Antitumor antibiotic fostriecin covalently binds to cysteine-269 residue of protein phosphatase 2A catalytic subunit in mammalian cells

Article abstract of DOI:10.1016/j.bmc.2009.09.050

Fostriecin is a phosphate monoester with excellent antitumor activity against mouse leukemia, and it is a potent inhibitor of protein phosphatase (PP) 2A. This compound has been predicted to covalently bind to the Cys269 residue of the PP2A catalytic subu

Full text of DOI:10.1016/j.bmc.2009.09.050

Bioorganic & Medicinal Chemistry 17 (2009) 8113–8122
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Bioorganic & Medicinal Chemistry
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Antitumor antibiotic fostriecin covalently binds to cysteine-269 residue
of protein phosphatase 2A catalytic subunit in mammalian cells
Toshifumi Takeuchi a, Noriyuki Takahashi a, Kazutomo Ishi b, Tomoe Kusayanagi a, Kouji Kuramochi a,
Fumio Sugawara a,b,
*
a Department of Applied Biological Science, Tokyo University of Science (RIKADAI), 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan
b Genome and Drug Research Center, Tokyo University of Science (RIKADAI), 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Fostriecin is a phosphate monoester with excellent antitumor activity against mouse leukemia, and it is a
potent inhibitor of protein phosphatase (PP) 2A. This compound has been predicted to covalently bind to
the Cys269 residue of the PP2A catalytic subunit (PP2Ac) at the a,b-unsaturated lactone via a conjugate
addition reaction. However, this binding has not yet been experimentally proven. To confirm such bind-
ing, we synthesized biotin-labeled fostriecin (bio-Fos), which has an inhibitory activity against the pro-
liferation of mouse leukemia cells. We showed that fostriecin directly binds to PP2Ac in HeLa S3 cells by
pull-down assays using bio-Fos. Moreover, we directly demonstrated that fostriecin covalently binds to
the Cys269 residue of PP2Ac by matrix assisted laser desorption/ionization time-of-flight mass spectrom-
etry analysis. From these results, the inhibitory mechanism of fostriecin on PP2A activity is discussed.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 3 September 2009
Revised 25 September 2009
Accepted 26 September 2009
Available online 3 October 2009
Keywords:
Fostriecin
Protein phosphatase 2A catalytic subunit
Covalent bond
a,b-Unsaturated lactone
1. Introduction
lactone via conjugate addition: (1) The a,b-unsaturated d-lactone
of fostriecin has been observed for a potential Michael accep-
tor.20–25 (2) The binding site of the Cys269 residue is unique to
PP2A and PP4 and absent in PP1, PP2B, PP5 and PP7, and fostriecin
selectively inhibits PP2A as against PP1 (>104–105),15,27 PP2B
(>105)15 and PP5 (>104–105).27 (3) The b12–b13 loop (256 VVTIF
SAPNYCYRCG NQAAIMEL 278) of PP2Ac including the Cys269 res-
idue (underlined) is essential for the inhibition of PP2Ac activity by
fostriecin.28 (4) PP2A C269S and C269F mutants in yeast are much
less sensitive against fostriecin (>10).28 (5) 2,3-Dihydrofostriecin
has 200-fold less inhibitory activity against PP2A than fostriecin.27
(6) Phoslactomycin, a member of the naturally occurring fostriecin
family, has been reported to covalently bind to the Cys269 residue
of PP2Ac, resulting in the selective inhibition of PP2A.24 (7) Chime-
ras of PP1 and PP5, in which key residues predicted for inhibitor
contact with PP2A (267 YRCG 270) were introduced into PP1 and
PP5 using site-directed mutagenesis, are much more sensitive
against fostriecin (>600 and ꢀ200, respectively).29 However, the
covalent binding between fostriecin and PP2Ac has not yet been
experimentally demonstrated.24,26,29
Fostriecin (Fig. 1) is a phosphate monoester isolated in 1983 by
Tunac and co-workers from a fermentation broth of Streptomyces
pulveraceus subsp. fostreus ATCC 31906.1–5 It exhibits antitumor
activity against a wide spectrum of tumor cells in vitro and has
excellent inhibitory activities against mouse leukemia cell lines
L1210 and P388 in vivo.6–10 Initially, its antitumor effect was sug-
gested to be due to the inhibition of topoisomerase II.11 However,
fostriecin has recently been shown to interrupt a cellular mitotic
entry checkpoint via a more potent and selective inhibition of
the enzymatic activity of protein phosphatase (PP) 2A versus PP1
(inhibitory activity: PP1, IC50 = 45 lM; PP2A, IC50 = 1.5 nM; selec-
tivity: PP2A/PP1 >104–105).12–17 Thus, fostriecin is widely used as
a selective PP2A inhibitor in studies of the phosphorylation signal-
ing pathway.18,19
It has been reported that some biologically active compounds
containing a,b-unsaturated carbonyl moiety can react with biolog-
ical nucleophiles such as the sulfhydryl group of the cysteine
residue.20–25 Recently, we reported that monoacetylcurcumin,
which selectively inhibits DNA polymerase k, covalently binds to
the cysteine residue of the BRCT domain in DNA polymerase k.25
The following previous reports have predicted that fostriecin cova-
In this study, we investigated the covalent binding of fostriecin
to PP2Ac in HeLa S3 cells by pull-down assays using synthetically
prepared biotin-labeled fostriecin (bio-Fos). We were able to
directly demonstrate that fostriecin covalently binds to the
Cys269 residue of PP2Ac by matrix assisted laser desorption/ion-
ization time-of-flight mass spectrometry (MALDI-TOF MS)
analysis.
lently binds to the Cys269 residue of PP2Ac at the a,b-unsaturated
* Corresponding author. Tel.: +81 4 7124 1501x3400; fax: +81 4 7123 9757.
E-mail address: sugawara@rs.noda.tus.ac.jp (F. Sugawara).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.09.050

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T. Takeuchi et al. / Bioorg. Med. Chem. 17 (2009) 8113–8122
Figure 1. Structure of fostriecin (1) and biotin-labeled fostriecin (bio-Fos, 2).
stannane derivative 13,41,42 yielded triene 15 in 79% yield over
2. Results
three steps. After phosphorylation43,44 of alcohol 15, deprotection
of the fluorenylmethyl and silyl group provided bio-Fos 2 as a tri-
ethylammonium salt in 36% yield over three steps.
2.1. Synthesis of biotin-labeled fostriecin
To identify the direct binding proteins of fostriecin by affinity
matrix-based protein purification,30–33 we chemically synthesized
bio-Fos (Fig. 1). Synthesis started from the introduction of imidate
6 to alcohol (12Z)-7 (Scheme 1), which was reported in our previ-
ous work.34 Imidate 6 was synthesized by the tosylation of com-
mercially available alcohol 3, followed by the alkylation of p-
hydroxybenzyl alcohol with tosylate ester 4 to provide benzylic
alcohol 5 in 56% yield over two steps, which was subsequently con-
verted into imidate 6. Treatment of alcohol (12Z)-7 with imidate 6
in the presence of triphenylmethyl (Tr) BF4 furnished benzylic
ether 8, followed by the selective removal of the C8 triethylsilyl
(TES) group to give alkyne 9 in 76% yield over two steps. For assem-
bly of the probe molecule, biotin derivative 12 was prepared by the
mono reduction of commercially available ether 10 and the subse-
2.2. Effects of biotin-labeled fostriecin on cell viability
The cytotoxicity of bio-Fos was evaluated on mouse leukemia
cells. The 50% inhibitory concentration (IC50) of bio-Fos was
58
Fos had weaker cytotoxic activity than fostriecin (IC50 = 0.46
l
M (Fig. 2), as determined by WST-8 assay.45,46 Although bio-
l
M),10
it still retained cytotoxic activity toward the L1210 mouse leukemia
cell line. Thus, we decided that 2 could be used for affinity matrix-
based protein purification.
