Design and synthesis of caged ceramide: UV-responsive ceramide releasing system based on UV-induced amide bond cleavage followed by O-N acyl transfer

Article abstract of DOI:10.1016/j.tet.2011.04.048

Sphingolipids, recognized as membrane constructs and as key signaling molecules, have been studied to examine intracellular function. Some caged sphingolipids that release parent sphingolipids after exposure to UV-irradiation have been previously developed, but caged ceramide has yet to be reported. In this study, we report the design and synthesis of a caged ceramide. Photo-irradiation experiment clarified that the caged ceramide can be successfully converted to the parent ceramide by UV-irradiation. Introduction of an alkyne-handle moiety for further modification of the caged ceramide is also reported.

Full text of DOI:10.1016/j.tet.2011.04.048

Tetrahedron 67 (2011) 3984e3990
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Design and synthesis of caged ceramide: UV-responsive ceramide releasing
system based on UV-induced amide bond cleavage followed by OeN acyl transfer
,
a
a
a
a
a
Akira Shigenaga a , Hiroko Hirakawa , Jun Yamamoto , Keiji Ogura , Masaya Denda , Keiko Yamaguchi ,
*
Daisuke Tsuji b, Kohji Itoh b, Akira Otaka a
,
*
a Institute of Health Biosciences and Graduate School of Pharmaceutical Sciences, The University of Tokushima, Shomachi, Tokushima 770-8505, Japan
b Department of Medicinal Biotechnology, Institute for Medicinal Research, Graduate School of Pharmaceutical Sciences, The University of Tokushima, Shomachi,
Tokushima 770-8505, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Sphingolipids, recognized as membrane constructs and as key signaling molecules, have been studied to
examine intracellular function. Some caged sphingolipids that release parent sphingolipids after expo-
sure to UV-irradiation have been previously developed, but caged ceramide has yet to be reported. In this
study, we report the design and synthesis of a caged ceramide. Photo-irradiation experiment clarified
that the caged ceramide can be successfully converted to the parent ceramide by UV-irradiation. In-
troduction of an alkyne-handle moiety for further modification of the caged ceramide is also reported.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 16 March 2011
Received in revised form 9 April 2011
Accepted 12 April 2011
Available online 20 April 2011
Keywords:
Amide bond cleavage
Caged compound
Ceramide
Sphingolipid
UV-responsive
1. Introduction
which the ceramide activity is recovered not via polarity change but
via structural change. In this paper, we report development and
In the field of cell biology, sphingolipids have been recognized
not only as membrane constructs but also as key signaling mole-
cules in recent years.1 In an effort to study on the mechanism of
sphingolipid mediated biological phenomena, caged sphingolipids,
which release sphingolipids after exposure to UV-irradiation are
needed to enable spatiotemporal control of the function of sphin-
golipids. Recently, some caged sphingolipids, such as ceramide
1-phosphate,2 sphingosine 1-phosphate,2,3 sphingosine,4,5 dehy-
drosphingosine,5 phychosine,5 and glycosphingolipids6 were
reported (Scheme 1). In these molecules, a highly polar phosphate
or amine moiety was temporarily masked by a less polar photo-
responsive protective group to suppress their biological activity.
UV-irradiation induces release of the phosphate or the amine
moiety followed by drastic polarity change to recover their bio-
activity. Moreover, ceramide is a member of the sphingolipids and
is also involved in critical cellular events such as apoptosis.7,1 To our
knowledge, however, a caged ceramide has not been reported so far
presumably due to its lack of highly polar functional group available
for caging. Therefore, we decided to design a caged ceramide in
photo-reactivity of a caged ceramide that generates a parent
ceramide by acyl transfer-mediated structural change after expo-
sure to UV-irradiation.
Scheme 1. Representative caged sphingolipids previously reported (PG: protective
* Corresponding authors. E-mail addresses: ashige@ph.tokushima-u.ac.jp
group removable by UV-irradiation).
(A. Shigenaga), aotaka@ph.tokushima-u.ac.jp (A. Otaka).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.04.048

A. Shigenaga et al. / Tetrahedron 67 (2011) 3984e3990
3985
Previously, we reported the development of stimulus-responsive
amino acids and their application to control peptidyl function in
living cells (Scheme 2).8 In this system, an amide bond in peptide 1
at a C-terminal position of the stimulus-responsive amino acid is
cleaved by stimulus-induced removal of PG (protective group re-
movable by a stimulus) followed by lactonization of trimethyl lock
acyl moiety and O-stearyl moiety at 1,2-position. Upon UV-induced
removal of o-nitrobenzyl group and subsequent lactonization fol-
lowed by amide bond cleavage, generated O-stearyl intermediate 5
should easily isomerize to afford ceramide 6 via OeN acyl transfer.
Because intracellular distribution of ceramide is a key factor in its
biological activity,11 we planned to introduce a handle at R position
for facile attachment of a functional moiety such as an intracellular
localization signal peptide for controlling the distribution of the
caged ceramide in living cells.
2. Results and discussion
2.1. Synthesis of caged ceramide
Model caged ceramide 11 was synthesized as shown in Scheme 4.
For simplification, an acetyl group was introduced at N-terminal
position of UV-responsive amino acid. Starting from known sphin-
gosine derivative 7 (R¼Boc),12 the Boc group was removed under
acidic conditions, and the generated amino group of 7 (R¼H) was
acylated with racemic Fmoc protected UV-responsive amino acid
128e using O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium
hexafluorophosphate (HBTU). The obtained product was diaster-
eomerically purified, and each diastereomer 8a or 8b was subjected
to the following reactions, respectively. The hydroxyl group of 8 was
acylated with stearic acid to afford 9. The Fmoc group of 9 was
replaced with acetylgroupbytreatment with piperidine followed by
acylation with acetic acid. Finally, the TBDPS group was removed
using HF/pyridine to generate model caged ceramide 11.
