Design and physicochemical properties of new fluorescent ligands of protein kinase C isozymes focused on CH/π interaction

Article abstract of DOI:10.1016/j.bmc.2007.10.045

Phorbol ester-type tumor promoters such as indolactam-V (IL-V, 1) bind to the C1 domains of protein kinase C (PKC) isozymes. A more convenient method to investigate the interaction between each tumor promoter and PKC C1 domain is needed. Focusing on our recent finding that the indole ring of IL-V is involved in the CH/π interaction with Pro-11 of the PKCδ-C1B domain, we developed new fluorescent probes (2-4) from IL-V by forming a pyrroloindazole ring. Compound 2 without a substituent at the pyrroloindazole ring bound most strongly to PKC C1 domains with a potency similar to IL-V, but its fluorescent intensity was the weakest of any of the probes. Although the binding affinity of 3 with a methyl group was significantly weaker than that of IL-V, 4 with a trifluoromethyl group showed moderate affinity and the most potent fluorescence intensity. The fluorescence intensity and emission maxima of 4 changed significantly when bound to the PKCδ-C1B peptide in both the presence and absence of phosphatidylserine. These results suggest that 4 could be a useful probe for analyzing the interaction of tumor promoters with PKC C1 domains.

Full text of DOI:10.1016/j.bmc.2007.10.045

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 650–657
Design and physicochemical properties of new fluorescent ligands
of protein kinase C isozymes focused on CH/p interaction
Takuya Sugimoto, Koji Itagaki and Kazuhiro Irie*
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho,
Sakyo-ku, Kyoto 606-8502, Japan
Received 3 August 2007; revised 13 October 2007; accepted 16 October 2007
Available online 22 October 2007
Abstract—Phorbol ester-type tumor promoters such as indolactam-V (IL-V, 1) bind to the C1 domains of protein kinase C (PKC)
isozymes. A more convenient method to investigate the interaction between each tumor promoter and PKC C1 domain is needed.
Focusing on our recent finding that the indole ring of IL-V is involved in the CH/p interaction with Pro-11 of the PKCd-C1B
domain, we developed new fluorescent probes (2–4) from IL-V by forming a pyrroloindazole ring. Compound 2 without a substi-
tuent at the pyrroloindazole ring bound most strongly to PKC C1 domains with a potency similar to IL-V, but its fluorescent inten-
sity was the weakest of any of the probes. Although the binding affinity of 3 with a methyl group was significantly weaker than that
of IL-V, 4 with a trifluoromethyl group showed moderate affinity and the most potent fluorescence intensity. The fluorescence inten-
sity and emission maxima of 4 changed significantly when bound to the PKCd-C1B peptide in both the presence and absence of
phosphatidylserine. These results suggest that 4 could be a useful probe for analyzing the interaction of tumor promoters with
PKC C1 domains.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
tains two highly conserved C1 domains (C1A and
C1B),11 zinc finger motifs with cysteine-rich sequences
that act as the binding sites for DAG and tumor pro-
moters.12,13 When DAG or tumor promoters bind to
the C1 domains of PKCs, they translocate to the mem-
brane to be activated.14 Since the translocation and acti-
vation of PKCs are characteristic of each isoform and
activator,15,16 a precise and intimate analysis of the
binding between each tumor promoter and C1 domain
is needed to understand the mechanism of PKCs activa-
tion both in vivo and in vitro.
The members of a protein kinase C (PKC) family of
serine/threonine kinases play critical roles in complex
signal transduction networks that regulate a diverse
range of cellular processes, such as cell proliferation,
differentiation, and apoptosis.1,2 They are activated
by phorbol ester-type tumor promoters3 as well as
the endogenous second messenger sn-1,2-diacylglyc-
erol (DAG).4,5 Recent investigations revealed that
PKC isozymes (PKCs) are important therapeutic tar-
gets because they are involved in various diseases such
as diabetes, nerve pain, AIDS, and tumor promo-
tion.6–10
Conventional PKC
C2
C1A C1B
Catalytic domain
(α, βI, βII, γ)
The PKCs comprise classical PKCs (a, bI, bII, c) that
are calcium-dependent, novel PKCs (d, e, g, h) that
are calcium-independent, and atypical PKCs (f, i/k) that
are not activated by DAG or tumor promoters
(Fig. 1).4,5 Each of the classical and novel PKCs con-
Tumor Promoters
Novel PKC
(δ, ε, η, θ)
C2-like
Catalytic domain
Catalytic domain
C1A C1B
C1
Keywords: CH/p interaction; Fluorescent probe; Indolactam-V; Pro-
tein kinase C; Pyrroloindazole ring.
Atypical PKC
(ζ, ι, λ)
*
Corresponding author. Tel.: +81 75 753 6281; fax: +81 75 753
6284; e-mail: irie@kais.kyoto-u.ac.jp
Figure 1. Structure of PKC isozymes.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.10.045

T. Sugimoto et al. / Bioorg. Med. Chem. 16 (2008) 650–657
651
The most reliable method of analyzing the binding of a
2. Results and discussion
ligand to PKC C1 domains is the competitive assay using
[3H]phorbol 12,13-dibutyrate (PDBu) established by
Sharkey and Blumberg.17 As a more convenient way to
assess the ability of a ligand to bind PKCs, a modified
binding assay using synthetic peptides of each PKC C1
domain was developed in our laboratory.18,19 However,
the use of radioisotopes is troublesome, and the assay
using [3H]PDBu cannot estimate weak binding, espe-
cially in the absence of phosphatidylserine, because of
difficulty in separating the bound [3H]PDBu from free
[3H]PDBu. A fluorescent PKC ligand has the potential
to circumvent these problems. For example, sapintoxin-
D (SAPD), a fluorescent phorbol ester, is used to estimate
binding affinity to PKC C1 domains.20 Resonance energy
transfer between the tryptophan residue in each PKC C1
domain and the SAPD fluorophore allows direct determi-
nation of SAPD binding without separating the bound
SAPD from free SAPD, as is required in the assay using
[3H]PDBu. However, this assay is not valid when using
C1 domains without a tryptophan residue such as the
C1B domains of classical PKCs. A new fluorescent probe
with a fluorophore that can interact directly with any res-
idue of the PKC C1 domains is thus strongly desired.
2.1. Synthesis of new fluorescent derivatives of IL-V (2–4)
It is quite difficult to anticipate whether a designed com-
pound will fluoresce or not. However, elongation of the
conjugation is an effective way to make it fluoresce. Pre-
vious structure–activity studies on IL-V revealed that a
hydrogen atom at position 1 is not necessary for binding
to PKC C1 domains, and hydrophobic substituents at
position 7 enhanced the binding.23,24,30,31 We introduced
an additional ring between positions 1 and 7 to elongate
the conjugation. A pyrroindazole ring was selected be-
cause of ease of synthetic accessibility. In order to tune
the PKC binding ability and fluorescence intensity, three
kinds of compounds with distinct substituents (2–4)
were synthesized (Scheme 1).
