LETTER
Haptens and Conjugates for Antibody Production against Kainoids
1945
Intergovernmental Oceanographic Commission
(UNESCO): Paris, 2003, 247–266.
Although it is generally admitted that the length of the
linker is important in the design of the bioconjugate,24
there is a relative dearth of publications relating to the ef-
fects of the length of the linker on the performance of the
conjugates. To study these effects, the HWE reaction was
carried out (Scheme 3) using phosphonocrotonate.25 Con-
jugated esters 11 (Z/E and E/E isomers) were purified by
HPLC. Hapten 12 was obtained according the same ex-
perimental procedure as described for hapten 8.26 Immu-
noconjugate 13 was obtained under the same
experimental procedure as described for 9 and coupling
efficiency was estimated to be 4:1 by MALDI–MS analy-
sis. After deprotection with H2–Pd/C, the conjugates-to-
BSA ratio for 14 was found to be 3.7:1.
(7) Ciminiello, P.; Dell’Aversano, C.; Fattorusso, E.; Forino,
M.; Magno, G. S.; Tartaglione, L.; Quilliam, M. A.; Tubaro,
A.; Poletti, R. Rapid Commun. Mass Spectrom. 2005, 19,
2030.
(8) Leftley, J. W.; Hannah, F. In Natural Toxicants in Food;
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Sauvant, P.; Vergne, S.; Degueil, M.; Babin, P.; Bennetau,
B.; Bennetau-Pelissero, C. J. Agric. Food Chem. 2008, 56,
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Foster, D.; Wilkins, A. L. Nat. Toxins 1998, 6, 93.
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S. I.; Nilsen, M. V.; Goksoyr, A.; Briggs, L.; Holland, P.;
McNabb, P. J. AOAC Int. 2007, 90, 1011.
Haptens 10 and 14 were designed with short linkers to
minimize the influence of the chain (length, stereochem-
istry) at C-4 position for a specific antibodies production.
The length of the alkyl chain is long enough to place the
carrier protein a greater distance away from the common
part of kainoid analogues (pyrrolidine ring, carboxylic
and amino groups) but short enough to mask the alkyl
chain at C-4 position.
(14) Lefebvre, K. A.; Robertson, A. Toxicon 2010, 56, 218.
(15) Baldwin, J. E.; Fryer, A. M.; Pritchard, G. J. Bioorg. Med.
Chem. Lett. 2000, 10, 309.
(16) Ganorkar, R.; Natarajan, A.; Mamai, A.; Madalengoitia, J. S.
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M. W.; Wood, M. E. Tetrahedron 1997, 53, 5233.
(18) Mauger, A. B.; Irreverre, F.; Witkop, B. J. Am. Chem. Soc.
1996, 88, 2019.
In summary, new haptens for the generation of specific
antibody against kainoids have been synthesized from
commercially available trans-4-hydroxy-L-proline. To
the best of our knowledge, the synthesis of haptens in ASP
family, with a linker at C-4 position is reported for the first
time. Good antibody titers were observed after immuniza-
tion, as measured by ELISA (see Supporting Informa-
tion). It is expected that antibodies induced from these
new haptens will recognize kainoid analogues better than
those generated using the carboxylic or amino groups of
the pyrrolidine ring. Development of an immunoassay us-
ing these antibodies will be reported in due course.
(19) 2-{(4S,5S)-4-[2-(Benzyloxy)-2-oxoethyl]-1,5-
bis(benzyloxycarbonyl)pyrrolidin-3-ylidene} Acetic
Acid (8): To a solution of compound 7 (0.71 g, 1.18 mmol)
in anhyd CH2Cl2 (2 mL), triethylsilane (0.34 g or 0.47 mL,
2.92 mmol) and trifluoroacetic acid (1.73 g or 1.14 mL,
15.19 mmol) were added under argon. The reaction mixture
was stirred at r.t. for 16 h. Removal of the solvent in vacuo
afforded the product as a yellow oil (0.5 g, 80%). The
compound 8 was used in the next step without further
purification. 1H NMR (300 MHz, CDCl3): d (mixture of
stereoisomers and conformers) = 2.61–2.89 (m, 2 H), 3.40–
3.58 (m, 0.65 H), 4.25–4.35 (m, 0.35 H), 4.42–4.83 (m, 3 H),
5.07–5.28 (m, 6 H), 5.88, 5.94 (2 × s, 1 H), 7.23–7.55 (m, 15
H), 11.00 (br s, 1 H). 13C NMR (75 MHz, CDCl3): d (mixture
of stereoisomers and conformers) = 37.5, 38.9 (CH2), 42.7,
43.6, 45.1, 46.0 (CH), 50.4, 51.0, 51.4, 51.8 (CH2), 62.8,
63.1, 63.9, 64.2 (CH), 66.7–67.5 (CH2), 114.3, 114.6 (CH),
127.5–129.2 (CH), 135.0–136.1 (C), 154.4–155.0 (C),
159.9, 160.9 (C), 169.4–170.8 (C). HRMS (ES): m/z [M +
Na+] calcd for C31H29NO8Na: 566.1785; found: 566.1770.
(20) Metha, A.; Jaouhari, R.; Benson, T. J.; Douglas, K. T.
Tetrahedron Lett. 1992, 33, 5441.
Supporting Information for this article is available online at
experimental procedures and spectroscopic data for compounds 6,
7, 9, 10, 11, 13 and 14.
Acknowledgment
We gratefully thank the Conseil Régional d’Aquitaine for a stu-
dentship (E.B.) and for financial support.
References and Notes
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(24) Szurdoki, F.; Bekheit, H. K. M.; Marco, M.; Goodrow,
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Synlett 2010, No. 13, 1943–1946 © Thieme Stuttgart · New York