Designed hapten aimed at anti-ciguatoxin monoclonal antibody: Synthesis, immunization and discrimination of the C2 configuration

Article abstract of DOI:10.1055/s-1999-3646

The ABC-ring fragment of ciguatoxin was synthesized as a C2 epimeric mixture (1:1). The mixture, which was designed as a possible hapten for preparing an anti-ciguatoxin monoclonal antibody was conjugated with a carrier protein (KLH) and then used for immunization of mice. The three monoctonal antibodies (mAbs) obtained have shown an appreciable reactivity to the hapten. Each mAb discriminated configuration of the C2 hydroxy group of the ABC-ring fragment. The mAb which showed reactivity to the 2S-fragment was cross-reacted with ciguatoxin.

Full text of DOI:10.1055/s-1999-3646

PAPER
1431
Designed Hapten Aimed at Anti-ciguatoxin Monoclonal Antibody: Synthesis,
Immunization and Discrimination of the C2 Configuration
Hiroki Oguri,a Shin-ichiro Tanaka,a Shojiro Hishiyama,a Tohru Oishi,a Masahiro Hirama,*a Takeshi
Tumuraya,b,1Yoshihisa Tomioka,c Michinao Mizugakic
a Department of Chemistry, Graduate School of Science, Tohoku University, and CREST, Japan Science and Technology Corporation
(JST), Sendai 980-8578, Japan
Fax+81(22)2176566; E-mail: hirama@ykbsc.chem.tohoku.ac.jp
b Kao Institute for the Fundamental Research, Tochigi 321-3426, Japan
c Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai 980-0872, Japan
Received 22 February 1999
H
Abstract: The ABC-ring fragment of ciguatoxin was synthesized
5R
A
H
C
O
2S
OH
HO
B
H
as a C2 epimeric mixture (1:1). The mixture, which was designed as
a possible hapten for preparing an anti-ciguatoxin monoclonal anti-
body was conjugated with a carrier protein (KLH) and then used for
immunization of mice. The three monoclonal antibodies (mAbs)
obtained have shown an appreciable reactivity to the hapten. Each
mAb discriminated configuration of the C2 hydroxy group of the
ABC-ring fragment. The mAb which showed reactivity to the 2S-
fragment was cross-reacted with ciguatoxin .
O
H
H
H
11
H
O
H
O
H
HO
H
H
H
32
O
I
OH
D
H
O
E
G
O
H
H
O
OH
J
O
O
H
F
H
H
47
H
H
K
H
H
O
H
O
L
O
Ciguatoxin (CTX1B, 1)
55
M
OH
H
H
H
H
O
O
Key words: ciguatoxin, anti-ciguatoxin antibody, synthetic hapten,
artificial antigen, immunization.
A
C
B
A
C
B
16
11
16
11
5R
5R
(1 : 1)
2
O
O
O
O
1
2
H
H
H
H
H
H
H
OH
CO2R
OH
HO
HO
R2
OH
R1
2 : R = H
3 : R = KLH
4 : R = BSA
5-(2R) : R1=H, R2=OH
6-(2S) : R1=OH, R2=H
Over twenty thousand people in the tropics and subtropics
suffer annually from ciguatera, a widespread human food
poisoning that causes gastrointestinal, neurological and
cardiovascular disorders.2 Ciguatoxin (CTX1B, 1),3,4 the
most potent ciguatoxin, which is isolated from the moray
eel, is regarded as the principal causative toxin of
ciguatera. Considerable attention has been directed to the
development of accurate methods for detecting ciguatox-
ins.5 Although Hokama et al. prepared a monoclonal anti-
body (mAb) using CTX1B (1)6 itself, the antibody
showed a high cross-reactivity to okadaic acid. The LM-
ring moiety was believed to be an antigenic determinant.6b
Due to the extremely low accessibility of 1, immunization
with a protein conjugate composed of a structurally de-
fined synthetic hapten should prove useful for the prepa-
ration of the antibody.7 We focused on the ABC-ring
fragment 2 (Figure 1) as a haptenic group with a carboxy-
lic acid linker. Synthesis of the artificial antigen, immuni-
zation and reactivities of mAbs are described herein.
Figure 1. Structures of Ciguatoxin (1), hapten 2, hapten-protein con-
jugates 3 and 4, and ABC-ring fragments 5 and 6
group and deprotection of MPM group produced the diol
13. The diol 13 was treated with K2CO3 at room tempera-
ture for 2.5 hours and then at 50 °C for 4 hours to give the
trans/syn-fused (7,6,6)-tricyclic ethers, 14 and 15 in 72%
yield and a 6:1 ratio, respectively, via hetero-conjugate
addition and epimerization processes.11,12 p-Bromobenz-
oylation of 14 followed by deprotection of TIPS groups
yielded 16. Construction of the A-ring side chain from 16
was achieved by applying the reported protocol8 to furnish
a 1:1 epimeric mixture of the allylic alcohol 17. Stepwise
removal of the protecting groups8 followed by hydrolysis
of the methyl ester produced the haptenic group 2.
