I want to bind two amino acids (COOH from a Fmoc protected and NH2 from another one) via PyBOP coupling agent. After this, I add TAEA (Tris(2-aminoethyl)amine) to the reaction flask to deprotect Fmoc-amino acid. The reactions are simply done immediately. However, in workup (after 2 described reaction steps), I cannot remove ByPOB by-product ((pyridoline)3 P=O) via washing with saturated saline solution and 3Xphosphate buffer extraction.
I think you should do the purification with the Fmoc on first, then do the depretection of the pure stuff!
What solvent are you using for your extraction? I'm also interested in how you're monitoring your reaction and what these results look like (e.g., TLC results). Phosphoramides are water soluble but will stay in the organic layers in certain solvents like DCM.
You should be able to hydrolyze your phosphoramide in acidic work up conditions with a few washes of 0.5 to 2N HCl, so long as your peptidyl substrate tolerates this (some residues are more prone to epimerization, so that depends on what exactly you're coupling); there are a few other alternatives including 10% citric acid, NH4Cl, or sodium thiophosphate. However, you will need to carry out your amide coupling first, do several acidic washes and confirm removal of your phosphoramide, then deprotect your Fmoc. I'm not quite sure why you are not losing a significant amount of your product to the aqueous layer as is, to be honest, given that the free amino acid will preferentially go into the aqueous layer.
Next, I would suggest that you should purify by flash column chromatography as the Rf's for your product and byproduct should be substantially different. Again though, this would make the most sense if you coupled, purified, and subsequently deprotected Fmoc (as suggested above). In general, it is best to isolate your intermediates so you can get a better idea of where your reaction is going wrong, and then see if there's ways to do a one-pot reaction like you're trying (i.e., coupling and deprotection).
Finally, there are many other ways to carry out a coupling reaction with many robust and powerful coupling reagents that might be easier for you to purify. HATU is particularly strong and tends to prevent epimerization. There are also carbodiimides you could try, such as EDC and HOBt.
Hi Grant
Thank you so much for your time, consideration and also great information.
Great to hear! Glad that I could be helpful. Do let me know if you still run into problems with this synth. Good luck.
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