Caged compounds with a steroid skeleton: Synthesis, liposome-formation and photolysis

Article abstract of DOI:10.1016/S0040-4020(02)00077-7

The synthesis of two key precursors for caging bioactive compounds, an o-nitrobenzyl p-nitrophenyl carbonate and an o-nitrobenzyl diazo compound with a steroid skeleton, is reported. The former is a suitable caging group for an amino group, while the latter is appropriate for a carboxylic acid. The resulting caged compounds were irradiated at 350nm to afford the corresponding amine and carboxylic acid. A caged compound released bioactive compounds more efficiently when embedded in liposome than when in methanol.

Full text of DOI:10.1016/S0040-4020(02)00077-7

TETRAHEDRON
Pergamon
Tetrahedron 58 (2002) 1685±1691
Caged compounds with a steroid skeleton: synthesis,
liposome-formation and photolysis
Soichiro Watanabe, Remi Hiratsuka, Yosuke Kasai, Kazunori Munakata, Yasuhiro Takahashi
and Michiko Iwamurap
Department of Biomolecular Science, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan
Received 16 November 2001; accepted 23 January 2002
AbstractÐThe synthesis of two key precursors for caging bioactive compounds, an o-nitrobenzyl p-nitrophenyl carbonate and an
o-nitrobenzyl diazo compound with a steroid skeleton, is reported. The former is a suitable caging group for an amino group, while the
latter is appropriate for a carboxylic acid. The resulting caged compounds were irradiated at 350 nm to afford the corresponding amine and
carboxylic acid. A caged compound released bioactive compounds more ef®ciently when embedded in liposome than when in methanol.
q 2002 Published by Elsevier Science Ltd.
1. Introduction
also to other bioactive compounds with other functional
groups such as a carboxylic acid. In this paper, we describe
detailed methods for the synthesis of steroid-substituted
o-nitrobenzyl- and o-nitrophenethyl-type caged compounds
and their photochemical reactivities.
Caged compounds1 are biologically inert molecules that can
release bioactive compounds upon photolysis. They are
used to investigate and control the function of biological
systems. The most popular caging groups (photolabile
protecting groups) are an o-nitrobenzyl group and its
derivatives. Although other photolabile protecting groups,
such as desyl, p-hydroxyphenacyl2 and coumarinylmethyl,3
have been reported, o-nitrobenzyl-type caging groups have
been widely used because of the ease with which derivatives
can be synthesized. Some of them have electron-donating
substituents on their aromatic rings to make their absorption
maxima longer, and others have side chains as a linker to
connect to another component.4
2. Results and discussion
We designed two key precursors; an o-nitrobenzyl p-nitro-
phenyl carbonate for an amino caging group and an o-nitro-
benzyl diazo compound for a carboxylic acid caging group.
Pregnenolone acetate was used as a starting material for
both types of caging groups. The ketone moiety was
converted to an ole®n by Wittig reaction, followed by
methylation of a hydroxyl group to afford 3. Nitrophenyl
groups, a 5-hydroxy-2-nitrobenzaldehyde for an o-nitro-
benzyl caging group and a 5-hydroxy-2-nitroacetophenone
for an o-nitrophenethyl caging group, were introduced to the
steroid skeleton after hydroboration of the ole®n moiety and
tosylation of an alcohol. After the reduction of a carbonyl
moiety at a benzyl position to an alcohol, the mixed car-
bonate 8 was obtained by reacting 7 and p-nitrophenyl
chloroformate. In this reaction, an alcohol-free chloroform
should be used because ethyl p-nitrophenyl carbonate was
obtained as a main product when we used commercially
available chloroform containing ethylalcohol as a stabilizer.
The mixed carbonate 8 is a useful caging group for bioactive
molecules with amino groups. As a model case, a
LeuLeuOMe was caged using 8. An o-nitrophenethyl
caged compound 9b was obtained as a diastereomeric
mixture (about 1:1). An o-nitrophenyl diazo compound 11
was obtained from 6a via a tosylhydrazone 10, the structure
of which was con®rmed by IR absorption at 2076 cm21 due
to the diazo group. The diazo compound 11 is considered to
We have been studying the caged compounds of an
l-leucyl-l-leucine methyl ester (LeuLeuOMe),5 which has
been reported to induce apoptosis in NK cells and macro-
phages.6 Although an o-nitrobenzyl caged LeuLeuOMe has
suf®cient photochemical properties for caged compounds,
some additional modi®cations should be made for practical
biological application. For example, since bioassays are
carried out in aqueous buffered solution, water-soluble
compounds are desirable. For this purpose, we developed
a sugar-modi®ed caged compound.5c Liposome-forming
caged compounds with a lipophilic substituent such as a
steroid unit are also considered to be useful because macro-
phages are capable of endocytosis.5d This strategy can be
applied not only to LeuLeuOMe with a free amino group but
Keywords: biologically active compounds; photochemistry; steroids and
sterols.
Corresponding author. Tel.: 181-47-472-7562; fax: 181-47-475-1855;
e-mail: michiko@biomol.sci.toho-u.ac.jp
p
0040±4020/02/$ - see front matter q 2002 Published by Elsevier Science Ltd.
PII: S0040-4020(02)00077-7

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S. Watanabe et al. / Tetrahedron 58 (2002) 1685±1691
Figure 1. Time-dependent decrease in caged compounds and release of
LeuLeuOMe. 9a in MeOH: X, 9b in MeOH: B, 9a in liposome: O, 9b
in liposome: P, LeuLeuOMe from 9a in MeOH: W, LeuLeuOMe from 9b
in MeOH: A, LeuLeuOMe from 9a in liposome: K, LeuLeuOMe from 9b in
liposome: L.
Ê
out with a Rayonet photochemical reactor (RPR 3500 A£4).
First, a photochemical reaction of 9a was monitored by
UV±VIS absorption spectra, which showed an isosbestic
point for up to 40 min of irradiation.5d Since we intend to
irradiate caged compounds for 5 min in a bioassay,5c the
present photochemical properties are suf®cient for our
system.