2.2. UV-irradiation experiment
First, UV-irradiation experiment of caged ceramide 11a was ex-
amined. Caged ceramide 11a in i-PrOH13 with 0.5% (v/v) triethyl-
amine was irradiated by UV (>365 nm) for 1 h, and the reaction
mixture was stirred overnight (Scheme 5). Although generation of
ceramide 6 was confirmed by ESI-MS and TLC analysis of the crude
reaction mixture, isolation of that was quite difficult because
a complex mixture was obtained after evaporation. We thought that
the complication after evaporation was caused by a side reaction of
highly reactive o-nitrosobenzaldehyde or its derivative under con-
centrated conditions. Therefore, HPLC analysis of the crude material
without evaporation was attempted. However, detection of an au-
thentic sample of ceramide 6 by UV-absorptionwas difficult because
of its low extinction coefficient. For sensitive HPLC analysis of the
reaction, analogous 10a possessing TBDPS group that absorbs UV
efficiently was used rather than original 11a, and ceramide analogue
13 was easily detected by UV-absorption. Therefore, we used caged
ceramide analogue 10a for HPLC monitoring of the UV-irradiation
experiment as shown below. Caged ceramide analogue 10a in
i-PrOH/H2O¼1:1 with 0.5% (v/v) triethylamine was irradiated by UV
(>365 nm) for 3 min,14 and the reaction mixture was incubated at
37 ꢀC. Reaction progress was monitored by HPLC, and ceramide
analogue 13 was identified by ESI-MS and comparison of retention
time with that of an authentic sample.15 After UV-irradiation and
subsequent 5 h incubation, caged ceramide analogue 10a was
completely converted to ceramide analogue 13 as shown in Fig. 1.
This result suggests that caged ceramide 11 should also be converted
to ceramide 6 in high purity.
Scheme 2. Stimulus-responsive controlling system of peptidyl function (PG: pro-
tective group removable by a stimulus).
moiety9 to generate isopeptide 2. In a manner similar to a click
peptide or a switch peptide,10 isopeptide 2 can easily isomerize via
OeN acyl transfer to yield a linear bioactive peptide under physi-
ological conditions. Ceramide 6 also possesses an N-acyl 1,2-ami-
noalcohol unit similar to peptide 3 (Scheme 3). Therefore, we
designed caged ceramide 4 composed of N-UV-responsive amino
2.3. Introduction of alkyne handle on caged ceramide
Intracellular distribution of ceramide is one of the key factors in
its biological activity.11 Therefore, introduction of a handle to en-
able facile attachment of a functional moiety, such as an in-
tracellular localization signal peptide, is of value for spatiotemporal
control of ceramide function. In this context, we planned to
Scheme 3. Design of caged ceramide (R¼functional moiety).

3986
A. Shigenaga et al. / Tetrahedron 67 (2011) 3984e3990
Scheme 4. Reagents and conditions: (i) TFA, CH2Cl2, 76%; (ii) 12, HBTU, N,N-diisopropylethylamine, DMF, 39% for 8a, 41% for 8b. Chemical yields of 8a derivatives are presented in
following text; (iii) stearic acid, N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride (EDC$HCl), Et3N, DMAP, CH2Cl2, 99%; (iv) piperidine, DMF; (v) AcOH, EDC$HCl, 1-
hydroxybenzotriazole hydrate (HOBt$H2O), Et3N, DMF, 78% (two steps); (vi) HF, pyridine, 73%.
ceramide was also examined, and the caged ceramide was suc-
cessfully coupled with an azide derivative by click chemistry. In-
troduction of a functional moiety and biological application of the
caged ceramide in living cells are underway.
Scheme 5. Reagents and conditions: 11a to 6 (R¼H): UV-irradiation (>365 nm, 1 h) in
i-PrOH with 0.5% (v/v) Et3N followed by stirring at room temperature. 10a to 13
(R¼TBDPS): UV-irradiation (>365 nm, 3 min) in H2O/i-PrOH (1:1 (v/v)) with 0.5% (v/v)
Et3N followed by incubation at 37 ꢀC.
introduce an alkyne handle on the caged ceramide for click
chemistry with a functional moiety (Scheme 6).16 Caged ceramide
14 possessing an alkyne handle was synthesized starting from
Fmoc derivative 9. Briefly, the Fmoc group of 9 was removed by
piperidine treatment, and the generated amine was acylated with
4-pentynoic acid. The obtained material was subjected to TBAF and
acetic acid in THF to remove TBDPS group, and alkyne derivative 14
was synthesized successfully.
Next, we examined ligation of caged ceramide 14b with a func-
tional moiety (Scheme 7). For simplification of the reaction system,
benzyl azide was used as a model of the functional moiety. To caged
ceramide 14b in H2O/dichloromethane (1:1 (v/v)) were added
benzyl azide, CuSO4, sodium ascorbate, and tris(1-benzyl-1H-
[1,2,3]triazol-4-ylmethyl)amine (TBTA) 16;17 ligated product 15b
was obtained in 91% isolated yield. This result suggests that the
alkyne handle on the caged ceramide is potentially applicable for
introduction of a functional moiety.
3. Conclusion
Fig. 1. HPLC profiles of UV-induced uncaging of caged ceramide analogue 10a. Reaction
conditions are shown in Scheme 5. (a) Before UV-irradiation. (b) After UV-irradiation
In conclusion, a caged ceramide was developed. UV-irradiation
experiment clarified that the caged ceramide with TBDPS group
(>365 nm, 3 min) followed by incubation at 37 ꢀC for 5 h. Compounds eluted at
can be completely converted to the corresponding ceramide de-
3e4 min are photo-cleaved derivatives of UV-responsive amino acid. HPLC conditions:
rivative by 3 min of UV-irradiation followed by 5 h of incubation at
TOSOH TSK-GEL (4.6ꢁ250 mm) with hexane/i-PrOH¼99:1 (v/v) at
a flow rate of
37 ꢀC. Introduction of an alkyne-handle moiety on the caged
1.0 mL/min, detection at 220 nm.

A. Shigenaga et al. / Tetrahedron 67 (2011) 3984e3990
3987
135.4; HRMS (ESI-TOF) calcd for
538.4080, found: 538.4078.
C
34H56NO2Si ([MþH]þ):
4.2.2. {1-[(1S,2R,3E)-1-(tert-Butyldiphenylsilanyloxymethyl)-2-hy-
droxyheptadec-3-enylcarbamoyl]-2-[2,4-dimethyl-6-(2-nitro-
benzyloxy)phenyl]-2-methylpropyl}carbamic acid 9H-fluoren-9-ylmethyl
ester (8a, b). N,N-Diisopropylethylamine (107 mL, 0.610 mmol)
and HBTU (201 mg, 0.530 mmol) were added to a solution of
carboxylic acid 128e (332 mg, 0.560 mmol) in DMF (1.0 mL) at
room temperature. After 30 min of stirring at room temperature,
TBDPS sphingosine 7 (R¼H) (300 mg, 0.56 mmol) was added to the
solution. The resulting mixture was stirred at room temperature
Scheme 6. Reagents and conditions: (i) piperidine, DMF; (ii) 4-pentynoic acid,
EDC$HCl, HOBt$H2O, Et3N, DMF; (iii) TBAF, AcOH, THF, 76% for 14a (three steps).
