Sequence Determination One Terminal Unit at a Time

Uploaded by lamechivanes on 12.03.2010


In order to determine the sequence of a polypeptide
beyond the N-terminal sequence,
one needs to perform a sequential degradation.
And so here’s a polypeptide
showing the last three residues.
In the sequential degradation known as the Edman degradation,
the last group will be removed and analyzed first,
then the second-to-last group will be removed
and analyzed second,
and then the third group will be removed and analyzed third.
This is the process known as Edman degradation,
and this webcast covers the chemistry that goes on
in the Edman degradation process.
It involves the reagent phenylisothiocyanate,
whose structure is shown here,
and phenyliso isothiocyanate is electrophilic at that carbon.
So with a nucleophilic amino group,
the first step involves nucleophilic attack
of that amino group onto the electrophilic carbon
of the phenylthio isothiocyanate
to generate first this intermediate shown here.
That sulfur group basically will do a rearrangement
where there’ll be an internal proton transfer
– that is essentially a tautomerization kind of step
– to make this thiocarbamate structure that’s shown here,
or it’s sometimes called a thiourea.
What we see now is, um, this is the, ah,
the completion of the step one of this repetitive process.
At this point, the reaction conditions would be changed
to, ah, acidic conditions often by the addition of HF.
And what HF will do is first,
it will protonate that oxygen of the, of the amide carbonyl.
Once that oxygen is protonated,
we have an internal nucleophile
that will allow a 5-membered ring to form.
And so with the nitrogen lone pair,
ah, attack basically doing a, a donation
into the carbon- sulfur π bond,
and that doing an addition into the carbonyl group
that’s been protonated, this 5-membered ring can form.
Now with general acid catalyzed assistance,
this oxygen can do a β elimination
eliminating the bond between the carbon and nitrogen group.
And, again, there will be a general acid.
HF we’ll use as our general acid
to facilitate that β elimination step.
That generates this 5-membered ring known as a thiazoline.
It contains that one amino acid
that was originally on the terminus of the chain,
and then we’re all set up to repeat the process again.
We’ve got the second-to-last amino acid residue
ready to be re-subjected to the first step
of the Edman degradation process
or Edman sequencing method.