I want to lower the loading of Knorr resin (Rink amide). I am thinking of using 0.5 eq of the 1st amino acid then cap the resin after coupling. I am hoping that this will lower the loading of the resin and allow me to increase the yield of my peptide. I suspect that my peptide is aggregating during the synthesis which is why my yields are low. Does anybody know if this approach will work?
That sounds like a good place to start. Perhaps combining that with use of a solvent that does not cause the resin to swell with the first coupling reaction like THF which may minimize exposure to the inner reaction sites. Then when capping use solvent that causes swelling like dichloromethane to expose and cap remaining active sites. Good luck!
u can use lower substitution of resin like 0.7 mmol/g for long residue peptides and washing in solid phase very important to inhibit aggregation.
The resin loading efficiency and load value which you want to have at 0.5 First AA equivalent is important to consider in synthesis plan. The capping group and swellability are other factors to weigh in. Anyway, both the suggestions by Adak and Maloney also need to be given consideration in your plan.
Yes it will work. You should also look at why your synthesis is aggregating there are a number of possibilities you could try to prevent aggregation. Do you get a expected crude peptide yield or a poor crude peptide HPLC profile.
I have been successful in lowering the resin loadings. All you have to do is cap the resin in the first step (before loading the first amino acid) by using the capping reagent in appropriate equivalent [eg capping reagent (eq 0.6) = resin loading (1.0 eq) - required loading is 0.4 eq].
Secondly, you could confirm if aggregation is the problem by LC-MS analysis. If you observe truncated/deletion sequences, then it certainly is aggregation. To circumvent aggregation, use chaotropic salts in the solvents for coupling/deblocking reagents, also microwave heating facilitates synthesis.
Additionally, you could program capping step after every cycle, this would simplify purification at the end.
If you use Fmoc chemistry I suggest to mix your first Fmoc-aa with Boc-Ala-OH or Boc-Gly-OH, 1:1 (or less) molar ratio, this prevents aggregation better than the suggested previously.
Hi sometimes low loaded resins are commercially available also. Bachem used to sell it.
I strongly support the proposal by Krzysztof Krajewski!
This is the best was to adjust your resin loading.
Thank you for all the suggestions, I will definitely try these suggestions. For Paul Jones question, our lab does not look at crude peptide yield but overall yield of the peptide.
I have a question for Krzysztof Krajewski and Ronald Frank, does the addition of Boc-Ala-OH or Boc-Gly-OH act like a cap? Your responses will be greatly appreciated.
Anthony the Boc-Gly will act as a cap. I have used Z-Gly successfully in the past for both Boc and Fmoc resins.
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