Scheme 7. Reagents and conditions: (i) benzyl azide, 0.1 equiv CuSO4, 0.3 equiv sodium ascorbate, 0.1 equiv TBTA (16), H2O/CH2Cl2¼1:1 (v/v), 91%.
4. Experimental section
for 3 h and was quenched by the addition of 5% (w/v) aqueous
solution of KHSO4. The mixture was extracted with CH2Cl2, and the
organic layer was washed with brine, dried over MgSO4, and
concentrated in vacuo. The obtained crude product was purified by
column chromatography (hexane/AcOEt¼10:1 (v/v)) and 243 mg
of 8a (39%) and 253 mg of 8b (41%, less polar diastereomer of 8a)
4.1. General methods
All reactions were carried out under a positive pressure of ar-
gon. For column chromatography, silica gel (KANTO KAGAKU N-
60) was employed. Exact mass spectra were recorded on a Waters
MICROMASSÒ LCT PREMIERÔ or a Bruker Esquire200 T. NMR
spectra were measured using a JEOL GSX400 or a JEOL GSX300
spectrometer. For HPLC analysis, a TSK-GEL Silica-60 analytical
column (TOSOH, 4.6ꢁ250 mm, flow rate at 1.0 mL/min) was
employed, and eluting products were detected by UV at 220 nm
(flow rate: 1.0 mL/min; solvent system: hexane and i-PrOH).
Photolysis was performed using Moritex MUV-202U with the fil-
tered output (>365 nm) of a 3000 mW/cm2 HgeXe lamp. Optical
rotations were measured using a JASCO P-2200 polarimeter
(concentration in g/100 mL).
were obtained as a colorless amorphousness. Compound 8a (polar
19
diastereomer): [
400 MHz)
a
]
þ0.88 (c 1.02, CHCl3); 1H NMR (CDCl3,
D
d
¼0.88 (3H, t, J¼6.8 Hz), 1.00 (9H, s), 1.18e1.31 (22H, m),
1.50 (3H, s), 1.55 (3H, s), 1.91 (2H, dt, J¼6.6 and 7.1 Hz), 2.00 (3H, s),
2.28 (3H, s), 2.97 (1H, br s), 3.14 (1H, dd, J¼7.3 and 9.5 Hz), 3.42
(1H, dd, J¼3.2 and 9.5 Hz), 3.97 (1H, m), 4.15 (2H, m), 4.20 (1H, m),
4.34 (1H, m), 5.29 (1H, dd, J¼6.6 and 15.4 Hz), 5.32 (1H, d,
J¼8.3 Hz), 5.38 (1H, d, J¼14.6 Hz), 5.48 (1H, d, J¼8.3 Hz), 5.60 (1H,
d, J¼14.6 Hz), 5.64 (1H, dt, J¼7.1 and 15.4 Hz), 5.70 (1H, d,
J¼8.3 Hz), 6.30 (1H, s), 6.47 (1H, s), 7.29 (2H, d, J¼7.8 Hz), 7.34e7.51
(10H, m), 7.53e7.66 (6H, m), 7.75 (2H, d, J¼7.8 Hz), 7.94 (1H, d,
J¼7.8 Hz), 8.16 (1H, d, J¼7.8 Hz); 13C NMR (CDCl3, 75 MHz)
¼14.1,
d
19.0, 20.6, 22.7, 25.6, 26.7, 28.0, 29.1, 29.3, 29.5, 29.6, 29.7, 31.9,
32.3, 45.1, 47.1, 54.4, 59.5, 62.8, 67.0, 68.8, 73.4, 113.2, 119.9, 125.1,
125.2, 127.0, 127.6, 127.8, 128.5, 128.8, 129.4, 129.6, 129.9, 132.5,
132.6, 133.4, 134.2, 134.4, 135.5, 137.2, 138.5, 141.2, 143.9, 146.9,
156.2, 157.8, 171.0; HRMS (ESI-TOF) calcd for C69H88N3O8Si
([MþH]þ): 1114.6341, found: 1114.6370. Compound 8b (less
4.2. Synthesis of caged ceramide
4.2.1. (2S,3R,4E)-2-Amino-1-(tert-butyldiphenylsilanyloxy)octadec-
4-en-3-ol (7 (R¼H)). Trifluoroacetic acid (2.0 mL) was added to
a solution of N-Boc derivative 7 (R¼Boc)12 (1.50 g, 2.35 mmol) in
CH2Cl2 (2.0 mL) at 0 ꢀC. The resulting mixture was stirred at room
temperature for 5 h and was quenched by the addition of satu-
rated aqueous solution of NaHCO3. The mixture was extracted
with CH2Cl2, and the organic layer was washed with brine, dried
over Na2SO4, and concentrated in vacuo. The obtained crude
material was purified by column chromatography (hexane/
AcOEt¼2:1 (v/v)) and 0.960 g of amine 7 (R¼H) (76%) was
polar diastereomer): [
400 MHz)
a
]
18 ꢂ1.11 (c 1.09, CHCl3); 1H NMR (CDCl3,
D
d
¼0.88 (3H, t, J¼6.8 Hz), 0.93 (9H, s), 1.26 (22H, m), 1.58
(3H, s), 1.67 (3H, s), 1.96 (2H, q, J¼6.8 Hz), 2.21 (3H, s), 2.53 (3H, s),
2.60 (1H, d, J¼8.5 Hz), 3.59 (1H, m), 3.63 (1H, m), 3.72 (1H, dd,
J¼3.9 and 10.3 Hz), 3.78 (1H, m), 4.24 (2H, m), 4.46 (1H, m), 5.20
(1H, dd, J¼5.6 and 15.4 Hz), 5.48 (1H, d, J¼9.0 Hz), 5.55 (1H, d,
J¼15.1 Hz), 5.60 (1H, dt, J¼6.8 and 15.4 Hz), 5.69 (1H, d, J¼15.1 Hz),
5.75 (1H, d, J¼9.0 Hz), 5.94 (1H, d, J¼7.6 Hz), 6.64 (1H, s), 6.66 (1H,
s), 7.26e7.44 (10H, m), 7.48e7.65 (8H, m), 7.77 (2H, d, J¼7.8 Hz),
8.10 (1H, d, J¼7.8 Hz), 8.15 (1H, d, J¼7.8 Hz); 13C NMR (CDCl3,
obtained as a colorless oil; [
(CDCl3, 400 MHz)
a
]
18 þ8.80 (c 1.02, CHCl3); 1H NMR
D
d
¼0.88 (3H, t, J¼6.8 Hz), 1.06 (9H, s), 1.25 (22H,
m), 2.01 (2H, dt, J¼6.6 and 6.8 Hz), 2.93 (1H, q, J¼5.6 Hz), 3.67 (1H,
dd, J¼5.4 and 10.0 Hz), 3.70 (1H, dd, J¼5.9 and 10.0 Hz), 4.09 (1H,
dd, J¼5.6 and 6.8 Hz), 5.40 (1H, dd, J¼6.8 and 15.4 Hz), 5.73 (1H, dt,
J¼6.6 and 15.4 Hz), 7.26e7.47 (6H, m), 7.64e7.69 (4H, m); 13C NMR
75 MHz)
d
¼14.1, 18.9, 20.7, 22.7, 25.8, 26.4, 28.3, 29.1, 29.3, 29.5,
29.6, 29.7, 31.9, 32.3, 45.5, 47.2, 53.9, 59.4, 63.4, 67.1, 68.9, 73.3,
113.5, 119.9, 125.2, 127.0, 127.7, 127.8, 128.3, 128.8, 129.4, 129.7,
129.9, 132.2, 132.4, 133.1, 133.6, 134.6, 135.4, 137.4, 138.7, 141.2,
(CDCl3, 75 MHz)
d
¼14.0, 19.1, 22.6, 26.7, 29.1, 29.2, 29.3, 29.4, 29.5,
29.6, 31.8, 32.2, 56.4, 65.9, 74.0, 127.6, 129.1, 129.6, 133.1, 133.6,

3988
A. Shigenaga et al. / Tetrahedron 67 (2011) 3984e3990
143.9, 146.7, 156.2, 158.2, 170.9; HRMS (ESI-TOF) calcd for
chromatography (hexane/AcOEt¼5:1 (v/v)) and 100 mg of com-
pound 10a (78%) was obtained as a colorless oil. Compound 10a:
C69H87N3NaO8Si ([MþNa]þ): 1136.6160, found: 1136.6167.