2.3. Fostriecin covalently binds to PP2Ac in HeLa S3 cells
We carried out pull-down assays using bio-Fos to confirm
whether fostriecin covalently binds to PP2Ac.24 HeLa S3 cells were
quent condensation of the resulting amine with D-biotin. Huisgen
cycloaddition35–39 of alkyne 9 with azide 12, followed by a Stille
coupling reaction40 of the resulting vinyl iodide 14 with the known
incubated with 20
lM bio-Fos or biotin for 2 h. After washing with
phosphate buffered saline (PBS), the cells were lysed. Both proteins
Scheme 1. Synthesis of bio-Fos (2). Reagents and conditions: (a) TsCl, DMAP/CH2Cl2 88%; (b) p-hydroxybenzyl alcohol, K2CO3/MeCN, 64%; (c) Cl3CCN, NaH/CH2Cl2; (d)
separation, 78%; (e) TrBF4/Et2O; (f) HCl aq, THF, MeCN, 69% in two steps; (g) PPh3/EtOAc, HCl aq, 79%; (h) i-Pr2NEt, HBTU/DMF, 89%; (i) CuI, Et3N/THF, 98%; (j) 13,
Pd(MeCN)2Cl2/DMF, 81%; (k) i-Pr2NP(OFm)2, tetrazole/CH2Cl2 then TBHP; (l) Et3N/MeCN; (m) Et3Nꢁ3HF, Et3N/MeCN, 36%. Ts = p-toluenesulfonyl, DMAP = N,N-dimethy-4-
laminopyridine, Tr = triphenylmethyl, THF = tetrahydrofuran, HBTU = O-benzotriazole-N,N,N’,N’-tetramethyl-uronium-hexafluoro-phosphate, DMF = N,N-dimethylformam-
ide, TBHP = tert-butyl hydroperoxide.

T. Takeuchi et al. / Bioorg. Med. Chem. 17 (2009) 8113–8122
8115
2.4. Fostriecin covalently binds to Cys269 residue of PP2Ac
in vitro
To identify the binding site of fostriecin on PP2Ac by covalent
addition, native and fostriecin-treated PP2Ac was digested with
trypsin and analyzed by MALDI-TOF MS analysis.21,23,25 The human
PP2A, consisting of a 36 kDa catalytic subunit (PP2Ac), a 55 kDa
regulatory subunit (PP2Ab), and a 65 kDa structural subunit
(PP2Aa), was treated with or without fostriecin (0.6 lM) for 2 h.
The resulting PP2A was electrophoresed on 10% acrylamide gel, fol-
lowed by band visualization with silver staining. Each band corre-
sponding to PP2Ac was reduced with the addition of dithiothreitol
and alkylated with acrylamide (71.0 Da), followed by in-gel diges-
tion with trypsin and by MALDI-TOF MS analysis (Fig. 4A). The
analysis of fostriecin-treated PP2Ac showed the presence of the
fragment P-20 with a mass of 2069.9 Da (Fig. 4A (lower panel)),
whereas this fragment was not observed in native PP2Ac (Fig. 4A
(upper panel)).
Figure 2. Dose-dependant effects of bio-Fos on L1210 cell viability as determined
by WST-8 assay. Dose–response curve of growth inhibition induced by fostriecin-
biotin (h) in L1210 cells. Data are presented as means SEM of three independent
experiments.
binding to bio-Fos or biotin were precipitated with streptavidin
beads and analyzed by sodium dodecyl sulfate polyacrylamide
gel electrophoresis (Fig. 3A). Silver staining revealed the presence
of several proteins in the sample derived from bio-Fos-treated cells
(Fig. 3Aa, arrows). Detection of biotinylated proteins using alkaline
phosphatase-conjugated avidin (AP-avidin) demonstrated a biotin-
ylated 36 kDa protein in the sample from bio-Fos-treated cells
(Fig. 3Ab, arrow). This protein was identified as PP2Ac by Western
blot analysis using anti-PP2Ac antibody (Fig. 3Ac). This result indi-
cates that PP2Ac was covalently biotinylated by bio-Fos addition,
implying that bio-Fos covalently binds to PP2Ac in living cells.
To determine the binding specificity between bio-Fos and
PP2Ac (Fig. 3B), we performed competition assay with fostriecin.
After pretreatment of HeLa S3 cells with free fostriecin at several
The peptides derived from native PP2Ac after trypsin digestion
were assigned from the theoretical molecular masses of the frag-
ments in PP2Aca, one of the isomers of PP2Ac (Fig. 4B). Approxi-
mately 80% of the digested peptides, including nine cysteine-
containing fragments, were identified (Table 1). Because the cys-
teine residues were alkylated with acrylamide (71.0 Da), the
modification of one cysteine residue results in an additional mass
of 71.0 Da. The calculated original (non-alkylated) masses of the
nine cysteine (underlined)-containing fragments are as follows:
1478.8 (P-8, Q22LSESQVKSLCEK34), 1621.8 (P-10, C269GNQAAI-
MELDDTLK283), 1646.8 (P-11, N255VVTIFSAPNYCYR268), 1647.8
(P-12, E9LDQWIEQLNECEK21), 1734.8 (P-14, Q122ITQVYGFYDECLR135),
1858.8 (P-15, A240HQLVMEGYNWCHDR254), 1862.9 (P-16, Q122ITQ-
VYGFYDECLRK136), 2452.1 (P-17, L186QEVPHEGPMCDLLWSDPD-
DR206) and 2403.1 (P-18, C50PVTVCGDVHGQFHDLMELEFR70) Da.
By comparing the original (non-alkylated) mass of P-10 with that
of P-20, the difference of 448.1 Da between the mass of P-20
(2069.9 Da) and that of P-10 (1621.8 Da) possibly represented
the molecular weight of hydrolyzed fostriecin (exact
mass = 18.0 + 430.1 = 448.1). In our previous work, the lactone
opening of PD 116,251, which is one of the members of the fostrie-
cin family, occurred in H2O at room temperature within 2 h (data
concentrations (0, 0.01, 0.1, and 1
lM) for 1.5 h, the cells were
incubated with 20 M bio-Fos or biotin for 1 h. Biotin-containing
l
proteins were isolated with streptavidin beads and analyzed by
Western blot analysis using AP-avidin and anti-PP2Ac antibody.
The bands obtained from PP2Ac were reduced by the addition of
fostriecin, indicating that the binding between bio-Fos and PP2Ac
was competitively blocked by fostriecin in a dose-dependent man-
ner. These observations strongly suggest that fostriecin competes
with bio-Fos in the same binding site of PP2Ac.
Figure 3. Fostriecin covalently binds to PP2Ac in HeLa S3 cells. (A) HeLa S3 cells were incubated with 20 lM bio-Fos or biotin for 2 h. After washing with phosphate buffered
saline, the cells were lysed and bio-Fos- or biotin-binding proteins were recovered with streptavidin beads. After thorough washing of the beads, bound proteins were
analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by silver staining (a). Biotinylated proteins with bio-Fos or biotin were visualized by
Western blot analysis using alkaline phosphatase-conjugated avidin (AP-avidin) (b). Proteins binding to bio-Fos or biotin were visualized by Western blot analysis using anti-
PP2Ac antibody (c). (B) HeLa S3 cells were incubated in cell medium containing bio-Fos or biotin in the absence or presence of several concentrations of free fostriecin. After
washing with phosphate buffered saline, the cells were lysed, and proteins bound to bio-Fos or biotin were recovered with streptavidin beads. Then, the beads were washed
and bound proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by visualization by Western blot analysis using AP-avidin or anti-
PP2Ac antibody.

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T. Takeuchi et al. / Bioorg. Med. Chem. 17 (2009) 8113–8122
Figure 4. Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis of native or fostriecin-treated PP2Ac digested with trypsin.
