C3 unit addition reaction accompanied by formation of C-C bond in phytotoxin biosynthesis of phytopathogen.

Sorokinianin是由一株（OB-25-1） sorokiniana产生的一种植物毒素，是一种引起小麦和其他禾草的斑疹病或足腐病和根腐病的病原体。这种毒素被认为是由线虫孢醇型倍半萜和C_3部分组成的。先前进行的饲养实验表明，由前helminthosporol通过添加TCA循环成员，脱羧和内酯化反应合成了sorokinianin。L-[羟基-^<18>]苹果酸投喂试验表明，直接C_3单元前体不是苹果酸/草酸，而是富马酸/琥珀酸。本研究的目的是鉴定富马酸/琥珀酸加入前孢菌素后形成的产物。为此，我们从含有^<14>C标记的葡萄糖培养基上培养的sorokiniana中制备了^<14>C标记的前叶孢素（1.7 mg，比放射性35 KBq/mg）。在用^<14>C标记的前孢菌素和^3H标记的琥珀酸孵育得到的菌丝体中，我们发现了一个同时具有^<14>C和^3H放射性的部分。然后我们分析了经乙酰化和甲基化后，在冷运行中得到的与放射性同位素实验中相同的组分。

Sorokinianin is a phytotoxin produced by a strain (OB-25-1) of Bipolaris sorokiniana, a pathogen causing spot blotch or foot and root rot diseases of wheat and other grasses. This toxin is assumed to be composed of a helminthosporol-type sesquiterpene and a C_3 portion. The feeding experiments conducted previously suggested that sorokinianin was synthesized from prehelminthosporol by reactions that include the addition of a member of the TCA cycle, decarboxylation and lactonization. A feeding experiment with L-[hydroxy-^<18>O] malic acid indicated that the direct C_3 unit precursor was not malic acid/oxalacetic acid but fumaric acid/succinic acid. The aim of this research is to identify the product formed just after addition of fumaric acid/succinic acid to prehelminthosporol. For this purpose we prepared ^<14>C labeled prehelminthosporol (1.7 mg, specific radioactivity 35 KBq/mg) from B.sorokiniana cultured on the medium containing ^<14>C labeled glucose. Among the fractions obtained from the mycelium incubated with both ^<14>C labeled prehelminthosporol and ^3H labeled succinic acid we found a fraction which had both ^<14>C and ^3H radioactivity. Then we analyzed the same fraction obtained in the cold run as in the radio isotopic experiment by GCMS after acetylation and methylation.