1904 Edman Degradation

ignoring the side chain of lysine the in terminal amino group of a polypeptide chain is by far the most nucleophilic nitrogen containing group in a polypeptide and we can take advantage of the nucleophilic reactivity of the n-terminus to selectively cleave a polypeptide at the n-terminus and determine its sequence by degrading the inter man I one by one walking along the polypeptide chain until we get to the other end the essential idea is to treat the polypeptide with a reagent that attaches to the in terminus and subsequently reacts with the carbonyl carbon to release the next nitrogen along the backbone as a leaving group creating a new end terminus this also creates a hetero cycle which can be isolated to determine the identity of r1 the in terminal amino acid once we do that we can repeat the process to determine r2 r3 r4 so on and so forth the reagent used to do this is phenol isothiocyanate or P I see you'll also sometimes see it written as P ITC structurally P IC is analogous to carbon dioxide and the central carbon here is electrophilic as it is in carbon dioxide the nitrogen and sulphur atoms link to that central carbon or potential nucleophiles and with this pattern of nucleophilicity electrophilicity we have a pattern that can be used to form bonds to both the electrophilic carbonyl carbon and the nucleophilic amino carbon of the internal amino acid within this heterocyclic product we can see the elements of p IC which i've highlighted in blue right here here's the sulphur atom central carbon nitrogen and phenyl ring and we can also see the elements of the amino acid and let's highlight that in red the entire in terminal amino acid becomes incorporated into this hetero cycle two key bonds are formed in this process there's a bond formed via donation of a pair from the amino nitrogen in the amino acid to the central carbon of p IC that's formed through a nucleophilic addition process and there's a bond formed via donation of an electron pair from the nitrogen and p IC back to the carbonyl car of the n-terminal amino acid and that's formed through ultimately a nucleophilic acyl substitution process note also that what's missing is this nitrogen the nitrogen of the next amino acid in the sequence and that remains incorporated into a shortened polypeptide chain so notice that now we have a new in terminal residue with r2 as the amino acid residue too now hanging off the end of this remaining polypeptide chain this hints at the idea that we can repeat this sequence of reactions with piz followed by hydrofluoric acid - once again cleave off the n-terminal residue which is now residue - and characterize it the analysis on the last slide gave us the basic idea that the Edman degradation generates a hetero cycle involving only the in terminus that can be analyzed to determine the in terminus and repetitively applied to determine the sequence of a polypeptide here I wanted to look at this reaction a little more mechanistic detail we won't go through the individual elementary steps but we'll look at some key intermediates phenol isothiocyanate is electrophilic at its central carbon atom flanked by nitrogen and sulfur atoms and really the key first step in the mechanism of formation of this hetero cycle is nucleophilic addition of the amino nitrogen to this carbon NPIC through something like acid or base catalyzed nucleophilic addition to a polarized PI bond the nucleophilic amino nitrogen the terminal amino nitrogen adds to the electrophilic carbon and P IC the next stage involves nucleophilic acyl substitution this initially occurs through nucleophilic action of the sulphur atom which adds to the carbonyl carbon to give after addition and elimination of the amino group a structure containing a thio ester known as a thiazole anote this electron flow has done two important things it's established a bond between sulfur and the carbonyl carbon and it's resulted in the cleavage of this carbon nitrogen bond which releases the remainder of the polypeptide from the end terminus now we have our new in terminus and we're ready to repeat the Edman degradation again to identify the next residue the thiazole enone is isomeric with this final hetero cycle that you see in the product sign here and ultimately what happens to generate this final head cycle is an isomerization process that's mediated by HF and heat and this essentially involves opening of the ring and use of nitrogen as a nucleophile toward the carbonyl carbon to reclose the ring this resulting hetero cycle is known as a phenol bio hi Dan - and it's a hydantoin with sulfur replacing oxygen and a phenyl group linked to nitrogen we'll just call it PTH for short and the PTH may be analyzed chromatic graphically or spectroscopically to determine the identity of this art group and thus the identity of the n-terminal residue to fully complete the sequencing process we just repeat the previous steps treatment with p IC and HF until we get to the c-terminal residue at which point we form one more PTH and there's no material left so the beauty of the Edman degradation is that it takes advantage of the unique nucleophilic reactivity of the n-terminal amino group which is not found anywhere else along the polypeptide backbone this allows us to capture a molecule like P I see an electrophilic molecule with the potential to open the first amide along the polypeptide backbone and release a new interest repeated application of p IC and HF allows us to determine the sequence of the polypeptide one by one