New Directions in Flow-based Peptide and Protein Synthesis
Nina Hartrampf, Ph.D.,
Assistant Professor, Department of Chemistry,
University of Zurich
Peptide makers struggling with aggregation and solubility caused by difficult to make sequences should consider flow-based synthesis methods say researchers from the University of Zurich.
Most peptides are made using “solid phase” synthesis methods in which one end of the molecule being made is bound to a support structure and then built up step-by-step in a single reaction vessel.
While these methods are effective, the purity and yield, of the finished product are strongly dependent on the peptide sequence. Some sequences are straightforward to make, others – “difficult sequences” – are harder to produce at scale and often manufacturers struggle to stop the product from aggregating or coming out of solution.
A flow chemistry method run in a continuous stream through multiple reactors offers a solution according to Dr Nina Hartrampf, Ph.D., an assistant Professor at Department of Chemistry at the University of Zurich in Switzerland.
Undruggable Cancer Target
Development of the method – details of which were presented at TIDES Europe – was prompted by a desire to know more about a protein called MYC which, while known to play a role in many cancers, has proved hard to synthesize and study.
“So when I started my lab in 2020 we were really interested in a protein called MYC, which is a so-called un-druggable cancer target.
“We know that it is heavily controlled through post-translational modifications [PTMS], many of which have not been studied - there are two at position 58 and 62 that have been described - but the other ones have never really been looked at so much.”
The team focused on PTMS in the trans-activation domain, the first 143 amino acids of the MYC protein. The plan was to synthesise the sequence in two parts using a flow chemistry method and combine them for analysis.
“We started synthesizing MYC one to 84 and this actually looked quite nice using flow-based peptide synthesis. We can synthesize this in just a couple of hours. But then when we tried to synthesize the second fragment - a peptide of 58 amino acids - we were not even able to find the mass in the LCMS sample that we looked at.
“We determined that approximately 40% of our peptides were aggregating during synthesis. And what that means is that while we were synthesizing, the resin was actually collapsing and the peptides are really aggregating on the beads.”
But aggregation was not the only problem the researchers encountered.
Hartrampf told delegates “The second challenge that we had was solubility. And so once we synthesize the peptide and then we want to purify it. And at low pH, the peptide fragment was unfortunately very poorly soluble.
“Afterwards we have to bring it to a different pH and even there we also saw it didn't really help so much. So we tried all kind of methods and it would just not really go into solution.”
Flow Chemistry Solution
Despite these issues, the use of flow-based chemistry rather than a solid phase approach prove useful when the team came to identify the causes of the challenges Hartrampf said.
“So usually when you synthesize on resin, it's quite difficult to see where aggregation is actually occurring and where it starts. However, with flow chemistry we can actually really easily see this.”
The real advantage is the speed of flow based synthesis reactions compared to solid phase reactions according to Hartrampf, who said “we can actually get a lot of data and look into this problem a lot more.”
