Development of biotin-avidin technology to investigate okadaic acid-promoted cell signaling pathway

Article abstract of DOI:10.1016/S0040-4020(00)00752-3

Four biotin conjugates of okadaic acid were synthesized for evaluating their interactions with protein phosphatase 2A (PP2A) by surface plasmon resonance (SPR). C7-biotinylated okadaic acid exhibited the strongest binding affinity to the enzyme, while Cl-biotinylated derivative was devoid of affinity. C24- or C27-biotinylated okadaic acid showed moderate affinity to the enzyme. In the wake of this finding, a biotinyl photoaffinity probe was introduced into 7-OH of okadaic acid. Photoaffinity labeling followed by SDS-PAGE analysis indicated that the okadaic acid derivative clearly labeled PP2A. Furthermore, three proteins were also labeled in crude extracts of a marine sponge Halichondria okadai. All these results imply that the C7-biotin conjugate is a versatile reagent for biochemical studies of okadaic acid-binding proteins including PP2A. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00752-3

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 9003±9014
Development of Biotin±Avidin Technology to Investigate Okadaic
Acid-Promoted Cell Signaling Pathway
Keiichi Konoki,a,b Naoyuki Sugiyama,a Michio Murata,a,b,² Kazuo Tachibanaa,b,
*
and Yasumaru Hatanakac
aDepartment of Chemistry, School of Science, The University of Tokyo, Hongo, Bunkyo-Ku, Tokyo 113-0033, Tokyo, Japan
bCREST, Japan Science and Technology Corporation (JST), Hongo, Bunkyo-Ku, Tokyo 113-0033, Tokyo, Japan
cResearch Institute for Wakan-yaku, Toyama Medical and Pharmaceutical University, Sugitani, Toyama 930-0194, Japan
Received 17 May 2000; accepted 24 July 2000
AbstractÐFour biotin conjugates of okadaic acid were synthesized for evaluating their interactions with protein phosphatase 2A (PP2A) by
surface plasmon resonance (SPR). C7-biotinylated okadaic acid exhibited the strongest binding af®nity to the enzyme, while C1-biotinylated
derivative was devoid of af®nity. C24- or C27-biotinylated okadaic acid showed moderate af®nity to the enzyme. In the wake of this ®nding,
a biotinyl photoaf®nity probe was introduced into 7-OH of okadaic acid. Photoaf®nity labeling followed by SDS-PAGE analysis indicated
that the okadaic acid derivative clearly labeled PP2A. Furthermore, three proteins were also labeled in crude extracts of a marine sponge
Halichondria okadai. All these results imply that the C7-biotin conjugate is a versatile reagent for biochemical studies of okadaic acid-
binding proteins including PP2A. q 2000 Elsevier Science Ltd. All rights reserved.
Introduction
pseudocyclic conformation formed by hydrogen bonding
between C1 carboxylic acid and 24-OH, also reduce the
potency. While these preceding studies convince impor-
tance of the pseudocyclic structure for its recognition by
PP2A, it still remains vague which side of this structure is
recognized except for an observation on okadaic acid
acylated at 7-OH retaining a moderate binding ability to
PP2A.
Reversible phosphorylation of proteins is catalyzed by both
protein kinases and protein phosphatases, through which
intracellular events are regulated in eukaryotic cells accord-
ing to extracellular signals and their transduction. Several
natural products such as okadaic acid (Fig. 1, 1),1 micro-
cystin-LR, tautomycin and calyculin A are known to bind to
protein phosphatases 1 (PP1) and 2A (PP2A), and inhibit
their enzymatic activity, resulting in accumulation of phos-
phorylated proteins in cells of various organs.2 The binding
of these inhibitors to protein phosphatases has been mainly
investigated by replacement experiments using a radio-
ligand such as 27-[3H]okadaic acid3 or by monitoring the
enzymatic activity.4 Among them, okadaic acid (1) is the
most potent inhibitor of PP2A, and now regarded as a stan-
dard reagent for biological studies of protein phosphatases.2
Recently, optical-biosensing techniques have become
potential methods to investigate kinetics of diverse inter-
actions between two or more molecules including proteins,
peptides, nucleic acids, carbohydrates, lipids, and low
molecular weight molecules such as signaling substances
and drug candidates.6 Surface plasmon resonance (SPR) is
one of these apparatus. Once a ligand is immobilized to a
sensor plate, binding with its receptor can be monitored in
real time as the local change in refractive index of a solution
passing near the surface of a sensor plate, or the change in
mass concentration of the surface layer. This change is
re¯ected on resonance between the evanescent wave of
the probe light, irradiated from the other side of the sensor
plate at the total re¯ection condition, and free electron
plasmon at the surface of the plate made from gold foil. In
order to evaluate applicability of this method on more
precise studies on the molecular mechanism of okadaic
acid inhibiting action of the protein phosphatase, immobili-
zation of okadaic acid was undertaken.
The structure±activity relationship of okadaic acid has been
investigated in detail by many groups.5 Substitution at the
C1 carboxylic acid or 24-OH greatly reduces the activity.
Other structural changes such as hydrogenation at C14±C15
ole®nic bond or deoxidation at C2, both of which affect the
Keywords: surface plasmon resonance; photoaf®nity labeling; okadaic acid;
protein phosphatase.
* Corresponding author. Department of Chemistry, School of Science, The
University of Tokyo, Hongo, Bunkyo-Ku, Tokyo 113-0033, Tokyo, Japan.
Tel.: 181-3-5841-4366, fax: 181-3-5841-8380;
e-mail: ktachi@chem.s.u-tokyo.ac.jp
Present address: Department of Chemistry, Graduate School of Science,
Osaka University, Osaka, Japan.
²
The immobilization of a ligand can be achieved by in situ
formation of a covalent bond such as amide, disul®de, and
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00752-3

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K. Konoki et al. / Tetrahedron 56 (2000) 9003±9014
Figure 1. Okadaic acid and its biotinylated derivatives.
hydrazide, or a strong noncovalent bonding such as biotin±
avidin complexation with a suitable kind and length of a
spacer attached at a desired site of the ligand molecule.
Since optimization of the solid-phase reaction for covalent
immobilization would require a large excess of okadaic acid
derivatives and possibly be costing expenses for wasted
sensor plates, the non-covalent method was adopted in the
present study. Namely, okadaic acid was biotinylated in
liquid phase at its C1, C7, C24, or C27 positions through
a linker chosen according to precedents (Fig. 1), and these
derivatives were respectively immobilized to the same
sensor plate where streptavidin was pre-immobilized.
Besides the utility in SPR sensing, biotinylation of a ligand
enables us to adopt methodologies widely used in bio-
chemistry and molecular biology such as sensitive detection
of proteins or protein capture applications.7 Among them,
photoaf®nity labeling has become a powerful tool for

K. Konoki et al. / Tetrahedron 56 (2000) 9003±9014
9005
Scheme 1. (a)t-Butyl N-(5-aminopentyl)carbamate, EDC´HCl, HOBt, Et3N, DMF, rt, 52%; (b) CF3CO2H±CH2Cl2 (1:2), rt, quantitative; (c) 7, EDC´HCl,
HOBt, DMF, rt, 40%; (d) p-BrC6H4COCH2Br, Et3N, acetone, 408C, 94%; (e) N,N0-Disuccinimidyl carbonate, Et3N, DMF; (f) 7, Et3N, DMF, rt; (g) Zn/AcOH,
rt, 54% in 3 steps; (h) TMSCHN2, C6H6±MeOH (1:1), rt, quantitative; (i) BzCl, Et3N, CH2Cl2, rt; (j) TBDMSCl, imidazole, DMF, rt; (k) Ac2O, pyridine, rt,
68% in 3 steps; (l) 47% aq. HF±CH3CN (1:4), rt, 85%; (m) N,N0-Disuccinimidyl carbonate, Et3N, CH3CN, rt; (n) 7, Et3N, DMF, rt, 85% in 2 steps; (o) 1 M aq.
NaOH±THF (1:1), 408C; (p) 5% NaOH/MeOH, 608C; (q) TBDMSCl, imidazole, DMF, rt, 95%; (r) N,N0-Disuccinimidyl carbonate, Et3N, CH3CN, rt; (s) 6,
Et3N, DMF, rt, 28% in 2 steps; (t) 47% aq. HF±CH3CN (1:4), rt; (u) 1 M aq. NaOH±THF (1:1), 408C, 71% in 2 steps.

9006
K. Konoki et al. / Tetrahedron 56 (2000) 9003±9014
generally needed to ease the biotin for complex formation,
d-biotin was treated with t-butyl N-(5-aminopentyl)carba-
mate in the presence of EDC´HCl and Et3N, and deprotec-
tion in an acidic condition gave N-biotinyl-1,5-
pentanediamine 7 (Scheme 1).