Synthesis of the haptenic group 2 is shown in Scheme 1.8,9
The AB-ring fragment 7, which is readily available from
D-glucose8 was subjected to the protecting group manipu-
lation to yield the primary alcohol 8. Swern oxidation of 8
followed by Wittig reaction yielded the a,b-unsaturated
ester 9. 1,4-Reduction of 9 with L-Selectride in the pres-
ence of tert-butyl alcohol produced the ester 10,10 fol-
lowed by reduction with DIBAL which yielded the
alcohol 11. Oxidation of 11 and subsequent Wittig reac-
tion afforded the a,b-unsaturated ester 12. Removal of si-
lyl groups of 12 with TBAF followed by selective
protection of the resulting primary alcohol with TIPS
The haptenic group 2 was conjugated with keyhole limpet
hemocyanine (KLH) and bovine serum albumin (BSA)
through their lysine residue using carbodiimide and N-hy-
droxysuccinimide.13 The resulting hapten-protein conju-
gates 3 and 4 were purified by dialysis. The haptenic
density in the conjugates was estimated by 2,4,6-trinitro-
phenylation to be 137/246 and 19/30 for KLH and BSA,
respectively.14
The artificial antigen 3 was emulsified in RIBI adjuvant
and was injected in five Balb/c mice intraperitioneally at
three week intervals. After four injections, the spleen was
exiled from the mice and the cells were fused with myelo-
Synthesis 1999, No. SI, 1431–1436 ISSN 0039-7881 © Thieme Stuttgart · New York

1432
H. Oguri et al.
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Reagents and conditions: (a) TBPSCl/imidazole/DMF, 84%; (b) PPTs/MeOH, 98%; (c) p-MeOC6H4CH(OMe)2/PPTs/CH2Cl2, 96%; (d)
K2CO3/MeOH, 84%; (e) TBSCl/imidazole, 87%; (f) DIBAL/CH2Cl2, –30 °C, 99%; (g) (COCl)2/DMSO/CH2Cl2, –78 °C, then Et3N, –78° to
0 °C, then Ph3PCHCO2Me, 0 °C to r.t., 90%; (h) L-Selectride/t-BuOH/THF, –78 °C, 89%; (i) DIBAL/Et2O, –78 °C, 88%; (j) SO3∑Py/DMSO/
CH2Cl2, 0 °C to r.t., then Ph3PCHCO2Me, 93%; (k) TBAF/THF, 63%; (l) TIPSCl/Et3N/DMAP/CH2Cl2, 74%; (m) DDQ/H2O/CH2Cl2, 89%;
(n) K2CO3/MeOH, r.t., 2.5 h, then 50 °C, 4 h, 72%; (o) p-BrBzCl/Et3N/DMAP/CH2Cl2, 95%; (p) HF/MeCN, 99%; (q) Dess–Martin periodi-
nane/CH2Cl2, r.t.; (r) Ph3PCHCOCH2OMPM/toluene, r.t.; (s) NaBH4/CeCl3∑7 H2O, –78 °C, 83% (3 steps); (t) p-BrBzCl/Et3N/DMAP/CH2Cl2,
84%; (u) DDQ/CH2Cl2, 85%; (v) LiOH/H2O/t-BuOH, >90%
Scheme 1
ma cells (P3X63-Ag8.653). Three hybridomas, which inhibition of the binding of 6H7 and 6F12 to the conjugate
were designated as 4H2, 6F12, and 6H7, were obtained af- 4 by 2 was observed in more than 90 and 70%, respective-
ter the subcloning.15
ly, while weak inhibition of 4H2 binding was seen. The
order of reactivity of mAbs to 2 was estimated to be 6H7
> 6F12 > 4H2.
