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Fun With Tunichromes

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.

17 comments on “Fun With Tunichromes”

  1. eugene says:

    Ack! vanadium is not next to iron and is not the next one down. That would be ruthenium.
    Do you think that isolations, the way they are done in the Nakinishi lab, may become a lost art in the future? Is someone out there still teaching how to do all this stuff? Besides the now retired Nakinishi of course. Although, I bet if we really needed to, we could re-invent the technique. It’s just that to stick with a project that you realize at one point will require so much extra trouble, is rather commendable. I don’t know how much he had to threaten the grad students though.

  2. Derek Lowe says:

    Fixed! I remember thinking that I was totally wrong on that, but posted without correcting it; thanks.

  3. MTK says:

    I one time saw a kilo-scale countercurrent extraction apparatus. It took up a whole room and consisted of what looked to me like several hundred extraction chambers. I didn’t see it in operation, but it looked like the chambers rocked back and forth like those old school compressed air driven Kugelrohr gizmos. The raffinate would go one way and the extract the other. It was one the most complex pieces of glassware, I’d ever seen.

  4. Jose says:

    There are also some odd V enzymes from the same species.
    http://en.wikipedia.org/wiki/Vanabins
    Vanadium Bromoperoxidase-Catalyzed Biosynthesis of Halogenated Marine Natural Products, J. N. Carter-Franklin and Alison Butler, J. Am. Chem. Soc. 2004, 126, 15060-15066.

  5. CMC guy says:

    Cool post and admirable undertaking- I have done isolations/separations in dark with photosensitive compounds and also performed chromatography (IEX) in a cold room but never dealt with the combo so do salute the achievement. Anything known about the toxicity as suspect vanadium and/or phenols could be deadly? Another likely point of appreciation.
    eugene’s comment is interesting as natural products isolation does seem to be becoming more a lost art/science. 20-30 years ago larger schools had at least one prof in this area but they likely have retired by now with no replacements. Industry too had such groups that think no longer exists although have seen some biotechish companies promoting such discovery efforts. Still greater interest in Japan I guess but taking 2K Kg of Sea-slime to get a few mgs of isolated molecule lacks appeal to most chemists.

  6. howard says:

    Just skimming the experimental portion of the isolation article shows two other really, really horrible things about this –
    1) “Uncontaminated blood was obtained from healthy individuals (as determined by olfactory inspection)…”
    2) Reference 26 – It was determined that this powder(ed blood from tunicates) possessed strong allergenic properties. Exposure produced severe hay fever symptoms in on worker (R.C.B.).
    So not only did they weed out the bad organisms by smelling which ones were rotten, one of the authors was allergic to the blood of the tunicates.

  7. Jose says:

    From the isolation of Et 743 (JOC; 1990; 55(15) 4512 – 4515)
    Key techniques employed for the isolation
    and preliminary characterization included centrifugal countercurrent chromatography, tissue culture bioautography, moving belt liquid chromatography/fast atom bombardment mass spectrometry (LC/FABMS) [snip]

  8. ZAL says:

    I had no clue that there were organisms on this planet with vanadium in their blood – fascinating stuff, thanks for making me learn something new, once again!
    Plus, it’s amazing to learn that you are an old colleague of Carsten Bolm, I know him very well too – small world!

  9. Sili says:

    I vaguely recall learning about V enzymes and Cu containing blood (cephalopods or crustaceans?), but this is the first I hear of the combination.
    Damn, that’s some hard work.

  10. anon says:

    wow, they couldn’t find, well, ANYTHING better than t-butyl thiol to inhibit oxidation???!!!
    ugh.
    Another awesome tidbit:
    Preparation of An-1 Me. A number of conditions (i.e. Mitsonobu, dimethyl sulfate, DBU/MeI, and dethyl-p-toluatriazene/Al(O-r-Bu),) were employed in unsuccessful attempts to prepare An-1 Me. Only
    diazomethane/MeOH successfully provided the desired product, but all attempts to improve the yield of this reaction with various catalysts failed (i.e. SiO,, BF3.Et20, and ZnCI,).
    I DEFINITELY have diazomethane to the list of sh*t I won’t work with. . .

  11. Pat Frank says:

    Thanks for writing about tunicates Derek. I’m one of the people working on the vanadium in tunicates. Early-on, people thought the vanadium was in an oxygen-carrying protein, but it’s not. Tunicates rely strictly on mere diffusion for respiration, and can survive long periods of anoxia.
    We have a paper just out now in J. Inorg. Biochem. showing evidence for a vanadium reductase in tunicates, opening the entirely new field of vanadium redox enzymology.
    Jose in #4, be careful of the vanabins. It’s not at all likely that these proteins are associated with vanadium metabolism in tunicates. Vanadium is a very sticky metal and will bind to any protein it encounters.
    In tunicates, most of the vanadium exists as the free V3+ ion in strong acid, inside of blood cells called signet-ring cells. Lysis of these blood cells liberates the V3+ and will produce a raft of non-physiological vanadium complexes. The vanabin workers haven’t protected against that possibility in their work, and it’s almost certainly true that they are pursuing an artifact.

  12. CMC guy says:

    #10 anon diazomethane well deserves a healthy respect with double whammy of potential tox and explosive (exacerbated by customary Et2O solution) consequences however can be prepared and used in limited amounts with appropriate precautions. In fact there were (and assume still are) a couple specialized vendors who could do larger scale CH2N2 reactions; one made weapon explosives so treated like a bomb operation and the other engineered a continuous process so never had large quantity at any one time. There was always nasty stuff in syn labs I accepted as nature of chosen field but I was more frightened walking into biochemistry areas which seemed short on hoods and had lots of open containers of god knows what.

  13. MolecModeler says:

    Could these things be engineered to extract Au out of sea water?
    Hmm

  14. daen says:

    I am again reminded of John Thomson freezing and blending kilograms of frozen calf thymus at Vertex in Barry Werth’s book, “The Billion-Dollar Molecule”.

  15. Paul F. Dietz says:

    Could these things be engineered to extract Au out of sea water?
    The concentration of gold in seawater is several hundred times lower than that of vanadium.
    I am reminded of the amidoxime polymers developed in Japan that selectively absorb uranium and vanadium from seawater. Concentrating uranium from 3 ppb in seawater to a loading of 1% in the adsorber is a nice feat.

  16. Gerald Smith says:

    Fly mushrooms (Amanita muscaria) those (usually) bright red ones with the white spots are also widely reported to concentrate vanadium. I wonder why?

  17. Mona Albano says:

    Fascinating!
    The DOI link for “horrible undertaking” is broken. This gets you closer: pubs.acs.org/doi/abs/10.1021/ja00226a035. However, the actual paper is behind a paywall at $35 U.S. for 48 hours’ access.

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