Access to Nitrogen–nitrogen Bond-Containing Heterocycles Through Substrate Promiscuity of Piperazate Synthases

The nitrogen–nitrogen (N–N) bond motif comprises an important class of compounds for drug discovery. Synthetic methods are primarily based on the modification of N–N or N═N precursors, whereas selective methods for direct N–N coupling offer advantages in terms of atom economy and yield. In this context, enzymes such as piperazate synthases (PZSs), which naturally catalyze the N–N cyclization of l-N⁵-hydroxyornithine to the cyclic hydrazine l-piperazate, may allow an expansion of the current narrow range of chemical approaches for N–N coupling. In this study, we demonstrate that PZSs are able to catalyze the conversion of various N-hydroxylated diamines, which are different from the natural substrate. The N-hydroxylated diamines were obtained in situ using N-hydroxylating monooxygenases (NMOs), allowing subsequent cyclization by PZS, ultimately forming the N–N bond to yield various N–N bond-containing heterocycles. Using bioinformatic tools, we identified NMO and PZS homologues that exhibit distinct activity and stereoselectivity profiles. The screened panel yielded 17 hydroxylated diamines and more promiscuous NMOs, thereby expanding the substrate range of NMOs, resulting in the formation of previously poorly accessible N-hydroxylated products as substrates for PZS. The investigated PZSs led to a series of 5- and 6-membered cyclic hydrazines, and the most promiscuous catalysts were used to scale up and optimize the synthesis, yielding the desired N–N bond-containing heterocycles with up to 45% isolated yield. Overall, our data provides essential insights into the substrate promiscuity and activity of NMOs and PZSs, further enhancing the potential of these biocatalysts for an expanded range of N–N coupling reactions.