Rapid Peptide Cyclization Inspired by the Modular Logic of Nonribosomal Peptide Synthetases.

Nonribosomal cyclic peptides (NRcPs) are structurally complex natural products and a vital pool of therapeutics, particularly antibiotics. Their structural diversity arises from the ability of the multidomain enzyme assembly lines, nonribosomal peptide synthetases (NRPSs), to utilize bespoke nonproteinogenic amino acids, modify the linear peptide during elongation, and catalyze an array of cyclization modes, e.g., head to tail, side chain to tail. The study and drug development of NRcPs are often limited by a lack of easy synthetic access to NRcPs and their analogues, with selective macrolactamization being a major bottleneck. Herein, we report a generally applicable chemical macrocyclization method of unprecedented speed and selectivity. Inspired by biosynthetic cyclization, it combines the deprotected linear biosynthetic precursor peptide sequence with a highly reactive C-terminus to produce NRcPs and analogues in minutes. The method was applied to several NRcPs of varying sequences, ring sizes, and cyclization modes including rufomycin, colistin, and gramicidin S with comparable success. We thus demonstrate that the linear order of modules in NRPS enzymes that determines peptide sequence encodes the key structural information to produce peptides conformationally biased toward macrocyclization. To fully exploit this conformational bias synthetically, a highly reactive C-terminal acyl azide is also required, alongside carefully balanced pH and solvent conditions. This allows for consistent, facile cyclization of exceptional speed, selectivity, and atom efficiency. This exciting macrolactamization method represents a new enabling technology for the biosynthetic study of NRcPs and their development as therapeutics.

Discovery of New Carbonyl Reductases Using Functional Metagenomics and Applications in Biocatalysis.

Enzyme discovery for use in the manufacture of chemicals, requiring high stereoselectivities, continues to be an important avenue of research. Here, a sequence directed metagenomics approach is described to identify short chain carbonyl reductases. PCR from a metagenomic template generated 37 enzymes, with an average 25% sequence identity, twelve of which showed interesting activities in initial screens. Six of the most productive enzymes were then tested against a panel of 21 substrates, including bulkier substrates that have been noted as challenging in biocatalytic reductions. Two enzymes were selected for further studies with the Wieland Miescher ketone. Notably, enzyme SDR-17, when co-expressed with a co-factor recycling system produced the anti-(4aR,5S) isomer in excellent isolated yields of 89% and 99% e.e. These results demonstrate the viability of a sequence directed metagenomics approach for the identification of multiple homologous sequences with low similarity, that can yield highly stereoselective enzymes with applicability in industrial biocatalysis.

Conversion of Alcohols to Phosphorothiolates Using a Thioiminium Salt as Coupling Agent.

We report a method for the direct and rapid conversion of primary and secondary alcohols to the corresponding phosphorothiolates in yields ranging from 64% to 97%, using as a coupling agent the iminium salt prepared from N,N-dimethylthioformamide and Meerwein's salt. Selective reaction of primary alcohols in the presence of secondary alcohols is possible. The reaction of secondary alcohols proceeds stereospecifically with inversion of configuration.

Biochemical characterization and selective inhibition of ß-carotene cis-trans isomerase D27 and carotenoid cleavage dioxygenase CCD8 on the strigolactone biosynthetic pathway.

The first three enzymatic steps of the strigolactone biosynthetic pathway catalysed by ß-carotene cis-trans isomerase Dwarf27 (D27) from Oryza sativa and carotenoid cleavage dioxygenases CCD7 and CCD8 from Arabidopsis thaliana have been reconstituted in vitro, and kinetic assays have been developed for each enzyme, in order to develop selective enzyme inhibitors. Recombinant OsD27 shows a UV-visible λmax at 422 nm and is inactivated by silver(I) acetate, consistent with the presence of an iron-sulfur cluster that is used in catalysis. OsD27 and AtCCD7 are not inhibited by hydroxamic acids that cause shoot branching in planta, but OsD27 is partially inhibited by terpene-like hydroxamic acids. The reaction catalysed by AtCCD8 is shown to be a two-step kinetic mechanism using pre-steady-state kinetic analysis. Kinetic evidence is presented for acid-base catalysis in the CCD8 catalytic cycle and the existence of an essential cysteine residue in the CCD8 active site. AtCCD8 is inhibited in a time-dependent fashion by hydroxamic acids D2, D4, D5 and D6 (> 95% inhibition at 100 µm) that cause a shoot branching phenotype in A. thaliana, and selective inhibition of CCD8 is observed using hydroxamic acids D13H and D15 (82%, 71% inhibition at 10 µm). The enzyme inhibition data imply that the biochemical basis of the shoot branching phenotype is due to inhibition of CCD8.