Protein kinase C translocation by modified phorbol esters with functionalized lipophilic regions

Article abstract of DOI:10.1021/jo030029w

Several novel phorbol esters were prepared with polar functional groups terminating their C12 and/or C13 acyl chains. Designed to be inhibitory protein kinase C (PKC) ligands, these phorbol analogues contain various polar functional groups (amide, ester,

Full text of DOI:10.1021/jo030029w

P r otein Kin a se C Tr a n sloca tion by Mod ified P h or bol Ester s w ith
F u n ction a lized Lip op h ilic Region s
Thomas M. Bertolini,† J ennifer Giorgione,‡ Daniel F. Harvey,† and Alexandra C. Newton*,‡
Departments of Chemistry and Pharmacology, University of California, San Diego,
La J olla, California, 92093
anewton@uscd.edu
Received J anuary 23, 2003
Several novel phorbol esters were prepared with polar functional groups terminating their C12
and/or C13 acyl chains. Designed to be inhibitory protein kinase C (PKC) ligands, these phorbol
analogues contain various polar functional groups (amide, ester, carboxylic acid, or quaternary
ammonium salt) to prevent membrane insertion of the PKC-phorbol ester complex. All phorbol
derivatives were synthesized with use of diterpene starting materials obtained from croton oil, the
seed oil of Croton tiglium. The ability of these derivatives to recruit PKC to the lipid bilayersa
usual requirement for enzyme activationswas determined by using a sucrose-loaded vesicle assay.
Phorbol 12-octanoate-13-acetate derivatives translocate PKC-âII to increasing degrees as the
functionality on the C12 ester becomes more hydrophobic. Likewise, PKC translocation by carboxylic
acid-containing phorbol esters was dependent upon length and saturation of the hydrocarbon tether.
The most promising PKC inhibitors had short carboxylic acids capping their C12 and C13 acyl
chains, since these compounds did not recruit PKC to any appreciable extent.
In tr od u ction
Conversely, PMA is a stronger PKC activator11 that is
hardly metabolized by the cell.12-14 Hence, phorbol ester
binding to PKC hyperactivates the kinase, triggering cell
proliferation and amplifying the efficacy of carcinogens
(promotion or cocarcinogenesis).
The phorbol esters are tetracyclic diterpenes isolated
from the seeds of Croton tiglium, a member of the
Euphorbiaceae (or spurge) plant family.1 Although they
have been consumed medicinally for two millennia,2 these
natural products are the most powerful tumor promoters
known. For example, application of nanomolar concen-
trations of phorbol 12-myristate-13-acetate3 (PMA) causes
100% of initiated mice to develop papillomas, with an
average of 14 tumors per survivor.4 In 1982, Nishizuka
and co-workers discovered that phorbol esters bind to and
activate protein kinase C (PKC).5 The ubiquitious PKC
family of serine/threonine kinases6,7 plays a critical role
in signal transduction pathways regulating cell growth
and differentiation.8,9 During normal signaling processes
this enzyme is transiently activated by sn-1,2-diacyl-
glycerol (DAG), which is then rapidly hydrolyzed.10
Since DAG is a competitive inhibitor of the phorbol
esters,15 it was initially thought that both ligands bind
* Address correspondence to this author at the Department of
Parmacology.
† Department of Chemistry.
(8) Clemens, M. J .; Trayner, I.; Menaya, J . J . Cell Sci. 1992, 103,
881-887.
‡ Department of Pharmacology.
(1) Hecker, E.; Schmidt, R. Fortschr. Chem. Org. Naturst. 1974, 31,
377-467.
(2) Singer, C.; Underwood, E. A. A Short History of Medicine; Oxford
University Press: New York, 1962; p 674.
(9) Nishizuka, Y. Science 1992, 258, 607-614.
(10) Sando, J . J .; Maurer, M. C.; Bolen, E. J .; Grisham, C. M. Cell.
Signalling 1992, 4, 595-609.
(11) Mosior, M.; Newton, A. C. J . Biol. Chem. 1995, 270, 25526-
(3) The IUPAC name for phorbol is [1aR-(1aR,1bâ,4aR,7aR,7bR,8R,9â,-
9aR)]-1,1a,1b,4,4a,7a,7b,8,9,9a-decahydro-4aâ,7bR,9â,9aR-tetrahydroxy-
3-(hydroxymethyl)-1,1,6,8R-tetramethyl-5H-cyclopropa[3,4]benz(1,2-
e)azulen-5-one. The older, trivial phorbol numbering and nomenclature
is more common in the literature and will be used herein for simplicity
(see: Blumberg, P. M. Crit. Rev. Toxicol. 1980, 8, 153-197).
(4) Yamasaki, H.; Weinstein, I. B.; Van Duuren, B. J . Carcinogenesis
1981, 2, 537-543.
(5) Castanga, M.; Takai, Y.; Kaibuchi, K.; Sano, K.; Kikkawa, U.;
Nishizuka, Y. J . Biol. Chem. 1982, 257, 7847-7851.
(6) Newton, A. C. Chem. Rev. 2001, 101, 2353-2364.
(7) Nishizuka, Y. Science 1986, 233, 305-312.
25533.
(12) Segal, A.; Van Duuren, B. L.; Mate´, U. Cancer Res. 1975, 35,
2154-2159.
(13) Kreibich, G.; Witte, I.; Hecker, E. Z. Krebsforsch. 1971, 76, 113-
123.
(14) Hecker, E. Structure-Activity Relationships in Diterpene
Esters Irritant and Cocarcinogenic to Mouse Skin. In Carcinogenesis:
A Comprehensive Survey, Mechanisms of Tumor Promotion and
Cocarcinogenesis; Slaga, T. J ., Sivak, A., Boutwell, R. K., Eds.; Raven:
New York, 1978; Vol. 2, pp 11-48.
(15) Sharkey, N. A.; Leach, K. L.; Blumberg, P. M. Proc. Natl. Acad.
Sci U.S.A. 1984, 81, 607-610.
10.1021/jo030029w CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/22/2003
5028
J . Org. Chem. 2003, 68, 5028-5036

Protein Kinase C Translocation
to PKC via their structurally similar vic-diesters.16
A
therefore a rationally designed class of PKC inhibitors,
because they should bind strongly to the enzyme without
activating it.
1995 crystal structure of phorbol ester-bound PKC re-
vealed instead that a different oxygen-rich edge of the
phorbol molecule was responsible for enzyme binding.
Specifically, oxygen atoms at phorbol carbons 3, 4, and
20 form a hydrogen-bonding network to three highly
conserved PKC amino acid residues.17 Twenty years ago,
the laboratories of Hecker and Van Duuren established
the significance of this oxygenation through structure-
activity studies: synthetic modifications to C3, C4, or C20
(e.g., deoxygenation, epimerization, oxidation, reduction,
or esterification) abolished or greatly diminished phorbol
ester potency.14,18 Importantly, crystallography also re-
vealed that the phorbol C12 and C13 esters are not
hydrogen-bound to PKC, and the C12 oxygen atom is
found on a hydrophobic surface of the protein.17 These
data support the role of the long-chain C12 and C13
esters as lipophilic anchors facilitating membrane
insertionsan absolute requirement for PKC activation19s
of phorbol ester-bound PKC. The process of relocating
inactive, cytosolic PKC to the plasma membrane is known
as membrane recruitment or translocation of the kinase,
and can be achieved with either phorbol esters or DAG.11
Phorbol esters are invaluable biochemical probes that
have been used to elucidate signal transduction path-
ways, the mechanism of carcinogenesis, and the diverse
biological activity of PKC. Due to their extremely potent
cocarcinogenic activity, it is surprising that these natural
products have an enormous (and largely unexplored)
potential as pharmaceutical agents. For example, some
naturally occurring phorbol esters are tumor inhibi-
tors,20,21 inhibit HIV replication,22-25 or display antileu-
kemic activity.26,27 With this in mind, we are currently
focused on the development of synthetic, phorbol-based
inhibitors of PKC. Herein, we describe the preparation
and PKC-recruiting activity of phorbol analogues that
have polar functional groups terminating their C12 and/
or C13 acyl chains. With the C3-C4-C20 binding region
intact, these derivatives should remain strong PKC
ligands; PKC translocation (and hence PKC activation)
by these phorbol esters is expected to diminish because
the added hydrophilic functionality will not insert well
into the lipid bilayer. These novel phorbol esters are
Resu lts
Syn th esis of P h or bol Der iva tives. The most potent
tumor-promoting phorbol esters are 12,13-diesters with
unsubstituted 4, 9, and 20 hydroxyls. Phorbol, the parent
diterpene of phorbol esters, contains five hydroxy groups
with different reactivities toward acylation.1 Due to
accessibility differences, the alcohols are acylated in the
following order: 20-OH > 13-OH > 12-OH . 9-OH ≈
4-OH. To selectively acylate the C12 or C13 hydroxyls,
the primary allylic alcohol at C20 must first be protected.