(A) MALDI-TOF MS analysis of trypsin-digested PP2Ac. PP2A was incubated in the absence (upper panel) or presence (lower panel) of fostriecin (0.6 M) at 30 °C for 2 h. After
l
electrophoresis on 10% acrylamide gel, each band corresponding to PP2Ac was reduced with dithiothreitol and alkylated with acrylamide, followed by digestion with trypsin
and analysis by MALDI-TOF MS. In the upper panel (fostriecin-untreated), the fragment with a mass of 1692.8 Da corresponds to Cys269-Lys283 conjugated with acrylamide.
In the lower panel (fostriecin-treated), the fragment with a mass of 2069.9 Da was newly observed and this fragment corresponds to Cys269-Lys283 conjugated with the
lactone-hydrolyzed fostriecin. (B) Tryptic digest fragments of native PP2Ac. The fragments were assigned by comparing the observed masses with those of the theoretical
fragments. (C) Possible structure of fostriecin-modified Cys269-Lys283 fragment.
not shown). Hydrolysis of the lactone of phoslactomycin B, one of
the members of the fostriecin family, under basic conditions has
been reported by Reynolds and co-workers.47 Taken together, these
results suggest that the lactone opening of fostriecin occurs under
the digesting process (Fig. 4C). MALDI post source decay MS/MS
(MALDI-PSD-MS/MS) analysis of the fragments at m/z = 2069.9

T. Takeuchi et al. / Bioorg. Med. Chem. 17 (2009) 8113–8122
8117
Table 1
Peptides identified by MALDI-TOF MS from PP2Ac
Position
Peptide sequence
Calculated mass
MS
Peak
1–8
9–21
22–34
30–36
MDEKVFTK
997.5
997.8
P-5
P-12
P-8
ELDQWIEQLNECEK
QLSESQVKSLCEK
SLCEKAK
1718.8
1549.8
849.4
1718.8a
1549.8a
849.5
P-2
37–49
50–70
75–89
EILTKESNVQEVR
1544.8
1544.8
2545.2a
1792.8
1655.8
900.5
P-7
CPVTVCGDVHGQFHDLMELEFR
SPDTNYLFMGDYVDR
GYYSVETVTLLVALK
ERITILR
QITQVYGFYDECLR
QITQVYGFYDECLRK
YGNANVWK
LQEVPHEGPMCDLLWSDPDDR
GGWGISPR
GAGYTFGQDISETFNHANGLTLVSR
AHQLVMEGYNWCHDR
NVVTIFSAPNYCYR
CGNQAAIMELDDTLK
(fostriecin-modified)CGNQAAIMELDDTLK
YSFLQFDPAPR
RGEPHVTR
RTPDYFL
2545.2
1792.8
1644.9
900.6
1805.9
1933.9
951.5
P-18
P-13
P-9
90–104
109–115
122–135
122–136
137–144
186–206
207–214
215–239
240–254
255–268
269–283
269–283
284–294
295–302
303–309
P-3
1805.9a
1933.9a
951.3
P-14
P-16
P-4
P-17
P-1
P-19
P-15
P-11
P-10
P-20
P-6
2523.1
829.4
2523.1a
830.3
2655.3
1929.8
1717.8
1692.8
2069.9
1340.6
951.3
2656.3
1929.8a
1717.9a
1692.8a
2069.9
1340.7
951.5
P-4
P-3
911.5
911.3
a
Acrylamide modification of cysteine results in a peptide mass increase of 71 Da per cysteine residue.
was performed to confirm the binding site of fostriecin. However,
we obtained only the original fragment of peptides without fostrie-
cin, suggesting that fostriecin moiety might be decomposed or re-
moved by retro-Michael reaction under the MALDI-PSD-MS/MS
conditions.
PP2Ac via the conjugate addition reaction of its
a
,b-unsaturated
lactone. However, this conjugate addition has not yet been exper-
imentally confirmed.24,26,29
In this study, we chemically synthesized bio-Fos in order to
determine whether fostriecin covalently binds to PP2Ac. Because
several antitumor natural products containing 4-substituted
a,b-
2.5. Fostriecin-binding model
unsaturated lactone have been reported, such as phoslactomycin
A,49,50 kazusamycin A,51 discodermolide52,53 and pironetin A,54,55
the introduction of biotin moiety at the C-4 position of fostriecin
might not affect biological activity. The obtained bio-Fos was pro-
duced via a seven-step synthesis process with an overall yield of
22% from our previously synthesized compound (12Z)-7 (Scheme
1).34 We found that bio-Fos shows cytotoxicity against the L1210
After confirming the binding of fostriecin to the Cys269 residue
of PP2Ac, we simulated a binding model of PP2Ac with fostriecin
using the X-ray crystal structure of the PP2A core enzyme.48 Be-
cause there are two cases in which the sulfhydryl group of the
Cys269 residue in PP2Ac attacks from either si- or re-face at the
C-3 position of fostriecin to provide (3S)- or (3R)-PP2Ac-conjugated
fostriecin, respectively (Fig. 5A and B, respectively), we constructed
two binding models, namely, (3S)- and (3R)-models, respectively
(Fig. 5C and D, respectively). A 3D structure of fostriecin-conju-
gated PP2Ac was generated using energy minimization and molec-
ular dynamics. The initial structure of PP2Ac with fostriecin was
based on PP2Ac manually connected to fostriecin via si- or re-face
at the C-3 position of fostriecin. The combined structure was opti-
mized in vacuum using DISCOVERY STUDIO (DS) 2.0 program. The result-
ing (3S)- and (3R)-models are shown in Figure 5. In either case
(Fig. 5E and F), fostriecin located over the active site of PP2Ac. In
the (3S)-model (Fig. 5G), three amino acids in PP2Ac, namely,
Ile123, Tyr127 and Gly215, formed a hydrophobic cage, including
the hydrophobic triene moiety of fostriecin. On the other hand,
in the (3R)-model (Fig. 5H), this hydrophobic cage was not buried.
Multiple van der Waals interactions with the hydrophobic part of
fostriecin appeared in either model (Fig. 5E–H) constructed with
Tyr265, Arg268 and Leu243, and in the (3S)-model constructed
with Gln242 and His241. The C9-phosphate esters of fostriecin
interacted with the active site of manganese cations, and fostriecin
blocked the paths toward these metal cations in either model.
mouse leukemia cell line with an IC50 of 58
the cytotoxicity of bio-Fos was less than that of fostriecin
(IC50 = 0.46
M),10 bio-Fos still retained cytotoxicity against
lM (Fig. 2). Although
l
L1210 and proved useful for PP2Ac isolation from living cells
(Fig. 2).
We also demonstrated that PP2Ac directly bound to bio-Fos in
HeLa S3 cells, and the resulting biotinylated PP2Ac was isolated
from streptavidin-immobilized beads. In addition to PP2Ac, 65
and 130 kDa proteins were also isolated by immunoprecipitation
using bio-Fos (Fig. 3A). We confirmed that PP2A covalently binds
to bio-Fos by immunoblotting using alkaline phosphatase-conju-
gated avidin (Fig. 3B). However, the 65 and 130 kDa proteins were
not detected by the immunoblotting, suggesting that these two
proteins did not directly bind to bio-Fos. PP2A consists of PP2Ac,
PP2Ab, and PP2Aa. Thus, the 65 kDa protein might be PP2Aa, which
can be isolated from lysates as a complex with the PP2Ac-bio-Fos
conjugate. Although the action of the 130 kDa protein remains un-
clear, both the 65 and 130 kDa proteins might interact with bio-Fos
or PP2Ac by non-covalent binding.12,15–17 Here, we confirmed that
fostriecin binds to PP2Ac at the same binding site of bio-Fos since
fostriecin competed with bio-Fos for binding to PP2Ac (Fig. 3).
