Photoaffinity labeling of PKC isozymes by phorbol ester derivatives

Article abstract of DOI:10.1016/S0968-0896(98)00068-6

Photoaffinity probes PPDA and PPTD, which have diazoacetyl and trifluorodiazopropionyl group at C-13 position in phorbol, respectively, were synthesized. Photoaffinity labeling of protein kinase C isozymes by both the probes resulted in specific cross-linking. Copyright (C) 1998 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00068-6

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 1117±1126
Photoanity Labeling of PKC Isozymes by
Phorbol Ester Derivatives
Koichiro Uotsu, Mikiko Sodeokay and Masakatsu Shibasaki*
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113, Japan
Received 3 April 1998; accepted 6 April 1998
AbstractÐPhotoanity probes PPDA and PPTD, which have diazoacetyl and tri¯uorodiazopropionyl group at C-13
position in phorbol, respectively, were synthesized. Photoanity labeling of protein kinase C isozymes by both the
probes resulted in speci®c cross-linking. # 1998 Elsevier Science Ltd. All rights reserved.
Introduction
of phorbol esters, which are essential for PKC activa-
Phorbol esters are tumor-promoting diterpene esters
isolated from croton oil.1±3 They are known to bind to
and activate isozymes of the protein kinase C (PKC)
family,4±6 serine±threonine phosphorylating enzymes
which play central roles in intracellular signal transduc-
tions. The structure of protein kinase C can be divided
into two domains, regulatory and catalytic (Figure 1).
The catalytic domain, which phosphorylates the sub-
strate proteins, is homologous with that of other ATP
dependent kinases such as protein kinase A (PKA). On
the other hand, the structure of the regulatory domain,
which consists of C1 and C2 regions, is characteristic of
PKC. The C1 region of cPKC (conventional PKC; PKC
a, bI/II, and g) and nPKC (novel PKC; PKC d, ", Z,
and y) has two repeats of a cysteine rich domain (CRD)
whereas aPKC (atypical PKC; PKC z and l/i), which is
not activated by phorbol esters, has only one CRD. The
CRDs have been shown to be the binding site of protein
kinase C activators such as diacylglycerol, phorbol
esters, and teleocidin.7±10 NMR studies11±13 and X-ray
analysis14 have revealed the structure of the second
CRD and the mode of interaction with phorbol esters.
However, further studies are still necessary to under-
stand the mechanism of PKC activation and to design
new PKC modulators, since most of the previous studies
such as the X-ray analysis14 were carried out in the
absence of phospholipids and/or the hydrophobic portion
tion. Moreover, peptides containing only one repeat of
CRDs were employed in those studies. Although it has
been proposed that only one CRD repeat is responsible
for phorbol ester binding,15,16 Slater et al. recently
reported that diacylglycerol and phorbol esters can bind
simultaneously to PKCa, suggesting more complicated
function of each CRD in native PKC.17 We therefore
focused on the photoanity labeling experiments as a
method for analyzing interactions between native PKC
and phorbol esters in the presence of phospholipids
to clarify the role of individual CRDs in PKC. The
development of an ecient photoanity probe of
phorbol ester derivatives is very important for such
an analysis.
Although there have been several reports in which the
photoanity labeling of PKC was carried out using
phorbol ester derivatives,18,19 cross-linking to PKC was
not, to our knowledge, achieved until we reported the
synthesis of a novel photoanity probe PPDA(5) and
cross-linking to crude PKC mixtures.20 Wender and Irie
et al. independently reported the photoanity labeling
of PKC model peptides.21 We suppose that the location
of the photolabile group in the complexes is important:
most previous ligands possessed an azidobenzoyl group
at C12 in phorbol. Previous structure±activity relation-
ship models22±29 have proposed that a hydrophobic
ester group is required at the C12 position in phorbol
esters, suggesting that these groups are incorporated
into phospholipid membranes. These models are con-
sistent with the unsuccessful photoanity labeling
*Corresponding author. E-mail: mshibasa@mol.f.u-tokyo.ac.jp
yCurrent address: Sagami Chemical Research Center, Nishi-
Ohnuma, Sagamihara, Kanagawa 229, Japan.
0968-0896/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00068-6

1118
K. Uotsu et al./Bioorg. Med. Chem. 6 (1998) 1117±1126
and interacts with PKC in phorbol ester±PKC±phos-
pholipid complexes. In the course of our investigations
towards the clari®cation of these interactions, we herein
report the design and synthesis of the two photoanity
probes and the photoanity labeling of pure native
PKC isozymes in detail, which is essential for further
analyses.
Results and Discussion
A photoanity probe [3H]PPDA (phorbol 12-(1-pyrene-
butyrate) 13-diazoacetate, 5) was synthesized as
shown in Scheme 1. After successful synthesis of
[3H]PPDA, we also designed the photoanity probe
[3H]PPTD (11) in a attempt to develop an even more
Figure 1. Structure of PKC family.
experiments of PKC by 12-azidobenzoyl phorbol deri-
vatives, resulting in speci®c cross-linking not to proteins
but only to phosphatidylserine. Our photoanity probe
PPDA, and Wender and Irie's probe possessed a photo-
labile diazoacetyl group at the C13 position in phorbol.
