PURINE AND PYRIMIDINE ANALOGUES 29 1 



inhibited cell wall mucopeptide synthesis: 5-bromouracil, 5-fluorouracil, 

 6-azauracil, 6-azathymine, 8-azaguanine and its riboside, 2 : 6-diamino- 

 purine, and 6-mercaptopurine. At a concentration of o-i jumole/ml. of 

 5-fluorouracil the biosynthesis of cell wall mucopeptide was inhibited 

 30-60%. The other compounds were tested in concentrations up to i 

 /xmole/ml. without effect upon biosynthesis. A separate section will be 

 devoted to the examination of the effect of certain substituted benzimida- 

 zoles which have also been examined. 



The accumulation within the cells of compounds soluble in trichloroace- 

 tic acid and which contain N-acetylhexosamine in a bound form, was 

 examined by the technique previously used by Strominger [18] for penicil- 

 lin-treated cells. It was found that a considerable accumulation of such 

 compounds occurred. For example, in the presence of 0-4 jumole of 5- 

 fluorouracil/ml. the cells accumulated 600 /xg. of N-acetylhexosamine/ 100 

 mg. of bacterial dry wt. during i hr. incubation, whilst the control cells 

 incubated without 5-fluorouracil had accumulated only 50 60 /xg. of bound 

 N-acetylhexosamine. The amino sugar concentration is expressed in terms 

 of N-acetylglucosamine. The amount present in the cells increased accor- 

 ding to the concentration of fluorouracil added to the incubation medium 

 up to the highest concentration of inhibitor examined (o •4/zmole/ml.). This 

 accumulation could be prevented equally by the simultaneous presence 

 of either uridine or uracil; thymidine was about a third as effective as 

 these two compounds on a molar basis. 



The nature of compounds containing N-acetylhexosamine 



Washed cells of Staphylococcus aureus 524/SC were incubated at 35° 

 in the solution used previously which contained the four cell wall amino 

 acids, glucose, phosphate, and chloramphenicol. A concentration of 0-4 

 /imole/ml. of [2-^^C]-5-fluorouracil was included. Incubation was con- 

 tinued for I hr. at 35° with aeration. At the end of this time the cells 

 were removed by centrifugation and washed with cold o • i m sodium- 

 potassium phosphate buffer at pH 7-0. They were then extracted with 

 cold 10% trichloroacetic acid [18] as before. The extract was freed from 

 trichloroacetic acid by ether extraction, the residual ether removed by 

 aeration and the extract then chromatographed on a column of Dowex-i 

 (X2, chloride form, 50-100 mesh). The eluents used were those described 

 by Strominger [18]. The emerging samples were examined for bound 

 N-acetylhexosamine by the usual technique [18] and for radioactivity. Two 

 peaks containing both N-acetylhexosamine and [2-^^C]-5-fluorouracil were 

 found corresponding in position to the bound N-acetylhexosamine peaks 

 obtained when extracts from penicillin-treated cells were examined in a 

 similar manner. Both these peaks appeared likely to contain more than 



