Development of a biotechnological toolkit for the synthesis of diverse cyclic peptides

Cyclic peptides--Synthesis;Biosynthesis;Macrocyclic compounds;Macrocyclic compounds;Natural products

Mann, Gregory ; Naismith, James ; Naismith, James

University:University of St Andrews

Department:Chemistry (School of)

关键词: Cyclic peptides--Synthesis;    Biosynthesis;    Macrocyclic compounds;    Macrocyclic compounds;    Natural products;   

Others  :  https://research-repository.st-andrews.ac.uk/bitstream/handle/10023/10826/GregMannPhDThesis.pdf?sequence=2&isAllowed=y

来源: DR-NTU

【 摘 要 】

Cyclic peptides possess desirable characteristics as potential pharmaceutical scaffolds.The cyanobactin family of cyclic peptide natural products boast diverse structures andbioactivity. Exemplars are the patellamides, which have attracted attention due totheir ability to reverse the effects of multi-drug resistance in human leukemia cells. Inaddition to their macrocyclic architecture patellamides contain azol(in)e heterocyclesand d-amino acids. This structural complexity makes them challenging targets forchemical synthesis. Understanding their biosynthesis will enable the developmentof a biotechnological ‘toolkit’ for the synthesis of new pharmaceutical compounds.Patellamides are ribosomally-synthesised and post-translationally modified peptides(RiPPs) and much of their biosynthesis has been elucidated, however there are stillelements of their biosynthesis that are not yet fully understood.PatA and PatG contain C-terminal domains of unknown function (DUFs). Thecrystal structure of PatG-DUF has been solved and subsequent to biochemical andbiophysical investigation PatG-DUF was found not to constitute an essential part of thebiotechnological ‘toolkit’ and can be excluded from in vitro enzyme-based synthesis ofcyanobactin-like cyclic peptides.The cyanobactin heterocyclases are able to introduce heterocycles into a peptidebackbone, seemingly irrespective of the neighbouring residues; however a molecularrational governing substrate recognition is unknown. Additionally the mechanism ofheterocyclisaton is disputed. Analysis of crystal structures of LynD in complex withcofactor and substrate (solved by Dr Jesko Koehnke) enabled the active site andsubstrate recognition site to be located. A new mechanism for heterocyclisation hasbeen proposed. Guided by the substrate recognition observed in complex structuresa constituently active heterocyclase (AcLynD) has been engineered, which is able toprocess short, leaderless peptide substrates.Epimerisation in cyanobactin biosynthesis is believed to be spontaneous, but its precisetiming is uncertain. NMR analysis of selectively labelled peptide substrates processed bythe modifying enzymes, identified epimerisation to be spontaneous on the macrocycle,regardless of whether the neighbouring heterocycles have been oxidised.A one-pot in vitro synthesis of cyanobactins has been developed, and employed to createa number of patellamide D analogues to ascertain structural-activity relationships.