Concise synthesis of ciguatoxin ABC-ring fragments and surface plasmon resonance study of the interaction of their BSA conjugates with monoclonal antibodies

Article abstract of DOI:10.1016/S0960-894X(01)00358-4

Monoclonal antibodies (mAbs), 4H2 and 6H7, were prepared previously using a protein conjugate of a 1:1 epimeric mixture of the synthetic ABC-ring fragments of ciguatoxin (CTX), 3 and 4. Here, the interactions of these mAbs with the fragments of CTX and CTX3C, 3 and 5, were investigated by surface plasmon resonance (SPR) spectroscopy in an attempt to clarify an antigenic determinant. Compared with the previous synthesis, the fragment 3 possessing the 2S configuration was synthesized from tri-O-acetyl-D-glucal much more effectively. The mAb 4H2 was already known to show a dose-dependent binding to the bovine serum albumin (BSA) conjugate of 3, but not to that of 5. The present SPR study of 4H2 demonstrates that the A-ring side chain of 3 plays a decisive role as an epitope. Therefore, SPR can effectively replace the ELISA method for the analysis of mAbs.

Full text of DOI:10.1016/S0960-894X(01)00358-4

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2037–2040
Concise Synthesis of Ciguatoxin ABC-Ring Fragments
and Surface Plasmon Resonance Study of the Interaction
of Their BSA Conjugates with Monoclonal Antibodies
Yoko Nagumo,a Hiroki Oguri,a Yumi Shindo,a Shin-ya Sasaki,a Tohru Oishi,a
Masahiro Hirama,a,* Yoshihisa Tomioka,b Michinao Mizugakib and Takeshi Tsumurayac
aDepartment of Chemistry, Graduate School of Science, Tohoku University, and CREST,
Japan Science and Technology Corporation (JST), Sendai 980-8578, Japan
bDepartment of Pharmaceutical Sciences, Tohoku University Hospital, Sendai 980-0872, Japan
cBiomolecular Engineering Research Institute, Suita, Osaka 565-0874, Japan
Received 9 April 2001; accepted 19 May 2001
Abstract—Monoclonal antibodies (mAbs), 4H2 and 6H7, were prepared previously using a protein conjugate of a 1:1 epimeric
mixture of the synthetic ABC-ring fragments of ciguatoxin (CTX), 3 and 4. Here, the interactions of these mAbs with the fragments
of CTX and CTX3C, 3 and 5, were investigated by surface plasmon resonance (SPR) spectroscopy in an attempt to clarify an
antigenic determinant. Compared with the previous synthesis, the fragment 3 possessing the 2S configuration was synthesized from
tri-O-acetyl-d-glucal much more effectively. The mAb 4H2 was already known to show a dose-dependent binding to the bovine
serum albumin (BSA) conjugate of 3, but not to that of 5. The present SPR study of 4H2 demonstrates that the A-ring side chain of
3 plays a decisive role as an epitope. Therefore, SPR can effectively replace the ELISA method for the analysis of mAbs. # 2001
Elsevier Science Ltd. All rights reserved.
Introduction
investigate the binding affinity more rapidly than affor-
More than 20,000 people in the tropics and subtropics
suffer annually from ciguatera, a disease that causes
gastrointestinal, neurological and cardiovascular dis-
orders.1 Ciguatoxin (1: CTX)2 and its congener (2:
CTX3C)3 are the principal causative toxins of ciguatera.
Considerable effort has been directed toward the devel-
opment of accurate detection methods for ciguatoxins,
particularly to those involving the application of anti-
ciguatoxin monoclonal antibodies.4 We previously pre-
pared three monoclonal antibodies (mAbs), 4H2, 6H7,
and 6F12, using a synthetic ABC-ring fragment of 1 as a
haptenic group, which was a 1:1 C2-epimeric mixture (3
and 4).5,6 Preliminary ELISA analysis indicated that
these mAbs differentiate the configuration of C2 carbi-
nol between 3 and 4: 4H2 binds to 3 which possesses the
2S-configuration and 6H7 binds to 4 but not to 3.5 To
ded by the ELISA method, interactions between the
mAbs and the bovine serum albumin (BSA) conjugates
of synthetic fragments, 3 and 5, were investigated using
surface plasmon resonance (SPR) spectroscopy. In
addition, concise syntheses of 3 and 5 were achieved.
*Corresponding author. Tel.:+81-22-217-6563; fax:+81-22-217-6566;
e-mail: hirama@ykbsc.chem.tohuoku.ac.jp
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00358-4