Possible phorbol 20-OH protecting groups include simple
aliphatic esters,28,29 triphenylmethyl ethers,30 and silyl
ethers.31 We first employed phorbol 20-(p-methoxy)trityl
ether as a starting material because it is commercially
available (albeit very expensive) and the mono(p-meth-
oxy)trityl (MMTr) protecting group is easily removed in
80% acetic acid.32 Since several of the phorbol ester
derivatives in this study contain free carboxylic acids,
concentration often resulted in the intermolecular re-
moval of the MMTr group (see Supporting Information).
Instead, the more robust triphenylmethyl (Tr) ether was
used due to its greater stability toward carboxylic acids
and silica gel chromatography. Substitution at the 20-
position also prevents autoxidation of phorbol, a problem
commonly encountered when the C20 hydroxyl is unpro-
tected.33
(16) Nishizuka, Y. Nature 1984, 308, 693-698.
(17) Zhang, G.; Kazanietz, M. G.; Blumberg, P. M.; Hurley, J . H.
Cell 1995, 81, 917-924.
(18) Van Duuren, B. L.; Tseng, S.-S.; Segal, A.; Smith, A. C.;
Melchionne, S.; Seidman, I. Cancer Res. 1979, 39, 2644-2646.
(19) Szallasi, Z.; Smith, C. B.; Blumberg, P. M. J . Biol. Chem. 1994,
269, 27159-27162.
(20) Schmidt, R.; Hecker, E. Cocarcinogenesis and Biological Effects
of Tumor Promoters; Hecker, E., Fusenig, N. E., Kunz, W., Marks, F.,
Thielmann, H. W., Eds.; Raven Press: New York, 1982; Vol. 7, pp 57-
63.
(21) Szallasi, Z.; Krsmanovic, L.; Blumberg, P. M. Cancer Res. 1993,
53, 2507-2512.
There are several published protocols1,34,35 for the
isolation of phorbol from hydrolyzed croton oil; however,
its crystallization is slow and decomposition often occurs
(22) Gustafson, K. R.; Cardellina, J . H. II; McMahon, J . B.; Gula-
kowski, R. J .; Ishitoya, J .; Szallasi, Z.; Lewin, N. E.; Blumberg, P. M.;
Weislow, O. S.; Beutler, J . A.; Buckheit, R. W., J r.; Cragg, G. M.; Cox,
P. A.; Bader, J . P.; Boyd, M. R. J . Med. Chem. 1992, 35, 1978-1986.
(23) El-Mekkawy, S.; Meselhy, M. R.; Nakamura, N.; Hattori, M.;
Kawahata, T.; Otake, T. Phytochemistry 2000, 53, 457-464.
(24) Erickson, K. L.; Beutler, J . A.; Cardellina, J . H., II; McMahon,
J . B. J . Nat. Prod. 1995, 58, 769-772.
(25) Gulakowski, R. J .; McMahon, J . B.; Buckheit, R. W., J r.;
Gustafson, K. R.; Boyd, M. R. Antiviral Res. 1997, 33, 87-97.
(26) Kupchan, S. M.; Uchida, I.; Branfman, A. R.; Dailey, R. G., J r.;
Yu, F. B. Science 1976, 191, 571-572.
(27) Handa, S. S.; Kinghorn, A. D.; Cordell, G. A.; Farnsworth, N.
R. J . Nat. Prod. 1983, 46, 123-126.
(28) Kreibich, G.; Hecker, E. Z. Krebsforsch. 1970, 74, 448-456.
(29) Irie, K.; Ishii, T.; Ohigashi, H.; Wender, P. A.; Miller, B. L.;
Takeda, N. J . Org. Chem. 1996, 61, 2164-2173.
(30) Sorg, B.; Fu¨rstenberger, G.; Berry, D. L.; Hecker, E.; Marks,
F. J . Lipid Res. 1982, 23, 443-447.
(31) Wender, P. A.; Kirschberg, T. A.; Williams, P. D.; Bastiaans,
H. M. M.; Irie, K. Org. Lett. 1999, 1, 1009-1012.
(32) Declos, K. B.; Yeh, E.; Blumberg, P. M. Proc. Natl. Acad. Sci.
U.S.A. 1983, 80, 3054-3058.
(33) Schmidt, R.; Hecker, E. Cancer Res. 1975, 35, 1375-1377.
(34) Crombie, L.; Games, M. L.; Pointer, D. J . J . Chem. Soc. (C) 1968,
1347-1362.
(35) Kauffmann, T.; J asching, W.; Schulz, J . Chem. Ber. 1959, 92,
1715-1721.
J . Org. Chem, Vol. 68, No. 13, 2003 5029

Bertolini et al.
SCHEME 1. Isola tion of Tr ityla ted P h or bol Ester s fr om Cr oton Oila
a
R1 and R2 are different alkyl groups. R3 is a proton or an acyl group.
SCHEME 2. Ba se-Ca ta lyzed C4 Ep im er iza tion of P h or bol1
before satisfactory crystals are obtained. Fortunately, Dr.
Bernd Sorg of the Deutsches Krebsforschung Zentrum
graciously gave us his procedure for the isolation of
tritylated phorbol derivatives (Scheme 1). Croton oil,
which contains various phorbol 12,13-diesters and phor-
bol 12,13,20-triesters, was treated with dilute perchloric
acid in methanol to transesterify the C20 esters. The
resulting phorbol 12,13-diesters (1) were then protected
at 20-OH by using trityl chloride in pyridine, giving a
complex mixture of 20-tritylated phorbol 12,13-diesters
(2). Hydrolysis of 2 with sodium methoxide produced
phorbol 20-trityl ether (3), which was selectively acety-
lated at the 13-OH to yield phorbol 13-acetate-20-trityl
ether (4).30 3 can be purified by silica gel chromatography
and 4 is a highly crystalline solid. Using this protocol,
1.6 g of pure 4 were obtained from 100 g of croton oil.
Acylations of phorbol have traditionally been conducted
with acid chlorides in pyridine,36 but these reactions are
often low-yielding and slow. Instead, C12 and/or C13
esterifications of phorbol were performed with 10 equiv
of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride (EDCI),37 10 equiv of the desired carboxylic acid,
and a stoichiometric amount of 4-(dimethylamino)pyri-
dine (DMAP). All esterifications were purified via chro-
matography on silica gel, and yields were consistently
good (71-96%). Allyl esters38 were employed to protect
the carboxylic acid-containing phorbol derivatives. Re-
F IGURE 1. PKC recruitment by phorbol derivatives with
different functional groups.
moval of the allyl moiety was accomplished with tetrakis-
(triphenylphosphine)palladium(0) and morpholine in yields
dependent upon catalyst quality (26-99%). A 10% solu-
tion of TFA in anisole at 0 °C was used to remove the
trityl ether. The structures of the novel phorbol deriva-
tives prepared in this study are summarized in Table 1,
and their syntheses are included in the Supporting
Information.
An artifact of phorbol chemistry is the inevitable
contamination of phorbol ester samples with 2-5% of a
diastereomeric impurity. When exposed to organic or
inorganic bases, phorbol esters epimerize to their 4R-
epimer39,40 via a thermodynamically favorable rearrange-
ment (Scheme 2). Since the undesirable 4R-epimer is
difficult to separate by silica gel chromatography, all final
(36) von Szczepanski, C.; Schairer, H. U.; Gschwendt, M.; Hecker,
E. Liebig Ann. Chem. 1967, 70, 199-210.
(37) Uotsu, K.; Sodeoka, M.; Shibasaki, M. Bioorg. Med. Chem. 1998,
6, 1117-1126.
(38) Deziel, R. Tetrahedron Lett. 1987, 28, 4371-4372.
5030 J . Org. Chem., Vol. 68, No. 13, 2003

Protein Kinase C Translocation
F IGURE 2. PKC recruitment by phorbol derivatives with
different tether lengths. The binding curves for 9 and 10 are
nearly identical.
F IGURE 3. PKC recruitment by phorbol derivatives with
aromatic versus aliphatic acids.
TABLE 1. Ap p a r en t KD for Mem br a n e Bin d in g of
P h or bol Ester Der iva tivesa
compd
R1
R2
KD (µM)
5
6
7
8
-(CH2)6CO2CH3
-(CH2)6CONH2
-CH3
-CH3
0.024 ( 0.003
1.3 ( 0.1
12 ( 1
-(CH2)7N(CH3)3+ I- -CH3
-(CH2)6CO2H
-CO2H
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
1.6 ( 0.3
34 ( 7
F IGURE 4. PKC recruitment by diacid phorbol derivatives.