The MALDI-TOF MS analysis demonstrating that fostriecin binds
to the Cys269 residue (Fig. 4) strongly supports the result that the
loop domain (260 F SAPNYCYRCG NQ 272) between the b12 (256
VVTI 259) and b13 (273 AAIMEL 278) loop serves as the critical re-
gion for PP2Ac inhibition by fostriecin.28 This result also supports
the finding that the PP2A C269S mutant in yeast is much less sen-
sitive against fostriecin.28 The fragment with a mass of 2069.9 Da
3. Discussion
The inhibitory mechanism of fostriecin on PP2Ac has been iden-
tified in this study. This mechanism was previously suggested to be
due to the covalent binding of fostriecin to the Cys269 residue of

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T. Takeuchi et al. / Bioorg. Med. Chem. 17 (2009) 8113–8122
Figure 5. Proposed binding models of fostriecin–PP2Ac complex. (A and B) Structure of (3S)- (A) or (3R)-PP2Ac-conjugated fostriecin (B). (C–H) Docking model of PP2Ac with
fostriecin, which was attacked from the si-face (C, E, G) or re-face (D, F, H) of fostriecin. Carbon, oxygen, hydrogen, nitrogen, sulfur and manganese are indicated in gray, red,
white, blue, yellow and violet, respectively. PP2Ac residues near fostriecin are indicated in green. (C and D) Connolly surfaces and electronstatic potentials of fostriecin-
conjugated PP2Ac. Blue denotes positive charge and red indicates negative charge. (E and F) Protein backbone (indicated in yellow) of fostriecin-conjugated PP2Ac. (G and H)
Secondary structure of fostriecin-conjugated PP2Ac around fostriecin. The colors of the three-dimensional structure of the domain consisting of
flexible loop are red, blue, green and white, respectively.
a-helix, b-sheet, turn, and
corresponding to fostriecin-modified Cys269-Lys283 (269
C
On the other hand, the peptide fragment conjugated with the
lactone-opening derivative of fostriecin has been detected by MAL-
DI-TOF MS analysis. However, we consider that hydrolysis of the
lactone occurs during the tryptic digestion process, not within
the cells. Furthermore, it is reported that fostriecin is unstable
GNQAAIMELD DTLK 283) clearly appeared; however, the fragment
with a mass of 1692.8 Da corresponding to Cys269-Lys283 did not
completely disappear, suggesting that fostriecin might reversibly
conjugate PP2Ac.

T. Takeuchi et al. / Bioorg. Med. Chem. 17 (2009) 8113–8122
8119
and needs to be stored frozen in buffer.2,8 Boger and co-workers re-
ported that the lactone-opening derivative of fostriecin is less ac-
tive against PP2A inhibition and loses cytotoxic activity.27 Thus,
we consider that fostriecin, occurring in the lactone-closed form,
binds to PP2Ac and exerts inhibitory activity against this enzyme.
From these data, we constructed binding models of PP2Ac and
conducted binding simulation of lactone-closed fostriecin with
PP2Ac. Because of the two possibilities that the sulfhydryl group
of the Cys269 residue of PP2Ac will bind to fostriecin via the si-
or re-face of fostriecin to give (3S)- or (3R)-PP2Ac-conjugated fost-
riecin, respectively, we constructed two binding models of PP2Ac,
namely, the (3S)- and (3R)-models, respectively (Fig. 5). In both
models, the C9-phosphates bind or interact with the active site
of manganese cations in PP2Ac, and conjugates fostriecin to pre-
vent the substance of PP2Ac from entering the active site. This ac-
counts for the decreased inhibitory activity of dephosphofostriecin
(>105-fold) against PP2A.27 The largest differences in the two mod-
els appeared in the surrounding area with the triene moiety of fost-
riecin. In the (3S)-model (Fig. 5E and G), the triene moiety of
fostriecin is stabilized by a hydrophobic cage with Ile123, Tyr127
and Gly215.
bined organic layers were washed with H2O and saturated NaCl
solution, dried over Na2SO4 and concentrated under reduced pres-
sure. The resultant residue was purified by silica gel column chro-
matography (hexane/EtOAc = 4/1–1/1) to afford 4 as a colorless oil
(20.1 g, 88%): IR, 13C NMR and MS data were consistent with re-
ported values.56
5.1.1.2. (4-{2-[2-(Prop-2-yn-1-yloxy)ethoxy]ethoxy}phenyl)me-
thanol (5). To a stirred solution of 4 (111 mg, 0.35 mmol) in MeCN
(0.7 mL) were added p-hydroxybenzylalcohol (30 mg, 0.24 mmol)
and K2CO3 (48 mg, 0.35 mmol), and the mixture was refluxed over-
night. After the addition of H2O, the reaction mixture was extracted
with EtOAc. The combined organic layers were washed with H2O
and saturated NaCl solution, dried over Na2SO4, and concentrated
under reduced pressure. The resultant residue was purified by sil-
ica gel column chromatography (hexane/EtOAc = 2/1–1/1) to afford
5 as a white amorphous solid (56 mg, 64%): 1H NMR (600 MHz,
CDCl3) d 7.28 (2H, d, J = 8.5 Hz), 6.91 (2H, d, J = 8.5 Hz), 4.62 (2H,
s), 4.21 (2H, d, J = 2.4 Hz), 4.14 (2H, t, J = 4.9 Hz), 3.86 (2H, t,
J = 4.9 Hz), 3.72–3.77 (4H, m), 2.43 (1H, t, J = 2.4 Hz); 13C NMR
(100 MHz, CDCl3) dC 158.3, 133.3, 128.5 (2C), 114.6 (2C), 79.5,
74.6, 70.6, 69.7, 69.0, 67.4, 64.9, 58.4; IR mmax (neat) 3257, 2920,
2122, 1739, 1611, 1511 cmꢂ1
(C14H18O4+Na+ requires 273.1097).
; ESI-TOF HRMS m/z 273.1104
4. Conclusion
Using an affinity matrix-based protein purification ap-
proach,30–32 we directly demonstrated that fostriecin covalently
binds to PP2Ac in HeLa S3 cells, specifically to the Cys269 residue
of PP2Ac in vitro. The present findings may provide valuable infor-
mation for the design and development of new biological tools
and novel drugs for the treatment or prevention of cancer.
5.1.1.3. (5S,6S)-6-[(1E,3R,4R,6R,7Z)-6-(tert-Butyldimethylsilyloxy)-
8-iodo-3-methyl-3,4-bis(triethylsilyloxy)-octa-1,7-dien-1-yl]-5-
hydroxy-5,6-dihydro-2H-pyran-2-one ((12Z)-7). (5S,6S)-6-[(1E,
3R,4R,6R,7EZ)-6-(tert-Butyldimethylsilyloxy)-8-iodo-3-methyl-3,4-
bis(triethylsilyloxy)-octa-1,7-dien-1-yl]-5-[(4-methoxybenzyl)
oxy]-5,6-dihydro-2H-pyran-2-one (12EZ)-734 (E:Z = ca. 1:4)
(801 mg, 0.92 mmol) was carefully purified by silica gel column
chromatography (hexane/EtOAc = 9/1–6/1) to afford the (12Z)-7
5. Experimental
as a colorless oil (514 mg, 78%): ½a D24
ꢃ
+70.3 (c 0.22, CHCl3); 1H
5.1. Synthesis of biotin-labeled fostriecin (bio-Fos, 2)
NMR (600 MHz, CDCl3) d 6.99 (1H, dd, J = 9.7, 5.5 Hz), 6.21 (1H, d,
J = 7.7 Hz), 6.18 (1H, dd, J = 7.7, 7.7 Hz), 6.14 (1H, d, J = 9.7 Hz),
6.08 (1H, dd, J = 15.9, 1.3 Hz), 5.68 (1H, dd, J = 15.9, 5.7 Hz), 4.94
(1H, ddd, J = 5.7, 2.8, 1.3 Hz), 4.43 (1H, ddd, J = 10.4, 7.7, 2.9 Hz),
4.13 (1H, dd, J = 5.5, 2.8 Hz), 3.76 (1H, dd, J = 8.2, 1.5 Hz), 2.31
(1H, br s, OH), 2.03 (1H, ddd, J = 13.9, 10.4, 1.5 Hz), 1.38 (3H, s),
1.13 (1H, ddd, J = 13.9, 8.2, 2.9 Hz), 1.00 (9H, t, J = 8.0 Hz), 0.96
(9H, t, J = 8.0 Hz), 0.88 (9H, s), 0.73 (6H, q, J = 8.0 Hz), 0.62 (6H, q,
J = 8.0 Hz), 0.13 (3H, s), 0.06 (3H, s); 13C NMR (100 MHz, CDCl3) dC
163.1, 144.8, 144.1, 140.0, 123.1, 122.1, 80.0, 80.0, 77.5, 75.6,
72.9, 62.4, 40.7, 25.9 (3C), 21.0, 18.1, 7.3 (3C), 7.2 (3C), 6.7 (3C),
5.8 (3C), -3.0, -4.0; IR mmax (neat) 3428, 2955, 2877, 1729,
1253 cmꢂ1; ESI-TOF HRMS m/z 775.2721 (C32H61O6Si3I+Na+ re-
quires 775.2712).
