A simple procedure for sulfation and 35S radiolabelling of paralytic shellfish poisoning (PSP) gonyautoxins

A method is described to sulfate PSP toxins at various positions in the molecule and to prepare 35S labelled compounds using H2(35)SO4 in the presence of dicyclohexylcarbodiimide (DCC). The 11-sulfates of saxitoxin and neosaxitoxin, known as gonyautoxins, are often the most abundant of the PSP toxins in algae and contaminated shellfish. Receptor site binding and antibody assays based on these analogues should, therefore, better reflect toxicity than those in which saxitoxin is used. Although the specific activity of 35S-gonyautoxins is lower than that of commercially available 3H-saxitoxin, the label is strongly bound and is not lost through proton exchange with water as occurs with tritiated saxitoxin. The labelling procedure is rapid, inexpensive and can be done on a small scale. Sulfate can be removed from the 11-position of GTX's in methanolic-HCl and from the 21-position by mild acid hydrolysis and H2(35)SO4 added in 5-10-fold excess. Addition or exchange occurs rapidly on mixing DCC in dimethylformamide with dry toxin and sulfate. Reaction conditions were optimized and reaction products identified by capillary electrophoresis, autoradiography and ionspray mass spectrometry. Together with methods for selective removal of sulfate, the sulfation reaction provides an additional way to prepare some of the naturally occurring derivatives of saxitoxin, many of which are sulfates.