9
10
11
12
13
14
-(CH2)2CO2H
-(CH2)4CO2H
-(CH2)8CO2H
-(CH2)12CO2H
-(CH2)2CHdCH-
(CH2)2CO2H
-(p-C6H4)CO2H
-(p-C6H4)CO2H
-(CH2)2CO2H
-CH3
33 ( 6
43 ( 9
1.1 ( 0.2
0.024 ( 0.002
19 ( 2
15
16
17
18
19
20
21
-CH3
39 ( 13
500 ( 200
700 ( 300
500 ( 200
160 ( 20
0.035 ( 0.005
0.24 ( 0.02
-(p-C6H4)CO2H
-(CH2)2CO2H
-(CH2)2CO2H
-(p-C6H4)CO2H
-(CH2)2CO2H
-(CH2)12CO2H
-CH3
-(CH2)12CH3
-CH3
a
PMA was found to have a KD value of 0.025 ( 0.004 µM.
products were purified to >99% purity via reverse-phase
HPLC. Removal of 4R-phorbol 12,13-diesters is critical
for biological studies because 4R-phorbol derivatives do
not activate PKC.14
F IGURE 5. Effect of unsaturation on PKC recruitment.
µM PMA.41 The apparent KD for membrane bindingsthe
concentration of phorbol ester required to translocate 50%
of PKC to the lipid bilayersfor each phorbol derivative
is also listed in Table 1. The PKC-catalyzed substrate
phosphorylation (activity) was also measured (see Ex-
perimental Section). The PKC activity observed in the
presence of either PMA or analogue 8 is shown in Figure
7. Activity curves mimic binding curves with half-
maximal activation at roughly the same value as the KD
shown in Table 1. Half-maximal activation is at 15 and
800 nM for PMA and analogue 8, vs KD values of 25 and
1600 nM, respectively. Table 2 shows the percentage of
PKC activity obtained at the KD of each remaining
P KC Tr a n sloca tion Stu d ies of P h or bol Der iva -
tives. Phorbol ester analogues were assayed for their
ability to translocate PKC-âII to lipid vesicles by using
a sucrose-loaded vesicle assay (see Experimental Section).
Membrane recruitment of PKC-âII by these derivatives
is shown graphically in Figures 1-6. Each figure contains
a binding curve for PMA with a KD value of 0.025 ( 0.004
(39) J acobi, P.; Ha¨rle, E.; Schairer, U.; Hecker, E. Liebigs Ann.
Chem. 1970, 741, 13-32.
(40) Tseng, S.-S.; Van Duuren, B. L.; Solomon, J . J . J . Org. Chem.
1977, 42, 3645-3649.
J . Org. Chem, Vol. 68, No. 13, 2003 5031

Bertolini et al.
F IGURE 6. Nonequivalence of C12 and C13 esters.
F IGURE 8. Relative PKC activity at high concentrations of
diacid 17.
tions, and quaternary ammonium salt 7 is less effective
than PMA by about 2 orders of magnitude.
Another set of analogues (8-13) with the same func-
tional groupsa carboxylic acidswere only different due
to the length of their C12 acyl chains (Figure 2). These
phorbol derivatives became more efficient at translocat-
ing PKC as their C12 side chains increased in length.
Although none can recruit PKC to the lipid bilayer more
efficiently than PMA, the more potent phorbol acids 12
and 13 resemble PMA in structure. Indeed, the strongest
known phorbol ester promoters have C12 and C13 acyl
groups with a combined total of 14 to 20 carbon atoms43
(such as PMA, which has 16).
At cytosolic pH, carboxylic acids equilibrate between
unprotonated and protonated states but only the latter
is appreciably lipophilic. Benzoic diacids are about an
order of magnitude more acidic than aliphatic acids44 and
therefore more likely to be ionized at physiological pH.
As shown in Figure 3, we compared PKC recruitment by
phorbol esters with aromatic (15 and 19) versus aliphatic
acids (10 and 18). No difference in PKC translocation was
observed between 10 and 15; surprisingly, 19 (a consti-
tutional isomer of 15) very poorly translocated PKC and
18 (a constitutional isomer of 10) did not recruit PKC at
all. Incidentally, when the 12-acetate moiety of 18 is
replaced with tetradecanoate (20), PKC translocation is
completely restored. The presence of two short carboxylic
acids, whether aromatic or aliphatic, in the lipophilic
region of a phorbol ester is sufficient to prevent any
translocation of PKC to lipid vesicles. As shown in Figure
4, phorbol diacids 16 and 17 are completely unable to
membrane-recruit PKC even at 0.1 mM concentrations.
Unsaturation of the C12 and C13 acyl groups impairs
both the cocarcinogenic activity45,46 and PKC binding47
of a phorbol ester. To examine the effect of unsaturation
F IGURE 7. PKC activity in the presence of PMA or acid 8.
TABLE 2. P er cen t P KC Activity a t th e Ap p a r en t KD for
Mem br a n e Bin d in g of P h or bol Ester Der iva tives
compd
% max activity
compd
% max activity
5
6
7
9
10
11
12
13
59 ( 4
50 ( 2
70 ( 2
67 ( 6
41 ( 3
31 ( 7
61 ( 10
26 ( 3
14
15
16
17
18
19
20
21
62 ( 1
54 ( 9
0
0
63 ( 6
31 ( 8
51 ( 1
38 ( 5
analogue. Figure 8 shows the relative activity obtained
with high concentrations of analogue 17. Activity is
shown relative to vesicles containing no analogue. PMA
(1 nM) was included in all vesicles in Figure 8.
Discu ssion
A series of phorbol 12-octanoate-13-acetates (com-
pounds 5-8) were synthesized based on natural phorbol
esters, which always contain one short and one long acyl
group.42 These analogues are identical to each other
except for the functional group substitution at the C12
ester. Redistribution of PKC to the lipid bilayer dimin-
ishes as these functional groups increase in polarity
(Figure 1): methyl ester 5 displays similar activity to
PMA, amide 6 and acid 8 recruit PKC at 1 µm concentra-
(43) Thielmann, H. W.; Hecker, E. In Fortschritte der Krebsfors-
chung; Schmidt, C. G., Wetter, O., Eds.; Schattauer: Stuttgart,
Germany, 1969; Vol. 7, pp 171-179.
(44) The pKa values for succinic acid, methylsuccinic acid, and
terephthlalic acid at 25 °C are 4.16, 5.64, and 3.51, respectively.
From: CRC Handbook of Chemistry and Physics, 70th ed.; Weast, R.
C., Ed.; CRC Press Inc.: Boca Raton, FL, 1989; pp D163-D164.
(45) Fu¨rstenberger, G.; Berry, D. L.; Sorg, B.; Marks, F. Proc. Natl.
Acad. Sci. U.S.A. 1981, 78, 7722-7726.
(46) Marks, F.; Berry, D. L.; Bertsch, S.; Fu¨rstenberger, G.; Richter,
H. Carcinogenesis; Hecker, E., Ed.; Raven Press: New York, 1982; pp
331-346.
(41) Giorgione et al., submitted for publication.
(42) Hecker, E. Naturwissenschaften 1967, 282-284.
(47) Dunn, J . A.; Blumberg, P. M. Cancer Res. 1983, 43, 4632-4637.
5032 J . Org. Chem., Vol. 68, No. 13, 2003

Protein Kinase C Translocation
on PKC recruitment, we compared the activity of two
phorbol analogues (8 and 14) that were only different by
one degree of unsaturation. As shown in Figure 5, the
presence of a single Z-alkene in compound 8 diminishes
membrane recruitment by more than an order of mag-
nitude. Conceivably, it is the conformational rigidity
imposed by the added alkene that encumbers transloca-
tion of the phorbol-PKC complex.
Since phorbol esters bind to PKC within the chiral
milieu of the enzyme, substitution of a carboxylic acid at
the C12 or C13 acyl chain might produce two phorbol
esters with unique biological activities. As shown in
Figure 6, phorbol 12-tetradecandioate-13-acetate (13) is
about three times more strongly recruiting than phorbol
12-acetate-13-tetradecandioate (21). After also comparing
constitutional isomers 10 versus 18 and 15 versus 19
(Figure 3), we consistently observed that phorbol deriva-
tives with free carboxyl groups on the C12 acyl chain
were several times more potent than their constitutional
isomers.
et al. synthesized a phorbol ester analogue with a
hydrophilic 12-ester chain.48 This compound was able to
inhibit PKC activity by about 30%, and could compete
with PMA for binding to PKC. Its effect on membrane
translocation of PKC was not tested, nor were potential
inhibitory effects of the compound on the direct catalytic
activity of PKC assessed. Nonetheless, this study sup-
ports the potential for the design of translocation inhibi-
tors as a mechanism for preventing PKC activation.
Con clu sion
The phorbol ester lipophilic domain, largely comprised
of the C12/C13 diacyl moiety, is thought to be responsible
for redistributing the PKC-phorbol complex to the
plasma membrane. Crystallographic data show that a
large, exposed area of the phorbol-binding domain of PKC
contains only uncharged, nonpolar amino acid residues.
Phorbol ester binding is thought to create a continuous
hydrophobic region at this surface that facilitates mem-
brane association of the PKC-phorbol ester complex.17
Apparently, membrane insertion of this complex is so
thermodynamically favorable that the energetic cost of
introducing a single hydrophilic functional group can be
overcome. The presence of two short carboxylic acids,
however, is sufficient to abolish PKC translocation
altogether, and when present at very high concentrations
can inhibit up to 90% of the measured PKC activity.
