There are any side reactions that may occur during SPPS. Most have been well documented in literature and there mechanisms are known. Listed below are some of the most common side reactions known to occur.
Racemization is always a consideration in peptide synthesis. Naturally occurring biologically active amino acids are almost exclusively found in the L-conformation at their α-carbon, however racemization can convert the L-conformation to the D-conformation. Glycine with an R group equal to hydrogen does not have a chiral α-carbon.
Removal and reattachment of the hydrogen atom attached to the α-carbon represents a potential mechanism for enantiomerization. During amino acid activation, the acidity of the α-carbon proton increases. However, this does not appear to be significant mechanism of enantiomerization. Another route to enantiomerization is deprotonation and oxazolone formation.
In general, enantiomerization through oxazolone formation is rarely encountered with stepwise synthesis except for histidine and cysteine. However, special coupling chemistries for these amino acids have largely circumvented the problem. Aspartimide Formation
This very common side reaction occurs with aspartic acid or asparagine. The reaction occurs from formation of a five-membered imide. This intermediate can undergo a series of fates that result in various side products as shown below.
This side reaction occurs most frequently with peptides containing the Asp(OtBu)-X sequence, where X = Asn(Trt), Gly, Ser, Thr. Guanidinium Formation
A capping reaction can form when excess uronium activator versus amino acid is added to the reaction vessel, leading to guanidinium formation. Thus, when using HBTU activation, a 0.9/1 ratio of activator/amino acid is recommendedDehydration
The amino acids asparagine and glutamine have been shown to undergo dehydration at elevated temperatures. This side reaction involves the loss of a water molecule from the side chain groups of these two amino acids. With microwave peptide synthesis, the deprotection and coupling reaction temperatures are elevated and can cause dehydration of the side chains of these two amino acids when they are exposed. For this reason it is necessary to use side chain protecting groups on these two amino acids with microwave peptide synthesis. Both of these amino acids are commonly available with side chain protecting groups. Below are the recommended side chain protecting groups that will prevent dehydration during the peptide chain assembly. These protecting groups will be removed during the final acid cleavage of the peptide; therefore, it is essential that this reaction temperature be minimized. Experts in the CEM peptide synthesis laboratory have designed appropriate cleavage methods that prevent dehydration.
When creating a new cleavage method for a particular peptide, an excessive amount of power delivered to the peptide can increase the bulk temperature of the reaction enough to cause dehydration of a peptide containing asparagine or glutamine. For assistance with cleavage methods, contact the CEM peptide application support group.
Recommended protection of Asparagine and Glutamine:
This side reaction can occur if Glycine or Proline amino acid is at the first or second position on the peptide chain from the resin. This side reaction leads to cleavage from the resin of the first two amino acids in what is called a diketopiperazine.