Ever hear of tunicates? They’re these little sea squirt creatures that sit around day and night, filtering sea water for food. The weird thing about them is that although they have blood, it isn’t red, but in many species a Mr. Spock shade of bluish green. That’s because they don’t concentrate iron there, but rather vanadium, in the same row of the periodic table.
You don’t hear much about vanadium. Not even chemists hear all that much about it, at least not since the 1980s heyday of one of the Sharpless epoxidation reactions that uses it as a catalyst. (An old colleague of mine, Carsten Bolm, has been doing his best to revive that one). For one thing, it’s not a particularly abundant element – which led to questions about just how the tunicates were getting so much of it in their greenish blood. They had to be sequestering it from seawater, but that meant that they had to have some very efficient way to extract it.
Well, it turns out that they produce compounds now called tunichromes, unusual cyclic peptide catechol beasts that have a high affinity for vanadium. But finding and figuring out the structure of these things was a horrible undertaking by the Nakanishi group at Columbia, no strangers to ugly natural products. (He's now a professor emeritus, but I believe he's still hard at work).
The problem is, tunichromes are rather sensitive - all those phenols, y’know. They don’t like light (it’s pretty dim down in tunicate country), they don’t like heat (it’s not real warm there, either), and they don’t even like oxygen much, at least not the amounts found up here above the waves. And, as it turned out, they most definitely don’t like any form of silica gel, or any of the other solid supports used in chromatography. That ruled out HPLC, after what you have to gather was much heartbreak, because giving up HPLC means giving up an awful lot of separating power.
The group ended up using a technique you hardly see used any more, countercurrent chromatography. That requires a special apparatus where two immiscible solvents flow past each other, stage by stage. You’re extracting components from one into the other as things go along – it’s like regular chromatography, in a way, except you’re not using a solid powdery matrix to flow solvent over. You’re using another solvent. It takes, I believe, a delicate hand (I’ve never had the pleasure of doing it).
The sea squirt extract definitely got the kid glove treatment. Nakanishi’s folks ended up doing their countercurrent work with added t-butyl thiol in all their solvents, to guard against oxidation. That made things smell like the biggest natural gas leak in New York, I’m sure, but at least the smell was confined. That’s because they were doing all this in a cold room (a glorified meat locker), in the dark (with occasional darkroom lights when needed), and in inert-atmosphere bags and glove boxes. What a joy that must have been.
As it turns out, there's still a lot of controversy (PDF) about tunichromes and what they're doing in the live organisms. (That extends to the whole topic of metal concentration by marine organisms). When they were isolated, it looked like a good bet that they were the vanadium-concentrating substances, but later work has shown that they're not actually found in the same cells as the high vanadium concentrations. The whole reason that tunicates like vanadium so much is still something of a mystery - perhaps it's used in forming their characteristic outer tunic, and might serve to keep predators away.
But whenever things are going poorly for me in the lab, I consider that I could be sitting there in the reeking dark in my winter coat, hour after hour, grinding up dead tunicates and trying to keep the countercurrent apparatus working. Cheers me right up.
May 2, 2006
The natural products topic (which I'll return to in a couple of days) has me starting up a companion to the "Things I Won't Work With" category. This is the first in the new category of "Things I'm Glad I Don't Do".
I mentioned ciguatoxin, and I notice that one of the comments to that post was from someone who'd had a brush with the stuff. My sympathies - it's supposed to be really awful. A number of warm-water fish species can have dangerous concentrations of the compound in them, and it's probably one of the most common non-bacterial sources of food poisoning. The thing is, the fish themselves don't make the stuff. They concentrate it from marine algae, who produce a lot of extravagantly crazy molecules.
So if you want some ciguatoxin yourself, you fool, you, a good source is an organism near the top of the food chain. Moray eels turn out to be a good bet. But you don't just turn one of them upside down over a beaker and squeeze his tail. No, the isolation is a bit more involved:
The moray eels (ca. 4000 kg) were collected from the Tuamotu Archipelago and from the Island of Tahiti in French Polynesia. The viscera (125 kg) were homogenized and extracted with two volumes of acetone twice. After filtration, the extract was left at -20 "C for 1 day to precipitate oily residue. The supernatant was evaporated to dryness and partitioned between diethyl ether and water. The ether layer was condensed and suspended in aqueous 80% MeOH, followed by defatting with hexane. The methanolic layer was condensed, dissolved in acetone. . .
The prep goes on in this vein, through six different columns, one after the other. Now, imagine joining this research group (which was Yasumoto's, in Japan). It's your first day in the lab, and here comes one of the post-docs carrying a couple of blenders in his arms. Behind him, another one is wheeling in the bags of frozen eel guts. It's moray margarita time, and will be for some time to come.
One other aspect of this isolation deserves comment, because I don't think you could do it like this today. In the final column or two, the paper outlines a brutal but effective method for cutting fractions to get the ciguatoxin: take a sample from each cut of the column and inject it into a mouse. If it doesn't die immediately, that fraction doesn't have any ciguatoxin in it. Gloves recommended.
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