Preventing aspartimide rearrangements during Fmoc-based solid phase peptide synthesis

Aspartimide rearrangements are a particularly nasty side reaction that can occur during fmoc-based solid phase peptide synthesis.  Not only is this a mass-neutral side reaction, chromatographically resolving the undesired, rearranged product can be particularly difficult.  To make matters worse, this side reaction can occur at any point during the synthesis after the Asp has been incorporated into the peptide.

In a prevous post, I described method that I have found useful for identifying whether or not an aspartimide rearrangment as occured during synthesis of a peptide that contains an aspartimide-susceptible sequence.  In today’s post, I’ll discuss some strategies that can be used to suppress, or even eliminate this side reaction.

Because an aspartimide rearrangement is so problematic, there has been a lot of work done of the years research different strategies to prevent the side reaction all together (check out this great review for more info).

One of the simplest strategies for limiting aspartimide formation is to change the Fmoc-removal conditions.  Simply adding 0.1 M hydroxybenzotriazole (HOBt) to the piperidine solution has been shown to reduce aspartimide formation significantly.  However, HOBt is an explosive substance when in its anhydrous state, and is exclusively sold today wetted, introducing nucleophilic water into the deprotection solution.  Alternatively, piperizine, a weaker base, has been shown to be effective at removing the Fmoc group, while simultaneously suppressing aspartimide formation.  It is important to note however, than neither of these methods eliminates rearranged products.

There has also been significant effort developing alternative protecting group strategies that can potentially eliminate this side reaction.  One of the most obvious strategies is to develop a different protecting group for the Asp side chain carboxylic acid.  Increasing the steric bulk of the side chain protecting group can effectively block succinimide ring formation, preventing the rearrangement.  Many different protecting groups have been evaluated to help alleviate this problem.  Some including, 3-methylpent-3-yl (Mpe), 2,3,4-trimethylpent-3-yl (Die), have shown improvements over t-butyl protected Asp while others, like Dmab, have actually shown an increase in aspartimide propensity.  Interestingly, the protecting group needs to be not only bulky, but also somewhat flexible to ensure protection from the aspartimide rearrangement.  Large, conformationally restrained protecting groups have shown little success.

The only method thusfar shown to eliminate rearranged products is protection of the adjacent building block amine.  Converting the standard, Fmoc-protected secondary amine to a tertiary amine removes the reactive free lone pair of electrons.  The Dmb protecting group is almost exclusively used at this point.  Just like with proline, the secondary amine negatively impacts the coupling effciency, so care must be taken when optimizing the incoming Asp amino acid coupling.  To this end, amino acid suppliers routinely stock the Asp-Gly dipeptide, the most susceptible sequence, which can be readily incorporated into a growing peptide chain.

Each of these strategies comes with increased cost.  The alternatively protected building blocks are absolutely more expensive than the standard Fmoc-Asp(OtBu)-OH, Table 1.  But considering the alternative (think painful purification or repeated synthesis), the extra cost may be worth it.

NameQuantity (g)Price (USD)Structure
Fmoc-Asp (OtBu)-OH25$70.50
Fmoc-Asp(Ompe)-OH1$267.00
Fmoc-(Dmb)Gly-OH1$295.00
Fmoc-Asp(OtBu)-(Dmb)Gly-OH1$305.00
Price comparison for a variety of Asp building blocks differing by protecting group strategy. Note: retail price listed from a single supplier.

For my past work, I found great success using the Asp(OMpe) protection strategy.  My sequence contained an Asp residue adjacent to a Peg building block.  This combination is reminiscent of the Asp-Gly sequence, and often yielded a significant amount of rearranged product.  The Asp(OMpe) residue was readily incorporated into my peptides even when using a reduced number of equivalents, yielding peptides of much greater purity (and biological activity!).

What strategy have you used to prevent aspartimide formation during your peptide synthesis?

 

 

I have had a lot of fun exploring these different challenges in the peptide space, but I know I have barely begun to scratch the surface.  Please complete our survey and let me know if there’s anything you’d like to see discussed in the future.

If you’re interested, check my colleague’s new white paper, A Holistic Approach to the Peptide Workflow in Drug Discovery

 

4 thoughts on “Preventing aspartimide rearrangements during Fmoc-based solid phase peptide synthesis”

  1. “Many different protecting groups have been evaluated including, but certainly not limited to, 3-methylpent-3-yl (Mpe), 2,3,4-trimethylpent-3-yl (Die), and more recently Dmab and all have shown improvements over the standard t-butyl protection strategy.”

    Dmab is actually an exceptionally poor Asp protecting group and has significant aspartimide formation. In fact, your Dmab citation characterizes this problem. Dmab was originally synthesized as an orthogonal carboxylic acid protecting group that would be labile to hydrazine – however elimination of the amino benzyl alcohol moiety is problematic as well!

    1. Hi James,
      Thanks for your comment and careful reading of the post. I had misread, and hence mis-cited the paper referenced above. The document has been corrected based on your comments and a more careful reading of the paper.

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