Chemical Forums
Chemistry Forums for Students => Organic Chemistry Forum => Organic Chemistry Forum for Graduate Students and Professionals => Topic started by: Babcock_Hall on August 29, 2017, 11:48:58 AM
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We synthesized an adduct between cysteine and bromoacetylpyridine, which is commercially available as the hydrobromide salt. The adduct is a ketone with a sulfide at the alpha-carbon (see attached). We typically add two equivalents of a base, although we have not settled on the best base. The product has bromide salt as one impurity. I suspect that we have unreacted bromoacetylpyridine or its derivative as one impurity on the basis of H-1 NMR. Our typical means of purifying amino acid derivatives is Dowex-50 using water followed by ammonia, but this standard method has not produced good results for our compound. Sometimes compounds with aromatic rings are retained on Dowex, presumably via hydrophobic interactions with the polystyrene beads.
We are trying reversed phase HPLC on C-18, starting at 3% acetonitrile and running a gradient, although I believe that we have two isocratic holds in the protocol ATM. We see a mixture in the presence of TFA, but we are not 100% certain which peak is which, lacking a pure product to use as a standard. We are considering using triethylamine as the base in the synthesis and also as an ion-pair reagent in RP HPLC, although its water solubility is not great. I know just enough about ion pairing and buffering in RP HPLC to be dangerous.
Does anything come to mind as a good separation strategy? We have several C-18 columns, which makes this approach a bit more attractive. We only need to purify a few milligrams for NMR, and then we can write this up (phew!). Ideally any buffer or ion-pair reagent present in excess should be volatile (or easily removable at least).
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What about acetate as a base or ion pair reagent? I've heard of ammonium acetate being used as a buffer since it is actually volatile at low pressures. Ammonium formate has this same property I think (BP 180C ... via decomposition to ammonia and formaldehdye).
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1). You need thee equivalents of base because the carboxylate anion that is formed in situ, inhibits the complete neutralization of the pyridinium salt.
2). How can you be sure (on the basis of 1H-NMR) that it is not a mixture of the desired compound and it’s zwitterion salt?
3). In reverse phase chromatography the most polar ingredient (amine salt) is the first eluted one, which means that the desired product is probably the one with the longer retention time.
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One of my students found a purification of nicotinic acid derivatives in 20 mM ammonium acetate at the Phenominex website. They used a gradient of acetonitrile. So acetate as a base is certainly possible.
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One thing to look out for is the potential forming of a six-membered imine-ring with the ketone and the amino-group.
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Good point. Until now I had been liking the idea of making the triethylammonium salt as the crude product and using TEA as the ion pair reagent, but an adduct forms, that might complicate the analysis.
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One thing to look out for is the potential forming of a six-membered imine-ring with the ketone and the amino-group.
Now that you mention it, I believe that I have see such additions with other electrophilic groups that are conjugated onto cysteine.