Some unexpected lessons

It is not that often that I pick up an interesting purification trick from a chemical biology paper. Yet, this is exactly what happened to me while I was reading the ACS Chemical Biology manuscript by Bernat et al. This intriguing recent work documents the use of boronic acid probes of allosteric modulation of the chemokine CXCR3 receptor. Biological merits of the manuscript aside, I was drawn to the way the authors purified their boronic acid inhibitors. Their solution to the problem involves dry column flash chromatography, the description of which even made its way into the paper’s abstract. Given the focus of the journal, this probably means that the authors were really satisfied with the results. The technique consists of placing a dry bed of silica gel in a sintered glass funnel followed by fraction elution using suction. I have not attempted this methodology myself in the good old days when I was still doing somewhat meaningful research at the bench. I did a bit of digging and discovered a nice paper in the Journal of Chemical Education detailing this method. I actually think my students use this all the time, but I do not recall anything extraordinary about this tool. Now it turns out that it might be just what we need for some of our annoyingly polar boron-containing molecules. By the way, whenever there is something I want to learn in detail, I turn to the Journal of Chemical Education. Therein lies a treasure trove of information because the idea is to publish methods and procedures that are adaptable in undergraduate settings.

 http://pubs.acs.org/doi/abs/10.1021/cb500678c

http://pubs.acs.org/doi/abs/10.1021/ed074p1222

One more item of substance that relates to aliphatic boronic acids: during lyophilization, don’t go too crazy in your attempts to rigorously remove water from the purified compound. This is a bad idea as the Le-Chatelier principle will shift things in the direction of boraxine, which is notoriously sensitive to oxidative cleavage of the C-B bond. We like to keep our products somewhat wet. I picked up this tip from Professor Singaram while lecturing at University of California, Santa Cruz, last year.

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11 thoughts on “Some unexpected lessons

  1. “In the good old days when I was still doing somewhat meaningful research at the bench…”

    An entry at Curly Arrow blog post called Dry Column Vacuum Chromatography starts with similar words “Some years ago when I was silly enough to have a real job in the chemical industry…”
    (I think he is a guy with lots of experience on this, you may want to drop him an e-mail.)

    http://curlyarrow.blogspot.com/2006/10/dry-column-vacuum-chromatography.html

    http://www.sejergroup.org/curriculum-vitae.html

    • That’s exactly where the first author of the ACS ChemBio paper (me) took the inspiration from!
      The trick was to figure out that aprotic solvents do not elute boronic acids from the TLC-grade silica regardless of polarity (but elute pretty much everything else if strong enough). The most extreme case of crude mixture is on the picture which didn’t make it to the SI but ended up in my thesis. This is sequential elution with acetonitril and then with chloroform/ethanol mixtures, arrow shows the desired product: https://www.dropbox.com/s/n9hdj0t1vbnx05b/tlc.png?dl=0

      Notably, the trick didn’t work only for one boronic acid – the one with orto fluorine atom.

      • This is pretty interesting, that boronic acids would hold onto silica even in straight acetonitrile, but 5% of alcohol would release them from silica – I wonder, do you think it is possible that boronic acid could be complexing to silanol groups present on silica, in the same way they form complexes with polyols?

  2. Thanks a lot, it is indeed nicely described (with pictures) there! My point was also about the applicability of this method to boronic acids. It appears that this could be an interesting solution. We should give it a shot…

    • with regards to boraxine formation due to overdrying, which complicates NMRs and leads to decomposition, I wonder if one could add some strongly basic volatile amine, someting innocuous that is water soluble, i.e. diethylamine, to the purified product before lyophilization or evaporation of the purified material. I realize that it would require change in mobile phase for the prep HPLC as TFA salt of diethylamine from the mobile phase would probably not be desirable in the product.

  3. @milkshake It was my bet, that boron will complex with silanol groups, and I’m happy that it worked. Was it for that reason? Maybe, maybe not – I didn’t have enough persistence and means to test it! TLC grade of silica was for sure important. Burk’s catch-and-release of MIDA boronates with THF is much bigger enigma for me.

    @ayudin2013 Thanks for bringing up the paper!

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