IF. CIIKMISTHY 113 



can 1)0 readily (U'tciiniiuul, since it is a t'nnclion ot the lieij^lit of the wave 

 lietween inflection points. Control e.\|)eriinents showed that neither the 

 pyrimidine ring nor p-aminohenzoylglutaniic acid is reduced under the 

 same polarographic conditions. In this manner it was demonstrated that 

 the crude product formed by the reduction of P(IA or lO-formyl PCI A 

 under the conditions used in the synthesis'^- "^ is a tetrahydropteridine 

 derivative (XIV) because it produces the typical tetrahydropteridine wave 

 in the polarograi)h at pll 9.0. Leucovorin was found to give no wave in the 

 polarograph at pU 9.0, but after the mild acid treatment which destroj^ed 

 activity for Le. citrovorum but not for *S. faecalis R the tj^ical tetrahy- 

 dropteridine wave was found. This observation, discussed in greater detail 

 below, not only adds to the proof that leucovorin is a tetrahydropteridine 

 derivative but serves as the basis for a polarographic assay of leucovorin. 

 Thus, determinations are made before and after mild acid treatment, and 

 the difference in height of the tetrahydro wave forms a measure of the 

 concentration of leucovorin. 



Available evidence indicates that it is the pyrazine ring which undergoes 

 reduction during hydrogenation of pteridines. Hydrogen and platinum 

 readily reduce pyrazine derivatives to the corresponding piperazines,^" ■ ^' 

 whereas the pyrimidine nucleus is more resistant to catalytic hydrogena- 

 tion.82 



It has been shown by Waller et al.^ that treatment of PGA with sul- 

 furous acid results in cleavage to 2-amino-4-hydroxypteridine-6-carboxalde- 

 hyde, probably through the corresponding intermediate dihydropteridine. 

 I'nder similar conditions leucovorin is partially destroyed.*^ Since unsatura- 

 tion of the pyrazine ring appears necessary for the occurrence of this re- 

 action, the stability of leucovorin is a further indication of the 5,6,7,8- 

 tetrahydro structure (XII). 



Additional evidence that the four hydrogen atoms of leucovorin reside in 

 the 5, 6, 7, and 8 positions of the pyrazine ring and not the pyrimidine ring 

 is provided l)y electrometric titration data.*^ Thus, leucovorin exhibits a 

 phenolic hydroxyl group with a pKa of 10.4 which is comparable to the 

 value of 8.2 found for the hydroxyl group in the 4 position of PCJ A. If reduc- 

 tion of the pyrimidine ring had occurred, no enolic group would be found. 



'9 B. Roth, M. E. Hultquist, M. J. Fahrenbach, D. Ji. Cosulich, H. P. Broquist, 



J. A. Brockman, Jr., J. M. Smith, Jr., R. P. Parker, E. L. R. Stokstad, and T. H. 



Jukes, J. Am. Chem. Soc. 74, 3247 (1952). 

 80 F. B. Kipping, J. Chem. Soc. 1929, 2889. 

 «' F. B. Kipping, J. Chem. Soc. 1932, 1336. 



'2 E. B. Brown and T. B. Johnson, J. Am. Chem. Soc. 45, 2702 (1923). 

 " C W. Waller, A. A. Goldman, R. B. Angier, J. H. Boothe, B. L. Hutchings, J. II. 



Mowat, and J. Semb, J. Ayn. Chem. Soc. 72, 4630 (1950). 

 «< A. Pohland, B. H. Flynn, R. G. Jones, and W. Shive, /. Am. Chem. Soc. 73, 3247 



(1951). 