The mAbs were tritrated with hapten-BSA conjugate 4 by
ELISA, and dose-response curves were obtained as shown
in Figure 2. The cross-reactivities of the mAbs to the car- The mAbs to the synthetic ABC-ring fragment were thus
rier proteins, KLH and BSA, were not detectable (data not prepared using the hapten-KLH conjugate 3 as an artifi-
shown). The ABC-ring moiety is most likely to act as an cial antigen. In order to evaluate the binding selectivity of
antigenic determinant in the mAbs (4H2, 6F12 and 6H7). these mAbs to the C2 stereoisomers, the epimers,
(2R,5R)-5 and (2S,5R)-6, were then synthesized from the
To evaluate the relative affinity of these three mAbs to the
haptenic group, competitive inhibition with the epimeric
mixture (2) was then investigated (Figure 3). Satisfactory
tetraol 18,8 separated and used as competitive inhibitors
(Scheme 2). Two-carbon elongation from 18 followed by
manipulation of the protecting groups and tosylation
yielded 21. Ring closure of the tosylate 219a upon treat-
ment with TBAF produced the BC ring fragment 22 in
Figure 3. Competitive inhibition of the binding of mAbs to hapten-
Figure 2. Titration curves of mAbs by hapten-BSA conjugate 4
BSA conjugate 4 by 2
Synthesis 1999, No. SI, 1431–1436 ISSN 0039-7881 © Thieme Stuttgart · New York

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Designed Hapten Aimed at Anti-ciguatoxin Monoclonal Antibody
1433
H
H
H
H
H
H
H
H
H
H
O
O
O
TBPSO
OH
TBPSO
CN
TBPSO
e
a, b
c, d
HO
OH
18
BnO
H
OMPM
BnO
OTBS
H
BnO
H
OBn
OH
19
20
H
H
H
H
H
H
H
H
H
O
O
O
O
HO
TBPSO
TBPSO
OTs
HO
B
i, j
C
g, h
f
BnO
O
O
O
O
BnO
OTBS
O
H
H
OBn
H
H
H
H
OBn
Bu3Sn
OH
OBz-Br-p
23
24
21
22
H
O
H
H
H
O
H
H
H
H
7
H
O
k
l, m
n, o
HO
A
C
B
5R
O
O
O
O
O
O
H
H
H
H
1
2
HO
H
H
H
OBz-Br-p
OBz-Br-p
OBz-Br-p
p
25
26
HO
R2
27-(2R): R1=H, R2=OBz-Br-p
28-(2S): R1=OBz-Br-p, R2=H
5
R1
p
6
Reagents and conditions: (a) p-MeOC6H4CH(OMe)2/PPTs/CH2Cl2, r.t., then BnBr/NaH/THF/DMF (2:1), 0 °C to r.t., 90% (2 steps); (b) DI-
BAL/CH2Cl2, –20 °C, then TsCl/Py/DMAP/CH2Cl2, r.t., NaCN/18-crown-6/DMF, 80 °C, 73% (3 steps); (c) DDQ/H2O/CH2Cl2, r.t., TBSOTf/
2,6-lutidine/CH2Cl2, 0 °C to r.t., 66% (2 steps); (d) DIBAL/CH2Cl2, –78 °C, (TMS)2NNa/Ph3PMeBr/THF, 0 °C, 90% (2 steps); (e) 9-BBN/
THF, sonication, then NaOH/H2O, then TsCl/Py/, r.t., 85% (2 steps); (f) TBAF/THF, r. t., 91%; (g) TBPSCl/imidazole/DMF, r.t., then H2/
Pd(OH)2/EtOH/EtOAc (1:1), 81% (2 steps); (h) PivCl/Py/DMAP, 0 °C, then Pd (dba)2 (10 mol%)/dppb (40 mol%)/CH2 = CHCH2 CO2Me/
THF, 65 °C, then DIBAL/CH2Cl2, –78 °C, 87% (3 steps); (i) BuLi/HMPA/THF, –78 °C, then Bu3SnCl, 29% (23 60% recovered); (j) p-BrBzCl/
Et3N/DMAP/CH2Cl2, r.t.; then TBAF/THF, r.t., 93% (2 steps); (k) SO3∑Py/DMSO/Et3N/CH2Cl2, 0 °C to r.t.; then BF3∑OEt2/CH2Cl2, –78 °C,
84% (2 steps); (l) O3, CH2Cl2/MeOH (3:1), –78 °C, then NaBH4, –78 to 0 °C, then TBSCl/Et3N/DMAP/CH2Cl2, r. t. 82% (2 steps); (m) MsCl/
Et3N/CH2Cl2, 0 °C to r.t., then DBU/toluene, 100 °C, TBAF/THF, r.t. 63% (3 steps); (n) Dess–Martin periodinane/CH2Cl2, r.t., then
Ph3PCHCOCH2OMPM/benzene, r.t., then NaBH4/CeCl3∑7H2O/MeOH, –78 °C, 34% (3 steps); (o) pBrBzCl/Et3N/DMAP/CH2Cl2, r.t., then
DDQ/H2O/CH2Cl2, r.t., 85%; (p) K2CO3/MeOH, r.t., 92% for 5 and 72% for 6
Scheme 2
91% yield. To construct A-ring, 22 was converted to the group of the ABC-ring fragment. The mAb which showed
allylic tin compound 24. Oxidation of 24 followed by reactivity to the fragment (2S)-6 also showed a cross-reac-
treatment with BF3∑OEt2 yielded the ABC-ring fragment tivity with CTX1B (1). Affinity maturation of the mAbs
25 in 84% yield. Construction of the C1-C7 functional and further efforts to prepare the highly sensitive anti-
groups from 25 was achieved according to the reported ciguatoxin antibodies are currently underway in our labo-
protocol to yield a 1:1 mixture of 27 and 28. At this stage ratory.
the epimers were separated. Methanolysis of 27 and 28
produced 5 and 6, respectively. The C2 configuration of 5
and 6 was determined by the CD spectra of the corre-
sponding tris-p-bromobenzoates.16,17
Binding of the mAb 6H7 to the conjugate 4 was inhibited
by (2R)-5 more effectively than by (2S)-6 (Figure 4). In
contrast, 4H2 and 6F12 were selectively bound to (2S)-6,
but not to (2R)-5. Thus, each mAb distinguished the C2
configuration of the epimeric ABC-ring fragments,
though the affinities to 5 and 6 were not high. As CTX1B
(1) has a (2S)-configuration,4 competitive inhibition of the
binding of 6F12 to the conjugate 4 by CTX1B was then
examined. Figure 5 shows the observed weak but definite
cross-reactivity of 6F12 with CTX1B (1).