Thus, we have shown the ability of a phorbol ester to
membrane-recruit PKC can be gradually decreased or
completely abolished depending on the polarity and
amount of functional group substitution. These data are
entirely consistent with the role of the C12 and/or C13
acyl chains as lipophilic anchors that permit membrane-
insertion of phorbol ester-bound PKC.
The ability of phorbol analogues to translocate PKC
to the membrane varies according to the hydrophobicity
of their side chains, and the ability of the compounds to
incorporate into the membrane. Analogues that do not
translocate PKC well are likely not incorporated into the
membrane to a full extent.
Our studies do not address whether the analogues
directly bind to PKC either in solution or while in the
membrane. However, the basic structure of the phorbol
molecule was not changed, and all binding sites for PKC
were unaltered. Since most of the analogues could recruit
PKC to the membrane at least partially, this suggests
that they could in fact bind directly to the enzyme while
partitioned in the membrane. While in solution, there is
an orientation issue. It would be much less likely for PKC
to bind to the analogues in the correct way, since they
are not presented on a 2-dimensional membrane, but
rather in 3-dimensional solution. Also, the concentration
of analogues sensed by PKC in solution would be much
lower than the concentration in a membrane. Therefore,
the amount of soluble phorbol ester required to bind PKC
would be orders of magnitude higher in solution. Any
phorbol inhibitor would have to be present in much
higher amounts than the competing membrane-bound
phorbol ester.
Since most of the analogues tested were still able to
recruit PKC to the membrane, it was not surprising that
their relative strengths at activating the enzyme coin-
cided with this (Figure 7 and Table 2). Half-maximal
activity was seen at about the same value as the
measured KD for binding. Porbol diacids 16 and 17 did
not activate PKC at their measured KD. Slight activation
was observed at 0.8-2 mM, followed by complete inhibi-
tion at higher concentrations (Figure 8). A promising
possibility is that the unincorporated analogue is able
to bind to PKC in solution and thus prevent PKC from
binding to the membrane. However, the high concentra-
tion required for inhibition could reflect nonspecific
effects of the compound on the kinase activity since such
high concentrations cause some inhibition using a cofac-
tor-independent activity assay (data not shown). None-
theless, these data suggest some promise for the ability
to design translocation inhibitors that trap PKC away
from its membrane activators. In a recent study, Wada
Exp er im en ta l P r oced u r es
Ca u tion ! Phorbol esters are powerful tumor promoters that
are rapidly absorbed through the skin. They are also severely
irritating to skin, eyes, mucous membranes, and lungs. Care
should be exercised when handling croton oil and all phorbol
ester derivatives.
Gen er a l Meth od s. Due to the air-sensitivity of phorbol
esters, reactions were degassed via 3 freeze-pump-thaw
cycles and then conducted under nitrogen. Phorbol ester
derivatives were stored in CH2Cl2 or ethyl acetate33 and never
chloroform nor ethereal solvents. All reagents were obtained
from commercial suppliers and used as received unless oth-
erwise indicated. Tetrahydrofuran (THF) was glass-distilled
from potassium and benzophenone ketyl under a nitrogen
atmosphere. Dimethylformamide (DMF) and methylene chlo-
ride (CH2Cl2) were glass-distilled from calcium hydride under
a nitrogen atmosphere. Melting points are uncorrected. Mass
spectrometry was performed by the Scripps Research Institute
in La J olla, CA. Reverse-phase HPLC was performed on a
semipreparative C8 column with a photodiode array detector
at 254 nm; samples were dissolved in 200 µL of 50% MeOH
and centrifugally filtered (0.45 µm nylon).
(48) Wada, R.; Suto, Y.; Kanai, M.; Shibasaki M. J . Am. Chem. Soc.
2002, 124, 10658-10659.
(49) Orr, J . W.; Keranen, L. M.; Newton, A. C. J . Biol. Chem. 1992,
267, 15263-15266.
(50) Bartlett, G. R. J . Biol. Chem. 1959, 234, 466-468.
(51) Rebecchi, M.; Peterson, A.; McLaughlin, S. Biochemistry 1992,
31, 12742-12747.
(52) Mosior, M.; Epand, R. M. Biochemistry 1993, 32, 65-75.
(53) Mosior, M.; Epand, R. M. J . Biol. Chem. 1994, 269, 13798-
13805.
J . Org. Chem, Vol. 68, No. 13, 2003 5033

Bertolini et al.
Isola tion of P h or bol Sta r tin g Ma ter ia ls 3 a n d 4 fr om
Cr oton Oil. Croton oil (100 g, Sigma) was added to 350 mL
of a 0.01 M HClO4 solution in MeOH and vigorously stirred
so that a brown emulsion formed. After 40 h, this mixture was
neutralized with 1 g of NaOAc and partitioned between
hexanes and MeOH. The MeOH layer was washed with
hexanes (3 × 500 mL) and concentrated in vacuo to give a
reddish-brown syrup. The syrup was dissolved in 100 mL of
pyridine and 30 g of trityl chloride was added. The solution
became a cloudy beige color and was stirred for 72 h, upon
which it was concentrated in vacuo and partitioned between
EtOAc and saturated aqueous NaHCO3. The organic layer was
washed successively with saturated aqueous NaHCO3 (400
mL), 1:1 brine:1.0 M HCl (400 mL), and saturated aqueous
NaHCO3 (400 mL). Concentration of the organic layer in vacuo
gave an orange paste that was triturated with MeOH (150 mL)
and filtered to remove precipitated triphenylmethanol. To the
clear, amber filtrate was added portionwise 0.81 g of NaOMe
and the reaction was stirred for 22 h. Addition of a 10%
solution of AcOH in MeOH to achieve neutrality, concentration
in vacuo, and chromatography on silica gel (using an eluant
gradient of 10% acetone in CH2Cl2, EtOAc, and 1:1 acetone:
EtOAc) gave 1.85 g of p h or bol 20-tr ityl (3) as an off-white
resin. A small sample (isolated as a hydrate) was purified by
RP-HPLC (65% MeOH in H2O, tR ) 19.0 min) for characteriza-
6H), 1.36-1.31 (m, 4H), 1.23 (s, 3H), 1.20 (s, 3H), 1.09 (d, J )
2.6 Hz, 1H), 0.88 (d, J ) 6.3 Hz, 3H) ppm; 13C NMR (125 MHz,
CDCl3) δ 208.5, 173.9, 173.5, 173.3, 160.4, 140.3, 132.7, 128.9,
78.1, 76.6, 73.7, 68.0, 65.6, 56.2, 51.6, 43.0, 39.1, 38.7, 36.3,
34.6, 34.0, 28.9, 28.7, 25.7, 25.0, 24.9, 24.0, 21.2, 17.0, 14.6,
10.3 ppm; IR (neat) 3410, 1723, 1653 cm-1; HRMS (MALDI)
calcd for C31H44O10Na (MNa+) 599.2832, found 599.2831.
P h or bol 12-O-Su ber a m id e-13-a ceta te (6). Clear oil; [R]D
+5.0° (c 0.3, MeOH); 1H NMR (400 MHz, CDCl3) δ 7.58 (br s,
1H), 5.68 (d, J ) 4.9 Hz, 1H), 5.51 (br s, 1H), 5.41 (d, J ) 10.3
Hz, 1H), 5.40 (br s, 2H), 4.02 (AB, J ) 13.0 Hz, 2H), 3.24 (s,
1H), 2.70 (s, 1H), 2.56 (d, J ) 19.0 Hz, 1H), 2.48 (d, J ) 19.0
Hz, 1H), 2.34 (t, J ) 7.3 Hz, 2H), 2.21 (t, J ) 7.6 Hz, 2H), 2.14
(dq, J ) 10.3, 6.3 Hz, 1H), 2.09 (s, 3H), 2.05 (br s, 1H), 1.78-
1.70 (m, 3H), 1.68-1.58 (m, 4H), 1.39-1.33 (m, 4H), 1.24 (s,
3H), 1.21 (s, 3H), 1.09 (d, J ) 5.4 Hz, 1H), 0.90 (d, J ) 6.3 Hz,
3H) ppm; 13C NMR (125 MHz, CDCl3) δ 208.6, 176.2, 173.5,
173.2, 160.4, 140.4, 132.8, 128.8, 78.1, 76.8, 73.7, 68.0, 65.6,
56.2, 43.0, 39.1, 38.6, 36.3, 35.7, 34.5, 28.9, 28.6, 25.8, 25.3,
25.0, 24.0, 21.2, 17.0, 14.7, 10.3 ppm; IR (neat) 3373, 3223,
1710, 1663 cm-1; HRMS (MALDI) calcd for C30H43NO9Na
(MNa+) 584.2835, found 584.2826.
