In the first 2 years of this project, we have made great progress towards our ambitious research vision of establishing N-N bond forming enzymes as versatile biocatalysts in organic synthesis (ACS Catal. 2025, 15, 310-342, 10.1021/acscatal.4c05268). We have generated libraries of NNzymes from three different families by genome mining and identified new interesting candidates for further biocatalytic applications. To unravel the potential of candidate enzymes, we extensively screened them by simple plate assays or LC/GC-MS analysis for substrate scope and identification of potential products/reactions. To demonstrate substrate promiscuity in the piperazate synthase (PZS) family, we analyzed various N-hydroxylated diamines as substrates other than the natural substrate. The N-hydroxylated diamines were obtained in situ using a panel of N-hydroxylating monooxygenases (NMOs), allowing subsequent cyclization by PZS to yield various N-N bond-containing heterocycles. The screened panel yielded 17 hydroxylated diamines and new promiscuous NMOs, thereby expanding the substrate set of poorly accessible hydroxylated products. The investigated PZSs led to a series of 5- and 6-membered N-N bond-containing heterocycles, 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 (DOI: 10.26434/chemrxiv-2025-f1l90). Overall, our data provide 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.
Screening of the remaining NNzymes from our libraries also revealed an unexpectedly broad substrate range, leading to additional N-N bonded products as pharmaceutical building blocks (unpublished data). In parallel, we were able to perform structural and mechanistic studies based on either structural models generated with AlphaFold or X-ray crystallography, and developed biocatalytic cascades involving NNzymes to demonstrate their synthetic potential for pharmaceutical synthesis (unpublished data).
Overall, in the first years of this ERC project, we have made significant progress in establishing NNzymes as versatile biocatalysts for applications in organic synthesis. The NNzyme candidates identified so far in our toolbox provide access to cyclic hydrazines, aromatic diazo compounds and aromatic hydrazones, thus offering a novel biocatalytic methodology to access these precursors. In particular, PZS provide a particularly attractive route to the synthesis of hydrazines as the enzymes do not require any cofactor and produce only water is a by-product of the reaction.
