116 MICROSOMAL PARTICLES 



We began this work by attempting to prepare a number of aminoacyl adenyl- 

 ates in a reasonable state of purity and in good yield in order to make possi- 

 ble the synthesis of radioactive aminoacyl adenylates. It is not unusual in mod- 

 ern biochemical research to synthesize and study compounds that are believed 

 to be intermediates in biochemical reactions. In the present instance this ap- 

 proach may suffer from a possible difficulty in that the intermediates may be 

 bound to enzymes and therefore not be in equilibrium with an external source 

 of intermediate. It has been observed, however, that chemically synthesized acyl 

 adenylate derivatives are enzymatically active in systems that catalyze activa- 

 tion of acetate [5] and fatty acids [6, 7] and the synthesis of phenylacetyl- 

 glutamine and hippurate [8]. 



Two procedures have been described for the preparation of aminoacyl adenyl- 

 ates. One of these [9] involves the condensation of the acid chloride of an 

 amino acid with silver adenylate. DeMoss et al. [9] obtained leucyl adenylate 

 in 9 per cent yield by this procedure. The other method involves condensa- 

 tion of the free amino acid and adenylic acid in the presence of N,N'-dicyclo- 

 hexylcarbodiimide, and precipitation of the product by addition of acetone [2]. 

 In our hands, these methods suffered from shortcomings often encountered in 

 the attempted synthesis of highly reactive molecules; thus, we obtained very 

 low yields of products of very low purity, and experienced great difficulty in 

 attempts at purification of the anhydrides. 



In an effort to solve these problems, we investigated a number of synthetic 

 approaches, two of which have proved successful. The first consisted of con- 

 densing an N-carbobenzoxyamino acid anhydride with adenylic acid in aque- 

 ous pyridine; the N-carbobenzoxyaminoacyl adenylate was isolated, and the 

 blocking group was removed by catalytic hydrogenation with palladium [10]. 



Subsequently, an alternative synthesis was developed which has proved to be 

 more convenient; it will therefore be described here in greater detail. Equi- 

 molar quantities of N-carbobenzoxyamino acid and adenylic acid were shaken 

 in aqueous pyridine with an excess of N,N'-dicyclohexylcarbodiimide for sev- 

 eral hours. N,N'-Dicyclohexylurea was removed by filtration, and the filtrate 

 was treated with acetone to precipitate the product. Treatment with acetone 

 removed unreacted N-carbobenzoxyamino acid, N,N'-dicyclohexylcarbodi- 

 imide, and most of the pyridine and water. The precipitate was extracted 

 with ethylene glycol monomethyl ether; the product is soluble, and adenylic 

 acid is insoluble in this solvent. The N-carbobenzoxyaminoacyl adenylate was 

 precipitated from the extract by addition of ether. After catalytic hydrogena- 

 tion of the carbobenzoxy compound and removal of the catalyst, the super- 

 natant solution was lyophilized. The free aminoacyl adenylate was obtained 

 as a white powder. 



The yields varied from 40 to 80 per cent; for example, glycyl-l-C 14 -adenylate 

 was obtained in 75 per cent yield, and the yield of DL-tryptophanyl-3-C 14 -adenyl- 

 ate was 44 per cent. The final products are estimated to be 70 to 80 per cent 