Concerning the C13 position in phorbol esters, we have
pointed out the possible importance of the C13 ester
group by synthesis and biological evaluation of
13-deoxyphorbol esters.30 Some recent reports on
molecular modeling supported the importance of the
C13 ester group,31,32 although models by other groups
including X-ray data14 have not suggested the
importance of this group. Our results, the biological
evaluation of 13-deoxyphorbol esters and photo-
anity labeling by 13-diazoacetyl phorbol ester deriv-
ative 5 (PPDA), suggest that the ester group at C-13
in phorbol esters is located in close proximity to PKC
ecient probe. [3H]PPTD (11) has
a
tri¯uoro-
diazopropionyl group in place of a diazoacetyl group at
the C-13 position in phorbol. It has been reported that
the tri¯uorodiazopropionyl group is superior to the
diazoacetyl group in that the tri¯uorodiazopropionyl
group does not su€er from an undesirable Wol€
rearrangement of the carbene intermediate generated by
irradiation.33 Although there have been several reports
of successful photoanity labeling using tri¯uoro-
diazopropionyl derivatives,34 direct comparisons of the
diazoacetyl and tri¯uorodiazopropionyl groups in bio-
logical systems are scarce. Therefore we are interested in
evaluating these two groups in our systems as well. The
photoanity probe [3H] PPTD (phorbol 12-(1-pyr-
enebutyrate) 13-(3,3,3-tri¯uoro-2-diazo-propionate), 11)
was synthesized as shown in Scheme 2.
.
Scheme 1. (a) BocNHCH2CO2H, DCC, Et3N, THF, r.t. (77%); (b) 1-pyrenebutyric acid, EDC HCl, DMAP, CH2Cl2, r.t. (91%); (c)
TFA, 0 ꢀC (100%); (d) isoamyl nitrite, AcOH, CHCl3, isoamyl alcohol, r.t. (58%); (e) MnO2, CH2Cl2, r.t. (60%); (f) [3H]NaBH4,
MeOH, 0 ꢀC (65%).

K. Uotsu et al./Bioorg. Med. Chem. 6 (1998) 1117±1126
1119
First, the binding anity of both ligands 5 (PPDA) and
11 (PPTD) to PKC bI was measured by evaluating their
ability to inhibit [3H]PDBu binding competitively using
a reported procedure with minor modi®cations.35 Both
of the ligands showed a very strong anity, which is
comparable to PMA (phorbol 12-myristate 13-acetate,
the most potent phorbol ester) (Figure 2(A)). The binding
anity to PKCa was also measured, and similar results
were obtained. Thus con®rming that these photoanity
probes have a very strong binding anity, we next inves-
tigated their ability to activate PKC catalytic activities
(Figure 2(B)). It was also shown that these probes activate
PKC bI with almost the same potency as PMA. It is
very favorable that incorporation of photolabile groups
was achieved without decreasing biological activities.
CaCl2 (1 mM), [3H]PPDA or [3H]PPTD (1 mM), and
PKC bI (280 nM) in a 50 mM Tris±HCl bu€er (pH 6.8).
After incubating for 3 min at 30 ꢀC and for 2 min at
0 ꢀC, the mixture was irradiated at 254 nm for 10 s at
0 ꢀC. After separation of PKC bI by SDS±PAGE and
silver staining, the gel was sliced every 3 mm and the
radioactivity in each slice was measured after dissolving
the gels in acidic H2O2. Radioactivities were detected
only in the fraction containing PKC bI, indicating cross-
linking of the probes to PKC bI (Figure 3). When the
reaction was carried out in the presence of excess cold
PMA (40 mM), the radioactivity in the fraction contain-
ing PKC bI was suppressed, suggesting that these cross-
linking is speci®c. The yield for speci®c cross-linking
by 5 and 11 was 1.2% and 1.3%, respectively. After
investigations, it was observed that a small amount of
cross-linking occurred also even in the absence of
photolysis, and this cross-linking was not suppressed
by the presence of excess cold PMA, suggesting that it
was nonspeci®c (Figure 4, lane 3, 4, 7 and 8). The degree
of the UV-independent cross-linking was larger in the
case of [3H]PPTD (11) than [3H]PPDA (5). It has been
reported that the diazoacetyl group can react with the
SH group of a cysteine residue in cysteine protease
and that the tri¯uorodiazopropionyl group can react
with thiols such as mercaptoethanol and dithiothreitol
to yield hydrazones in the dark.36,37 Thus it is likely
We next investigated conditions for the photolysis of the
probes. The half-life of probe 5 and 11 irradiated at
254 nm was measured to be 5 and 20 s respectively, by
following UV absorption. However, irradiation of PKC
for longer than 30 s resulted in signi®cant damage of
PKC, judged by SDS±PAGE. To avoid this PKC
damage, the reaction mixture was irradiated for only
10 s in the following experiments.