A strategy for synthesizing these derivatives is to intro-
duce each acyl chain at position 7 of IL-V and then to
form a pyrroloindazole ring. After the protection of
the hydroxyl group of IL-V with an acetyl group (5,
91%), acyl substituents were introduced at position 7
of 5. For the introduction of a formyl group, the Vilsme-
ier–Haack reaction of 5 with N-methylformamide gave 6
(54%). For the introduction of an acetyl or trifluoroace-
tyl group, the Friedel–Crafts acylation of 2 with acetic
anhydride and trifluoroacetic anhydride gave 7 and 8,
respectively (21% and 40%). Resultant compounds (6,
7, and 8) were deprotected and treated with t-butyldi-
methylsilyl chloride (TBDMS-Cl) for reprotection to af-
ford 9, 10, and 11, respectively (79%, 76%, and 82%).
The carbonyl groups of 9, 10, and 11 were converted
to the corresponding oximes (12, 13, and 14) with
hydroxyammonium chrolide (67%, 64%, and 51%,
respectively) and were tosylated in the presence of
dimethylaminopyridine (DMAP) and triethylamine
(TEA). Resultant tosylated compounds were very labile
and a ring-closing reaction at position 1 proceeded suc-
cessively to yield 15, 16, and 17 (35%, 31%, and 48%,
respectively). A nitrogen atom at position 1 would have
attacked nucleophilically the nitrogen atom bonded with
the tosyl group as a good leaving group. Final deprotec-
tion of the hydroxyl groups with tetrabutylammonium
fluoride (TBAF) gave 2, 3, and 4 (85%, 87%, and 88%,
To develop the new fluorescent probe, we selected (À)-
indolactam-V21 (IL-V) as a lead compound which is
the basic structure of tumor-promoting teleocidins3
and was isolated from Streptomyces blastmyceticum.22
Because of its stability and relatively simpler structure,
many IL-V derivatives have been synthesized for struc-
ture–activity studies.23–27 Since the indole ring of IL-V
has recently been shown to be involved in the CH/p
interaction with Pro-11 of the PKCd-C1B domain
(Fig. 2),28 transformation of the indole ring to a fluoro-
fore is preferable. Although we previously introduced
various substituents into the indole ring of IL-V to find
that 2,7-diacylindolactam-Vs fluoresced strongly, their
binding affinity for PKCs was extremely low.29
In this study, we transformed the indole moiety of IL-V
to a pyrroloindazole ring to give three fluorescent
probes (2, 3, and 4). Compound 4 with a trifluoro-
methyl group showed moderate binding affinity to
PKC C1 domains and the most potent fluorescence
intensity. The fluorescence intensity and emission max-
ima of 4 changed significantly when bound to the
PKCd-C1B peptide in both the presence and absence
of phosphatidylserine. The results suggest that 4 could
be a useful tool for analyzing the interaction of ligands
with PKC C1 domains.
1
respectively). The H NMR spectra showed that 2, 3,
and 4 existed only as the twist conformer with a cis
amide32 at room temperature. All proton and carbon
atoms in 2, 3, and 4 were assigned in CDCl3 or CD3OD
using 1H–1H COSY and scalar heteronuclear experi-
ments (HMBC and HMQC). Compounds 2, 3, and 4
showed strong fluorescence on TLC plates under light
at 365 nm. Black et al.33 reported a synthesis of the pyr-
roloindazole ring that shows fluorescence.
O
Pro-11 of
PKCδ-C1B
N
CH/π interaction
2.2. Fluorescent properties of the IL-V derivatives with a
pyrroloindazole ring (2–4)
H
H
Indole ring
of IL-V
The data on the fluorescence of these derivatives (2–4)
are summarized in Table 1. The fluorescent intensity
of all derivatives was considerably higher in EtOH than
in Tris–maleate buffer. Since the fluorescence intensity
of 4 both in EtOH and in Tris–maleate buffer was high-
N
H
Figure 2. CH/p interaction between IL-V and Pro-11 of the PKCd-
C1B domain.

652
T. Sugimoto et al. / Bioorg. Med. Chem. 16 (2008) 650–657
H
N
H
N
N
OAc
N
OH
O
a) N-Me-formamide
POCl3
O
HONH3+Cl—
Ac2O
1) NaOH / MeOH
5
9-11
N
H
EtOH
2) TBCMS-Cl
b) Ac2O / AlCl3
or
Pyridine
N
H
Pyridine
Imidazole / DMF
O
c) (CF3CO)2O / AlCl3
R
(-)-Indolactam-V (1, IL-V)
R = H (6), CH3 (7), CF3 (8)
H
H
H
N
N
N
N
N
OTBDMS
N
OH
OTBDMS
O
O
O
TBAF
TsCl
N
N
N
DMAP / NEt3
Ether
THF
N
H
N
R
R
R
N OH
R = H (12), CH3 (13), CF3 (14)
R = H (15), CH3 (16), CF3 (17)
R = H (2), CH3 (3), CF3 (4)
Scheme 1. Synthesis of fluorescent IL-V derivatives (2–4).
Table 1. Effects of solvents on fluorescent IL-V derivatives (2–4)a
Compound Solvent kmax kmax Relative
cence, and the fluorescence intensity of 2 was similar
to that of 3. These results suggest that the electron-with-
drawing group in the pyrroloindazole ring increases the
intensity. In addition, the fluorescence intensity of these
probes greatly increased when 1,2-dioleoyl-sn-glycero-3-
phospho-L-serine (PS), a co-activator of PKC, was
added (data not shown). This reflects the strong fluores-
cence of these probes in EtOH compared that to in Tris–
maleate buffer.
(excitation) (emission) fluorescence
intensityb
2
3
4
EtOH
Buffer
EtOH
Buffer
EtOH
Buffer
340
343
362
361
376
378
379
390
422
432
422
437
4.22
1.00
3.81
1.64
9.18
3.47
a Measurements were carried out at 25 ꢁC at a concentration of
10À6 M. Each sample was excited at the wavelength of its excitation
maximum.
2.3. Binding affinity of the fluorescent IL-V derivatives
(2–4) for PKC C1 domains
b Compound 2 in buffer was chosen as the standard for the relative
fluorescence intensity.