5.1.1. General chemistry methods
1H and 13C NMR were recorded on a BRUKER DRX400 or
DRX600. Chemical shifts were reported in d, parts per million
(ppm), relative to TMS as an internal standard or calibrated using
residual undeuterated solvent as an internal reference. IR spectra
were recorded on a JASCO FT/IR-410 spectrometer. Mass spectra
were obtained on API QSTAR Pulsar i spectrometer. Optical rota-
tions were measured on a JASCO P-1030 digital polarimeter. Col-
umn chromatography was carried out on Fuji Silisia PSQ100B.
C18-reverse phase silica gel column chromatography was carried
out on Fuji Silisia Chromatorex ODS (100–200 mesh). Analytical
thin-layer chromatography (TLC) was performed on precoated
Merck Silica Gel 60 F254 plates, and compounds were visualized
by UV illumination (254 nm) or heating 150 °C after spraying phos-
phomolylbdic acid in ethanol. THF was distilled from sodium/ben-
zophenone. CH2Cl2 was distilled from P2O5. All other solvent and
reagents were obtained from commercial sources and used with-
out further purification. Organic extracts were dried over Na2SO4,
filtered, concentrated using a rotary evaporator. Involatile oils
and solids were vacuum dried.
5.1.1.4. (5S,6S)-6-[(1E,3R,4R,6R,7Z)-6-(tert-Butyldimethylsilyloxy)-
4-hydroxy-8-iodo-3-methyl-3-(triethylsilyloxy)-octa-1,7-dien-1-yl]
-5-(4-{2-[2-(prop-2-yn-1-yloxy)ethoxy]ethoxy}benzyloxy)-5,6-
di-hydro-2H-pyran-2-one (9). To a stirred solution of NaH (60%,
100 mg, 2.5 mmol) in CH2Cl2 (6 mL) was added 5 (2.5 g, 10.0 mmol),
and the mixture was stirred for 30 min at room temperature. After
the mixture was stirred for 15 min at 0 °C, a solution of Cl3CCN
(1.0 mL, 10.0 mmol) in CH2Cl2 (4 mL) was added dropwise at the
same temperature, and the reaction mixture was stirred for 1 h at
room temperature. After the addition of saturated NaHCO3 solution,
the reaction mixture was extracted with EtOAc. The combined or-
ganic layers were washed with H2O and saturated NaCl solution,
dried over Na2SO4, and concentrated under reduced pressure. The
resultant imidate 6 as a violet oil was used directly to the next step
without further purification.
5.1.1.1. 2-[2-(Prop-2-yn-1-yloxy)ethoxy]ethyl 4-methylbenzene
sulfonate (4). To a stirred solution of commercially available 3
(11.0 g, 76.3 mmol) in CH2Cl2 (150 mL) were added Et3N
(31.9 mL, 229 mmol) and DMAP (93 mg, 0.76 mmol) at 0 °C. The
mixture was stirred for 15 min at the same temperature and a
solution of TsCl (16.0 g, 83.9 mmol) in CH2Cl2 (150 mL) was added
dropwise at the same temperature. The mixture was stirred at
room temperature for 12 h. After the addition of saturated NaHCO3
solution, the reaction mixture was extracted with EtOAc. The com-
To a stirred solution of (12Z)-7 (45 mg, 0.060 mmol) and the
crude
6 above (118 mg) in Et2O (0.6 mL) was added TrBF4

8120
T. Takeuchi et al. / Bioorg. Med. Chem. 17 (2009) 8113–8122
(0.6 mg, 1.8
l
mol) at room temperature, and the mixture was stir-
(H2O/MeOH = 3/1–1/1) to afford 12 as a white amorphous solid
red at the same temperature for 20 min. After the addition of
(HOCH2CH2)2O (115 l, 1.2 mmol) at the same temperature, the
(252 mg, 89%): ½a D24
ꢃ
+49.4 (c 0.93, H2O); 1H NMR (600 MHz, D2O)
l
d 4.43 (1H, dd, J = 7.9, 5.0 Hz), 4.25 (1H, dd, J = 7.9, 4.6 Hz), 3.54
(2H, t, J = 4.9 Hz), 3.48 (2H, t, J = 5.3 Hz), 3.31 (2H, t, J = 4.9 Hz),
3.23 (2H, t, J = 5.3 Hz), 3.20 (1H, ddd, J = 9.8, 4.6, 4.6 Hz), 2.82 (1H,
dd, J = 13.1, 5.0 Hz), 2.61 (1H, d, J = 13.1 Hz), 2.11 (2H, t,
J = 7.3 Hz), 1.38–1.59 (4H, m), 1.29–1.24 (2H, m); 13C NMR
(100 MHz, MeOD) dC 176.9, 166.1, 70.6, 70.1, 63.2, 61.4, 56.7,
51.5, 40.9, 40.1, 36.7, 29.4, 29.2, 26.5; IR mmax (KBr) 3288, 2925,
mixture was stirred at the same temperature for 10 min. Then sat-
urated NaHCO3 solution was added to the mixture. The reaction
mixture was extracted with EtOAc and the combined organic lay-
ers were washed with H2O and saturated NaCl solution. The resul-
tant organic layer was dried over Na2SO4 and concentrated under
reduced pressure. The resultant residue was roughly purified by
silica gel column chromatography (hexane/EtOAc = 6/1–3/1) to
provide the corresponding crude 8 as a colorless oil which was car-
ried on directly to the next step without further purification.
To a stirred solution of the crude 8 above in MeCN (2.4 mL) and
2105, 1696, 1551 cmꢂ1
;
ESI-TOF HRMS m/z 379.1531
(C14H24N6O3S+Na+ requires 379.1522).
5.1.1.7. N-{2-[2-(4-{[2-(2-{4-[({(2S,3S)-2-[(1E,3R,4R,6R,7Z)-6-(tert-
Butyldimethylsilyloxy)-4-hydroxy-8-iodo-3-methyl-3-(triethylsily-
loxy)-octa-1,7-dien-1-yl]-6-oxo-3,6-dihydro-2H-pyran-3-yl}oxy)
methyl]phenoxy}ethoxy)ethoxy]methyl}-1H-1,2,3-tri-azol-1-yl)
ethoxy]ethyl}-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]
THF (1.2 mL) was added 1 M HCl aq (400
lL) at ꢂ10 °C, and the mix-
ture has stirred at the same temperature for 2.5 h. After the addition
of NaHCO3 solution, the reaction mixture was extracted with EtOAc.
