Synthesis and mutagenicity of the two stereoisomers of an azide metabolite (azidoalanine).

The L- and D-isomers of azidoalanine (azide metabolite) have been chemically synthesized with 60% yield using corresponding N-(tert-butoxycarbonyl)-serine as starting materials. The mutagenic properties of synthesized L-azidoalanine are very similar to those of azide and in vivo synthesized azidoalanine. Synthetic D-azidoalanine shows very low mutagenic activity on Salmonella typhimurium TA1530 strain compared to that of the L-isomer. Thus a stereoselective process is involved in azidoalanine mutagenicity. The data presented in this study suggest that further biochemical activation is required for L-azidoalanine to produce its mutagenic activity.

A mutagenic metabolite synthesized by Salmonella typhimurium grown in the presence of azide is azidoalanine.

A mutagenic azide metabolite was purified from the medium in which Salmonella typhimurium cells were grown in the presence of azide. This metabolite was identified to be azidoalanine based on infrared and mass spectroscopy and elemental analysis. This compound appeared to be identical to the mutagenic compound synthesized in vitro from azide and O-acetylserine by partially purified O-acetylserine sulfhydrylase. The metabolite (azidoalanine) mutagenic efficiency and spectrum in S. typhimurium was similar to that of inorganic azide. The compounds 2-azidoethylamine, 2-bromoethylamine, 3-bromopropionic acid and N-(azidomethyl) phthalimide were also mutagenic with a similar spectrum to azide and azidoalanine, but with lower efficiency. The compounds 3-azidopropylamine, 4-azidobutylamine, 3-chloroalanine and ethylamine were only weakly or nonmutagenic. Numerous other chloro, bromo and azido phthalimide derivatives tested were nonmutagenic. It is suggested that the lack of azide mutagenicity (and perhaps carcinogenicity) in mammalian cells may be due to their inability to convert azide to azidoalanine.

Isolation of an azide mutagenic metabolite in Salmonella typhimurium.

A scheme that employs a cation-exchange column and high-pressure liquid chromatography (HPLC) is devised to isolate and process large quantities of azide metabolite produced by S. typhimurium TA1530 strain. The mutagenic metabolite adheres strongly to the cation-exchange column, thus providing a convenient way to separate the metabolite from unreacted azide (N3-). The metabolite is very polar and only sparingly soluble in most organic solvents. Recrystallization in a methanol-carbon tetrachloride solvent system gave rise to microcrystalline material that decomposes with charring and gas evolution at 173-176 degrees C. The infrared spectrum indicates the presence of a covalently bound azide moiety.

Grahamimycins: antibiotics from Cytospora sp. Ehrenb. W.F.P.L. 13A.

Aerobic fermentation of cultures of Cytospora sp. Ehrenb. W.F.P.L. 13A produces a group of novel, broad-spectrum, dilactone, macrocyclic antibiotics. These compounds, named grahamimycins, were isolated from the culture medium by extraction with organic solvents and separated by chromatography. Three active components have been obtained in crystalline form and have been designated grahamimycins A, A1, and B. The most active of these, grahamimycin A, exhibited activity against 36 species of bacteria, 8 species of blue-green algae (cyanobacteria), and 2 species of green algae, and antifungal activity against 5 fungi. Studies on the growth of the Cytospora with respect to formation of antibiotic activity, and the isolation, characterization, and structure elucidation of the grahamimycins are described. These studies indicate that the grahamimycins are distinct from other known antibiotics and represent a new class of antibiotic structures.

Homogeneous enzyme immunoassay for cannabinoids in urine.

We describe a homogeneous enzyme immunoassay for measurement of cannabinoid metabolites, as well as delta9-tetrahydrocannabinol (I) in urine. Malate dehydrogenase from pig heart mitochondria was labeled with a derivative of I. The compound used to calibrate the assay was the I metabolite, 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid (II). With 15 microgram of II per liter of urine as the cutoff concentration, the assay can detect 25 microgram of II per liter with greater than 95% confidence. A positive response was obtained for urine specimens assayed within 30 min after exposure to cannabinoids. However, the persistence of metabolites of I in urine indicates that assay of this fluid is useful as an indicator of cannabinoid use but not as an indicator of intoxication.