We then carried out photoanity labeling of PKC bI in
the presence of phosphatidylserine vesicles (100 mg/ml),
ꢀ
.
Scheme 2. (a) 1-Pyrenebutyric acid, EDC HCl, DMAP, CH2Cl2, r.t. (97%); (b) K2CO3, MeOH, THF, 0 C (44%); (c)
CF3C(N2)COCl, DMAP, CH2Cl2, r.t. (63%); (d) TFA, anisole, CH2Cl2, 0 ꢀC (100%); (e) TPAP, NMO, CH2Cl2, r.t. (80%); (f)
[3H]NaBH4, MeOH, 0 ꢀC (63%).

1120
K. Uotsu et al./Bioorg. Med. Chem. 6 (1998) 1117±1126
that the UV-independent cross-linking was the result
of a nonspeci®c reaction with the cysteine residues in
PKC.
phosphatidylserine(PS)-Triton X-100 mixed micelles
(PS/Triton X-100=1/4) were prepared according to the
method of Bell et al., followed by the addition of CaCl2,
[3H]PPDA or PPTD (1 mM), and PKC bI (280 nM). The
mixture was irradiated for 10 s, and after separation by
SDS±PAGE, the radioactivities in the gel were mea-
sured as above. Under these conditions, interestingly,
the results were somewhat di€erent from those in the PS
vesicle systems. Although [3H]PPTD (11) resulted in
speci®c cross-linking with PKC bI (1.1% yield),
[3H]PPDA (5) resulted only in a very low cross-linking
yield (0.4%). The PS/Triton X-100 mixed micelles con-
taining phorbol esters seem to have weaker interaction
with PKC than PS vesicles containing phorbol esters
because of lower surface concentration of PS. Assuming
that a ketene intermediate was formed by a Wol€ re-
arrangement of carbene spices generated from [3H]PPDA
(5), it is likely that less reactive, and long-living ketene
Photoanity labeling of other subtypes of PKC (a and
", 140nM) also gave similar results (Figure 5). These
results are consistent with the observation that both
cPKC and nPKC have similar binding anity to phor-
bol esters. Further analysis of the cross-linking site
would reveal the role of individual CRD in either cPKC
or nPKC. Photoanity labeling experiments were also
carried out in other phospholipid systems (Figure 6). The
Figure 2. Biological activities of PPDA and PPTD. (A)
%[3H]PDBu bound to PKC bI was measured in the presence of
various concentrations of ligands. (B) [32P]Phosphate group
incorporated into substrate peptides by PKC bI activated by
various concentrations of ligands were measured.
Figure 3. Photoanity labeling of PKC bI in PS vesicles by
[3H]PPDA (A) and [3H]PPTD (B). Fourteen picomoles of PKC
bI was used.

K. Uotsu et al./Bioorg. Med. Chem. 6 (1998) 1117±1126
1121
intermediates might be able to form cross-linking only
in PS vesicle systems, where PKC has a stronger inter-
action with phospholipids containing phorbol esters. On
the contrary, it is possible that the carbene adduct
derived from [3H]PPTD (11), which does not undergo a
rearrangement to a ketene, can form cross-linking with
PKC in both the PS vesicle and Triton X-100 mixed
micelle systems. The lower degree of UV-independent
nonspeci®c cross-linking in the PS-Triton X-100 mixed
micelles system (Figure 6, lane 3, 4, 7 and 8) compared
to the PS vesicle systems (Figure 4), in case of both
probes, can also be interpreted as a result of a weaker
interaction between PKC and PS-Triton X-100 mixed
micelles containing phorbol esters. Thus, the results of
photoanity labeling experiments were in¯uenced by
both the nature of the photolabile groups within the
probes and the phospholipid systems employed.
Although PS vesicle systems were essential for ecient
cross-linking in the case of [3H] PPDA (5) possibly
because of formation of a less reactive ketene inter-
mediate, it was shown that [3H]PPTD (11), which had
tri¯uorodiazopropionyl group in place of diazoaetyl
group, resulted in cross-linking with similar eciency in
both PS vesicles and PS/Triton X-100 mixed micelle
systems.
Figure 5. Photoanity labeling of PKC isozymes by [3H]
PPDA (lanes 1, 2, 5, 6, 9, and 10) or [3H]PPTD (lanes 3, 4, 7, 8,
11, and 12) in PS vesicles. Reactions were carried out using
7 pmol of PKC a (lanes 1±4), PKC bI (lanes 5±8), and PKC "
(lanes 9±12), respectively.
Figure 4. Photoanity labeling of PKC bI in PS vesicles.
Reactions were carried out with (lanes 1, 2, 5, and 6) or without
(lanes 3, 4, 7, and 8) UV irradiation (10 s) in the presence (lanes
2, 4, 6, and 8) or absence (lanes 1, 3, 5, and 7) of 40-fold excess
of cold PMA. Fourteen picomoles of PKC bI was used in each
experiment. Radioactivities found in the fractions containing
PKC were shown.
