Traditionally peptides have been isolated by extraction from natural product sources, but typically this approach is limited to food-based products, not API molecules. Peptides can be made by fermentation with recombinant technologies used to express a particular peptide sequence in an industrial producer host like e. coli or yeast. This can be operated on scale, but separation of the product from biomass can be resource intensive and problems may arise from degradation of the product from host proteases. Another major drawback is that unnatural or non-canonical amino acids cannot be incorporated in the peptide. Molecular biology techniques do exist to insert unnatural amino acids but are expensive and currently used only at small scale, and in the future, cell-free synthesis might prove workable at scale.
It has been known for some time that isolated enzymes can synthesize amide bonds & peptides from esters and amines. Typically, natural or mutant proteases are used that have little activity to hydrolyze the peptide, either due to specific binding and recognition of certain ester groups, or by avoiding the hydrolytic reaction by removing the product from the enzyme solution as it is formed – usually by crystallization. Certain solvent additives can also promote amide formation over amide hydrolysis. Probably the most applied use of isolated enzymes in peptide synthesis is targeted ligation of specific shorter peptide chains. In this case two or three short chains are constructed using traditional LPPS or SPPPS then ligating by using an enzyme to join an activate ester at the C-terminus with the N -terminal amine of a second fragment.
There is currently a lot of work being published on natural and evolved lipases and esterases that have very hydrophobic active sites and favor ester/amide formation over hydrolysis and ligase enzymes with ATP recycling systems for the direct synthesis of amides from acids and amines.
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