Okadaic acid 1 was reacted with freshly prepared 7 in the
presence of EDC´HCl and HOBt to furnish 2 in 40% yield.11
The structural resemblance between 2 and methyl okadaate
1
10 was evident in the H NMR spectra, and additional
signals observed in the spectrum of 2 at d 4.51 (±S±
CH2±CH±), 4.31 (±S±CH±CH±), 3.15 (±S±CH±), and
2.91/2.71 (±S±CH2±) were attributed to the biotin moiety.
Since nucleophilicity of 7-OH is known to be higher than
the other secondary hydroxyl groups, selective biotinylation
at this position was envisaged by controlling quantity and
concentration of reagents.12 Okadaic acid biotinylated at
7-OH (3) was synthesized as follows.11 After the carboxyl
group in okadaic acid was protected as the p-bromophenacyl
ester (8), the ester was reacted with N,N0-disuccinimidyl
carbonate in the presence of Et3N to give an intermediate
mixed carbonate.13 Without further puri®cation, this inter-
mediate was treated with 7 in the presence of Et3N, and
deprotected under Zn/AcOH at 408C to afford 3. The reac-
tion of 8 with N,N0-disuccinimidyl carbonate was turned out
not to be so selective, and thus needs to be controlled in
relatively diluted condition at this stage. Otherwise, the
following reaction with 7 and deprotection of p-bromo-
phenacyl substituent resulted in simultaneous production
of regioisomers such as 4 or 5, which was observed by
LC/MS analysis (data not shown). Introduction of the biotin
unit at 7-OH in 3 was con®rmed by 1H NMR spectroscopy,
where 7-H, 24-H, and 27-H were assigned by tracing the
COSY spectrum. The signal for 7-H in 3 appeared at d 4.57,
being downshifted from that in 1 at d 3.35.1 Since 1 and 3
exhibited the closely resembling spectra except for the
above signals for 7-H, the signals at d 4.07 and d 4.05
assigned to 24-H and 27-H, respectively, indicated that
neither of corresponding hydroxyls is acylated.
Figure 2. Interaction of biotinylated okadaic acids and protein phosphatase
2A. A streptavidin-immobilized sensor chip was loaded with the bio-
tinylated okadaic acid 2, 3, 4 or 5 (100 nM) in 100 mL of a running buffer
at a ¯ow rate of 5.0 mL min21 for 180 s. After a PP2A solution (12.5 nM) in
the running buffer containing 0.02% w/v BSA was let ¯ow at 10 mL min21
for 180 s, dissociation of the bound protein was monitored for 180 s by
washing the sensor chip with the same buffer. The sensor chip was
regenerated by passing 100 mM Na2CO3 at a ¯ow rate of 5.0 mL min21
for 180 s. All experiments were carried out at 258C.
investigating ligand/receptor interactions at various labels.
To screen target molecules or identify binding domains,
3
radiochemical probes bearing H or 125I were commonly
used before.8 Because of requirements for streamlining the
tedious process, however, detection techniques with ¯uores-
cence or chemiluminescence have been developed, acquir-
ing similar sensitivity to those for the radioligands to date.
In this course, a new photoaf®nity ligand 20 (Scheme 2) was
devised by Hatanaka et al., and successfully used in identi-
®cation of binding domain on GalT (b-1,4-galactosyltrans-
ferase).9 This tridentate ligand bears a biotin moiety for
detection, a 3-tri¯uoromethyl-3-diazirinyl moiety for photo-
labeling, and a carboxyl group for linkage to ligands. The
photoreactive moiety has provided outstanding photo-
crosslinking results,10 because irradiation at 360 nm would
not destroy proteins, and it is also stable to mild reductive,
acidic, and basic conditions. In their study, the binding
proteins on an SDS-PAGE gel were captured by streptavidin
conjugated to horseradish peroxidase (HRP), and thus were
24-O-Biotinylated okadaic acid (4) was synthesized based
on difference in nucleophilicity between 24-OH and 27-OH,
which has been indicated in a modi®ed preparation of 27-
[3H]okadaic acid via methyl 7-O,24-O-disilylokadaic acid,
vide infra (Ojika, M. Personal communication). Namely, the
7-, 24-, and 27-hydroxyl groups in methyl okadaate (10)
were protected as benzoate, TBDMS ether, and acetate,
respectively in this order, to yield 13. Selective deprotection
at 24-OH in 13 by aq. HF±AcOH, carbonation of 24-OH in
14 with N,N0-disuccinimidyl carbonate and Et3N, and
substitution by 7 furnished 15. Successive deprotection
with aq. NaOH±THF followed by NaOH/MeOH produced
4. Introduction of the biotin unit at 24-OH in 4 was
con®rmed by the 1H NMR signal at d 5.31 for 24-H, instead
of that at d 4.12 in 1.1 Virtually unchanged chemical shifts
of 7-H and 27-H at d 3.37 and d 4.15, respectively, from 1
ruled out their acylations.
detected by chemiluminescence. After
a proteolytic
digestion, the peptidic fragments were subjected to amino
terminal sequence analysis, and the labeled sites were
con®ned within fragments of ten amino acid levels. In this
article, we report our design and synthesis of biotinylated
okadaic acids (Fig. 1) for SPR measurements, and then
photoaf®nity probe based on the result from the above.
Labeling experiments using this probe successfully labeled
PP2A, as well as a few proteins in crude extracts of a marine
sponge Halichondria okadai, known to contain high content
of okadaic acid. These new proteins are suspected to be
involved in self-resistance mechanism of the sponge against
okadaic acid.
Results and Discussion
27-O-Biotinylated okadaic acid 5 was synthesized from
methyl okadaate (10). After protection of the 7- and 24-
hydroxyl groups as TBDMS ethers, the disilyl ether 17
was then treated with N,N0-disuccinimidyl carbonate and
Synthesis of biotinylated okadaic acids
To insert a spacer between biotin and okadaic acid, which is

K. Konoki et al. / Tetrahedron 56 (2000) 9003±9014
9007
Figure 3. Dependency of the SPR sensorgram on the concentration of PP2A in the running buffer with the sensor chip with immobilized 3 (A) or 5 (B), and
their derived kinetic parameters (C). Experimental conditions are the same as in Fig. 2.
Et3N, and the mixed carbonate was reacted with 7 to give
18. Deprotection with aq. HF±AcOH and alkaline hydroly-
sis gave 5, where introduction of the biotin unit at 27-OH
of 5 to 1 gave the signals at d 3.33 and d 4.09 for 7-H and
24-H, respectively.
1
was con®rmed by the H NMR signal at d 5.32 for 27-H,
Each of the four biotin conjugates prepared above (2±5)
gave a single spot on silica gel thin-layer chromatogram
instead of that at d 4.08 in 1.1 The otherwise close spectrum

9008
K. Konoki et al. / Tetrahedron 56 (2000) 9003±9014
Scheme 2. (a) t-Butyl N-(5-aminopentyl)carbamate, EDC´HCl, HOBt, DMF, rt, 86%; (b) TFA±CH2Cl2 (1:1), rt, 71%; (c) N,N0-Disuccinimidyl carbonate,
Et3N, CH3CN, rt; (d) 22, Et3N, DMF, rt, quantitative in 2 steps; (e) 1 M aq. NaOH±THF (1:1), 408C, 75%.
(10% methanol/chloroform) at different Rf values. This and
their H NMR spectra justi®ed purity of these samples,
which were used in the following analysis.
con®rming this binding to be speci®c. C24-Biotin conjugate
(4) and C27-biotin conjugate (5) exhibited moderate
binding af®nity to PP2A. The af®nity of the immobilized
conjugates to PP2A was in the order of 3.5.4.2.
1
Surface plasmon resonance analysis
As shown in Fig. 3, this complex formation was dependent
on the concentration of PP2A. Kinetic analysis of the
sensorgrams revealed the association rate constant (ka)
between PP2A and the immobilized 3 and 5 to be
9.3£105 M21 s21 and 1.8£105 M21 s21, and their dissocia-
Each biotin conjugate was captured on a streptavidin-coated
sensor chip (SA) placed in a ¯ow cell of an SPR instrument
(BIACOREe X, Biacore AB) through binding between
biotin and streptavidin, and the unbound conjugate was
washed out by the running buffer (data not shown). After
an equilibrium was reached, PP2A was introduced to the
¯ow cell from the time zero onward, and then the interaction
between immobilized okadaic acid and the enzyme was
monitored as the change in resonance unit (Fig. 2).11
tion rate constants (kd) to be 9.6£1024 s21 and 8.4£1024 s21
,
respectively. They gave rise to the calculated dissociation
constants in equilibrium (Kd) of 1.0 nM for 3 and 4.8 nM for
5. Substantial close values of calculated Kd and maximum
binding (Rmax), i.e. the amount of the immobilized ligand,
regardless of varied PP2A concentrations warrant relia-
bility of these kinetic data. These results indicate that
these two biotinylated okadaic acids retained binding
af®nity to PP2A to different degrees,14 and the extent of
interaction was successfully evaluated by SPR, supporting
the previous observation on structure±activity relationships
in reference to the derivatized position. Thus this assay
system would be applicable to the binding studies of the
okadaic acid derivatives to other protein phosphatase
preparations in conjunction with their inhibitory action
and also the search for other binding proteins of okadaic
acid as described below.
