Hi all,
What is in Your opinion the best reagent for activation of carboxyl group of Histidine (His(Trt))? In my routinely synthesis I used COMU in coupling step. But in the case of peptide containing Histidine residue, I didn't get satisfactory result. Maybe something else causes the problem, so I want to repeat the synthesis, also optimize the coupling conditions.
I'll be very gratefull for all Your suggestions.
Dear Sunit,
thank You for Your answer to my question.
I would like to ask You another question. Have You ever used DIC/HOBt as reagent for activating the -COOH group of Histidine? I ask, because at the moment I have run out of ethyl chloro formate and I think about using DIC/HOBt in my next try, but I don't know if it would give good results.
I'll be really gratefull for Your answer.
I have problems to couple fmod-his (boc) in solid phase. I used several coupling reagent but the reaction seems to work very bad. do you think the mixed anydride method can be also used in solid phase?
Adriano, did You also use DIC/HOBt as coupling reagent ? If yes, what is Your opinion?
What do you mean by unsatisfactory result? Incomplete coupling or perhaps recemisation? Are you using solid- or solution-phase? HCTU/DIEA normally works fine unless you've hit a 'difficult' (e.g. due to chain aggregation) part in your sequence. Are you certain that the preceding Fmoc group was fully removed? If not, using DBU/piperidine might help, unless Asp(OtBu) is also present. If racemisation is problem consider switching to His(Mbom) or His(Bum).
Zoltan, I used solid-phase synthesis.
After synthesis, on MS spectrum I didn't see the mass of my compound (only mass of my compound plus 113, the MS was in positive ion mode). When I synthesized the peptides which has on Histidine's residue another amino acids, the results was really good. To cleavage the peptide from the resin I used this cocktail: TFA/phenol/water/TIPS (88,5,5,2; v/v/v/v). The time of coupling (COMU/DIPEA) was 2 hours.
with the carbodiimide coupling reagents, you will always run the risk of racemisation. In addition, if you work in solution, DIC gives a soluble urea which can be difficult to remove, the water soluble EDC (CAS 1892-57-5) is much easier to use than DIC or DCC.
In my experience PyBrop and HATU usually give the best results (yields and low racemisation).
Either way, Tetrahedron, 2005, 10827-10852, is an excellent review on amide coupling
Hi Kinga, this doesn't sound histidine related. Is it possible you have added an extra Leu/Ile? Unwanted insertions like that can occur if there are traces of piperidine present during coupling (not enough washing) and the coupling time is extended too long (i.e. multiple hours).
Or it could just be an MS artifact - give the ion source a good clean and increase the DP then see if anything changes.
We did a lot of work on this at Bachem. We published a report that using DEPBT/DIEA was the best reagent for minimizing racemization and still economical. You may find the following poster useful.
http://www.bachem.com/fileadmin/user_upload/pdf/TechLib_X2_Coupling_Methods_for_Fmoc-His-Trt--OH_Resulting_in_Minimal_Racemization.pdf
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