Demethylation, ergosterol biosynthesis

Triadimefon (Triazole) C —O - CH—COC(CHj)3. N, N— Steroid demethylation (ergosterol biosynthesis) inhibitor... 

Although termed "SI" or "EBI" compounds, the latter referring to ergosterol biosythesis inhibitors, these compounds do not all inhibit ergosterol biosynthesis at the same metabolic site (Fig. 2). For instance, the fungicide tridemorph, unlike most EBI compounds, does not inhibit demethylation at C-14 but rather it apparently prevents the A A isomerization resulting in the accumulation of A containing sterols in treated cells (3). 

In ergosterol biosynthesis, side chain alkylation of lanosterol normally takes place to build 24-methylenedihydrolanosterol, which itself is then the substrate for demethylation reactions at and C. The C -demethylation has been studied in detail. It is an oxidative demethylation catalyzed by a cytochrome P -system. The first step involved in this reaction is the hydroxylation of the Cj -methy1-group to form the C -hydroxymethyl derivative. A second hydroxylation and loss of water lead to the C -formyl intermediate, which is hydroxylized a third time to form the corresponding carboxylic acid. Decarboxylation does not directly take place, but proceeds instead by abstraction of a proton from C, followed by elimination and formation of a A 4-double bond. The NADPH-dependent reduction of the A14 -double bond finishes the demethylation reaction. Subsequently, demethylation at has to take place twice, followed by a dehydrogenation reaction in A" -position and isomerization from A8 to A7 and A24(28) to A22. respectively. 

Sterol biosynthesis inhibiting insecticides. The earliest examples of sterol biosynthesis inhibitors were triadimefon, imazalil, and triarimol. These compounds act by blocking the C14 alpha demethylation step in ergosterol biosynthesis [54], Further work in this area has resulted in numerous fungicides, a number of them containing aromatic fluorine, perfluoroalkyl ethers, and the trifluoromethylphenyl group (Fig. 11). In this section, we will discuss the trifluoromethylphenyl sterol biosynthesis inhibitors, such as triflumizole and fluotrimazole. 

Rahier and his co-workers also characterized the activities of a sterol C-4 methyl oxidase (SMO), a 4-carboxysterol-3-hydroxysteroid dehydrogenase/ C-4 decarboxylase (3-HSD/D) and an NADPH-dependent 3-oxosteroid reductase in order to define the steps involved in C-4 demethylation in plants (Pascal et al, 1993 Rondet et al, 1999). Only recently, they have isolated two cDNAs from Arabidopsis thaliana encoding bifunctional 3-HSD/D. Transformation of a yeast ergosterol auxotroph mutant, which lacks 3-HSD/D activity, with either of these cDNAs restored ergosterol biosynthesis in the yeast mutant (Rahier et al, 2006). 

These 1,2,4-triazole fungicides share a common mode of action by inhibiting the C-14a-demethylation step in ergosterol biosynthesis (Figure 1) ( 3 ) between 24-methylenedihydrolanosterol and 4,4-dimethylergosta-8,14,24(28)-trienol. 

In summary, prochloraz, like other azoles, is an ergosterol biosynthesis inhibitor (i.e. it is an EB1) and it does this by blocking 14-demethylation (such compounds are sometimes referred to as demethylation inhibitors, or DMIs). As is now well known, these molecules bind to the sterol binding site of the demethylase enzyme in such a way as to allow the azole to bind to an iron atom in the active site (see below). The normal physiological reaction is therefore prevented. 

Since ergosterol is used in the formation of the leishmanial cell membrane, inhibition of ergosterol biosynthesis has been considered as a useful target for chemotherapeutic attack. Allylamines (eg. terbinafine) and imidazole antifungals (eg. ketoconazole) have been found to interfere with different steps in the biosynthetic pathway of C28 sterols in leishmania and fungi. Allylamines inhibit the microsomal squalene 2,3-epoxidase and, therefore, inhibit the synthesis of squalene epoxide, the precursor of lanosterol. Imidazoles, on other hand, inhibit cytochrome P-450 dependent C-14 demethylation of lanosterol leading to decreased or no synthesis of ergosterol [30]. 

Many fungicides [15] act by inhibiting sterol biosynthesis. More specifically, they block the C-14a demethylation step in ergosterol biosynthesis [16]. In many of these compounds (Flutriafol [17], Flusilazole [18], Triflumizole [19], Fluotri-... 

Ergosterol synthesis is inhibited differently by another class of fungicides, namely by preventing demethylation at C-14 during biosynthesis. As a result, useless sterols accumulate such as 14-methylfecosterol. Examples of this class are fenarimol 6.64), which is l-(2,4 -dichlorodiphenyl)-l-pyrimidin-5-yl-methanol, and triadimefon 6.65) which is l-(4-chlorophenoxy-3,3-dimethyl-l-(l,2,4-triazol-l-yl)butan-2-one. These are somewhat wedge-shaped molecules with alternating lipophilic and hydrophilic areas. The heterocyclic fragment can be varied to be pyridine, piperazine, or imidazole as in buthiobate, triforine, or imazilil, respectively (Kato, 1983). 

CYP51 14a-sterol demethylation Biosynthesis of membrane ergosterol E.g., C. albicans, S. cercvisiae E.g., [452]... 

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