The combined organic layers were washed with H2O and saturated
NaCl solution, dried over Na2SO4, and concentrated under reduced
pressure. The resultant residue was purified by silica gel column
chromatography (hexane/EtOAc = 4/1–2/1) to afford 9 as a colorless
imidazol-4-yl)pentanamide (14). To
a
stirred solution of
9
(66 mg, 0.075 mmol), 12 (108 mg, 0.3 mmol) and Et3N (315
l
L,
2.3 mmol) in THF (8 mL) was added CuI(I) (144 mg, 0.75 mmol)
at room temperature. The mixture was stirred at the same temper-
ature for 1 h, then 12 (108 mg, 0.3 mmol) was added, and stirred
for 20 min at the same temperature. After the addition of saturated
NH4Cl solution (1 mL), H2O (4 mL) and MeOH (1 mL) in this order,
the reaction mixture was purified directly by C18-reverse phase sil-
ica gel column chromatography (H2O only to MeOH only) to afford
oil (36 mg, 69%): ½a D24
ꢃ
+49.8 (c 0.13, CHCl3); 1H NMR (600 MHz,
CDCl3) d 7.22 (2H, d, J = 8.6 Hz), 6.90 (2H, d, J = 8.6 Hz), 6.77 (1H,
dd, J = 9.9, 4.3 Hz), 6.32 (1H, dd, J = 7.6, 7.6 Hz), 6.21 (1H, dd,
J = 7.6, 0.9 Hz), 6.09 (1H, d, J = 9.9 Hz), 6.00 (1H, dd, J = 15.7,
4.3 Hz), 5.97 (1H, d, J = 15.7 Hz), 4.92 (1H, dd, J = 4.3, 4.3 Hz), 4.66
(1H, dddd, J = 7.6, 7.6, 3.0, 0.9 Hz), 4.57 (1H, d, J = 11.6 Hz), 4.51
(1H, d, J = 11.6 Hz), 4.22 (2H, d, J = 2.4 Hz), 4.14 (2H, t, J = 4.8 Hz),
4.08 (1H, t, J = 4.3 Hz), 3.87 (2H, t, J = 4.8 Hz), 3.73–3.78 (4H, m),
3.66 (1H, ddd, J = 10.8, 2.8, 1.2 Hz), 2.99 (1H, br d, J = 2.8 Hz, OH),
2.43 (1H, t, J = 2.4 Hz), 1.77 (1H, ddd, J = 14.0, 7.6, 1.2 Hz), 1.36
(1H, ddd, J = 14.0, 10.8, 3.0 Hz), 1.36 (3H, s), 0.94 (9H, t, J = 8.0 Hz),
0.88 (9H, s), 0.58 (6H, q, J = 8.0 Hz), 0.10 (3H, s), 0.06 (3H, s); 13C
NMR (100 MHz, CDCl3)dC 162.8, 158.7, 144.0, 143.6, 139.5, 129.5,
129.3 (2C), 123.3, 122.8, 114.6 (2C), 80.0, 79.8, 79.5, 77.6, 74.8,
74.5, 74.1, 71.4, 70.6, 69.7, 69.1, 69.1, 67.4, 58.4, 37.0, 25.7 (3C),
22.8, 17.9, 7.1 (3C), 6.7 (3C), ꢂ4.5, ꢂ5.1; IR mmax (neat) 3494,
14 as a colorless oil (91 mg, 98%): ½a D24
ꢃ
+71.0 (c 0.75, CHCl3); 1H
NMR (600 MHz, MeOD) d 7.97 (1H, s), 7.24 (2H, d, J = 8.6 Hz),
7.02 (1H, dd, J = 9.9, 4.9 Hz), 6.91 (2H, d, J = 8.6 Hz), 6.22 (1H, d,
J = 7.6 Hz), 6.15 (1H, dd, J = 7.6, 7.6 Hz), 6.08 (1H, d, J = 9.9 Hz),
6.05 (1H, d, J = 15.7, 4.0 Hz), 6.02 (1H, dd, J = 15.7, 2.3 Hz), 5.00
(1H, ddd, J = 4.0, 3.6, 2.3 Hz), 4.64 (2H, s), 4.64 (1H, ddd, J = 9.5,
7.6, 2.9 Hz), 4.53–4.56 (4H, m), 4.46 (1H, dd, J = 7.8, 4.8 Hz), 4.26
(1H, dd, J = 7.8, 4.6 Hz), 4.10–4.13 (2H, m), 4.08 (1H, dd, J = 4.9,
3.6 Hz), 3.81–3.85 (4H, m), 3.68–3.74 (4H, m), 3.66 (1H, dd,
J = 10.7, 1.2 Hz), 3.49 (2H, t, J = 5.4 Hz), 3.29–3.32 (2H, m), 3.16
(1H, ddd, J = 9.1, 5.6, 4.6 Hz), 2.89 (1H, dd, J = 12.8, 4.8 Hz), 2.68
(1H, d, J = 12.8 Hz), 2.18 (2H, t, J = 7.4 Hz), 1.89 (1H, ddd, J = 13.8,
9.5, 1.2 Hz), 1.52–1.74 (4H, m), 1.38–1.43 (2H, m), 1.28 (3H, s),
1.23 (1H, ddd, J = 13.8, 10.7, 2.9 Hz), 0.97 (9H, t, J = 7.9 Hz), 0.89
(9H, s), 0.64 (6H, q, J = 7.9 Hz), 0.13 (3H, s), 0.06 (3H, s); 13C NMR
(100 MHz, MeOD) dC 176.1, 166.1, 165.6, 160.2, 146.1, 146.1,
145.9, 140.4, 131.4, 130.6 (2C), 125.8, 124.8, 123.5, 115.6 (2C),
82.1, 80.0, 78.8, 75.6, 74.3, 72.9, 71.8, 70.9, 70.9, 70.7, 70.5, 70.1,
68.7, 65.1, 63.4, 61.6, 57.0, 51.4, 41.0, 40.2, 39.4, 36.7, 29.8, 29.5,
26.8, 26.5 (3C), 25.6, 19.0, 7.9 (3C), 7.7 (3C), ꢂ3.8, ꢂ4.4; IR mmax
(neat) 3299, 2927, 2108, 1698, 1252 cmꢂ1; ESI-TOF HRMS m/z
1249.4571 (C54H87N6O12Si2SI+Na+ requires 1249.4578).
3307, 2928, 1731, 1513 cmꢂ1
; ESI-TOF HRMS m/z 893.2944
(C40H63O9Si2I+Na+ requires 893.2947).
5.1.1.5. 2-(2-Azidoethoxy)ethanaminium chloride (11). To
a
stirred solution of 10 (11.7 g, 75 mmol) in EtOAc (600 mL) and
1 M HCl aq (120 mL) was added PPh3 (20.6 g, 79 mmol) at room
temperature, and the mixture was stirred for 4 h at the same tem-
perature. After the addition of HCl (1 M in H2O), the organic layer
was extracted with H2O. The combined aqueous layers were
washed with EtOAc and concentrated under reduced pressure.
The resultant residue was purified by C18-reverse phase silica gel
column chromatography (H2O only to H2O/MeOH = 1/1) to afford
11 as a white amorphous solid (9.9 g, 79%): 1H NMR (600 MHz,
D2O) d 3.66 (2H, t, J = 4.8 Hz), 3.61 (2H, t, J = 4.8 Hz), 3.40 (2H, t,
J = 4.8 Hz), 3.09 (2H, t, J = 4.8 Hz); 13C NMR (100 MHz, D2O+MeOD)
dC 70.5, 67.3, 51.62, 40.3; IR mmax (film) 3114, 2113, 1625,
1407 cmꢂ1; ESI-TOF HRMS m/z 131.0927 (C4H10N4O+H+ requires
131.0927).