Fig. 1 shows a time-dependent decrease in 9a and 9b and a
concomitant increase in released LeuLeuOMe during
photolysis. The amounts of caged LeuLeuOMe and released
LeuLeuOMe during photolysis were estimated by HPLC
analysis, and the latter was monitored by the intensity of
¯uorescence derived from adduct of ¯uorescamine8 with
released LeuLeuOMe. The o-nitrophenethyl caged
compound 9b showed effective photoinduced bond
cleavage compared with the o-nitrobenzyl derivative 9a,
which is consistent with other caged compounds reported
previously.7
Scheme 1. Synthesis of steroid-substituted caged compounds. (a):
(i) Ph3CH31Br2, KOtBu, THF, re¯ux 12h, (ii) K 2CO3, MeOH, 15 h,
95%; (b): KOH, MeI, DMSO, rt, 10 h, 88%; (c): (i) 9-BBN, THF, re¯ux,
13 h, (ii) 2N NaOH, 30% H2O2, rt, 16 h, 90%; (d): TsCl, Py, rt, 12h, 96%;
(e): 5-hydroxy-2-nitrobenzaldehyde, K2CO3, DMF, 1108C, 15 h, 77% for
6a; 5-hydroxy-2-nitroacetophenone, K2CO3, DMF, 72% for 6b; (f): NaBH4,
EtOH, rt, 20 min, 95% for 7a, 91% for 7b; (g): 4-nitrophenyl chloro-
formate, DMAP, CHCl3, rt, 30 h, 61% for 8a, 59% for 8b; (h):
LeuLeuOM´TFA, DMAP, CHCl3, 4 d, 58% for 9a, 42% for 9b; (i):
p-toluenesulfonyl hydrazide, EtOH, rt, overnight, 92%; (j): Et3N, MeOH,
rt, overnight, 84%; (k): p-bromophenylacetic acid, CHCl3, re¯ux, over-
night, 69% for 12a; p-tolylacetic acid, CHCl3, rt, 12% for 12b.
be a useful caging group for carboxylic acids, such as the
widely used o-nitrobenzyl diazomethanes.7 Since the diazo
compound 11 is a highly reactive precursor for caged
compounds, we used it for the next synthetic step without
extensive puri®cation. We synthesized caged compounds of
a p-bromophenyl and p-tolylacetic acid as model
compounds (12a and 12b).
The structures of the target molecules were con®rmed by 1H
and 13C NMR spectra, and solutions of 9a, 9b, 12a and 12b
in methanol exhibited absorption maxima at 309, 304, 310
and 310 nm (1 12400, 10300, 9940 and 6970) in their
UV±VIS spectra, respectively.
Figure 2. Time-dependent decrease in caged carboxylic acids. 12a: B,
12b: X.
Photolysis of the caged compounds in methanol was carried

S. Watanabe et al. / Tetrahedron 58 (2002) 1685±1691
1687
Fig. 2shows a time-dependent decrease in 12a and 12b.
Unfortunately, the appearance of released acids could not
be quanti®ed because of overlapping of the signals due to
other photoproducts in HPLC analysis. The ef®ciency of the
photodegradation of starting materials was almost the same
for 12a and 12b (Scheme 1).
under argon atmosphere unless otherwise noted. Preparative
gel permeation liquid chromatography was performed by
LC-908 with JAIGEL 1H12H columns (Japan Analytical
Industry) with chloroform as solvent. Column chromato-
1
graphy was performed with Merck Kieselgel 60. H NMR
(270 MHz) and 13C NMR (67.5 MHz) spectra were
measured in CDCl3 or CD3OD with a JEOL JNM-GSX
270 spectrometer using tetramethylsilane as an external
standard, or CHCl3 or CH3OH as an internal standard. EI
mass spectral data were obtained on a HITACHI M-80 mass
spectrometer. High resolution mass spectral data were
obtained on a JEOL SX-102mass spectrometer. Electronic
spectra were recorded on a HITACHI U3210 UV/VIS
spectrometer. Infrared spectra were recorded with a
JASCO FT/IR 5000 spectrometer. Elemental analyses
were performed by the Instrument Analysis Center of
School of Pharmaceutical Sciences, Toho University.
Photochemical reaction was carried out with a Rayonet
Photochemial Chamber Reactor Model RPR-200 (RPR
Liposomes containing the caged LeuLeuOMe 9, phospha-
tidyl choline, and dicetyl phosphate in phosphate-buffered
saline (PBS) were prepared by the usual method. This
combination of phospholipids was selected for liposome
formation because liposomes consisting of these phospho-
lipids have been reported to be effectively incorporated into
macrophage via phagocytosis.9 About 80% of the caged
compound 9a was considered to be incorporated in lipo-
somes because only 20% of 9a was detected in the super-
natant after centrifugation of the liposome suspension. The
caged compound 9a embedded in liposomes is fairly stable;
more than 95% of 9a remained intact even after standing for
10 d in the dark (by HPLC analysis).
Ê
3500 A£4). Analytical HPLC was performed with a
JASCO Crestpak C18 S or a MERCK LiChrosorb RP-18.
The photochemical reaction of 9a and 9b in liposome as
well as the release of LeuLeuOMe are shown in Fig. 1
together with those in methanol. Interestingly, the rate of
photocleavage for 9a was faster in liposome than in
methanol during the initial 60 min, although the intensity
of UV light is considered to be reduced in the former case
because of the scattering of light by phospholipids in lipo-
some. The time-dependent release of LeuLeuOMe in the
photoreaction of 9a and 9b shows that the reaction
proceeded more ef®ciently in liposome than that in metha-
nol. The present results indicates that caged compound 9a
and 9b work well as effective precursors of the bioactive
molecule even in the presence of phospholipids. The
ef®cient photoreaction in liposome compared with that in
methanol may be caused by the in¯uence of the reaction
®eld due to phospholipid or the desirable conformation of
9 for photoreaction in liposome.
4.1.1. 20-Methyl-pregna-5,20-dien-3b-ol (2). A solution
of methyltriphenylphosphonium bromide (16.09 g,
45.0 mmol) and potassium t-butoxide (5.07 g, 45.2mmol)
in THF (50 mL) was stirred for 50 min at rt under argon
atmosphere. To this reaction mixture was added a solution
of pregnenolone acetate (1; 5.18 g, 14.5 mmol) in THF
(35 mL) at rt and the mixed solution was stirred for 2h.
The solution was warmed to re¯ux and stirred for additional
12h. After cooled to rt, the reaction mixture was washed
with water, 1N HCl, then aq. NaHCO3. The organic layer
was dried over anhydrous MgSO4, ®ltered and the solvent
was evaporated under reduced pressure. The residual solid
was subsequently treated by K2CO3 (3.35 g, 24.2 mmol) in
methanol (150 mL) for 15 h at rt. The reaction mixture was
®ltered through celite, and the ®ltrate was concentrated
under reduced pressure. The residual solid was chromato-
graphed (SiO2/hexane±ethyl acetateˆ5:1) to afford 2
(4.31 g, 13.7 mmol, 94.7%). 2: white crystals, mp 129.0±
1
131.08C; H NMR (270 MHz, CDCl3) d 0.59 (s, 3H), 1.01
3. Conclusion
(s, 3H), 1.76 (s, 3H), 0.91±2.29 (m, 20H), 3.53 (m, 1H), 4.71
(br s, 1H), 4.85 (br s, 1H), 5.36 (d, Jˆ5.4 Hz, 1H); 13C NMR
(67.5 MHz, CDCl3) d 12.8 (q), 19.5 (q), 21.2 (t), 24.3 (t),
24.7 (q), 25.5 (t), 31.6 (t), 31.9 (t), 32.2 (d), 36.6 (s), 37.3 (t),
38.7 (t), 42.2 (t), 43.1 (s), 50.3 (d), 56.5 (d), 57 (d), 71.7 (d),
110.6 (t), 121.5 (d), 140.6 (s), 145.5 (s). Anal. calcd for
C22H34´H2O: C, 84.02; H, 10.90; found: C, 81.51; H,
10.66%.
