554 BIOTIN 



mina; partition with butanol and cresol; countercurrent distribution; and 

 crystallization from water or from aqueous methanol or aqueous acetone. 

 When the biocytin was allowed to crystallize rapidly from aqueous meth- 

 anol or acetone the product obtained melted at 228 to 230° (dec. micro- 

 block). After slow crystallization from aqueous acetone, biocytin melted 

 at 245 to 252° (dec). 



Acid hydrolysis of recrystallized biocytin yielded about 00% of biotin 

 measured by microbiological assay. The same result was obtained when 

 alkaline hydrolysis was used, followed by reaction with phosgene to convert 

 the biotindiamine to biotin. Microanalysis of the free base gave the com- 

 position C16H28H4O4S. Paper chromatography of the hydrolyzate revealed 

 one ninhydrin-reacting spot, which was identified as lysine by its Rj value. 

 Microbiological assay confirmed the presence of lysine in the hydrolyzate. 

 In biocytin a free amino group was found as evidence by reaction with 

 ninhydrin and 2,4-dinitro-fluorobenzene. Nitrous acid interacts with the 

 lysine, furnishing after hydrolysis a derivative which did not react with 

 ninhydrin under the test conditions. Thus, biocytin appears to be c-N- 

 biotinyl-L-lysine.^"' ^- 



CO 



/ \ 

 NH NH 



I I 



CH CH 



I I 



CH2 CH(CH2)4CONH(CH2)4-CH-COOH 



\ / I 



S NHj 



Biocytin 



The structure of biocytin was confirmed by its synthesis from synthetic 

 biotin acid chloride^^ and the copper chelate complex of L-lysine. Reaction 

 of biotin acid chloride with a-N-formyl-L-lysine to give e-N-biotinyl-a-N- 

 formyl-L-lysine, followed by hydrolytic removal of the formyl group, also 

 yielded e-N-biotinyl-L-lysine.^^ 



Biocytin isolated from yeast extract and the synthetic compound were 

 compared by a number of chemical and biological tests and were found in 

 all respects identical.^^- *^ This comparison included infrared absorption 

 spectra, microbiological activities, stimulation of specific enzymatic reac- 



82 R. L. Peck, D. E. Wolf, and K. Folkers, /. Am. Chem. Soc. 74, 1999 (1952). 

 " D. E. Wolf, J. Valiant, and K. Folkers, J. Am. Chem. Soc. 73, 4142 (1951) 

 8< D. E. Wolf, J. Valiant, R. L. Peck and K. Folkers, J. Am. Chem. Soc. 74, 2002 



(1952). 

 86 L. D. Wright, E. L. Cresson, K. V. Liebert, and H. R. Skeggs, J. Am. Chem. Soc. 



74,2004 (1952). 



