What do you do when your peptide synthesis fails?

 It never fails. A sequence looks relatively straightforward on paper and it's of moderate length so you start synthesizing without too much thought into the protocol. Your synthesis is finished and you run the analytical HPLC but your product is nowhere to be found (or only very small quantities). So what went wrong? And what can you do differently to increase your crude yield and purity?

This is one of those questions that comes up frequently, unfortunately the answer is - it depends. But don't fret! There are some strategies that can help you move forward and improve your crude yield and purity. In today's post, I'll offer some suggestions for a path forward after a peptide synthesis has failed.

The first and most important thing to do when a synthesis fails is identify the major species present in the sample. Are you seeing major deletion products? Is your desired product present but now inadvertently modified as a result of the cleavage reaction or storage (think methionine oxidation)?Once this analysis is complete there are a couple of strategies that I'd try first which are outlined below.

• Use a sequence predictor tool to identify less obvious, but specific regions that may prove difficult

There are features that render a peptide as a "difficult" sequence and many of which can be overcome improving the overall synthesis efficiency. More often than not, a synthesis fails due to hydrophobicity within the sequence causing inter- or intramolecular aggregation or secondary structure formation. These interactions were well defined by Milton et al and have since been used to create prediction tools readily available online (I like this one in particular).

• Consider changing your primary system solvent

While DMF is the most common solvent used in solid phase peptide synthesis, there are alternatives. Many groups that I work with routinely choose NMP, particularly for synthesis of peptides with greater hydrophobic content. NMP doesn't decompose to dimethylamine like DMF can and also solvates both the amino acids and the growing peptide chain to a greater extent than DMF can. If NMP isn't a good option for your group, mixtures of solvents have also been used successfully to improve the outcome of solid phase peptide synthesis.

• Consider a different resin type

Several new polymers have been introduced for Fmoc-based peptide synthesis over the last few years. While traditional polystyrene-based resins will certainly perform as expected for most peptides, the apparent hydrophobic outer sphere of the resin polymer can encourage peptide aggregation on the resin itself. More recently, Peg-based resins have demonstrated to yield peptides with greater yield and crude purity with higher loading levels, particularly for peptides of greater length or high hydrophobic amino acid content.

• Change the chemistry

While this may be one of the easiest strategies to employ immediately, it is also the most difficult to predict. Changing coupling reagents, adding in double coupling steps strategically (or not), increasing your reaction time (or temperature) or even changing the composition of your deprotection solution have been shown to improve the synthesis outcomes. This strategy can quickly become one of the most time consuming strategies of the above discussed as you'll essentially be optimizing the coupling chemistry of every single amino acid in the peptide sequence.

This is just a short list of some of the strategies that could be deployed to improve the outcome of a peptide synthesis, but they have all been proven to improve synthesis outcomes. Remember, changing even one amino acid in a known peptide sequence can have serious ramifications in the outcome of your synthesis and one (or more) of these strategies may still be required.

If you're new to solid phase peptide synthesis, click the link below to learn about Beth's top tips and tricks for successful syntheses.

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