In accordance with marked reduction in af®nity to PP2A
previously reported on C1 esters of okadaic acid (2), no
signi®cant change in sensorgram was observed for the
chip bearing the C1-biotin conjugate. On the other hand,
when the C7-biotin conjugate (3) was loaded on the sensor
chip, the change of resonance (Y-axis) kept increasing until
the PP2A solution was replaced with the running buffer at
180 s. The PP2A solution preincubated with 1 did not cause
any change in the refractive index on the sensorgram,
Photoaf®nity labeling
Based on the above results, a biotinyl photoaf®nity probe
2010a was introduced into 7-OH of okadaic acid (Scheme 2).
First to insert a spacer between okadaic acid and the probe,
20 was conjugated with t-butyl N-(5-aminopentyl)carba-
mate, and deprotection with tri¯uoroacetic acid gave 22.
Methyl okadaate (10) was activated with N,N0-disuccinimi-
dyl carbonate in the presence of Et3N, and then treated with
22. Deprotection in aqueous basic condition and puri®cation
by ¯ash chromatography afforded okadaic acid bearing the
photoaf®nity probe (24). The substitution at 7-OH was
con®rmed in the same manner as the above with observation
of the 7-H NMR signal at d 4.60.
Figure 4. Electrophoregram of photolabeled PP2A by photoaf®nity probe
24. A PP2A solution (100 mL) in the absence (lane 1) or presence (lane 2) of
okadaic acid 1 (12.5 mM) was incubated with 24 (330 nM) for 1 h, and
irradiated at 360 nm for 1 h. After the SDS-PAGE gel was blotted to a
nitrocellulose membrane, the membrane was immersed in T-PBS buffer
containing HRP-conjugated streptavidin for 1 h, and chemiluminescence
was exposed to an autoradiography ®lm.
After the compound 24 was incubated with PP2A in a buffer

K. Konoki et al. / Tetrahedron 56 (2000) 9003±9014
9009
Figure 5. Electrophoregram of photolabeled proteins in H. okadai by photoaf®nity probe 24. Proteins from crude extracts from H. okadai (60 g) were
separated by stepwise centrifugation to give: (A) three precipitates (P1±P3) and one supernatant (S3), (B) and protein solutions P1±P3 and S3 in the absence or
presence of okadaic acid (12.5 mM) were photolabeled in the same manner as in Fig. 4.
solution at 48C for 1 h, the reaction mixture was irradiated at
365 nm for 40 min. To identify possible labeling due to
nonspeci®c binding, the same procedure was repeated in
the presence of excess okadaic acid. Each mixture was solu-
bilized in aqueous SDS, and subjected to SDS-PAGE.
Proteins on the gel were blotted to a nitrocellulose
membrane, and biotin conjugates were bound to horseradish
peroxidase (HRP)-conjugated streptavidin. The membrane
was then incubated with a substrate of the enzyme, and the
chemiluminescence was exposed to a ®lm. As shown in
Fig. 4, the probe was found to photolabel PP2A (ca.
37 kDa for the catalytic subunit) in lane 1 but not in lane
2. The comparison between the two lanes indicated that the
binding of 24 to PP2A is completely competitive. Though
Nishiwaki et al. reported that methyl 7-O-(4-azidobenzoyl)-
27-[3H]okadaate labeled the catalytic subunit of PP2A, the
amino acid sequence of the labeled domain has not been
clari®ed.3b Since the present results were obtained by a
radiochemical-free detection of labeled proteins, LC-MS/
MS techniques will be applicable to precise identi®cation
of the labeled region.9,15
okadaic acid was originally isolated.1 As widely accepted
at the present, okadaic acid must not originate from the
sponge but from symbiotic or ingested microbe. Since
both phosphorylation and dephosphorylation of proteins
are inevitable process in cell signaling pathway, amino
acid sequences for the catalytic subunit of protein phos-
phatases are highly conserved.16 To consider toxicity of
okadaic acid, a simple question has been raised how the
sponge protects itself from such a potent phosphatase
inhibitor. Putting aside the biological roles of okadaic acid
in the sponge which has been obscure so far in spite of a lot
of investigations,17 we suspected the marine sponge has
binding proteins to reduce free concentration of okadaic
acid in their cells, and carried out photoaf®nity labeling
experiments by using crude extracts from the sponge.
Sixty grams of H. okadai was homogenized in Waring
blender, and centrifuged at 600£g for 10 min. The obtained
supernatant was successively centrifuged at 8000£g for
10 min and 75,600£g for 1 h (Fig. 5A). The fractionated
precipitates (P1±P3) and one supernatant (S3) were
subjected to photoaf®nity labeling experiments. As shown
in Fig. 5B, three proteins in S3 were speci®cally labeled by
the ligand, while labeling was not observed in the other
fractions. Ammonium sulfate precipitation, ion exchange
This photoaf®nity labeling was next applied to possible
identi®cation of okadaic acid-binding protein(s) hitherto
unknown in a sponge Halichondria okadai, from which

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K. Konoki et al. / Tetrahedron 56 (2000) 9003±9014
1
its tri¯uoroacetic salt (136.8 mg, quantitative): H NMR
chromatography, hydrophobic gel chromatography, and gel
®ltration chromatography so far made us purify two okadaic
acid binding proteins. Further characterizations of these
proteins, including sequential analysis, are currently under-
way and will be reported elsewhere in due course.
(CD3OD) d 4.45 (dd, Jˆ8.0, 5.5 Hz, 1H), 4.26 (dd,
Jˆ7.5, 4.5 Hz, 1H), 3.18±3.12 (m, 3H), 2.90±2.86 (m,
3H), 2.66 (d, Jˆ13.0 Hz, 1H), 2.16 (t, Jˆ7.5 Hz, 2H),
1.71±1.48 (m, 8H), 1.42±1.33 (m, 4H).
In conclusion at the present, we believe that the okadaic acid
photoaf®nity probe 24, together with 7-biotinylated okadaic
acid 3, are evidently versatile reagents to investigate
okadaic acid binding proteins.
N-(5-Biotinamidopentyl)okadaamide (2). A solution of
okadaic acid (1; 500 mg), 7 (1.0 mg), HOBt (1.0 mg),
EDC´HCl (1.0 mg) in DMF (50 mL) was treated with Et3N
(50 mL), and stirred for 4 h at room temperature. After the
solvent was removed under vacuum, the concentrate was
dissolved in H2O (500 mL), and extracted with EtOAc
(500 mL£3). The organic layer was concentrated, and
subjected to reversed-phase HPLC (YMC Pack ODS-AM
250, f 10£250 mm, 60% aq. CH3CN). The eluate at 30±
Experimental
Materials
1
33 mL were collected based on absorption at 210 nm: H
The marine sponge Halichondria okadai was collected off at
the coast of Aburatsubo Bay in Kanagawa Prefecture.
Okadaic acid was isolated from the sponge as described in
the literature.1b Protein phosphatase 2A was purchased from
Wako Pure Chemicals. BIACOREe X (Biacore AB,
Uppsala) was used as the surface plasmon resonance
apparatus. Sensor plates SA, with preimmobilized strepta-
vidin, were purchased from Biacore AB. Stepwise syntheses
NMR (CDCl3) d 5.45 (dd, Jˆ15.0, 8.0 Hz, 1H), 5.39 (s,
1H), 5.35 (dd, Jˆ15.0, 9.0 Hz, 1H), 5.31 (s, 1H), 5.04 (s,
1H), 4.51 (m, 2H), 4.31 (dd, Jˆ7.5, 4.0 Hz, 1H), 4.09±4.03
(m, 2H), 3.97±3.93 (m, 2H), 3.72 (m, 1H), 3.67±3.60 (m,
2H), 3.53 (m, 1H), 3.38±3.28 (m, 4H), 3.26 (dd, Jˆ10.5,
2.0 Hz, 1H), 3.15 (td, Jˆ8.0, 4.5 Hz, 1H), 3.08 (br, 2H),
2.91 (dd, Jˆ12.5, 5.0 Hz, 1H), 2.71 (d, Jˆ13.0 Hz, 1H),
2.33 (t, Jˆ7.5 Hz, 2H), 2.30±1.20 (m, 49H), 1.04 (d,
Jˆ6.5 Hz, 3H), 0.94 (d, Jˆ6.5 Hz, 3H), 0.90 (d,
Jˆ7.0 Hz, 3H); HRMS (FAB) calcd for C59H94O14SN4Na
([M1Na]1) 1137.6385, found 1137.6371.