The affinity of 2, 3, and 4 for the PKC C1 peptides was
evaluated by inhibition of the specific binding of
[3H]phorbol 12,13-dibutyrate (PDBu) to these peptides
with a method reported previously.17–19 Table 2 shows
the inhibition constant (Ki) values of 2, 3, and 4 for
the PKC C1 peptides. The binding affinity of 2 was sim-
ilar to that of IL-V, suggesting that steric hindrance does
not exist at positions 1 and 7 of IL-V as previously re-
ported.23,24,30,31 However, the affinity of 3 and 4 was sig-
nificantly lower than that of IL-V and 2. This is an
unexpected result since the introduction of a hydropho-
bic substituent at position 7 of IL-V enhanced the bio-
logical activities related to PKCs activation.24 The
pyrroloindazole ring with a substituent is more rigid
than the indole ring with a substituent at position 7 of
IL-V. This might produce some steric hindrance to re-
duce the binding of these derivatives.
est, the excitation and emission spectra of 4 are shown in
Figure 3 as representative. Compound 4 with an elec-
tron-withdrawing group showed the strongest fluores-
250
excitation emission
200
EtOH
150
100
50
0
Buffer
However, an electronic factor is also deduced to play a
critical role because the nitrogen atoms on the pyorro-
loindazole ring may have some polarity. Since the substit-
uents at positions 1 and 7 of IL-V are involved in the
hydrophobic interaction with phosphatidylserine when
bound to PKC C1 domains,23,24,27 the polar nitrogens
would decrease the binding ability. Electron-donating
400
Wavelength (nm)
500
300
Figure 3. Fluorescence spectra of 4. Fluorescent excitation and
emission spectra of 4 (10À6 M) are shown with solid lines (in EtOH)
and dotted lines (in 50 mM Tris–maleate buffer).

T. Sugimoto et al. / Bioorg. Med. Chem. 16 (2008) 650–657
Table 2. Ki values for the inhibition of the specific binding of [3H]PDBu by 3, 4, and 5
653
PKC C1 peptide
Ki (nM)
2 (R = H)
3 (R = CH3)
4 (R = CF3)
IL-V
a-C1A (72-mer)a
a-C1B
b-C1A (72-mer)
b-C1B
c-C1A
c-C1B
d-C1B
e-C1B
g-C1B
h-C1B
85.5 (14)b
5960 (730)
88.0 (6.4)
357 (4.3)
61.3 (3.2)
225 (6.4)
17.7 (3.0)
22.5 (1.9)
9.9 (1.2)
1090 (180)
>10,000
112 (13)
9380 (32)
127 (31)
21
4000
19
1100 (650)
2620 (350)
1510 (220)
2580 (20)
128 (5.4)
407 (34)
721 (110)
234 (3.7)
1100 (65)
52.1 (1.7)
80.1 (9.6)
29.7 (7.3)
51.1 (15)
140
140
210
11
7.7
5.5
8.7
223 (4.5)
149 (24)
18.6 (0.1)
a Ten residues from both the N- and C-terminal of a-C1A and b-C1A were elongated because the solubility of the original 52-mer peptides was
extremely low.16
b Standard deviation of at least two separate experiments.
methyl group might make the nitrogens more polar to
reduce the binding affinity while electron-withdrawing
trifluoromethyl group might delocalize the electrons on
the nitrogen to increase the binding affinity. The fact that
the binding affinity of 4 was lower than 2 could be
explained by the polality of the trifluoromethyl group
itself.
PKCd-C1B. In the presence of PKCd-C1B, the fluores-
cence intensity of 4 increased and the emission maxi-
mum moved to a lower wavelength. Since the indole
ring of IL-V is involved in the CH/p interaction,28 bind-
ing to PKCd-C1B would have changed the electron den-
sity of the pyrroloindazole ring to vary the fluorescence
property of 4. This result corresponds with the fact that
the fluorescence intensity of 4 increased in EtOH com-
pared to that in Tris–maleate buffer since the binding
pocket of PKCd-C1B was thought to have a hydropho-
bic nature.
These results suggest that 2, 3, and 4 have potential as
fluorescent probes of PKCs since they showed signifi-
cant ability to bind most PKC C1 peptides.
2.4. Fluorescence spectra of 4 when bound to PKCd-C1B
On the other hand, the fluorescence intensity of 4 de-
creased when bound to PKCd-C1B in the presence of
phosphatidylserine (Fig. 4b). This would be because
the nature inside the micelle of phosphatidylserine vesi-
cle is more hydrophobic than that in the binding pocket
of PKCd-C1B. To examine whether these changes in the
fluorescence reflect the specific binding of 4 to PKCd-
C1B, the competition assay using PDBu was carried
out. Some part of the changed fluorescence intensity of
4 recovered when added excess amount of PDBu both
in the presence and absence of phosphatidylserine
The above data indicate that 4 would be the most suit-
able fluorescent ligand among the three compounds be-
cause it showed the strongest fluorescence both in EtOH
and in Tris–maleate buffer and a moderate affinity for
most PKC C1 peptides. To examine the potential of 4
as a new fluorescent probe for PKCs, the fluorescence
spectra of 4 in the presence of a PKC C1 peptide with
or without phosphatidylserine were recorded. Figure 4
shows the emission spectrum of 4 when bound to
b
a
100
100
50
0
50
0
400
500
400
450
500
450
Wavelength (nm)
Wavelength (nm)
Figure 4. The change in the fluorescent property of 4 when bound to PKCd-C1B peptide in 50 mM Tris–maleate buffer. Fluorescence emission
spectra of 4 in the absence of PKCd-C1B peptide are shown with solid lines, those in the presence of PKCd-C1B peptide with dotted lines, and those
in the presence of both PKCd-C1B peptide and excess amount (10 lM) of PDBu with dashed lines. (a) In the absence and (b) in the presence of
phosphatidylserine. Excitation wavelength was 377 nm. The concentrations of 4 were 10À6.5 M (a) and 10À7 M (b), respectively. In both experiments,
200 nM of PKCd-C1B was employed since the Bmax value in the Scatchard analysis was 20–30% as reported previously.19

654
T. Sugimoto et al. / Bioorg. Med. Chem. 16 (2008) 650–657
(Fig. 4a and b, dashed lines), suggesting that at least
some part of the change in the fluorescence intensity
of 4 could reflect the specific binding. These results sug-
gest that 4 could be used to estimate the ability of a li-
gand to bind PKC C1 domains both in the presence
and in the absence of phosphatidylserine.
4.2.4. (À)-14-O-Acetyl-7-trifluoroacetylindolactam-V (8).
To a mixture of 5 (77.2 mg, 0.23 mmol) and trifluoroace-
tic anhydride (150 ll) in nitrobenzene (1 ml) was added
AlCl3 (33.6 mg) at room temperature. The reaction mix-
ture was stirred for 7 h at room temperature and parti-
tioned between EtOAc and H2O. The collected EtOAc
layer was washed with brine, dried over Na2SO4, and
concentrated. The residue was purified by column chro-
matography on Wakogel C-200 using hexane and
increasing amounts of EtOAc, followed by HPLC on
YMC SH-342-5 using 60%, 70%, and 100% MeOH to
give 8 (39.8 mg, 0.091 mmol, 40%).
3. Conclusion
In summary, new fluorescent probes (2, 3, and 4) with a
pyrroroindazole group as a fluorophore were designed
from IL-V, to focus on the CH/p interaction of the aro-
matic ring with PKC C1 domains. These probes bound
significantly to most of the PKC C1 peptides. Since the
fluorophore of these probes can directly interact with
Pro-11 of the PKC C1 domains, the probes could be use-
ful for analyzing the direct interaction between each
PKC ligand and C1 domain. Notably, 4 could be most
useful since 4 with moderate binding affinity for PKC
C1 peptides fluoresced most strongly among them. A
combination of 4 and PKC C1 peptides would be useful
for estimating the binding ability of various PKC
ligands.