Figure 6. Photoanity labeling of PKC bI in PS-Triton X-100
mixed micelles. Reactions were carried out with (lanes 1, 2, 5,
and 6) or without (lanes 3, 4, 7, and 8) UV irraditation (10 s) in
the presence (lanes 2, 4, 6, and 8) or absence (lanes 1, 3, 5, and
7) of 40-fold excess of cold PMA. Fourteen picomoles of PKC
bI was used in each experiment.

1122
K. Uotsu et al./Bioorg. Med. Chem. 6 (1998) 1117±1126
Conclusion
m), 3.99 (1H, m), 4.98 (1H, m), 5.57 (1H, dd, J=1.5,
5.0 Hz), 7.20±7.50 (15H, m), 7.56 (1H, dd, J=1.4, 3.0 Hz).
It was demonstrated that photoanity probes, which
have photolabile groups at the C13 position in phorbol,
can form cross-linking with native pure isozymes of
PKC by irradiation. Although the cross-linking yield is
not very high, we suppose that determination of the
cross-linking site can be realized by taking advantage of
subpicomole order analysis by mass spectroscopy and
other methodologies to clarify the role of individual
CRD in native PKC. Further studies along these lines
are now in progress together with studies to develop
more ecient photoanity probes.
FAB HRMS (NBA) m/z 786.3565, (M+Na+) calcu-
lated for C46H53O9NNa: 786.3618.
Preparation of compound 3
To a solution of compound 2 (54.8 mg, 71.7 mmol) in
.
methylene chloride (1.5 mL) were added EDCI HCl
(27.5 mg, 144 mmol), DMAP (8.8 mg, 71.7 mmol), and
1-pyrenebutyric acid (41.4 mg, 144 mmol) at 0 ꢀC and the
mixture was allowed to warm to r.t. The mixture was
stirred for 3 h and ®ltered through SiO2 (AcOEt/
Hex=1/1), and the ®ltrate was concentrated. The residue
was puri®ed by silica gel column chromatography (AcOEt/
benzene=1/12) to give compound 3 (67.2 mg, 91%).
Experimental
General procedures
Infrared (IR) spectra were recorded on a Perkin Elmer
1600 FT IR spectrometer. 1H NMR spectra were
measured with a JEOL JNM-EX 270 spectrometer.
Chemical shifts are reported on the d scale relative to
CHCl3 as an internal reference (7.26 ppm). Mass spectra
(MS) were measured on a JEOL JMS-SX 102A instrument.
In general, reactions were carried out in dry solvents
under an argon atmosphere, unless otherwise mentioned.
All phorbol esters should be handled with care, because
they are potent tumor promoters and strong irritants.
3,3,3-Tri¯uoro-2-diazopropionyl chloride was purchased
from Pierce. Phorbol was purchased from LC laboratories.
[3H]NaBH4 (488.4 GBq/mmol) was purchased from
NEN. Each isozyme of PKC (human, recombinant) was
purchased from Calbiochem. Phosphatidylserine was
purchased from SIGMA. The binding assay and PKC
assay were carried out using reported procedure with
minor modi®cations.35,38 The results shown in the Figures
are representative of three or four sets of experiments.
1
IR (neat) 3422, 2974, 1708, 1448, 1367, 1161, 845 cm
.
1H NMR (CDCl3, 270 MHz) d 0.95 (3H, d, J=6.1 Hz),
1.15 (1H, d, J=5.0 Hz), 1.27 (3H, s), 1.29 (3H, s), 1.47
(9H, s), 1.82 (3H, dd, J=1.1, 2.6 Hz), 2.10 (1H, s), 2.16
(1H, m), 2.21 (2H, m), 2.46 (1H, d, J=18.0 Hz), 2.53
(2H, t, J=7.1 Hz), 2.57 (1H, d, J=18.0 Hz), 3.21 (1H,
dd, J=5.0, 5.0 Hz), 3.30 (1H, m), 3.46 (2H, t,
J=7.6 Hz), 3.57 (2H, s), 3.93 (1H, dd, J=18.0, 5.1 Hz),
4.02 (1H, dd, J=18.0, 6.5 Hz), 5.03 (1H, m), 5.19 (1H,
s), 5.43 (1H, d, J=10.5 Hz), 5.63 (1H, d, J=5.0 Hz),
7.20±7.50 (15H, m), 7.56 (1H, m), 7.80±9.30 (9H, m).
FAB HRMS (NBA) m/z 1033.4677, (M+) calculated for
C66H67O10N: 1033.4765.
Preparation of compound 4
To compound 3 (31.9 mg, 30.8 mmol) was added TFA
(0.5 mL) at 0 ꢀC, and the mixture was stirred for 0.5 h.
The mixture was concentrated, and the residue was
puri®ed by silica gel column chromatography (MeOH/
CHCl3=1/10) to give compound 4 (21.3 mg, 100%).