1
of biotinylated okadaic acids were monitored by H NMR
spectroscopy (500 MHz) on Brucker DRX-500 or JEOL
A-500, and FABMS on JEOL SX102L or ESIMS on Micro-
mass Q-tof 2. Chemical shifts in the NMR data are refer-
enced to CHCl3 at d 7.24, or CHD2OD at d 3.30. Photo-
crosslinking was performed by an XX-15L lamp (UVP,
Inc.). Analytical thin-layer chromatography (TLC) was
performed using silica gel 60 F254 precoated plates
(0.25 mm thickness; E. Merck). Column chromatography
in the normal phase was on 230±400 mesh silica gel 60 (E.
Merck), and that in the reversed-phase on YMC gel ODS-AM
120-S50 (YMC). Other reagents and solvents were used as
supplied from the vendors unless otherwise noted.
p-Bromophenacyl okadaate (8). A solution of 1 (3.0 mg)
and bromophenacyl bromide (2.0 mg) in 4.6% Et3N/acetone
(500 mL) was stirred at 408C for 3 h. Concentration and ¯ash
chromatography (0±5% MeOH/CHCl3) gave p-bromo-
1
phenacyl okadaate 8 (3.5 mg, 94%): H NMR (CDCl3) d
7.76 (dt, Jˆ8.5, 2.0 Hz, 2H), 7.64 (dt, Jˆ8.5, 2.0 Hz, 2H),
5.56 (dd, Jˆ15.0, 7.5 Hz, 1H), 5.50 (d, Jˆ16.5 Hz, 1H),
5.43 (dd, Jˆ15.0, 7.5 Hz, 1H), 5.34 (s, 1H), 5.32 (s, 1H),
5.23 (d, Jˆ16.5 Hz, 1H), 5.03 (s, 1H), 4.41 (td, Jˆ8.0,
7.0 Hz, 1H), 4.13±4.02 (m, 3H), 3.91 (d, Jˆ9.5 Hz, 1H),
3.74 (m, 1H), 3.65 (td, Jˆ11.0, 3.0 Hz, 1H), 3.59±3.50 (m,
2H), 3.46±3.38 (m, 2H), 3.26 (dd, Jˆ10.0, 2.0 Hz, 1H),
2.30±1.23 (m, 37H), 1.03 (d, Jˆ7.0 Hz, 3H), 0.99 (d,
Jˆ6.5 Hz, 3H), 0.90 (d, Jˆ7.0 Hz, 3H); FABMS 1023.4,
1025.4 [M1Na]1.
Synthesis
t-Butyl 5-biotinamidopentylcarbamate (6). A solution of
d-biotin (150 mg, 610 mmol), t-butyl N-(5-aminopentyl)-
carbamate (148 mg, 732 mmol), HOBt (100 mg,
732 mmol), and EDC´HCl (141 mg, 732 mmol) in DMF
(2.5 mL) was treated with Et3N (1 mL), and stirred at
room temperature for 3 h. After the solution was concen-
trated under vacuum, it was diluted with EtOAc (100 mL),
and washed with brine (20 mL£2). Drying over MgSO4,
concentration, and ¯ash chromatography (5±10% MeOH/
CHCl3) gave 6 (136 mg, 52%): 1H NMR (CDCl3) d 6.42 (br,
1H), 6.25 (br, 1H), 5.60 (br, 1H), 4.69 (br, 1H), 4.49 (dd,
Jˆ7.7, 5.0 Hz, 1H), 4.29 (dd, Jˆ7.5, 4.8 Hz, 1H), 3.19 (td,
Jˆ6.7, 6.2 Hz, 2H), 3.12 (td, Jˆ7.5, 4.6 Hz, 1H), 3.08 (brtd,
Jˆ6.6, 6.3 Hz, 2H), 2.88 (dd, Jˆ12.8, 4.9 Hz, 1H), 2.71 (d,
Jˆ12.8 Hz, 1H), 2.18 (t, Jˆ7.5 Hz, 2H), 1.75±1.61 (m, 6H),
1.51±1.43 (m, 6H), 1.41 (s, 9H).
p-Bromophenacyl 7-O-(5-biotinamidopentylcarbamoyl)-
okadaate (9). A solution of 8 (1 mg) and N,N0-disuccini-
midyl carbonate (2 mg) in CH3CN (50 mL) was treated with
Et3N (5 mL), and stirred at room temperature for 5 h. After
the reaction was quenched with saturated aq. NaHCO3
(20 mL), the solvent was removed under stream of N2 gas.
The residue was suspended in saturated aq. NaHCO3
(500 mL), and extracted with EtOAc (500 mL£3). The
organic layer was washed with brine (200 mL), and the
solvent was evaporated under vacuum. The mixed carbonate
in DMF (50 mL) was added dropwise with 5-biotinamido-
amylamine (7; 10 mg) in 2.8% Et3N/DMF (150 mL), and the
mixture was stirred at room temperature for 12 h. After the
solvent was evaporated under vacuum, the residue
suspended in saturated aq. NaHCO3 (500 mL) was extracted
with EtOAc (500 mL£3). Washing with brine (200 mL),
concentration, and ¯ash chromatography (0±10% MeOH/
CHCl3) gave 9: 1H NMR (CDCl3) d 7.75 (dt, Jˆ8.5, 2.0 Hz,
2H), 7.63 (dt, Jˆ8.5, 2.0 Hz, 2H), 5.62 (dd, Jˆ15.0, 7.5 Hz,
5-Biotinamidopentylamine (7). To a solution of 6
(136 mg) in CH2Cl2 (2.0 mL), tri¯uoroacetic acid
(1.0 mL) was added dropwise at 08C, and the mixture was
stirred at room temperature for 2 h. After the solvent was
removed under vacuum, reversed-phase chromatography
(0±50% aq. MeOH) followed by lyophilization gave 7 as

K. Konoki et al. / Tetrahedron 56 (2000) 9003±9014
9011
1H), 5.50 (d, Jˆ16.5 Hz, 1H), 5.45 (dd, Jˆ15.0, 8.5 Hz,
1H), 5.34 (s, 1H), 5.32 (s, 1H), 5.23 (d, Jˆ16.5 Hz, 1H),
5.03 (s, 1H), 4.60 (dd, Jˆ11.8, 4.2 Hz, 1H), 4.54 (dd, Jˆ8.0,
5.5 Hz, 1H), 4.34 (dd, Jˆ7.5, 4.5 Hz, 1H), 4.24 (d,
Jˆ10.0 Hz, 1H), 4.13 (d, Jˆ10.0 Hz, 1H), 4.06 (m, 1H),
3.91 (d, Jˆ10.0 Hz, 1H), 3.85±3.45 (m, 4H), 3.45±3.35
(m, 3H), 3.26 (dd, Jˆ10.0, 2.0 Hz, 1H), 3.20±3.12 (m,
3H), 2.91 (dd, Jˆ12.5, 5.0 Hz, 1H), 2.72 (d, Jˆ13.0 Hz,
1H), 2.36±1.18 (m, 51H), 1.03 (d, Jˆ7.0 Hz, 3H), 0.99 (d,
Jˆ6.5 Hz, 3H), 0.90 (d, Jˆ7.0 Hz, 3H); FABMS 1377.7,
1379.7 [M1Na]1.
A solution of 12 in pyridine (100 mL) was treated with Ac2O
(5 mL) at 08C, and stirred at room temperature for 14 h.
After the reaction was quenched with MeOH (1 mL), the
solution was diluted with H2O (500 mL), and extracted
with 25% EtOAc/hexane (500 mL£3). Concentration and
¯ash chromatography (25% EtOAc/hexane) gave 13
1
(470 mg, 68% in 3 steps): H NMR (CDCl3) d 7.92 (d,
Jˆ7.0 Hz, 2H), 7.50 (t, Jˆ7.5 Hz, 1H), 7.39 (t, Jˆ7.5 Hz,
2H), 5.75 (dd, Jˆ15.5, 7.5 Hz, 1H), 5.55 (d, Jˆ16.0, 7.0 Hz,
1H), 5.52 (t, Jˆ10.0 Hz, 1H), 5.36 (s, 1H), 5.21 (s, 1H), 5.02
(s, 1H), 4.92 (dd, Jˆ11.5, 4.5 Hz, 1H), 4.62 (br, 1H), 4.51
(td, Jˆ9.0, 7.0 Hz, 1H), 4.12±4.02 (m, 4H), 3.83 (m, 1H),
3.74 (s, 3H), 3.72±3.52 (m, 3H), 3.38 (t, Jˆ10 Hz, 1H), 3.26
(dd, Jˆ10.0, 2.0 Hz, 1H), 2.06 (s, 3H), 2.42±1.24 (m, 37H),
1.13 (d, Jˆ7.0 Hz, 3H), 1.04 (d, Jˆ6.5 Hz, 3H), 0.90 (s,
3H), 0.87 (d, Jˆ6.5 Hz, 3H), 0.08 (s, 3H) 0.05 (s, 3H);
FABMS m/z 1101.5 [M1Na]1.
