20 



reduction with hydrogen and palladium/BaSO of N-(4-nitro- 

 benzoyl) -N ' -BOC-hexamethylenediamine yielded N-(4-amino- 

 benzoyl) -N ' -BOC-hexamethylenediamine which was subjected to 

 diazotization with a-amanitin . In a typical reaction 34mg of 

 NaNO„ were added to 112mg of N- (4-aminobenzoyl) -N ' -BOC- 

 hexamethylenediamine in 7ml of 50% acetic acid (5°C) and incu- 

 bated 10 minutes at 5°C. This diazonium cation containing 

 solution was combined with 65mg of a-amanitin dissolved in 

 4.5ml of pyridine (5°C) . After 15 minutes of reaction at 5°C, 

 the mixture was flash evaporated to dryness (40°C with vacuum 

 for rapid removal of pyridine and other solvents) , resolu- 

 bilized in spectral grade methanol and applied to a column 

 of Sephadex LH-20 in methanol (4.25 x 95cm). The a-amanitin 

 derivative , a-amanitin-diazobenzoyl-N-N ' -BOC-hexamethylene- 

 diamine (ADBH) was identified as a reddish-purple band 

 eluting in 0.54 column volumes by its typical mobility 

 during TLC in methanol: methylethylketone (Faulstich and 

 Trischmann, 1973) . The ADBH was further characterized by 

 absorption and NMR spectra and stored at room temperature, 

 dried, in darkness until used (Hencin and Preston, 1979). 



Free carboxyl group containing a-amanitin derivatives 

 were produced by diazotization of a-amanitin with p-amino- 

 benzoylglycylglycine (Preston and Hencin, manuscript in 

 preparation) . p-Aminobenzoylglycylglycine was prepared 

 by catalytic hydrogenation of p-nitrobenzoylglycylglycine 

 with Pd/BaSO . . For coupling to a-amanitin, 30mg of p-amino- 

 benzoylglycylglycine dissolved in 1.0ml of 50% acetic acid 



