SPPS of a 16 aa peptide was performed on a 0.15 mmol scale. Resin gained a minimal amount of weight: starting weight of 224 mg and a final weight of 280.I believe my peptide failed to elongate but im not sure why.
Rink amide MBHA resin was swollen, deprotected by 20% Piperidine/DMF prior to addition of an Fmoc protected Diaminobenzoic acid linker (Fmoc-Dbz; Peptide Intl). After coupling of Fmoc-Dbz for 40 min, a Kaiser test indicates a 99.8% coupling yield. I proceeded with the synthesis and ended with a standard Dbz --> Nbz derivitization (acylation of Dbz with p-nitrochloroformate, followed by base-catalyzed cyclization; Dawson 2008).
Ive performed many synthesis and have never failed to elongate. I will attempt to cleave the product soon and analyze by LCMS but I need to wait through the holiday. Does anyone have any experience with this sort of failed synthesis?
Hi i agree with Saraniya Bharathi Based individual amino acid combination - the peptide validated as "Easy" "Hard" to synthesis.
You may synthesized many peptides but this was failed due to sequence difficult - if you provide sequence means i will validate it and give you clear reply.
Some Points :
1. You used Rink amide resin - so here there was no problem found
2. After first amino acid coupling - amino acids solvation effect and coupling deficiency issues synthesis goes to wrong direction .
3. Each amino acids have different internal properties - Neutral and Hydrophobic amino acids create trouble - so before planning to synthesis - you write protocol for each "Hard" amino acid.
If sequence easy means no issues - if "difficult sequences" means should be careful and modify your procedure in difficult red-zone area of the sequence.
Hope some of my publications have details about this - may helpful for your research.
All The Best For Your Research and Success
- Dr.R.Selvam - Custom Peptide Synthesis Expert
Grey Matter Research Foundation.
Erick Delgado @ You have not written the peptide sequence, but the reason may be in it. Certain peptides can be "difficult" to elongate because of their ability to form inter- and intramolecular H-bonds, resulting in difficult Fmoc-deprotection and chain elongation. In any case, you should analyze your product by LC-MS; this analysis will show you, what steps have been difficult to proceed. To overcome elongation difficulties, you can use either more efficient reagent for Fmoc-deprotection (2% DBU + 2% piperidine in DMF, but it is not recommended if you have Asp residues in your peptide, except Asp-Pro, because of the possibility of aspartimide formation) or employ more efficient activation reagents (HCTU, HATU, PyAOP). I strongly recommend using Oxyma Pure instead of HOBT, basing on the experience of our lab works.
Besides that, we observed some acylation of the second amino group of diaminobenzoic acid residue while performing a similar synthesis, be aware of this side-reaction.
Hi Erick,
I completely agree with Ekaterina Kolesanova 's statement about certain peptides being difficult to elongate, due to aggregation of the sequence.
As for your Kaiser test, I'm unaware of what this would look like in quantitative fashion. If you want to be certain of your resin loading, I'd recommend taking a small amount of your resin after coupling with Fmoc-Dbz, deprotecting the small amount of resin, and using UV to determine the dibenzofullvene concentration to figure out your resin loading. After derivatization, repeat this step to confirm the issue doesn't lie with your derivatization protocol, but the actual synthesis of your peptide.
In principle, you could repeat this procedure after every coupling step, but that might be rather time-consuming. Hope this helps!
Kindly check https://onlinelibrary.wiley.com/doi/full/10.1002/psc.2836
Also check https://www.sigmaaldrich.com/technical-documents/articles/chemistry/overcoming-aggregation-in-spps.html
Hi sir
Seen your question and details - Not all peptides in same nature and easy to do, some peptides based on amino acid combination hard to synthesis comes under "Difficult sequences" so based on amino acid combination you slightly modify synthesis method means you will get this peptide.
especially hydrophobic amino acids combination and Fmoc De-protection drawn back to fail. if need more details show me your sequence so we can give complete answer why failed.
Hi i agree with Saraniya Bharathi Based individual amino acid combination - the peptide validated as "Easy" "Hard" to synthesis.
You may synthesized many peptides but this was failed due to sequence difficult - if you provide sequence means i will validate it and give you clear reply.
Some Points :
1. You used Rink amide resin - so here there was no problem found
2. After first amino acid coupling - amino acids solvation effect and coupling deficiency issues synthesis goes to wrong direction .
3. Each amino acids have different internal properties - Neutral and Hydrophobic amino acids create trouble - so before planning to synthesis - you write protocol for each "Hard" amino acid.
If sequence easy means no issues - if "difficult sequences" means should be careful and modify your procedure in difficult red-zone area of the sequence.
Hope some of my publications have details about this - may helpful for your research.
All The Best For Your Research and Success
- Dr.R.Selvam - Custom Peptide Synthesis Expert
Grey Matter Research Foundation.
i agree with R. Selvam & Saraniya Bharathi
have question - in final stage anything you got as a peptide? if you analyse the final product with HPLC and LCMS means easy to find in sequence which area red line. if you revel sequence means will give clear answer.
Shiab K P Subhair R. Selvam Saraniya Bharathi
thank you very much for the feedback. Ive used online "sequence difficulty" calculators and I did not observe this sequence to be particularly difficult
Dbz-ASWCEKSVSCDVDTFZ
Hi sir, you are right , based on your sequence there was no difficulty found.
Sir what is Z in C-Terminal? Found Two Cysteine Group but this won't make any issues. Sir keep try one more time with different raw materials.
Just take care the Cys part - monitor each and ever coupling.
Sure you will get this peptide.
All the best "Happy Year"
Sir had seen all discussion here
Sequence not have any issues , try once again monitor each and every step
sure you should get this
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Peptides