7-O-(5-Biotinamidopentylcarbamoyl)okadaic acid (3). A
solution of 9 in AcOH (100 mL) was stirred over activated
Zn powder (20 mg) at room temperature for 5 h. The Zn
powder was ®ltered, and the solvent was evaporated from
the ®ltrate. The residue suspended in H2O (500 mL) was
extracted with 1% AcOH/EtOAc (500 mL). Concentration
and ¯ash chromatography (CHCl3±MeOH±AcOH,
87:10:3) gave 3 (625 mg, 54% in 3 steps): 1H NMR
(CDCl3, 500 MHz) d 5.63 (dd, Jˆ15.0, 8.5 Hz, 1H),
5.52±5.46 (m, 2H), 5.36 (s, 1H), 5.23 (s, 1H), 5.03 (s,
1H), 4.57 (dd, Jˆ12.0, 4.5 Hz, 1H), 4.52±4.45 (m, 2H),
4.31 (dd, Jˆ7.5, 4.5 Hz, 1H), 4.12±4.02 (m, 3H), 3.91 (d,
Jˆ10.0 Hz, 1H), 3.75±3.45 (m, 4H), 3.42±3.32 (m, 3H),
3.27 (dd, Jˆ10.0, 2.0 Hz, 1H), 3.20±3.10 (m, 3H), 2.91
(dd, Jˆ12.5, 5.0 Hz, 1H), 2.72 (d, Jˆ13.0 Hz, 1H), 2.36±
1.18 (m, 51H), 1.03 (d, Jˆ6.5 Hz, 3H), 0.99 (d, Jˆ6.5 Hz,
3H), 0.90 (d, Jˆ7.0 Hz, 3H); HRMS (FAB) calcd
for C60H94O16SN4Na ([M1Na]1) 1181.6283, found
1181.6283.
24-O-(5-Biotinamidopentylcarbamoyl)okadaic acid (4).
A solution of 13 (470 mg) in 47% aq. HF±AcOH
(100 mL, 1:4) was stirred at room temperature for 75 min.
The reaction was quenched with saturated aq. NaHCO3
(500 mL), and the product was extracted with CHCl3
(500 mL£3). Flash chromatography of the concentrate
(25% AcOEt/hexane) gave alcohol 14 (360 mg, 85%).
A solution of alcohol 14 (360 mg) and N,N0-disuccinimidyl
carbonate (40 mg) in CH3CN (100 mL) was treated with
Et3N (20 mL), and stirred at room temperature for 24 h.
After evaporation of the solvent under stream of N2 gas,
the residue suspended in saturated aq. NaHCO3 (750 mL)
was extracted with EtOAc (750 mL£3). The organic layer
was washed with brine (500 mL), and the solvent was
evaporated under vacuum. To a solution of the concentrate
in DMF (20 mL) was added a solution of amine 7 (10 mg) in
20% Et3N/DMF (100 mL), and the mixture was stirred at
room temperature for 7 h. After the solvent was removed
under stream of N2 gas, the residue was partitioned between
H2O (750 mL) and EtOAc (750 mL). Flash chromatography
(5% MeOH/CHCl3) of the organic concentrate afforded 15
(425 mg, 85% in 2 steps).
Methyl okadaate (10). A solution of 1 (3.0 mg) in benzene±
MeOH (150 mL, 1:1) was treated with 2.0 M TMSCHN2 in
hexane (18 mL), and stirred at room temperature for 10 min.
The reaction was terminated with AcOH (3 drops). Concen-
tration and ¯ash chromatography (5% MeOH/CHCl3) gave
methyl ester 10 (3.2 mg, quantitative): 1H NMR (CDCl3) d
5.54 (dd, Jˆ15.0, 8.0 Hz, 1H), 5.48 (dd, Jˆ15.0, 7.0 Hz,
1H), 5.38 (s, 1H), 5.28 (s, 1H), 5.04 (s, 1H), 4.46 (dd,
Jˆ8.5, 7.0 Hz, 1H), 4.11±4.01 (m, 2H), 3.95±3.88 (m,
2H), 3.79 (s, 3H), 3.67±3.51 (m, 4H), 3.42±3.35 (m, 2H),
3.27 (dd, Jˆ10.0, 2.0 Hz, 1H), 2.32±1.22 (m, 37H), 1.04 (d,
Jˆ6.5 Hz, 3H), 1.01 (d, Jˆ6.5 Hz, 3H), 0.90 (d, Jˆ6.5 Hz,
3H); FABMS 841.5 [M1Na]1.
A solution of 15 (425 mg) in THF (50 mL) was added
with 1 M aq. NaOH (50 mL) at 08C, and stirred at
408C for 3 h. It was cooled to 08C, and then neutralized
with saturated aq. NH4Cl (500 mL). The solution was
extracted with 1% AcOH/EtOAc (500 mL£5), and the
solvent was removed to give 16: 1H NMR (CDCl3) d
7.91 (d, Jˆ7.0 Hz, 2H), 7.51 (t, Jˆ7.5 Hz, 1H), 7.38
(t, Jˆ7.5 Hz, 2H), 6.38 (br, 1H), 6.32 (br, 1H), 5.97
(br, 1H), 5.67 (dd, Jˆ15.0, 8.5 Hz, 1H), 5.57 (t,
Jˆ10.0 Hz, 1H), 5.51 (dd, Jˆ15.0, 8.5 Hz, 1H), 5.28±5.20
(m, 3H), 5.18 (s, 1H), 5.06 (br, 1H), 5.01 (s, 1H), 4.90
(dd, Jˆ11.5, 4.5 Hz, 1H), 4.52 (dd, Jˆ7.5, 4.5 Hz, 1H),
4.42 (td, Jˆ9.0, 7.0 Hz, 1H), 4.38 (d, Jˆ7.5, 4.5 Hz, 1H),
4.20 (t, Jˆ10.5 Hz, 1H), 4.09 (d, Jˆ10 Hz, 1H), 3.82
(td, Jˆ10.0, 4.5 Hz, 1H), 3.51 (m, 1H), 3.45 (m, 1H),
3.44 (t, Jˆ10.0 Hz, 1H), 3.29 (dd, Jˆ11.0, 2.0 Hz, 1H),
3.15 (brtd, Jˆ6.5, 5.5 Hz), 3.08 (m, 1H), 2.90 (dd,
Jˆ13.0, 5.0 Hz, 1H), 2.69 (d, Jˆ13.0 Hz, 1H), 2.32 (t,
Jˆ7.5 Hz, 2H), 2.07 (s, 3H), 2.25±1.15 (m, 49H), 1.06 (d,
Jˆ6.5 Hz, 3H), 1.02 (d, Jˆ6.5 Hz, 3H), 0.86 (d, Jˆ7.0 Hz,
3H).
Methyl 27-O-acetyl-7-O-benzoyl-24-O-(t-butyldimethyl-
silyl)okadaate (13). A solution of methyl ester 10
(500 mg) and Et3N (5 mL) in CH2Cl2 (50 mL) was treated
with benzoyl chloride (5 mL) in CH2Cl2 (50 mL), and stirred
at room temperature for 20 h. After the solution was cooled
to 08C, MeOH (20 mL) was added to terminate the reaction.
It was diluted with CHCl3 (500 mL), washed with H2O
(500 mL), and the solvent was evaporated to afford 11 to
be directly used in the next step. To a solution of the above
concentrate (11) and imidazole (11.5 mg) in DMF (100 mL),
t-butyldimethylsilyl chloride (12.6 mg) was added portion-
wise at 08C, and the solution was stirred at room tempera-
ture for 1 h. The reaction was terminated with H2O
(500 mL), and the mixture was extracted with 25%
EtOAc/hexane (500 mL). Evaporation of the solvent under
vacuum gave 12, which was again used in the following
reaction without further puri®cation.

9012
K. Konoki et al. / Tetrahedron 56 (2000) 9003±9014
A solution of the methyl ester 16 in 5% w/v NaOH/MeOH
(100 mL) was stirred at 608C for 3 h. The solution was
neutralized with AcOH (3 drops) at 08C, and the solvent
was removed under stream of N2. The concentrate was
diluted with H2O (500 mL), extracted with EtOAc
(500 mL£5), and the solvent was evaporated under vacuum
(dd, Jˆ11.0, 4.0 Hz, 1H), 3.36 (t, Jˆ9.5 Hz, 1H), 3.28±3.23
(m, 3H), 3.20±3.13 (m, 3H), 2.93 (dd, Jˆ13.0, 5.0 Hz, 1H),
2.72 (d, Jˆ12.0 Hz, 1H), 2.39±2.30 (m, 3H), 2.25±1.20 (m,
48H), 1.07 (m, 6H), 0.91 (m, 9H), 0.86 (d, Jˆ6.5 Hz, 3H),
0.77 (s, 9H), 0.08 (s, 3H), 0.05 (s, 3H), 20.05 (s, 3H), 20.09
(s, 3H); FABMS m/z 1423.9 [M1Na]1.