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Y. Nagumo et al. / Bioorg. Med. Chem. Lett. 11 (2001)2037–2040
Synthesis
Construction of the C-ring and the synthesis of 3 were
achieved through an intramolecular conjugate addition
of 18 mediated by tetrabutylammonium fluoride
(TBAF) according to our reported protocol5 (Scheme
2). An established concise synthetic route to 3 from 6
includes only 17 steps and accelerated the preparation
of the synthetic samples for the SPR study.
Since the previous synthesis of 3 was lengthy and
laborious (47 steps from d-glucose),5,7 we established a
more concise and convergent route to the ABC-ring
fragment.8 The new synthesis of 3 began with commer-
cially available tri-O-acetyl-d-glucal (6) (Scheme 1).
Protecting groupmanipulation and stereoselective ally-
lation using Spilling’s protocol9 gave the C-glycoside
(9). The acid-sensitive key fragment (14), which has a 2S
configuration, was synthesized from 9 via transition
metal ([Pd], [Ru], and [Ni])-catalyzed sequential process
only by altering the nature of the protecting groups
from the previously reported synthesis.10
On the other hand, the ABC-ring fragment (5) of
CTX3C (2) was synthesized from an intermediate 20,
which is readily available from 6 (Scheme 3).11 One
carbon homologation of 20 and conjugate addition to
12
ethyl propiolate gave 22. Cyclization of 22 with SmI2
furnished steroselectively a trans-fused tricyclic ether
Scheme 1. Reagents and conditions: (a) K2CO3 (cat), MeOH; (b) Cl(iPr)2SiOSi(iPr)2Cl, pyridine, 73% (two steps); (c) NaH, THF/DMF=2:1, 0 ꢀC,
1 h, then MPMCl, 0 ꢀC to rt, 42%; (d) N-bromosuccinimide, THF/H2O=10:1; (e) K[N(SiMe3)2], 18-crown-6, toluene, ꢁ78 ꢀC, 11 h, then
CH2¼CHCH2MgBr, ꢁ20 ꢀC to rt, 33% (two steps); (f) H2CCCHOPh (8 equiv), Pd(OAc)2 (10 mol%), Ph2P(CH2)3PPh2 (10 mol%), K2CO3 (1.5
equiv), CH3CN, reflux, 10 (24%; 45% based on recovery of 9), 11 (17%; 32%, based on recovery of 9), 9 recovered (47%); (g) Pd(OAc)2
(10 mol%), Ph2P(CH2)3PPh2 (10 mol%), PhOH (1 equiv), K2CO3 (1.5 equiv), CH3CN, reflux, 10:11=2:1, 64% based on recovery of 11: (h)
(PCy3)2Cl2Ru¼CHPh (7 mol%), CH2Cl2, rt to 40 ꢀC, 79%; (i) 13 (1.8 equiv), (PPh3)2NiCl2 (10 mol%), NaI (1 equiv), tBuCN (2 equiv), THF, 0 ꢀC
to rt, 14 (39%), 15 (49%).
Scheme 2. Reagents and conditions: (a) 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), CH2Cl2/H2O=10:1, 92%; (b) (COCl)2, dimethyl sulf-
oxide (DMSO), Et3N, CH2Cl2, ꢁ78 ꢀC to rt; (c) Ph3PCHCO2Me, CH2Cl2, 99% (two steps); (d) L-Selectride (2 equiv), tBuOH (5 equiv), THF,
ꢁ70 ꢀC, then NaOH aq, H2O2 aq, 0 ꢀC, 81%; (e) diisobutylaluminium hydride (DIBAL), Et2O, ꢁ78 to ꢁ40 ꢀC, 73%; (f) Dess–Martin periodinane,
CH2Cl2, rt; (g) Ph3PCHCO2Me, CH2Cl2, rt; (g) Ph3PCHCO2Me, CH2Cl2, 84% (two steps); (h) TBAF, THF, then Ac2O, pyridine, 87%; (i)
LiOHꢂ H2O, tBuOH/H2O=4:1; (j) EDCꢂ HCl, NHS, DMF, then BSA in phosphate buffer (pH 7.4).

