in. INDUSTRIAL PREPARATION 121 



sis of this coinijouiul a new asymmetric center has been created at position 

 () in the pyrazine riii<>;. Another point of (UfTerence between the natural and 

 synthetic citrovorum factor is that, when the two substances are exposed 

 to the action of ihhite acid, the natural material loses about one-third of its 

 l)otency as a source of folic acid for S. faecalis whereas the synthetic 

 material following acid treatment increases 11 % in activity for S. jaecalisP 



Synthetic leucovorin has been sepai'ated into its two diastereoisomers bj^ 

 Cosulich cl a(.^^'^ by utilizinjj; the di(ferenc(> in solubility of the calcium salts. 

 The /l form is the less solul)le form, and it has an [ajp of —15.1 compared 

 with +10.3 for the original mixture of the two diastereoisomers. The cal- 

 t'ium /L-leuco\orin has biological activities for S. faecalis and Levconostoc 

 cilrovorum strictly comparable to those for the natural material and is de- 

 creased in activity by acid treatment. The more soluble calcium c?L-leuco- 

 \orin has not been obtained in a pure state. 



The existence of different forms of the citrovorum factor in nature was 

 first clearly demonstrated by Winsten and Eigen.^"* When various crude 

 materials such as j^east extract or liver were chromatographed on paper and 

 the strips placed on an agar medium deficient only in CF and seeded with 

 Leuconostoc citrovorum, several distinct zones of growth appeared. It was 

 suggested that the chromatographically slower factors represent conjugated 

 forms of the faster forms. Different forms of the synthetic factor have also 

 been prepared corresponding to the mono-, di-, and triglutamic acid deriva- 

 tives of PGA. 



III. Industrial Preparation 



ROBERT S. HARRIS 



Because of the intense activity of commercial groups in the development 

 of inexpensive and effective methods for the synthesis of pteroylglutamic 

 acid, it is not possible at this time to describe the methods now being used 

 in the industrial i)reparation of this vitamin. Instead it is necessary to 

 describe some of the synthetic methods first reported in the scientific litera- 

 ture and to list a few of the patents which have been issued. 



The chemical synthesis of PGA was first reported by Angier ct al.,^ who 



'"^ W. A. Winsten and E. lOigen, J. Biol. Chem. 184, 155 (1950). 



»»< D. B. Cosulich, J. M. Smith, Jr., and 11. P. Bnuiuist, J. Am. Chem. Soc. 74, 1215 



(1952). 

 ' R. B. Angier, J. H. Boothe, B. L. Hutchings, J. II. Mowat, J. Semb, E. L. R. 



Stokstad, Y. SubbaRow, C. W. Waller, D. B. Cosulich, M. J. Fahronbach, M. E. 



Hultquist, E. Kuh, E. H. Northey, D. R. Seeger, J. P. Sickels, and J. M. Smith, 



Jr., Science 103, 667 (1946); Ann. N. Y. Acad. Sci. 48, 283 (1946). 