1
to afford 4 (260 mg, 71% in 2 steps): H NMR (CDCl3) d
6.43 (br, 2H), 5.93 (br, 1H), 5.82 (br, 1H), d 5.65 (dd,
Jˆ15.0, 9.0 Hz, 1H), 5.42 (dd, Jˆ15.0, 9.0 Hz, 1H), 5.33
(m, 1H), 5.31 (d, Jˆ9.0 Hz)), 5.18 (s, 1H), 5.05 (s, 1H), 4.99
(s, 1H), 4.50 (dd, Jˆ7.5, 4.5 Hz, 1H), 4.31 (m, 2H), 4.15 (t,
Jˆ9.5 Hz, 1H), 4.02 (t, Jˆ11.0 Hz, 1H), 3.91 (d,
Jˆ10.5 Hz, 1H), 3.70±3.60 (m, 3H), 3.52±3.43 (m, 4H),
3.37 (dd, Jˆ11.5, 4.5 Hz, 1H), 3.27 (dd, Jˆ11.0, 2.0 Hz,
1H), 3.14 (m, 1H), 3.06 (m, 1H), 2.90 (dd, Jˆ13.0,
5.0 Hz, 1H), 2.69 (d, Jˆ13.0 Hz, 1H), 2.33 (t, Jˆ7.5 Hz,
2H), 2.30±1.20 (m, 49H), 1.07 (d, Jˆ6.5 Hz, 3H), 0.99 (d,
Jˆ6.5 Hz, 3H), 0.90 (d, Jˆ7.0 Hz, 3H); HRMS (FAB) calcd
for C60H94O16SN4Na ([M1Na]1) 1181.6283, found
1181.6293.
27-O-(5-Biotinamidopentylcarbamoyl)okadaic acid (5).
A solution of 18 in 47% aq. HF±CH3CN (40 mL, 1:4) was
stirred at room temperature for 45 min, and the solution was
neutralized with saturated aq. NaHCO3 (750 mL). Extrac-
tion with EtOAc (750 mL£4) and concentration gave 19,
which was used in the following reaction without further
puri®cation: 1H NMR (CDCl3) d 5.56 (dd, Jˆ15.0, 8.0 Hz,
1H), 5.47 (dd, Jˆ15.0, 8.0 Hz, 1H), 5.38 (s, 1H), 5.34 (m,
1H), 5.30 (s, 1H), 5.04 (s, 1H), 4.51±4.47 (m, 2H), 4.32 (dd,
Jˆ7.5, 5.0 Hz, 1H), 4.12±4.07 (m, 2H), 3.94 (t, Jˆ10.5 Hz,
1H), 3.79 (m, 1H), 3.72 (s, 3H), 3.65±3.45 (m, 3H), 3.37±
3.34 (m, 2H), 3.30±3.15 (m, 5H), 3.09 (m, 1H), 2.91 (dd,
Jˆ13.0, 5.0 Hz, 1H), 2.71 (d, Jˆ13.0 Hz, 1H), 2.33 (t,
Jˆ7.5 Hz, 2H), 2.30±1.20 (m, 49H), 1.06 (d, Jˆ6.5 Hz,
3H), 1.01 (d, Jˆ6.5 Hz, 3H), 0.86 (d, Jˆ7.0, 3H);
FABMS m/z 1195.6 [M1Na]1.
Methyl 7-O,24-O-bis(t-butyldimethylsilyl)okadaate (17).
A solution of methyl okadaate (10; 2.0 mg) and imidazole
(11.5 mg) in DMF (100 mL) was added portionwise with
t-butyldimethylsilyl chloride (12.6 mg) at 08C, and stirred
at room temperature for 2 h. The reaction was quenched
with H2O (1.0 mL), and extracted with 25% EtOAc/hexane
(700 mL£4). Concentration and ¯ash chromatography (25%
EtOAc/hexane) gave 17 (2.9 mg, 95%): 1H NMR (CDCl3) d
5.70 (dd, Jˆ15.5, 7.5 Hz, 1H), 5.49 (dd, Jˆ15.5, 7.0 Hz,
1H), 5.36 (s, 1H), 5.12 (s, 1H), 5.00 (s, 1H), 4.89 (s, 1H),
4.47 (td, Jˆ7.5, 7.0 Hz, 1H), 4.10±4.04 (m, 2H), 3.95±3.88
(m, 2H), 3.73 (m, 1H), 3.72 (s, 3H), 3.65 (td, Jˆ11.5,
2.5 Hz, 1H), 3.54±3.50 (m, 2H), 3.45±3.41 (m, 2H), 3.27
(dd, Jˆ10.0, 2.0 Hz, 1H), 2.35±1.23 (m, 37H), 1.07 (d,
Jˆ6.5 Hz, 3H), 1.04 (d, Jˆ7.0 Hz, 3H), 0.92 (s, 9H), 0.90
(d, Jˆ7.0 Hz, 3H), 0.77 (s, 9H), 0.08 (s, 3H), 0.05 (s, 3H),
20.04 (s, 3H), 20.08 (s, 3H); FABMS m/z 1069.5
[M1Na]1.
To a solution of 19 in THF (500 mL) was added 1 M aq.
NaOH (500 mL), and the mixture was stirred at 408C for 3 h.
The solution was neutralized with saturated aq. NH4Cl
(750 mL) at 08C, and extracted with 1% AcOH/EtOAc
(750 mL£4). The solvent was removed under vacuum to
give 5 (480 mg, 71% in 2 steps): 1H NMR (CDCl3,
500 MHz) d 5.65 (dd, Jˆ15.0, 8.0 Hz, 1H), 5.43 (dd,
Jˆ15.0, 8.0 Hz, 1H), 5.40 (s, 1H), 5.36 (m, 1H), 5.29 (s,
1H), 5.07 (s, 1H), 4.57 (td, Jˆ7.5, 4.5 Hz, 1H), 4.49 (dd,
Jˆ7.5, 5.0 Hz, 1H), 4.37 (dd, Jˆ7.5, 5.0 Hz, 1H), 4.11 (d,
Jˆ10.0 Hz, 1H), 4.09 (d, Jˆ10.0 Hz, 1H), 3.94 (t,
Jˆ11.5 Hz, 2H), 3.77 (m, 1H), 3.64±3.51 (m, 3H), 3.38±
3.27 (m, 5H), 3.23 (td, Jˆ6.6, 6.3, 2H), 3.14 (m, 1H), 2.91
(dd, Jˆ13.0, 5.0 Hz, 1H), 2.71 (d, Jˆ13.0 Hz, 1H), 2.33 (t,
Jˆ7.5 Hz, 2H), 2.20±1.20 (m, 49H), 1.06 (d, Jˆ6.5 Hz,
3H), 1.01 (d, Jˆ6.5 Hz, 3H), 0.85 (d, Jˆ7.0 Hz, 3H);
HRMS (FAB) calcd for C60H94O16N4SNa ([M1Na]1)
1181.6283, found 1181.6268.
Methyl 27-O-(5-biotinamidopentylcarbamoyl)-7-O,24-
O-bis(t-butyldimethylsilyl)okadaate (18). A solution of
17 (2.9 mg) and N,N0-disuccinimidyl carbonate (20 mg) in
CH3CN (100 mL) was treated with Et3N (20 mL), and stirred
at room temperature for 18 h. After evaporation under
stream of N2, the residue was diluted with saturated aq.
NaHCO3, and extracted with EtOAc (1 mL£5). Washing
with brine (1.0 mL) and concentration gave a mixed
carbonate.
5-[2-(8-Biotinamido-3,6-dioxaoctyl)-4-(3-tri¯uoromethyl-
3H-diazirin-3-yl)benzamido]pentylamine (22). A solu-
tion of benzoic acid 20 (5 mg, 82 mmol), t-butyl N-(5-
aminopentyl)carbamate (20 mg, 99 mmol), HOBt (13 mg,
95 mmol) and EDC (30 mg, 156 mmol) in DMF (150 mL)
was treated with Et3N (20 mL, 156 mmol) at 08C, and stirred
at room temperature for 72 h. After the solvent was
evaporated under vacuum, the residue dissolved in
EtOAc (1.0 mL) was washed with brine (1.0 mL).
Concentration and ¯ash chromatography (CHCl3±MeOH±
AcOH, 80:17:3) gave 21 (5.63 mg, 86%): 1H NMR
(CDCl3) d 8.17 (d, Jˆ8.0 Hz, 1H), 8.00 (br, 1H), 6.88
(d, Jˆ8.5 Hz, 1H), 6.67 (s, 1H), 6.32 (br, 1H), 5.89
(br, 1H), 5.10 (br, 1H), 4.77 (br, 1H), 4.45 (dd, Jˆ8.0,
5.0 Hz, 1H), 4.30±4.20 (m, 3H), 3.87 (t, Jˆ4.5 Hz,
2H), 3.67 (br, 2H), 3.61 (br, 2H), 3.51 (t, Jˆ5.0 Hz, 2H),
3.45±3.35 (m, 4H), 3.10 (br, 3H), 2.88 (dd, Jˆ13.0, 5.0 Hz,
1H), 2.70 (d, Jˆ12.5 Hz, 1H), 2.15 (t, Jˆ7.5 Hz, 2H),
1.75±1.45 (m, 12H), 1.40 (s, 9H); FABMS m/z 810.38
[M1Na]1.