P h or b ol 12-[8-(Tr im et h yla m m on iu m )oct a n oa t e]-13-
a ceta te Iod id e (7). Off-white powder, mp 243.5 °C dec; Rf
0.16 (8:6:6:3 CHCl3:MeOH:acetone:concd NH4OH); [R]D +12.3°
1
1
tion. [R]D +3.3° (c 0.6, MeOH); H NMR (500 MHz, CDCl3) δ
(c 1.1, MeOH); H NMR (400 MHz, CD3OD) δ 7.55-7.54 (m,
7.56-7.53 (m, 1H), 7.42 (d, J ) 7.3 Hz, 6H), 7.29 (t, J ) 7.3
Hz, 6H), 7.23 (t, J ) 7.2 Hz, 3H), 5.55 (d, J ) 4.0 Hz, 1H),
4.14-4.10 (m, 1H), 3.59 (AB, J ) 12.5 Hz, 2H), 3.04 (t, J )
5.1 Hz, 1H), 2.92-2.87 (m, 1H), 2.45 (d, J ) 20.5 Hz, 1H), 2.35
(d, J ) 19.0 Hz, 1H), 2.12 (s, 1H), 1.86 (dq, J ) 11.0, 6.4 Hz,
1H), 1.79-1.76 (m, 3H), 1.32 (s, 3H), 1.19 (s, 3H), 1.07 (d, J )
6.6 Hz, 3H), 0.80 (d, J ) 5.1 Hz, 1H) ppm; 13C NMR (100 MHz,
CDCl3) δ 208.3, 159.7, 143.9, 139.0, 133.0, 128.7, 128.5, 127.7,
126.9, 87.0, 81.5, 78.6, 73.6, 68.8, 62.9, 57.6, 45.2, 39.5, 39.0,
36.2, 26.8, 23.4, 17.1, 15.2, 10.3 ppm; IR (neat) 3412, 1697,
1H), 5.62 (d, J ) 4.4 Hz, 1H), 5.44 (d, J ) 10.3 Hz, 1H), 3.94
(AB, J ) 13.0 Hz, 2H), 3.37-3.30 (m, 2H), 3.14 (s, 9H), 2.51
(AB, J ) 18.9 Hz, 2H), 2.44-2.32 (m, 2H), 2.21 (dq, J ) 10.4,
6.4 Hz, 1H), 2.06 (s, 3H), 1.83-1.76 (m, 2H), 1.74 (dd, J ) 2.8,
1.3 Hz, 3H), 1.66 (pentet, J ) 7.3 Hz, 2H), 1.42 (br s, 6H),
1.25 (s, 3H), 1.21 (s, 3H), 1.16 (d, J ) 5.1 Hz, 1H), 0.88 (d, J
) 6.2 Hz, 3H) ppm; 13C NMR (100 MHz, CD3OD) δ 210.5,
175.7, 175.4, 160.7, 143.0, 134.7, 129.4, 79.8, 78.4, 74.7, 68.0,
67.8, 67.1, 53.6, 52.3, 44.3, 40.0, 38.4, 37.0, 35.2, 29.8, 27.22,
27.15, 25.9, 24.1, 23.8, 21.0, 17.3, 14.8, 10.2 ppm; IR (KBr)
3396, 1708, 1631 cm-1; HRMS (MALDI) calcd for C33H52NO8
(M+) 590.3687, found 590.3663.
1628 cm-1
.
Following a literature procedure,30 phorbol 20-trityl (1.85
g, 3.05 mmol) was dissolved in CH2Cl2 (120 mL) and THF (120
mL) and to this mixture were sequentially added Et3N (14 mL)
and Ac2O (8 mL). After 4 h the reaction was quenched with
saturated aqueous NaHCO3 (200 mL), stirred 1 h, and
partitioned between EtOAc and saturated aqueous NaHCO3.
The organic layer was washed successively with saturated
aqueous NaHCO3 (200 mL), 1:1 brine:0.1 M HCl (200 mL), and
saturated aqueous NaHCO3 (200 mL). Concentration of the
organic layer in vacuo and recrystallization from warm CH2-
Cl2 gave 1.61 g (2.48 mmol, 81%) of p h or bol 13-a ceta te-20-
tr ityl (4) as a white solid. For characterization, a small sample
was purified by RP-HPLC (65% MeOH in H2O, tR ) 19.0 min)
to give white crystals, mp 219-220 °C; [R]D +8.4° (c 0.6,
MeOH); 1H NMR (400 MHz, CDCl3) δ 7.58-7.56 (m, 1H), 7.42
(d, J ) 7.3 Hz, 6H), 7.29 (t, J ) 4.0 Hz, 6H), 7.23 (t, J ) 7.2
Hz, 3H), 5.60 (d, J ) 4.4 Hz, 1H), 3.99 (dd, J ) 9.9, 3.3 Hz,
1H), 3.56 (s, 2H), 3.11 (t, J ) 5.1 Hz, 1H), 3.07 (br s, 1H), 2.49
(d, J ) 19.0 Hz, 1H), 2.37 (d, J ) 19.0 Hz, 1H), 2.13 (s, 3H),
2.04 (s, 1H), 1.98 (dq, J ) 10.4, 6.4 Hz, 1H), 1.80-1.77 (m,
3H), 1.27 (s, 3H), 1.23 (s, 3H), 1.09 (d, J ) 5.1 Hz, 3H), 1.02
(d, J ) 6.6 Hz, 1H) ppm; 13C NMR (100 MHz, CDCl3) δ 208.4,
173.9, 160.1, 143.8, 138.6, 132.8, 128.8, 128.5, 127.7, 126.9,
86.9, 78.2, 77.4, 73.6, 69.0, 68.4, 56.9, 45.0, 39.3, 39.1, 35.4,
26.8, 23.8, 21.2, 17.0, 15.3, 10.3 ppm; IR (neat) 3386, 1720,
1695 cm-1; LRMS (ES) 671 (MNa+, 100) 243 (13); HRMS
P h or bol 12-Su ber a te-13-a ceta te (8). White solid, mp
139.5 °C; [R]D +17.7° (c 0.3, MeOH); 1H NMR (400 MHz,
CDCl3) δ 7.59 (s, 1H), 5.68 (d, J ) 4.8 Hz, 1H), 5.58 (br s, 1H),
5.40 (d, J ) 10.3 Hz, 1H), 4.01 (AB, J ) 13.2 Hz, 2H), 3.25 (br
s, 2H), 2.56 (d, J ) 19.0 Hz, 1H), 2.48 (d, J ) 19.0 Hz, 1H),
2.36-2.29 (m, 4H), 2.14 (dq, J ) 9.9, 6.6 Hz, 1H), 2.09 (s, 3H),
1.77-1.75 (m, 3H), 1.68-1.60 (m, 4H), 1.37-1.32 (m, 4H), 1.24
(s, 3H), 1.21 (s, 3H), 1.09 (d, J ) 5.5 Hz, 1H), 0.89 (d, J ) 6.2
Hz, 3H) ppm; 13C NMR (100 MHz, CDCl3) δ 208.9, 177.0, 173.5,
173.3, 160.7, 140.3, 132.8, 129.0, 78.2, 76.4, 73.7, 68.0, 65.6,
56.1, 42.9, 39.1, 38.5, 36.3, 34.5, 33.6, 28.7, 25.8, 25.0, 24.6,
23.9, 21.2, 17.0, 14.6, 10.3; IR (neat) 3400, 1712, 1629 cm-1
;
HRMS (MALDI) calcd for C30H42O10Na (MNa+) 585.2676,
found 585.2688.
P h or bol 12-Oxa la te-13-a ceta te (9). Clear oil; [R]D +12.2°
(c 0.5, CH2Cl2); 1H NMR (400 MHz, CDCl3) δ 7.58 (s, 1H), 5.67
(d, J ) 4.9 Hz, 1H), 5.54 (br s, 1H), 5.48 (d, J ) 10.2 Hz, 1H),
4.01 (AB, J ) 13.1 Hz, 2H), 3.29-3.22 (m, 2H), 2.51 (AB, J )
19.5 Hz, 2H), 2.27 (dq, J ) 10.4, 6.4 Hz, 1H), 2.12 (s, 3H),
2.10 (s, 3H), 1.77 (d, J ) 1.6 Hz, 3H), 1.20 (s, 3H), 1.14 (d, J
) 5.2 Hz, 1H), 0.93 (d, J ) 6.3 Hz, 3H) ppm; 13C NMR (100
MHz, CDCl3) δ 208.9, 173.6, 160.4, 140.6, 133.0, 128.5, 81.2,
78.2, 73.6, 67.9, 65.0, 56.1, 42.9, 39.0, 38.5, 36.8, 26.4, 24.0,
21.2, 16.6, 14.6, 10.3 ppm; IR (neat) 3402, 1767, 1706, 1628
cm-1; LRMS (ESI) 429 (MNa+ - C2O3).
(MALDI) calcd for
671.2999.
