Chemistry Forums for Students > Organic Chemistry Forum for Graduate Students and Professionals
Phenylfluorenyl PG removal + isolation of small aminoacid
Babcock_Hall:
We deprotect our amino acids using TFA, and we purify them over Dowex-50 using water first, then HCl. We have not seen loss of the double bond.
rolnor:
I guess you see coupling pattern on the double bond, if this is intact you just have to run the deprotection for shorter time as you suggest. Whats happening with the trityl is hard to say but it seems like a dead end.
kriggy:
--- Quote from: Babcock_Hall on November 10, 2021, 11:17:23 AM ---We deprotect our amino acids using TFA, and we purify them over Dowex-50 using water first, then HCl. We have not seen loss of the double bond.
--- End quote ---
Hm I dont think we have dowex but im going to look around if I can get some. Also, how do you know how much water / HCl to use? I suppose you cant realy TLC it to see how it moves on dowex column?
--- Quote from: rolnor on November 10, 2021, 11:36:59 AM ---I guess you see coupling pattern on the double bond, if this is intact you just have to run the deprotection for shorter time as you suggest. Whats happening with the trityl is hard to say but it seems like a dead end.
--- End quote ---
I guess. Im worried that the double bond moved around but thats not something I can figure out now without having the purified amino acid.
Thanks for help guys
rolnor:
TFA will not make a double bond move, if there is a double bond I think its OK.
Babcock_Hall:
For Dowex-50 we use step gradients, such as water then 4 M HCl. We make large pools, comprising several column volumes. We often load the amino acid in 0.5 column volumes of water, then use 1.5 column volumes of water as a wash. Then we use 2-3 column volumes of HCl. If an amino acid elutes slowly (and when there is a hydrophobic side-chain, it has in our hands), then the product still make show up in more than one pool. We take several pools, rotovap/vacuum, and check masses.
Dowex-50 is an old-school technique for purifying amino acids. Often people used water followed by 2 M ammonia , and I have used this many times myself in the past. Although I have seen people back the ammonia down to 0.5 M once or twice (presumably for sensitive side chains), I decided to avoid any possibility of its attacking the amino acid, and that is why I chose to elute with HCl. 4 M HCl is probably overkill, but people have used it before, so I just decided to try it. We only occasionally use TLC on amino acids, but Stahl's venerable TLC book gives five common solvents. 70:30 ethanol/water is one of them. There are both acidic and basic conditions.
We occasionally use Dowex-1 in the acetate form, but it does not always work in our hands for reasons that are not known to me. The idea is for the carboxylate group of the amino acid to displace an acetate ion on the resin. Acetate is less strongly bound to the resin than chloride; the conversion of the resin from the chloride form to the acetate form is lengthy but not difficult. If you need any references or protocols, just let me know.
This brings me to a question. Someone once asked me if I knew of any papers on the purification of amino acids by ion-exchange. I said that I did not, but that I could almost write one. Since that time my students and I worked out one additional method that is applicable when the side chain carries a charge. Would any journal or serial publication (by which I mean something like Methods in Enzymology) be interested in such a work?
EDT
Just FYI, there are some reviews (I have not read them in a while) of unsaturated amino acids that deals a little with isotope labeling:
Kaiser J et al., Org. Biomol. Chem. 2005 3:3435-3467. (a review which covers 2000-2005)
DOI: 10.1039/b001538p (a review which covers 1990-2000 IIRC)
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