II. CHEMISTRY 



533 



amino nitrogen procedure.-^ These results show that biotin contains neither 

 an amino group nor basic ring nitrogen atoms. 



Through drastic hydrolysis of biotin with hydrochloric acid'"* or barium 

 hydroxide'-* an optically active diaminocarboxylic acid, 09111802X28, was 

 obtained which was found to contain two primary amino groups by the 

 Van Slyke nitrous acid method, ^''arious derivatives of this compound were 

 prepared, such as dibenzoate, diacetate, sulfate, dihydrochloride, and di- 

 picrolonate. The formation of the strongly basic diaminocarboxylic acid, 

 C9H18O2N2S, from the weakly basic biotin, C10H16O3N2S, with the re- 

 placement of one CO group by two hydrogen atoms, pointed to a cyclic 

 urea structure. This assumption was well supported by the resynthesis 

 of biotin from the diaminocarboxylic acid with phosgene.^" 



CsHisS ' 



-NH 

 \ 



CO 



— NH 



Ba(OH)i 



Phosgene 



CgHisb 



— NH2 

 — NH2 



— COOH 



^ —COOH 

 Biotin 



Diaminocarboxylic acid 



The methyl ester of the resynthesized product was identical with the 

 original natural biotin methyl ester. Their yeast growth activities were 

 also equal. 



2, The Nature of the Sulfur in Biotin 



The sulfur of biotin appeared to be stable. No H2S was liberated when 

 biotin was treated with zinc dust and HCl, and after treatment with 

 bromine no inorganic sulfate could be detected. The nitroprusside test for 



CgHn ■ 



— NH 



\ 



( 



/ 

 — NH 



CO 



CsHia 



-COOH 



\ 



s 



[/ 



Biotin 



-NH 



CO 



/ 



— NH 



—COOH 



SO, 



Biotin sulfone 



" G. B. Brown and V. du Vigneaud, J. Biol. Chem. 141, 85 (1941). 



»» D. B. Melville, K. Hofmann, and V. du Vigneaud, /. Biol. Chem. 145, lUl (1942). 



