Macrocyclization and Fatty Acid Modification during the Synthesis of Nonribosomal Peptides
Nonribosomal peptides (NRPs) constitute a large and diverse class of pharmacologically important natural products that find useful therapeutic application as immunosuppressants, antibiotics, or anticancer agents. The biological activity of many of these compounds relies on the macrocyclic structure...
|Online Access:||PDF Full Text|
No Tags, Be the first to tag this record!
|Summary:||Nonribosomal peptides (NRPs) constitute a large and diverse class of pharmacologically important natural products that find useful therapeutic application as immunosuppressants, antibiotics, or anticancer agents. The biological activity of many of these compounds relies on the macrocyclic structure of their peptide backbone and the incorporation of a wide assortment of building blocks including proteinogenic and nonproteinogenic amino acids as well as modified fatty acid moieties. Particularly, these structural features are key determinants of nonribosomal lipopeptide antibiotics that are in the focus of this thesis.
To provide rapid access to these structurally demanding compounds, a chemoenzymatic approach towards the synthesis of the lipopeptide antibiotics daptomycin and A54145 was developed, based on the combined utilization of powerful solid phase peptide synthesis and the recombinant daptomycin and A54145 thioesterase (TE) domains. In vitro studies with these so-called peptide cyclases revealed their ability to catalyze the macrocyclization of linear peptidyl-thiophenol substrates with relaxed specificity for the cyclization nucleophile and electrophile. Ten lipopeptide variants were synthesized in order to explore the relatively sparse known acidic lipopeptide structure-activity relationship. Remarkably, this small library included a lipopeptide hybrid with a minimal inhibition concentration close to that of chemoenzymatic derived daptomycin as well as a bioactive macrolactam variant of A54145. Thus, single amino acid residues within the daptomycin and A54145 peptide sequences could be identified that are crucial for their antimicrobial potency.
Additionally, a unique and hitherto unknown type of imine macrocyclization as found for the cyanobacterial nostocyclopeptide (ncp) was investigated during the course of these studies. Experiments with ncp-CoA substrate mimics showed that a reductase (R) domain located at the C-terminal end of the ncp nonribosomal peptide synthetase (NRPS) is responsible for the reductive release of a reactive peptide aldehyde. Subsequently, imine macrocyclization occurs enzyme-independent under physiological pH conditions as proven with synthetic analogs of the ncp peptide aldehyde. An alanine scan experiment elucidated structural elements within the linear heptapeptide precursor that are essential for imine macrocyclization. Further, the biochemical characterization of ncp R also revealed its broad tolerance towards the C- and N-terminal amino acids of ncp substrate mimics.
In the third part of this work, the tailoring enzymes HxcO and HcmO from the calcium dependent antibiotic (CDA) trans 2,3 epoxyhexanoic acid biosynthetic pathway were chosen as a model system to investigate fatty acid modification during nonribosomal lipopeptide synthesis. While HxcO was characterized as a novel type of enzyme with dual function as an FAD-dependent fatty acid oxidase paired with intrinsic epoxidase activity, HcmO could be identified as a second epoxidase acting on 2,3-unsaturated fatty acids. Experiments with acyl-CoAs, acyl CoAs loaded onto an acyl carrier protein (ACP), and chemoenzymatically synthesized CDA variants revealed that both enzymes only accept ACP-bound substrates. To compare these ACP-bound HxcO and HcmO reaction products with synthetic standards a novel experimental approach had to be developed. Based on the thermodynamic activation inherent to thioester derivatives, the enzymatic products were cleaved from the ACP under mild conditions utilizing an amide ligation reaction and directly transformed into derivatives of smaller size suitable for HPLC-MS analysis. By the application of this versatile method the trans 2,3 epoxyhexanoic acid products of HxcO and HcmO were ascertained to have opposite absolute configuration, namely (2R,3S) and (2S,3R), respectively. In general, the established experimental approach holds great potential for the detailed analysis of all biochemical systems involving carrier protein-bound intermediates. These include integrated enzymes from NRPS and polyketide synthase (PKS) assembly lines or in trans acting tailoring enzymes|