19 



Wieland, 1968) by other investigators. Additional details 

 of the experiments resulting in the final purification 

 procedure are contained in a manuscript currently in 

 preparation for publication (Preston et al., in preparation 

 for submission to Lloydia) . 

 Derivatization 



The absence of either a free carboxyl or amino group 

 on a-amanitin necessitated the development of methods for 

 modifying the basic amanitin structure to allow direct 

 coupling of a-amanitin to proteins. The basic concept was 

 derived from the work of Faulstich and Trischmann (1973) 

 in which a-amanitin was coupled via diazotization to an 

 aromatic group linked to a six carbon spacer molecule 

 containing a free terminal amino group. Their procedure 

 was used with some modification to produce a-amanitin deri- 

 vatives with free amino groups. A derivative containing a 

 free carboxyl group was obtained by using the diazonium 

 coupled spacer molecule approach with new procedures developed 

 by J. F. Preston (Preston and Hencin, 1979). Details of both 

 of these syntheses are presented below. 



For production of a-amanitin derivatives containing ter- 

 minal amino groups, a-amanitin was diazotized to N-(4-amino- 

 benzoyl) -N ' -BOC-hexamethylenediamine . Mono-BOC-1 , 6-diamino- 

 hexane was prepared according to procedures described by 

 Faulstich and Trischmann (1973). N- (4-nitrobenzoyl) -N ' -BOC- 

 hexamethylenediamine was prepared by acylation of mono-BOC- 

 1 , 6-diaminohexane with 4-nitrobenzoyl chloride. Catalytic 