[a
]
2D0þ4.33 (c 0.84, CHCl3); 1H NMR (CDCl3, 400 MHz)
¼0.88 (6H, t,
d
4.2.3. Octadecanoic acid (1R,2E)-1-{(1S)-2-(tert-butyldiphenylsila-
nyloxy)-1-[3-[2,4-dimethyl-6-(2-nitrobenzyloxy)phenyl]-2-(9H-fluo-
ren-9-ylmethoxycarbonylamino)-3-methylbutyrylamino]ethyl}
hexadec-2-enyl ester (9a, b). Typical procedure. Triethylamine
J¼6.8 Hz), 1.01 (9H, s), 1.25 (52H, m), 1.45 (3H, s), 1.47 (3H, s), 1.87
(2H, dt, J¼6.3 and 6.8 Hz), 1.98 (3H, s), 1.99 (3H, s), 2.16 (3H, s), 2.19
(2H, dt, J¼2.7 and 7.6 Hz), 2.79 (1H, dd, J¼9.0 and 10.0 Hz), 3.18 (1H,
dd, J¼4.6 and 10.0 Hz), 4.19 (1H, dddd, J¼3.4, 4.6, 9.0 and 9.3 Hz),
5.14 (1H, dd, J¼7.3 and 15.4 Hz), 5.31 (1H, d, J¼9.3 Hz), 5.37 (1H, d,
J¼15.1 Hz), 5.44 (1H, dd, J¼3.4 and 7.3 Hz), 5.59 (1H, d, J¼9.0 Hz),
5.62 (1H, d, J¼15.1 Hz), 5.64 (1H, dt, J¼6.8 and 15.4 Hz), 6.19 (1H, s),
6.31 (1H, d, J¼9.0 Hz), 6.45 (1H, s), 7.36e7.46 (6H, m),7.49 (1H, t,
J¼7.8 Hz), 7.58e7.65 (4H, m), 7.72 (1H, t, J¼7.8 Hz), 8.07 (1H, d,
(56.0
mL, 0.400 mmol), EDC$HCl (56.8 mg, 0.300 mmol), and DMAP
(0.8 mg, 7
m
mol) were added to a solution of stearic acid (77.0 mg,
0.270 mmol) in CH2Cl2 (0.50 mL) at room temperature. After
30 min of stirring at room temperature, alcohol 8a (150 mg,
0.130 mmol) was added to the reaction mixture. The resulting so-
lution was stirred at the same temperature for 3 h and was
quenched by the addition of saturated aqueous solution of NaHCO3.
The obtained mixture was extracted with CH2Cl2, and the organic
layer was washed with brine, dried over MgSO4, and concentrated
in vacuo. The obtained residue was purified by column chroma-
J¼7.8 Hz), 8.14 (1H, d, J¼7.8 Hz); 13C NMR (CDCl3, 75 MHz)
¼14.1,
d
19.1, 20.6, 22.7, 23.5, 24.8, 25.4, 26.4, 26.7, 28.1, 28.9, 29.1, 29.3, 29.5,
29.6, 29.7, 31.9, 32.4, 34.3, 45.3, 52.5, 57.2, 61.4, 68.9, 73.3, 113.2,
123.2, 125.0, 127.7, 127.8, 128.4, 128.7, 129.5, 129.8, 132.8, 133.5,
134.5, 135.5, 135.6, 136.5, 137.0, 138.5, 146.7, 157.9, 169.7, 170.5,
172.8; HRMS (ESI-TOF) calcd for C74H113N3NaO8Si ([MþNa]þ):
tography (hexane/AcOEt¼10:1 (v/v)) and 185 mg of ester 9a (99%)
19
was obtained as a colorless amorphousness. Compound 9a: [
a
]
1222.8195, found: 1222.8179. Compound 10b: [
CHCl3); 1H NMR (CDCl3, 400 MHz)
a
]
19 ꢂ6.72 (c 0.81,
D
D
þ2.22 (c 1.02, CHCl3); 1H NMR (CDCl3, 400 MHz)
d
¼0.88 (6H, t,
d
¼0.88 (6H, t, J¼6.8 Hz), 0.97
J¼6.8 Hz), 1.01 (9H, s), 1.18ꢂ1.31 (52H, m), 1.50 (3H, s), 1.58 (3H, s),
1.88 (2H, dt, J¼6.6 and 7.1 Hz), 1.99 (3H, s), 2.17 (2H, dt, J¼2.7 and
7.6 Hz), 2.20 (3H, s), 2.88 (1H, m), 3.25 (1H, m), 4.17 (2H, m), 4.24
(1H, m), 4.37 (1H, m), 5.19 (1H, dd, J¼7.1 and 15.4 Hz), 5.36 (1H, d,
J¼8.5 Hz), 5.41 (1H, d, J¼15.4 Hz), 5.34e5.49 (2H, m), 5.62 (1H, d,
J¼15.4 Hz), 5.67 (1H, dt, J¼7.1 and 15.4 Hz), 5.71 (1H, d, J¼8.5 Hz),
6.23 (1H, s), 6.45 (1H, s), 7.29 (2H, q, J¼7.8 Hz), 7.35e7.53 (10H, m),
7.55e7.67 (6H, m), 7.75 (2H, d, J¼7.8 Hz), 8.00 (1H, d, J¼7.8 Hz), 8.16
(9H, s), 1.25 (52H, m),1.53 (3H, s), 1.62 (3H, s),1.89 (2H, dt, J¼6.8 and
7.6 Hz), 1.92 (3H, s), 2.15 (2H, dt, J¼7.3 and 8.3 Hz), 2.18 (3H, s), 2.51