In conclusion, the ABC-ring fragment 2 of CTX1B (1)
was synthesized as a 1:1 C2 epimeric mixture. The mix-
ture, which was designed as a haptenic group was conju-
gated with a carrier protein (KLH) and then used in
immunization as an artificial antigen. The three mAbs ob-
tained discriminated the configuration of the C2 hydroxy BSA conjugate 4 by (2R)-5 and (2S)-6
Figure 4. Competitiv inhibition of the binding of mAbs to hapten-
Synthesis 1999, No. SI, 1431–1436 ISSN 0039-7881 © Thieme Stuttgart · New York

1434
H. Oguri et al.
PAPER
4.62 (1 H, d, J = 11.0 Hz), 4.75 (1 H, d, J = 11.0 Hz), 4.94 (1 H, d,
J = 11.0 Hz), 4.99 (1 H, d, J = 11.0 Hz), 7.2–7.4 (10 H, m).
IR (film): n = 3418, 3030, 2854, 1453, 1097, 1028, 736, 698 cm-1.
ABC-Ring Moiety 25
Oxidation of the Primary Alcohol 24 to Aldehyde
A solution of the alcohol 24 (510 mg, 0.685 mmol) and Et3N (955
mL, 6.85 mmol) in DMSO (1.5 mL) and CH2Cl2 (6 mL) was treated
with SO3•pyridine (435 mg, 2.74 mmol) at 0 °C and allowed to
stand at r.t. for 40 min. The mixture was quenched with H2O (2 mL)
and extracted with EtOAc (200 mL). The separated organic layer
was washed with H2O, brine and dried (Na2SO4). The filtrate was
concentrated and subjected to flash column chromatography (silica
gel, hexane/EtOAc/Et3N, 100:25:1) to give the corresponding alde-
hyde (473 mg, 93%).
1H NMR (200 MHz, CDCl3/TMS): d = 0.60–0.90 (15 H, m), 1.15–
1.65 (20 H, m), 1.81 (1 H, m), 2.16 (1 H, m), 2.57 (2 H, m), 3.09 (1
H, t, J = 9.5 Hz), 3.24 (2 H, m), 3.48 (2 H, m), 3.92 (1 H, m), 4.33
(1 H, td, J = 9.0, 6.0 Hz), 5.38 (1 H, t, J = 9.0 Hz), 5.77 (1 H, dt,
J = 6.0, 1.5 Hz), 7.57 (2 H, m), 7.90 (2 H, m), 9.78 (1 H, t, J = 1.5
Hz).
Figure 5. Competitive inhibition of the binding of mAb 6F12 to hap-
ten-BSA conjugate 4 by CTX1B
Hapten 2
Colorless solid.
Cyclization of Aldehyde to 25
1H NMR (600 MHz, CD3OD/TMS): d = 1.59 (1 H, m, H-15), 1.67
(1 H, m, H-14), 2.05 (1 H, m, H-15), 2.21 (1 H, m, H-14), 2.54 (1
H, br, H-17), 2.57 (1 H, m, H-8), 2.71 (1 H, br, H-17), 2.78 (1 H, m,
H-8), 3.19 (1 H, t, J = 9 Hz, H-12), 3.27 (1 H, m, H-13), 3.41 (1 H,
ddd, J = 9.7, 8.8, 3.8 Hz, H-9), 3.52 (1 H, m, H-10), 3.66 (1 H, dd,
m, H-1), 3.69 (1 H, dd, m, H-1), 3.71 (1 H, m, H-11), 3.97 (1 H, br,
H-16), 4.31 (1 H, m, H-2), 4.76 (1 H, m, H-5), 5.98 (2 H, m, H-6,7),
5.98 (1 H, m, H-3), 6.05 (1 H, m, H-4).
To a stirred solution of the resultant aldehyde (473 mg, 0.637 mmol)
in CH2Cl2 (30 mL) was added dropwise BF3•OEt2 (8.2 mL of 0.1 M
CH2Cl2 solution, 0.82 mmol) over a period of 20 min at –78 °C. Af-
ter 40 min, the mixture was quenched with aq satd NaHCO3 solution
(2 mL) and extracted with Et2O and EtOAc. The organic layer was
washed with brine and dried (MgSO4). Filtration, concentration and
flash column chromatography (silica gel, hexane/EtOAc, 2:1) af-
forded 25 (259 mg, 90%) as a colorless oil; [a]D24+5.55 (c = 1.03,
CHCl3).
