Generating and transferring nitrenes enables unnatural biosynthesis in living cells

Nitrenes are among chemistry’s most useful and most difficult-to-control intermediates. These nitrogen-centred species can insert into carbon–hydrogen bonds, add across carbon–carbon double bonds, and rearrange molecular frameworks with remarkable speed. Yet the same reactivity that makes nitrenes attractive for synthesis also makes them dangerous inside a living cell, where they can attack proteins, nucleic acids and membranes before reaching their intended target. A study by Ian Donnell, Alex Quest, Jian Tang and colleagues, published in Nature Chemistry, reports a strategy for generating and transferring nitrenes inside living cells to produce molecules that biology does not ordinarily make.

The work addresses a central problem in synthetic biology: how to combine the selectivity of enzymes with chemical reactions that have no natural biological equivalent. Microorganisms are extraordinarily capable molecular factories, but their natural metabolic pathways are constrained by the reactions encoded in their genomes. Introducing an abiological transformation could expand the chemical structures that cells are able to manufacture, potentially creating new pharmaceuticals, advanced materials and specialty chemicals. Nitrene transfer is particularly appealing because it can form carbon–nitrogen bonds directly, often in a single step, without the lengthy sequence of reactions required by conventional organic synthesis.

A nitrene is commonly described as the nitrogen counterpart of a carbene. It contains an electron-deficient nitrogen atom with only six electrons in its valence shell, allowing it to react rapidly with nearby chemical bonds. Depending on its electronic state and environment, a nitrene may behave as a highly reactive singlet species or a less tightly paired triplet species. In practical biocatalysis, researchers often work with metal-bound “nitrenoids,” in which a metal centre and a nitrogen-containing reagent cooperate to control the intermediate. This coordination can channel the reactive nitrogen toward a selected substrate instead of allowing it to react indiscriminately with the contents of the cell.

Donnell and colleagues’ study focuses on making that control possible in living systems. Rather than attempting to release a free nitrene throughout the cellular environment, the researchers developed a process in which a biological catalyst generates the reactive nitrogen species and transfers it to an appropriate molecular partner. The concept separates two chemical tasks that are often difficult to perform simultaneously: activating a relatively stable nitrogen source and directing the resulting nitrogen fragment to a useful bond. By placing these steps under enzymatic control, the system aims to reduce unwanted side reactions while retaining the characteristic power of nitrene chemistry.

The approach is significant because living cells are chemically crowded reaction vessels. They contain millimolar concentrations of water, reducing agents, nucleophiles, unsaturated metabolites and thousands of proteins. Any unprotected nitrene would have many potential targets. Cellular metabolism also imposes strict constraints on oxygen levels, pH, cofactors and the availability of energy-rich molecules. A successful intracellular reaction therefore has to operate under mild conditions, tolerate biological components and avoid destroying the host cell. The reported platform demonstrates that nitrene transfer can be integrated into this environment rather than being restricted to a purified enzyme in a laboratory flask.

At the heart of the strategy is the use of biological machinery to control the timing and location of nitrogen activation. Enzymes achieve selectivity through three-dimensional binding pockets that position substrates and reactive cofactors with atomic precision. They can also use hydrophobic cavities, charged residues and hydrogen-bonding networks to stabilize transition states that would otherwise be too energetic for a cell to support. In a nitrene-transfer reaction, such features may determine whether nitrogen is inserted into a carbon–hydrogen bond, added to an alkene to form an aziridine, or diverted into an unwanted decomposition pathway. The study therefore represents not simply the introduction of a new reagent into cells, but the construction of a reaction environment around a highly reactive intermediate.

The researchers further show how the chemistry can be connected to biosynthetic production. In an engineered microorganism, the cell supplies the biological components needed to express the catalyst, while externally provided or metabolically generated precursors feed the abiological reaction. The resulting products can then be detected and analysed using analytical methods such as chromatography and mass spectrometry. This arrangement creates a hybrid manufacturing system: conventional metabolism provides the starting materials and cellular infrastructure, while nitrene transfer supplies a new chemical transformation. Such systems could eventually be expanded by modifying enzyme sequences, changing substrate-binding pockets or linking the reaction to pathways that make more complex precursors.

The ability to form new carbon–nitrogen bonds inside cells could have broad consequences for synthetic biology. Nitrogen-containing structures are common in medicines, agrochemicals, natural products and functional materials, but they are often difficult to assemble selectively. Direct C–H amination could convert a previously unreactive position in a molecule into a valuable amine or nitrogen heterocycle, reducing the need for protecting groups and repeated purification steps. Aziridination could create strained three-membered rings that serve as versatile intermediates for further chemical diversification. If these reactions can be directed toward chosen substrates, living cells might produce molecular architectures that are inaccessible through their natural enzymatic repertoire.

The research also highlights the importance of chemical containment. In conventional organic synthesis, a reactive intermediate can be generated under an inert atmosphere, surrounded by carefully selected solvents and protected from biological contaminants. Inside a cell, containment must be achieved through molecular design. The catalyst, precursor and target substrate have to work together so that nitrene formation occurs only when the desired reaction is possible. The study’s contribution lies in showing that this level of control is achievable sufficiently to support unnatural biosynthesis, while also identifying the practical boundaries that future systems will need to overcome, including catalyst efficiency, substrate transport, product toxicity and competition from native cellular reactions.

Although the technology remains at an early stage, its most important message is conceptual: living cells can host chemical reactions that evolution never selected, provided that the reactive intermediates are generated and directed with enough precision. Nitrene chemistry has long been associated with high-energy laboratory synthesis, but the new work places it within the toolkit of engineered metabolism. Future developments may combine nitrene-transfer catalysts with automated protein evolution, pathway engineering and real-time control of precursor delivery. Such advances could turn cells into programmable factories for nitrogen-rich compounds, allowing researchers to design not only biological pathways, but entirely new forms of chemistry that operate under the gentle conditions of life.

Subject of Research: Nitrene generation and transfer for unnatural biosynthesis in living cells

Article Title: Nitrene generation and transfer for unnatural biosynthesis in living cells

Article References: Donnell, I., Quest, A., Tang, J. et al. “Nitrene generation and transfer for unnatural biosynthesis in living cells.” Nature Chemistry (2026). https://doi.org/10.1038/s41557-026-02224-4

Image Credits: AI Generated

DOI: https://doi.org/10.1038/s41557-026-02224-4

Keywords: nitrene transfer, unnatural biosynthesis, synthetic biology, biocatalysis, living cells, carbon–nitrogen bond formation, enzyme engineering, metabolic engineering

Tags: bio-orthogonal chemical reactionsbioengineering of abiological reactionscarbon-nitrogen bond formationchemical reactivity control in cellsenzyme-inspired chemical transformationsexpanding metabolic pathwaysin vivo chemical synthesismicrobial engineering for chemical productionNitrene transfer in living cellsnitrogen-centered reactive intermediatessynthetic biologyunnatural biosynthesis