5.1.1.8. N-{2-[2-(4-{[2-(2-{4-[({(2S,3S)-2-[(1E,3R,4R,6R,7Z,9Z,-
11E)-6-(tert-Butyldimethylsilyloxy)-13-(tert-butyldiphenylsilyl-
oxy)-4-hydroxy-3-methyl-3-(triethylsilyloxy)-trideca-1,7,9,11-
tetraen-1-yl]-6-oxo-3,6-dihydro-2H-pyran-3-yl}oxy)methyl]phe-
noxy}ethoxy)ethoxy]methyl}-1H-1,2,3-triazol-1-yl)ethoxy]ethyl}-
5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)
pentanamide (15). To a stirred solution of stannane 1341,42
5.1.1.6. N-[2-(2-Azido-ethoxy)ethyl]-5-((3aS,4S,6aR)-2-oxohexa-
hydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (12). To
stirred solution of biotin (200 mg, 0.82 mmol), 11 (264 mg,
1.6 mmol) and i-Pr2NEt (820 l, 4.8 mmol) in DMF (6.5 mL) was
added HBTU (308 mg, 0.81 mmol) at room temperature, and the
mixture was stirred overnight at the same temperature. After the
addition of saturated NaHCO3 solution, the reaction mixture was
extracted with EtOAc. The combined organic layers were washed
with H2O and saturated NaCl solution, dried over Na2SO4, and
concentrated under reduced pressure. The resultant residue was
purified by C18-reverse phase silica gel column chromatography
a
(29 mg, 0.047 mmol) and 14 (9.6 mg, 7.8
lmol) in DMF (500 ll)
was added Pd(MeCN)2Cl2 (0.2 mg, 0.77 mol) at 0 °C. The resultant
l
l
mixture was stirred at the same temperature for 10 h. After the
addition of saturated NaHCO3 solution, the reaction mixture was
extracted with CHCl3. The combined organic layers were washed
with H2O and saturated NaCl solution, dried over Na2SO4 and con-
centrated under reduced pressure. The resultant residue was
directly purified by silica gel column chromatography (CHCl3 only
to CHCl3/MeOH = 4/1) to afford 15 as a colorless oil (9.0 mg, 81%):
½
a 2D4 +21.5 (c 0.35, CHCl3); 1H NMR (600 MHz, CDCl3) d 7.66–7.70
ꢃ

T. Takeuchi et al. / Bioorg. Med. Chem. 17 (2009) 8113–8122
8121
(5H, m), 7.35–7.44 (6H, m), 7.22 (2H, d, J = 8.7 Hz), 6.89 (2H, d,
J = 8.7 Hz), 6.77 (1H, dd, J = 9.9, 4.5 Hz), 6.74 (1H, ddd, J = 15.0,
11.3, 1.0 Hz), 6.33 (1H, dd, J = 11.3, 11.3 Hz), 6.19 (1H, t,
J = 4.9 Hz, NH), 6.14 (1H, dd, J = 11.3, 11.3 Hz), 6.08 (1H, dd,
J = 9.9, 0.6 Hz), 6.04 (1H, dd, J = 11.3, 11.3 Hz), 5.99 (1H, dd,
J = 16.4, 3.8 Hz), 5.95 (1H, dd, J = 16.4, 2.1 Hz), 5.83 (1H, dt,
J = 15.0, 4.5 Hz), 5.62 (1H, br s, NH), 5.53 (1H, dd, J = 11.3, 8.4 Hz),
4.89–4.94 (2H, m), 4.76 (1H, br s, NH), 4.71 (2H, s), 4.57 (1H, d,
J = 11.7 Hz), 4.51 (2H, t, J = 5.0 Hz), 4.50 (1H, d, J = 11.7 Hz), 4.48
(1H, dd, J = 7.3, 5.3 Hz), 4.31 (1H, dt, J = 7.3, 4.9 Hz), 4.29 (2H, dd,
J = 4.5, 1.0 Hz), 4.12 (2H, t, J = 4.8 Hz), 4.08 (1H, ddd, J = 4.5, 3.9,
0.6 Hz), 3.83–3.86 (4H, m), 3.73–7.76 (4H, m), 3.70 (1H, br dd,
J = 11.5, 2.7 Hz), 3.52 (2H, t, J = 4.9 Hz), 3.38 (2H, dt, J = 4.9,
4.9 Hz), 3.14, (1H, td, J = 7.3, 4.9 Hz), 2.99 (1H, br d, J = 2.7 Hz,
OH), 2.90 (1H, dd, J = 12.4, 5.3 Hz), 2.70 (1H, d, J = 12.4 Hz), 2.20
(2H, t, J = 7.1 Hz), 1.67–1.76 (1H, m), 1.57–1.76 (4H, m), 1.40–
1.47 (2H, m), 1.33–1.40 (1H, m), 1.34 (3H, s), 1.08 (9H, s), 0.92
(9H, t, J = 7.9 Hz), 0.88 (9H, s), 0.57 (6H, q, J = 7.9 Hz), 0.06 (3H,
s), 0.03 (3H, s); 13C NMR (100 MHz, CDCl3) dC 173.1, 163.0, 162.9,
158.7, 145.1, 143.7, 139.7, 135.6, 135.5 (4C), 134.3, 133.6, 133.5,
130.0, 129.7, 129.7 (2C), 129.4 (2C), 127.7 (4C), 124.5, 123.7,
123.3, 123.2, 122.8, 122.2, 114.6 (2C), 80.1, 77.7, 74.9, 71.5, 70.8,
69.8, 69.7, 69.7, 69.2, 68.8, 67.5, 67.0, 64.7, 64.2, 61.8, 60.0, 55.4,
50.1, 40.6, 39.0, 38.9, 35.6, 28.0, 27.9, 26.8 (3C), 25.8 (3C), 25.4,
22.6, 19.2, 18.1, 7.1 (3C), 6.7 (3C), ꢂ4.3, ꢂ5.0; IR mmax (neat)
J = 7.7, 4.7 Hz), 4.10–4.14 (4H, m), 4.08 (1H, dd, J = 5.1, 3.2 Hz),
3.80–3.84 (4H, m), 3.68–3.73 (4H, m), 3.48 (2H, t, J = 5.4 Hz),
3.25–3.33 (2H, m), 3.15 (1H, dt, J = 10.1, 4.7 Hz), 3.12 (6H, q,
J = 10.9 Hz, Et3N), 2.89 (1H, dd, J = 12.8, 5.0 Hz), 2.68 (1H, d,
J = 12.8 Hz), 2.18 (2H, t, J = 7.4 Hz), 1.52–1.74 (4H, m), 1.60–1.70
(1H, m), 1.42–1.51 (1H, m), 1.29–1.42 (2H, m), 1.35 (3H, s), 1.27
(9H, t, J = 10.9 Hz, Et3N); 13C NMR (100 Hz, MeOD) dC 176.2,
166.1, 165.7, 160.2, 146.1, 145.9, 140.8, 135.9, 135.6, 131.5, 130.9
(2C), 130.8, 126.6, 125.8, 125.3, 124.5, 124.2, 123.2, 115.6 (2C),
82.3, 79.2 (JC–P = 7 Hz), 75.7 (JC–P = 1.7 Hz), 73.3, 71.8, 70.9, 70.9,
70.6, 70.5, 70.1, 68.7, 65.1, 64.6, 63.4, 63.3, 61.6, 57.0, 51.4, 47.0
(3C, Et3N), 41.1, 40.9 (JC–P = 1.4 Hz), 40.2, 36.7, 29.8, 29.5, 26.8,
24.6, 9.24 (3C, Et3N); 31P NMR(162 MHz, MeOD) dP 3.6; IR mmax
(KBr) 3429, 2925, 1697, 1514, 1458 cmꢂ1; ESI-TOF HRMS m/z
1136.5335 (C53H82N7O16PS+H+ requires 1136.5349).
5.2. Cell culture
Murine leukemia L1210 cells (provided by Cell Resource Center
for Biomedical Research, Tohoku University) were cultured in RPMI
1640 medium (Nacalai Tesque) with 10% fetal calf serum (TRACE
SCIENTIFIC LTD.), 2 mM
L-glutamine, penicillin (100 units/mL),
and streptomycin (100 g/mL) at 37 °C in 5% CO2.
l
5.3. Growth inhibition assay
3299, 2927, 2108, 1698, 1252 cmꢂ1
;
ESI-TOF HRMS m/z
1443.7170 (C75H112N6O13Si3S+Na+ requires 1443.7208).