Compound 8: [a]D À340ꢁ (c = 0.80, MeOH, 23.8 ꢁC);
UV kmax (MeOH) nm (e): 339 (22,000), 300 (2800), 266
(9200); 1H NMR d (500 MHz, 0.043 M, CDCl3,
295 K) ppm: 0.59 (3H, d, J = 6.8 Hz), 0.96 (3H, d,
J = 6.4 Hz), 2.11 (3H, s), 2.62 (1H, m), 3.04 (3H, s),
3.19 (1H, dd, J = 17.5, 3.5 Hz), 3.15 (1H, d,
J = 17.5 Hz), 4.00 (1H, dd, J = 11.3, 8.4 Hz), 4.17 (1H,
m), 4.22 (1H, dd, J = 11.3, 3.6 Hz), 4.58 (1H, d,
J = 10.2 Hz), 6.18 (1H, s), 6.56 (1H, d, J = 8.8 Hz),
7.06 (1H, d, J = 1.6 Hz), 7.84 (1H, d, J = 8.8 Hz), 10.7
(1H, br s); 13C NMR d (125 MHz, 0.043 M, CDCl3,
295 K) ppm: 18.9, 20.8, 21.0, 28.6, 33.1, 34.6, 52.7,
65.7, 71.3, 106.0, 106.4, 114.5, 116.4, 117.5 (q,
J = 290 Hz), 122.8, 129.9, 140.3, 155.4, 171.0, 171.2,
177.9 (q, J = 34 Hz); HR-EI-MS m/z: 439.1734 (M+,
calcd for C21H24F3N3O4, 439.1719).
4. Experimental
4.1. General remarks
The following spectroscopic and analytical instruments
were used: UV, Shimadzu UV-2200A; [a]D, Jasco DIP-
1000; 1H NMR, Bruker AVANCE 400 (reference to
TMS); HPLC, Waters Model 600E with Model 2487
UV Detector; (HR) EIMS, JEOL JMS-600H; fluores-
cence spectra, JASCO FP-6500.
4.2.5. (À)-14-O-TBDMS-7-formyl-indolactam-V (9). To
a solution of 6 (27.8 mg, 0.075 mmol) in MeOH (1 ml)
was added 2 M NaOH (100 ll). The reaction mixture
was stirred for 30 min at room temperature and parti-
tioned between EtOAc and saturated aqueous NaHCO3.
The collected EtOAc layer was washed with brine, dried
over Na2SO4, and concentrated to give crude (À)-7-for-
myl-indolactam-V. To this compound dissolved in dry
DMF (0.1 ml) were added imidazole (10.5 mg) and
TBDMS-Cl (22.4 mg) under cooling with a water bath.
The reaction mixture was stirred for 40 min at room
temperature and partitioned between EtOAc and H2O.
The collected EtOAc layer was washed with brine, dried
over Na2SO4, and concentrated. The residue was puri-
fied by column chromatography on Wakogel C-200
using hexane and increasing amounts of EtOAc to give
9 (26.0 mg, 0.059 mmol, 79% for two steps).
HPLC was carried out on a YMC-packed SH-342-5
(ODS, 20 mm id · 150 mm) column and YMC-packed
SH-042-5 (SIL, 20 mm id · 150 mm) column (Yamam-
ura Chemical Laboratory). Wakogel C-200 (silica gel,
Wako Pure Chemical Industries) and YMC A60-350/
250 gel (ODS, Yamamura Chemical Laboratory) were
used for column chromatography. [3H]PDBu (15.6 Ci/
mmol) was purchased from Perkin-Elmer Life Science.
1,2-Dioleoyl-sn-glycero-3-phospho-L-serine and c-glob-
ulin were purchased from Sigma. All the other chemicals
and reagents were purchased from Wako and Aldrich,
and used without further purification.
Compound 9: [a]D À560ꢁ (c = 1.15, MeOH, 20.0 ꢁC); UV
kmax (MeOH) nm (e): 371 (25,000), 261 (16,100); 1H
NMR d (400 MHz, 0.047 M, CDCl3, 298 K) ppm: 0.03
(3H, s), 0.06 (3H, s), 0.57 (3H, d, J = 6.8 Hz), 0.88 (9H,
s), 0.94 (3H, d, J = 6.3 Hz), 2.62 (1H, m), 2.97 (1H, dd,
J = 17.7, 3.2 Hz), 3.00 (3H, s), 3.15 (1H, d, J = 17.7 Hz),
3.49 (1H, dd, J = 10.1, 9.7 Hz). 3.65 (1H, dd, J = 10.1,
4.4 Hz), 3.97 (1H, m), 4.56 (1H, d, J = 10.2 Hz), 6.23
(1H, s), 6.56 (1H, d, J = 8.3 Hz), 6.99 (1H, d, J = 1.6 Hz),
7.51 (1H, d, J = 8.3 Hz), 9.83 (1H, s), 10.7 (1H, br s); 13C
NMR d (100 MHz, 0.047 M, CDCl3, 298 K) ppm: À5.4,
4.2. Synthesis of the fluorescent IL-V derivatives (2–4)
4.2.1. (À)-14-O-Acetylindolactam-V (5).26 (À)-Indolac-
tam-V (1, 213 mg, 0.71 mmol) was treated as reported
to give 5 (211 mg, 0.65 mmol, 91%).
4.2.2. (À)-14-O-Acetyl-7-formylindolactam-V (6).26
Compound 5 (48.5 mg, 0.14 mmol) was treated as re-
ported to give 6 (27.8 mg, 0.075 mmol, 54%).
˚
Àa5.3, 18.3, 19.0, 21.1, 25.8 (3C), 28.6, 32.9, 34.0, 54.8,
4.2.3. (À)-14-O-Acetyl-7-acetylindolactam-V (7).26
Compound 5 (219.2 mg, 0.64 mmol) was treated as re-
ported to give 7 (51.8 mg, 0.134 mmol, 21%).
65.3, 71.2, 105.4, 113.9, 115.1, 114.6, 116.2, 122.4, 132.2,
138.1, 154.1, 171.6, 190.4; HR-EI-MS m/z: 443.2604 (M+,
calcd for C24H37N3O3Si, 443.2604).

T. Sugimoto et al. / Bioorg. Med. Chem. 16 (2008) 650–657
655
4.2.6. (À)-14-O-TBDMS-7-acetyl-indolactam-V (10).
Compound 7 (47.7 mg, 0.12 mmol) was treated similarly
as described for the synthesis of 9 to give 10 (41.7 mg,
0.091 mmol, 76% for two steps).
