Frontier in Structure Elucidation of Natural Products

精子在受精过程中对卵子的趋化是物种保护的关键事件，特别是对生活在水生环境中的动物来说。从海胆和珊瑚等少数海洋生物中发现了相关的化学引诱剂。我们先前的研究表明，海鞘的精子被激活，然后被从卵子释放的一种因子吸引到卵子，从而从海鞘的卵子条件培养液中纯化出精子激活和吸引因子(SAAF)。根据2D-1HNMR和FAB-MS/MS分析，SAAF的结构被认为是一种新的聚羟基甾醇硫酸酯，使用不超过4微克的标本。然而，关于生物活性可能归因于一种次要成分的说法仍然很小，但仍存在严重的含糊性。因此，合成对于明确地确定SAAF的活性成分是必不可少的，对于包括C25的立体化学在内的完整结构的阐明也是必不可少的。以鹅去氧胆酸为原料，经19步反应合成了SAAF及其C-25同分异构体，并对其激活和吸引精子的活性进行了生物测定。人工合成的SAAF在3.7 nM激活海鞘的精子，同时在10 nM处表现出引诱活性，从而明确了SAAF的结构并验证了其双重活性。值得注意的是，25-epi-SAAF具有与SAAF类似的活性。

Chemotaxis of sperm toward eggs during fertilization is a crucial event for species conservation, particularly for animals living in aquatic environments. Relevant chemical attractants have been found from a few marine organisms such as sea urchins and corals. We previously showed that sperm of the ascidian Ciona intestinalis is activated and then attracted toward the egg by a factor released from the egg, which led to the purification of sperm-activating and attracting factor (SAAF) from the egg-conditioning medium of Ciona intestinalis.The structure of SAAF was proposed to be a novel polyhydroxysterol sulfate on the basis of 2D-^1H NMR and FAB-MS/MS analysis using no more than approximately 4 microgram of a specimen. However, there still remained small but serious ambiguity that the biological activity might be attributed to a minor constituent. Synthesis is, therefore, essential for the unequivocal identification of SAAF as the active principle, and for the complete structure elucidation including the stereochemistry at C25. SAAF and its C-25 epimer were synthesized from chenodeoxycholic acid in 19 steps via the pivotal intermediate and both the sperm-activating and attracting activity of them were bioassayed. Synthetic SAAF activated the sperms of ascidian Ciona intestinalis at 3.7 nM and concurrently exhibited the attracting activity at<10 nM, which led to the unambiguous structure determination of SAAF and the verification of its dual activity. It is noteworthy that 25-epi-SAAF turned out to possess the comparative activities as those of SAAF.