Anhydroleucovorin forms

Second, leucovorin is known to dehydrate under acidic conditions to form anhydroleucovorin, 5,10-methenyl H, F, which absorbs at 352-353 nm. In the absence of interfering species, leucovorin may be analyzed by acidification with 0.1N HC1 followed by uv measurement after 1.5-2.0 hours. Purity may be determined relative to a sample of known purity or relative to literature values e352 = 2.39-2.41 x 10 M 1 cm"1.21... 

When formiminoglycine (FIG) was broken down to glycine, derivatives of folic acid were involved and ATP formation occurred (H, 297). These reactions are shown in Fig. 17. The first of these reactions was catalyzed by FIG formimino transferase FIG reacted with FH4, free glycine was formed, and iV -formimino-FH now carried the formimino grouping. In the second step, NH3 was released from iNT -formimino-FEU and the cyclic iVs. Vio-imidazolinium derivative of iV -formyl-FH4 resulted. The latter compound was also called lV ,Ari >-methenyl-FH4, anhydroleucovorin, or anhydrocitrovorum factor the enzyme responsible for the formation of the imidazolinium derivative from A -formimino-FH4 was named formimino-FH4 cyclodeaminase. In the third reaction, methenyl-FH4 cyclohydrolase catalyzed the conversion of lV ,iW -methenyl-FH4 to JV -formyl-FH4. The fourth step, which resulted in ATP formation, was catalyzed by FH4-formylase. ADP and P reacted with JW -formyl-FH4 and produced FH4, free formic acid, and ATP. The reverse of this reaction resulted in the activation of free formic acid [Eq. (5)]. 

An important chemical property of leucovorin is its ready conversion to 5,10-roethenyl-FH4 (anhydroleucovorin, 5,10-CH=FH4+) by the action of dilute acid. This transformation may also be brought about enzymically by an enzyme citrovorum factor cyclodehydrase which was separated from sheep liver by Peters and D. M. Greenberg (SB). 5,10-CH=FH4+ is also active as a single-carbon donor in crude enzyme systems and may be in equilibrium with 10-formyl-FH4 (S3) which may in turn be formed enzymically from FH4 and formate. 

When the purified material was treated with alkali at 100°C. (conditions which destroy the cofactor activity), a fluorescent compound was formed which had an activation peak at 380-400 m/t and a fluorescent peak at 470-480 m/A. Since these fluorescent characteristics resembled those reported for folic acid and folinic acid, various folic acid derivatives were tested for cofactor activity. Tetrahydrofolate was found to be highly active, while folic acid, dihydrofolic acid, and 5-formyltetrahydrofolate were completely inactive. Anhydroleucovorin (5, 10-methenyltetra-hydrofolate), which would be expected to hydrolyze to 10-formyltetra-hydrofolate under the conditions of the assay, showed some activity (Kaufman, 1958b). 

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