(1H, dd, J = 10.1, 4.4 Hz), 4.13 (1H, m), 4.46 (1H, d,
J = 10.2 Hz), 6.28 (1H, s), 6.50 (1H, d, J = 8.1 Hz),
6.95 (1H, s), 7.08 (1H, d, J = 8.1 Hz), 7.85 (1H, br s),
8.28 (1H, s), 9.96 (1H, br s); 13C NMR d (100 MHz,
0.12 M, CDCl3, 298 K) ppm: À5.4, À5.3, 14.1, 18.2,
19.3, 21.4, 25.8 (3C), 28.6, 32.9, 34.0, 55.0, 65.3, 71.2,
105.6, 108.1, 114.5, 117.2, 121.9, 126.5, 136.2, 149.9,
151.4, 172.8; HR-EI-MS m/z: 458.2714 (M+, calcd for
C24H38N4O3Si, 458.2713).
Compound 10: [a]D À480ꢁ (c = 0.92, MeOH, 24.7 ꢁC);
UV kmax (MeOH) nm (e): 370 (19,500), 257 (14,500);
1H NMR d (400 MHz, 0.040 M, CDCl3, 297 K) ppm:
0.02 (3H, s), 0.05 (3H, s), 0.57 (3H, d, J = 6.8 Hz),
0.87 (9H, s), 0.94 (3H, d, J = 6.4 Hz), 2.61 (1H, m),
2.61 (3H, s), 2.95 (1H, dd, J = 17.6, 3.2 Hz), 2.99 (3H,
s), 3.15 (1H, d, J = 17.6 Hz), 3.48 (1H, dd, J = 10.2,
9.7 Hz). 3.64 (1H, dd, J = 10.2, 4.4 Hz), 4.01 (1H, m),
4.53 (1H, d, J = 10.2 Hz), 6.21 (1H, s), 6.48 (1H, d,
J = 8.5 Hz), 6.98 (1H, s), 7.70 (1H, d, J = 8.5 Hz), 10.8
(1H, br s); 13C NMR d (100 MHz, 0.040 M, CDCl3,
297 K) ppm: À5.4, À5.3, 18.2, 19.1, 21.1, 25.8 (3C),
28.6, 32.9, 34.1, 54.8, 65.3, 71.2, 104.8, 113.2, 114.1,
116.7, 122.4, 128.3, 139.1, 153.4, 171.9, 198.0; HR-EI-
MS m/z: 454.2774 (M+, calcd for C25H39N3O3Si,
454.2761).
4.2.9. Compound 13. Compound 10 (41.7 mg, 0.091 mmol)
was treated similarly as described for the synthesis of 12 to
give 13 (27.4 mg, 0.058 mmol, 64%).
Compound 13: [a]D À320ꢁ (c = 1.5, MeOH, 26.6 ꢁC);
UV kmax (MeOH) nm (e): 339 (21,200), 250 (15,800);
1H NMR d (400 MHz, 0.11 M, CDCl3, 298 K) ppm:
0.01 (3H, s), 0.03 (3H, s), 0.61 (3H, d, J = 6.7 Hz),
0.86 (9H, s), 0.94 (3H, d, J = 6.3 Hz), 2.39 (3H, s),
2.68 (1H, m), 2.94 (1H, dd, J = 17.3, 3.4 Hz), 2.95
(3H, s), 3.17 (1H, d, J = 17.3 Hz), 3.47 (1H, dd,
J = 10.2, 9.7 Hz). 3.63 (1H, dd, J = 10.2, 4.4 Hz), 4.15
(1H, m), 4.45 (1H, d, J = 10.2 Hz), 6.25 (1H, s), 6.51
(1H, d, J = 8.3 Hz), 6.92 (1H, s), 7.33 (1H, d,
J = 8.3 Hz), 7.64 (1H, s), 10.5 (1H, br s); 13C NMR d
(100 MHz, 0.11 M, CDCl3, 298 K) ppm: À5.4, À5.3,
18.2, 19.1, 21.1, 25.8 (3C), 28.6, 32.9, 34.1, 54.8, 65.3,
71.2, 104.8, 113.2, 114.1, 116.7, 122.4, 128.3, 139.1,
153.4, 171.9, 198.0; HR-EI-MS m/z: 472.2850 (M+, calcd
for C25H40N4O3Si, 472.2870).
4.2.7. (À)-14-O-TBDMS-7-trifluoroacetyl-indolactam-V
(11). Compound 8 (312.2 mg, 0.71 mmol) was treated
similarly as described for the synthesis of 9 to give 11
(296.6 mg, 0.580 mmol, 82% for two steps).
Compound 11: [a]D À620ꢁ (c = 1.28, MeOH, 24.5 ꢁC);
UV kmax (MeOH) nm (e): 399 (22,000), 300 (3200),
267 (9800); 1H NMR d (500 MHz, 0.065 M, CDCl3,
298 K) ppm: 0.03 (3H, s), 0.05 (3H, s), 0.57 (3H, d,
J = 6.8 Hz), 0.87 (9H, s), 0.95 (3H, d, J = 6.4 Hz),
2.61 (1H, m), 2.98 (1H, dd, J = 17.5, 3.1 Hz), 3.03
(3H, s), 3.15 (1H, d, J = 17.5 Hz), 3.49 (1H, dd,
J = 9.9, 9.7 Hz). 3.64 (1H, dd, J = 9.9, 4.5 Hz), 3.90
(1H, m), 4.60 (1H, d, J = 10.2 Hz), 6.25 (1H, s), 6.54
(1H, d, J = 8.8 Hz), 7.02 (1H, s), 7.82 (1H, d,
J = 8.8 Hz), 10.7 (1H, br s); 13C NMR d (125 MHz,
0.065 M, CDCl3, 298 K) ppm: À5.4, À5.3, 18.3, 18.9,
20.9, 25.8 (3C), 28.6, 33.0, 34.1, 54.5, 65.3, 71.3,
105.9, 106.2, 115.1, 115.9, 117.6 (q, J = 289 Hz),
122.6, 130.0, 140.3, 155.6, 171.1, 177.7 (q, J = 33.4 Hz);
HR-EI-MS m/z: 511.2479 (M+, calcd for C25H36F3
N3O3Si, 511.2478).
4.2.10. Compound 14. Compound 11 (99.6 mg, 0.195 mmol)
was treated similarly as described for the synthesis of 12 to
give 14 (52.8 mg, 0.100 mmol, 51%).
