Hi,
According to a protocol −obtained from aapptec company− 1 equivalent of the first amino acid has to be coupled to the 2-chlorotrityl chloride resin (CTC) for synthetizing peptide (Fmoc strategy, solid phase method).
For example if the loading capacity of the CTC was 1 mmol/gr, the first amino acid (AA1) by the amount of 1 mmol has to be coupled on 1 gr of the CTC followed by end-capping with methanol.
I followed the protocol accordingly but at the end, the reaction yield was far too little than what I expected.
To make sure that I have occupied all of the binding side on the resin particles with the AA1, can I couple my first amino acid twice (do the famous double coupling)?
Or alternatively, despite the protocol, can I take 2eq of AA1 for coupling to 1eq of the CTC resin to make sure that the entire binding site has occupied with the AA1?
Many thanks for your helps.
All the best,
Amir
Dear Amir,
there can be two issues for the lower yield. Incomplete coupling of the first amino acid or your 2Cl-Trt is degraded and it simply has no more Cl left. so you could use two equivalent of amino acids and if this does not help then its a sign that you resin has taken up moisture or in other words will not be suitable for loading the first amino acid. you could use Wang resin if you don't need protected peptide in the end or purchase a per-loaded ClTrt resin. have you determined the level of attachment by quantifying Fmoc release?
best regards,
Parvesh
Dear Parvesh,
Many thanks for your reply. I must use CTC to obtained protected peptide after cleavage. I have quantify the amount of attached amino acid by weighting the resin before and after coupling with Trp. Despite the loading capacity of 1.12 mmol/gr reported by the company, it is only 0.6 mmol/gr according to my measurement.
I just found an article (Int. J. Peptide Protein Res. 37, 1991, 513-520) which emphasizes that the first amino acid has to be coupled ,stoichiometrically, in fewer amount relative to the binding sites on the resin. Are you sure using two equivalent of the first amino acid is good?
All the best,
Amir
Dear Amir,
you are right that maximum only 1.2 eq excess is suggested but that is in the ideal case...in your case Trp as first residue as its own share of additional issues but if the yield is low there is nothing against it. good luck ( i would buy a Fmoc or just H-Trp (boc)-clTrt resin from irisbiotech (RAA1305.0005 H-L-Trp(Boc)-2CT Resin 5 g
€80.00 ) and you can trust the resin loading as well.
best regards,
Parvesh
Hallo Amir,
in our lab we usually use 1.0-1.2 eq of amino acid and reaction times of 1-4 hours depending on the amino acid used. Under these conditions we also achieve resin loadings between 0.5 and 0.8 mmol/g (determined by UV-Spectroscopy after Fmoc-removal).
Why do you want to achieve such high resin loadings? Probably, these wouldn't be beneficial for the synthesis of your peptides, especially in the case of long sequences or difficult sequences with high aggregation tendencies. Therefore I would recommend to use your loaded resin.
Best regards,
Matthias
Thanks guys. By summarizing your comments, now I know what to do. For my next synthesis, I will apply more than 1 equivalent of the AA1 and also I will increase the coupling time to hours. These are the ideas everybody agrees upon.
Dear Amir,
as Parvesh already mentioned in his first answer it might be possible that you resin has taken up moisture and hydrolyzed to 2-chlorotrityl alcohol resin. This happens very quickly if the resin is stored longer than a month in the fridge. You can bring it back to activated chloro resin by following this activation protocol:
Suspend the resin in DCM (approximately 10 mL per gram of resin). Cool the suspension in an ice bath. Add 2.4 equivalents of pyridine and 1.2 equivalents of thionyl chloride (SO2Cl2). Caution: Thionyl chloride is corrosive and reacts with moisture to release HCl. Wear safety goggles and chemical resistant gloves. Perform this reaction in an efficient fume hood.Heat at reflux for 2 to 4 hours. Filter the resin. Wash it 6 times with DCM. Dry the resin in vacuo.
After that you can follow the usual procedure for loading the first amino acid.
Best,
Philipp
Hi Amir,
I can concur will all of the above. We had some 2-chlorotrityl resin that had an apparent loading of 1.53 mmo/g but when fully loaded with the first amino acid the maximum I could get was 0.3 mmol/g even with excess reagents. This points to hydrolysis of the Cl to OH, deactivating the resin.
Also note that the loading will decrease from the initial loading even if is quantitative so your loading of 0.6 mmol/g is pretty ok (if you are talking about after the loading of the first amino acid)
cheers
Paul
Just to add to this, there are reports of using 2 equivalents of amino acid for 6 hours.[1] I have definitely used up to 4 equivalents in a loading step. The rational being that the first amino acid was far cheaper than the resin, which is worth considering and hence me deciding to comment. I've even heard of using up to 10 equivalents of amino acid. There was even a recent paper in Nature Chemical Biology [2] that used a high excess of AA's to achieve 7 second coupling times, and 40 second overall synthesis times, though this was in the setting of flow chemistry which is admittedly out of your scope.
[1] ChemMedChem. 2011 Sep 5;6(9):1739-45. doi: 10.1002/cmdc.201100113. Epub 2011 Jul 14. "Unexpected opioid activity profiles of analogues of the novel peptide kappa opioid receptor ligand CJ-15,208."
[2] A fully automated flow-based approach for accelerated peptide synthesis
Nature Chemical Biology (2017)
DOI:10.1038/nchembio.2318
Hi Philipp,
Could you please provide me the reference for the method you suggested? I experience the same issue. Thanks
Guys have you ever tried capping by ethanol instead of methanol? does it affect the loading?
cheers
It is very difficult to get high loading efficiency with 2-chlorotrityl chloride resin. But, if you can follow few steps then you will get maximum loading efficiency.
1. Before start your synthesis let it dry in a desiccator for 2-4h;
2. Swell the resin for at least 1h in dry DCM;
3. If your Fmoc-amino acid ( AA1 ) is soluble in DCM then do the 1st coupling in DCM instead of DMF;
4. Use 3 equivalents of amino acid ( AA1 ) with overnight reaction at room temperature.
All the best.
Hi,
Have some of you any experience with loading Fmoc-Cys(Trt)-OH on the 2-Cl Trt resin, or its methylated analogue, Fmoc-Pen(Trt)-OH? I've tried some different strategies (0.7-1.2 equivalents of amino acid relative to resin, 1-2 h up til 24 h reaction time), but I still get low loading (less than 0.3 mmol/g). I've read that if the loaded resin is stored with the Fmoc-group still attached to the first amino acid, the amino acid-resin bond is more susceptible to break, hence, the Fmoc group should be removed immediately after esterification and before drying. I'm currently using a protocol from Aapptec, but I've tried others as well to compare. It could be that the resins has hydrolyzed to 2-chloro alcohol resin (even tough it's not old and kept dry as far as I know).
Best,
Ingrid
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