.9, 124.0, 128.4, 129.7, 129.8,
130.0, 133.1, 137.2, 155.3, 165.4, 166.5; HR-MS [FAB, MNa+]
m/z calcd for C33H45NO7Na 590.3094, found 590.3069.
(2S,3R)-1,3-O-Diben zoyl-2-N-(ter t-bu toxyca r bon yla m i-
d o)-14-O-((4′-t r iflu or oa cet yl)p h en yl)-(4E)-t et r a d ecen e-
1,3,14-tr iol (13). To a solution of DIAD (277 mg, 1.37 mmol)
in dry CH2Cl2 (10 mL) at 0 °C was added a solution of Ph3P
(395 mg, 1.51 mmol) in dry CH2Cl2 (5 mL). After the mixture
was stirred for 10 min, a solution of 4-hydroxytrifluoroac-
etophenone (22) (260 mg, 1.37 mmol) in dry CH2Cl2 (5 mL)
was added over a 20 min period. The reaction mixture was
stirred for another 10 min, and a solution of 9 (521 mg, 0.92
mmol) in dry CH2Cl2 (10 mL) was added over a 20 min period.
After the mixture was stirred for 10 min at 0 °C, the ice bath
was removed, and the mixture was stirred at rt overnight. TLC
(EtOAc/hexane, 1:6) indicated that 9 had disappeared. The
organic solution was concentrated, and the residue was
purified by chromatography (elution with EtOAc/hexane, 1:6),
(2S,3R)-1,3-O-Diben zoyl-2-N-(ter t-bu toxyca r bon yla m i-
d o)-14-O-(4′-ben zoylp h en yl)-(4E)-tetr a d ecen e-1,3,14-tr i-
ol (10). To a solution of DIAD (50 mg, 0.25 mmol) in dry
CH2Cl2 (10 mL) at 0 °C was added a solution of Ph3P (70 mg,
0.28 mmol) in dry CH2Cl2 (5 mL). After the mixture was stirred
for 10 min, a solution of 4-hydroxybenzophenone (56 mg, 0.28
mmol) in dry CH2Cl2 (5 mL) was added over a period of 20
min. The reaction mixture was stirred for another 10 min, and
a solution of alcohol 9 (94 mg, 0.17 mmol) in dry CH2Cl2 (10
mL) was added over a period of 20 min. After the mixture was
stirred for 10 min at 0 °C, the ice bath was removed, and the
mixture was stirred at rt overnight. TLC indicated that 9 had
disappeared. The organic solution was concentrated, and the
resulting residue was purified by chromatography (elution
with EtOAc/hexane, 1:6) to afford 10 (93 mg, 76%) as a white
wax: [R]25D -2.71° (c 2.8, CHCl3); Rf 0.42 (EtOAc/hexane, 1:3);
1H NMR δ 1.27-1.58 (m, 21H), 1.77 (m, 2H), 2.04 (q, 2H, J )
7.2 Hz), 4.02 (t, 2H, J ) 6.4 Hz), 4.42 (m, 1H), 4.50 (m, 2H),
4.87 (d, 1H, J ) 9.2 Hz), 5.54 (dd, 1H, J ) 7.2, 15.2 Hz), 5.67
(m, 1H), 5.89 (td, 1H, J ) 6.4, 15.6 Hz), 6.93 (m, 2H), 7.45 (m,
6H), 7.57 (m, 3H), 7.75 (d, 2H, J ) 7.2 Hz), 7.80 (d, 4H, J )
8.8 Hz), 8.04 (m, 4H); 13C NMR δ 22.0, 26.0, 28.3, 28.7, 29.1,
29.3, 29.4, 30.9, 32.3, 40.4, 52.3, 63.6, 75.1, 79.9, 114.0, 124.1,
128.2, 128.4, 129.7, 129.9, 130.0, 131.8, 132.6, 133.2, 137.2,
138.4, 155.3, 162.9, 165.4, 166.5, 195.6.
giving 13 (491 mg, 74%) as a colorless wax: [R]25 -2.82° (c
D
5.0, CHCl3); Rf 0.41 (EtOAc/hexane, 1:3); 1H NMR δ 1.27-1.58
(m, 21H), 1.79 (m, 2H), 2.04 (q, 2H, J ) 7.2 Hz), 4.02 (t, 2H,
J ) 6.4 Hz), 4.42 (m, 1H), 4.50 (m, 2H), 4.87 (d, 1H, J ) 9.2
Hz), 5.54 (dd, 1H, J ) 7.2, 15.2 Hz), 5.67 (m, 1H), 5.89 (dt,
1H, J ) 6.4, 15.6 Hz), 6.97 (d, 2H, J ) 9.2 Hz), 7.43 (m, 4H),
7.57 (m, 2H), 8.02 (m, 6H); 13C NMR δ 25.2, 27.5, 28.0, 28.2,
28.3, 28.6, 28.7, 31.6, 31.9, 38.9, 51.7, 62.9, 67.9, 74.5, 79.2,
114.2, 114.8 (q, J CF ) 290 Hz), 121.8, 123.4, 127.7, 129.0, 129.2,
132.0, 133.4, 136.5, 154.6, 164.4, 164.7, 165.7, 178.3 (q, J CF
34 Hz); MS (ESI) m/z 757.2 (MNH4+).
)
Ack n ow led gm en t . We thank Dr. George Kaysen
(University of California at Davis) for providing the
analbuminemic rat plasma samples. This work was
supported in part by USPHS Grants HL-16660 (R.B.)
and CA-92160 (G.T.).
(2S,3R)-2-N-(ter t-Bu toxyca r bon yla m id o)-14-O-(4′-ben -
zoylp h en yl)-(4E)-tetr a d ecen e-1,3,14-tr iol (11). To a solu-
tion of 1 M NaOH (10 mL) in methanol was added 10 (57 mg,
0.076 mmol). After the solution was stirred overnight at rt,
the volatiles were evaporated under vacuum. The residue was
purified by chromatography (elution with EtOAc/hexane, 1:6),
providing diol 11 (38.7 mg, 70%) as a white wax: [R]25D -1.21°
Su p p or tin g In for m a tion Ava ila ble: Preparation of com-
1
1
pounds 14-22 and H and 13C NMR spectra for compounds 4
(c 2.4, CHCl3); Rf 0.38 (EtOAc/hexane, 1:1); H NMR δ 1.26-
1.50 (m, 21H), 1.79 (m, 2H), 2.04 (q, 2H, J ) 7.2 Hz), 3.60 (m,
1H), 3.71 (dd, 1H, J ) 3.6, 11.6 Hz), 3.92 (dd, 1H, J ) 3.6,
11.6 Hz), 4.04 (t, 2H, J ) 6.4 Hz), 4.31 (m, 1H), 5.34 (m, 1H),
5.51 (dd, 1H, J ) 6.4, 15.6 Hz), 5.75 (dt, 1H, J ) 8.0, 15.6 Hz),
and 7-19. This material is available free of charge via the
Internet at http://pubs.acs.org.
J O034828Q
7050 J . Org. Chem., Vol. 68, No. 18, 2003