Compound 14: [a]D À230ꢁ (c = 0.90, MeOH, 26.1 ꢁC);
1
UV kmax (MeOH) nm (e): 333 (9800), 245 (13,000); H
NMR d (500 MHz, 0.032 M, CDCl3, 300 K) ppm: 0.04
(3H, s), 0.05 (3H, s), 0.62 (3H, d, J = 6.8 Hz), 0.87
(9H, s), 0.94 (3H, d, J = 6.3 Hz), 2.63 (1H, m), 2.95
(3H, s), 2.99 (1H, dd, J = 17.6, 3.3 Hz), 3.16 (3H, d,
J = 17.6 Hz), 3.51 (1H, dd, J = 9.8, 9.4 Hz). 3.66 (1H,
dd, J = 9.8, 4.6 Hz), 4.16 (1H, m), 4.46 (1H, d,
J = 10.2 Hz), 6.39 (1H, s), 6.57 (1H, d, J = 8.4 Hz),
6.93 (1H, s), 7.26 (1H, d, J = 8.4 Hz), 8.24 (1H, s),
10.6 (1H, br s); 13C NMR d (125 MHz, 0.032 M, CDCl3,
300 K) ppm: À5.5, À5.3, 18.2, 19.3, 21.4, 25.8 (3C), 28.6,
32.9, 34.0, 54.9, 65.3, 71.3, 102.3, 106.0, 114.7, 117.8,
121.3 (q, J = 274 Hz), 122.0, 123.8, 136.9, 146.8 (q,
J = 32 Hz), 150.6, 173.0; HR-EI-MS m/z: 526.2576
(M+, calcd for C25H37F3N4O3Si, 526.2587).
4.2.8. Compound 12. To a solution of 9 (33.7 mg,
0.076 mmol) in dry pyridine (0.3 ml) were added
hydroxyammonium chloride (31.7 mg) and EtOH
(0.1 ml). The reaction mixture was stirred for 3 h at
70 ꢁC and partitioned between EtOAc and H2O. The
collected EtOAc layer was washed with brine, dried over
Na2SO4, and concentrated. The residue was purified by
HPLC on YMC SH-342-5 using 85% MeOH, then on
YMC SH-042-5 (hexane/CHCl3/i-PrOH = 90:8:2) to
give 12 (23.5 mg, 0.051 mmol, 67%).
4.2.11. Compound 15. To a mixture of 12 (23.5 mg,
0.051 mmol), TEA (18 ll), and DMAP (310 lg) in
diethyl ether (1 ml) was added p-toluenesulfonyl chlo-
ride (11.4 mg) at 0 ꢁC. The reaction mixture was stirred
at 0 ꢁC for 10 min and then at room temperature for
58 h. Diethyl ether was removed by an Ar atmosphere
and the residue was partitioned between EtOAc and
H2O. The collected EtOAc layer was washed with brine,
dried over Na2SO4, and concentrated. The residue was
purified by HPLC on YMC SH-342-5 using 85% MeOH
to give 15 (8.0 mg, 0.018 mmol, 35%).
Compound 12: [a]D À400ꢁ (c = 2.7, MeOH, 20.0 ꢁC);
UV kmax (MeOH) nm (e): 343 (27,600), 255 (17,300);
1H NMR d (400 MHz, 0.12 M, CDCl3, 298 K) ppm:
0.01 (3H, s), 0.04 (3H, s), 0.60 (3H, d, J = 6.7 Hz),
0.87 (9H, s), 0.94 (3H, d, J = 6.3 Hz), 2.64 (1H, m),
2.95 (1H, dd, J = 17.5, 3.4 Hz), 2.95 (3H, s), 3.16 (1H,
d, J = 17.5 Hz), 3.50 (1H, dd, J = 10.1, 9.6 Hz), 3.65

656
T. Sugimoto et al. / Bioorg. Med. Chem. 16 (2008) 650–657
Compound 15: [a]D À630ꢁ (c = 1.6, MeOH, 20.0 ꢁC);
UV kmax (MeOH) nm (e): 340 (31,200), 244 (43,100);
1H NMR d (400 MHz, 0.036 M, CDCl3, 297 K) ppm:
0.03 (3H, s), 0.06 (3H, s), 0.56 (3H, d, J = 6.7 Hz),
0.88 (9H, s), 0.93 (3H, d, J = 6.3 Hz), 2.61 (1H, m),
2.94 (3H, s), 2.95 (1H, dd, J = 17.6, 3.3 Hz), 3.14 (1H,
d, J = 17.6 Hz), 3.50 (1H, dd, J = 10.2, 9.6 Hz). 3.64
(1H, dd, J = 10.2, 4.4 Hz), 4.00 (1H, m), 4.43 (1H, d,
J = 10.2 Hz), 6.20 (1H, s), 6.49 (1H, d, J = 8.3 Hz),
6.99 (1H, s), 7.40 (1H, d, J = 8.3 Hz), 8.69 (1H, s); 13C
NMR d (100 MHz, 0.036 M, CDCl3, 297 K) ppm:
À5.4, À5.3, 18.2, 19.1, 21.2, 25.8 (3C), 28.6, 32.9, 34.0,
54.7, 65.3, 71.3, 85.7, 106.0, 115.7, 117.0, 118.5, 122.3,
128.6, 139.7, 152.2, 171.7; HR-EI-MS m/z: 440.2607
(M+, calcd for C24H36N4O2Si, 440.2608).
over Na2SO4, and concentrated. The residue was purified
by HPLC on YMC SH-342-5 using 60% MeOH to give 2
(3.6 mg, 0.011 mmol, 85%).
Compound 2: [a]D À420ꢁ (c = 2.3, MeOH, 16.6 ꢁC); UV
kmax (MeOH) nm (e): 340 (20,700), 244 (28,100); 1H
NMR d (400 MHz, 0.037 M, CDCl3, 297 K) ppm: 0.57
(3H, d, J = 6.7 Hz), 0.94 (3H, d, J = 6.4 Hz), 2.58 (1H,
m), 2.94 (3H, s), 3.09 (1H, dd, J = 17.6, 3.4 Hz), 3.16
(1H, d, J = 17.6 Hz), 3.60 (1H, dd, J = 11.4, 8.5 Hz).
3.75 (1H, dd, J = 11.4, 3.6 Hz), 4.11 (1H, m), 4.47
(1H, d, J = 10.2 Hz), 6.48 (1H, d, J = 8.4 Hz), 7.01
(1H, s), 7.39 (1H, d, J = 8.4 Hz), 7.47 (1H, s), 8.89
(1H, s); 13C NMR d (100 MHz, 0.037 M, CDCl3,
297 K) ppm: 14.2, 20.0, 21.3, 26.0, 33.1, 34.0, 55.5,
65.0, 71.2, 106.1, 115.7, 117.1, 118.6, 122.5, 128.6,
139.8, 152.2, 173.1; HR-EI-MS m/z: 326.1751 (M+, calcd
for C18H22N4O2, 326.1743).
4.2.12. Compound 16. Compound 13 (22.6 mg, 0.048 mmol)
was treated similarly as described for the synthesis of 15 to
give 16 (6.8 mg, 0.015 mmol, 31%).
4.2.15. Compound 3. Compound 16 (6.8 mg, 0.015 mmol)
was treated similarly as described for the synthesis of 2 to
give 3 (4.5 mg, 0.013 mmol, 87%).