(3H, s), 3.44 (1H, dd, J¼6.6 and 10.3 Hz), 3.55 (1H, dd, J¼4.6 and
10.3 Hz), 4.23 (1H, m), 5.02e5.12 (2H, m), 5.48 (1H, d, J¼15.4 Hz),
5.57 (1H, dt, J¼6.6 and 13.9 Hz), 5.65 (1H, d, J¼9.3 Hz), 5.65 (1H, d,
J¼9.0 Hz), 5.66 (1H, d, J¼15.4 Hz), 6.36 (1H, d, J¼9.0 Hz), 6.53 (1H,
s), 6.57 (1H, s), 7.32e7.44 (7H, m), 7.54e7.60 (4H, m), 7.63 (1H, t,
J¼7.8 Hz), 8.05 (1H, d, J¼7.8 Hz), 8.13 (1H, d, J¼7.8 Hz); 13C NMR
(1H, d, J¼7.8 Hz); 13C NMR (75 MHz, CDCl3)
d¼14.1, 19.1, 20.6, 22.7,
(CDCl3, 75 MHz)
d¼14.1, 19.0, 20.8, 22.7, 23.3, 24.8, 25.8, 26.7, 27.2,
24.8, 25.5, 26.7, 27.9, 28.9, 29.1, 29.2, 29.3, 29.5, 29.6, 29.7, 31.9, 32.4,
34.3, 45.2, 47.1, 52.4, 59.3, 61.5, 67.0, 68.8, 73.4, 113.2, 119.9, 123.5,
125.0, 125.1, 127.0, 127.6, 127.7, 127.8, 128.3, 128.7,129.3, 129.4, 129.7,
129.8,132.7,133.1,133.6,134.4,135.5,135.6,136.6,137.0,138.5,141.2,
143.9, 146.7, 156.3, 157.8, 170.3, 172.8; HRMS (ESI-TOF) calcd for
C87H121N3NaO9Si ([MþNa]þ): 1402.8770, found: 1402.8751. Com-
27.9, 29.0, 29.1, 29.2, 29.3, 29.5, 29.6, 29.7, 31.9, 32.3, 34.3, 45.1, 53.1,
57.6, 62.2, 69.0, 74.1, 113.4, 124.3, 124.9, 127.7, 128.3, 128.6, 129.5,
129.6, 129.7, 132.8, 133.9, 134.4, 135.5, 136.6, 137.0, 138.3, 146.8,
158.3, 169.4, 170.6, 172.6; HRMS (ESI-TOF) calcd for C74H113KN3O8Si
([MþK]þ): 1238.7934, found: 1238.7924.
pound 9b: [
a
]
19 ꢂ6.63 (c 1.43, CHCl3); 1H NMR (CDCl3, 400 MHz)
4.2.5. Octadecanoic acid (1R,2E)-1-((1S)-1-{2-acetylamino-3-[2,4-
dimethyl-6-(2-nitrobenzyloxy)phenyl]-3-methylbutyrylamino}-2-hy-
droxyethyl)hexadec-2-enyl ester (11a, b). Typical procedure. To a
D
d¼0.88 (6H, t, J¼6.6 Hz), 0.97 (9H, s), 1.26 (52H, m), 1.56 (3H, s), 1.65
(3H, s), 1.90 (2H, dt, J¼6.8 and 7.1 Hz), 2.16 (3H, s), 2.16 (2H, t,
J¼7.3 Hz), 2.53 (3H, s), 3.47 (1H, dd, J¼6.6 and 10.3 Hz), 3.59 (1H, dd,
J¼4.4 and 10.3 Hz), 4.14 (2H, m), 4.26 (1H, m), 4.33 (1H, m), 5.10
(2H, m), 5.40 (1H, d, J¼9.0 Hz), 5.45e5.73 (4H, m), 5.76 (1H, d,
J¼9.0 Hz), 6.51 (1H, s), 6.58 (1H, s), 7.27 (2H, t, J¼7.8 Hz), 7.31e7.43
(9H, m), 7.48e7.63 (7H, m), 7.74 (2H, d, J¼7.8 Hz), 7.97 (1H, d,
solution of silylether 10a (80.0 mg, 66.0
were added TBAF in THF (1 M, 130 L, 130
130
mol) at 0 ꢀC, and the mixture was stirred overnight. The re-
mmol) in THF (0.50 mL)
m
m
mol) and AcOH (7.6 L,
m
m
action was quenched with saturated aqueous solution of NaHCO3
and the obtained mixture was extracted with CHCl3. The extract
was washed with brine, dried over MgSO4, and concentrated in
vacuo to give a crude product, which was purified by column
chromatography (hexane/AcOEt¼4:1 (v/v)) and 47.0 mg of alcohol
J¼7.8 Hz), 8.12 (1H, d, J¼7.8 Hz); 13C NMR (CDCl3, 75 MHz)
¼14.0,
d
19.1, 20.7, 22.6, 24.8, 25.8, 26.8, 27.2, 27.9, 29.0, 29.2, 29.3, 29.5, 29.6,
29.7, 31.9, 32.3, 34.3, 45.1, 47.3, 53.3, 59.9, 62.3, 67.0, 68.9, 74.0,
113.5, 119.8, 119.9, 124.6, 124.9, 125.1, 125.2, 127.0, 127.6, 127.7, 128.3,
128.8, 129.6, 129.7, 133.1, 133.8, 134.2, 135.5, 136.4, 137.0, 138.5,
141.2, 141.3, 144.0, 147.1, 156.1, 158.3, 170.5, 172.5; HRMS (ESI-TOF)
calcd for C87H122N3O9Si ([MþH]þ): 1380.8950, found: 1380.8928.