C
41H44O7Na (MNa+) 671.2985, found
P h or bol 12-Su ccin a te-13-a ceta te (10). Clear oil; [R]D
1
+1.9° (c 0.13, CH2Cl2); H NMR (400 MHz, CDCl3) δ 7.59 (s,
P h or bol 12-(m eth yl su ber a te)-13-a ceta te (5). White
film, mp 28.5 °C dec; [R]D +14.7° (c 0.2, MeOH); 1H NMR (300
MHz, CDCl3) δ 7.59 (s, 1H), 5.68 (d, J ) 5.7 Hz, 1H), 5.54 (br
s, 1H), 5.40 (d, J ) 10.4 Hz, 1H), 4.01 (AB, J ) 12.0 Hz, 2H),
3.66 (s, 3H), 3.24-3.23 (m, 2H), 2.51 (AB, J ) 7.7 Hz, 2H),
2.31 (apparent quartet, J ) 6.9 Hz, 4H), 2.14 (dq, J ) 10.4,
6.0 Hz, 1H), 2.09 (s, 3H), 1.78-1.77 (m, 3H), 1.66-1.58 (m,
1H), 5.66 (d, J ) 5.1 Hz, 1H), 5.59 (br s, 1H), 5.41 (d, J ) 10.3
Hz, 1H), 4.01 (AB, J ) 13.0 Hz, 2H), 3.24 (br s, 2H), 2.73-
2.62 (m, 4H), 2.55 (d, J ) 19.0 Hz, 1H), 2.48 (d, J ) 19.0 Hz,
1H), 2.15 (dq, J ) 10.3, 6.6 Hz, 1H), 2.08 (s, 3H), 1.76 (d, J )
1.8 Hz, 3H), 1.23 (s, 3H), 1.21 (s, 3H), 1.08 (d, J ) 5.1 Hz,
1H), 0.88 (d, J ) 6.6 Hz, 3H) ppm; 13C NMR (100 MHz, CDCl3)
δ 208.9, 175.8, 173.6, 171.7, 160.7, 140.4, 132.7, 129.0, 78.3,
5034 J . Org. Chem., Vol. 68, No. 13, 2003

Protein Kinase C Translocation
77.7, 73.6, 68.0, 65.5, 56.1, 43.0, 39.0, 38.5, 36.3, 29.2, 28.8,
25.9, 23.9, 21.2, 16.9, 14.5, 10.3 ppm; IR (neat) 3406, 1714,
1630 cm-1; LRMS (ES) 505 (MH-, 100%); HRMS (MALDI)
calcd for C26H34O10Na (MNa+) 529.2050, found 529.2026.
1708, 1626 cm-1; LRMS (ES-neg ion) 553 (M-H, 100); HRMS
(MALDI) calcd for
577.2017.
C
30H34O10Na (MNa+) 577.2044, found
P h or bol 12,13-Diter ep h th a la te (16). Viscous yellow oil;
[R]D +3.6° (c 0.3, MeOH); 1H NMR (400 MHz, CD3OD) δ 8.14-
8.08 (m, 8H), 7.62 (s, 1H), 5.86 (d, J ) 10.3 Hz, 1H), 5.71 (d,
J ) 4.4 Hz, 1H), 3.97 (AB, J ) 13.0 Hz, 2H), 3.46 (t, J ) 5.1
Hz, 1H), 3.25 (t, J ) 2.7 Hz, 1H), 2.59 (d, J ) 19.0 Hz, 1H),
2.53 (d, J ) 19.0 Hz, 1H), 2.48 (dq, J ) 10.3, 6.3 Hz, 1H), 1.75
(d, J ) 1.5 Hz, 3H), 1.48 (s, 3H), 1.40 (d, J ) 5.4 Hz, 1H), 1.31
(s, 3H), 0.98 (d, J ) 6.3 Hz, 3H) ppm; 13C NMR (100 MHz,
CD3OD) δ 210.0, 177.8, 169.0, 168.6, 167.0, 160.2, 142.8, 134.6,
134.5, 134.2, 130.70, 130.66, 130.5, 129.0, 79.9, 79.8, 74.7, 68.1,
68.0, 57.5, 44.8, 40.2, 38.5, 37.6, 28.4, 24.3, 17.8, 15.1, 10.3
ppm; IR (neat) 3405, 1785, 1706 cm-1; HRMS calcd for
P h or bol 12-Ad ip a te-13-a ceta te (11). Clear, pale yellow
oil; [R]D +7.9° (c 0.1, MeOH); 1H NMR (400 MHz, CDCl3) δ
7.58 (br s, 1H), 5.68 (d, J ) 5.1 Hz, 1H), 5.54 (br s, 1H), 5.41
(d, J ) 10.6 Hz, 1H), 4.02 (AB, J ) 13.0 Hz, 2H), 3.26-3.23
(m, 2H), 2.54 (d, J ) 19.8 Hz, 1H), 2.47 (d, J ) 19.6 Hz, 1H),
2.41-2.35 (m, 4H), 2.18-2.11 [m, 2H containing 2.14 (dq, J )
10.6, 6.6 Hz, 1H)], 2.09 (s, 3H), 1.78-1.77 (m, 3H), 1.71-1.69
(br m, 4H), 1.24 (s, 3H), 1.20 (s, 3H), 1.09 (d, J ) 5.1 Hz, 1H),
0.89 (d, J ) 6.2 Hz, 3H) ppm; 13C NMR (100 MHz, CDCl3) δ
173.6, 172.9, 160.8, 140.3, 132.7, 128.9, 78.1, 76.5, 73.7, 68.0,
56.2, 42.9, 39.1, 38.6, 36.3, 34.3, 33.1, 25.8, 24.6, 24.1, 23.9,
21.2, 16.9, 14.6, 10.3; IR (neat) 3415, 1712 cm-1; HRMS
C
36H34O12Na [(M - 2H+)Na+] 681.1953, found 681.1926.
C
28H38O10Na (MNa+) 557.2363, found
P h or bol 12,13-Disu ccin a te (17). Clear, yellow oil; [R]D
(MALDI) calcd for
557.2344.
1
+22.0° (c 0.2, MeOH); H NMR (400 MHz, CD3OD) δ 7.56 (s,
1H), 5.62 (d, J ) 4.9 Hz, 1H), 5.44 (d, J ) 10.7 Hz, 1H), 3.94
(AB, J ) 13.2 Hz, 2H), 3.30 (t, J ) 5.4 Hz, 1H), 3.17-3.14 (m,
1H), 2.62-2.60 (m, 8H), 2.54 (d, J ) 19.5 Hz, 1H), 2.48 (d, J
) 19.0 Hz, 1H), 2.23 (dq, J ) 10.5, 6.6 Hz, 1H), 2.03 (s, 1H),
1.74 (dd, J ) 2.9, 1.0 Hz, 3H), 1.26 (s, 3H), 1.22 (s, 3H), 1.15
(d, J ) 5.4 Hz, 1H), 0.90 (d, J ) 6.8 Hz, 3H) ppm; 13C NMR
(100 MHz, CD3OD) δ 210.6, 177.0, 175.9, 174.5, 160.7, 142.9,
134.7, 129.4, 79.9, 78.8, 74.7, 68.0, 67.2, 57.3, 44.3, 39.9, 38.4,
36.9, 30.4, 30.3, 29.9, 29.6, 27.5, 23.9, 17.3, 14.7, 10.1 ppm; IR
P h or bol 12-Seba ca te-13-a ceta te (12). Clear oil; [R]D +3.1°
(c 0.1, CH2Cl2); 1H NMR (400 MHz, CDCl3) δ 7.60 (s, 1H), 5.68
(d, J ) 4.8 Hz, 1H), 5.59 (br s, 1H), 5.40 (d, J ) 10.3 Hz, 1H),
4.02 (AB, J ) 12.8 Hz, 2H), 3.30-3.20 (m, 2H), 2.57 (d, J )
19.0 Hz, 1H), 2.48 (d, J ) 19.0 Hz, 1H), 2.35-2.31 (m, 4H),
2.17 (s, 1H), 2.15 (dq, J ) 10.6, 6.4 Hz, 1H), 2.09 (s, 3H), 1.77
(dd, J ) 2.6, 1.1 Hz, 3H), 1.66-1.58 (m, 4H), 1.67-1.58 (m,
4H), 1.37-1.28 (m, 4H), 1.24 (s, 3H), 1.21 (s, 3H), 1.09 (d, J )
5.1 Hz, 1H), 0.89 (d, J ) 6.2 Hz, 3H) ppm; 13C NMR (100 MHz,
CDCl3) δ 173.6, 173.4, 161,0, 140.4, 132.8, 129.1, 78.3, 76.6,
73.7, 68.0, 65.7, 42.9, 39.1, 38.4, 36.3, 34.6, 33.9, 31.7, 29.13,
29.06, 29.01, 28.99, 25.8, 25.2, 24.7, 23.9, 21.2, 17.0, 14.6, 10.2
ppm; IR (neat) 3410, 1712, 1629 cm-1; HRMS (MALDI) calcd
for C32H46O10Na (MNa+) 613.2989, found 613.2997.
(neat) 3405, 1715 cm-1; HRMS (MALDI) calcd for C28H36O12
Na (MNa+) 587.2099, found 587.2107.