Cell proliferation was evaluated using Cell Count Reagent SF
(Nacalai Tesque). Cells are inoculated into 96-well plates contain-
5.1.1.9. N,N-Diethylethanaminium (1E,3R,4R,6R,7Z,9Z,11E)-3,6,
13-trihydroxy-1-{(2S,3S)-3-[(4-{2-[2-({1-[2-(2-{[5-((3aS,4S,6aR)-2-
oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl] amino}
ethoxy)ethyl]-1H-1,2,3-triazol-4-yl}methoxy)ethoxy] ethoxy} ben-
zyl)oxy]-6-oxo-3,6-dihydro-2H-pyran-2-yl}-3-methyl-trideca -1,
7,9,11-tetraen-4-yl hydrogen phosphate (2). To a stirred solution
ing 5000 cells in total volume of 100
bation with bio-Fos (0, 5, 10, 25, 50, 100, 250, 500 and 1000
for 72 h, 10 l of Cell Count Reagent was added to each well 2 h be-
fore evaluating cell proliferation on ARVO SX microplate reader
(WALLAC) at 450 nm. Each experimental condition was assayed
in tetralicate, and all experiments were performed at least three
times. Percentage growth inhibition is calculated from time zero,
control growth, and the each concentration level of absorbance.
l
L in each well. After the incu-
lM)
l
of 15 (7.6 mg, 5.3
lmol) and tetrazole (5.6 mg, 0.080 mmol)
43,44
in CH2Cl2 (500 L) was added i-Pr2NP(OFm)2
l
(28 mg,
0.053 mmol) at room temperature, and the mixture was stirred
at the same temperature for 20 min. After the addition of TBHP
5.4. In situ fostriecin-binding assay
(3.2 M in CH2Cl2, 60 lL, 0.96 mmol) at 0 °C, the resulting mixture
was stirred at the same temperature for 1 h. Following addition
of MeOH (0.2 mL) and CHCl3 (0.5 mL), the reaction mixture was di-
rectly roughly purified by silica gel column chromatography (CHCl3
only to CHCl3/MeOH = 9/1) to give roughly purified 16, which was
used directly to the next step.
To a stirred solution of crude 16 above in MeCN (0.5 mL) was
added Et3N (0.1 mL, 0.71 mmol) at 0 °C, and the mixture was stir-
red at room temperature for 17 h. The mixture was concentrated
under reduced pressure to give crude phosphate monoester 17,
which was used directly to the next step.
Full confluent HeLa S3 cells (ca. 1 g of wet weight) adherent to
wells were incubated in medium (see above) containing 20 lM of
bio-Fos or biotin at 37 °C for 2 h. After the wash of the cells with
phosphate buffered saline (PBS) (H3PO4 137 mM NaCl, 118 mM
[pH 7.4]) three times, cells were scraped from the wells in PBS,
followed by transferring the cells to a clean Eppendorf tube, cen-
trifugation at 350g and discard of the supernatant. The cells were
treated with 100 lL of the lysis buffer (50 mM Tris–HCl, 150 mM
NaCl, 1 mM EDTA, 2.5 mM EGTA, 0.1% NP-40, 0.1 mM PMSF, 1%
Protease Inhibitor Cocktail (EDTA free) (nacalai tesque) [pH 7.5])
and placed on ice for 1 h. After centrifugation at 16,000g, strepta-
The reaction was performed in a plastic vessel. To a stirred solu-
tion of the crude phosphate monoester 17 above in MeCN (0.5 mL)
vidin–agarose (20 lL, Aldrich) were added to supernatant of the
were added Et3Nꢁ3HF (26
l
L, 0.16 mmol) and Et3N (11
l
L,
lysate, followed by the mixing with rotator at 4 °C for 30 min.
Then the beads, together with bound proteins, were washed with
1 ml of beads-washing buffer (25 mM Tris–HCl, 137 mM NaCl,
0.079 mmol) at 60 °C, and the mixture was stirred at the same tem-
perature for 2 h. After the addition of 0.5 M NaHCO3 aq (1.9 mL,
0.95 mmol) at 0 °C and H2O (720
l
L), the reaction mixture was di-
0.05% Tween 20 [pH 7.5]) 10 times and boiled in 20 ll of SDS-
rectly purified by C18-reverse phase silica gel column chromatogra-
loading buffer (50 mM Tris–HCl, 10% glycerol, 2.0% SDS, 5% 2-
mercaptoethanol, and a few drops of bromophenol blue [pH
6.8]). The resultant mixtures were electrophoresed on 10% acryl-
amide gels, followed by analyze by silver stain or western blot-
ting using an alkaline phosphatase-conjugated avidin (1:500
dilution, Zymed) or anti-PP2Ac antibody (1:500 dilution, rabit
polyclonal, Calbiochem). The alkaline phosphatase-conjugated
anti-rabit IgG (1:2000 dilution, Sigma) was used as secondary
antibody. The protein bands were visualized by the use of CDP-
Star (GE Healthcare Bio-Sciences).
phy (H2O only to H2O/MeCN = 9/1) to afford 2 as white crystals
(2.2 mg, 37%): ½a D24
ꢃ
+43.2 (c 0.08, MeOH); 1H NMR (MeOD,
600 MHz) d 7.96 (1H, s), 7.27 (2H, d, J = 8.6 Hz), 6.99 (1H, dd,
J = 9.8, 5.1 Hz), 6.91 (2H, d, J = 8.6 Hz), 6.73 (1H, dd, J = 15.0,
12.2 Hz), 6.43 (1H, dd, J = 11.6, 11.6 Hz), 6.37 (1H, dd, J = 11.6,
11.6 Hz), 5.99–6.11 (4H, m), 5.84 (1H, dt, J = 15.0, 5.7 Hz), 5.48
(1H, dd, J = 11.6, 9.8 Hz), 5.02 (1H, dd, J = 9.8, 9.8 Hz), 4.99 (1H,
dd, J = 6.6, 3.2 Hz), 4.64 (2H, s), 4.49–4.58 (4H, m), 4.56 (1H, dd,
J = 7.7, 5.0 Hz), 4.30 (1H, ddd, J = 11.3, 11.3, 1.8 Hz), 4.26 (1H, dd,

8122
T. Takeuchi et al. / Bioorg. Med. Chem. 17 (2009) 8113–8122
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To the solution of fostriecin (0.5 mM in H2O, 2
H2O (2 l) in 13 L of tris–HCl buffer (25 mM Tris–HCl, 137 mM
NaCl [pH 7.5]) was added 5 l of PP2A (0.5 unit, Upstate). After
the incubation of the mixtures at 30 °C for 2 h, 6
ing buffer (250 mM Tris–HCl, 50% glycerol, 10% SDS, 25% 2-
mercaptoethanol, and a few drops of bromophenol blue [pH
lL, 1 nmol) or
l
l
l
l
l of 5ꢄ SDS-load-
6.8]), H2O (2.5 lL) and dithiothreitol (1 M in H2O, 1.5 lL) were
added, followed by boiling for 1 min. The resulting samples were
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with dithiothreitol and the alkylation with acrylamide. After the
digestion with trypsin (Promega) at 37 °C for 16 h, the resulting
peptides (0.5 lL) were spotted onto the MALDI target with a MAL-
DI matrix solution (2.5-dihydroxybenzoic acid), followed by co-
crystallization of the sample and matrix at room temperature.
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by a reflex IV MALDI-TOF mass spectrometer (Bruker Daltonics,
Billerica, MA, USA).
5.6. Protein-inhibitor docking modeling
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We thank Cell Resource Center for Biomedical Research, Insti-
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.09.050.
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