II. ('iii;Misrio 09 



the aromatic amino and a ivduccd ptcMidinc.'' Attcr ivoxidation with 

 manganese (hoxide, the i)tei"i(hne ()l)tained l)y zinc icduclion \\;is identified 

 as 2-amin()-4-hy(h()xy-()-met hyli)teridine. 



The foregoing evidence iiuhcated the following conclusions regarding Ihe 

 structure of pteroylglutamic acid. 



1. Aerobic alkaline hydrolysis, sulfurous acid cleavage, and chemical or 

 catalytic reduction each yielded a pteridine and a primary aromatic amine. 

 This indicated tlu^ linkage of the pteridine to the nitrogen of the aiomatic 

 amine. 



2. The aromatic amine formed during sulfurous acid cleavage of pteroyl- 

 trigliitamic acid was a tetrapeptide, p-aminobenzoj'ldiglutamylglutamic 

 acid. The aromatic amme from pteroylglutamic acid was p-aminohenzoyl- 

 glutamic acid. 



3. There was a single-carbon atom linkage between the pteridine and the 

 aromatic amine. This was indicated by the fact that only pteridines with a 

 single-carbon atom side chain were obtained and that no other 2-carbon 

 fragments could be detected in the two degradation reaction products. 

 The evidence also indicated that this single-carbon atom is present in a 

 methylene link. If this carbon atom were present in an amide linkage, the 

 cleaN-age would be hydrolytic and would not require oxygen. The formation 

 of 2-amino-4-hydroxy-6-methylpteridine by reduction also constitutes evi- 

 dence for the methylene linkage. 



h. Degradation of Vitamin Be {Pteroylglutamic Acid) 



Wittle et al.-^ described the oxidative degradation of vitamin Be (pteroyl- 

 glutamic acid) which had been isolated from liver. Oxidation with alkaline 

 permanganate or chloric acid gave a fluorescing pigment with a charac- 

 teristic absorption spectrum which was identified as 2-amino-4-hydroxy-6- 

 carboxypteridine. The chloric acid oxidation mixture also yielded an ethyl 

 acetate-soluble crystalline material which proved to be 3,5-dichloro-4- 

 aminobenzoylglutamic acid. This compound decomposed at a temperature 

 slightly above the melting point to yield two lower molecular weight frag- 

 ments which were identified as 3,5-dichloro-4-aminol)enzoic acid and dl-pyr- 

 rohdonecarboxylic acid. 



P.. PHYSICAL AM) CHEMICAL PROPERTIES 



Pteroylglutamic acid when crystallized from watei- forms yellow, sjiear- 

 shaped leaflets (Fig. 2). On heating it darkens, and it chars at around 250° 

 without melting (Pfiffner ct a/.'"). Its solubility in water as the free acid is 

 10 mg. per liter at 0' and over 500 mg. per liter at 100". The disodiuni salt 



" E. L. Wittlo. B. L. ()'I)('11..J. .M. \ •unlcnhclt . and .J. .1. IMilTiKT. ./. Am. Chew. Soc. 

 69, 1786 (1947). 



