Exploring how nature builds and uses the smallest, most powerful rings in its chemical arsenal
In the sophisticated chemical arsenal of nature, some of the most powerful tools come in the smallest packages. Cyclopropane, epoxide, and aziridine—three-membered rings of carbon, or carbon and oxygen, or carbon and nitrogen—are deceptively simple structures. Their geometric strain makes them unstable and highly reactive, perfect for organisms to use as molecular crossbows, ready to snap open and interact with precise biological targets 1 .
3 carbon atoms
2 carbon atoms + 1 oxygen atom
2 carbon atoms + 1 nitrogen atom
These unique rings are the warheads in many natural products, granting exceptional antibiotic, antitumor, and neurological activities to compounds like the antitumor agent duocarmycin and the antibiotic mitomycin 1 . For decades, chemists have struggled to synthesize these rings under mild conditions. Yet, nature's enzymes, or biocatalysts, perform these feats with ease at room temperature and in water.
The immense reactivity of three-membered rings stems from their fundamental geometry. The bond angles in these rings are forced to about 60 degrees, a significant deviation from the ideal 109.5 degrees for a tetrahedral carbon. This angle strain creates a "spring-loaded" system, storing energy that is released when the ring opens 1 .
Three-membered rings have bond angles of ~60° compared to the ideal 109.5° for sp³ hybridized carbon, creating significant ring strain.
The stored energy from ring strain makes these compounds highly reactive, perfect for biological targeting mechanisms.
In biological systems, this opening is precisely controlled. An enzyme can trigger a cyclopropane, epoxide, or aziridine ring to spring open at the exact moment to alkylate, or covalently bind, a target like DNA or a protein, disrupting cellular function and leading to the death of a pathogen or cancer cell 1 .
Enzymes like squalene synthase generate stable allylic carbocation intermediates that attack double bonds to form cyclopropane rings 1 .
Radical SAM enzymes use iron-sulfur clusters to generate carbon-free radicals for challenging cyclopropanations 5 .
For aziridine rings, sulfotransferase enzymes activate molecules for intramolecular nucleophilic attack 4 .
For years, the biosynthesis of the cyclopropane "warhead" in the potent antitumor compound CC-1065 was a mystery. Genetic studies pointed to a radical SAM enzyme, C10P, being essential, but its activity in the lab was puzzling. A breakthrough came when researchers discovered that a second enzyme, a methyltransferase called C10Q, was equally essential 5 . This led to the identification of a remarkable two-component cyclopropanase system.
The following table outlines the key reagents that made this experiment possible:
| Research Reagent | Function in the Experiment |
|---|---|
| Radical SAM Enzyme (C10P/Swoo_2002) | Catalyzes the radical-based reaction; contains a [4Fe-4S] cluster for reductive cleavage of SAM 5 . |
| Methyltransferase (C10Q) | Catalyzes the final intramolecular cyclization step to form the cyclopropane ring 5 . |
| S-adenosyl-L-methionine (SAM) | Serves a dual role: as a source of a 5'-deoxyadenosyl radical, and as the source of the methylene carbon for the ring 5 . |
| Substrate 6 | The late-stage biosynthetic intermediate of CC-1065 that lacks the cyclopropane ring 5 . |
| Sodium Dithionite | A reducing agent required to activate the [4Fe-4S] cluster in the radical SAM enzyme 5 . |
The genes for C10P and C10Q were expressed in E. coli, and the corresponding proteins were purified.
The radical SAM enzyme C10P was "reconstituted" with iron and sulfide to ensure its [4Fe-4S] cluster was intact and functional.
The complete reaction mixture was assembled under strictly anaerobic conditions with all necessary components.
Products were analyzed using high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy.
The results were clear and profound. Only when both C10P and C10Q were present in the reaction mixture was the final product, CC-1065, formed. All control reactions missing either enzyme failed, confirming their synergistic partnership 5 .
The two-enzyme system (C10P + C10Q) is essential for cyclopropane formation in CC-1065 biosynthesis.
Compound 7 was identified as an "off-pathway" product that helped illuminate the mechanism 5 .
C10P uses its [4Fe-4S] cluster to cleave a first SAM molecule, generating a highly reactive 5'-deoxyadenosyl radical. This radical abstracts a hydrogen atom from the methyl group of a second SAM molecule, producing a SAM methylene radical 5 .
This methylene radical adds directly to an sp²-hybridized carbon (C-11) on the substrate, forming a covalent SAM-substrate adduct. The methyltransferase C10Q then catalyzes an intramolecular SN2 reaction, where a carbon on the substrate attacks the methylene carbon, displacing S-adenosylhomocysteine and forging the cyclopropane ring 5 .
The study of these complex enzymatic pathways relies on a suite of specialized reagents and techniques. The table below details some of the most critical tools used by researchers in this field.
| Tool / Reagent | Explanation of its Role |
|---|---|
| S-adenosyl-L-methionine (SAM) | The "Swiss Army knife" of enzymatic chemistry; used as a methyl donor, a radical generator, and a source of one-carbon units for ring construction 4 5 . |
| Deuterium/Labeled Amino Acids | Isotope-labeled building blocks (e.g., D,L-[2,3,3-²H₃]serine) are fed to producing organisms to trace the origin of atoms in the final natural product, elucidating biosynthetic pathways . |
| Isothermal Titration Calorimetry (ITC) | A technique used to measure the binding affinity between two molecules, such as confirming the physical interaction between partner enzymes like C10P and C10Q 5 . |
| Heterologous Expression | The process of cloning a gene from its native organism (e.g., an actinomycete) into a lab-friendly host like E. coli to produce and study the encoded enzyme more easily 5 . |
| Alpha-Ketoglutarate (αKG) | A crucial cofactor for a family of non-heme iron enzymes; recently found to drive the oxidative cyclization that forms aziridine rings in enzymes like TqaL 6 . |
| 3'-phosphoadenosine-5'-phosphosulfate (PAPS) | The universal sulfonate donor for sulfotransferase enzymes; used to activate alcohols as leaving groups to facilitate aziridine ring closure, as in ficellomycin biosynthesis 4 . |
The world of enzymatic small-ring biosynthesis is a vivid demonstration of nature's chemical ingenuity. From the carbocation-driven cyclizations of terpenoid pathways to the radical-mediated collaborations and sulfation-triggered cyclizations, enzymes have mastered the art of building and deploying these strained, high-energy systems.
The discovery of the two-component cyclopropanase system for CC-1065 is just one example of the novel biochemistry waiting to be uncovered. The implications of this research extend far beyond understanding natural product biosynthesis.
Enzymes can be engineered and harnessed for the environmentally friendly production of pharmaceuticals.
Understanding how nature constructs these warheads enables design of targeted therapies like antibody-drug conjugates.
These enzymatic tools expand the capabilities of synthetic biology for creating new-to-nature compounds.
As research continues, the line between what nature can build and what chemists can create continues to blur, driven by the power of nature's tiniest rings.