To a solution of the above concentrate in DMF (20 mL) was
added 5-biotinamidopentylamine (7; 10 mg) in Et3N±DMF
(90 mL, 1:8). After stirred at room temperature for 43 h, the
solvent was evaporated under stream of N2 gas, and the
residue suspended in H2O (1.0 mL) was extracted with
EtOAc (1.0 mL£5). Concentration and ¯ash chromato-
graphy (5% MeOH/CHCl3) gave 18 (900 mg, 28% in 2
1
steps): H NMR (CDCl3) d 5.69 (dd, Jˆ15.5, 7.5 Hz, 1H),
5.50 (dd, Jˆ15.5, 7.5 Hz, 1H), 5.40±5.32 (m, 2H), 5.12 (s,
1H), 5.00 (s, 1H), 4.51 (dd, Jˆ7.5, 5.0 Hz, 1H), 4.45 (td,
Jˆ15.0, 7.5 Hz, 1H), 4.33 (dd, Jˆ7.5, 5.0 Hz, 1H), 4.09±
4.04 (m, 2H), 3.92 (brt, Jˆ11.5 Hz, 1H), 3.75 (m, 1H), 3.70
(m, 1H), 3.62 (td, Jˆ11.5, 2.0 Hz, 1H), 3.53 (m, 1H), 3.42

K. Konoki et al. / Tetrahedron 56 (2000) 9003±9014
9013
A solution of amine 21 in CH2Cl2 (150 mL) was treated with
tri¯uoroacetic acid (150 mL) at 08C, and stirred at room
temperature for 30 min. Concentration and ¯ash chromato-
graphy (CHCl3±MeOH±AcOH, 80:17:3) gave 22 (3.45 mg,
71%): 1H NMR (CD3OD) d 7.96 (d, Jˆ8.5 Hz, 1H), 7.03 (d,
Jˆ8.0 Hz, 1H), 6.86 (s, 1H), 4.46 (dd, Jˆ7.5, 5.0 Hz, 1H),
4.30±4.20 (m, 3H), 3.90 (br, 2H), 3.71 (br, 2H), 3.63 (br,
2H), 3.51 (br, 2H), 3.42 (br, 2H), 3.35±3.25 (m, 2H), 3.15
(m, 1H), 2.95±2.87 (m, 3H), 2.69 (d, Jˆ12.5 Hz, 1H), 2.17
(t, Jˆ7.5 Hz, 2H), 1.75±1.40 (m, 12H).
3H), 0.90 (d, Jˆ6.50 Hz, 3H); ESIMS m/z 1517.6
[M2H]2.
SPR analysis on interaction of immobilized okadaic
acids with protein phosphatase 2A
SPR analysis carried out on BIACOREe X were as follow-
ing. A streptavidin-immobilized sensor chip was loaded in a
¯ow cell, and contacted with a solution of a biotinylated
okadaic acid (4.3 nmol) in a running buffer (100 mL)
consisting of 150 mM NaCl, 3.4 mM EDTA, 0.01%
Tweenw 20, and 10 mM Hepes±NaOH (pH 7.4) at a ¯ow
rate of 5.0 mL min21 for 180 s. After a protein solution of a
known concentration in the running buffer containing 0.02%
(w/v) BSA was injected at 10 mL min21 for 180 s, dissocia-
tion of the bound PP2A was monitored for 180 s by washing
the sensor chip with the same buffer. The sensor chip was
regenerated to be free of bound PP2A by passing 100 mM
Na2CO3 at a ¯ow rate of 5.0 mL min21 for 180 s. All experi-
ments were carried out at 258C.
Methyl 7-O-[5-[2-(8-biotinamido-3,6-dioxaoctyl)-4-(3-tri-
¯uoromethyl-3H-diazirin-3-yl)benzamido]pentylcarba-
moyl]okadaate (23). A solution of methyl okadaate (10;
500 mg) and N,N0-disuccinimidyl carbonate (8 mg,
31 mmol) in CH3CN (100 mL) was treated with Et3N
(20 mL, 156 mmol) at room temperature, and stirred for
3.5 h. After the reaction was quenched with saturated aq.
NaHCO3 (100 mL), the solution was concentrated under
stream of N2 gas, diluted again with saturated aq.
NaHCO3 (500 mL), and extracted with EtOAc
(750 mL£5). Washing with brine (500 mL) and concen-
tration gave a mixed carbonate.
Data transformation and interaction analyses were
performed by BIAevaluatione software equipped to the
instrument, with algorithms for numerical integration and
global analysis linked to the software. With this software,
nonlinear curve ®ttings were performed to calculate kinetic
parameters of ka and kd.
To a solution of the mixed carbamate in DMF (50 mL) was
added 22 (3.4 mg) in 10% Et3N/DMF (110 mL), and the
mixture was stirred at room temperature for 29 h. After
the solvent was evaporated under vacuum, the concentrate
was diluted with H2O (500 mL), and extracted with EtOAc
(700 mL£3). Concentration and ¯ash chromatography
Photoaf®nity labeling experiments
1
(10% MeOH/CHCl3) gave 23 (900 mg, quantitative): H
A protein solution (100 mL) was incubated with 24
(330 nM) for 1 h, and irradiated at 360 nm by an XX-15L
lamp (UVP, Inc.) for 1 h. After the proteins were denatured
with a buffer consisting of 2.0% SDS, 10% glycerol, 5.0%
w/v dithiothreitol, bromophenol blue (a tracking dye,
10 m mL21), and 50 mM Tris±HCl (pH 6,8), they were
subjected to SDS-PAGE (Ready Gel J 10±20%, BioRad),
and electroblotted to a nitrocellulose membrane (Hybond
ECL, Amersham Pharmacia). The membrane was blocked
by 2.0% BSA in T-PBS buffer (PBS buffer containing 0.1%
Tweenw 20) for 1 h, and washed with the buffer (15 min£1,
and 5 min£2). It was then immersed in T-PBS buffer
containing horseradish peroxidase (HRP)-conjugated
streptavidin (Amersham Pharmacia) for 1 h, and washed
with the same buffer (15 min£1, and 5 min£2). After the
membrane was reacted with a substrate for the enzyme in
aq. H2O2 (ECL Western Blotting Reagent, Amersham
Pharmacia), chemiluminescence was exposed to an auto-
radiography ®lm (Hyper®lm-ECL, Amersham Pharmacia)
for a desired period, and the ®lm was developed as per
manufacturer's recommendation.
NMR (CDCl3) d 8.17 (d, Jˆ8.0 Hz, 1H), 6.88 (d,
Jˆ8.0 Hz, 1H), 6.68 (s, 1H), 5.58 (dd, Jˆ14.5, 7.5 Hz,
1H), 5.49 (dd, Jˆ14.5, 7.5 Hz, 1H), 5.37 (s, 1H), 5.23 (s,
1H), 5.03 (s, 1H), 4.59 (dd, Jˆ12.0, 4.5), 4.51±4.45 (m,
2H), 4.30 (br, 1H), 4.25 (t, Jˆ4.5 Hz, 2H), 4.10 (d,
Jˆ10 Hz, 1H), 4.06 (t, 9.5 Hz, 1H), 4.00 (t, Jˆ10 Hz,
1H), 3.93 (d, Jˆ10.0 Hz, 1H), 3.88 (t, Jˆ4.5 Hz, 2H),
3.77 (s, 3H), 3.69±3.37 (m, 15H), 3.27 (dd, Jˆ10.0,
2.0 Hz, 1H), 3.15±3.02 (m, 3H), 2.90 (dd, Jˆ13.0, 5.0 Hz,
1H), 2.71 (d, Jˆ12.5 Hz, 1H), 2.33 (t, Jˆ7.5 Hz, 2H), 2.40±
1.20 (m, 40H), 1.04 (d, Jˆ6.5 Hz, 3H), 1.00 (d, Jˆ7.0 Hz,
3H), 0.91 (d, Jˆ6.50 Hz, 3H).
