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Synlett 2010(13): 1943-1946  
DOI: 10.1055/s-0030-1258479
LETTER
© Georg Thieme Verlag Stuttgart ˙ New York

Synthesis of Novel Haptens and Conjugates for Antibody Production against Kainoid Family

Etienne Bacoa, Luc Vellutinia, Jean-Paul Pillota,b, François-Xavier Felpina, Jean-Marie Schmittera,c, Bernard Bennetau*a,b, Marie Degueila,b
a Université de Bordeaux, UMR 5255 ISM, Talence 33405, France
b CNRS, UMR 5255 ISM, Talence 33405, France
Fax: +33(5)40006994; e-Mail: b.bennetau@ism.u-bordeaux1.fr;
c CNRS, UMR 5248 CBMN, Talence 33405, France
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Publication History

Received 11 May 2010
Publication Date:
09 July 2010 (online)

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Abstract

Amnesic shellfish poisoning (ASP) is caused by consumption of contaminated seafood that has accumulated kainic acid or kainoid analogues such as domoic acid. Among the different ASP bioassays, immunoassays are an attractive alternative to the in vivo mouse bioassay. Herein, we report the synthesis and bioconjugation of two new haptens for the generation of a specific antibody against members of the kainoid family.

Key words

ASP - kainoids - haptens - antibody - ELISA

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2-{(4 S ,5 S )-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): δ (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): δ (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.

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(2 S ,3 S )-Dibenzyl 3-[2-(Benzyloxy)-2-oxoethyl]-4-[4-( tert -butoxy)-4-oxobut-2-en-1-ylidene]pyrrolidine-1,2-dicarboxylate (12): The same experimental procedure as described for hapten 8 was used for compound 12 (yield: 80%). ¹H NMR (200 MHz, CDCl3): δ (mixture of stereo-isomers and conformers) = 2.56-2.84 (m, 2 H), 3.31-3.52 (m, 0.7 H), 3.65-3.90 (m, 0.3 H), 4.32-4.57 (m, 3 H), 5.01-5.23 (m, 6 H), 5.79 (d, J = 14.9 Hz, 1 H), 6.07 (d, J = 12.2 Hz, 1 H), 7.18-7.42 (m, 16 H), 10.00 (br s, 1 H). ¹³C NMR (50 MHz, CDCl3): δ (mixture of stereoisomers and conformers) = 38.9 (CH2), 44.9, 45.8 (CH), 48.3, 48.9 (CH2), 63.7 (CH), 66.5-67.8 (CH2), 121.9 (CH), 128.4-128.7 (CH), 135.4, 136.0 (C), 141.3 (CH), 147.7, 148.7 (C), 154.7, 155.2 (C), 170.2-171.4 (C). HRMS (ES): m/z [M + Na+] calcd for C33H31NO8Na: 592.1941; found: 592.1926.