-
P h or bol 12-Aceta te-13-su ccin a te (18). Clear, viscous
1
liquid; [R]D +7.7° (c 0.3, CH2Cl2); H NMR (400 MHz, CDCl3)
δ 7.55 (s, 1H), 5.80 (br s, 1H), 5.64 (d, J ) 4.4 Hz, 1H), 5.33 (d,
J ) 10.3 Hz, 1H), 3.97 (AB, J ) 13.0 Hz, 2H), 3.26-3.22 (m,
2H), 2.84-2.75 (m, 1H), 2.70-2.60 (m, 1H), 2.59-2.48 (m, 4H),
2.19-2.14 [m, 2H containing 2.16 (s, 1H)], 2.09 (br s, 1H), 2.06
(s, 3H), 1.73 (d, J ) 1.5 Hz, 3H), 1.23 (s, 3H), 1.22 (s, 3H),
1.07 (d, J ) 4.9 Hz, 1H), 0.86 (d, J ) 6.3 Hz, 3H) ppm; 13C
NMR (100 MHz, CDCl3) δ 209.7, 176.0, 175.2, 170.9, 161.0,
140.7, 132.9, 128.4, 78.6, 76.9, 73.7, 67.3, 66.0, 55.9, 42.8, 38.8,
38.2, 36.1, 29.1, 28.6, 26.2, 23.8, 21.2, 17.0, 14.6, 10.3 ppm; IR
(neat) 3401, 1725, 1629 cm-1; HRMS (MALDI) calcd for
P h or bol 12-Tetr a d eca d ioa te-13-a ceta te (13). Yellow oil;
[R]D +5.4° (c 0.25, CH2Cl2); 1H NMR (400 MHz, CDCl3) δ 7.63-
7.60 (m, 1H), 5.67 (d, J ) 4.4 Hz, 1H), 5.61 (br s, 1H), 5.40 (d,
J ) 10.3 Hz, 1H), 4.01 (AB, J ) 12.8 Hz, 2H), 3.30-3.24 (m,
2H), 2.58 (d, J ) 19.0 Hz, 1H), 2.48 (d, J ) 19.0 Hz, 1H), 2.36-
2.31 (m, 4H), 2.172 (s, 1H), 2.167 (dq, J ) 10.3, 6.6 Hz, 1H),
2.09 (s, 3H), 1.77-1.76 (m, 3H), 1.67-1.57 (m, 4H), 1.35-1.22
(m, 19H), 1.21 (s, 3H), 1.08 (d, J ) 5.1 Hz, 1H), 0.89 (d, J )
6.6 Hz, 3H) ppm; 13C NMR (100 MHz, CDCl3) δ 210.0, 173.93,
173.85, 161.5, 140.6, 133.0, 129.3, 78.3, 76.6, 73.7, 68.0, 65.6,
56.1, 42.8, 38.9, 38.3, 36.1, 34.6, 29.4, 29.3, 29.2, 29.13, 29.08,
29.04, 28.9, 28.8, 25.6, 25.0, 24.5, 23.7, 21.0, 16.7, 14.3, 10.0
ppm; IR (neat) 3400, 1713, 1628 cm-1; HRMS (MALDI) calcd
for C36H54O10Na (MNa+) 669.3609, found 669.3594.
C
26H34O10Na (MNa+) 529.2044, found 529.2026.
P h or bol 12-Aceta te-13-ter ep h th la te (19). White powder,
mp 189-190 °C; [R]D +17.5° (c 0.1, CH2Cl2); 1H NMR (400
MHz, acetone-d6) δ 8.18-8.08 (m, 4H), 7.56 (s, 1H), 5.68 (d, J
) 4.4 Hz, 1H), 5.57 (d, J ) 10.3 Hz, 1H), 5.33 (br s, 1H), 3.96
(AB, J ) 13.0 Hz, 2H), 3.36 (t, J ) 5.0 Hz, 1H), 3.22 (t, J )
2.2 Hz, 1H), 2.57 (d, J ) 19.4 Hz, 1H), 2.51 (d, J ) 19.4 Hz,
1H), 2.32 (dq, J ) 10.3, 6.6 Hz, 1H), 2.14 (s, 1H), 2.08 (s, 3H),
1.71 (d, J ) 1.5 Hz, 3H), 1.40 (d, J ) 5.1 Hz, 1H), 1.37 (s, 3H),
1.35 (s, 3H), 1.19 (s, 1H), 0.93 (d, J ) 6.6 Hz, 3H) ppm; 13C
NMR (100 MHz, acetone-d6) δ 208.8, 171.8, 169.0, 160.0, 143.4,
134.1, 131.2 (br), 129.0, 79.5, 78.4, 75.2, 68.5, 68.3, 57.8, 44.9,
40.7, 39.2, 37.7, 28.1, 25.0, 21.8, 18.2, 15.7, 11.2 ppm; IR (neat)
3412, 1729, 1703, 1628 cm-1; HRMS (MALDI) calcd for
P h or bol 12-[(Z)-4-Octen ed ioa te]-13-a ceta te (14). Yellow
1
oil; [R]D +10.2° (c 0.5, MeOH); H NMR (400 MHz, CDCl3) δ
7.59 (s, 1H), 5.67 (d, J ) 5.1 Hz, 1H), 5.45-5.38 (m, 3H), 4.00
(AB, J ) 13.0 Hz, 2H), 3.31-3.22 (m, 2H), 2.56 (d, J ) 19.0
Hz, 1H), 2.48 (d, J ) 19.0 Hz, 1H), 2.38 (br s, 8H), 2.16 (dq, J
) 10.3, 6.6 Hz, 1H), 2.09 (s, 3H), 1.75 (d, J ) 1.8 Hz, 3H), 1.24
(s, 3H), 1.20 (s, 3H), 1.08 (d, J ) 5.1 Hz, 1H), 0.87 (d, J ) 6.2
Hz, 3H) ppm; 13C NMR (100 MHz, CDCl3) δ 209.3, 177.6, 173.6,
172.8, 161.0, 140.4, 132.8, 129.0, 128.9, 128.8, 78.4, 76.9, 73.7,
68.0, 65.6, 56.0, 42.9, 38.9, 38.3, 36.3, 34.5, 34.0, 25.9, 23.9,
23.0, 22.7, 21.2, 16.9, 14.6, 10.3 ppm; IR (thin film) 3407, 1713,
1628 cm-1; HRMS (MALDI-FTMS) calcd for C30H40O10Na
(MNa+) 583.2514, found 583.2538.
C
30H34O10Na (MNa+) 577.2044, found 577.2070.
P h or bol 12-Myr ista te-13-su ccin a te (20). Colorless oil;
[R]D +6.2° (c 0.1, CH2Cl2); 1H NMR (400 MHz, CDCl3) δ 7.63-
7.60 (m, 1H), 5.67 (d, J ) 4.4 Hz, 1H), 5.61 (br s, 1H), 5.40 (d,
J ) 10.3 Hz, 1H), 4.01 (AB, J ) 12.8 Hz, 2H), 3.30-3.24 (m,
2H), 2.58 (d, J ) 19.0 Hz, 1H), 2.48 (d, J ) 19.0 Hz, 1H), 2.36-
2.31 (m, 4H), 2.172 (s, 1H), 2.167 (dq, J ) 10.3, 6.6 Hz, 1H),
2.09 (s, 3H), 1.77-1.76 (m, 3H), 1.67-1.57 (m, 4H), 1.35-1.22
(m, 19H), 1.21 (s, 3H), 1.08 (d, J ) 5.1 Hz, 1H), 0.89 (d, J )
6.6 Hz, 3H) ppm; 13C NMR (100 MHz, CDCl3) δ 210.0, 173.93,
173.85, 161.5, 140.6, 133.0, 129.3, 78.3, 76.6, 73.7, 68.0, 65.6,
56.1, 42.8, 38.9, 38.3, 36.1, 34.6, 29.4, 29.3, 29.2, 29.13, 29.08,
29.04, 28.9, 28.8, 25.6, 25.0, 24.5, 23.7, 21.0, 16.7, 14.3, 10.0
ppm; IR (neat) 3400, 1713, 1628 cm-1; HRMS (MALDI) calcd
for C36H54O10Na (MNa+) 669.3609, found 669.3594.