Preparation of compound 2
To a solution of phorbol 20-trityl ether39 (11.8 mg,
19.4 mmol) in THF (0.3 mL) were added DCC (20.1 mg,
97.2 mmol), triethylamine (5.9 mg, 58.3 mmol), and N-
tert-butoxycarbonylglycine (17.0 mg, 97.2 mmol) at 0 ꢀC
and the mixture was allowed to warm to r.t. After stir-
ring for 2 h, the mixture was ®ltered, and the ®ltrate was
concentrated. The residue was puri®ed by silica gel col-
umn chromatography (AcOEt/Hex=1/2) to give com-
pound 2 (11.4 mg, 77%).
1
IR (neat) 3393, 2923, 1704, 1376, 1207, 1140, 844 cm
.
1H NMR (CDCl3, 270 MHz) d 0.95 (3H, d, J=6.3 Hz),
1.22 (1H, d, J=5.2 Hz), 1.23 (3H, s), 1.28 (3H, s), 1.69
(3H, dd, J=1.2, 1.9 Hz), 2.19 (2H, m), 2.29 (1H, m),
2.47 (2H, m), 2.56 (2H, t, J=6.9 Hz), 3.17 (1H, m), 3.31
(1H, m), 3.46 (2H, dd, J=6.1, 8.5 Hz), 3.92 (1H, d,
J=13.0 Hz), 3.98 (1H, d, J=13.0 Hz), 4.12 (1H, d,
J=16.0 Hz), 4.20 (1H, d, J=16.0 Hz), 5.59 (1H, d,
J=10.5 Hz), 5.63 (1H, d, J=5.5 Hz), 7.52 (1H, m),
7.90±8.60 (9H, m).
IR (neat) 3412, 2978, 1702, 1215, 1164, 1054 cm-1.
1H NMR (CDCl3, 270 MHz) d 1.04 (1H, d, J=5.4 Hz),
1.08 (3H, d, J=6.5 Hz), 1.22 (3H, s), 1.27 (3H, s), 1.46
(9H, s), 1.78 (3H, dd, J=1.5, 3.0 Hz), 1.99 (1H, m), 2.36
(1H, br-s), 2.37 (1H, d, J=19.0 Hz), 2.48 (1H, d,
J=19.0 Hz), 3.00 (1H, br-s), 3.06 (1H, m), 3.11 (1H, dd,
J=5.0, 5.4 Hz), 3.30 (1H, br-s), 3.56 (2H, s), 3.98 (2H,
FAB HRMS (NBA) m/z 692.3250, (M+H+) calculated
for C42H46O8N: 692.3223.

K. Uotsu et al./Bioorg. Med. Chem. 6 (1998) 1117±1126
1123
Preparation of compound 5 (PPDA)
the reaction mixture. The organic layer was dried over
Na2SO4 and concentrated after ®ltration. The residue
was puri®ed by silica gel column chromatography
(AcOEt/Hex=1/1) to give [3H] 5 (1.33 mg, 50 GBq/
mmol, 65%). The radiochemical purity of the product
was con®rmed by radioscanning of the TLC plate.
To a solution of compound 4 (5.3 mg, 7.7 mmol) in
CHCl3 (0.19 mL) and isoamyl alcohol (0.02 mL) were
added isoamyl nitrite (27.1 mg, 232 mmol), 40 mM acetic
acid/CHCl3 (0.19 mL, 7.7 mmol) at 0 ꢀC and the mixture
was allowed to warm to r.t. The mixture was stirred for
1 h and K2CO3 (3.0 mg) was added. After ®ltration, the
®ltrate was concentrated. The residue was puri®ed by
silica gel column chromatography (AcOEt/hexane=1/1)
to give compound 5 (3.2 mg, 58%).
Preparation of compound 8
To a solution of phorbol 20-monomethoxytrityl ether18
(31.3 mg, 49.2 mmol) in methylene chloride (1 mL) were
.
added EDCI HCl (28.3 mg, 147 mmol), DMAP (18.0 mg,
147 mmol), and 1-pyrenebutyric acid (34.0 mg, 118 mmol)
at 0 ꢀC and the mixture was allowed to warm to r.t. The
mixture was stirred for 12 h and ®ltered through SiO2
(AcOEt/Hex=1/1), and the ®ltrate was concentrated.
The residue was puri®ed by silica gel column chroma-
1
IR (neat) 3397, 2118, 1670, 1370, 845 cm
.
1H NMR (CDCl3, 270 MHz) d 0.93 (3H, d, J=6.6 Hz),
1.18 (1H, m), 1.19 (3H, s), 1.22 (3H, s), 1.78 (3H, dd,
J=1.1, 2.6 Hz), 2.18 (1H, m), 2.19 (1H, s), 2.20 (2H, m),
2.48 (2H, m), 2.51 (2H, t, J=7.5 Hz), 3.25 (2H, m), 3.40
(2H, t, J=7.9 Hz), 4.02 (2H, s), 4.84 (1H, br-s), 5.57 (1H,
d, J=10.2 Hz), 5.69 (1H, br-s), 5.70 (1H, d, J=5.0 Hz),
7.59 (1H, m), 7.80±8.40 (9H, m).
tography (AcOEt/Hex=1/3) to give compound
(57.9 mg, 100%).