1H NMR (200 MHz, CDCl3/TMS): d = 1.52 (1 H, m), 1.72 (2 H, m),
1.95 (4 H, m), 2.14 (1 H, m), 3.14 (1 H, t, J = 9.5 Hz), 3.28 (2 H, m),
3.42 (1 H, m), 3.52 (1 H, t, J = 9.0 Hz), 3.78 (2 H, m), 3.93 (1 H, m),
4.92 (1 H, dt, J = 11.0, 1.5 Hz), 5.08 (1 H, dt, J = 17.0, 1.5 Hz), 5.32
(1 H, t, J = 9.0 Hz), 5.72 (1 H, ddd, J = 17.0, 11.0, 5.0 Hz), 7.57 (2
H, m), 7.82 (2 H, m).
IR (film): n = 3348, 2928, 2860, 2366, 1727, 1578, 1439, 1094, 752,
615 cm-1.
MALDI-TOFMS: m/z calcd for C18H26O8Na (M+Na+) 393.1526,
found 393.1517.
Hapten-Protein Conjugates 3 and 4
To a stirred mixture of 2 (4.0 mg, 10 mmol) and N-hydroxysuccin-
imide (23.0 mg, 200 mmol) in DMF (200 mL) was added 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide hydrochloride (19.2 mg,
100 mmol). The mixture was stirred at r.t. for 5 h to give 200 mL of
DMF solution of the crude activated ester (ca 10 mmol).
IR (film): n = 3496, 2946, 2870, 1727, 1591, 1487, 1458, 1400,
1332, 1272, 1174, 1100, 1054, 1013, 946, 843, 754, 685, 623, 596
cm-1.
MALDI-TOFMS: m/z calcd for C21H2579BrO6Na (M+Na+)
475.0733, found 475.0815.
To a stirred solution of keyhole limpet hemocyanine (5 mg) in PBS
aqueous solution (800 mL) was added the resultant crude activated
ester (ca. 7 mmol) in DMF (140 mL) and the mixture was allowed to
stand at r.t. for 3.5 h without stirring. The resultant mixture was di-
alyzed against PBS aqueous solution (1 L) at 4 °C to give hapten-
KLH conjugate 3.
Allylic p-Bromobenzoates 27, 28
A solution of 26 (70.6 mg, 0.161 mmol) in CH2Cl2 (4 mL) was treat-
ed with Dess–Martin periodinane (340 mg, 0.803 mmol) and the
mixture was stirred at r.t. for 45 min. The mixture was quenched
with aq sat. Na2S2O3 solution (1 mL) at 0 °C, diluted with Et2O (20
mL) and stirred at r.t.for 20 min. The organic layer was separated,
washed with aq sat. NaHCO3 solution, dried (Na2SO4) and filtered.
The filtrate was concentrated in vacuo and treated immediately with
a solution of Ph3PCHCOCH2OMPM (731 mg, 1.61 mmol) in benz-
ene (3 mL) at r.t. After stirring for 2 h, the mixture was concentrated
and subjected to column chromatography on florisil (hexane/
EtOAc, 2:1) to give a crude enone. To a stirred mixture of
CeCl3•7H2O (300 mg, 0.803 mmol) in MeOH (2 mL) was added a
solution of the enone in MeOH (3 mL) at –78 °C. The mixture im-
mediately treated with NaBH4 (30 mg, 0.803 mmol) and was al-
lowed to warm to –35 °C for 40 min. The mixture was quenched
with aq sat. NH4Cl (1 mL) and diluted with EtOAc. The organic lay-
er was concentrated, diluted with EtOAc, washed with H2O, aq sat.
NaHCO3 and dried (MgSO4). Filtration, concentration and flash
column chromatography (silica gel, hexane/EtOAc, 2:1) afforded
the crude allylic alcohol as a diastereomeric mixture (1:1) at C2
(33.8 mg, 34% from 26).
To a stirred solution of bovine serum albumin (2 mg, 2 mmol) in
PBS aqueous solution (500 mL) was added the resultant the crude
activated ester (ca. 3 mmol) in DMF (60 mL) was added and the mix-
ture was allowed to stand at r.t. for 3.5 h without stirring. The result-
ant mixture was dialyzed against PBS aqueous solution (1 liter) at
4 °C to give hapten-BSA conjugate 4.
BC-Ring Fragment 22
A solution of 21 (142 mg, 0.152 mmol) in THF (5 mL) was treated
with TBAF (608 mL of 1.0 M THF solution, 0.608 mmol) and the
mixture was stirred at r.t. for 12 h. Concentration and flash column
chromatography (silica gel, hexane/EtOAc, 2:1) gave 22 (56.9 mg,
91%) as a colorless solid; [a]D28 –8.73 (c = 1.00, CHCl3).