P h or bol 12-Ter ep h th a la te-13-a ceta te (15). Clear liquid;
[R]D +18.1° (c 0.5, CH2Cl2); 1H NMR (400 MHz, CDCl3) δ 8.17
(d, J ) 8.3 Hz, 2H), 8.11 (d, J ) 8.3 Hz, 2H), 7.65 (s, 1H), 5.71
(d, J ) 5.4 Hz, 1H), 5.69 (d, J ) 10.3 Hz, 1H), 4.04 (AB, J )
18.8 Hz, 2H), 3.35 (t, J ) 4.9 Hz, 1H), 3.31 (t, J ) 2.4 Hz, 1H),
2.59 (d, J ) 18.8 Hz, 1H), 2.53 (d, J ) 18.8 Hz, 1H), 2.36 (dq,
J ) 10.3, 6.3 Hz, 1H), 2.13 (s, 3H), 2.11 (s, 1H), 1.78-1.76 (m,
3H), 1.38 (s, 3H), 1.20 (s, 3H), 1.16 (d, J ) 4.9 Hz, 1H), 0.94
(d, J ) 6.3 Hz, 3H) ppm; 13C NMR (100 MHz, CDCl3) δ 210.2,
174.0, 169.6, 165.7, 161.7, 140.7, 134.4, 133.1, 130.3, 129.8,
129.2, 78.40, 78.37, 73.8, 68.0, 65.6, 56.2, 43.1, 39.0, 38.3, 36.5,
26.0, 23.7, 21.0, 17.0, 14.4, 10.0 ppm; IR (neat) 3396, 1716,
P h or bol 12-Aceta te-13-tetr a d eca n ed ioa te (21). Yellow
1
oil; [R]D +2.4° (c 0.1, CH2Cl2); H NMR (300 MHz, CDCl3) δ
J . Org. Chem, Vol. 68, No. 13, 2003 5035

Bertolini et al.
7.59 (s, 1H), 5.82 (br s, 1H), 5.68 (d, J ) 5.0 Hz, 1H), 5.38 (d,
J ) 10.3 Hz, 1H), 4.01 (AB, J ) 12.3 Hz, 2H), 3.28 (t, J ) 2.6
Hz, 1H), 3.25 (t, J ) 5.3 Hz, 1H), 2.52 (br s, 2H), 2.34-2.26
(m, 4H), 2.15 (dq, J ) 10.3, 6.2 Hz, 1H), 2.07 (s, 3H), 1.77 (d,
sulfate, as described previously.53 This substrate is phospho-
rylated by PKC in
a
cofactor (eg. lipids, PMA, Ca2+)-
independent manner. Therefore, the ratio of activity obtained
in the supernatant and pellet will not be affected by phorbol
analogues. To further ensure this, equal amounts of vesicles/
analogues are added back to the supernatant to provide
identical conditions. The phosphorylation reaction was initi-
ated by the addition of 15 µL of 0.5 mM [γ-32P]ATP (75 µCi/
mL), 25 mM MgCl2, 20 mM HEPES (pH 7.5), and 0.5 mg mL-1
protamine sulfate to a 60 µL reaction volume of supernatant
or pellet fraction. Phosphorylation was carried out for 10 min
at 25 °C and quenched with 25 µL of 0.1 M ATP and 0.1 M
EDTA (pH 8.0). Samples (85 µL) were spotted on P-81 ion-
exchange paper and subsequently washed with 0.4% (v/v)
phosphoric acid for 20 min. Radioactivity associated with
papers was determined by liquid scintillation counting in
Biosafe II. The vesicle-associated kinase activity, Av, was
calculated according to eq 1, where Ab and At are the measure
activities of the bottom and top fractions, respectively:
J
) 1.5 Hz, 3H), 1.67-1.58 (m, 4H), 1.42-1.20 [m, 22H,
containing 1.24 (s, 3H), 1.22 (s, 3H)], 1.05 (d, J ) 5.3 Hz, 1H),
0.89 (d, J ) 6.4 Hz, 3H) ppm; 13C NMR (100 MHz, CDCl3) δ
209.5, 176.6, 171.1, 161.1, 140.6, 133.0, 129.3, 78.4, 77.1, 73.7,
67.9, 65.3, 56.0, 42.9, 38.9, 38.4, 36.3, 34.2, 29.6, 29.2, 29.1,
29.0, 28.9, 25.8, 24.7, 24.4, 23.8, 20.9, 16.7, 14.3, 10.0 ppm; IR
(thin film) 3401, 1713 cm-1; HRMS (MALDI-FTMS) calcd for
C
36H54O10Na (MNa+) 669.3609, found 669.3621.
P r otein Kin a se C âII. Protein kinase C âII from the
baculovirus expression system was purified as described
previously49 and stored at -20 °C in 10 mM Tris buffer, pH
7.5 (4 °C), 0.5 mM EDTA, 0.5 mM EGTA, 0.5 mM dithiothrei-
tol, and 50% (v/v) glycerol.
Su cr ose-Loa d ed Vesicles. A lipid mixture composed of 30
mol % of PS and 70 mol % of POPC in chloroform/methanol
was dried under a stream of dry nitrogen and subsequently
concentrated in vacuo (0.1 mmHg). Lipids were then resus-
pended in a sucrose buffer (pH 7.5) containing 0.170 M sucrose,
5 mM MgCl2, and 20 mM HEPES. Aliquots (0.5 mL) of 5 mM
lipid were subjected to 5 freeze-pump-thaw cycles in liquid
nitrogen, followed by 21 rounds of extrusion through two
stacked 100-nm polycarbonate filters in a microextruder to
form large unilamellar vesicles. Both extrusion of vesicles and
subsequent incorporation of phorbol esters into them (see
below) was accomplished at 20 °C. The vesicles were suspended
in 100 mM KCl, 20 mM HEPES (pH 7.5) and centrifuged at
100 000 × g for 30 min. The pelleted sucrose-loaded vesicles
were resuspended in buffer to achieve a concentration of 1.0
mM lipid (all phospholipid concentrations in stock solutions
were determined by phosphate analysis). At later stages of
experiments, the lipid concentration was calculated from the
amount of [3H]DPPC that was routinely included in trace
quantities in lipid mixtures.50
Av)[âAb + (â - 1)At]/[R + â - 1]
(1)
The fraction of sedimented vesicles, R, was calculated from
the distribution of [3H]-labeled PC, which was included in trace
amounts in all lipid mixtures. The fraction of kinase activity
found in the supernatant in the absence of lipid, â, was equals
within the limits of experimental errorsto the value expected
for a nonsedimenting protein (i.e. 0.73 under the experimental
conditions used). All experiments were performed in a stan-
dard solution composed of 100 mM KCl, 5 mM MgCl2, 20 mM
HEPES (pH 7.5), 200 µM Ca2+, and 0.3 mg mL-1 of BSA.
Concentrations of additions that varied depending on experi-
ment are given with appropriate results. The fraction of
membrane-bound enzyme was calculated as [Av/(Ab + At)]. The
distribution of PKC between the supernatant and pellet
measured by the sucrose-loaded vesicle assay is the same as
that obtained with use of a Western Blot or silver stain to
physically determine the distribution of the protein.
In cor p or a tion of P h or bol Ester s in to Vesicles. Stock
solutions of all phorbol esters were prepared as DMSO
solutions degassed by 3 freeze-pump-thaw cycles under
vacuum (0.1 mmHg) and stored at -22 °C in the dark.33
Appropriate aliquots of concentrated PMA or the synthetic
phorbol ester in dimethyl sulfoxide were injected into vigor-
ously vortexed suspensions of large unilamellar vesicles.
Subsequently, vesicles were incubated for 30 min at 20 °C with
occasional gentle vortexing. The partitioning of PMA into
vesicles was complete (>98%) as assessed by centrifugation
of sucrose-loaded vesicles and analysis of bound [20-3H]PMA.
Appropriate aliquots of suspensions of vesicles containing the
phorbol esters were diluted several-fold in binding assay
samples. The final content of DMSO did not exceed 0.5% (v/v)
and did not change the association of the enzyme with vesicles
to any appreciable degree.
P r otein Kin a se C-âII Recr u itm en t Assa y. The sucrose-
loaded vesicle assay was based on the procedure of Rebecchi
et al.51 adapted for protein kinase C as previously described,52
except final volumes were 525 µL. Membrane-bound enzyme
was separated by centrifugation at 100 000 × g for 30 min at
25 °C to ensure g95% sedimentation of large unilamellar
vesicles. Both the supernatant and the pellet were assayed
under identical conditions for kinase activity toward protamine
The PKC-catalyzed phosphorylation of the peptide ac-
FKKSFKL-amide was determined in the presence of PMA or
analogue. The assay consisted of 100 mM KCl, 5 mM MgCl2,
20 mM HEPES (pH 7.5), 200 µM Ca2+, 0.3 mg/mL of BSA, 0.2
mg/mL of peptide, and 0.1 mM ATP. The reaction was carried
out for 6 min at 25 °C. Samples were treated as above for all
remaining steps. Activity is reported as a percentage of that
obtained with 1 µM PMA.
Ack n ow led gm en t. We are extremely grateful to Dr.
Bernd Sorg of the Deutsches Krebsforschung Zentrum
(Heidelberg, Germany) for his donation of authentic
samples of phorbol 13-acetate-20-trityl and for invalu-
able advice on the isolation of phorbol esters from croton
oil. J .G. is funded in part by the Canadian Institute of
Health Research and T.M.B. is a Lucille Markey scholar.
Su p p or tin g In for m a tion Ava ila ble: Complete experi-
1
mental procedures and H and 13C NMR spectra for compounds
3-21 and their precursors. This material is available free of
charge via the Internet at http://pubs.acs.org.
J O030029W
5036 J . Org. Chem., Vol. 68, No. 13, 2003