8
1
IR (neat) 3404, 1711, 1604, 1080 cm
.
FAB HRMS (NBA) m/z 702.2906, (M+) calculated for
C42H42O8N2: 702.2941.
1H NMR (270 MHz, CDCl3) d 0.93 (3H, d, J=6.1 Hz),
1.05 (1H, d, J=5.2 Hz), 1.07 (3H, s), 1.24 (3H, s), 1.78
(3H, m), 2.10±2.30 (5H, m), 2.35±2.65 (6H, m), 3.17
(1H, m), 3.28 (1H, m), 3.30±3.50 (4H, m), 3.52 (2H, s),
3.76 (3H, s), 5.55 (1H, s), 5.57 (1H, d, 10.5 Hz), 5.66
(1H, m), 6.80±7.40 (14H, m), 7.6 (1H, m), 7.78±8.40
(18H, m).
Preparation of compound 6
To a solution of compound 5 (5.6 mg, 7.9 mmol) in
methylene chloride (1.0 mL) was added MnO2 (6.9 mg,
79 mmol) at r.t., and the mixture was stirred for 3 h.
After ®ltration through SiO2 (acetone) and concentra-
tion, the residue was puri®ed by silica gel column chro-
matography (AcOEt/Hex=1/1) to give 6 (3.3 mg, 60%).
FAB HRMS (NBA) m/z 1176.5143, (M+) calculated for
C80H72O9: 1176.5176.
Preparation of compound 9
IR (neat) 3383, 2924, 2118, 1676, 1545, 1373, 1193, 909,
845.
To a solution of compound 8 (5.8 mg, 4.9 mmol) in THF
(0.25 mL) and MeOH (0.25 mL) was added K2CO3 at
10 ꢀC, and the mixture was stirred for 4.5 h. The reac-
tion mixture was poured into cold water and extracted
with AcOEt, washed with brine, and dried over Na2SO4.
After ®ltration and concentration, the residue was pur-
i®ed by silica gel column chromatography (AcOEt/
Hex=1/3) to give compound 9 (2.5 mg, 56%).
1H NMR (CDCl3, 270 MHz) d 0.93 (3H, d, J=6.2 Hz),
1.23 (6H, s), 1.34 (1H, d, J=5.1 Hz), 1.78 (3H, m), 2.20
(1H, m), 2.22 (2H, m), 2.37 (1H, s), 2.47 (1H, d,
J=19.6 Hz), 2.49 (2H, t, J=7.0 Hz), 2.94 (1H, d,
J=19.6 Hz), 3.06 (1H, m), 3.41 (2H, t, J=7.6 Hz), 3.62
(1H, dd, J=5.1, 5.1 Hz), 4.88 (1H, br-s), 5.59 (1H, d,
J=10.3 Hz), 5.91 (1H, br-s), 6.73 (1H, dd, J=2.0,
5.1 Hz), 7.54 (1H, m), 7.80±8.40 (9H, m), 9.43 (1H, s).
IR (neat) 3426, 2922, 1702, 1250, 1217, 1180, 1076,
1
755 cm
.
FAB HRMS (NBA) m/z 700.2891, (M+) calculated for
C42H40O8N2: 700.2785.
1H NMR (270 MHz, CDCl3) d 0.90 (1H, d, J=6.0 Hz),
1.02 (3H, d, J=6.3 Hz), 1.06 (3H, s), 1.22 (3H, s), 1.78
(3H, m), 2.10 (1H, s), 2.10 (1H, m), 2.21 (2H, m), 2.36
(1H, d, J=19.0 Hz), 2.46 (1H, m), 2.51 (2H, t,
J=7.0 Hz), 3.04 (2H, m), 3.41 (2H, t, J=5.0 Hz), 3.53
(1H, d, J=12.0 Hz), 3.58 (1H, d, J=12.0 Hz), 3.8
(3H, s), 4.71 (1H, s), 4.89 (1H, d, J=9.5 Hz), 5.63 (1H,
m), 6.80±7.40 (14H, m), 7.58 (1H, m), 7.82±8.36
(9H, m).
Preparation of compound [3H]5 ([3H]PPDA)
To a solution of [3H]NaBH4 (0.76 mmol, 370 MBq) in
50 mM aqueous NaOH (30 mL) was added a solution of
5 (2.6 mg, 3.6 mmol) in 5 mM AcOH/MeOH (300 mL) at
0 ꢀC. After stirring for 20 min, 40 mM AcOH/CHCl3
(160 mL), and phosphate bu€er (pH 6.8) were added to

1124
K. Uotsu et al./Bioorg. Med. Chem. 6 (1998) 1117±1126
FAB HRMS (NBA) m/z 906.4180, (M+) calculated for
C60H58O8: 906.4132.
