Why does capping after coupling improve results during solid phase peptide synthesis (SPPS)?

The Case: I have conducted a lab experiment where we coupled three Fmoc-protected amino acids, and did a final capping (acetylate) at the end of the synthesis. However, I was told that one can theoretically cap after every single amino acid coupling to ensure that the final product is correct. That is, one can "eliminate" or terminate the synthesis of peptides where the amino acids are innocently coupled (?).

I don't really understand why, because if you cap then you "block" all of the peptides that you are synthesizing (?). Therefore, how can you add further onto the chain if you cap after each amino acid? This seems counterintuitive, or I might be a bit confused. If you are able to de-protect the cap to further couple on more amino acids, then what is the point of capping?

Is there someway that capping only “caps” onto amino acids that are not coupled or that did not couple the previous amino acid somehow?

  • $\begingroup$ I'd guess that it means that at each step if you capped the amino acid by acetylation that you could purify each intermediate product. Thus in the end you'd have a higher purity product, but probably less of it. $\endgroup$
    – MaxW
    Oct 31, 2015 at 20:48

1 Answer 1


if you cap then you “block” all of the peptides that you are synthesizing.(?)

No, you don’t, the order of steps is important. First you try to couple your new amino acid to the solid phase. You will end up with uncoupled ends with free amino groups and coupled ends with a protected amino group. Then you cap, e.g. with phenoxyacetic acid anhydride. This will only cap the uncoupled ends with free amino groups. Then the Fmoc or whatever protective group is removed rendering free amino groups for new couplings.

Say you wanted to create the sequence $\ce{CHEMISTRY}$. And say at one specific spot on the resin, you failed to couple the serine residue; you have $\ce{H2N-TRY-CO-resin}$ at that one spot. If you now do not cap, you might generate the sequence $\ce{CHEMITRY}$ because isoleucine will couple to threonine’s free amino group etc. By capping, you essentially generate $\ce{Ac-NH-TRY-CO-resin}$ which won’t disturb you anymore.

And the benefits don’t stop there. Without capping, at the end you would end up with the sequences $\ce{CHEMISTRY}$ (the one you want), $\ce{HEMISTRY}$, $\ce{CEMISTRY}$, $\ce{CHMISTRY}$, $\ce{CHEISTRY}$, $\ce{CHEMSTRY}$, $\ce{CHEMITRY}$, $\ce{CHEMISRY}$, $\ce{CHEMISTY}$ and maybe even $\ce{CHEMISTR}$. These are all of similar length to your desired product and hard to purify away via HPLC. But if you cap, you can only get $\ce{HEMISTRY}$, $\ce{EMISTRY}$, $\ce{MISTRY}$, $\ce{ISTRY}$, $\ce{STRY}$, $\ce{TRY}$, $\ce{RY}$, and $\ce{Y}$ — most of these are significantly shorter and easier to purify away.

Also, assume a constant amount of amino acids always being added to the resin to couple. If only the so far correctly built sequences are available, these will ‘see’ a higher ‘concentration per site’ of their coupling partners maybe resulting in slightly increased yield, too. (Note that this paragraph used layman’s terminology.)


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