In this paper, we reported a method for synthesizing a lipo-
philic caging group with a steroid skeleton that can convert
a variety of bioactive compounds to their caged forms. For
example, biomolecules with an amino group or a carboxyl
group can be converted to caged compounds via o-nitro-
benzyl p-nitrophenyl carbonate or a diazo compound,
respectively. The resulting caged compound formed a lipo-
some with phospholipids and underwent photoreaction
more ef®ciently in liposome than in methanol.
4.1.2. 3b-Methoxy-20-methyl-pregna-5,20-dien (3). To a
solution of 2 (335 mg, 1.13 mmol) in DMSO (30 mL) was
added powdered potassium hydroxide (2.53 g, 45.0 mmol)
and methyl iodide (0.70 mL, 11 mmol), and the solution
was stirred for 10 h at rt. The reaction mixture was poured
into ice-water and the aqueous layer was extracted with
CHCl3. The organic layer was washed with brine, dried
over anhydrous MgSO4, ®ltered, and the solvent was
evaporated under reduced pressure. The crude mixture
was separated by column chromatography (SiO2/hexane±
CH2Cl2ˆ2:1) to give 3 (327 mg, 1.0 mmol, 88%) with
recovery of 2 (17 mg, 0.054 mmol, 4.8%). 3: white crystals,
4. Experimental
4.1. General remarks
Melting points were determined on a MEL-TEMP II
(Mitamura Riken Kogyo) or a Yanaco micro melting
point apparatus. All melting points were uncorrected. All
solvents used in the reactions were puri®ed by the reported
methods. THF was puri®ed by distillation from benzo-
phenone ketyl before use. All reactions were carried out
1
mp 96.0±97.08C; H NMR (270 MHz, CDCl3) d 0.59 (s,

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S. Watanabe et al. / Tetrahedron 58 (2002) 1685±1691
3H), 1.01 (s, 3H), 1.76 (s, 3H), 1.04±2.38 (m, 20H), 3.07 (m,
1H), 3.35 (s, 3H), 4.71 (br s, 1H), 4.85 (br s, 1H), 5.36 (d,
Jˆ5.1 Hz, 1H); 13C NMR (67.5 MHz, CDCl3) d 12.7 (q),
19.4 (q), 21.1 (t), 24.2 (t), 24.6 (q), 25.4 (t), 28.0 (t), 31.9 (t),
32.2 (d), 36.9 (s), 37.2 (t), 38.7 (t£2), 43.1 (s), 50.4 (d), 55.6
(q), 56.5 (d), 57.3 (d), 80.3 (d), 110.7 (t), 121.5 (d), 140.9
(s), 145.6 (s). Anal. calcd for C23H36O: C, 84.09; H, 11.04;
found: C, 83.94; H, 11.19%.
cooled to rt, the solution was ®ltered through celite and the
®ltrate was concentrated under reduced pressure. To the
residue was added CH2Cl2 and water, and the solution was
stirred for a few hours. The aqueous layer was extracted
with CH2Cl2, and the combined organic layer was washed
with brine, dried over anhydrous MgSO4, ®ltered, and the
solvent was evaporated under reduced pressure. The crude
mixture was puri®ed by column chromatography (SiO2/
CH2Cl2) to give 6a (570 mg, 1.15 mmol, 77.2%). 6a: pale
green crystals, mp 182.0±183.08C (decomp.); 1H NMR
(270 MHz, CDCl3) d 0.75 (s, 3H), 1.01 (s, 3H), 1.15 (d,
Jˆ6.6 Hz, 3H), 0.88±2.16 (m, 20H), 2.37 (m, 1H), 3.05
(m, 1H), 3.36 (s, 3H), 3.84 (dd, Jˆ8.9, 7.0 Hz, 1H), 4.04
(dd, Jˆ8.9, 3.3 Hz, 1H), 5.36 (d, Jˆ5.1 Hz, 1H), 7.13 (dd,
Jˆ9.0, 2.8 Hz, 1H), 7.30 (d, Jˆ2.8 Hz, 1H), 8.12 (d,
Jˆ9.0 Hz, 1H), 10.49 (s, 1H); 13C NMR (67.5 MHz,
CDCl3) d 11.9 (q), 17.3 (q), 19.4 (q), 21.0 (t), 24.3 (t),
27.8 (t), 28.0 (t), 31.9 (t), 31.9 (d), 36.3 (d), 36.9 (s), 37.2
(t), 38.7 (t), 39.6 (t), 42.5 (s), 50.1 (d), 52.4 (d), 55.6 (q),
56.4 (d), 74.4 (t), 80.3 (d), 113.8 (d), 118.8 (d), 121.4 (d),
127.2 (d), 134.4 (s), 140.9 (s), 141.9 (s), 163.9 (s), 188.7 (d).
Anal. calcd for C30H41NO5: C, 72.70; H, 8.34; N, 2.83;
found: C, 72.42; H, 8.53; N, 2.69%.
4.1.3. 20a-Hydroxymethyl-3b-methoxy-5-pregnen (4).
To a solution of 3 (1.40 g, 4.26 mmol) in THF (40 mL)
was added a 0.5 M solution of 9-BBN in THF (30 mL,
15.0 mmol) at 08C during 15 min under argon atmosphere.
The reaction mixture was stirred at rt for 1 h, then warmed
to re¯ux and stirred for additional 13 h. To the reaction
mixture was added 2N NaOH (40 mL) and 30% H2O2
(40 mL) at 08C, then the reaction mixture was stirred for
16 h at rt. After the aqueous layer was extracted with ether,
the organic layer was washed with brine, dried over
anhydrous MgSO4, ®ltered, and the solvent was evaporated
under reduced pressure. Chromatographic separation (SiO2/
hexane±ethyl acetateˆ4:1) of the crude mixture afforded 4
(1.32g, 3.82mmol, 89.6%). 4: white crystals, mp 153.0±
1
154.08C; H NMR (270 MHz, CDCl3) d 0.71 (s, 3H), 1.00
(s, 3H), 1.05 (d, Jˆ6.6 Hz, 3H), 0.89±2.42 (m, 21H), 3.06
(m, 1H), 3.35 (s, 3H), 3.35 (m, 1H), 3.64 (dd, Jˆ10.5,
3.2Hz, 1H), 5.36 (d, Jˆ5.1 Hz, 1H); 13C NMR
(67.5 MHz, CDCl3) d 11.9 (q), 16.8 (q), 19.4 (q), 21.1 (t),
24.4 (t), 27.7 (t), 28.0 (t), 31.9 (d), 31.9 (t), 36.9 (s), 37.2 (t),
38.7 (t), 38.7 (d), 39.6 (t), 42.4 (s), 50.2 (d), 52.4 (d), 55.6
(q), 56.5 (d), 68.0 (t), 80.3 (d), 121.5 (d), 140.9 (s). Anal.
calcd for C23H38O2: C, 79.71; H, 11.05; found: C, 79.70; H,
11.31%.