1.4 mmol) and NMO (1.2 mg, 10 mmol) at r.t., and the
mixture was stirred for 30 min. After ®ltration through
SiO2 (acetone) and concentration, the residue was pur-
i®ed by silica gel column chromatography (AcOEt/
Hex=1/3) to give compound 12 (5.6 mg, 70%).
Preparation of compound 10
To a solution of compound 9 (24.2 mg, 26.7 mmol) in
methylene chloride (0.5 mL) was added DMAP (3.9 mg,
32.0 mmol) and 2-diazo-3,3,3-tri¯uoropropionyl chlor-
ide (4.6 mg, 26.7 mmol) at 0 ꢀC. The mixture was allowed
to warm to r.t. and stirred for 2.5 h. The mixture was
concentrated, and the residue was puri®ed by silica gel
column chromatography (AcOEt/Hex=1/4) to give
compound 10 (17.5 mg, 63%).
IR (neat) 3422, 2925, 2142, 1702, 1459, 1351, 1142,
1
846 cm
.
1H NMR (270 MHz, CDCl3) d 0.95 (3H, d, J=6.1 Hz),
1.24 (3H, s), 1.26 (3H, s), 1.40 (1H, d, J=6.9 Hz), 1.78
(3H, m), 2.15±2.30 (3H, m), 2.40 (1H, d, J=1.2 Hz),
2.47 (1H, d, J=25.0 Hz), 2.48 (2H, t, J=9.0 Hz), 2.94
(1H, d, J=25.0 Hz), 3.07 (1H, m), 3.41 (2H, t,
J=9.5 Hz), 3.63 (1H, m), 5.40 (1H, s), 5.58 (1H, d,
J=13.0 Hz), 6.72 (1H, dd, J=2.5, 7.0 Hz), 7.52 (1H, m),
7.80±8.32 (9H, m), 9.42 (1H, s).
1
IR (neat) 3434, 2928, 2141, 1701, 1348, 1143, 1063 cm
.
1H NMR (270 MHz, CDCl3) d 0.92 (3H, d, J=6.3 Hz),
1.23 (3H, s), 1.24 (3H, s), 1.25 (1H, m), 1.77 (3H, m),
2.10 (1H, s), 2.10±2.30 (3H, m), 2.40 (1H, d), 2.48 (2H, t,
J=7.5 Hz), 2.52 (1H, d, J=18.9 Hz), 3.15±3.28 (2H, m),
3.40 (2H, t, J=7.1 Hz), 3.46 (2H, s), 3.80 (3H, s), 5.08
(1H, s), 5.56 (1H, d, J=10.5 Hz), 5.67 (1H, m), 6.80±
7.22 (14H, m), 8.58 (1H, m), 7.85±8.35 (9H, m).
FAB HRMS (NBA) m/z 768.2722, (M+) calculated for
C43H39F3N2O8: 768.2659.
Preparation of compound [3H]11 ([3H]PPTD)
To a solution of [3H]NaBH4 (0.76 mmol, 370 MBq) in
50 mM aqueous NaOH (30 mL) was added a solution of
12 (3.1 mg, 4.1 mmol) in 5 mM AcOH/MeOH (300 mL) at
0 ꢀC. After stirring for 20 min, 40 mM AcOH/CHCl3
(160 mL), and phosphate bu€er (pH 6.8) were added to
the reaction mixture. The organic layer was dried over
Na2SO4 and concentrated after ®ltration. The residue
was puri®ed by silica gel column chromatography
(AcOEt/Hex=1/1) to give [3H] 11 (PPTD)(1.48 mg,
50 GBq/mmol, 63%). The radiochemical purity of the
product was con®rmed by radioscanning of the TLC plate.
FAB HRMS (NBA) m/z 1042.3922, (M+) calculated for
C63H57F3N2O9: 1042.4016.
Preparation of compound 11 (PPTD)
To a solution of compound 10 (10.1 mg, 9.7 mmol) in
methylene chloride (0.5 mL) was added anisole (10.0 mg,
92.0 mmol) and TFA (10 mL) at 0 ꢀC, and the mixture
was stirred for 1 h. To the mixture was added saturated
aqueous NaHCO3, and the resulting mixture was
extracted with AcOEt, washed with brine, and dried
over Na2SO4. After ®ltration and concentration, the
residue was puri®ed by silica gel column chromato-
graphy to give compound 11 (7.7 mg, 100%).
