98 PTEROYLGLUTAMIC ACID 



aromatic amine and a fluorescent pigment. This pigment reacted rapidly 

 with typical aldehyde reagents, indicating the presence of an aldehyde 

 group. This fluorescent pigment did not possess a carboxyl group, as evi- 

 denced by the fact that its distribution coefficient between water and 

 butanol was the same at pH 3.0 as at pH 7.0. When the pigment obtained 

 by sulfurous acid hydrolysis was treated anaerobically with dilute sodium 

 hydroxide, approximately equal amounts of 2-amino-4-hydroxypteridine 

 6-carboxylic acid (V) and 2-amino-4-hydroxy-6-methylpteridine (VII) were 

 formed. The formation of approximately equal molal quantities of carboxy 

 and methyl derivatives from what is apparently an aldehyde probably 

 involves a Cannizzaro type of reaction, although the mechanism of this 

 reaction is obscure. 



Prolonged aqueous hydrolysis of pteroyltriglutamic acid at pH 4 yielded 

 1-pyrrolidonecarboxylic acid, which on hydrolysis with alkali yielded l( + )- 

 glutamic acid."^ 



The aromatic amine which was produced during sulfurous acid hydrolysis 

 was isolated as the barium salt. This compound when diazotized and 

 coupled with N-(l-naphthyl)ethylenediaminedihydrochloride yielded a red 

 pigment,^^ which indicated a primary aromatic amine with a highly negative 

 substituent group. The aromatic amine nitrogen as measured by the method 

 of Bratton and MarshalP^ constituted approximately 25% of the total 

 nitrogen. The remaining 75% of the nitrogen could be converted into 

 a-amino acid nitrogen by alkaline hydrolysis. From such hydrolyzates the 

 aromatic amine was isolated and identified as p-aminobenzoic acid. Micro- 

 biological assay of the hydrolyzate indicated the presence of 3 moles of 

 glutamic acid. The peptide linkage of the glutamic acid to p-aminobenzoic 

 acid must involve the carboxyl group of the latter as a primary aromatic 

 amine is required for reaction in the Bratton and Marshall test.-- 



The diazotizable aromatic amine obtained by aerobic alkaline hydrolysis 

 of racemic pteroylglutamic acid was found to contain 2.1 atoms of nitrogen 

 for each atom of aromatic amino nitrogen. On hydrolysis with 2 N sulfuric 

 acid it yielded p-aminobenzoic acid and 45 % of the total nitrogen appeared 

 as a-amino acid nitrogen. This was later identified as glutamic acid, and 

 thus the diazotizable amine was identified as p-aminobenzoylglutamic acid. 



Evidence regarding the mode of linkage is furnished by the results of 

 alkaline hydrolysis.^^ The absence of fluorescence and of the free aromatic 

 amine in the original pteroylglutamic acid, and the simultaneous ap])carance 

 of these two during aerobic alkaline hydrolysis, suggested that the pteridine 

 is linked to the aromatic amine nitrogen. As hydrolysis proceeded, the 

 lil)eration of ptei'idine and aromatic amine appeared at a])i)r()\iniately the 

 same rate. 



Reduction in acid solution, either catalytieallv or with zinc dust, vielded 



