Next generation chemoenzymatic peptide synthesis (NXPEP)

Recently the Micklefield lab has made major breakthroughs in the discovery, characterisation and engineering of novel enzymes for use in synthesis. In addition, we have pioneered new methods for merging enzymes with chemocatalysis (integrated catalysis). The Procter lab has also developed ground-breaking, cleaner, and more efficient synthetic methods.7-9 In this project we aim to develop the first chemoenzymatic methods for peptide synthesis (NXPEP). Peptides perform many vital functions in life and their bioactivity has been exploited to provide essential medicines including insulin derivative for treating diabetes, antibiotics, anticancer agents and antiviral drugs, such as nirmatrelvir, Pfizer's recently approved treatment for COVID-19. 10-12 Synthetic peptides are also widely used as vaccines, drug delivery systems (cell-penetrating peptides), scaffolds for tissue engineering and other biomaterials. Despite their fundamental importance in life and diverse applications, the synthesis of peptides is reliant on old technologies which have major limitations, such as solid phase peptide synthesis (SPPS awarded the Nobel Prize 1984) where amino acids are coupled together on a solid support. SPPS and other chemical methods are difficult to scale-up, costly, atom-inefficient, rely on heavily protected amino acids, toxic/undesirable reagents and large volumes of solvents that are damaging to the environment and increasingly unsustainable.13-16 NXPEP will solve this long-standing problem by developing the first chemoenzymatic approach for more sustainable peptide synthesis, using ligase enzymes to couple amino acids together under benign aqueous conditions. By virtue of their mild operating conditions, selectivity, and evolvability,17 enzymes could provide more efficient and sustainable routes, to peptides making essential medicines and other valuable products more widely available.
We will initially focus on using two types of ligase enzymes, the amide bond synthases (ABS) and ATP-grasp ligases (AGL), which are present in biosynthetic pathways to various peptide natural products. In addition to these existing enzymes, we will also search for new ligase enzymes using bioinformatics techniques. The new enzymes will then be characterised, and their substrate scope tested with various natural and synthetic amino acid substrates. We will determine X-ray structures of the most interesting and promising ligase enzymes, which can be used to guide engineering improving catalytic activity and broadening substrate scope. Recently we showed that we can combine two mutually orthogonal ligases in one reaction, to produce tripeptides of defined peptide sequences controlled by the substrate selectivity of the individual enzymes. We aim to expand this solution phase enzymatic peptide synthesis approach, using a wider range of improved orthogonal ligases, to synthesise peptide-based drug targets in a single-step (one-pot) ligase cascade reaction, without any protecting group manipulation. We also aim to develop ligases for the synthesis of longer peptides sequences using a novel chemoenzymatic approach. In this case we envisage growing longer peptide chains on a cheap, water-soluble polyethylene glycol (PEG) support (carrier). Unlike SPPS, the synthesis of longer peptides will be in solution phase allowing higher substrate loading (concentrations) to be achieved. We will use membrane filtration technology (in collaboration with a company Exactmer) to separate the soluble growing peptide (attached to the carrier), from any smaller unreacted monomers. Our initial approach relies on the use of a ligase to couple azido acids, as the azido group is small and atom efficient, easily installed from amino acids.21 Following ligation the azido group can be reduced to the amine under very mild conditions using TCEP, which is a biocompatible water-soluble phosphine.