In Fmoc-based solid phase peptide synthesis of sequences containing secondary amines (like Proline), coupling with the next amino acid may not be complete. Any unreacted proline residues can lead to impurities with delete sequences. Thus, acetic anhydride is often used to cover any unreacted proline residues.
I work in India and Acetic anhydride is unavailable here due to some national restrictions.
Are there any alternative chemicals that can be used for easily covering unreacted secondary amines like proline?
And, can I use other simple anhydrides like propionic anhydride for the same purpose?
Smriti
Acetic anhydride is also under control in China, you need permit to purchase it.
I believe you can use propionic anhydride, maybe with a little longer reaction time.
Good luck!
Hi Smriti,
In place of acetic anhydride, you can use Acetic acid (0.5M), DIPEA (0.56 M), DIC (0.5M), or HBTU (0.45M) for 30 min at RT, or you can elevate the temperature to increase the rate of the reaction. Like the way, we do in normal Fmoc-based SPPS.
You can look at the following link for the complete protocol.
Article Benzimidazolinone-Free Peptide o -Aminoanilides for Chemical...
Thank you for your response, Guobing Xiang. I will try using propionic anhydride.
Thank you for your response, Sameer Singh.
I found the protocol in the linked article. Can you please share the quantitative effectiveness of this protocol?
You also can double-couple after Pro, to improve efficiency. (Run the coupling reaction a second time without deprotecting in between.)
And as Sameer Singh indicated, for capping via acetylation, you can just couple HOAc like you would couple an AA, although that's much more expensive than AcOAc and base.
Raising the temperature dramatically increases any coupling reaction, but it also increases racemization of AA.
Hi Smriti,
We have been following this protocol for the last 5 years.
The quantitative effect of that protocol is as effective as normal AA coupling; actually, it's more effective than AA coupling.
Since AcOH does not have any chiral center, You can heat the reaction to increase the efficiency in a reduced time (10 min at 60 degC) without worrying about racemization.
As Sameer Singh indicates, coupling HOAc is quite facile - much easier than coupling a significantly more sterically hindered AA.
And I admit he's right about safely heating for Ac coupling; it can't racemize. I'm just nervous about heating.
Sameer,
Do you typically double (or triple) couple difficult ones? I once had to make a series of (naturally occurring) P-P, P-P-P, and even P-P-P-P peptides. I found double coupling was critical. Do you just heat them?
Hi Jhon
I have made one peptide with sequence of PPPPPPPPP-OH without any double coupling. But I heated at 50 degC for 20 min each coupling in continuous stirring. The synthesis was really good.
We do use heating till 75 °C (some people do heat till 90°C also) for almost every AA except Cys and, His which are highly prone to racemize. And Arg forms delta lactum upon heating, resulting in incomplete coupling, so Arg coupling we do at RT. Normally we don't do double coupling. But if any beta branch residue and Arg is after Pro then we do couple two times.
What did your poly-Pro peptide look like on RPHPLC? As you like heating, did you heat the column? I found Pro cis-trans isomerization made funny cups on HPLC until I used the column oven.
I have a minor racemized product in the poly-Pro peptide sequence, and we use 40 degC in RP-HPLC.
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