11a (73%) was obtained as a colorless oil. Compound 11a: [
a
]
2D0þ11.1
(c 1.73, CHCl3); 1H NMR (CDCl3, 400 MHz)
d¼0.88 (6H, t, J¼6.8 Hz),
1.25 (52H, m), 1.50 (3H, s), 1.61 (3H, s), 1.84 (1H, m), 1.92 (2H, dt,
J¼6.8 and 7.1 Hz), 2.00 (3H, s), 2.23 (2H, t, J¼7.6 Hz), 2.24 (3H, s),
2.51 (3H, s), 3.02 (1H, m), 3.25 (1H, m), 3.84 (1H, m), 5.07 (1H, dd,
J¼6.1 and 7.6 Hz), 5.24 (1H, dd, J¼7.6 and 15.4 Hz), 5.47 (1H, d,
J¼14.4 Hz), 5.60 (1H, d, J¼9.0 Hz), 5.65 (1H, dt, J¼6.8 and 15.4 Hz),
5.74 (1H, d, J¼14.4 Hz), 5.78 (1H, d, J¼8.5 Hz), 6.25 (1H, d, J¼9.0 Hz),
6.64 (1H, s), 6.69 (1H, s), 7.54 (1H, t, J¼7.8 Hz), 7.80 (1H, t, J¼7.8 Hz),
8.11 (1H, d, J¼7.8 Hz), 8.22 (1H, d, J¼7.8 Hz); 13C NMR (CDCl3,
4.2.4. Octadecanoic acid (1R,2E)-1-[(1S)-1-{2-acetylamino-3-[2,4-
dimethyl-6-(2-nitrobenzyloxy)phenyl]-3-methylbutyrylamino}-
2-(tert-butyldiphenylsilanyloxy)ethyl]hexadec-2-enyl ester (10a,
b). Typical procedure. Fmoc derivative 9a (150 mg, 110 mmol) was
treated with 20% (v/v) piperidine/DMF (1.0 mL) at room tempera-
ture. After 30 min of stirring, the reaction mixture was evaporated
to remove piperidine and DMF. To the residue dissolved in DMF
(0.50 mL) was added a preactivated acetylation reagent (a 30-min
75 MHz)
d
¼14.1, 20.7, 22.7, 23.5, 24.8, 25.7, 26.3, 28.3, 28.8, 29.1,
29.2, 29.3, 29.4, 29.5, 29.6, 29.7, 31.9, 32.2, 34.3, 45.4, 53.5, 57.2, 61.3,
68.9, 73.8, 113.2, 124.3, 125.2, 128.6, 128.7, 129.6, 129.9, 133.2, 134.6,
136.8, 137.5, 138.7, 147.0, 158.2, 169.7, 171.2, 173.4; HRMS (ESI-TOF)
calcd for C58H96N3O8 ([MþH]þ): 962.7197, found: 962.7186. Com-
stirred solution of acetic acid (31.0
mL, 540
mmol), HOBt$H2O
(92.0 mg, 600 mol), and EDC$HCl (100 mg, 540
m
mmol) in DMF
(0.50 mL)). The resulting mixture was stirred at room temperature
for 3 h and was quenched by the addition of saturated aqueous
solution of NaHCO3. The mixture was extracted with ether, and the
organic layer was washed with brine, dried over MgSO4, and con-
centrated in vacuo. The obtained residue was purified by column
pound 11b: [
a
]
20 ꢂ4.43 (c 1.02, CHCl3); 1H NMR (CDCl3, 400 MHz)
D
d
¼0.88 (6H, t, J¼6.8 Hz), 1.25 (52H, m), 1.53 (3H, s), 1.58 (3H, s), 1.96
(2H, dt, J¼5.4 and 6.8 Hz), 1.96 (3H, s), 2.21 (2H, t, J¼7.8 Hz), 2.23
(3H, s), 2.49 (3H, s), 3.03 (1H, br s), 3.48 (1H, dd, J¼3.9 and 11.7 Hz),
3.59 (1H, m), 3.80 (1H, m), 4.71 (1H, dd, J¼4.2 and 6.8 Hz), 5.16

A. Shigenaga et al. / Tetrahedron 67 (2011) 3984e3990
3989
(1H, dd, J¼7.1 and 15.4 Hz), 5.46 (1H, d, J¼14.4 Hz), 5.58 (1H, d,
J¼8.8 Hz), 5.59 (1H, dt, J¼7.1 and 15.4 Hz), 5.74 (1H, d, J¼14.4 Hz),
6.03 (1H, d, J¼7.1 Hz), 6.30 (1H, d, J¼8.8 Hz), 6.60 (1H, s), 6.66 (1H,
s), 7.54 (1H, t, J¼7.8 Hz), 7.79 (1H, t, J¼7.8 Hz), 8.06 (1H, d, J¼7.8 Hz),
stirred at room temperature for 3 h and was quenched by the ad-
dition of saturated aqueous solution of NaHCO3. The mixture was
extracted with ether, and the organic layer was washed with brine,
dried over MgSO4, and concentrated in vacuo. The obtained residue
was purified by column chromatography (hexane/AcOEt¼5:1 (v/v))
and 74.0 mg of the alkyne derivative was obtained as a colorless oil.
8.20 (1H, d, J¼7.8 Hz); 13C NMR (CDCl3, 75 MHz)
¼14.1, 20.8, 22.7,
d
23.4, 24.8, 25.8, 26.8, 28.2, 28.9, 29.1, 29.2, 29.3, 29.5, 29.6, 29.7, 31.9,
32.2, 34.3, 45.1, 55.6, 57.6, 61.9, 68.8, 74.5, 113.1, 124.5, 125.2, 128.6,
128.7, 129.3, 130.0, 133.4, 134.5, 136.4, 137.2, 138.4, 147.2, 158.1,
169.8, 172.0, 173.4; HRMS (ESI-TOF) calcd for C58H96N3O8
([MþH]þ): 962.7197, found: 962.7183.
