114 PTEROYLGLUTAMIC ACID 



The ultraviolet spectra of a number of P(>A derivatives,^'' pyrimi- 

 dines,^^'^* and their reduction products provides further support for the 

 hypothesis that all three double bonds in the pyrimidine ring of PCiA remain 

 intact in its conversion to leucovorin. Thus, dihydropyrimidines such as 

 dihydrouracil or thymine glycol demonstrate little if any absorption in the 

 ultraviolet region.^® On the other hand, the ultraviolet absorption of tetra- 

 hydro PGA (XVI), 10-formyltetrahydro PGA (XIV), leucovorin (XII), 

 and 5,10-diformyltetrahydro PGA (XVII) closely resembles those of cer- 

 tain model pyrimidines. Furthermore, it is highly improbable that the 

 benzene ring of the p-aminobenzoyl portion of PGA would be reduced 

 under the relatively mild conditions employed in the synthesis of leucovorin. 



d. Location of the Formyl Group 



Since the reduction of 10-formyl PGA (XV) to a tetrahydro derivative 

 (XIV) does not result in appreciable leucovorin activity initil the reaction 

 mixture has been allowed to stand or is poured into dilute alkali and heated, 

 the development of activity appears to depend on a subsequent rearrange- 

 ment. The resulting leucovorin (XII) has several iniique chemical proper- 

 ties. Thus, it is cjuite stable in dilute alkali but is quickly inactivated in 

 dilute acid;^*' ^*' ^^"^^ the acid inactivated product, however, retains activity 

 for S. faecalis R. 



Many possibilities have been considered to explain the pronounced 

 changes in biological activity and stability exhibited by leucovorin and the 

 various intermediates betw^een PGA and leucovorin which have been iso- 

 lated. These include the formation of a new ring linking the N'" position 

 by a 1-carbon bridge to position 5, 7, or 8 in the tetrahydropyrazine ring; 

 a shift of the N'°-formyl group to the pyrazine ring; and the introduction 

 of an additional formyl group (or groups) in the tetrahydropyrazine ring 

 with subsequent removal of the N^^-formyl group, by hydrolysis in the 

 alkaline treatment. 



The possibility that the formyl group in leucovorin exists as a bridge 

 linking the 10 position with the 7 position of the tetrahydropteridine ring 

 was investigated^" by means of oxidation with alkaline permanganate. Such 

 treatment converts 2-amino-4-hydroxy-6- or 7-alkvlpteridines, including 

 PGA and its analogs, to the corresponding G- or 7-carboxypteridines.-^ 



^* L. F. Cavalieri, A. Beiidich, J. F. Tinker, and G. H. Brown, ,/. Am. Cheni. Soc 



70, 3875 (1948) 

 8« L. F. Cavalieri and A. Bendich, J. Am. Chem. Soc. 72, 2587 (1950). 

 " T. H. Jukes, H. P. Broquist, and E. L. R. Stokstad, Arch. Biochem. 26, 157 (1950). 

 s« J. C. Keresztesy and M. Silverman, J. Biol. Chem. 183, 473 (1950). 

 ^^ M. Silverman and J. C. Keresztesy, J. Am. Chem. Soc. 73, 1897 (1951). 

 8»D. B. Cosulich, B. Roth, J. M. Smith, Jr., M. E. Hultquist, and R. P. Parker, 



/. Am. Chem. Soc. 74, 3252 (1952). 



J 



