γ-linolenic acid biosynthesis:: Cryptoregiochemistry of Δ6 desaturation
γ-linolenic acid biosynthesis:: Cryptoregiochemistry of Δ6 desaturation
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DOI：
10.1021/jo001294
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发表时间：
2001-02-23
影响因子：
3.6
通讯作者：
Buist, PH
中科院分区：
文献类型：
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作者：
Fauconnot, L;Buist, PH
有价值的营养品γ-亚麻酸（GLA，（6 Z，9 Z，12 Z）-十八碳三烯酸）是通过一系列区域和立体选择性脱氢反应生物合成的，所述脱氢反应由一组称为脂肪酸去饱和酶的酶催化。作为正在进行的研究这些显着的催化剂的机制的一部分，我们已经检查了密码区域化学（初始氧化的网站）的Delta（6）去饱和，因为它发生在原生动物四膜虫嗜热。两个互补的方法，解决这个问题的描述。在第一组实验中，我们测量了与C-6和C-7处的C-H键裂解相关的单个初级氘动力学同位素效应。用适当氘标记的4-硫代底物进行的竞争实验表明，在C-6处观察到C-H键断裂步骤的大KIE（k（H）/k（D）= 7.1 +/-0.5），而在C-7处观察到C-H键断裂对氘取代不敏感（k（H）/k（D）= 1.04 +/-0.05）。这些结果指出C-6是Delta（6）去饱和中的初始氧化位点，因为这种类型的反应中的第一个化学步骤是强的未活化的C-H键的断裂，这是一个能量上困难的过程，通常表现出大的KIE。我们的第二种方法的结果支持这一结论，该方法涉及通过监测氧代转移到一系列硫杂脂肪酸探针的效率来定位推定的二铁氧代氧化剂相对于底物的位置。因此，仅6-硫杂-类似物转化为显著量的相应亚砜（9%产率）。用（S）-MPAA作为手性位移试剂，确定该产物的绝对构型为S。综上所述，这些结果表明，C-6亲S氢的提取是Δ（6）去饱和的初始事件，正如它在T中发生的那样。嗜热菌
The valuable nutraceutical gamma -linolenic acid (GLA, (6Z,9Z,12Z)-octadecatrienoic acid) is biosynthesized by a series of regio- and stereoselective dehydrogenation reactions that are catalyzed by a set of enzymes known as fatty acid desaturases. As part of ongoing research into the mechanism of these remarkable catalysts, we have examined the cryptoregiochemistry (site of initial oxidation) of Delta (6) desaturation as it occurs in the protozoan Tetrahymena thermophila. Two complementary approaches that address this issue are described. In the first set of experiments, we measured the individual primary deuterium kinetic isotope effects associated with the C-H bond cleavages at C-6 and C-7. Competition experiments using appropriately deuterium-labeled 4-thiasubstrates revealed that a large KIE (k(H)/k(D) = 7.1 +/- 0.5) was observed for the C-H bond-breaking step at C-6, whereas the C-H bond cleavage at C-7 was insensitive to deuterium substitution (k(H)/k(D) = 1.04 +/- 0.05). These results point to C-6 as the site of initial oxidation in Delta (6) desaturation since the first chemical step in this type of reaction is rupture of a strong, unactivated C-H bond, an energetically difficult process that typically exhibits a large KIE. This conclusion was supported by the results of our second approach, which involved locating the position of the putative diiron oxo oxidant with respect to substrate by monitoring the efficiency of oxo transfer to a series of thia fatty acid probes. Thus only a 6-thia-analogue is converted to significant amounts of the corresponding sulfoxide (9% yield). The absolute configuration of this product was determined to be S using(S)-MPAA as a chiral shift reagent. Taken together, these results point tb the abstraction of the C-6 pro S hydrogen as the initial event in Delta (6) desaturation as it occurs in T. thermophila.