4.1.6. 20a-(3-Acetyl-4-nitrophenoxymethyl)-3b-methoxy-
5-pregnen (6b). The procedure similar to 6a but using a
solution of 5 (473 mg, 0.95 mmol), 5-hydroxy-2-nitro-
acetophenone (205 mg, 1.13 mmol), and K2CO3 (653 mg,
4.73 mmol) in DMF (30 mL), gave 6b (345 mg,
0.68 mmol, 71.6%). 6b: yellow crystals mp 128.0±
1
129.08C; H NMR (270 MHz, CDCl3) d 0.74 (s, 3H), 1.01
(s, 3H), 1.14 (d, Jˆ6.9 Hz, 3H), 1.20±2.42 (m, 21H), 2.53
(s, 3H), 3.07 (m, 1H), 3.36 (s, 3H), 3.83 (m, 1H), 4.00 (m,
1H), 5.35 (d, Jˆ4.3 Hz, 1H), 6.75 (d, Jˆ2.6 Hz, 1H), 6.96
(dd, Jˆ2.6, 9.2 Hz, 1H), 8.13 (d, Jˆ9.2Hz, 1H); 13C NMR
(67.5 MHz, CDCl3) d 11.8 (q), 17.2(q), 19.2(q), 20.9 (t),
24.2 (t), 27.6 (t), 27.8 (t), 30.3 (q), 31.7 (t), 31.8 (d), 36.2 (d),
36.7 (s), 37.0 (t), 38.5 (t), 39.4 (t), 42.4 (s), 45.0 (d), 52.3 (d),
55.4 (q), 56.3 (d), 74.2(t), 80.2(d), 112.2(d), 115.0 (d),
121.3 (d), 126.9 (d), 137.6 (s), 140.7 (s), 141.1 (s), 164.2 (s),
200.2 (s). Anal. calcd for C31H43NO5: C, 73.05; H, 8.50; N,
2.75; found: C, 73.07; H, 8.57; N, 2.80%.
4.1.4.
20a-(p-Toluenesulfonylmethyl)-3b-methoxy-5-
pregnen (5). To a solution of 4 (64 mg, 0.18 mmol) in
pyridine (5 mL) was added tosyl chloride (1.68 g,
8.80 mmol) at 08C and the reaction mixture was stirred for
12h at rt. The solution was poured into ice-cooled diluted
HCl, and a white precipitate was ®ltered with suction. The
precipitates was dissolved in CH2Cl2 and the solution was
dried over anhydrous MgSO4, ®ltered, and the solvent was
evaporated under reduced pressure. The crude mixture was
separated by gel permeation liquid chromatography to
4.1.7. 20a-(3-Hydroxymethyl-4-nitrophenoxymethyl)-
3b-methoxy-5-pregnen (7a). To a solution of 6a (39 mg,
0.079 mmol) in ethanol (5 mL) was added a solution of
sodium borohydride (11 mg, 0.29 mmol) in ethanol
(1 mL) at rt and the reaction mixture was stirred for
20 min. Water was added to the reaction mixture and the
solution was extracted with CH2Cl2. The organic layer was
washed with brine, dried over anhydrous MgSO4, ®ltered,
and the solvent was evaporated under reduced pressure. The
residual solid was pure 7a (37 mg, 0.074 mmol, 94.5%). 7a:
1
afford 5 (89 mg, 0.18 mmol, 96%). 5: colorless syrup; H
NMR (270 MHz, CDCl3) d 0.64 (s, 3H), 0.99 (s, 3H), 0.99
(d, Jˆ6.6 Hz, 3H), 0.87±1.95 (m, 19H), 2.15 (m, 1H), 2.38
(m, 1H), 2.45 (s, 3H), 3.06 (m, 1H), 3.35 (s, 3H), 3.39 (dd,
Jˆ9.3, 6.4 Hz, 1H), 3.97 (dd, Jˆ9.3, 3.2Hz, 1H), 5.34 (d,
Jˆ5.1 Hz, 1H), 7.34 (d, Jˆ8.3 Hz, 2H), 7.78 (d, Jˆ8.3 Hz,
2H); 13C NMR (67.5 MHz, CDCl3) d 11.8 (q), 16.8 (q), 19.3
(q), 21.0 (t), 21.6 (q), 24.2 (t), 27.4 (t), 28.0 (t), 31.8 (t), 31.9
(d), 36.2(d), 36.8 (s), 37.1 (t), 38.7 (t), 39.4 (t), 42.4 (s), 50.0
(d), 51.7 (d), 55.6 (q), 56.3 (d), 75.6 (t), 80.3 (d), 121.4 (d),
127.9 (d), 129.7 (d), 133.1 (s), 140.9 (s), 144.6 (s). Anal.
calcd for C30H44O4´H2O: C, 69.46; H, 8.94; found: C, 69.79;
H, 8.74%.
1
yellow crystals, mp 186.5±188.58C; H NMR (270 MHz,
CDCl3) d 0.74 (s, 3H), 1.01 (s, 3H), 1.15 (d, Jˆ6.6 Hz,
3H), 0.92±2.10 (m, 20H), 2.38 (m, 1H), 2.70 (br s, 1H),
3.07 (m, 1H), 3.36 (s, 3H), 3.82(dd, Jˆ9.0, 6.8 Hz, 1H),
4.02(dd, Jˆ9.0, 3.4 Hz, 1H), 4.99 (s, 2H), 5.36 (d,
Jˆ5.1 Hz, 1H), 6.87 (dd, Jˆ9.2, 2.8 Hz, 1H), 7.20 (d,
Jˆ2.8 Hz, 1H), 8.17 (d, Jˆ9.2Hz, 1H); 13C NMR
(67.5 MHz, CDCl3) d 11.9 (q), 17.3 (q), 19.4 (q), 21.0 (t),
24.4 (t), 27.8 (t), 28.0 (t), 31.9 (t), 31.9 (d), 36.3 (d), 36.9 (s),
37.2 (t), 38.7 (t), 39.6 (t), 42.5 (s), 50.1 (d), 52. 5 (d), 55.6
4.1.5. 20a-(3-Formyl-4-nitrophenoxymethyl)-3b-methoxy-
5-pregnen (6a). A solution of 5 (746 mg, 1.49 mmol),
5-hydroxy-2-nitrobenzaldehyde (505 mg, 3.02 mmol), and
K2CO3 (1.03 g, 7.46 mmol) in DMF (20 mL) was warmed
to 1108C and stirred for 15 h under argon atmosphere. After

S. Watanabe et al. / Tetrahedron 58 (2002) 1685±1691
1689
(q), 56.5 (d), 63.1 (t), 73.9 (t), 80.3 (d), 113.5 (d), 114.8 (d),
121.5 (d), 128.1 (d), 140.0 (s), 140.2 (s), 140.9 (s), 164.2 (s).