To a solution of the alkyne derivative (74.0 mg, 60.0
mmol) in THF
were added TBAF in THF (1 M, 120 L, 120
m
m
mol) and AcOH at 0 ꢀC,
and the mixture was stirred overnight. The reaction was quenched
with saturated aqueous solution of NaHCO3 and the mixture was
extracted with CHCl3. The extract was washed with brine, dried over
MgSO4, and concentrated in vacuo to give a crude product, which
was purified by column chromatography (hexane/AcOEt¼3:1 (v/v))
and 55.0 mg of 14a (76%) was obtained as a colorless oil. Compound
4.3. UV-irradiation experiment
4.3.1. UV-irradiation experiment of 11a. To caged ceramide 11a
(10.5 mg, 10.9
v) Et3N, and the obtained mixture was irradiated by UV light
>365 nm) for 1 h. After stirring overnight at room temperature,
mmol) was added i-PrOH (400
mL) containing 0.5% (v/
14a: [a]
20 þ0.26 (c 0.62, CHCl3); 1H NMR (CDCl3, 400 MHz)
d¼0.88
D
(6H, t, J¼6.8 Hz), 1.25 (52H, m), 1.53 (3H, s), 1.63 (3H, s), 1.93 (2H, dt,
J¼6.8 and 7.1 Hz), 2.00 (1H, t, J¼2.4 Hz), 2.22 (2H, t, J¼7.6 Hz), 2.23
(3H, s), 2.41e2.48 (2H, m), 2.48e2.55 (2H, m), 2.52 (3H, s), 3.06 (1H,
br d, J¼11.7 Hz), 3.26 (1H, dd, J¼3.7 and 11.7 Hz), 3.85 (1H, m), 5.08
(1H, dd, J¼1.0 and 7.3 Hz), 5.24 (1H, dd, J¼7.3 and 15.4 Hz), 5.52 (1H,
d, J¼14.9 Hz), 5.63 (1H, d, J¼8.8 Hz), 5.65 (1H, dt, J¼6.6 and 15.4 Hz),
5.73 (1H, d, J¼14.9 Hz), 5.75 (1H, d, J¼7.3 Hz), 6.43 (1H, d, J¼8.8 Hz),
6.75 (1H, s), 6.78 (1H, s), 7.53 (1H, t, J¼7.8 Hz), 7.78 (1H, t, J¼7.8 Hz),
8.08 (1H, d, J¼7.8 Hz), 8.21 (1H, d, J¼7.8 Hz); 13C NMR (CDCl3,
(l
the reaction mixture was analyzed by ESI-MS and TLC to confirm
the generation of ceramide 6. MS (ESI-Ion Trap) calcd for [6þH]þ:
566.6, found 566.5. TLC (Merck, TLC Silica gel 60 F254) CHCl3/
MeOH¼9:1 (v/v), Rf¼0.4. The Rf value was identical to that of au-
thentic sample of ceramide.
4.3.2. UV-irradiation experiment of 10a. Caged ceramide 10a (30
0.025 mol) in i-PrOH (120 L) was added to H2O (120 L) contain-
ing 0.5% (v/v) Et3N and the obtained mixture was irradiated by UV
light ( >365 nm) for 3 min. The resulting mixture was incubated at
mg,
m
m
m
75 MHz)
d
¼14.1, 15.0, 20.7, 22.7, 24.8, 25.7, 26.3, 28.4, 28.8, 29.1, 29.2,
29.3, 29.4, 29.5, 29.6, 29.7, 31.9, 32.2, 33.0, 34.3, 35.6, 45.3, 53.6, 57.4,
61.3, 69.0, 69.5, 69.7, 73.8, 82.9, 113.4, 116.1, 124.3, 125.2, 128.6, 129.6,
129.7, 133.4, 134.6, 135.0, 136.8, 137.5, 138.7, 146.9, 158.2, 170.6, 171.0,
173.3; HRMS (ESI-TOF) calcd for C61H98N3O8 ([MþH]þ): 1000.7354,
l
37 ꢀC, and reaction progress was monitored by HPLC. HPLC condi-
tions: hexane/i-PrOH¼99:1 (v/v). Retention times, 10a: 17.2 min; 13:
9.6 min. MS (ESI-Ion Trap) calcd for C52H90NO3Si ([MþH]þ) 804.7,
found 804.6. The retention time and MS spectrum were identical to
that of an authentic sample, which was prepared as follows. Trie-
found: 1000.7365. Compound 14b: [
NMR (CDCl3, 400 MHz)
a
]
20 ꢂ1.22 (c 0.96, CHCl3); 1H
D
d
¼0.88 (6H, t, J¼6.8 Hz), 1.25 (52H, m), 1.56
(3H, s), 1.60 (3H, s), 1.97 (2H, dt, J¼6.8 and 7.3 Hz), 1.99 (1H, t,
J¼2.4 Hz), 2.21 (2H, t, J¼7.8 Hz), 2.23 (3H, s), 2.37e2.42 (2H, m),
2.44e2.49 (2H, m), 2.50 (3H, s), 3.01 (1H, br s), 3.48 (1H, dd, J¼5.9
and 11.7 Hz), 3.58 (1H, br dd, J¼2.4 and 11.7 Hz), 3.81 (1H, m), 4.71
(1H, dd, J¼4.2 and 6.8 Hz), 5.16 (1H, dd, J¼7.3 and 15.4 Hz), 5.49 (1H,
d, J¼14.6 Hz), 5.60 (1H, dt, J¼7.1 and 15.4 Hz), 5.62 (1H, d, J¼8.8 Hz),
5.74 (1H, d, J¼14.6 Hz), 6.04 (1H, d, J¼7.3 Hz), 6.50 (1H, d, J¼8.8 Hz),
6.60 (1H, s), 6.64 (1H, s), 7.53 (1H, t, J¼7.8 Hz), 7.77 (1H, t, J¼7.8 Hz),
8.05 (1H, d, J¼7.8 Hz), 8.20 (1H, d, J¼7.8 Hz); 13C NMR (CDCl3,
thylamine (26
mL, 0.19 mmol), EDC$HCl (21 mg, 0.11 mmol), and
HOBt$H2O (1.8 mg, 0.12
mmol) were added to a solution of stearic
acid (77 mg, 0.27 mmol) in CH2Cl2 (0.50 mL) at room temperature.
After 30 min of stirring at room temperature, 7 (R¼H) (50 mg,
93 mmol) was added to the reaction mixture. The resulting solution
was stirred at the same temperature for 3 h. The reaction mixture
was quenched by the addition of saturated aqueous solution of
NaHCO3. It was extracted with CH2Cl2, and the organic layer was
washed with brine, dried over MgSO4, and concentrated in vacuo.
