Biosynthesis of new diketopiperazine natural products from unnatural amino acids

Ryan Lopez, Chase Webb, Rajesh Viswanathan, Amy L. Lane

Address for Chase and Raj: Department of Chemistry and Small Molecule Drug Discovery Core, Case Western Reserve University, Cleveland, OH

Diketopiperazines offer many clinical applications. Nocardioazine A is an anticancer diketopiperazine natural product isolated by Capon and co-workers from a marine actinomycete Nocardiopsis. This molecule exhibited anticancer properties by inhibiting P-glycoprotein to reverse the drug resistance that handicaps the efficacy of many anticancer therapies. Previous work from the Lane and Viswanathan groups showed that in E. coli expressing genes from the hypothesized nocardioazine A pathway, two separate enzymes catalyzed the same reaction to produce the nocardioazine intermediate, cyclo(L-Trp-L-Trp), from L-Tryptophan (L-Trp). Thus, we are testing the hypothesis that these enzymes may accept unnatural amino acids to yield cyclo(L-Trp-L-Trp) analogs. To test this, the E. coli cells were fed unnatural amino acids, then analyzed with liquid chromatography-mass spectrometry (LC-MS) to evaluate the production of molecules containing the unnatural amino acids.  Cyclo(L-Trp-L-Trp) analogs containing CH₃-, Cl-, or F- substituted Trp were detected, suggesting that it may be possible to tailor nocardioazine A analogs biosynthetically to evaluate the relationship between its structure and anticancer function. Additionally, this study highlighted a strategy for biosynthesis of molecules containing amino acids beyond the canonical 20 amino acids, clearing a path for increasing diversity of diketopiperazine natural products.