Anal. calcd for C30H43NO5: C, 72.40; H, 8.71; N, 2.81;
found: C, 72.12; H, 8.89; N, 2.67%.
3.36 (s, 3H), 3.81 (m, 1H), 4.00 (m, 1H), 4.36 (m, 1H), 5.37
(m, 1H), 6.55 (d, Jˆ8.2Hz, 1H), 6.90 (m, 1H), 7.36 (d,
Jˆ8.6 Hz, 2H), 8.12 (d, Jˆ8.2Hz, 1H), 8.26 (d,
Jˆ8.6 Hz, 2H); 13C NMR (67.5 MHz, CDCl3) d {11.7,
12.1} (q), 17.2 (q), {19.2, 19.4} (q), {20.8, 20.9} (t), 21.9
(q), {23.9, 24.2} (t), 26.6 (t), {27.7, 27.8} (t), 31.7 (d), 35.2
(t), 36.1 (d), {36.6, 37.0} (t), 38.4 (t), {39.3, 39.4} (t), 40.7
(s), {42.3, 42.7} (s), 45.7 (d), 49.9 (d), {55.5, 55.9} (q), 56.3
(d), 63.5 (d), 73.8 (t), 80.3 (d), {112.4, 112.5} (d), {113.3,
113.5} (d), 121.2 (d), 121.6 (d), 125.2 (d), 127.8 (d), 139.6
(s), 139.7 (s), 140.7 (s), 145.2(s), 151.2(s), 155.1 (s),
{163.68, 163.73} (s). Anal. calcd for C38H48N2O9: C,
67.44; H, 7.15; N, 4.14; found: C, 67.73; H, 7.43; N, 4.01%.
4.1.8. 20a-[3-(10-Hydroxyethyl)-4-nitrophenoxymethyl]-
3b-methoxy-5-pregnen (7b). The procedure similar to 7a
but using a solution of 6b (345 mg, 0.67 mmol) in ethanol
(35 mL) and sodium borohydride (93 mg, 2.5 mmol) in
ethanol (8 mL), gave 7b (312mg, 0.61 mmol, 91.0%). 7b:
yellow crystals mp 62.0±63.08C; 1H NMR (270 MHz,
CDCl3) d 0.75 (s, 3H), 1.01 (s, 3H), 1.15 (d, Jˆ6.6 Hz,
3H), 1.20±2.37 (m, 21H), 1.78 (d, Jˆ6.3 Hz, 3H), 3.07
(m, 1H), 3.36 (s, 3H), 3.81 (m, 1H), 4.00 (m, 1H), 5.37 (d,
Jˆ3.6 Hz, 1H), 5.57 (m, 1H), 6.83 (dd, Jˆ2.3, 8.9 Hz, 1H),
7.30 (d, Jˆ2.3 Hz, 1H), 8.04 (d, Jˆ8.9 Hz, 1H); 13C NMR
(67.5 MHz, CDCl3) d 11.9 (q), 17.3 (q), 19.3 (q), 21.0 (t),
24.0 (q), {24.22, 24.27} (t), 27.6 (t), {27.7, 27.9} (t), 31.8
(t), 31.8 (d), 36.3 (d), 36.8 (s), 37.1 (t), 38.6 (t), 39.5 (t), 42.5
(s), 50.0 (d), 52.4 (d), 55.5 (q), {56.4, 56.5} (d), 65.7 (d),
73.4 (t), 80.3 (d), {112.1, 112.4} (d), {113.17, 113.24} (d),
121.38 (d), 127.44 (d), 140.0 (s), {140.7, 140.9} (s), 145.1
(s), {163.6, 163.8} (s). Anal. calcd for C31H45NO5: C, 72.76;
H, 8.86; N, 2.74; found: C, 72.82; H, 9.02; N, 2.82%.
4.1.11. 5-(3b-Methoxy-5-pregnen-20a-yl)methoxy-2-nitro-
phenylmethoxycarbonyl l-leucyl-l-leucine methyl ester
(9a). A solution of 8a (45 mg, 0.068 mmol), l-leucyl-l-
leucine methyl ester tri¯uoroacetate (31 mg, 0.083 mmol),
and DMAP (13 mg, 0.11 mmol) in CHCl3 (5 mL) was stir-
red at rt for 4 d under argon atmosphere. The reaction
mixture was washed with 1N HCl, aq. NaHCO3, then
brine, dried over anhydrous MgSO4, ®ltered, and the solvent
was evaporated under reduced pressure. The residual solid
was separated by gel permeation liquid chromatography to
afford 9a (31 mg, 0.040 mmol, 58.4%) with recovery of 8a
(5.0 mg, 0.0075 mmol, 11%). 9a: white crystals mp 79.0±
4.1.9. 3b-Methoxy-20a-(4-nitro-3-p-nitrophenoxycar-
bonyloxymethylphenoxymethyl)-5-pregnen (8a). A solu-
tion of 7a (98 mg, 0.20 mmol), 4-nitrophenyl chloroformate
(46 mg, 0.23 mmol), and DMAP (40 mg, 0.33 mmol) in
CHCl3 (10 mL) was stirred at rt for 30 h under argon
atmosphere. The reaction mixture was washed with 1N
HCl, aq. NaHCO3, then brine, dried over anhydrous
MgSO4, ®ltered, and the solvent was evaporated under
reduced pressure. The residual solid was separated by gel
permeation liquid chromatography to afford 8a (80 mg,
0.12mmol, 61.3%). 8a: white crystals, mp 153.0±155.08C
(decomp.); 1H NMR (270 MHz, CDCl3) d 0.75 (s, 3H), 1.01
(s, 3H), 1.16 (d, Jˆ6.6 Hz, 3H), 0.93±2.16 (m, 20H), 2.37
(m, 1H), 3.05 (m, 1H), 3.36 (s, 3H), 3.83 (dd, Jˆ8.8, 7.1 Hz,
1H), 4.03 (dd, Jˆ8.8, 3.2Hz, 1H), 5.36 (d, Jˆ4.6 Hz, 1H),
5.75 (s, 2H), 6.94 (dd, Jˆ9.2, 2.5 Hz, 1H), 7.15 (d,
Jˆ2.5 Hz, 1H), 7.43 (d, Jˆ9.0 Hz, 2H), 8.26 (d, Jˆ
9.2Hz, 1H), 8.31 (d, Jˆ9.0 Hz, 2H); 13C NMR
(67.5 MHz, CDCl3) d 11.9 (q), 17.3 (q), 19.4 (q), 21.0 (t),
24.3 (t), 27.8 (t), 28.0 (t), 31.9 (d), 31.9 (t), 36.3 (d), 36.9 (s),
37.2 (t), 38.6 (t), 39.6 (t), 42.5 (s), 50.1 (d), 52.4 (d), 55.6
(q), 56. 5 (d), 67.7 (t), 74.1 (t), 80.3 (d), 113.4 (d), 114.6 (d),
121.3 (d), 121.7 (d), 125.4 (d), 128.3 (d), 133.7 (s), 139.7
(s), 140.9 (s), 145.5 (s), 152.0 (s), 155.3 (s), 164.0 (s); High-
resolution FAB-MS: observed m/z 663.3304. Calcd for
C37H47N2O9 ([M1H]1) 663.3282. Anal. calcd for
C37H46N2O9: C, 67.05; H, 7.00; N, 4.23; found: C, 67.09;
H, 6.99; N, 4.29%.
