Agaritine

Agaritine (AGT) is an aromatic, antiviral, hydrazine-derivative mycotoxin and IARC Group 3 carcinogen that occurs in mushroom species of the genus "Agaricus".
Occurrence.
Studies have found significant (>1000 mg/kg) agaritine levels in fresh samples of at least 24 species of the genera "Agaricus", "Leucoagaricus", and "Macrolepiota". Mushrooms of these species are found around the world. They typically fruit from late spring through autumn, and are particularly prevalent in association with feces. These mushrooms grow in a wide range of habitats; indeed, one species alone, "Agaricus bisporus", is cultivated in over 70 countries and on every continent except Antarctica. "A. bisporus", also known as the common button mushroom, is of particular socio-economic importance because of both its prevalence in traditional cultural recipes and its booming cultivation industry in modernized countries.
Agaritine content varies between individual mushrooms and across species. Agaritine content (% fresh weight) in raw "Agaricus bisporus", for example, ranges from 0.033% to 0.173%, with an average of 0.088%. The highest amount of agaritine is found in the cap and gills of the fruiting body, and the lowest in the stem. Agaritine oxidizes rapidly upon storage, however, and is totally degraded after 48 hours in aqueous solution with exposure to air. It has also been shown to decompose readily upon cooking (up to 90% reduction) as well as upon freezing (up to 75% reduction).
Known to cause cancer and mutations in animals.
Agaritine has been shown to induce adenomas and adenocarcinomas in the lungs of mice when administered through drinking water. It has also been shown to cause bladder cancer in mice.
Agaritine has been claimed to be a weak carcinogen, with an estimate for cumulative lifetime risk from mushroom consumption at approximately 1 in 10,000. However, this claim is poorly supported, with little available data about toxicity and no published .
Agaritine has been shown to test positive as a mutagen in the Ames test and mutagenize DNA in the bacterium "Salmonella typhimurium". It has also been shown to covalently bind to DNA "in vivo".
Mechanism via toxic metabolites.
Agaritine has been shown to be broken down by enzymes in animal kidneys into the toxic metabolites 4-(hydroxymethyl)phenylhydrazine and 4-(hydroxymethyl)benzenediazonium ions.
These metabolites have been shown to cause stomach cancer in mice and cause lasting DNA damage after a single dose in mice.
The mutagenic activity of the diazonium ion is due to its reaction with oxygen to produce hydrogen peroxide, which then covalently modifies DNA through a radical mechanism.
Potential antiviral activity.
Extracts of mushrooms from the genus "Agaricus" have been used for generations as traditional Chinese herbal remedies. Some of these extracts have been shown to possess antiviral properties, and investigators have identified agaritine as a prominent compound in the extracts. This led researchers to investigate potential antiviral properties of agaritine, and docking assays have shown the molecule to be a potent inhibitor of HIV protease. Computer modelling research has been conducted in an attempt to optimize binding for potential use as an anti-HIV drug.
Biosynthesis.
Agaritine (1) was long thought by biologists to emanate from shikimate (4), with the glutamate moiety clearly originating from glutamic acid.
This assumption was made purely by inference: a similar compound, γ-glutaminyl-4-hydroxybenzene (5) is produced in the fruiting body of mushrooms in the genus "Agaricus" with similar abundance to agaritine and has been shown to be derived from the shikimate biosynthetic pathway. Recent work, however, has uncovered several problems with this hypothesis, of which inconsistencies in radiolabeling experiments are most notable. These recent efforts now assert that the molecule is synthesized in the vegetative mycelium and then translocated into the fruiting body. These researchers posit that the p-hydroxybenzoic acid moiety (6) is absorbed directly from the lignin on which the fungus feeds, not produced by the fungus itself (Figure 2).
Despite recent work, however, experts still acknowledge the nebulous origin of the hydrazine functionality. Two theoretical mechanisms are postulated: oxidative coupling of two amines via a phenolic radical mechanism or fixation of nitrogen via nitrogenase.
Synthesis.
Three total syntheses of agaritine have been completed. The first was performed in 1962 by R.B. Kelly "et al." (Figure 3). These researchers used as their key step the coupling of the γ-azide of N-carbobenzoxy-L-glutamic acid (9) with α- hydroxy-p-tolylhydrazine (8). But compound 8 proved difficult to produce, presumably because of the ease with which water can eliminate across the benzene ring. This was finally overcome by in situ formation by reduction of p-carboxymethylphenylhydrazine (7) with lithium aluminium hydride, followed by a pH-neutral workup using a small quantity of saturated sodium chloride as a drying agent. Neutral conditions were required because agaritine is sensitive to both acid and base. No satisfactory method was found to isolate and purify 8 from its side products, so this solution was treated directly with 9. This produced a mixture of compounds, one of which was the adduct 10. After deprotection by hydrogenolysis, agaritine was extracted by chromatography. The overall yield was 6%, of which half was isolated in pure crystalline form.
This synthesis could clearly be improved, and in 1979 L. Wallcave "et al." published a modified synthesis (Figure 4). These investigators began with a slightly different starting material, the diprotected hydrazine of L-glutamic acid (11) and reacted it with p-carboxyphenylhydrazine (12) to produce the N'-hydrazide (13). The limiting step in the first synthesis was the very imprecise reduction with LAH, which proceeded with several side reactions and little reaction specificity. Wallcave "et al." instead used diborane to selectively reduce the carboxylic acid and reach compound 14, with some over-reduction to 15. The benzyl ester protecting groups were then cleaved by final hydrogenolysis. This last step was initially performed in aqueous solution, but the over-reduction product 15 carried on to produce a 15% side product impurity. This impurity was reduced to less than 2% when the solvent was changed from water to tetrahydrofuran, as the agaritine precipitated out of solution as it formed. The overall yield for this synthesis was 25%.
This was still unsatisfactory, however, and in 1987 S. Datta and L. Hoesch devised the third and most recent synthesis of agaritine (partially upon claims that the synthesis by Wallcave "et al." could not be reproduced). The Datta and Hoesch synthesis (Figure 5) also used the joining of p-hydrazinobenzyl alcohol (8) with the 5-carboxy group of L-glutamic acid as its keystone, in the same vein as the initial Kelly synthesis. Unlike Kelly "et al.", however, these researchers achieved an efficient synthesis of 8 from 7 by using an even milder reducing agent than the diborane used by Wallcave "et al." – diisobutylaluminum hydride (DIBALH) in toluene at -70 °C. Additionally, compound 8 was found to be much more stable than Kelly "et al." had asserted. Mixture of 8 with the same diprotected L-glutamic acid 11 used by Wallcave "et al." produced the already-reduced adduct (16). Subsequent deprotection via hydrogenolysis using a 10% poisoned Pd/C catalyst (to minimize the over-reduced side product encountered by Wallcave et al.) yielded agaritine. The final step had 83% yield, and the overall yield for this synthesis was 33%.