Evaluation of binding anity of compound 5 and 11 to
PKC
Plastic tubes of each binding assay mixture (300 mL)
contained 50 mM Tris±HCl (pH 7.5), 1 mM CaCl2,
100 mg/mL phosphatidyl-l-serine (L-PS), 10 nM
[3H]PDBu (20.7 Ci/mmol, from NEN), 0.5% DMSO,
4 mg/mL bovine serum albumin, 0.8 mg/mL PKC bI and
2nM±20 mM cold PMA, PPDA(5), or PPTD(11). l-PS
was sonicated in 50 mM Tris±HCl (pH 7.5) at 0 ꢀC prior
to use. After incubation at 30 ꢀC for 30 min, the mixture
was ®ltered through a glass-®ber ®lter which was pre-
treated with freshly prepared 0.3% polyethyleneimine
for 1 h. The ®lter was washed three times with 3 mL of
ice cold 0.5% DMSO. The radioactivity of each ®lter
was counted in a scintillation vial with 10 mL of scintil-
lator (Aquasol-2, NEN) using a liquid scintillation
counter. The count in the presence of 10 mM of PMA
which accounted for nonspeci®c binding was subtracted
from each of the counts to account for the speci®c
bindings.
IR (neat) 3420, 2141, 1700, 1684, 1559, 1540, 1457,
1
1339, 1140, 1064 cm
.
1H NMR (270 MHz, CDCl3) d 0.92 (3H, d, J=6.1 Hz),
1.22 (3H, s), 1.24 (3H, s), 1.25 (1H, m), 1.78 (3H, m),
2.13 (1H, s), 2.13±2.30 (3H, m), 2.40±2.55 (4H, m), 3.23±
3.30 (2H, m), 3.40 (2H, t, J=7.0 Hz), 4.03 (2H, m), 5.15
(1H, s), 5.56 (1H, d, J=10.2 Hz), 5.69 (1H, m), 7.59
(1H, m), 7.80±8.31 (9H, m).
FAB HRMS (NBA) m/z 770.2805, (M+) calculated for
C43H41F3N2O8: 770.2815.
Preparation of compound 12
To a solution of compound 11 (8.0 mg, 10 mmol) in
methylene chloride (0.5 mL) were added TPAP (0.5 mg,

K. Uotsu et al./Bioorg. Med. Chem. 6 (1998) 1117±1126
1125
Evaluation of an ability of compound 5 and 11 to
activate PKC
Photoanity labeling of PKC in PS-Triton X-100 mixed
micelles
PKC activity was determined by measuring the incor-
poration of 32P from [g±32P]ATP into EGF-R fragment
peptide (651±658, BIOMOL) using the method reported
by Bell et al. with minor modi®cations. The amorphous
powder of l-PS (SIGMA) in 0.3% Triton X-100 solu-
tion in 50 mM Tris±HCl bu€er (pH 7.5) was vortexed
for 1 min, and incubated for 10 min at 25 ꢀC, followed
by the addition of CaCl2, DTT, and peptide. To this
mixture was added 0.5 mL of various concentrations of
PMA, PPDA(5), or PPTD(11) in DMSO, after which it
was vigorously vortexed. After incubation for 6 min at
25 ꢀC, 25 mL of the PKC solution in Tris bu€er was
added, and incubated for 7 min at 25 ꢀC. The enzyme
reaction was started by adding 25 mL of solution con-
taining ATP, [g±32P]ATP, and MgCl2. The ®nal reaction
mixture (75 mL) contained CaCl2 (1 mM), MgCl2
(15 mM), ATP (50 mM), [g±32P]ATP (6 mCi), peptide
(75 mM), 20 mol % of l-PS dispersed in 0.025% (w/v)
Triton X-100 in 50 mM Tris±HCl bu€er (pH 7.5), and
various concentration of activators. The reaction was
conducted for 15 min at 25 ꢀC, and stopped by the
addition of 100 mL ice-cold 25% trichloroacetic acid.
The resulting precipitate was collected on a binding
paper (P-81, Whatman Ltd., England) and washed twice
with 250 mL of 75 mM phosphoric acid. The ®lter was
placed in a scintillation vial containing 10 mL of Aqua-
sol-2 solution, and 32P radioactivities were quantitated
by a scintillation counter. The radiocount for the con-
trol assay without PMA was determined and was sub-
tracted from each of the radiocounts to account for
nonspeci®c kinase activities. The result shown is repre-
sentative of three sets of experiments.
The amorphous powder of l-PS (SIGMA) in 0.3% Tri-
ton X-100 solution in 50 mM Tris±HCl bu€er (pH 7.5)
was vortexed for 1 min, and incubated for 10 min at
25 ꢀC. The reaction mixture (50 mL) in a 2.0 mL plastic
tube contained PKC (280 nM), [3H]PPDA or PPTD
(1 mM), 20 mol% of l-PS dispersed in 0.025% (w/v)
Triton X-100, and CaCl2 (1 mM) in 50 mM Tris±HCl
(pH 6.8) in the presence or absence of cold PMA
(40 mM). After incubation for 3 min at 30 ꢀC and for
2 min at 0 ꢀC, photoanity labeling experiments were
carried out as described above.
Acknowledgements
This study was ®nanced with Special Coordination
Funds of the Science and Technology Agency of the
Japanese Government and Uehara Memorial Foundation.
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