Compound 16: [a]D À230ꢁ (c = 0.84, MeOH, 25.2 ꢁC); UV
kmax (MeOH) nm (e): 362 (15,800), 300 (4100), 248
(11,300); 1H NMR d (400 MHz, 0.030 M, CDCl3, 298 K)
ppm: 0.01 (3H, s), 0.05 (3H, s), 0.72 (3H, d, J = 6.7 Hz),
0.87 (9H, s), 0.99 (3H, d, J = 6.4 Hz), 2.63 (1H, m), 2.66
(3H, s), 2.92 (1H, dd, J = 17.0, 3.5 Hz), 3.04 (3H, s), 3.19
(1H, d, J = 17.0 Hz), 3.50 (1H, dd, J = 10.0, 9.0 Hz), 3.63
(1H, dd, J = 10.0, 4.3 Hz), 3.71 (1H, m), 4.59 (1H, d,
J = 10.4 Hz), 6.15 (1H, s), 6.68 (1H, d, J = 8.0 Hz), 7.32
(1H, d, J = 1.6 Hz), 7.59 (1H, d, J = 8.0 Hz); 13C NMR d
(100 MHz, 0.030 M, CDCl3, 298 K) ppm: À5.5, À5.4,
13.9,18.3, 19.0, 20.5, 25.8 (3C), 28.6, 32.0, 33.5, 55.4,
65.3, 73.0, 107.7, 109.6, 112.8, 120.0, 122.3, 122.9, 145.6,
151.9, 153.0, 172.2; HR-EI-MS m/z: 454.2772 (M+, calcd
for C25H38N4O2Si, 454.2764).
Compound 3: [a]D À340ꢁ (c = 2.0, MeOH, 26.6 ꢁC); UV
kmax (MeOH) nm (e): 361 (20,600), 300 (5500), 249
(14,800); 1H NMR d (400 MHz, 0.028 M, CDCl3,
297 K) ppm: 0.71 (3H, d, J = 6.7 Hz), 0.99 (3H, d,
J = 6.4 Hz), 2.60 (1H, m), 2.66 (3H, s), 2.95 (1H, br s),
3.01 (3H, s), 3.03 (1H, dd, J = 17.6, 3.8 Hz), 3.21 (1H,
d, J = 17.6 Hz), 3.61 (1H, m), 3.74 (1H, m), 3.80 (1H,
m), 4.60 (1H, d, J = 10.4 Hz), 6.67 (1H, d, J = 8.0 Hz),
7.10 (1H, s), 7.33 (1H, d, J = 1.5 Hz), 7.59 (1H,d,
J = 8.0 Hz); 13C NMR d (100 MHz, 0.028 M, CDCl3,
297 K) ppm: 13.8, 19.0, 20.5, 28.5, 32.1, 33.6, 55.9,
64.9, 73.1, 107.9, 109.6, 113.0, 120.0, 122.4, 122.9,
145.6, 152.0, 153.1, 173.2; HR-EI-MS m/z: 326.1751
(M+, calcd for C19H24N4O2, 326.1743).
4.2.13. Compound 17. Compound 14 (34.7 mg, 0.066 mmol)
was treated similarly as described for the synthesis of 15 to
give 17 (16.2 mg, 0.032 mmol, 48%).
4.2.16. Compound 4. Compound 17 (12.0 mg, 0.024 mmol)
was treated similarly as described for the synthesis of 2 to
give 4 (8.4 mg, 0.021 mmol, 88%).
Compound 17: [a]D À284ꢁ (c = 0.135, MeOH, 26.9 ꢁC);
UV kmax (MeOH) nm (e): 376 (20,000), 301 (5200), 250
(15,000); 1H NMR d (500 MHz, 0.032 M, CDCl3,
300 K) ppm: 0.03 (3H, s), 0.06 (3H, s), 0.73 (3H, d,
J = 6.7 Hz), 0.88 (9H, s), 1.00 (3H, d, J = 6.4 Hz), 2.64
(1H, m), 2.98 (1H, dd, J = 16.9, 3.2 Hz), 3.10 (3H, s),
3.22 (3H, d, J = 16.9 Hz), 3.54 (1H, dd, J = 10.7,
10.5 Hz). 3.65 (1H, dd, J = 10.7, 4.3 Hz), 3.65 (1H, m),
4.54 (1H, d, J = 10.4 Hz), 6.16 (1H, s), 6.87 (1H, d,
J = 8.3 Hz), 7.42 (1H, d, J = 1.3Hz), 7.73 (1H, d,
J = 8.3 Hz); 13C NMR d (125 MHz, 0.032 M, CDCl3,
300 K) ppm: À5.5, À5.3, 18.3, 18.9 20.2, 25.8 (3C),
28.6, 32.1, 33.4, 55.2, 65.4, 72.7, 105.7, 107.7, 113.1,
119.9, 123.5 (q, J = 244 Hz), 124.0, 125.6, 142.3, 142.4
(q, J = 35 Hz), 144.0, 152.8, 171.7; HR-EI-MS m/z:
508.2487 (M+, calcd for C25H35F3N4O2Si, 508.2481).
Compound 4: [a]D À330ꢁ (c = 4.11, MeOH, 25.6 ꢁC);
UV kmax (MeOH) nm (e): 376 (10,800), 302 (2700), 250
(8000); 1H NMR d (400 MHz, 0.053 M, CD3OD,
298 K) ppm: 0.76 (3H, d, J = 6.7 Hz), 1.01 (3H, d,
J = 6.4 Hz), 2.61 (1H, m), 3.12 (3H, s), 3.25 (1H, m),
3.31 (1H, m), 3.55 (1H, m). 3.64 (1H, m), 3.64 (1H,
m), 4.64 (1H, d, J = 10.4 Hz), 7.04 (1H, d, J = 8.4 Hz),
7.64 (1H, s), 7.75 (1H, d, J = 8.4 Hz); 13C NMR d
(100 MHz, 0.053 M, CD3OD, 298 K) ppm: 19.3, 20.5,
29.8, 32.7, 34.1, 57.3, 65.3, 73.7, 106.9, 108.6, 115.1,
121.3, 123.0 (q, J = 269 Hz), 124.7, 128.0, 142.8 (q,
J = 38.6 Hz), 145.3, 154.4, 174.1; HR-EI-MS m/z:
394.1616 (M+, calcd for C19H21F3N2O2, 394.1617).
4.3. Inhibition of specific binding of [3H]PDBu to the PKC
C1 peptides
4.2.14. Compound 2. Compound 15 (5.9 mg, 0.013 mmol)
was dissolved in THF (0.1 ml). To the solution, 90 ll of a
solution of TBAFÆ3H2O (13.5 mg) in THF (0.1 ml) was
added, and the mixture was stirred at 0 ꢁC for 40 min
and then partitioned between EtOAc and 5% KHSO4.
