Cyclic tetrapeptides (CTPs) have broad ranging bioactivities and are proteolytic stable, which often translates to enhanced bioavailability, biostability, and membrane permeability compared to linear bioactive peptides. Pseudoxylallemycins are a class of naturally occurring N-methylated CTPs with a symmetrical [phenylalanine–N-methylleucine]₂ primary sequence. Psuedoxylallemycins have displayed antibacterial activity against disease-relevant strains of Gram-negative bacteria, representing a promising development in antibacterial research. Despite this promise, synthesis of pseudoxylallemycin and CTPs in general is complicated by the final macrolactamization reaction, where the linear tetrapeptide forms a 12-membered ring. Indeed, unfavorable transannular interactions destabilize CTPs, and the low yielding desired cyclization is often accompanied by excess tetrapeptide polymerization to octa- or dodecapeptides among other decomposition pathways. For pseudoxylallemycin synthesis, cyclization was accompanied by C-terminal epimerization of the linear tetrapeptide, leading to a diastereomer of the natural product. Our studies demonstrated that traditional phosphonium and uronium peptide coupling reagents caused full C-terminal epimerization during macrolactamization while less utilized propylphosphonic anhydride (T3P^®) uniquely modulated the diastereoselectivity of macrolactamization depending on the organic base included in the reaction. Stronger bases formed more of the undesired diastereomer (e.g. diisopropyl ethyl amine DR 17:83) and weaker bases provided more of the natural product (e.g. di-tert-butyl pyridine DR 92:8). The optimized synthetic conditions were leveraged to prepare pseudoxylallemycin A analogues with unnatural stereochemistry and alanine incorporation, which were used to assess the structure activity relationships. The synthesis of analogues unveiled the temperamentality of tetrapeptide ring closure, where differences in stereochemistry or amino acids had enormous impact on the yield and stereocontrol of macrolactamization. This led to the development of a high-throughput optimization system whereby macrolactamization reactions were miniaturized to allow for the exploration of numerous combinations of peptide coupling reagent, organic base, and solvent, and the reaction outcome is analyzed using LC-MS. This system was further applied to the rapid construction of other CTP natural products and analogues thereof.

Matthew OReilly

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