75 



Other preparations in which Con A and ADH were coupled in 

 the presence of saturating amounts of specific ligand and 

 under varying salt and ion concentrations also failed to 

 absorb to Sephadex. Since it appeared likely that conju- 

 gation to carboxyl groups on Con A resulted in a loss of 

 ligand binding activity, investigations of these ADH-Con A 

 conjugates were discontinued. 



ADGG-BSA Conjugates 



The results of the ADH-Con A studies demonstrated the 

 need for an a-amanitin derivative containing a free car- 

 boxyl group. Such a derivative could be coupled to free 

 protein amino groups and possibly result in a derivative of 

 Con A that retained ligand binding properties as well as the 

 specificity of interaction of a-amanitin and RNA polymerase 

 II. 

 Synthesis 



The synthetic pathway for one derivative of this type 

 is shown in Figure 12 as developed by Dr. J. F. Preston. 

 The synthesis follows the same general pattern as that for 

 ADH conjugates. It results in the production by diazotiza- 

 tion of a-amanitin and an aromatic containing carbon chain 

 spacer molecule, p-aminobenzoylglycylglycine , of a free 

 carboxyl containing a-amanitin derivative, a-amanitin- 

 diazobenzoylglycylglycine (ADGG) . The ADGG was purified 

 from the diazotization reaction by chromatography on Sepha- 

 dex LH-20 in 80% methanol. The product was judged to be 