Y. Nagumo et al. / Bioorg. Med. Chem. Lett. 11 (2001)2037–2040
2039
Scheme 3. Reagents and conditions: (a) (CF3SO2)2O (1.1 equiv), 2,6-lutidine, CH2Cl2, ꢁ78 ꢀC to rt; (b) NaI, acetone, rt, 87% (two steps); (c) nBuLi
(4 equiv), 1,3-dithiane (5 equiv), THF/hexamethylphosphoramide (HMPA)=9:1, ꢁ30 to 0 ꢀC, 66%; (d) ethyl propiolate, N-methylmorpholine,
CH2Cl2, 94%; (e) MeI (10 equiv), NaHCO3 (5 equiv), CH3CN/H2O=4:1, rt, 78%; (f) SmI2 (2.2 equiv), MeOH (2.2 equiv), THF, rt, 94%; (g)
CH3OCH2Cl, (iPr)2NEt, Bu4NI; (h) DDQ (4 equiv), CH2Cl2/H2O=10:1, rt, 80%; (i) LiOHꢂ H2O, tBuOH/H2O=4:1; (j) EDCꢂ HCl, NHS, DMF,
then BSA in phosphate buffer (pH 7.4).
(23) in good yield. The synthesis of 5 in 17 steps from 6
was completed by protecting the hydroxy group of 23
and removing the benzyl ether with DDQ before
hydrolysis.
the BSA conjugate (19) was immobilized through its
amine groupto the sensor chip[CM5 sensor chisp
(BIACORE)], and the interactions of 19 with the mAbs,
4H2 and 6H7, were analyzed using BIACORE 2000
instruments.14 The mAbs diluted to a final concentra-
tion of 1.9 mg/mL in HBS buffer [10 mM Hepes (pH
7.4), 150 mM NaCl, 3 mM EDTA] were then passed
over the coated sensor tipat a constant flow of 20 mL/
min at 25 ꢀC (Fig. 1). The 4H2 was bound to the
immobilized 19 in a dose-dependent manner, although
the 6H7 was not bound at all. These affinities of the
mAbs with 19 are in agreement with the previous results
using ELISA: 4H2 binds to 3 and 6H7 binds to 4 but
not to 3.5 Thus, the SPR assay can effectively replace
the ELISA method for the screening of antibodies that
bind to the synthetic fragments.
The fragments, 3 and 5, were conjugated with BSA
using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
.
hydrochloride (EDC HCl) and N-hydroxysuccinimide
(NHS) (Schemes 2 and 3, respectively). The resulting
hapten–BSA conjugates, 19 and 24, were purified by gel
filtration (Amersham Pharmacia, PD-10 column), and
MALDI-TOF-MS analysis determined the average
number of the haptens attached to the BSA in the con-
jugates to be 14 and 15 for 19 and 24, respectively.13
Analysis of Surface Plasmon Resonance
Since epitope analyses of antibodies using SPR can be
performed by passing various conjugates over the
immobilized antibodies,15 we next examined the inter-
actions of immobilized 4H2 with the BSA conjugates,
19 and 24. As shown in Figure 2, 4H2 was bound to 19.
To developa raipd screening system for antibodies
using SPR spectroscopy in place of the ELISA protocol,
Figure 1. Sensorgrams obtained by passing mAbs [4H2 (A–E) and 6H7
(F)] over immobilized 19; [HBS buffer, 25 ꢀC, flow rate: 20 mL/min].
Figure 2. Sensorgrams obtained by passing 19, 24 and BSA over
immobilized 4H2; [HBS buffer, 25 ꢀC, flow rate: 30 mL/min].

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Y. Nagumo et al. / Bioorg. Med. Chem. Lett. 11 (2001)2037–2040
However, in the case of 24, which lacks the A-ring side
chain, 4H2 showed only negligible binding. Thus, the
side chain of 3 is an essential antigenic determinant for
4H2.
W. E.; Nishimura, K. L.; Ebesu, J. S. M. JAOAC Intern. 1998,
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Kinetic analysis for immobilized 4H2 by injection of 19
revealed the association rate constant (ka) to be 5.8ꢃ103
Mꢁ1 sꢁ1 and the dissociation rate constant (kd) to be
6. Carbon numbering corresponding to that of ciguatoxin has
been used throughout this paper.
3.5ꢃ10ꢁ3
s .
ꢁ1 16 Rapid association and slow dissociation
take place in the present system and the equilibrium
dissociation constant (Kd) is calculated to be 6.0ꢃ10ꢁ7
M.17
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Conclusion
Concise synthetic routes to the ABC-ring fragments 3
and 5 were established, accelerating the SPR study of
the interactions of mAbs, 4H2 and 6H7, with BSA
conjugates, 19 and 24. Binding analysis of 4H2 with 19
and 24 indicated that the A-ring side chain of 3 plays a
critical role as an epitope. The present SPR method was
shown to be convenient for not only epitope analysis
but also the screening of antibodies, and thus can effec-
tively replace the conventional ELISA protocol. Further
detailed SPR analyses of mAbs are currently being per-
formed at our laboratory.
13. Shoyama, Y.; Tanaka, H.; Fukuda, N. Cytotechnology
1999, 31, 9.
Acknowledgements
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The authors are grateful to Dr. Keiichi Konoki (The
University of Tokyo), Dr. Goh Matsuo (RIKEN), Mr.
Kazunobu Asano (BIACORE), and Dr. Junichi Ina-
gawa (BIACORE) for their helpful comments. A fel-
lowshipto Y.N. from the Jaapnese Society for the
Promotion of Science is gratefully acknowledged.
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