1
81.08C; H NMR (270 MHz, CD3OD) d 0.78 (s, 3H), 1.02
(s, 3H), 1.16 (d, Jˆ6.4 Hz, 3H), 0.85±2.16 (m, 38H), 2.36
(m, 1H), 3.06 (m, 1H), 3.34 (s, 3H), 3.69 (s, 3H), 3.91 (m,
1H), 4.01 (m, 1H), 4.22 (t, Jˆ7.4 Hz, 1H), 4.46 (dd, Jˆ5.4,
9.4 Hz, 1H), 5.36 (d, Jˆ4.0 Hz, 1H), 5.49 (s, 2H), 6.99 (d,
Jˆ9.2Hz, 1H), 7.14 (s, 1H), 8.18 (d, Jˆ9.2Hz, 1H); 13C
NMR (67.5 MHz, CD3OD) d 12.4 (q), 17.9 (q), 19.9 (q),
21.8 (q), 22.1 (q), 22.2 (t), 23.4 (q), 23.5 (q), 25.4 (t), 25.9
(d£2), 28.8 (t), 29.0 (t), 33.0 (t), 33.3 (d), 37.7 (d), 38.0 (s),
38.4 (t), 39.7 (t), 41.0 (t), 41.3 (t), 42.0 (t), 43.6 (s), 51.7 (d),
52.1 (d), 52.6 (q), 53.8 (d), 54.7 (d), 55.9 (q), 57.9 (d), 64.7
(t), 75.1 (t), 81.9 (d), 114.3 (d£2), 122.7 (d), 128.9 (d), 138.2
(s), 140.9 (s), 141.8 (s), 157.8 (s), 165.5 (s), 174.5 (s), 175.3
(s); UV lmax (methanol) 309 nm (1 12400). Anal. calcd for
C44H67N3O9: C, 67.58; H, 8.64; N, 5.37; found: C, 67.30; H,
8.65; N, 5.34%.
4.1.12. 1-[{5-(3b-Methoxy-5-pregnen-20a-yl)methoxy-2-
nitro}phenyl] ethoxy carbonyl l-leucyl-l-leucine methyl
ester (9b). The procedure for 9a but using a solution of 8b
(80 mg, 0.12mmol), l-leucyl-l-leucine methyl ester
tri¯uoroacetate (56 mg, 0.15 mmol), and DMAP (24 mg,
0.19 mmol) in CHCl3 (10 mL), gave 9b (40 mg,
0.05 mmol, 41.7%). 9b: yellow crystals mp 69.0±71.08C;
1H NMR (270 MHz, CD3OD) d 0.77 (s, 3H), 0.94±2.19 (m,
38H), 1.02(s, 3H), 1.14 (d, Jˆ4.3 Hz, 3H), 1.63 (d,
Jˆ6.3 Hz, 3H), 2.37 (m, 1H), 3.08 (m, 1H), 3.39 (s, 3H),
3.74 (s, 3H), 3.94 (m, 1H), 4.12(m, 1H), 4.35 (m, 1H), 4.69
(m, 1H), 5.20 (m, 1H), 5.35 (d, Jˆ3.6 Hz, 1H), 6.38 (m,
1H), 6.90 (d, Jˆ8.9 Hz, 1H), 7.02(m, 1H), 8.62 (d,
Jˆ8.9 Hz, 1H); 13C NMR (67.5 MHz, CD3OD) d {11.83,
11.87} (q), 17.3 (q), {19.3, 19.5} (q), 21.0 (t), {21.1, 21.4}
(q), {21.7, 21.8} (q), {21.9, 22.0} (q), {22.2, 22.6} (q),
{22.8, 23.1} (q), {24.0, 24.3} (t), {24.6, 24.7} (d), {24.8,
25.0} (d), 26.7 (t), {27.8, 27.9} (t), 31.8 (t), 31.8 (d), 36.3
(d), 36.8 (s), 37.1 (t), 38.5 (t), 39.5 (t), 39.6 (t), {40.9, 41.1}
(t), 42.5 (s), {50.0, 50.9} (d), 51.0 (d), {52.1, 52.3} (d),
4.1.10. 3b-Methoxy-20a-[4-nitro-3-{10-(p-nitrophenoxy-
carbonyloxy)ethyl}-phenoxymethyl]-5-pregnen
The procedure for 8a but using a solution of 7b (162mg,
0.32mmol), 4-nitrophenyl chloroformate (75 mg,
(8b).
0.37 mmol), and DMAP (65 mg, 0.53 mmol) in CHCl3
(10 mL), gave 8b (130 mg, 0.19 mmol, 59.4%). 8b: yellow
1
crystals, mp 76.0±77.08C; H NMR (270 MHz, CDCl3) d
{0.75, 0.77} (s, 3H), 1.02(s, 3H), 1.16 (d, Jˆ5.6 Hz, 3H),
1.25±2.46 (m, 21H), 1.78 (d, Jˆ6.3 Hz, 3H), 3.07 (m, 1H),

1690
S. Watanabe et al. / Tetrahedron 58 (2002) 1685±1691
{52.4, 52.5} (q), 53.4 (d), {55.5, 56.0} (q), 56.4 (d), 63.2 (d),
73.8 (t), 80.5 (d), 113.0 (d), 115.6 (d), 121.6 (d), 126.1 (d),
{127.5, 127.7} (s), 139.8 (s), 140.6 (s), 141.0 (s), 162.5 (s),
163.8 (s), 172.9 (s); UV lmax (methanol) 304 nm (1 10300).
Anal. calcd for C45H69N3O9´H2O: C, 66.39; H, 8.79; N, 5.16;
found: C, 66.77; H, 8.72; N, 5.10%.