The collected EtOAc layer was washed with brine, dried
The binding of [3H]PDBu to the PKC C1 peptides was
evaluated using the procedure of Sharkey and Blum-
berg17 with slight modifications, as reported previ-
ously,16 under the following conditions: 50 mM Tris–

T. Sugimoto et al. / Bioorg. Med. Chem. 16 (2008) 650–657
7. Lang, G. E. Ophthalomologica 2007, 221, 112.
657
maleate (pH 7.4 at 4 ꢁC), 50 lg/ml 1,2-dioleoyl-sn-glyce-
ro-3-phospho-L-serine, 3 mg/ml bovine c-globulin,
20 nM [3H]PDBu (15.6 Ci/mmol), 5–40 nM of each
PKC C1 peptide, and various concentrations of 2, 3,
or 4. The binding affinity was evaluated as the concen-
tration required to cause 50% inhibition of the specific
binding of [3H]PDBu, IC50, which was calculated by a
computer program (SAS) with a probit procedure.34
The binding constant, Ki, was calculated using the
method of Sharkey and Blumberg.17
8. Martin, W. J.; Malmberg, A. B.; Basbaum, A. I. . J.
Neurosci. 2001, 21, 5321.
9. Bocklandt, S.; Blumberg, P. M.; Hamer, D. H. Antiviral
Res. 2003, 59, 89.
10. Kedei, N.; Lundberg, D. J.; Toth, A.; Welburn, P.;
Garfield, S. H.; Blumberg, P. M. Cancer Res. 2004, 64,
3243.
11. Hurley, J. H.; Newton, A. C.; Parker, P. J.; Blumberg, P.
M.; Nishizuka, Y. Protein Sci. 1997, 6, 477.
12. Ono, Y.; Fujii, T.; Igarashi, K.; Kuno, T.; Tanaka, C.;
Kikkawa, U.; Nishizuka, Y. Proc. Natl. Acad. Sci. U.S.A.
1989, 86, 4868.
4.4. Fluorescence spectra of 4 when bound to PKCd-C1B
13. Quest, A. F. G.; Bardes, E. S. G.; Bell, R. M. J. Biol.
Chem. 1994, 269, 2961.
The basic protocol was similar to the method that was
used to evaluate the binding of 4 to the PKC C1 peptide.
The standard assay mixture (250 ll), in 1.5 ml Eppen-
dorf tubes, contained 50 mM Tris–maleate (pH 7.4 at
4 ꢁC), 200 nM of PKCd-C1B peptide, and 10À6.5 M of
4 in the absence of phospholipid. In the presence of
phospholipid, the assay mixture contained 50 mM
Tris–maleate (pH 7.4 at 4 ꢁC), 200 nM of PKCd-C1B
peptide, 10À7 M of 4, and 50 lg/ml 1,2-dioleoyl-sn-gly-
cero-3-phospho-L-serine. In the competition assay,
10 lM of PDBu was added to assay mixture. After
10 ll of the peptide solution was diluted with 990 ll of
helium-purged distilled water, the resultant solution
(58 ll) was added to the standard assay mixture de-
scribed above (192 ll), and the solution was incubated
at 4 ꢁC for 20 min. After the incubation, 50 ll of solu-
tion was removed and fluorescent spectra were moni-
tored. The excitation wavelength was 377 nm, the
excitation maximum of 4.
14. Buchner, K. Eur. J. Biochem. 1995, 228, 211.
15. Oancea, E.; Teruel, M. N.; Quest, A. F. G.; Meyer, T. J.
Cell. Biol. 1998, 140, 485.
16. Wang, Q. J.; Bhattacharyya, D.; Garfield, S.; Narco, K.;
Marquez, V. E.; Blumberg, P. M. J. Biol. Chem. 1999, 274,
37233.
17. Sharkey, N. A.; Blumberg, P. M. Cancer Res. 1985, 45, 19.
18. Irie, K.; Oie, K.; Nakahara, A.; Yanai, Y.; Ohigashi, H.;
Wender, P. A.; Fukuda, H.; Konishi, H.; Kikkawa, U. J.
Am. Chem. Soc. 1998, 120, 9159.
19. Shindo, M.; Irie, K.; Nakahara, A.; Ohigashi, H.; Konishi,
H.; Kikkawa, U.; Fukuda, H.; Wender, P. A. Bioorg.
Med. Chem. 2001, 9, 2073.
20. Slater, S. J.; Ho, C.; Stubbs, C. D. Chem. Phys. Lipids
2002, 116, 75.
21. Endo, Y.; Shudo, K.; Okamoto, T. Chem. Pharm. Bull.
1982, 30, 3457.
22. Irie, K.; Hirota, M.; Hagiwara, N.; Koshimizu, K.;
Hayashi, H.; Murao, S.; Tokuda, H.; Ito, Y. Agric. Biol.
Chem. 1984, 48, 1269.
23. Fujiki, H.; Suganuma, M.; Hakii, H.; Nakayasu, M.;
Endo, Y.; Shudo, K.; Irie, K.; Koshimizu, K.; Sugimura,
T. Proc. Jpn. Acad., Ser. B 1985, 61, 45.
Acknowledgments
24. Irie, K.; Hagiwara, N.; Tokuda, H.; Koshimizu, K.
Carcinogenesis 1987, 8, 547.
The authors thank Ms. Ayako Morishima and Mr. Tak-
ashi Yamaguchi at JASCO engineering for the fluores-
cent spectral measurements. This research was partly
supported by a Grant-in-Aid for Scientific Research
on Priority Areas (No. 18032041 to K.I.) from the Min-
istry of Education, Science, Culture, Sports, and Tech-
nology of the Japanese Government.
25. Endo, Y.; Sato, Y.; Shudo, K. Tetrahedron 1987, 43, 2241.
26. Irie, K.; Hagiwara, N.; Koshimizu, K. Tetrahedron 1987,
43, 5251.
27. Irie, K.; Koshimizu, K. Mem. Coll. Agric. Kyoto Univ.
1988, 132, 1.
28. Nakagawa, Y.; Irie, K.; Yanagita, R. C.; Ohigashi, H.;
Tsuda, K.-i. J. Am. Chem. Soc. 2005, 127, 5746.
29. Irie, K.; Okuno, S.; Koshimizu, K.; Tokuda, H.; Nishino,
H.; Iwashima, A. Int. J. Cancer 1989, 43, 513.
30. Nakagawa, Y.; Irie, K.; Komiya, Y.; Ohigashi, H.; Tsuda,
K.-i. Tetrahedron 2004, 60, 7077.
References and notes
31. Irie, K.; Nakagawa, Y.; Ohigashi, H. Chem. Rec. 2005, 5,
185.
1. Nishizuka, Y. FASEB J. 1995, 9, 484.
2. Newton, A. C. Chem. Rev. 2001, 101, 2353.
3. Fujiki, H.; Sugimura, T. Adv. Cancer Res. 1987, 49, 223.
4. Nishizuka, Y. Nature 1988, 334, 661.
5. Ron, D.; Kazanietz, M. G. FASEB J. 1999, 13, 1658.
6. Ishii, H.; Koya, D.; King, G. L. J. Mol. Med. 1998, 76, 21.
32. Endo, Y.; Shudo, K.; Itai, A.; Hasegawa, M.; Sakai, S.
Tetrahedron 1986, 42, 5905.
33. Wahyuningsih, T. D.; Pchalek, K.; Kumar, N.; Black, D.
S. Tetrahedron 2006, 62, 6343.
34. Sakuma, M. Appl. Entomol. Zool. 1998, 33, 339.