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We quantified the cyanamide content of young leaves of nine Vicia species, Lens culinaris, and Robinia pseudo-acacia using a modified analytical procedure that made it possible to measure the cyanamide content of a single leaf. Recent molecular phylogenetic analysis suggests that cyanamide is present in V. benghalensis, which is placed in a monophyletic group with cyanamide-biosynthesizing plants, V. villosa and V. cracca; this suggestion was verified.

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.

Cyanamide (NH2CN) has recently been proven to be a natural product, although it has been synthesized for over 100 years for agricultural and industrial purposes. The distribution of natural cyanamide appears to be limited, as indicated by our previous investigation of 101 weed species. In the present study, to investigate the distribution of natural cyanamide in Vicia species, we monitored the cyanamide contents in V. villosa subsp. varia, V. cracca, and V. amoena during their pre-flowering and flowering seasons. It was confirmed that V. cracca was superior to V. villosa subsp. varia in accumulating natural cyanamide, and that V. amoena was unable to biosynthesize this compound under laboratory condition examined. The localization of cyanamide in the leaves of V. villosa subsp. varia seedlings was also clarified. In a screening study to find cyanamide-biosynthesizing plants, only Robinia pseudo-acacia was found to contain cyanamide among 452 species of higher plants. We have investigated 553 species to date, but have so far found the ability to biosynthesize cyanamide in only three species, V. villosa subsp. varia, V. cracca and R. pseudo-acacia.

Cyanamide (NH2CN) is found in nature, although it has long been recognized as an industrial product. Distribution of cyanamide in the plant kingdom was investigated using a direct quantitative determination method to detect and measure cyanamide by stable isotope dilution gas chromatography-mass spectrometry (the SID-GC-MS method). The SID-GC-MS method proved to be a robust way to quantify cyanamide contents in the extracts of 101 species of herbaceous plants. The average recovery of cyanamide from all plants tested was 55.6+/-20.3%. Vicia villosa and V. cracca contained cyanamide at 369-498 microg/gFW and 3,460-3,579 microg/gFW respectively, while the other 99 species contained no detectable cyanamide (<1 microg/gFW). This result suggests that distribution of cyanamide in the plant kingdom is limited and uneven.

Cyanamide (NH(2)CN) has recently been isolated as a plant growth inhibitor from Vicia villosa, which is the first discovery of cyanamide from natural sources. To reveal the presence of the biosynthesized cyanamide in plants, 3.4 mM potassium ((15)N)nitrate was administered to 15- to 35-day-old plants of V. villosa, from which the cyanamide was purified and subjected to GC/MS analysis. The isotopic ratio (15)N/((14)N + (15)N) of the cyanamide was calculated to be 0.143, while that of the cyanamide extracted from V. villosa grown in the presence of a natural N source was 0.0065. The (15)N-enrichment proved de novo biosynthesis of cyanamide.

Cyanamide is a multifunctional agrochemical used, for example, as a pesticide, herbicide, and fertilizer. Recent research has revealed that cyanamide is a natural product biosynthesized in a leguminous plant, hairy vetch (Vicia villosa). In the present study, gas chromatography-mass spectrometry (GC-MS) equipped with a capillary column for amines was used for direct quantitative determination of cyanamide. Quantitative signals for ((14)N(2))cyanamide, ((15)N(2))cyanamide (internal standard for stable isotope dilution method), and m-(trifluoromethyl)benzonitrile (internal standard for correcting errors in GC-MS analysis) were recorded as peak areas on mass chromatograms at m/z 42 (A(42)), 44 (A(44)), and 171 (A(IS)), respectively. Total cyanamide content, ((14)N(2))cyanamide plus ((15)N(2))cyanamide, was determined as a function of (A(42)+A(44))/A(IS). Contents of ((14)N(2))cyanamide and ((15)N(2))cyanamide were then calculated by multiplying the total cyanamide content by A(42)/(A(42)+A(44)) and A(44)/(A(42)+A(44)), respectively. The limit of detection for the total cyanamide content by the GC-MS analysis was around 1ng. The molar ratio of ((14)N(2))cyanamide to ((15)N(2))cyanamide in the injected sample was equal to the observed A(42)/A(44) value in the range from 0.1 to 5. It was, therefore, possible to use the stable isotope dilution method to quantify the natural cyanamide content in samples; i.e., the natural cyanamide content was derived by subtracting the A(42)/A(44) ratio of the internal standard from the A(42)/A(44) ratio of sample spiked with internal standard, and then multiplying the resulting difference by the amount of added ((15)N(2))cyanamide (SID-GC-MS method). This method successfully gave a reasonable value for the natural cyanamide content in hairy vetch, concurring with the value obtained by a conventional method in which cyanamide was derivatized to a photometrically active compound 4-cyanimido-1,2-naphthoquinone and analyzed with reversed-phase HPLC (CNQ-HPLC method). The determination range of cyanamide in the SID-GC-MS method was almost the same as that in the CNQ-HPLC method; however, the SID-GC-MS method was much simpler than the CNQ-HPLC method.

Cyanamide was isolated from the leaves and stems of hairy vetch (Vicia villosa), guided by plant growth inhibitory activity against lettuce (Lectuca sativa) seedlings. A large proportion of the inhibitory activity in the crude extract was explained by the presence of cyanamide, suggesting it to be a possible allelochemical in this species. The amount in a 9-day-old seedling, which had been grown without nutrients, reached approx. 40 times that of a nongerminated seed, demonstrating cyanamide biosynthesis in the seedlings. This is the first report on the isolation of a possible allelochemical from hairy vetch and also of the finding of cyanamide as a natural product.

Natural cyanamide (NH2CN) 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. In the present study, we demonstrated the incorporation of 15N from [15N]nitrate and [15N]ammonium into cyanamide using shoots of V. villosa subsp. varia, which ruled out the possibility that nodules are essential in cyanamide biosynthesis. We also applied [15N2]cyanamide to shoots of V. villosa subsp. varia to monitor its turnover, and detected [15N2]cyanamide in the leaves within 4 h; it was present without detectable degradation for more than 4 days. In contrast, maximum incorporation of 15N into cyanamide molecules was observed after 4 days of feeding the shoots with 15N-labeled inorganic ions andl-[amide-15N]-glutamine, indicating that these nitrogenous compounds are distant precursors of cyanamide. Although the guanidino group ofl-arginine (-NH-C(NH2)=NH) and urea (NH2C(=O)NH2) were candidate precursors of cyanamide on the basis of structural similarity, direct incorporation of the guanidino group ofl-[13C6,15N4]-arginine and [13C,15N2]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. [ABSTRACT FROM AUTHOR]