1.11 (d, 3H, Jˆ7.0 Hz), 2.39 (d, 1H, Jˆ12.7 Hz), 3.07 (m,
1H), 3.36 (s, 3H), 3.72 (s, 2H), 3.63±3.84 (m, 2H), 5.36 (d,
1H, Jˆ3.3 Hz), 5.56 (s, 2H), 6.77 (d, 1H, Jˆ2.4 Hz), 6.84
(dd, 1H, Jˆ2.4, 8.9 Hz), 7.29 (d, 2H, Jˆ8.4 Hz), 7.47 (d,
2H, Jˆ8.4 Hz), 8.16 (d, 1H, Jˆ8.9 Hz); 13C NMR
(67.5 MHz, CDCl3) d 12.0 (q), 17.4 (q), 19.4 (q), 21.1 (t),
24.4 (t), 27.8 (t), 28.0 (t), 31.8 (t), 31.9 (d), 36.3 (d), 36.9 (s),
37.2 (t), 38.7 (t), 39.6 (t), 40.8 (t), 42.6 (s), 50.1 (d), 52.4 (d),
55.6 (q), 56.5 (d), 63.8 (t), 73.8 (t), 80.3 (d), 113.3 (d), 113.7
(d), 121.1 (s), 121.4 (d), 128.0 (d), 131.1 (d), 131.8 (d),
132.6 (s), 135.2 (s), 139.7 (s), 140.9 (s), 163.8 (s), 170.2
(s). Anal. calcd for C38H48BrNO6: C, 65.70; H, 6.96; N,
2.02; found: C, 65.97; H, 7.21; N, 1.99%. MS (70 eV): m/z
695 and 693 (M1 for C38H4879BrNO6 and C38H4881BrNO6,
respectively). UV/VIS (THF): lmax 310 nm (1 9940).
4.1.13. 3b-Methoxy-20a-(3-p-toluenesulfonylhydrazono-
methyl-4-nitro-phenoxymethyl)-5-pregnen (10). A solu-
tion
of
3b-methoxy-20a-(3-formyl-4-nitro-phenoxy-
methyl)-5-pregnen (6a: 391 mg, 0.789 mmol) and
p-toluenesulfonyl hydrazide (240 mg, 1.29 mmol) in
ethanol (100 mL) was stirred over night at rt. The solvent
was evaporated under reduced pressure and the residual
solid was puri®ed by column chromatography to give 10
(483 mg, 0.728 mmol, 92.2%). 10: yellow crystals mp
1
210.0±212.08C; H NMR (270 MHz, CDCl3) d 0.77 (s,
4.1.16.
20a-(3-p-Methylphenylmethylcarbonyloxy-
3H), 1.02 (s, 3H), 1.07±2.21 (m, 19H), 1.16 (d, 3H,
Jˆ6.5 Hz), 2.18 (s, 1H), 2.39 (s, 1H), 2.42 (s, 3H), 3.03±
3.12(m, 1H), 3.36 (s, 3H), 3.78±3.84 (m, 1H), 3.99±4.03
(m, 1H), 5.37 (d, 1H, Jˆ4.6 Hz), 6.94 (dd, 1H, Jˆ2.8,
9.1 Hz), 7.32(d, 2H, Jˆ8.1 Hz), 7.39 (d, 1H, Jˆ2.8 Hz),
7.87 (d, 2H, Jˆ8.1 Hz), 8.07 (d, 1H, Jˆ9.1 Hz), 8.45 (s,
1H); 13C NMR (67.5 MHz, CDCl3) d 12.0 (q), 17.4 (q),
19.4 (q), 21.1 (t), 21.7 (q), 24.4 (t), 27.9 (s), 28.0 (t),
31.90 (t), 31.94 (d), 36.4 (d), 36.9 (t), 37.2(t), 38.7 (t),
39.6 (t), 42.6 (s), 50.1 (d), 52.5 (d), 55.6 (q), 56.5 (d),
74.1 (t), 80.3 (d), 113.2 (d), 116.4 (d), 121.5 (d), 127.6
(d), 128.0 (d), 129.8 (d), 131.0 (s), 125.3 (s), 140.5 (s),
140.9 (s), 143.2(d), 144.5 (s), 163.4 (s). Anal. calcd for
C37H49N3O6S: C, 66.94; H, 7.44; N, 6.33; found: C, 66.65;
H, 7.46; N, 6.20%.
methyl-4-nitro-phenoxymethyl)-3b-methoxy-5-pregnen
(12b). The procedure for 12a but using a solution of 11
(63.6 mg; crude products) and p-tolylacetic acid (95 mg,
0.63 mmol) in CHCl3 (1 mL) at room temperature, gave
12b (11.1 mg, 0.0176 mmol, 11.7% from 10). 12b: yellow
crystals mp: 116.5±118.08C; 1H NMR (270 MHz, CDCl3) d
0.76 (s, 3H), 0.79±2.21 (m, 20H), 1.02 (s, 3H), 1.11 (d, 3H,
Jˆ6.6 Hz), 2.34 (s, 3H), 2.39 (m, 1H), 3.07 (m, 1H), 3.36 (s,
3H), 3.72 (s, 2H), 3.62±3.83 (m, 2H), 5.37 (d, 1H,
Jˆ3.3 Hz), 5.56 (s, 2H), 6.81 (d, 1H, Jˆ2.4 Hz), 6.84 (dd,
1H, Jˆ2.4, 8.9 Hz), 7.15 (d, 2H, Jˆ7.9 Hz), 7.23 (d, 2H,
Jˆ7.9 Hz), 8.15 (d, 1H, Jˆ8.9 Hz); 13C NMR (67.5 MHz,
CDCl3) d 12.1 (q), 17.4 (q), 19.5 (q), 21.1 (t), 21.2 (q), 24.4
(t), 27.9 (t), 28.1 (t), 31.9 (t), 32.0 (d), 36.4 (d), 36.9 (s), 37.2
(t), 38.7 (t), 39.7 (t), 41.1 (t), 42.6 (s), 50.2 (d), 52.4 (d), 55.6
(q), 56.5 (d), 63.5 (t), 73.7 (t), 80.3 (d), 113.2(d), 113.4 (d),
121.3 (d), 127.8 (d), 129.1 (d), 129.3 (d), 130.4 (s), 135.4
(s), 136.7 (s), 139.6 (s), 140.8 (s), 163.6 (s), 170.7 (s). Anal.
calcd for C39H51NO6: C, 74.37; H, 8.16; N, 2.22; found: C,
74.07; H, 8.35; N, 2.12%. UV lmax (THF) 310 nm (1 6970).