1H NMR (200 MHz, CDCl3/TMS): d = 1.48 (2 H, m), 1.70 (4 H, m),
1.98 (1 H, m), 2.14 (1 H, m), 3.12 (2 H, m), 3.26 (1 H, t, J = 9.5 Hz),
3.29 (1 H, t, J = 9.5 Hz), 3.40 (1 H, m), 3.62 (3 H, m), 3.98 (1 H, m),
Synthesis 1999, No. SI, 1431–1436 ISSN 0039-7881 © Thieme Stuttgart · New York

PAPER
Designed Hapten Aimed at Anti-ciguatoxin Monoclonal Antibody
1435
1H NMR (200 MHz, CDCl3 /TMS): d = 1.51 (1 H, m), 1.72 (2 H,
m), 2.13 (1 H, m), 2.38 (1 H, br, OH), 2.40 (1 H, m), 2.68 (1 H, m),
2.90–3.48 (5 H, m), 3.53 (1 H, t, J = 9.0 Hz), 3.59 (1 H, t, J = 9.0
Hz), 3.81 (3 H, s), 3.93 (1 H, m), 4.02–4.40 (3 H, m), 4.47 (1 H, m),
5.40 (1 H, t, J = 9.0 Hz), 5.47 (1 H, m), 5.72 (1 H, m), 5.80 (2 H, m),
6.88 (2 H, m), 7.22 (2 H, m), 7.57 (2 H, m), 7.92 (2 H, m).
13C NMR (150 MHz, Pyridine-d5/TMS): d = 25.65 (CH2, C-15),
29.83 (CH2, C-14), 34.81 (CH2, C-8), 67.39 (CH2, C-1), 67.59
(CH2, C-16), 73.26 (CH, C-2), 74.55 (CH, C-11), 75.24 (CH, C-13),
76.51 (CH, C-9), 78.63 (CH, C-5), 83.06 (CH, C-12), 88.16 (CH, C-
10), 127.16 (CH, C-7), 131.61 (CH, C-3 or C-4), 132.76 (CH, C-3
or C-4), 136.14 (CH, C-6).
IR (film): n = 3476, 2866, 2362, 1723, 1591, 1514, 1464, 1270,
MS (EI): m/z (rel. int.,%) = 294 (8), 282 (12), 281 (14), 280 (5), 278
(6), 263 (6), 253 (17), 252 (100), 251 (73), 238 (15), 139 (23), 138
(11), 127 (14), 113 (15), 109 (15), 101 (10), 97 (15), 71 (58).
1176, 1102, 843, 756, 667 cm-1.
A solution of the above allylic alcohol (23.1 mg, 37.6 mmol) and
Et3N (77 mL, 550 mmol) in CH2Cl2 (1 mL) was treated with p-
BrBzCl (24 mg, 110 mmol) and DMAP (1 mg, 5 mmol) at r.t. and the
mixture was stirred for 1.5 h, diluted with Et2O, washed with H2O
and dried (MgSO4). Filtration, concentration and flash column
chromatography (silica gel, hexane/EtOAc, 3:1) gave a mixture
(1:1) of benzoates. A mixture of the benzoates in CH2Cl2 (1 mL)/
H2O (50 mL) was treated with DDQ (18 mg, 75 mmol) and stirred at
r.t. for 1 h. The mixture was quenched with aq sat. Na2S2O3 (0.5
mL), diluted with Et2O, washed with H2O, brine and dried
(MgSO4). Filtration, concentration and flash column chromatogra-
phy (silica gel, hexane/EtOAc, 2:1) gave 27 (6.2 mg, 24% for 2
steps) and 28 (8.3 mg, 33% for 2 steps) along with a mixture of 27
and 28 (7.1 mg, 28% for 2 steps).
HRMS (EI): m/z calcd for C16H24O6 (M+) 312.5171, found
312.1571.
ABC-Ring Fragment 6
A solution of 28 (10.6 mg, 15.6 mmol) and anhyd K2CO3 (22 mg,
160 mmol) in absolute MeOH (1 mL) was stirred at r.t. for 23 h. The
mixture was concentrated and subjected to flash column chroma-
tography (silica gel, CH2Cl2/MeOH, 5:1) to give 6 (3.5 mg, 72%).
(2S)-6
Colorless solid.
1H NMR (600 MHz, Pyridine-d5): d = 1.46 (1 H, m, H-15), 1.48 (1
H, m, H-14), 1.56 (1 H, m, H-15), 2.04 (1 H, m, H-14), 2.53 (1 H,
m, H-8), 2.71 (1 H, ddd, J = 15.9, 8.0, 4.0 Hz, H-8), 3.18 (1 H, ddd,
J = 10.5, 9.1, 4.3 Hz, H-13), 3.26 (1 H, t, J = 9.1 Hz, H-12), 3.29 (1
H, m, H-16), 3.44 (1 H, td, J = 8.8, 4.0 Hz, H-9), 3.73 (1 H, t, J = 8.8
Hz, H-10), 3.88 (1 H, m, H-16), 3.96 (2 H, m, H-1), 4.04 (1 H, dd,
J = 9.1, 8.8 Hz, H-11), 4.68 (1 H, m, H-2), 4.84 (1 H, m, H-5), 5.74
(1 H, m, H-7), 5.88 (1 H, dt, J = 11.3, 3.2 Hz, H-6), 6.36 (2 H, m,
H-3,4), 6.43 (1 H, br, OH), 6.68 (1 H, br, OH), 7.14 (1 H, br, OH).
