Agaritine
Encyclopedia
Agaritine is an aromatic, antiviral
, hydrazine
-derivative mycotoxin
and IARC Group 3 carcinogen that occurs in mushroom
species of the genus Agaricus
.
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).
Figure 1. The bioactivation of agaritine to its diazonium ion.
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. Agaritine itself has also been shown to covalently bind to DNA in vivo. Agaritine is a weak carcinogen, however, and estimates for cumulative lifetime risk from mushroom consumption are approximately 1 in 10,000. There is little data about toxicity and there is no published LD50.
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 recently docking assays have shown the molecule to be a potent inhibitor of HIV protease. Computer modelling research is currently being conducted in an attempt to optimize binding for potential use as an anti-HIV drug.
Figure 2. Proposed biosynthesis of agaritine.
Figure 3. Kelley et al. synthesis of agaritine (1962).
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%.
Figure 4. Wallcave et al. synthesis of agaritine (1979).
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%.
Figure 5. Datta and Hoesch synthesis of agaritine (1987).
Antiviral drug
Antiviral drugs are a class of medication used specifically for treating viral infections. Like antibiotics for bacteria, specific antivirals are used for specific viruses...
, hydrazine
Hydrazine
Hydrazine is an inorganic compound with the formula N2H4. It is a colourless flammable liquid with an ammonia-like odor. Hydrazine is highly toxic and dangerously unstable unless handled in solution. Approximately 260,000 tons are manufactured annually...
-derivative mycotoxin
Mycotoxin
A mycotoxin is a toxic secondary metabolite produced by organisms of the fungus kingdom, commonly known as molds. The term ‘mycotoxin’ is usually reserved for the toxic chemical products produced by fungi that readily colonize crops...
and IARC Group 3 carcinogen that occurs in mushroom
Mushroom
A mushroom is the fleshy, spore-bearing fruiting body of a fungus, typically produced above ground on soil or on its food source. The standard for the name "mushroom" is the cultivated white button mushroom, Agaricus bisporus; hence the word "mushroom" is most often applied to those fungi that...
species of the genus Agaricus
Agaricus
Agaricus is a large and important genus of mushrooms containing both edible and poisonous species, with possibly over 300 members worldwide...
.
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).
Biological activity
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 mutagenize DNA in the bacterium Salmonella typhimurium. Upon mammalian ingestion, agaritine is metabolized into its highly reactive diazonium ion. This bioactivation is performed in two steps: it is first acted upon by a γ-glutamyltransferase (GGT) to produce 2 and then by a cytochrome P450-dependent monooxyengase to reach the diazonium ion 3 (Figure 1).Figure 1. The bioactivation of agaritine to its diazonium ion.
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. Agaritine itself has also been shown to covalently bind to DNA in vivo. Agaritine is a weak carcinogen, however, and estimates for cumulative lifetime risk from mushroom consumption are approximately 1 in 10,000. There is little data about toxicity and there is no published LD50.
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 recently docking assays have shown the molecule to be a potent inhibitor of HIV protease. Computer modelling research is currently being 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.Figure 2. Proposed biosynthesis of agaritine.
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.Figure 3. Kelley et al. synthesis of agaritine (1962).
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%.
Figure 4. Wallcave et al. synthesis of agaritine (1979).
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%.
Figure 5. Datta and Hoesch synthesis of agaritine (1987).
See also
- Agaritine gamma-glutamyltransferaseAgaritine gamma-glutamyltransferaseIn enzymology, an agaritine gamma-glutamyltransferase is an enzyme that catalyzes the chemical reactionThus, the two substrates of this enzyme are agaritine and acceptor, whereas its two products are 4-hydroxymethylphenylhydrazine and gamma-L-glutamyl-acceptor.This enzyme belongs to the family of...
- Genotoxicity
- GyromitrinGyromitrinGyromitrin is a toxin and carcinogen present in several members of the fungal genus Gyromitra, most notably the false morel G. esculenta. It is unstable and is easily hydrolyzed to the toxic compound monomethylhydrazine, a component of some rocket fuels. Monomethylhydrazine acts on the central...
- MonomethylhydrazineMonomethylhydrazineMonomethylhydrazine is a volatile hydrazine chemical with the chemical formula CH3 NH2. It is used as a rocket propellant in bipropellant rocket engines because it is hypergolic with various oxidizers such as nitrogen tetroxide and nitric acid...