The obtained residue was purified by column chromatography
(hexane/AcOEt¼5:1 (v/v)) and 50 mg of 13 (67%) was obtained as
75 MHz)
d
¼14.1, 14.9, 20.8, 22.6, 24.7, 25.8, 26.8, 28.2, 28.9, 29.1, 29.2,
29.3, 29.4, 29.6, 29.7, 31.9, 32.2, 34.2, 35.5, 45.1, 55.5, 57.6, 61.9, 68.8,
69.4, 74.4, 82.9, 113.2, 124.5, 125.2, 128.6, 129.3, 129.9, 133.5, 134.5,
136.4, 137.2, 138.4, 147.1, 158.1, 170.7, 171.8, 173.4; HRMS (ESI-TOF)
calcd for C61H98N3O8 ([MþH]þ): 1000.7354, found: 1000.7358.
a colorless oil; [
a
]
1D9 þ7.11 (c 1.19, CHCl3); 1H NMR (CDCl3, 400 MHz)
d
¼0.88 (6H, t, J¼6.8 Hz),1.07 (9H, s),1.26 (52H, m), 2.03 (2H, dt, J¼6.8
and 7.3 Hz), 2.15 (2H, t, J¼7.8 Hz), 3.56 (1H, d, J¼6.6 Hz), 3.76 (1H, dd,
J¼2.4 and 10.0 Hz), 3.95 (1H, dd, J¼3.2 and 10.0 Hz), 3.98 (1H, m),
4.20 (1H, m), 5.47 (1H, dd, J¼5.9 and 15.4 Hz), 5.77 (1H, dt, J¼6.8 and
15.4 Hz), 6.11 (1H, d, J¼7.8 Hz), 7.35e7.48 (6H, m), 7.59e7.65 (4H, m);
4.5. Click chemistry of caged ceramide
To a solution of alkynyl derivative 14b (8.9 mg, 8.9
CH2Cl2 (550 L) were added benzyl azide in CH2Cl2 (0.18 mM,
150 L, 27 mol), CuSO4 in H2O (4.0 M, 220 L, 0.89 mol), sodium
ascorbate in H2O (5.0 M, 530 L, 2.7
mol), and TBTA 1616 in
CH2Cl2 (19 M, 47 L, 0.89 mol) at room temperature, and the
mmol) in
13C NMR (CDCl3, 75 MHz)
d¼14.1, 19.1, 22.7, 25.8, 26.9, 29.2, 29.3,
m
29.4, 29.5, 29.7, 31.9, 32.3, 36.8, 54.0, 64.0, 74.2, 127.9, 129.0, 130.1,
132.4, 133.4, 135.5, 173.3; HRMS (ESI-TOF) calcd for C52H89NaNO3Si
([MþNa]þ): 826.6509, found: 826.6484.
m
m
m
m
m
m
m
m
m
m
m
mixture was stirred for 3 h. The reaction was quenched with sat-
urated aqueous solution of NaHCO3 and the mixture was extracted
with CHCl3. The extract was washed with brine, dried over Na2SO4,
and concentrated in vacuo. The obtained crude product was puri-
fied by column chromatography (hexane/AcOEt¼1:1 (v/v)) and
9.0 mg of ligated product 15b (91%) was obtained as a colorless oil;
4.4. Introduction of handle on caged ceramide
4.4.1. Octadecanoic acid (1R,2E)-1-((1S)-1-{3-[2,4-dimethyl-6-(2-ni-
trobenzyloxy)phenyl]-3-methyl-2-pent-4-ynoylaminobutyrylamino}-
2-hydroxyethyl)hexadec-2-enyl ester (14a, b). Typical procedure. Fmoc
derivative 9a (100 mg, 72.0
mmol) was treated with 20% (v/v) pi-
[a
]
1D9 þ1.05 (c 0.70, CHCl3); 1H NMR (400 MHz, CDCl3)
¼0.88 (6H, t,
d
peridine/DMF (1.0 mL) at room temperature. After 30 min of stirring,
the reaction mixture was evaporated to remove piperidine and DMF.
To the residue in DMF (0.50 mL) was added preactivated pentyny-
lation reagent (a 30-min stirred solution of 4-pentynoic acid
J¼6.8 Hz), 1.25 (52H, m), 1.44 (3H, s), 1.67 (3H, s), 1.95 (2H, dt, J¼6.8
and 7.1 Hz), 2.21 (2H, t, J¼7.6 Hz), 2.22 (3H, s), 2.47 (3H, s), 2.54 (2H,
m), 2.95 (2H, t, J¼7.1 Hz), 3.13 (1H, br s), 3.48 (1H, dd, J¼5.4 and
11.0 Hz), 3.58 (1H, dd, J¼4.9 and 11.0 Hz), 3.85 (1H, m), 4.76 (1H, dd,
J¼4.4 and 7.1 Hz), 5.14 (1H, dd, J¼7.1 and 15.4 Hz), 5.46 (1H, d,
J¼14.4 Hz), 5.47 (2H, s), 5.52 (1H, d, J¼8.8 Hz), 5.57 (1H, dt, J¼6.8
(21.0 mg, 210
(42.0 mg, 220
m
m
mol), HOBt$H2O (36.0 mg, 240
mol) in DMF (0.50 mL)). The resulting mixture was
mmol), and EDC$HCl

3990
A. Shigenaga et al. / Tetrahedron 67 (2011) 3984e3990
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and 15.4 Hz), 5.70 (1H, d, J¼14.4 Hz), 6.05 (1H, d, J¼7.6 Hz), 6.48
(1H, d, J¼8.8 Hz), 6.59 (1H, s), 6.61 (1H, s), 7.22 (1H, s), 7.26 (2H, m),
7.35 (3H, m), 7.50 (1H, t, J¼7.6 Hz), 7.73 (1H, d, J¼7.6 Hz), 7.98 (1H, d,
J¼7.6 Hz), 8.16 (1H, d, J¼7.6 Hz); 13C NMR (100 MHz, CDCl3)
¼14.2,
d
20.9, 21.6, 22.8, 24.9, 25.9, 26.9, 28.2, 29.0, 29.2, 29.3, 29.4, 29.7,
29.8, 32.0, 32.3, 34.3, 35.8, 44.8, 54.0, 55.4, 57.9, 61.9, 68.9, 74.4,
113.2, 121.2, 124.5, 125.2, 128.0, 128.5, 128.6, 128.9, 129.3, 129.8,
133.5, 134.4, 134.7, 136.3, 137.1, 138.4, 146.8, 147.0, 157.9, 171.6, 171.7,
173.2; HRMS (ESI-TOF) calcd for C68H104N6NaO8 ([MþNa]þ),
1155.7813; found 1155.7806.
Acknowledgements
This research was supported in part by a Grant-in-Aid for
Scientific Research (KAKENHI), Takeda Science Foundation, and
Astellas Foundation for Research on Metabolic Disorders. J.Y. is
grateful for a SUNBOR Scholarship.
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Supplementary data
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Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.tet.2011.04.048.
13. Because of low solubility of caged ceramide 11a in aqueous buffer, isopropanol
in which 11a is soluble was used as a solvent.
References and notes
14. Whereas removal of o-nitrobenzyl group of 11a required 1 h UV-irradiation,
that of 10a was completed within 3 min because the reaction scale of 10a
was smaller than that of 11a.
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