Dear researchgate community,
Is there any good method/idea to prevent aspartimide formation during Microwave-assisted peptide synthesis (e.g., on CEM Liberty Blue)? My peptide (28-mer) has ..."GDH" ... segment in the sequence and forms aspartimide (-18Da side reaction product).
Thanks a lot for your input.
Aspartimide formation is an important side reaction in the synthesis of N-protected beta-cyanoalanine (beta-cyanoalanine CAS number is 6232-19-5). Specifically, N-Boc, N-CBz, or N-4-methoxybenzenesulfonamide-protected asparagine dissolved in dichloromethane cyclizes in good yield when treated with p-toluenesulfonyl chloride and pyridine, but only if stirred. If the reactants are combined in methylene chloride ,and swirled once, then allowed to stand overnight, the corresponding N-protected beta-cyanoalanine forms instead. I can't find the reference for this synthesis of beta-cyanoalanine this way (I lost it), but the reference didn't talk about the formation of the imide anyway, if memory serves. The literature reference simply instructed letting the reaction stand overnight, without warning about what would happen if you stirred it. I had to find that out the hard way by stirring the reaction overnight, and getting the imide instead of the desired beta-cyanoalanine. So, you won't find this side reaction by searching the literature for aspartimide syntheses. FYI.
This begs the question: are you stirring or agitating the resin during microwave-assisted solid-phase protein synthesis? If so, maybe let it stand instead of mixing it during the protein synthesis.
Here are few methods.
Use high-quality reagents: Make sure you are using high-quality amino acids, solvents, and other reagents. Impurities in these materials can increase the likelihood of aspartimide formation.
Use a low microwave power: High microwave power can increase the likelihood of aspartimide formation. Use a lower power setting and/or shorter reaction times to reduce the risk of aspartimide formation.
Use a protecting group: A protecting group is a chemical group that is added to an amino acid during synthesis to protect it from unwanted reactions. Using a protecting group on the aspartic acid can help prevent aspartimide formation.
Use a slower-acting coupling reagent: Aspartimide formation is more likely to occur during rapid peptide synthesis. Using a slower-acting coupling reagent can help reduce the risk of aspartimide formation.
Optimize the synthesis conditions: Experiment with different reaction temperatures, solvent ratios, and other synthesis conditions to find the optimal combination for your particular peptide.
Use a solid-phase synthesis method: Solid-phase peptide synthesis (SPPS) is a method of synthesizing peptides on a solid support, such as a resin bead. This method can be used with microwave assistance and has been shown to be effective in reducing aspartimide formation.
Hi,
Trying to solve a similar problem I found the only method completely to prevent aspartimide formation is using of cianosulfurylides as PG for carboxylic acids.
Article Prevention of aspartimide formation during peptide synthesis...
Good luck!
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