Natural cyanamide (NH 2 CN) has recently been found in three Leguminosae plants: Vicia villosa subsp. varia, Vicia cracca and Robinia pseudo-acacia. As cyanamide has long been thought to be absent in nature, its physiological role and biosynthesis are totally unknown. N]-glutamine, indicating that these nitrogenous compounds are distant precursors of cyanamide. Although the guanidino group of l-arginine (-NH-C(NH 2 )=NH) and urea (NH 2 C(=O)NH 2 ) were candidate precursors of cyanamide on the basis of structural similarity, direct incorporation of the guanidino group of l-[ 13 C 6 , 15 N 4 ]-arginine and [ 13 C, 15 N 2 ]urea into cyanamide was not observed. These results eliminated the possibility that cyanamide is biosynthesized by the addition of ammonia to an electrophilic carbon or by the conversion of the tested compounds that were structurally relevant to cyanamide.

The ¹³C labels of [¹³C]carbon dioxide and D-[¹³C₆]glucose were incorporated into cyanamide (NH₂CN) when they were administered to Vicia villosa subsp. varia shoots. In contrast, the administration of sodium [2,3-¹³C₂]pyruvate did not affect the relative area of the [M + 1]+ ion of cyanamide in the gas chromatography-mass spectrometry analysis. [2,3-¹³C₂]pyruvate was incorporated into organic acids that are part of the citric acid cycle, such as succinate and fumarate, confirming that the shoots absorbed and metabolised it. These observations demonstrated that the carbon atom of cyanamide is derived from any of the carbohydrates that are present upstream of pyruvate in the metabolic pathway.