Yelow is quiet normal for Leucine or if your DIPEA is bit old.....It is also possible for Lysine to become Redish-Orangish when coupling on the resin.

What coupling you want to do exactly? may be I can help you with alternatives.

This is a glycine coupling in an oligoglycine (20 aa). I've seen yellow quite a bit, as well as the orange color for glutamine, but never for glycine. Have you seen this phenomenon?

• Have you checked the quality of the resulting peptide by HPLC and mass spec? Perhaps the unusual reaction color did not affect the final product quality.

• Are you using DMF or NMP? Did you change suppliers? Is the amine removal pouch in the DMF broken?

• Are you using shared reaction vessels? Has anyone else been working with dyes such as fluorescein, rhodamine, etc.?

• Are you using an automated synthesizer or manual reaction vessel? Contaminants from resin cleavage may become brightly colored in basic solutions.

• Are you pre-activating in an Erlenmeyer flask, glass scintillation vial, or disposable plastic tube? Certain leachables may become brightly colored in basic solutions.

• I've observed that certain Fmoc-amino acids such as Fmoc-Cys(Trt)-OH and Fmoc-Arg(PBf)-OH tend to yield intensely colored (yellow-orange) preactivation solutions using HOBt-related reagents. This coloration did not affect the qualities of the resulting peptide products. This coloration is perhaps caused by dipolar interactions between the aromatic rings of the activated ester and the sidechain protecting groups.

I coupled upto 6 glycines in a row but never came across glycine coupling turning orange. We use HCTU/DIPEA or DIC/HOBT couplings. Only reason I can think is Amine of glycine is free and not Fmoc protected.

Basically after adding DIEA the reaction mixture becomes yellowish or orange which is very common So I think there should not be any problem with you coupling reaction. This is due to the activation of amino acid by HBTU/HOBT which initiates after addition of DIEA.

I suggest first of all, to make a Kaiser test to see if amine is still free and see in this way the coupling reaction. Probably, the problem could be caused by a not correct washing procedure or the coupling ractives are not so fresh. Do you use them in solid or in solution?

I've seen faint pink colour often in premixed solutions-it often happens immediately - I use sieved, fresh, peptide-grade DMF and fresh reagents too. The colour never really worried me though, because it would always be reasonably faint and never seemed to cause big decreases in isolated peptide yield/purity (then again, I make glycopeptides so lots of other things caused decreases in isolated yield/purity). I normally do not pre-mix the base with the aa and the coupling agent-the base gets added when it goes into the reaction flask. I generally agree with Lael on all the comments, but it might just be the presence of minor impurities in any of the reagents that give that colour; probably nothing to be concerned with if everything else looks good.

I remember, I had this kind of colour using peptide synthesis grade DMF. Usually the quality of this grade is poor for almost all companies. I checked the quality with dinitrofluorbenzene. It was never sufficient. For easy sequences maybe. But for more diffecult ones I destilled the DMF over phthalic anhydride. Then the colour for the coupling mixture is allways more yellowish. The same kind accounts for DIPEA. If your base is a little older (the manufacturer can tell you the production date) you get a more intense colour. But anyway, I would not worry if the quality of your crude peptide looks reasonable.

Great ideas. I'm doing manual synthesis in a rxn vessel; it's not automated. Pre-activation is in glass scintillation vials. I've tried new reagents of everything except the DMF- so now I'm thinking that maybe distilling the DMF would help. The couplings seem to be more difficult now that I've started seeing the pinkish-orange color, and often double- or triple-coupling is necessary, and the last peptide I cleaved had quite a few deletions.

Just use properly purified solvents for the synthesis. Colors could be caused by presence of secondary amines into reaction mixture. In principle, this is a minor side reaction that does not influence significantly on the quality of the final product.

I have experience with the same low loaded Rink Amide resin and always upon mixing of Fmoc-AA, HBTU, HOBt and DIEA, the solutions turned differently coloured, but it has never been a problem. Always the coupling was performed as was expected. The activation time must not exceed 5 min. Did you try the coupling with that activated aminoacid?

Thanks, Nani. We continued to see this phenomenon for a while, but I'm not convinced that it had any negative outcome for our reaction.

Hi Erica,

I have the same problem with my HBTU. It is a 0.4 M solution in DMF. Over few days it turn organ-pink itself. I mean even with out using it for coupling! I guess it is impurities in the DMF. The following solution might help:

1- Purifying the DMF by distillation it.

2- Store the HBTU solution in 4 degree.

good luck

The colour is not as big an issue as impure DMF. As Nani was saying, you get some complexes with the reagents-but these are generally very minor concerns. Either buy peptide synthesis grade DMF or distill it prior to use. The main difference with the peptide synthesis is a guarantee of recent manufcature. DMF slowly decomposes to dimethylamine. This leads to unwanted Fmoc deprotections. This causes all sorts of problems of course. That is probably more responsible for any problems than any coloured minor contaminant. 

Did you get explanation for pinkish-orange color in peptide synthesis?