4.1.14. 20a-(3-Diazomethyl-4-nitro-phenoxymethyl)-3b-
methoxy-5-pregnen (11). To a solution of 10 (204 mg,
0.31 mmol) in methanol (10 mL) was added a triethylamine
(0.25 mL, 1.8 mmol) and stirred at rt over night. The yellow
precipitates was ®ltered by suction to afford 11 (131 mg,
0.26 mmol, 84%). 11: yellow crystals mp 140.0±142.08C;
1H NMR (270 MHz, CDCl3) d 0.74 (s, 3H), 0.95±2.21 (m,
20H), 1.01 (s, 3H), 1.14 (d, 3H, Jˆ6.5 Hz), 2.40 (d, 1H,
Jˆ10.5 Hz), 3.07 (m, 1H), 3.36 (s, 3H), 3.77 (m, 1H),
3.97 (m, 1H), 5.37 (s, 1H), 6.44 (d, 1H, Jˆ2.4 Hz), 6.58
(dd, 1H, Jˆ2.4, 9.5 Hz), 6.69 (s, 1H), 8.22 (d, 1H,
Jˆ9.5 Hz); 13C NMR (67.5 MHz, CDCl3) d 12.0 (q), 17.4
(q), 19.4 (q), 21.1 (t), 24.4 (t), 27.8 (t), 28.0 (t), 31.90 (t),
31.93 (d), 36.3 (d), 36.9 (s), 37.2(t), 38.7 (t), 39.6 (t), 42.5
(s), 47.3 (d), 50.1 (d), 52.5 (d), 55.6 (q), 56.5 (d), 73.7 (t),
80.2 (d), 106.7 (d), 110.2 (d), 121.3 (d), 129.4 (d), 131.5 (s),
135.4 (s), 140.7 (s), 163.5 (s). IR (KBr disk) 2076 cm21
(±N2).
4.2. Preparation of liposomes
Phosphatidylcholine (12.1 mg), dicetyl phosphate (4.5 mg),
and the caged LeuLeuOMe 9a (7.2mg, 9.2 mmol) was
dissolved in CHCl3 (10 mL) in a round-bottomed ¯ask. A
thin ®lm was formed on the interior of the ¯ask by the gentle
evaporation of the solvent under reduced pressure at 308C.
After the solvent was completely removed in vacuo, PBS
(Phosphate Buffered Saline, 10 mL) was added to the ¯ask.
This mixture was sonicated for 5 min to afford a suspension
of multilamellar vesicles.
4.3. Photolysis of the caged LeuLeuOMe in liposomes
4.1.15. 20a-(3-p-Bromophenylmethylcarbonyloxymethyl-
4-nitro-phenoxymethyl)-3b-methoxy-5-pregnen (12a). A
solution of 11 (42.8 mg, 0.084 mmol) and p-bromophenyl-
acetic acid (93.1 mg, 0.43 mmol) in CHCl3 (2mL) was
warmed to re¯ux and stirred over night. The solution was
washed with aq. NaHCO3, dried over anhydrous MgSO4,
®ltered and the solvent was evaporated under reduced pres-
sure. The residue was puri®ed by gel permeation liquid
chromatography to give 12a (40.1 mg, 0.058 mmol, 69%).
12a: yellow crystals mp: 94.0±96.08C; 1H NMR (270 MHz,
CDCl3) d 0.76 (s, 3H), 0.79±2.21 (m, 20H), 1.02 (s, 3H),
The liposomes prepared above was divided into several
portions and placed in test tube, the volume of each was
100 mL. After the sample was photolized by Rayonet Photo-
Ê
chemimal Reactor (RPR 3500 A£4), methanol (300 mL)
was added to the solution. After the sample became clear,
the amount of a remained starting material was estimated by
HPLC. The amount of a released LeuLeuOMe was also
estimated by HPLC after the photolized sample was treated
with a solution of ¯uorescamine in dioxane.

S. Watanabe et al. / Tetrahedron 58 (2002) 1685±1691
1691
4. (a) Olejnik, J.; Krzymanska-Olejnik, E.; Rothschild, K. J. Meth.
Enzymol. 1998, 291, 135. (b) Marriott, G.; Ottl, J. Meth.
Enzymol. 1998, 291, 155.
Acknowledgements
The authors are indebted to Professor Dr Takayuki
Kawashima and Dr Naokazu Kano, Department of
Chemistry, Graduate School of Science, The University of
Tokyo, for measuring high resolution mass spectra. This
work was supported by a Grant-in-Aid for Scienti®c
Research (No. 08680639) from the Ministry of Education,
Science, and Culture, Japan.
5. (a) Odaka, M.; Furuta, T.; Kobayashi, Y.; Iwamura, M.
Biochem. Biophys. Res. Commun. 1995, 213, 652. (b) Odaka,
M.; Furuta, T.; Kobayashi, Y.; Iwamura, M. Photochem.
Photobiol. 1996, 63, 800. (c) Sakauchi, H.; Furuta, T.;
Kobayashi, Y.; Iwamura, M. Biochem. Biophys. Res. Commun.
1997, 233, 211. (d) Watanabe, S.; Iwamura, M. J. Org. Chem.
1997, 62, 8616. (e) Watanabe, S.; Sato, M.; Sakamoto, S.;
Yamaguchi, K.; Iwamura, M. J. Am. Chem. Soc. 2000, 122,
12588.
References
6. Thiele, D. L.; Lipsky, P. E. J. Immunol. 1992, 148, 3950.
7. Photoactivable (Caged) Probes; Haugland, R. P. Ed.; Handbook
of Fluorescent Probes and Research Chemicals, 6th ed.;
Molecular Probes, Inc. 1996; p 447.
1. For leading reviews on caged compounds, see: (a) Kaplan, J. H.
Annu. Rev. Physiol. 1990, 52, 897. (b) Adams, S. R.; Tsien,
R. Y. Annu. Rev. Physiol. 1993, 55, 755. (c) Morrison, H.,
Ed.; Bioorganic Photochemistry, Wiley: New York, 1993;
Vol. 2.
È
8. (a) Udenfriend, S.; Stein, S.; Bohlen, P.; Dairman, W.;
Leimgruber, W.; Weigele, M. Science 1972, 178, 871.
(b) Weigele, M.; DeBernardo, S. L.; Tengi, J. P.; Leimgruber,
W. J. Am. Chem. Soc. 1972, 94, 5927.
2. Givens, R. S.; Weber, J. F.; Jung, A. H.; Park, C.-H. Meth.
Enzymol. 1998, 291, 1.
3. (a) Furuta, T.; Iwamura, M. Meth. Enzymol. 1998, 291, 50.
(b) Furuta, T.; Wang, S. S.-H.; Dantzker, J. L.; Dore, T. M.;
Bybee, W. J.; Callaway, E. M.; Denk, W.; Tsien, R. Y. Proc.
Natl. Acad. Sci. USA 1999, 96, 1193.
9. Naito, M.; Nagai, H.; Kawano, S.; Umezu, H.; Zhu, H.;
Moriyama, H.; Yamamoto, T.; Takatsuka, H.; Takei, Y.
J. Leukoc. Biol. 1996, 60, 337.