| The chemical stability of peptides is very dependent | | | | (DTT) or tris(2-carboxyethylphosphine) hydrochloride |
| on amino acid composition and sequence. Lyophilized | | | | (TCEP). Methionine oxidizes by both chemical and |
| peptides are generally more stable than their | | | | photochemical pathways to form methionine |
| counterparts in solution. The following are potential | | | | sulfoxide and further into methionine sulfone, both of |
| degradation pathways for peptides: | | | | which are almost impossible to reverse. |
| 1. Hydrolysis - This is generally a problem in peptides | | | | 4. Diketopiperazine and pyroglutamic acid formation - |
| containing Asp (D) in the sequence, which is very | | | | Diketopiperazine formation usually occurs when Gly is |
| susceptible to dehydration to form a cyclic imide | | | | in the third position from the N-terminus, and more |
| intermediate. For example, in the presence of | | | | especially if Pro or Gly is in position 1 or 2. The |
| Asp-Pro (D-P) in the sequence, the acid catalyzed | | | | reaction involves nucleophilic attack of the N-terminal |
| formation of cyclic imide intermediate can result to | | | | nitrogen on the amide carbonyl between the second |
| cleavage of the peptide chain. Similarly, in the | | | | and third amino acid, which leads to the cleavage of |
| presence of Asp-Gly (D-G) in the sequence, the cyclic | | | | the first two amino acids in the form of a |
| intermediate can be hydrolyzed either into the original | | | | diketopiperazine. On the other hand, pyroglutamic acid |
| Asp form (harmless) or into potentially inactive | | | | formation is almost inevitable if Gln is in the |
| iso-aspartate analog. Eventually, all of the aspartate | | | | N-terminus. This is an analogous reaction where the |
| form can be completely converted into the | | | | N-terminal nitrogen attacks the side chain carbonyl |
| iso-aspartate analog. To a lesser extent, sequences | | | | carbon of Gln to form a deaminated pyroglutamayl |
| containing Ser (S) can also form cyclic imide | | | | peptide analog. This conversion also occurs in peptide |
| intermediate that can end up cleaving the peptide | | | | containing Asn in the N-terminus, but to a much |
| chain. | | | | lesser extent. |
| 2. Deamidation - This base-catalyzed reaction | | | | 5. Racemization - This term is loosely used to refer |
| frequently occurs in sequences containing Asn-Gly | | | | to the overall loss of chiral integrity of the amino acid |
| (N-G) or Gln-Gly (Q-G) and follows a mechanism | | | | or peptide. Racemization involves the base-catalyzed |
| analogous to the Asp-Gly (D-G) sequence. The | | | | conversion of one enantiomer (usually the L-form) of |
| de-amidation (loss of amine) of the Asn-Gly sequence | | | | an amino acid into a 1:1 mixture of L- and |
| forms a cyclic imide intermediate that is subsequently | | | | D-enantiomers. This is more of a concern during |
| hydrolyzed to form the aspartate or iso-aspartate | | | | peptide synthesis, but a much lesser problem in the |
| analog of Asn. In addition, the cyclic imide | | | | finished peptide. In addition, this transformation is |
| intermediate can lead to racemization into D-Asp or | | | | very hard to detect and difficult to control. |
| D-iso-Asp analogs of Asn, all of which can potentially | | | | The general ways to prevent or minimize peptide |
| be inactive forms. | | | | degradation is to store the peptide in lyophilized form |
| 3. Oxidation - The Cys and Met residues are the | | | | at -20oC or preferably at -80oC (if available). If the |
| predominant residues that undergo reversible | | | | peptide is in solution, freeze-thaw cycles should be |
| oxidation. Oxidation of cysteine is accelerated at | | | | avoided by freezing individual aliquots. Exposure to |
| higher pH, where the thiol is more easily | | | | pH>8 should be avoided. However, if it is |
| deprotonated and readily forms intra-chain or | | | | necessary to dissolve peptides at pH>8, its |
| inter-chain disulfide bonds. Disulfide bonds can be | | | | exposure should be minimized and solutions should be |
| readily reversed by treatment with dithiothreitol | | | | chilled. |