13C NMR (150 MHz, Pyridine-d5): d = 25.66 (CH2, C-15), 29.84
(CH2, C-14), 34.82 (CH2, C-8), 67.42 (CH2, C-1), 67.59 (CH2, C-
16), 73.26 (CH, C-2), 74.56 (CH, C-11), 75.25 (CH, C-13), 76.53
(CH, C-9), 78.67 (CH, C-5), 83.07 (CH, C-12), 88.14 (CH, C-10),
127.15 (CH, C-7), 131.53 (CH, C-3 or C-4), 132.65 (CH, C-3 or C-
4), 136.23 (CH, C-6).
(2R)-27
[a]D24+16.8 (c = 0.31, CHCl3).
1H NMR (200 MHz, CDCl3): d = 1.50 (1 H, m), 1.70 (2 H, m), 2.14
(1 H, m), 2.43 (1 H, m), 2.68 (1 H, ddd, J = 16.0, 8.0, 4.0 Hz), 3.19
(1 H, t, J = 9.0 Hz), 3.20–3.56 (5 H, m), 3.58 (1 H, t, J = 9.0, 2.0
Hz), 3.92 (1 H, m), 4.52 (1 H, m), 5.40 (1 H, t, J = 9.0 Hz), 5.42 (1
H, m), 5.55–5.88 (3 H, m), 7.45 (2 H, m), 7.57 (2 H, m), 7.72 (2 H,
m), 7.87 (2 H, m).
IR (neat): n = 2956, 2868, 1725, 1591, 1487, 1464, 1439, 1400,
1338, 1270, 1176, 1102, 1071, 1044, 1013, 977, 847, 801, 756, 683,
667 cm-1;
(2S)-28
[a]D23+20.0 (c = 0.41, CHCl3).
MS (EI): m/z (rel. int.,%) = 294 (8), 282 (12), 281 (12), 263 (4), 253
(16), 252 (100), 251 (71), 238 (19), 139 (19), 138 (10), 127 (12),
113 (13), 109 (14), 97 (14), 84 (11), 83 (11), 71 (54).
HRMS (EI): m/z calcd for C16H24O6 (M+) 312.5171, found
312.1579.
1H NMR (200 MHz, CDCl3): d = 1.50 (1 H, m), 1.70 (2 H, m), 2.14
(1 H, m), 2.43 (1 H, m), 2.68 (1 H, ddd, J = 16.0, 8.0, 4.0 Hz), 3.20
(1 H, t, J = 9.0 Hz), 3.19–3.50 (5 H, m), 3.59 (1 H, t, J = 9.0 Hz),
3.94 (1 H, m), 4.50 (1 H, m), 5.40 (1 H, m), 5.41 (1 H, t, J = 9.0 Hz),
5.57–5.87 (3 H, m), 7.57 (2 H, m), 7.59 (2 H, m), 7.86 (2 H, m), 7.94
(2 H, m).
Acknowledgement
IR (neat): n = 3506, 2946, 2868, 1725, 1591, 1487, 1441, 1400,
1375, 1270, 1176, 1100, 1044, 1013, 977, 944, 847, 754, 683 cm-1;
We are grateful to Prof. Takeshi Yasumoto and Dr. Masayuki
Satake (Tohoku University) for their generous gift of CTX1B and
for their helpful comments.
ABC-Ring Fragment 5
A solution of 27 (12.1 mg, 17.8 mmol) and anhyd K2CO3 (22 mg,
160 mmol) in absolute MeOH (1 mL) was stirred at r.t. for 23 h. The
mixture was concentrated and subjected to column chromatography
on florisil (CH2Cl2/MeOH, 5:1) to give 5 (5.1 mg, 92%).
References
(1) Present address: Biomolecular Engineering Research
Institute, Suita 565.
(2) For recent reviews, see:
(a) Yasumoto T.; Murata, M. Chem. Rev. 1993, 93, 1897.
(b) Scheuer, P. J. Tetrahedron 1994, 50, 3.
(3) Relative structure:
(2R)-5
Colorless solid.