7-O-[5-[2-(8-Biotinamido-3,6-dioxaoctyl)-4-(3-tri¯uoro-
methyl-3H-diazirin-3-yl)benzamido]pentylcarbamoyl]-
okadaic acid (24). A solution of methyl ester 23 (950 mg) in
THF (100 mL) was treated with 1 M aq. NaOH (100 mL),
and stirred at 408C for 5 h. The solution was neutralized
with AcOH (5 drops) at 08C, and the solvent was removed
under stream of N2 gas. The concentrate was diluted with
H2O (500 mL), extracted with EtOAc (750 mL£3), and then
the organic layer was concentrated to give 24 (710 mg,
Extraction of proteins from a marine sponge
Halichondria okadai
1
75%): H NMR (CDCl3) d 8.17 (d, Jˆ8.5 Hz, 1H), 6.88
(d, Jˆ8.5 Hz, 1H), 6.68 (s, 1H), 5.67 (dd, Jˆ15.0, 8.5 Hz,
1H), 5.48 (dd, Jˆ15.0, 8.0 Hz, 1H), 5.40 (s, 1H), 5.24 (s,
1H), 5.03 (s, 1H), 4.60 (dd, Jˆ12.5, 5.0 Hz, 1H), 4.55±4.48
(m, 2H), 4.31 (br, 1H), 4.25 (t, Jˆ4.5 Hz, 2H), 4.10±4.04
(m, 3H), 3.92 (d, Jˆ9.5 Hz, 1H), 3.88 (t, Jˆ4.5 Hz,
2H), 3.70±3.30 (m, 15H), 3.27 (dd, Jˆ10.0, 2.0 Hz, 1H),
3.15±3.02 (m, 3H), 2.91 (dd, Jˆ13.0, 5.0 Hz, 1H), 2.71
(d, Jˆ12.5 Hz, 1H), 2.33 (t, Jˆ7.5 Hz, 2H), 2.30±1.20
(m, 49H), 1.03 (d, Jˆ6.5 Hz, 3H), 0.98 (d, Jˆ7.0 Hz,
A marine sponge Halichondria okadai (60 g, wet wt.),
collected at the coast of Aburatsubo Bay in Kanagawa
Prefecture, was washed with a buffer consisting of
540 mM NaCl, 7 mM (NH4)2SO4, 0.2 mM Na2CO3,
10 mM KCl, 20 mM EDTA, 0.5 mM phenylmethylsulfonyl
¯uoride, 1 m mL21 leupeptin hemisulfate monohydrate,
1 m mL21 pepstatin A, 0.5 mM dithiothreitol, and 20 mM
Tris±HCl (pH 8.2), cut into small pieces with a razor's edge,

9014
K. Konoki et al. / Tetrahedron 56 (2000) 9003±9014
Carcinogenesis 1990, 11, 1837±1841. (b) Takai, A.; Murata, M.;
Torigoe, K.; Isobe, M.; Mieskes, G.; Yasumoto, T. Biochem. J.
1992, 284, 539±544.
and homogenized in a Waring blender with the same buffer
(60 mL). The homogenate was ®ltered, and then the ®ltrate
was centrifuged at 600£g for 10 min. The supernatant was
successively centrifuged at 8000£g for 10 min, and then
75,600£g for 90 min.
È Ê
6. (a) Lofas, S.; Johnsson, B. J. Chem. Soc., Chem. Commun.
1990, 21, 1526±1528. (b) Karlsson, R.; Michaelsson, A; Mattsson,
È
L. J. Immunol. Methods 1991, 145, 229±240. (c) Sjolander, S.;
Urbaniczky, C. Anal. Chem. 1991, 63, 2338±2345. (d) Johnsson,
È Ê
B.; Lofas, S.; Lindquist, G. Anal. Biochem. 1991, 198, 268±277.
(e) Karlsson, R.; Michaelsson, A.; Mattsson, L. J. Immnol.
Acknowledgements
This work is supported in part by Grant-In-Aids from the
Ministry of Education, Science, Sports and Culture of Japan
(EYS10780349 to K. K.; SRPA08281105 and SR12470504
to Y. H.). The authors are grateful to Dr M. Ojika of Nagoya
University and Dr M. Sasaki in our laboratory of The
University of Tokyo for discussion on the derivatization
of okadaic acid, to Dr J. Inagawa of Biacore (K. K.) for
valuable discussions and suggestions on SPR analysis, Ms
M. Izumikawa in our laboratory of The University of Tokyo
for mass spectrometry, and to Messrs H. Kusuoku,
S. Matsuoka, H. Hori, and K. Otsuki in our laboratory of
The University of Tokyo for their help in collecting
H. okadai.
È
Methods 1991, 145, 229±240. (f) Fagerstam, L. G.; Karlsson,
Ê
È
A.-F.; Karlsson, R.; Persson, B.; Ronnberg, I. J. Chromatogr.
1992, 597, 397±410.
7. Wilchek, M.; Bayer, E. A. In Methods in Enzymology;
Academic: San Diego, 1990; Vol. 184, pp 5±13.
8. For example, see: (a) Rao, S.; He, L.; Chakravarty, S.; Ojima, I.;
Orr, G. A.; Horwitz, S. B. J. Biol. Chem. 1999, 274, 37990±37994.
(b) Wilson, C. J.; Husain, S. S.; Stimson, E. R.; Dangott, L. J.;
Miller, K. W.; Maggio, J. E. Biochemistry 1997, 36, 4542±4551.
9. (a) Hatanaka, Y.; Hashimoto, M.; Kanaoka, Y. J. Am. Chem.
Soc. 1998, 120, 453±454. (b) Hashimoto, M.; Hatanaka, Y. Chem.
Pharm. Bull. 1999, 47, 667±671.
10. (a) Hatanaka, Y.; Hashimoto, Kanaoka, Y. Bioorg. Med.
Chem. 1994, 2, 1367±1373. (b) Hatanaka, Y.; Hashimoto, M.;
Kurihara, H.; Nakayama, H.; Kanaoka, Y. J. Org. Chem. 1994,
59, 383±387.
References
11. Konoki, K.; Sugiyama, N.; Murata, M.; Tachibana, K.
Tetrahedron Lett. 1999, 40, 887±890.
1. (a) Tachibana, K.; Scheuer, P. J.; Tsukitani, Y.; Kikuchi, H.;
Engen D. V.; Clardy, J.; Gopichand, Y.; Shumitz, F. J. J. Am.
Chem. Soc. 1981, 103, 2469±2470. (b) Murata, M.; Shimatani,
M.; Sugitani, Y.; Oshima, Y.; Yasumoto, T. Bull. Jpn. Soc. Sci.
Fish. 1982, 48, 549±552.
12. Yanagi, T.; Murata, M.; Torigoe, K.; Yasumoto, T. Agric.
Biol. Chem. 1989, 53, 525±529.
13. Ghosh, A. K.; Duong, T. T.; McKee, S. P.; Thompson, W. J.
Tetrahedron Lett. 1992, 33, 2781±2784.
2. (a) Suganuma, M.; Fujiki, H.; Okabe, S.; Nishiwaki, S.;
Brautigan, D.; Ingebritsen, T. S.; Rosner, M. R. Toxicon 1992,
30, 873±878. (b) Fujiki, H.; Suganuma, M. Adv. Cancer Res.
1993, 61, 143±194.
14. Yasumoto, T.; Murata, M.; Oshima, Y.; Sano, M.; Matsumoto,
G. K.; Clardy, J. Tetrahedron 1985, 41, 1019±1025.
15. Fang, K.; Hashimoto, M.; Jockusch, S.; Turro, N. J.;
Nakanishi, K. J. Am. Chem. Soc. 1998, 120, 8543±8544.
16. (a) Shenolikar, S.; Nairn, A. C. Adv. Second Messenger 1991,
23, 1±121. (b) Cohen, P. Annu. Rev. Biochem. 1989, 58, 453±508.
(c) Shenolikar, S. Annu. Rev. Cell. Biol. 1994, 10, 55±86.
17. (a) Sugg, L. M.; VanDolah, F. M. J. Phycol. 1999, 35, 93±103.
(b) Barbier, M; Amzil, Z.; Mondeguer, F.; Bhaud, Y.; Soyer-
Gobillard, M. O.; Lassus, P. Phycologia 1999, 38, 41±46.
(c) Boland, M. P.; Holmes, C. F. B. Adv. Prot. Phosphatases
1994, 8, 325±342.
3. (a) Suganuma, M.; Suttajit, M.; Suguri, H.; Ojika, M.; Yamada,
K.; Fujiki, H. FEBS Lett. 1989, 250, 615±618. (b) Nishiwaki, S.;
Fujiki, H.; Suganuma, M.; Ojika, M.; Yamada, K.; Yasumoto, T.
Biochem. Biophys. Res. Commun. 1990, 170, 1359±1364.
4. (a) Bialojan C.; Takai, A. Biochem. J. 1988, 256, 283±290.
(b) Takai, A.; Mieskes, G. Biochem. J. 1991, 275, 233±239.
5. For example, see: (a) Nishiwaki, S.; Fujiki, H.; Suganuma, M.;
Suguri, H. F-, Matsushima, R.; Iida, Y.; Ojika, M.; Yamada, K.;
Uemura, D.; Yasumoto, T.; Schmitz, F. J.; Sugimura, T.