1H NMR (600 MHz, Pyridine-d5/TMS): d = 1.47 (1 H, m, H-15),
1.49 (1 H, m, H-14), 1.56 (1 H, m, H-15), 2.04 (1 H, m, H-14), 2.52
(1 H, m, H-8), 2.70 (1 H, ddd, J = 15.9, 8.0, 4.0 Hz, H-8), 3.18 (1 H,
ddd, J = 10.5, 9.1, 4.3 Hz, H-13), 3.26 (1 H, t, J = 9.1 Hz, H-12),
3.29 (1 H, m, H-16), 3.44 (1 H, td, J = 8.8, 4.0 Hz, H-9), 3.73 (1 H,
t, J = 8.8 Hz, H-10), 3.89 (1 H, m, H-16), 3.91 (2 H, m, H-1), 4.04
(1 H, dd, J = 9.1, 8.8 Hz, H-11), 4.67 (1 H, m, H-2), 4.82 (1 H, m,
H-5), 5.74 (1 H, m, H-7), 5.88 (1 H, dt, J = 11.3, 3.2 Hz, H-6), 6.33
(2 H, m, H-3,4), 6.43 (1 H, br, OH), 6.67 (1 H, br, OH), 7.13 (1 H,
br, OH).
(a) Murata, M.; Legrand, A. M.; Ishibashi, Y.; Yasumoto, T.
J. Am. Chem. Soc. 1989, 111, 8929.
(b) Murata, M.; Legrand, A. M.; Ishibashi, Y.; Fukui, M.;
Yasumoto, T. ibid. 1990, 112, 4380.
(4) Absolute configuration:
(a) Satake, M.; Morohashi, A.; Oguri, H.; Oishi, T.; Hirama,
M.; Harada, N.; Yasumoto, T. J. Am. Chem. Soc. 1997, 119,
11325.
(b) Oguri, H.; Hishiyama, S.; Sato, O.; Oishi, T.; Hirama, M.;
Synthesis 1999, No. SI, 1431–1436 ISSN 0039-7881 © Thieme Stuttgart · New York

1436
H. Oguri et al.
PAPER
Murata, M.; Yasumoto, T.; Harada, N. Tetrahedron 1997, 53,
(c) Kadota, I.; Park, C.-H.; Ohtaka, M.; Oguro, N.;
3057.
Yamamoto, Y. Tetrahedron Lett. 1998, 39, 6365.
(5) Yasumoto, T.; Fukui, M.; Sasaki, K.; Sugiyama, K. J. A. O. A.
C. Intern. 1995, 78, 574.
(12) The carbon numbering corresponding to that of CTX1B has
been used throughout this paper.
(6) (a) Hokama Y. Toxicon 1985, 23, 939.
(13) Kitagawa, T.; Miura, T.; Tanimiya, H. Chem. Pharm. Bull.
1972, 20, 2176.
(14) Shinoda T.; Tsuzukida, Y. J. Biochem. 1974, 75, 23.
(15) IgG subclass of 4H2, 6F12 and 6H7 was determined as IgG1l,
IgG3k and IgG1k, respectively.
(16) The 2,11-bis-p-bromobenzoates, 27 and 28, were converted to
the 1,2,11-tris-p-bromobenzoates, 29 and 30, which showed
the characteristic CD spectra as reported previously.
Assignment of the C2 configuration by CD spectra of the tris-
p-bromobenzoates is described in Ref 8.
(b) Hokama Y.; Hong T.W.P.; Isobe M.; Ichikawa Y.;
Yasumoto T. J. Clin. Lab. Analy. 1992, 6, 54.
(7) Production of mAbs to the synthetic JKLM-ring fragment-
carrier protein conjugates has been reported by K. Tachibana
and co-workers, see:
Pauillac, S.; Inoue, M.; Sasaki, M.; Naar, J.; Murata, M.;
Tachibana, K.; Chinain, M.; Legrand, A. M. Eur. J. Ichthyol.,
in press.
(8) Oguri, H.; Hishiyama, S.; Oishi, T.; Hirama, M. Synlett 1996,
1252, and Ref. 4b.
(17) (2R, 5R)-29
(9) For other synthetic studies of the ABC-ring fragment, see:
(a) Hosokawa, S.; Isobe, M. J. Org. Chem. 1999, 64, 37.
(b) Oka, T.; Fujiwara, K.; Murai, A. Tetrahedron 1998, 38, 21.
(10) Fortunato J. M.; Ganem, B. J. Org. Chem. 1976, 41, 2194.
(11) (a) Horita, K.; Hachiya, S.; Nagasawa, M.; Hikota, M.;
Yonemitsu, O. Synlett 1994, 38.
UV (EtOH): lmax = 245 nm (log e = 4.8).
CD (EtOH): lext = 257 nm (De = –1.5), 242 nm (+11.9).
(2S, 5R)
UV (EtOH): lmax = 245 nm (log e = 4.8).
CD (EtOH): lext = 253 nm (De+19.4), 236 nm (–4.6).
(b) Burke, S. D.; Jung, K. W.; Phillips, J. R.; Perri, R.E.
Tetrahedron Lett. 1994, 35, 703.
Article Identifier:
1437-210X,E;1999,0,SI,1431,1436,ftx,en;C01099SS.pdf
Synthesis 1999, No. SI, 1431–1436 ISSN 0039-7881 © Thieme Stuttgart · New York